CORNELL UNIVERSITY. THE BostueU $1. -jPCotxier Itibrarg THE GIFT OF ROSWELL P. FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE 1897 2757 .^JNELL UNIVERSITY LIBRARY 3 1924 104 224 476 Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924104224476 CLINICAL PATHOLOGY OF THE BLOOD A TREATISE ON THE GENERAL PRINCIPLES AND SPECIAL APPLICATIONS OF HEMATOLOGY. BY JAMES EWING, A.M., M.D.,.. PROFESSOR OF PATHOLOGY IN CORNELL UNIVERSITY MEDICAL COLLEGE, NEW YORK CITY. SECOND EDITION, REVISED AND ENLARGED. ILLUSTRATED WITH FORTY-THREE ENGRAVINGS, AND EIGHTEEN COLORED PLATES DRAWN BY THE AUTHOR. LEA BROTHERS & CO., NEW YORK AND PHILADELPHIA. 1903. T No. lA^V Entered according to the Act of Congress, in the year 1903, by LEA BROTHERS & CO., in the Office of the Librarian of Congress. All rights reserved. top. I DORNAN, PRINTER. PROFESSOR T. MITCHELL PRUDDEN, M.D., LL.D., IN APPRECIATION OP HIS CONSTANT AID, FREELY RENDERED TO THE AUTHOR DURING A TERM OF SERVICE IN THE LABORATORY OF THE NEW YORK COLLEGE OF PHYSICIANS AND SURGEONS, THIS WORK IS GRATEFULLY DEDICATED. PREFACE TO SECOND EDITION. Duking the past two years the contributions to the knowledge of the blood in both its clinical and its pathological aspects have been very numerous, and, in many instances, of fundamental importance. In the present edition of this work the author has endeavored to incorporate the results of these recent studies in such form as will be found available both for the medical practitioner and for the student of hematology. This undertaking has required references in the text to about four hundred new articles or monographs, which have been added to the bibliographical lists. Among the changes thus introduced are considerable additions to the chapter on Technics, the serum test for blood, and the subject of crioscopy. The discus- sion of the morphology of blood cells has been enlarged and a new plate added. Some noteworthy additions to the knowledge of leukemia have been included in that chapter. The essential features of Ehrlich's theories on immunity have been presented, but the proper limits of the volume do not permit full discussion of this subject. Four new plates and various other figures have been added to the illustrations. The author acknowledges with thanks numerous valuable sugges- tions of his critics, and in many instances has been able to follow these suggestions in the revision. J. E. PREFACE TO FIRST EDITION. The rapid advances in the knowledge of the pathology of the blood and the multitudinous applications of this knowledge in clinical diagnosis which the last decade has witnessed have brought forth several critical treatises on hematology, and still furnish, as the writer believes, abundant reason for the preparation of another such work in English. Although the clinical bearings of the subject have been partly or fully accessible to English readers in some recent text-books, many later contributions to the pathology of the blood and blood-forming organs have had to be sought elsewhere, often in the original articles. This omission the present work endeavors to supply. Much of the theoretical discussion in the volume, abstracts of special articles, and reports of cases, have been set in fine print, so as not to encumber the main text, which has been constructed for the student and general reader. The wants of the laboratory worker and special student of hematology have been partly con- sidered in the chapter on Technics and Chemistry, and in the limited references to pathological anatomy. There are probably always some deficiencies in a treatise closely relating to clinical medicine when that treatise emanates from a pathological laboratory. Yet a comparison of the various extant works on the so-called " clinical " pathology of the blood has convinced the writer that clinical pathology is pathology still, and that a wide experience at the autopsy table and in the micro- scopic examination of diseased tissues furnishes an absolutely essential standpoint from which to view pathological changes in the blood. The present volume aims therefore to associate changes in viii PREFACE TO FIRST EDITIOS. the blood as closely as possible with lesions in the viscera, without which combination the former are very often unintelligible. In the preparation of the work all available sources of informa- tion have been freely consulted, and the writer has profited espe- cially by the labors of Ehrlich, Limbeck, Hayem, Lukjanow, Lowit, Grawitz, Stengel, Cabot, and many others. An endeavor has been made to discriminate between authorities, and in all details of important subjects the author has invariably consulted the original sources of information. "Works without bibliography have been of little value in this task, and the considerable number of references involved have therefore been included as a feature of the present volume. J. E. CONTENTS. INTRODUCTORY. ON THE INTERPRETATION OF ANALYSES OF THE BLOOD. PART I. GENERAL PHYSIOLOGY AND PATHOLOGY. CHAPTER I. TECHNICS. PAGE Qualitative tests for blood 23 Volume of red cells and plasma 31 Enumeration of blood cells 35 Hemoglobin, Iron 43 Histological examination of blood 53 Specific gravity 57 Alkalescence 59 Osmotic tension 61 Crioscopy ... 62 Bacteriological examination 67 Bibliography 68 CHAPTER II. CHEMISTRY OF THE BLOOD. Red cells, Leucocytes 71 Serum 75 The Whole Blood. . 77 The Blood Ash ... 78 Urea, Uric Acid, Glucose, Glycogen . 80 Acetonemia, Lipacidemia, Cholemia 83 Specific gravity 84 Osmotic tension . 88 Alkalescence, Basic Capacity. 90 Bibliography . 93 CHAPTER III. MORPHOLOGY AND PHYSIOLOGY OF RED CELLS. Structure and staining reactions . . 96 Degenerative changes ... 100 Numbers, Polycythemia, Oligocythemia . . 104 Bibliography ..... . 120 x CONTENTS. CHAPTER IV. THE LEUCOCYTES AND LEUCOCYTOSIS . PAGE Morphology . . 123 Numbers and proportions . - • 128 Degenerative changes . .130 Leucocytosis, classification . . ■ 133 Relation to immunity • 138 Ehrlich's theories . . 141 Clinical types of Leucocytosis • 146 Experimental Leucocytosis . • 155 Bibliography . 158 Eosinophilia . 161 Lymphocytosis 169 Bibliography . • .173 CHAPTER V. DEVELOPMENT OF BLOOD CELLS. Erythrocytes • 175 Leucocytes . . • 180 Blood Plates 186 Bibliography • 188 PART II. SPECIAL PATHOLOGY OF THE BLOOD. CHAPTER VI. CHLOROSIS. Etiology . . 191 Changes in the Blood . . .193 Regeneration of Blood ... . 197 Varieties of Chlorosis, Pseudochlorosis 198 Bibliography . . . ... 200 CHAPTER VII. PROGRESSIVE PERNICIOUS ANEMIA. Historical . . 202 Etiology .... . . .204 Pathological changes in Viscera . . . 212 Pathogenesis . ... 215 Changes in the Blood ... . . 216 Resume . . . . 222 Bibliography . . . 224 CHAPTER VIII. LEUKEMIA. Historical 227 Etiology 230 CONTENTS. x i PAGE Pathological changes in Viscera 234 Pathogenesis 237 Changes in the Blood 239 Diagnosis of Leukemia . 249 Bibliography . 251 CHAPTER IX. PSEUDOLEUKEMIA. Historical . . 254 Anatomical characters ...... . 255 Relation to Leukemia, Pernicious Anemia, Tuberculosis 258 Changes in the Blood . 262 Bibliography . 266 CHAPTER X. ANEMIA INFANTUM PSEUDOLEUKEMIA. SPLENECTOMY. Historical, Etiology 268 Pathological changes in Viscera 269 Changes in the Blood . 269 Significance of v. Jaksch's anemia 270 Bibliography 272 Splenectomy. Effects of Splenectomy in Animals 273 Splenectomy in Man 274 Bibliography • 276 PART III. THE ACUTE INFECTIOUS DISEASES. INTRODUCTORY SECTION. THE BLOOD IN FEVER. Chemical changes, Resistance of red cells . 277 Febrile Hydremia 278 Action of Bacteria upon the Blood 279 Bibliography . . . 280 CHAPTER XI. PNEUMONIA. DIPHTHERIA. Pneumonia. Gross changes, Red cells, Leucocytes . . 282 Bacteriology. . . . 289 xii CONTENTS. Diphtheria. page Red cells, Leucocytes . 290 Effects of Antitoxin 292 CHAPTER XII. EXANTHEMATA. Variola . 294 Vaccinia, Varicella 297 Scarlatina 298 Measles 300 CHAPTER XIII. TYPHOID FEVER. Red cells, Leucocytes 302 Bacteriology . . 306 Bibliography, on Pneumonia, Diphtheria, Exanthems, and Typhoid Fever 308 CHAPTER XIV. widal's test. The reaction, etc. 311 Bibliography 319 CHAPTER XV. MISCELLANEOUS INFECTIOUS DISEASES. Septicemia, Pyemia, Osteomyelitis 321 Appendicitis, Abscess, Erysipelas 325 Acute Rheumatism, Tonsillitis, Whooping-cough 327 Inflammations of Serous Membranes 330 Gonorrhea, Yellow Fever 331 Typhus Fever, Influenza . 332 Tetanus, Plague, Malta Fever 333 Actinomycosis, Glanders, Anthrax 335 Bibliography 336 CHAPTER XVI. SYPHILIS, TUBERCULOSIS, LEPROSY. Syphilis, Grades of Anemia 338 Effects of Mercury 339 Congenital Syphilis 342 Tuberculosis, Grades of Anemia . . 343 Tuberculosis of Meninges, Bones and Joints 347 Chemistry, Bacteriology . 348 Leprosy . 349 Bibliography 350 CONTENTS. xiii PART IV. CONSTITUTIONAL DISEASES. CHAPTER XVII. HEMORRHAGIC DISEASES AND DIATHESIS. PAGE Purpura Hemorrhagica 353 Hemophilia . 356 Scurvy 357 Hemocytolysis 359 Methemoglobinemia 364 Paroxysmal Hemoglobinuria 366 Bibliography 369 CHAPTER XVIII. MISCELLANEOUS CONSTITUTIONAL DISEASES. Diabetes 372 Obesity . 374 Addison's Disease 375 Osteomalacia 377 Rachitis 378 Myxedema . . 380 Bibliography . 382 CHAPTER XIX. NERVOUS AND MENTAL DISEASES. Mania, General Paresis, Epilepsy, etc. . 384 Beriberi 386 Chorea, Graves' Disease 386 Bibliography . 387 PART V. GENERAL DISEASES OF VISCERA. CHAPTER XX. THE HEMOPOIETIC SYSTEM. Liver, general considerations, special diseases . . 389 Esophagus . 394 Stomach, general considerations, special diseases . 395 Intestines, absorption and depletion, special diseases . 402 Bibliography 405 XIV CONTENTS. CHAPTER XXI. LUNGS, HEART, KIDNEYS. Diseases of Lungs, Asphyxia . Diseases of Heart, Malignant Endocarditis Diseases of Kidneys, Uremia . Bibliography Carcinoma Sarcoma Bibliography CHAPTER XXII. MALIGNANT TUMORS. PAGE 407 410 414 418 421 427 428 PART VI. ANIMAL PARASITES. CHAPTER XXIII. MALARIA. Technics Morphology of Parasites Plurality of species in Estivo-autumnal group Crescents, Flagellate Bodies Development in Mosquito Conjugation Occurrence of Parasites in the Blood Malarial Anemia . Leucocytes in Malaria Bibliography CHAPTER XXIV. RELAPSING FEVER. Morphology of Spirillum Changes in the Blood Bibliography CHAPTER XXV. 431 435 446 450 453 454 455 459 462 464 466 468 469 MISCELLANEOUS PARASITIC DISEASES. Trichina spiralis . Distoma hematobium .... Ascaris lumbricoides, Anguillula stercoralis Ankylostoma duodenale, Bothriocephalus latus Filariasis Trypanosomiasis Bibliography 470 472 473 474 478 480 481 Appendix . 483 LIST OF PLATES. PLATE P AGE I. Normal blood. Triacid stain 97 II. Normal blood. Eosin and methylene blue 124 III. Normal blood. Fixed in alcohol. Stained two hours by the Nocht- Romanowsky method . . . . . . . . .127 IV. Mild chlorosis. Eosin and methylene blue 194 V. Severe chlorosis. Eosin and methylene blue 198 VI. Progressive pernicious anemia. Eosin and methylene blue . .218 VII. Secondary pernicious anemia. Eosin and methylene blue . . 220 VIII. Myelogenous leukemia. Triacid stain 240 IX. Lymphatic leukemia, Eosin and methylene blue .... 244 X. Degenerating leucocytes in myelogenous leukemia. Triacid stain . 242 XL Mast-cells. Ehrlich's dahlia stain 248 Xir. Glycogenic degeneration of leucocytes. Iodine and potassium iodide in mucilage of acacia 289 XIII. Agglutinated red cells . . 361 XIV. Developmental cycle of benign tertian parasite ... . 436 XV. Cycle of quartan parasite . . 443 XVI. Cycles of estivo-autumnal parasite ....... 445 XVII. Flagellating malarial parasites. Cerebral cortex in pernicious malaria 453 XVIII. Conjugating cycle of tertian malarial parasite 455 INTRODUCTORY. ON THE INTERPRETATION OF ANALYSES OF THE BLOOD. Plethora. Although the older physicians regarded the existence of a true plethora as an important and well-established fact in path- ology, this view, lacking proof, received a serious blow from the work of Ludwig's pupils, v. Lesser and Worm-Muller, and from the authoritative conclusion of Cohnheim. Lesser and Worm- Muller, attempting to produce artificial plethora by transfusion of blood in animals, found that when the red cells were thereby in- creased 28 per cent, the numbers fell to normal on the following day, and when increased 58 per cent, the blood became normal on the twenty-third day, while the total quantity of blood might be doubled without abnormal symptoms, owing to the rapid return to the normal quantity. Likewise, Hamburger was unable to produce a permanent plethora in horses, by the injection of large quantities (7 litres) of 5 per cent, solution of sodium sulphate, finding that the isotonic power of the blood, thus increased, became normal in one-half to two hours, while nearly all traces of the salt disappeared from the blood in twenty-four hours. Instead of drawing from these data the conclusion that continuous plethora cannot be induced artificially, the unwarranted claim was advanced that a true plethora does not exist (cf. v. Eecklinghausen, Lukjanow). Against this con- clusion stood the daily observation of clinicians and pathologists, at the bedside and in the dead-house, that there are extreme variations in the quantity of blood in the vessels of different subjects, in divers states or health and disease. Oertel especially strongly maintained, without being able to offer proof, that the volume of blood might be reduced in endocarditis and with good therapeutical effect. Positive data on the question were gathered by Bergmann and Heissler, pupils of Bollinger, and the fact established that there is, in general, a direct ratio between the volume of blood and size of the heart, and the muscular development of the subject, and an indirect ratio with the subject's fat deposits. Though lacking full experimental proof it is now generally accepted that the quantity of blood in the body is variable, may be increased by hygienic measures, and is diminished in many unhygienic and pathological conditions. Anhydremia, i. e., a reduction in the volume of blood with concen- tration of solids, must be admitted to result from loss of body fluids, as by sweating and diarrhea, or by diminished ingestion of water. Czerny exposed cats in a warm, dry chamber for thirty-six hours, finding that they lost 45 per cent, of their weight, the volume of 2 18 INTRODUCTORY. blood was greatly reduced, its viscosity increased, and the red cells rose, in one case, to ten millions per cubic millimetre. Grawitz found that profuse sweating is followed in the majority of cases by a concentration of the blood, in one instance from specific gravity 1040 to 1051. A few subjects showed a contrary reaction and diminution in gravity (1060 to 1057.5), which Grawitz refers to nervous influences. Limbeck examined the blood of a case of cirrhosis of the liver with extreme ascites, before and after tapping the abdomen. On the day after the removal of 18 litres of fluid the red cells had risen from 3,280,000 to 5,160,000. Stintzing and Gumprecht have made similar observations before and after the removal of large serous exudates. Grawitz found the specific gravity of the blood at first decreased by the intravenous injection of concentrated salt solutions (absorption of water), while the administration of the salt by mouth concentrated the blood. It therefore follows that the ingestion of water and the loss of body fluids always produces a more or less transitory effect on the volume of the blood. There can be no doubt also that the prolonged sweats of phthisis and the severe diarrhea of typhoid fever, dysentery, and cholera lead to a more or less continuous reduction in the volume of the blood and a concentration of blood cells. A very striking illustration of this principle was frequently encountered by the writer among the soldiers at Camp Wickoff (1898). When these patients, suffering from prolonged malaria with severe anemia, were attacked by typhoid fever or acute dysentery, the ordinary watery character of the expressed blood drop disappeared, and the blood soon became thick and deep red. Herz claims to have recognized an " acute swelling " of the red cells in febrile conditions, especially in typhoid fever, and v. Lim- beck, finding a considerable average increase in the volume of the red cells in eight febrile cases, believes that relative oligoplasmia with increased volume of red cells is of frequent occurrence in high fevers. The effect of nervous influences in altering the quality of the blood in the whole or a part of the circulation has been demonstrated by numerous studies. Cohnstein and Zuntz found that section of the cord above the origin of the splanchnic nerves is followed by general dilatation of bloodvessels, and reduction in the proportion of red cells. Grawitz and Knopfelmacher found, in general, that vasomotor paralysis is followed by local and general increase in the volume of blood, with diminished specific gravity and proportion of red cells, while the opposite effects follow vasomotor constriction of vessels. The nervous stimulus of cold baths has been found by Leichten- stern, Wick, Knopfelmacher, Winternitz, Thayer, and Grawitz to be followed by contraction of vessels and increase in the proportion of red and white cells, while the hot pack and the inhalation of amyl nitrite have an opposite effect. To a similar origin must be attributed the increased gravity of the blood observed by Grawitz after the injection of tuberculin, and INTERPRETATION OF ANALYSES OF THE BLOOD. 19 cultures of the bacillus of cholera and of diphtheria, and the oppo- site effect produced by streptococcus and staphylococcus pyogenes and B. anthracis. The influence of psychical emotions on the character of the blood in different regions of the body has been emphasized by Lloyd Jones, and is seen especially in the study of the blood in neurotic women. Jacobi, examining the blood of an hysterical woman, found on November 12th 3,892,000 red cells j on December 11th 8,084,000 red, 102,200 white cells ; on December 16th 3,393,000 red, 22,000 white. Grawitz refers to the remarkable increase seen in the specific gravity and proportion of cells in the blood of rabbits, in experi- ments conducted without anesthesia. The remarkable variations in the blood taken from the ears of rabbits, depending on the tempera- ture and blood-content of the member, are a matter of common observation. In the mechanism by which these changes are brought about there appear to be many factors. Heidenhain, Lowit, Gartner and Romer, find that many substances injected into the blood cause an increased flow of lymph. (Extract of cancerous tumors, peptone, tuberculin, toxins of B.pyocyaneus and pneumococcus, hemi-albumose, nuclein, uric acid, etc.) Heidenhain and Hamburger believe that some sub- stances excite a secretory action of the capillary endothelium, whereby the fluids of the blood are diminished. Grawitz explains the concentration of the blood under the influence of cold by the escape of fluids into the tissues, but the experiment of Cohnstein and Zuntz offers another explanation, since they found under the microscope that capillaries might be so reduced in calibre by irritation of vasoconstrictor nerves that no red cells, but only plasma, could pass in them. It then appears that many red cells may be caught in the contracted capillaries while the plasma passes on into the veins. Grawitz concluded from many comparative tests that the capillary blood is, in all ordinary conditions, richer in cells than that of the veins ; but Becker found no such differences in the cells of veins and capillaries, and thinks that the polycythemia of cold is referable both to the escape of fluids from the blood and to the sifting of cells by the contracted capillaries. It does not appear to have been proven that the vascular dilatation produced by heat is followed by the return of tissue fluids into the capillaries. It appears quite as likely that an irregular distribution of cells and plasma is to a larger extent responsible for such local variations in the composition of the blood (cf. Winternitz). The law that increased blood pressure leads to transudation, authoritatively stated by Ludwig and Landois, appears to have a less important application here than in more general and more prolonged processes. It is not likely that a cold bath can be followed by much exudation of serum into the tissues. Massage and electricity have been shown, by Mitchell and Cheron, to cause an immediate increase in the number of red cells in the blood of a part, an effect which may be explained according to the above data. 20 INTR OD UCTOB Y. Ekgren found that ten minutes of active massage of body or abdomen increased the peripheral leucocytes, chiefly the polynuclears, from 1000 to 3000, once 7000. The increase was not marked ten minutes after the massage. Oliver was able to lower the red cells of the finger 7 per cent, by elevating the arm, while elevation of the leg for fifteen minutes lowered the red cells of the toe 14 per cent., and raised them in the finger 3 per cent. Exercise and faradism caused an increase of 5 to 8 per cent, in the peripheral blood. A Turkish bath caused an increase of 15 per cent. The importance of the osmotic relations of the blood in controlling the volume of red cells and plasma is indicated from the discussion in Chapter II. In hydremia and anhydremia Hamburger has shown that the isotonic relations of the blood are maintained by a rapid interchange of salts and albumins between the cells and plasma, with frequent minor changes in their relative volume. From Lim- beck's analyses of venous and arterial blood it appears, also, that the imbibition of C0 2 causes swelling of red cells with absorption of water and salts and with relative diminution in the volume of plasma. The foregoing considerations are dwelt upon not only because of their theoretical interest, but because they deal with fundamental facts without the knowledge of which it is impossible to properly perform or sensibly interpret the results of an examination of the blood. To summarize the discussion, it has been shown that there are wide variations in the quantity and quality of the blood referable to diverse conditions other than disease. 1. There are considerable physiological variations in the volume and composition of the blood, according to the constitution of the individual (plethora), and the degree of muscular development. Here may be classed the variations between the sexes and between different periods of life. Such variations are permanent but usually not of extreme grade. 2. There is a great variety of physiological conditions producing marked but transitory changes in the blood, such as active digestion, muscular exertion, the ingestion of fluids, profuse perspiration, tem- porary cyanosis, etc. 3. The nervous system has a very striking temporary influence on the quality of the blood in local or general areas, acting through the cerebral (psychical) or medullary centres, or through local vasomotor nerves. 4. Various local influences may greatly change the quality of the blood specimen, as seen in the local and transient effects of cold, heat, massage, and electricity. 5. Many therapeutical procedures may temporarily alter the blood, as the aspiration of fluids, administration of diaphoretics, purges, vasodilators (amyl nitrite), vasoconstrictors, etc. INTERPRETATION OF ANALYSES OF THE BLOOD. 21 6. Various pathological conditions may partly or completely obscure the real status of the blood, as the sweats of phthisis ; the diarrhea of typhoid fever, dysentery, and cholera ; general cyanosis or local stasis ; the increased arterial tension of uremia ; the polyuria of diabetes and nephritis ; antemortem cardiac failure, etc. Having regard to the possible action of any of the above influences, one may avoid many of the local disturbances by observing special care in the manner of expressing the blood specimen. The blood should be expressed by very slight pressure, exerted at a distance, from a liberal puncture of the finger-tip or ear-lobe. The circulation in the part should be as nearly normal as possible and should be uniform. A cold, bloodless tissue is not suitable for fur- nishing a blood specimen, and if artificial means are taken to correct the condition a sufficient period must elapse to allow the accelerated circulation to subside. Blood should be taken not less tban four hours after a hearty meal, and when comparative tests are made the specimens should be taken at the same hour each day. The examination having been performed, its results are to be interpreted only in the light of the fullest possible clinical information. There can be no doubt, as Grawitz has pointed out, that the numer- ous contradictory results of hematological studies are largely referable to hasty conclusions drawn from figures without regard to the condi- tion of the patient or the stage of the disease under consideration. Likewise, hematological diagnosis has fallen into much discredit from the tendency to offer opinions from the isolated findings of the blood test. PART I. GENERAL PHYSIOLOGY AND PATHOLOGY. CHAPTER I. TECHNICS. QUALITATIVE TESTS FOR BLOOD. Demonstration of Red Cells. By far the most delicate for blood is the demonstration of red cells under the microscope, but as the cells are not always preserved in demonstrable form one must usually resort to tests for hemoglobin and its derivatives. To demonstrate red cells in dry blood stains one should soften the scrap- Re. 1. Hemin crystals. (Rieder.) ings in some agent which will not lake the corpuscles. Many of the fluids recommended for this purpose are active laking agents for dried red cells — e. g., 0.8 per cent, salt ; glycerin, 50 per cent.; sodium sulphate, 75 per cent. A strong alkaline solution, as KOH 30 per cent., is effective in separating the corpuscles and does not dissolve them. 24 GENERAL PHYSIOLOG Y AND PATHOLOG Y. The Guaiacum Test. To a watery solution of the suspected substance are added a few drops of tincture of guaiac, producing a milky precipitate. A few drops of hydrogen peroxide added will, in the presence of blood pigment, produce a distinct blue color. The tincture of guaiac should be freshly made, and diluted to the color of pale sherry wine. The guaiac must be added before the per- oxide, and the blue color must be developed immediately. Many substances turn tincture of guaiac blue, without the presence of per- oxide. This test is very delicate, demonstrating one part of fresh blood in several thousands of water. It is most effective with fresh specimens ; but after two or three years some specimens fail to react satisfactorily. Brandenburg finds that leucocytes of myelogenous origin, but not the lymphocytes, give this same reaction. The guaiac test demonstrates the presence of peroxidases, which exist in most animal and vegetable tissues. The Hemin Test (Teichmann's). A drop of blood or portion of suspected detritus is spread out on a glass slide, mixed with one drop of salt solution and dried at a low temperature. A cover-glass is then laid over the specimen, filled beneath with glacial acetic acid, and the specimen evaporated with higher heat, but without boiling. When the fluids have entirely evaporated the specimen may be mounted in distilled water and examined microscopically for the characteristic crystals of hemin (Teichmann's crystals). The hemin test, if successful, is absolutely reliable, but often fails, in unskilled hands, from (1) alteration of albumins by excessive heat, preventing the formation of crystals ; (2) excess of salt solu- tion ; (3) violent boiling which drives off free HO, thus preventing the formation of hemin crystals (hydrochloride of hematin). Hemin crystals often fail to form from old blood stains in which the -pigment has become comparatively insoluble from the formation of hematin. In such cases a successful result may sometimes be secured by allowing the specimen prepared in the usual way with excess of dilute acetic or hydrochloric acid to stand overnight (Richter). When the stain is very faint or diffuse, or washed, the blood pig- ment may be concentrated, as recommended by Wood, by dissolving the material in weak alkaline solution, precipitating the pigment by boiling after the addition of a few drops of saturated solution of sodium tungstate strongly acidified by acetic acid, collecting the brownish precipitate, and performing the usual hemin test upon it. MEDICO-LEGAL SERUM DIAGNOSIS OF BLOOD. One fruitful result of the recent trend of study of the mechanism of immunity has been the discovery of a delicate and reliable method of distinguishing human blood from that of lower animals. The TECHNICS. 25 first step in the elaboration of this method of serum diagnosis con- sisted in the discovery by Tschistowitsch that the serum of an animal treated by injections of defibrinated blood or of serum from another species of animal develops the power of agglutinating and dissolving the red cells or of causing a precipitate in the serum of the second animal. This effect was found to be limited to the species of animal whose blood was used in the injections, while on the blood of other animals the serum remained inert. It soon became apparent that the serum of an animal could be made to develop specific powers in relation to various animal tissues and albumins and even to vegetable albumins, and in 1900 Wasserman pointed out that the biological method was probably capable of separating between all albumins of whatever source. Almost simultaneously Deutsch, Uhlenhuth, Wasserman and Schutze, and Stern applied the method to the identification of blood for medico-legal purposes, and showed that it was possible thereby to determine the origin of old blood stains. Deutsch immunized rabbits by defibrinated human blood and pro- duced specific agglutination and solution of the red cells, finding that the test could be applied to minute quantities of blood cells prepared in the hanging drop. His method is of value in deal- ing with small amounts of blood in which the red cells are still intact, and, as will be seen, gives reliable information regarding the nature of more minute stains than can be treated by the method of precipitation of albumins. The other observers employed defibrin- ated blood and secured sera which caused precipitates in solutions of stains. Method of Preparation of the Serum. The rabbit is the animal usually employed, and three or four large healthy ones should be selected. They should be housed under the best hygienic condi- tions. The writer has had very satisfactory results with chickens, and there are theoretical reasons for preferring this animal to the rabbit. Defibrinated blood squeezed from the fresh placenta or drained from the umbilical cord should be used for the injections, although less potent sera have been obtained by the use of albuminous urine (Stern, Leclainche and Vallee) or of pleuritic exudate (Dinkel- spiel, Dieudonne), or of blood from the cadaver (Modica, Ziemke). The placental blood may be poured into 100 c.c. Erlenmeyer flasks containing broken glass or beads, and defibrination accomplished by shaking the flasks for a few minutes. Or the blood may be allowed to coagulate and the serum used which separates after forty-eight hours. The former method is preferable, as it yields a more hemo- lytic antiserum. From six to eight injections of 5 to 10 c.c. each, at intervals of three to five days, are required to develop a serum of sufficient potency for practical use. The intraperitoneal is the preferable route, and the animals usually tolerate the injections well. If they lose weight, treatment should be suspended for a week. Strict asepsis in the handling of the blood and in the injections is, of course, essential. 26 GENERAL PHYSIOLOGY AND PATROLOG Y. The serum may be tested from time to time by drawing a little blood from the ear vein, and after a sufficient course of treatment the animal may be exsanguinated by exposing the carotid and draw- ing the blood through a canula ; but it is well not to sacrifice the animal, but to bleed from the marginal ear vein, as a very little sub- sequent treatment serves to restore the potency of the serum in an animal which has once been bled. The drawn blood should be allowed to coagulate, and the serum drawn off as clear as possible, centrifuged, if necessary, sealed in glass tubes, and kept on ice. Preservation of the active agent may be secured by adding a few drops of chloroform, or by evaporating the serum on glass plates, the dry residue being dissolved in salt solution when needed (Modica and Nobell). In testing a suspected stain the material should be dissolved in a very little 0.6 per cent, solution of XaCl (c. p.), or in 0.1 per cent. ISTaOH, as preferred by Ziemke, or, if the stain is old and fails to dissolve readily, in concentrated potassium cyanide with subsequent neutralization with tartaric acid (Ziemke). The solution, if turbid, may usually be cleared by the centrifuge or by filtration through Schleicher's filter paper (blue ribbon), or through an asbestos filter. Small test tubes — diameter 0.5 cm. — are required in the work. To the filtrate should be added the active serum in proportion not greater than 1 : 50. In less dilutions spurious reactions may occur. The writer finds it advisable to prepare several controls in each test, one of known human blood, and one of the salt or other solu- tion used in dissolving the blood stain, one of blood from another animal, one of human blood to which normal rabbit serum is added, and one of the dissolved stain which shall remain untreated. With- out such controls a short experience with the method will convince one that errors will not infrequently arise from unknown factors. All of these specimens should be placed in the thermostat. The reaction is positive when a distinct turbidity appears instantly or within a few minutes, and when a flocculent precipitate forms within three hours. Less definite reactions, such as uniform turbidity, cannot at present be accepted (Ziemke), although sera of moderate potency give only turbidity at first and require more than three hours for precipitation. It is, therefore, important to secure a serum of high potency, while, on the other hand, the very powerful sera are more apt to cause turbidity in heterologous bloods (Strube, Lenossier and Lemoine). On the other hand, the more powerful sera can be safely diluted beyond the point where reactions in heterologous blood can occur. The reaction does not fail with very old specimens of blood, although it becomes less distinct the older the specimen. Ziemke obtained a cloudiness in three hours from a blood stain twenty-five years old ; from blood mixed with earth three years old ; from decomposed blood ; and from human blood highly diluted with six other kinds of blood. A solution of iron-rust gave no reaction. Uhlenhuth secured positive results with blood of the three months' TECHNICS. 27 decomposed cadaver, and with soapsuds containing washed blood. The reaction fails when dry blood has been heated one hour at 130° C. (Ferrai). Solutions of blood stains on leather may precipitate spontaneously, probably from the presence of tannin. The delicacy of the test is very great, a reaction having been obtained in solutions of blood too weak to give the absorption spectra, and in solutions of 1 of serum to 50,000 of blood (Stern). Limitations of the Test. The reaction is not, in one sense, specific, and there are only quantitative differences in the results obtained by adding humanized sera to various types of blood. However, these quantitative differences are very great, and in a large series of animals Stern, Wasserman and Schutze, and Nuttall, found none to react to humanized sera in any degree comparable to human blood except that of the monkey. There are several reports in the litera- ture showing that special sera added in slight dilution (1 : 5, 1 : 20) cause reactions in the blood of many animals not very closely related, but it has been fully shown that if the test serum is diluted up to 1 : 50 or 1 : 100 and added to blood diluted 1 : 20 or 1 : 100, the results become absolutely specific. It is necessary, therefore, to attend care- fully both to the dilution of the serum to be tested and to that of the active serum used in the tests. Kister and Wolff, and Strauss and the writer have found that most reactions in heterologous bloods may be avoided by diluting the test serum 1 : 50. Indeed, most of them fail to appear when the serum is diluted 1 : 30. The writer has compared the reactions of human and monkey blood (rhoesus) treated by several potent humanized rabbit sera, and finds that the reaction fails in this monkey blood when the test serum is diluted 1 : 50. The strength of the solution of the blood to be tested, if not greater than 1 : 1000, made comparatively little difference. The method is not capable of distinguishing the chemical variety of albumin, as Rostocki showed that rabbit serum potent against horse globulin precipitated also serum albumin of the horse. A more practical difficulty in identifying blood is found in the fact that the active serum precipitates albumins from exudates, urinary and pleuritic, etc., and from sputum. Strauss and the writer secured good precipitates from albuminous diarrheal stools not containing blood. It must, therefore, first be determined by the hemin test that the stain is blood. Contamination by pus, feces, sputum, or urine can usually be detected by appropriate tests. Sources of Error. 1. Turbidity or precipitate may occur in any blood from too slight dilution of serum. 2. Potent sera considerably diluted (1 : 20) cause turbidity in solutions of blood from closely related animals. 3. The chemicals used may be impure and cause precipitates. 4. Chloroform does not inhibit all bacterial growth, and precipi- tates occurring after three hours may be referable to this cause. 5. The reaction of the solution may be too acid or too alkaline, and the reaction fails or it occurs in heterologous blood (Ziemke, Kostocki). The reaction should be neutral or slightly alkaline (Lenossier, Lemoine), or slightly acid (Rostocki). 28 GENERAL PHYSIOLOG Y AND PATHOLOG Y. 6. The blood may be mixed with chemicals which cause precipi- tates, or human albuminous or mucous exudates may be present in the solution and react as blood albumins (cf. Graham-Smith and Sanger). From the foregoing discussion it is evident that full medico-legal requirements for the positive identification of blood stains may be met, under some conditions, by the biological test. These conditions are the positive proof by the hemin test that the material is blood and the occurrence of a flocculent precipitate appearing within one to three hours in the suspected specimen and in no other of the several controls when a potent serum is added in proportion of 1:50 of blood. While these conditions can usually be secured when dealing with fresh blood in considerable quantities, in the writer's opinion and experience the material submitted for medico-legal examination is more apt to be old, scanty, and impure, and the difficulty of secur- ing a fully satisfactory test by this method is thereby greatly increased. With such material one has often to be content with faint turbidities instead of flocculent precipitates, and in such cases it would appear, as Stoenesco maintains, that a guarded opinion be given and the claim made only that the specimen is probably human blood. It should be added that an absolutely faultless technique is required, and that this can be obtained only after considerable experience. SPECTROSCOPIC EXAMINATION OF BLOOD. In all cases where a sufficient quantity of dissolved blood can be obtained for examination the spectroscopic test is the best means of determining not only the presence of blood pigment but also its par- ticular form. Fig. 2. Browning's spectroscope. Of fresh blood a 1 per cent, solution yields very distinct absorption bands. Recently clotted blood dissolves readily in water. Old clots may usually be dissolved by maceration in acetic acid, after which the spectrum of acid hematin is obtained. Clots that have TECHNICS. 29 been exposed to heat must be macerated in ammonia, when the spectrum of reduced or of alkaline hematin will result. For ordinary clinical work Browning's spectroscope is very satis- factory, but absorption bands are more accurately located in larger instruments. The small instrument should be supported in a con- venient holder in strong daylight or gaslight. By means of a collar the width of the aperture may be varied according to the strength of the light and opacity of the fluid. By means of the sliding tube Fraunhofer's lines are brought into accurate focus. The fluids should be examined in small glass vials with flat sides. When dealing with fresh blood and unaltered Hb the spectrum is that of oxyhemoglobin, which shows two absorption bands between D and E, one rather thin and sharp near the orange, and the other broader near the green. The indigo and most of the blue is absorbed. In strong solutions the two bands of oxyhemoglobin may be united. Fig. S. Bed Orange Yellow Green Blue Indigo A a B C D Eb F ( "1 I m \ifh^^m,s^ 6 '■: c d II * : t. i e !;'/ ; j; i f iliiii .'if > h ' Oxyhozmoglobin Reduced Haemoglobin Beduced Hcematin Methoemoglobin .Acid Co-Haemoglobin Hcematin Alkaline Spectra of blood pigments. (After Limbeck.) If to the solution of oxyhemoglobin is added a little reducing sub- stance, such as ammonium sulphide, the color of the fluid becomes darker and the spectrum changes, becoming that of reduced hemo- globin, giving one broad absorption band between D and E. " The transformation of the spectrum of oxyhemoglobin to that of reduced Hb, by reducing agents, is the positive indication of the presence of blood. Cochineal and ammoniated carmine give spectra very similar to that of oxyhemoglobin, but on the addition of boric acid the spectra of these substances are displaced into the blue, while that of blood remains unaffected. The spectra of various other vegetable dyes simulate that of blood, but these are bleached by sodium bisulphite. Hematin is produced by the addition of acids or strong alkalies to reduced Hb. In acid solution its spectrum is similar to that of acid methemoglobin. In alkaline solution it gives a single rather broad band at D. 30 GENERAL PHYSIOLOGY AND PATHOLOGY. The end-product of the alteration of hemoglobin found in old blood stains is iron-free hematoporphyrin, which must be identified by the spectroscope. Kratter and Hammerl claimed to have ob- tained the spectrum of this pigment from specimens of incinerated blood. To prepare a solution of hematoporphyrin the suspected stain should be dissolved in concentrated sulphuric acid, which should yield a reddish violet fluid in one-half hour. The spectrum of hematoporphyrin resembles that of oxyhemoglobin, but the bands are displaced further to the left, especially the small band to the left of D. In alkaline solutions the spectrum resembles that of acid hematin (Fig. 4). Fig. 4. Spectra of hematoporphyrin. (1) in acid solution, (2) in alkaline solution. Clinically, the most important alteration of oxyhemoglobin is that into methemoglobin, sometimes detected by the chocolate color of the blood. In acid or neutral solution it gives four absorption bands, one quite sharp, between C and D ; a second, faint, in the yellow, immediately to the right of D ; a third, broad, rather distinct, between the yellow and green, and just to the left of E ; a fourth, broad, to the left of F, sometimes merged with a complete absorption of the blue end of the spectrum. The demonstration of carbonic-oxide-Hb is of great clinical im- portance in cases of gas poisoning. The blood drop has a rosy red tinge, seen alike in both venous and arterial blood. In h per cent, dilution a spectrum is obtained which resembles that of oxy- hemoglobin, but the bands are broader, and the D band is displaced slightly to the right. On the addition of ammonium sulphide the spectrum of oxyhemoglobin is replaced by that of reduced Hb, while Co-Hb remains unaltered. In applying this test clinically, since considerable oxyhemoglobin remains in the blood in most cases, the results of spectral analysis are not always clear, and corroborative tests are required. 1. Warm the specimen with equal parts of 10 per cent. NaOH ; normal blood becomes dark brownish green, the other becomes cloudy, then clear red, and red flakes gather on the surface (Hoppe- Seyler). 2. To a 2 per cent, solution of blood add a few drops of orange- colored ammonium sulphide containing an excess of sulphur, and faintly acidify with a few drops of dilute acetic acid, carefully shak- ing. Carbonic oxide blood then shows a beautiful rose-red color, TECHNICS. 31 with a flocculent precipitate, while normal blood becomes greenish or reddish gray. The test may be performed in a porcelain dish, adding a drop of blood to the mixed reagents (Katayama). Kunkel and Welzel employ a solution of zinc chloride, or very dilute solution of platinum chloride, which color carbonic oxide blood bright red, normal blood black. Rubner recommends that the suspected blood be diluted four to five times with acetate of lead, when normal blood becomes chocolate, carbonic oxide blood red. Watery neutral solutions of Co-Hb, boiled, yield a clear red coagu- lum, while oxyhemoglobin becomes grayish brown (Hoppe-Seyler). Estimation of Total Quantity of Blood. The limits of error in the methods of estimating the total quantity of blood, suggested by Valentin, Vierordt, Buntzen, and Thibault, probably exceed the physiological and pathological variations in the bulk of this tissue, and are, therefore, not available for clinical purposes. Haldane and Smith have determined the total quantity of blood in fourteen healthy subjects by a method based on the capacity of the blood to absorb CO^< In these subjects the total bulk of blood varied between 3.34 per cent, and 6.27 per cent, (average 4.88 per cent.) of the body weight (one-thirtieth to one-sixteenth). The lowest per- centage was obtained in a very fat subject. Most physiologists hold that the total blood in healthy animals is about one-thirteenth of the body weight. ESTIMATION OF THE VOLUME OF RED CELLS AND PLASMA. The Hematocrit. The idea that the centrifuge might give valu- able clinical information concerning the volume of red cells was original with Blix, while his instrument and method have been improved principally by Hedin, Gartner, and Daland. Although hand centrifuges have been used and recommended, the best results are obtained only with the electric centrifuge, which is at present in the market at a reasonable figure. The improved electric centrifuge consists of an iron-clad motor carrying a steel shaft and horizontal armature for urine tubes, which may be replaced by the hematocrit. A " speed indicator " may also be attached, which strikes a bell with every 100 revolutions. The instrument is practically noiseless even with a very high speed. A rheostat is used to control the current and speed. A speed of 8000 to 10,000 revolutions may be obtained by a small battery or from the street current. The hand centrifuge (Fig. 6) may be employed when it is not convenient to use electricity. The hematocrit attachment consists of two capillary tubes, grad- uated in 100 degrees, which are held in the armature by springs. Procedure. With walking patients fresh blood may be used. The capillary tubes may be filled automatically by holding the tube 32 GENERAL PHYSIOLOGY AND PATHOLOGY. Fig. 5. horizontally, or with one end slightly depressed, and touching it to the rather large drop of blood required. The tube is then imme- diately inserted in the armature, as in Fig. 6, the opposite tube hav- ing previously been filled with water, and the revolutions are begun before the blood coagulates. When the patient is at a distance the blood must be diluted, preferably in 2.5 per cent, solution potassium bichromate, as recom- mended by Daland. The blood should be diluted with equal parts of this solution, which may be accomplished in the red-cell or white-cell pipette of the hemo- cytometer. With the red-cell pipette the capillary tube is filled with blood to the mark 1, then a small air bubble is drawn in, followed by another tube-length of blood. Three or four tube- lengths of blood should be secured in this way and immediately mixed with an equal number of tube-lengths of diluent. With the white-cell pipette a single measure of blood and diluent is sufficient. The blood and diluent should then be mixed by gentle shaking, taking care not to en- close air bubbles. When diluted blood is used both tubes of the hematocrit should be filled with blood, which may be done by allowing the drops to flow in from the point of the pipette. The tubes are now revolved at a speed of eight to ten thousand revolutions per minute, for three minutes, after which the volume of the red cells has been found unalterable. The question now arises how many red cells are contained in one degree of the scale. Daland, working extensively with diluted blood, places the number at 99,390, practically 100,000. Cabot, in a series of 40 comparative tests, using undiluted blood in the hematocrit, found variations between 105,000 and 123,000, with an average of 112,000. Further observations on this point are required, but at present the above figures should be used for diluted and fresh blood respec- tively, multiplying the result by two when diluted blood has been used. Limitations of the Hematocrit. Since the centrifuge does not necessarily require the use of diluting fluids, a serious cause of error Improved electric hematocrit, with fender, rheostat, and speed indicator. The hematocrit attachment replaces the urine tubes seen in the revolving armature. TECHNICS. 33 in the method may be removed- by the use of fresh blood. It must be admitted, however, that the original volume of the red cells can- not always be exactly determined by this method, as in pathological conditions the compressibility of the corpuscles is not always uni- form. Moreover, in altered conditions of the plasma, it is uncertain how much change can be wrought in the natural volume of red cells Fig. 6. Daland's hematocrit. by violent centrifugal force. Some fragile cells are probably always destroyed during the centrifugal process. When diluting fluids are used the error is doubtless increased by alterations in the density and composition of the plasma and in the volume of the red cells caused by the action of the fluid. Never- theless Daland's claim must be admitted that the volume of the red 34 GENERAL PHYSIOLOGY AND PATHOLOGY. cells, except in leukemia, is determined by this method with accuracy sufficient for clinical purposes. The value of such information is, of course, quite evident. The further claim that the hematocrit may give more accurate estimates of the number of red cells than does the hematocytometer has not been confirmed. The volume of the red cells differs so markedly in both the chlorotic and the pernicious anemias that one cannot seriously consider the project of replacing the hematocytom- eter by the hematocrit. Only in the moderate secondary anemias, with, little change in the size and Hb-content of the cells, can the volume of the red corpuscles yield reliable indications of their number. In cases of leukemia and of extreme leucocytosis so many leucocytes are entangled with the red cells that even the volume of the red cells is not accurately told, much less their number. Each of these instruments has its proper field to which it should be restricted, and as the hematocrit is not overexact in its immediate object, it is unscientific to introduce a second source of error, as is done in attempting to compute the number of red cells from their volume. It may be added that the value of the hematocrit in estimating the character and severity of an anemia has not yet been as fully recog- nized by clinicians as it deserves, possibly because more attention has been paid to the number of red cells than to their functional capacity. The reliability of the centrifuge in determining even the volume of the red cells has been denied principally by the brothers Bleibtreu, and by Bleibtreu and Wendelstadt. These observers devised another method of determining the volume of the red cells, which they claim gives more trustworthy results than are obtained by the hematocrit. They employed 0.6 per cent, salt-solution to prevent coagulation, and allowed the blood to settle slowly. The nitrogen-content of the supernatant plasma was then determined by Kjeldahl's method, and from tables which these observers constructed the volume of the plasma, and hence that of the red cells, could be determined from the quantity of N obtained. While the results obtained with the hematocrit by several observers indicate that the normal volume of red cells varies between 40 and 66 per cent., Bleibtreu's method gave normal variations in cadaveric blood between 25.15 and 55.8 per cent. (Bleibtreu, Pfeiffer). v. Limbeck obtained very low volumes with Bleibtreu's method (24 to 28 per cent.), which he refers to the use of highly oxidized blood, in which he believes the red cells are reduced in volume. The lengthy discussion which has prevailed regarding the above points indicates that the volume of the red cells is subject to a considerable variety of changes, the origin and significance of which are little understood. It has been shown that in order to prevent N from leaving the red cells during sedimentation, the exact isotonic tension of the plasma must be determined in each instance, and a corresponding solution of salt used. The isotonic tension of plasma is rarely so low as 0.6 per cent. NaCl. Moreover, supposing that the red cells remain intact during sedi- mentation, the pathological variations in the N-content of the plasma, TECHNICS. 35 depending on several variable nitrogenous bodies, are too frequent and marked to permit of any fixed formula to give the volume of the serum from its content of nitrogen. (Bleibtreu's method has been sharply criticised by Hamburger, Eyckman, Hedin, Biernacki, and others.) The following table of results obtained by Biernacki well illus- trates the unreliability of comparisons between results obtained by different procedures and the general inaccuracy of all indirect methods of estimating the number of red cells or percentage of Hb from the volume of the cells and the specific gravity : Red cells. Content of water. Case. Hb. Number. Volume ji. 1. Normal 77. 18 % 5. 037 mil. 56.3 105 % 2. Normal .... . . 77.50 5.487 53.6 100 3. Cancer of esophagus 79.58 5.175 52.7 80 4. Rheumatism . . . 79.02 3.902 49.1 85 5. Chlorosis. ... . . 80.99 4.958 50.0 70 6. Phthisis 82.37 4.672 40.9 60 7. Nephritis. ... 82.73 4.800 40.0 70 8. Tabes . . . 83.09 4.512 47.6 65 9. Chlorosis . . 83.04 4.250 35.4 55 10. Phthisis .... 84.59 1.975 30.9 50 11. Ulcer of stomach . 85.43 3.825 37.1 45 12. Chlorosis 89.36 2.456 20.0 25 13. Nephritis . . . . 89.46 1.184 13.6 20 Grawitz determines the volume of red cells in blood drawn in considerable quantities by venesection. The specific gravities of the whole blood (D,), of the centrifuged serum (D 2 ), and of the sedi- mented red cells (D 3 ), are first determined, from which the volume percentage of red cells (x) may be computed by the following formula : X = 100 (D.-D,) . (D 3 -D 2 ) ESTIMATION OF THE NUMBER OF BLOOD CELLS. i The Hematocytometer. The instrument now in use for counting blood cells is that of Thoma, who combined and improved several features of instruments previously devised by Hayem, Gowers, and Malassez. This apparatus consists of a mixing pipette and a count- ing-chamber. (a) The pipette is a capillary tube graduated in ten equal divisions, surmounted by a bulb of exactly 100 times the capacity of the tube, and to which is attached a rubber tube and mouth-piece (Fig. 7). When the tube is filled with blood up to the mark 1, and this is mixed with a diluting fluid sucked up to the mark 101, a specimen of blood is obtained in the dilution of 1 : 100. By filling only one-half the tube with blood, up to the mark Q.,5, the subsequent dilution is in the proportion of 1 : 200. The bulb contains a glass ball to facilitate the mixing of the blood. 36 GENERAL PHYSIOLOGY AND PATHOLOGY. (b) The counting-chamber is constructed so as to secure a layer of diluted blood T J r millimetre in depth over a certain square area. On a thick glass slide is cemented a thinner glass plate, the cen- tral portion of which is cut out. In this central area is cemented a circular glass shelf the surface of which is exactly fa millimetre lower than the surface of the glass plate. When a drop of diluted blood is placed on the shelf and covered with a cover-glass, a layer of fluid is secured, which is exactly fa millimetre deep. Between the edge of the shelf and the surrounding plate is a moat into which the blood may run, but if the fluid should run over the = moat and beneath the cover-glass, the latter will be elevated and the resulting layer of fluid will be more than fa millimetre deep. The shelf is accurately ruled, as shown in Figs. 8 to 11. The entire ruled area is nine square millimetres, but only the cen- tral square millimetre is used in counting red cells, the others being Fie. 7. The Thoma hematocytometer. required in counting leucocytes. It will be seen that this central square millimetre is subdivided into 400 small squares (16 blocks of 25 each), so that each small square is T ^ 7 sq. mm. Beginning at the lower left-hand corner of this area, it will be seen that every fifth square, above and to the right, is subdivided by an extra line, which is added merely to assist in counting the squares. The out- lying square millimetres are variously ruled. The above description applies only to the so-called " Zappert " chamber which should always be secured, preferably of Zeiss' manu- facture. The older chambers cannot well be used for counting leucocytes. Diluting Fluids. Of the various diluting fluids, Toisson's Mix- ture is to be recommended : Sodium sulphate Sodium chloride Glycerin pur. . Aq. dest. , Methyl violet 8.0 grm. 1.0 " 30.0 160.0 ' 0.25 ' TECHNICS. 37 This fluid keeps well, stains the leucocytes, and is of high specific gravity solihat the red cells settle from it slowly. When counting leucocytes only, one may use with advantage a 0.6 per cent, solution of sodium chloride tinged with gentian violet (about 1 drop of saturated alcoholic solution gentian violet to 50 c.c. of salt solution). This fluid, while readily prepared, does not keep well, and the red corpuscles settle from it so rapidly that it ought not to be used in counting these cells. It permits, however, of the identification of eosinophile cells and of certain degenerative changes in leucocytes. A reliable fluid for diluting and permanently preserving blood is found in Hayem's Mixture : Hydrarg. bichlor 0.5 grm. Sod. sulphat 5.0 " Sod. ehlor . . 1.0 " Aq. dest. . 200.0 " Directions for Using the Hematocytometer. (a) Filling the Pipette. The finger-tip of the patient is cleansed with soap and water, dried with alcohol, and freely punctured with a needle or a specially prepared acne-lancet. Using very gentle pressure only, a compact drop of blood is then expressed and the capillary tube is filled to the mark 1 or 0.5. In doing this the pipette must be held between the thumb and forefinger and the hand steadied against the hand of the patient. In well-constructed pipettes the column of blood is easily controlled, and after filling the end of the tube may be cleansed of adherent blood. The diluting fluid is then sucked up to the mark 101, taking care that no blood runs out of the tube when it is immersed in the fluid. The specimen is then thoroughly mixed by shaking. (6) Filling the Counting-chamber. The counting-chamber and cover-glass are thoroughly dried and freed from particles of dust. One or two drops of diluted blood are first forced from the pipette, and a third drop, the size of which can be learned only by experience, is deposited on the central shelf. The cover-glass is then immediately adjusted, slipping one corner under the forefinger of the left hand and controlling the opposite corner with the second finger of the right hand, and lowering the glass slowly so as not to include air bubbles. Without raising the fingers, now quickly cover the other corners with the forefinger of the right and second finger of the left hand, and press the cover-glass firmly into position. If the application is successful and no dust particles have intervened, New- ton's color rings will appear beneath the cover-glass. The formation and permanency of these rings may be facilitated by breathing very gently on the specimen before applying the cover-glass. The specimen should now be held up to the light and examined closely to see that the red cells are evenly distributed. An uneven distribution is readily detected by the naked eye. After settling a few moments the specimen is ready for counting. The rapid and successful adjustment of the cover-glass is the most important detail in tJie process of counting blood cells. The cover-glass 38 GENERAL PHYSIOLOGY AND PATHOLOGY. must be rapidly adjusted, because from the moment the drop is placed upon the shelf there is a rain of cells upon 'the ruled area out of a layer of fluid which is more than ^ mm. deep, The specimen must be discarded : If Newton's rings do not appear. •', If any air bubbles are inclosed. If the fluid runs underneath the cover-glass. _. If the shelf is not well covered by fluid. •'■ If, on inspection, the cells are found unequally distributed. Fig. 8. Fig. 9. ifliBiKminiiiMMnia iiiiiii iiii iiii iiii liHiiaii iiiiiii iiii iiii iiii liiiiiiiiii iiBiBiaiililiiiliBiajajaJ iiiiiii iiii iiii iiii iiiiiiaiBii iiiiiii iiii iiii iiii iiiiiiiiiii iiiiiii iiii iiii iiii iiiiiiiiaii iiiiiii iiii iiii iiii iiiiiiaiBii Tiirck. Fig. 10. IIII IIII Iiii III IIII iiii iiii in; inn mi mi ■iiii muni mi mi mi mi lira mi mi mi mi Thoma.- Centre part. Fig. 11. Thoma. Blood-counting chambers. (c) Counting the Red Cells. The specimen proving satisfac- tory, the count may begin as soon as the cells have settled. Zeiss, D., Leitz, No. 7, Reichert's or Bausch and Lomb, \, are the lenses best suited ior this purpose, and a good mechanical stage is necessary for accurate work. Locate in the field the lower left-hand block of 25 small squares, begin at the lower and left square and passing to the right count all the cells lying in the first five squares. The fifth square will be found subdivided. 1 " In each square count all the cells lying on the lower and left side lines, leaving to be counted with the adjacent squares all the cells lying on the lines above and to the right. Proceed in this way until at least four blocks of 25 small squares and at least 1000 cells are enumerated. The more squares counted over the greater the accuracy, and when slight variations are to be de- monstrated the entire square millimetre must be covered. If the TECHNICS. 39 cells now appear to be unevenly distributed the specimen should be discarded and another prepared after thoroughly shaking the pipette. (d) Computation. Suppose that 128.0 cells are enumerated in 100 small squares, i. e., in | of the square millimetre. This number multiplied by 4 gives the number lying over one square millimetre. But the depth of the fluid is only y 1 ^ mm., so that we multiply again by 10 to get the number of cells in one cubic millimetre of fluid. Finally we must multiply by 100 because the blood is diluted in the proportion of 1 : 100. In short, after counting over 100 small squares the result is multiplied by IfiOO to give the number of cells per cubic millimetre (4 X 10 X 100 = 4000). If the capillary tube was originally filled to the mark 0.5, the dilution is 1 : 200, and the multiplier 8000. If 400 squares are counted over, the multiplier is 1000. (e) Sources of Error in the Use of the Hematocytometer. 1. In Securing the Drop of Blood. When much pressure is employed in expressing the drop of blood, tissue fluids are squeezed out with the blood and the number of red cells is reduced. Reinert found a reduction of 722,000 from this cause, which is especially potent in cases of dropsy and of severe anemia. When the finger is cold, the circulation poor, or local stasis is pro- duced, as by a ligature, the red cells are increased in number. To avoid errors of this class, the circulation in the hand should be as active as possible, the finger warm^ and the puncture liberal enough to permit the flow of Mood with little pressure applied at some dis- tance from the puncmre. Unless these conditions can be secured it is hardly worth while to count the blood cells. 2. In diluting the blood and in transferring it to the counting-chamber there are numerous plainly evident sources of error, such as the inaccurate filling of the capillary tube, the entrance of air with the blood column, the failure to cleanse the tip of adherent blood, the escape of blood into the diluting fluid, the overfilling of the bulb with diluting fluid, the inadequate mixture of the blood, the failure to discharge one or two drops before applying one to the shelf, the use of thin cover-glasses, and, above all, delay and inaccuracy in adjusting the cover-glass. A little experience and constant care serve to eliminate all these difficulties. 3. In the Construction and Condition of the Apparatus. The ten- dency to favor the Zeiss instruments is still probably well founded, although Leitz and Reichert are now making very excellent pipettes after Grawitz's model. Aside from inaccuracies in the graduation of the pipette and construction and ruling of the counting-chamber, which are now reduced to a minimum, some pipettes are still on sale which are too short, their calibre is too large, and is narrowed at the point so that the tubes cannot be cleaned, they require too much blood, and the short arm is so small that the mark 101 comes too close to the bulb. Accurate work cannot be performed with such instruments. The worker is at present advised to insist on having Grawitz's pipette made by Zeiss, Leitz, or Eeichert. 40 GEXERAL PHYSIOLOGY AXD PATHOLOGY. Many close observers find that their pipettes vary with the tem- perature. While accurate information on this point is not at hand it is just as well to avoid extremes of temperature in making the tests and in cleaning the instrument. It has been suggested that the polycythemia of high altitudes is partly referable to variations in the hematocytometer due to changes in atmospheric pressure, but this suspicion has not been confirmed. The condition of the pipette is of prime importance. Absolute dryness of the tube and bulb is essential. The collection of minute water drops in the tube and bulb is responsible for many of the shadow corpuscles sometimes seen in the counting-chamber. Every few weeks a pipette should be cleaned out with concentrated nitric acid. (/) Cleaning the Apparatus. After using the pipette the rubber tube may be transferred to the long arm and the remaining fluid expelled. The tube should then be cleaned thoroughly with water, then with alcohol and ether, or, better, with pure ether. It must be thoroughly dried before using again. The counting-chamber must be cleaned with water only, as alcohol and ether dissolve the cement under the shelf and plate. (g) The Limit of Ereor with the Hematocytometer. Lyon, Thoma (and Reinert), counting an average of 1141 cells in 100 squares with a dilution of 1 : 200, found an average error of 1.82 per cent, in 24 preparations of the same specimen, and in another case, counting an average of 934 cells in 100 squares, 1 : 200 dilution, an average variation of 2.71 per cent., in 12 preparations of the same specimen (Limbeck). These results in the hands of experts using special care indicate that a variation of 150,000 cells (3 per cent.) cannot be accepted as of any significance. More accurate data are, however, seldom required by the clinician. Oliver's Hematocytometer. The Principle. When a candle flame is viewed through a flat glass tube containing water a trans- verse line of bright light is seen which results from the blending of numerous images of the flame. The images are produced bv the minute longitudinal corrugations in the glass which reflect the light in various directions. When diluted blood is placed in the tube the fluid is sufficiently opaque to shut out the images until a certain dilution is reached, when a bright streak of light becomes rather suddenly visible. Oliver believes that the appearance of this bright horizontal line is a very sensitive indicator of the proportion of red cells in the fluid, and by a long series of observations has devised an instru- ment for determining the number of red cells in blood according to this principle. The apparatus consists of a measuring pipette (A) ; a drop- per for Hayem's fluid (B) ; a flat glass tube graduated in 120 degrees (C). The Procedure. The capillary pipette is carefully filled with blood and washed into the tube by means of Hayem's fluid. A proper amount of fluid is then added to the diluted blood, and TECHNICS. 41 Fig. 12. the two are mixed by inverting the tube closed by the thumb, care being taken not to remove any diluted blood with the thumb. The test should be made in a dark room, the light being fur- nished by a Christmas candle placed about ten feet from the operator. When the blood is insufficiently diluted the image of the candle is invisible when looking through the tube held horizontally (Fig. 12, D), but at a certain dilution the images begin to appear, and at the proper dilution a rather com- pact transverse line of light becomes visible. The bottom of the meniscus is then read off on the graduated scale. Each de- gree of the scale represents 100,- 000 red cells, the mark 100 cor- responding to 5,000,000 cells, 80 to 4,000,000, 60 to 3,000,000, etc. There are both theoretical and practical objections to the use of Oliver's instrument. Theoreti- cally, the method falls in the un- desirable class of indirect methods about which there are always a large series of unknown disturb- ing factors which can only be elim- inated by prolonged experience. Oliver's hematocytometer. A, measuring pi- pette. B, dropper. C, mixing tube graduated in percentages. D, mode of observation. Baumgarten finds that in addition to the variation in size of the corpuscles an important source of error is the coagulation of the serum by the Hayem's solution. He found variations of 20 to 30 per cent, from the results obtained by Thoma's instrument, and concludes that the method is of no value in clinical work. Scannell's report, however, is more favorable. Practically the difficulty of determining the exact dilution from the appearance of a compact line of light is very great. The method cannot be recommended until it has received much wider application than it has yet enjoyed. The Estimation of Leucocytes. The leucocytes may be counted by a method which requires a special mixing pipette, yielding a dilution of blood in the proportion of 1 : 10, and a diluting fluid (3 per cent, acetic acid, tinged with gentian violet) which dissolves the red cells, leaving only the stained leucocytes to be counted. The same chamber is used as for counting red cells, and the same procedure is followed. All the leucocytes in one square millimetre having been counted, the result is multi- plied by 100, giving the number of leucocytes per cubic millimetre. 42 GENERAL PHYSIOL OGY AND PA THOL OGY. The disadvantages early recognized in this method are the expense and inconvenience of an extra pipette, and a second diluting fluid, the time required in preparing a second specimen, the larger quantity of blood required, the difficulty sometimes encountered in distin- guishing leucocytes from the detritus of red cells, and the impossi- bility of separating and evenly distributing the cohesive leucocytes. This method has gradually been replaced to a large extent by the practice of counting leucocytes in the same specimen prepared for counting the red cells. In 1892 the writer found that he secured more uniform results with the latter method, and has since found no inducement to return to the former. The Counting' of Leucocytes in the Same Preparation with the Red. This method requires the Zappert Chamber, which was originally devised by Elsholz for the estimation of eosinophile cells in fresh blood. Varous modifications of the ruling in this chamber have been employed, one of which, made by Leitz, at the writer's suggestion, is represented in Fig. 10. With this chamber, using a Leitz lens No. 7, it is possible to count over nine square millimetres, which gives almost as many leucocytes as are counted in the other method. When the leucocytes are normal or reduced in number, it is necessary to count all there are in the available 9 sq. mm., and if the number is very low it is advisable to prepare, a second specimen in the chamber and count the white cells in 18 sq. mm. When the leucocytes are increased, 9 sq. mm., or in cases of leukemia, 6 sq. mm., will yield a number large enough to insure an accurate result. In order to make the leucocytes visible the Toisson's fluid or other solution should contain enough methyl violet to stain these cells dis- tinctly. With a little practice the eye very readily picks out the bluish highly refractive leucocytes. What has been said regarding the condition of the local circula- tion, and the effects of pressure in expressing the blood, is to be specially emphasized when estimating the number of leucocytes in a specimen of blood. Computation. Divide the number of leucocytes counted by the number of square millimetres traversed in the count and multiply by 1000. The result is the number/ of leucocytes per cubic millimetre of blood. If the original dilution is 1 : 200, which ought not to be employed except in cases of leukemia, the multiplier is 2000. Thus if 54 leucocytes are counted in 9 sq. mm. (dilution 1 : 100) the number per cubic mm. is 6000 (54 -=- 9 X 1000). The Enumeration of Eosinophile Leucocytes. (a)Jn the Same Preparation with the Red Cells. When the blood is diluted, 1 : 100, with 0.6 per cent, salt solution tinged with gentian violet, the leuco- cytes retain their natural size and shape and eosinophile cells can be readily identified by their large, greenish, refractive granules. In cases of myelogenous leukemia this method is satisfactory, but when the eosins are present in their usual numbers (1 to 5 per cent.) one must count a larger number than can be found by this method. The TECHNICS. 43 usual expedient is to estimate their percentage from a dried specimen of blood, and then to calculate their number from the total number of all leucocytes counted by other methods. Thus, if the count shows 12,000 leucocytes per cubic mm. and the dried blood slide shows 2 per cent, of eosins their number will be 240 per cubic millimetre. This method is sufficiently accurate for clinical purposes. (b) By Means of Thoma's Special Pipette for the Enumeration of Leucocytes. Klein, Mueller and Reider, and Elsholz, have employed methods for the accurate estimate of eosinophile cells adapted to finer clinical work and to experimental research. They use the large pipette of Thoma which gives a dilution of 1 : 10. The capillary tube is filled with blood to the mark 1, and the bulb is half filled with the following solution : watery eosin (2 per cent.), 7 c.c. ; glycerin, 45 c.c. ; aq. dest., 55 c.c. After shaking three to four minutes the bulb is filled to the mark 11 with the following staining fluid : aq. dest., 15 c.c. ; gentian violet, cone. aq. sol., 5 drops ; alcohol, d drop. In specimens thus prepared both neutrophile and eosinophile leucocytes are readily distinguished, the eosinophile cells being par- ticularly brilliant. The red cells are dissolved and the leucocytes concentrated so that a sufficient number of eosinophile cells may be counted. Zappert's extensive studies of eosinophile leucocytes were conducted with specimens diluted in the large pipette of Thoma, by the follow ing solution : 1 per cent, osmic acid sol., 5 c.c, to which are added 5 drops of a filtered mixture— aq. dest., 10 c.c. ; glycerin, 10 c.c. ; 1 per cent watery eosin, 5 c.c. THE ESTIMATION OF HEMOGLOBIN. 1. Gowers' Hemoglobinometer. This instrument has always been largely employed on account of its cheapness and simplicity, and the ease and rapidity with which its results are obtained. Except with low percentages of Hb, it is tolerably accurate, but much less reliable than Fleischl's instrument. With low percentages of Hb it is well to use a double quantity of blood, halving the result. Aside from any inaccuracy in the construction of the apparatus, errors arise chiefly from the difficulty of adding exactly the proper quantity of water to the blood, and the imperfect comparison of red colors in daylight. The apparatus consists of two glass tubes (A, B) of exactly equal calibre, one of which is partly filled with gelatin colored by picrocarmine so as to represent the color of a 1 per cent, sohitfcrn-ef- normal blood. The second tube carries a graduated scale from 10 to 120 and serves to hold the diluted blood. The capillary pipette C measures 20 c.mm., the quantity of blood to be used. In making the test the pipette is filled to the mark 20 c.mm. with blood obtained under the usual precautions. The specimen is quickly discharged into the tube B, in which a few drops of distilled water have previously been placed. v The pipette must then be washed once or twice into the tube, the distilled water removing all traces of 44 GENERAL PHYSIOLOGY AND PATHOLOGY. blood adherent to the inside of the capillary tube. Distilled water is now added drop by drop, until the solution of blood, carefully shaken and mixed, exactly matches the carmine gelatin. The percentage of Hb is indicated by the height of the solution on the scale, reading from the middle of the meniscus. The colors match in daylight, and the eye may be assisted by holding the tube in front of white paper. Haldane's Modification. The picrocarmine solution of Gowers' instrument is not stable and has been replaced by Haldane with a 1 per cent, solution of CO-Hb, which he has found to remain unchanged for months, even when exposed to heat and sunlight. The specimen of blood is drawn as usual and partly diluted. The air in the tube is then expelled by a current of illuminating gas introduced from a Fig. 13. Gowers' hemoglobiuometer. gas-burner by a rubber tube, and CO-Hb formed by inverting the tube, closed by the thumb, several times. The dilution and reading are then completed. Haldane claims very accurate results from this method. The apparatus with standard solution of CO-Hb is sup- plied by the makers of Gowers' hemoglobinometer. 2. Tallquist's Method of Estimation of Hemoglobin. Tallquist has recently described a rough method of estimating hemoglobin by comparison of a piece of filter paper stained by the blood with a care- fully prepared lithographic plate representing the colors of ten solu- tions of Hb, ranging from 10 to 100 per cent. A small piece of white filter paper of standard quality, such as is sold with the scale, is touched to a rather large drop of blood, which is allowed to diffuse TECHNICS. 45 slowly through the paper, so as to give as even a stain as possible. Immediately after the stain has lost its lustre and before drying or changing color by exposure the percentage of Hb is determined by matching with one of the colored areas in the scale. It is unneces- sary to mention the serious sources of error in this method, which hardly estimates but rather assists one to judge the percentage of lib. 3. Fleischl's Hemoglobinometer. (a) Apparatus. This appara- tus consists of a metal stand with plate, and plaster mirror (S) which casts diffused light through a circular opening in the plate. Beneath the plate, by means of a rack and wheel (T), slides a colored glass wedge fixed in a graduated frame (P). The glass wedge and gradu- ated scale are arranged so as to indicate the percentage of Hb corre- sponding to the different portions of the wedge. In the circular opening of the plate fits a cylindrical metallic cell ((?), with glass Fig. 14. bottom and metal partition, one compartment of which lies directly over the glass wedge. The other compartment (a) being filled with diluted blood, one is able to make a close comparison of the color of the dissolved blood with that of the glass wedge. The blood is measured by an automatic capillary pipette, while a slowly running dropper is provided with which to add distilled water. On the handle of each pipette is stamped a number, indicating the cubic content of the tube. On the stand of each instrument is also a number showing the capacity of the tubes with which it can be used. (£) Procedure. One should first see that the automatic pipette is in working order, by blowing it out several times with water until it fills instantly and completely, after which it must be thoroughly dried. A drop of blood having been expressed under very strict precautions against pressure, one end of the pipette is lightly touched 46 GENERAL PHYSIOLOGY AND PATHOLOGY. to the drop which instantly fills the tube. There should be neither negative nor positive meniscus to the column of blood, but the tube should be level full at either end. It should not be immersed in the blood drop, otherwise blood will adhere to the sides which cannot safely be removed. The tube of blood is immediately transferred to one compartment of the cell which has been half filled with dis- tilled water, and the blood is thoroughly dissolved by moving the tube rapidly from side to side. On withdrawing the tube it should be washed into the cell with a feAV drops of distilled water. With the handle of the pipette the blood which collects in the corners of the chamber is thoroughly mixed. Both chambers of the cell may now be filled level full with water. The thick round cover-glass should then be adjusted, avoiding the inclosure of air. Serious error may here result by forcing dissolved blood over into the water com- partment or over the side of the cell. If the reading is made promptly the cover-glass need not be used, but after an exposure of ten to fifteen minutes the oxidizing action of the air may darken the blood and increase the reading 10 to 15 per cent. The reading must be done in a dark room by means of candlelight or gaslight. The colors do not match in daylight. The best results are obtained by placing the candle about eighteen inches from the stand, and by looking through an improvised paper tube which exactly fits the cell. With low 'percentages of Hb a very dim, light is essential. When the cell is in place, and the light adjusted, the wedge is moved with quick rather than gradual turns until the color of the glass exactly matches that of the dissolved blood, when the percentage of Hb may be read on the scale. Several expedients are employed to assist the eye in the comparison of colors. It is well to relieve one eye with the other so as not to exhaust the color sense. The strength of the light may be varied by altering its distance, but the faintest distinct light is usually found to be the best. Two or three readings should always be made and the average taken. As the graduation of the instrument is more accurate and the matching of colors more exact in the middle of the scale, it is advisable to use a double quantity of blood when dealing with low percentages ofHb. (o) Limitations of Fleischl's Instrument. With considerable experi- ence and constant care Fleischl's instrument yields results which are sufficiently accurate for most clinical purposes. One cannot attach any significance, however, to a variation of 5 per cent., within which figure the ordinary error ought to be limited. All the causes of error mentioned as affecting the number of red cells may also disturb the percentage of Hb in the blood drop. In the procedure the principal difficulties relate to the even filling of the tube, the thorough cleaning of the tube, the overflow of blood solu- tion into the adjoining chamber, the proper adjustment and regula- tion of the light, and the accurate matching of colors. The chief sources of error in this method probably lie in the con- struction of the instrument. It is a common experience to find that different instruments give different results with the same specimen TECHNICS. 47 of blood, which Limbeck satisfied himself were referable principally to differences in the glass wedges. The writer tested his own blood with sixteen different instru- ments, two of which were old (1892), the others of more recent importation (1897). With the two old instruments the Hb regis- tered 87 per cent, and 90 per cent. ; with the newer instruments it varied between 97 per cent, and 105 per cent., except with one which gave 85 per cent, with different cells and different capillary tubes. The colored wedge with this instrument was broader and darker than usual, and when it was replaced by another wedge the instru- ment registered 100 per cent, with the same specimen. The error attributable to variations in different tubes and cells the writer finds is seldom greater than 5 per cent, among the newer instruments. Many old instruments read 10 to 15 per cent, lower than normal. Miescher's hemoglobinometer. The possible combination of these various defects in construction renders it desirable that one should test every new instrument for himself, using the blood of five or six healthy subjects. The newer instruments will not be found to require much correction. Miescher's Modification of Fleischl's Hemoglobinometer. Miescher's improvements have removed some important defects in Fleischl's instrument, and as it now stands the improved hemoglobinometer yields results that leave little to be desired in point of accuracy. With this apparatus the blood is diluted by means of a graduated pipette very similar to that of Thoma, but yielding dilutions of 1 : 200, 1 : 300, and 1 : 400, according as the tube is filled with blood to the mark 4-, f, or A. 48 GENERAL PHYSIOLOGY AND PATHOLOGY. Two cells are provided, one with a depth of 15 mm., the other with a depth of 12 mm., the percentage of Hb being obtained with the deeper cell, and the other being used as a control specimen and giving only f (i|) of the actual percentage of Hb. These cells have a projecting partition dividing the compartments, along which a grooved cover-glass (D) may be slid, without fear of mixing the blood and water. If an excess of the blood solution overflows the side of the cell during the adjustment of the cover, no harm results, as the blood is already properly diluted in the pipette, and the depth in the chamber will always be 15 mm. (or 12). These changes add very much to the facility and precision of the method. Finally, the cells are covered with diaphragms transmitting a ray of light which includes only three degrees on the scale, thus giving practically a single color of the wedge for comparison with the blood. In the procedure the blood is diluted in the pipette as with the hematocytometer, the diluting fluid being distilled water or a filtered 1 per cent, solution of sodium carbonate. After shaking and clear- ing the tube of diluent, one chamber in each cell is filled with diluted blood, the opposite chamber with distilled water, and cover-glasses and diaphragms are adjusted. Using a small candle and shielding the eyes from light, the readings with the two cells are carefully taken. The reading with the small cell should be | that with the larger. If there is any variation one reading may be used to correct the other. For example, suppose the readings to be : For the larger chamber (15 mm.) . ... . . . 64 For the smaller chamber (12 mm.) ... . . 50 If the first reading were absolutely correct the second reading should have been 51.2, since 64 X f = 51.2. Or, assuming the second to be correct, the first should have been 62.5, since 50 X | = 62.5. The mean of 64 and 62.5, i. e., 63.25, should be taken as the true value. If the original dilution was 1 : 200, the percentage of Hb is 63.25, the corrected result with the larger cell, but if the dilution has been 1 : 300, this result must be multiplied by 1 J, or if 1 : 400, by 2. The technical difficulties of this method are so slight and the results so accurate that the instrument may be recommended over any other yet devised for this purpose. 4. Oliver's Hemoglobinometer. This instrument is constructed on the excellent principle of the tintometer, which is extensively used in various arts and industries. It consists of a, series of six red glass disks (a) mounted upon white plaster mirrors in convenient frames. These disks represent the colors of twelve solutions of blood containing twelve different proportions of Hb. Two sets of disks are made, one for reading in daylight, the other for candlelight. The latter give more accurate results and should be chosen. The intermediate percentages of Hb are secured by means of " riders " of colored glass to be placed over the disks, and which represent respectively 2J and 5 per cent, of Hb TECHNICS. 49 in the upper half of the scale, but twice that amount in the lower half. The disks are graded according to the specific dilution-curve of Hb. Fig. 16. Oliver's hemoglobinometer. a, set of standard colored disks. 6, lancet, u, capillary pipette. d, dropper, e, mixing-chamber. The blood is measured in an automatic pipette, c, and diluted in a cell, e, provided with a white plaster, glass-covered bottom, and blue glass cover, which facilitates the reading. The blood is washed 4 50 GENERAL PHYSIOLOGY AND PATHOLOGY. from the tube by means of a pipette, d, with rubber nozzle. The tube may be cleaned by means of a thread and needle. Procedure. The capillary tube is filled by touching it to the blood drop, and the blood is washed into the cell by attaching the rubber nozzle of the pipette, filled with distilled water, taking care uot to aspirate any blood into the pipette. The handle of the tube is then used to mix the blood, and water is carefully added until the cell is level full. The cover is then applied and should inclose a Iminute air bubble, showing that the chamber has not been overfilled. The comparison of colors should be made by candlelight, the candle being placed at a convenient distance from the disks, and the eye shielded by means of a hood or paper tube. Oliver uses a special camera with green-glass eye-piece. If the color of the specimen matches any one of the disks the reading is completed. If it does not, the Fig. 17. H ^ Dare's hemoglobinometer (X %)• Parts in position. E, milled wheel rotating colored disk and scale. S. case inclosing disk. T, shield which receives the camera tube U V, visible portion of disk backed by white glass. W, white glass back of capillary chamber. Y, candle holder. Contents of case : E, colored disk. F, clear glass disk graduated along edge, H. I, white glass back. 6, opening for pivot. disks may be varied by using the " riders," adding a clear glass disk to the specimen to compensate for the thickness of the "rider." Oliver's instrument presents the advantage of a comparison with a single color instead of with a scale of 10 per cent. +, as in Fleischl's. This difficulty is slight, however, and is overcome in Miescher's modification of Fleischl's instrument. The color of Fleischl's glass wedge varies according to the specific dilution-curve of the colored glass, which differs from the specific dilution-curve of Hb. How serious an error may arise with Fleischl's instrument from this cause has not been clearly shown. It is probably not great, as it is not apparent in the use of Miescher's modification in which different parts of the scale are employed. Yet the principle of Oliver's instrument is, in this respect, a distinct improvement. TECHNICS. 51 The technical difficulties are considerably less with Miescher's hemo- globinometer. 5. Dare's Hemoglobinometer. This instrument consists of the following parts : 1. A capillary blood chamber composed of two rectangular pieces of glass which when clamped together leave a thin space at one end into which a layer of blood flows by capillary attrac- tion. One of the plates is of porcelain glass, which diffuses the light. This chamber, when filled and adjusted, lies over one of two aper- tures which meet the eye glancing through the camera tube of the instrument. 2. The color standard is a semicircular glass plate stained with Cassius' golden purple. The plate varies in thickness, giving colors which have been accurately adjusted to match various percentages of Hb. This plate is inclosed in a rubber case in which it rotates by means of a milled screw, carrying with it another glass plate with scale adjusted FlG - 18 - from 10° to 120°, which may be read on the left of the rubber case. 3. A detachable camera tube of black rubber screws into a movable shield at- tached to the case, and when the parts are in position this tube furnishes a dark chamber through which the operator com- pares the color of the blood with that of the standard scale, each of which is ex- posed by small circular openings in the shield. The light is provided by a candle fixed to the case. The wick should be trimmed low. Method of Use. One edge of the capil- lary chamber, firmly clamped, is touched to a rather large drop of blood sufficient to fill it rapidly and completely, after which the chamber is slipped into the holder, white glass outward. Rotating the shield and camera tube outward and pointing the instrument to- ward a dark background, one finds the color scale and blood film clearly outlined in the camera tube, and by rotating the scale is brought to match the blood. 0_ o 1 1 f pBMBM,W rjitiHi jgsjgggw^t^ • - "1 y l i M' M'/ N j Dare's hemoglobinometer. Hori- zontal section. Capillary chamber and clamp. J, candle. K, white glass back. L, colored disk. M, opening through which blood is viewed. M', opening in colored disk. N, camera tube. O P, clear and white glass plates of chamber between which lies the layer of blood. Q, metallic septum. milled wheel the color The instrument should be held steady, so that the flame does not flicker, and the read- ing should be completed promptly before the blood coagulates or evaporates. Dare's instrument is very cleverly devised and constructed. It requires the use of pure blood, offers ideal conditions for the com- parison of colors, and demands little space in transportation, little time in application, and little pains in cleansing, while in accuracy of results it stands next, but inferior, in the author's experience, to the Miescher-Fleischl apparatus. 52 GENERAL PHYSIOLOGY AND PATHOLOGY. ESTIMATION OF THE IRON OF THE BI^)OD. Jolles' Ferrometer. Jolles has devised a method and designed apparatus for the estimation of the iron of the blood, which is well adapted to clinical purposes. The apparatus is manufactured by Reichert, and full directions in German accompany each set. The procedure is as follows : : By means of a pipette 0.05 c.c. of blood is transferred to a plati- num crucible, and the adherent blood washed out with a few drops of water. The blood is then evaporated and incinerated over a Bunsen flame. The ash is melted with 0.1 gr. water-free potassium bisulphate, until white fumes of sulphuric anhydride cease to rise from the dish. Fig. 19. Papers containing the requisite amount of potassium bisulphate accompany the instrument. After cooling, the ash is washed into cylinder C (Fig. 20) with about 5 c.c. of hot distilled water, which is added until the whole quantity is 10 c.c. In cylinder C 1 c.c. of the standard solution of iron (0.00005 gr. iron oxide with potassium sulphate) is measured by a pipette and distilled water added to the mark lO c.c. Both cylinders are now placed in the stand (Fig. 20), and when at even temperature 1 c.c. of dilute HC1 (33 per cent.) is added to each. To cylinder C should then be added 4 c.c. of the solution of ammonium sulphocyanide, and to cylinder C about 3.5 c.c, and both are shaken after covering the ends with glass plates. Cylinder C, containing the blood, is now filled with sulphocyanide until it presents a positive meniscus, when it is permanently covered TECHNICS. 53 Fro. 20, and sealed by its glass plate. It may then be placed in the color- imeter. In cylinder C" the aluminum float is then adjusted, free from air bubbles, and it is also placed in position in the colorimeter. The further procedure consists in bringing the color of C to match that in C, when viewed from above in the colorimeter. For this purpose the fluid in C is allowed to run out drop by drop through a stopcock until the two colors are exactly alike. The comparison should be made in daylight. When the colors exactly match the height of the necessary column of fluid in cylinder C" may be read off and the percentage weight of iron de- termined by reference to a table ac- companying the instrument. The Hb may then be found according to v. Jaksch's formula, Hb = 100 X to 0.42 in which m = the percentage weight of metallic iron. In order to secure accurate re- sults with the f errometer it is neces- sary to observe the same care that is required in all quantitative chem- ical analyses, especially to avoid the loss of fluids by sputtering from the hot crucible, the use of wet and unclean instruments, and the inclosure of air bubbles, etc. Under most conditions the Hb may be accurately determined by means of the above formula, but from the considerations mentioned under " The Occurrence of Iron in the Blood," it will be seen that there may be considerable variations between the iron and the Hb-content. The ferrometer, therefore, usually gives a higher proportion of Hb than does Fleischl's instrument. THE HISTOLOGICAL EXAMINATION OF BLOOD. The greater part of the examination of blood is conducted with dry stained specimens. To prepare such specimens for staining one requires only polished glass slides and a Bunsen gas-burner. The glass slides must be thoroughly cleaned with soap and water, dried, and kept free from dust. Passing them a few times through a flame facilitates the even spreading of the cells. A rather small compact drop of blood expressed from the finger tip under the usual precautions is lightly scraped off with the polished edge of one slide and applied to one end of a second slide which should lie on firm support. When the blood has spread along the 54 GENERAL PHYSIOLOGY AND PATHOLOGY. edge of the smearer it should be slowly and firmly drawn over the surface of the receiving slide. The drop should, if possible, be small enough to be exhausted in the smearing, and the thickness of the layer can be fully controlled by the degree of pressure. The blood should be pushed before the smearer and not trailed after. (See Fig. 21.) Many prefer to use cover-glasses in spreading the blood. One polished cover-glass is touched to the drop of blood and applied to a second cover, all corners projecting. When the blood has spread to the edges the cover-glasses are gently slid apart without pressure. The cover-glasses should be handled with forceps, otherwise the moisture of the finger will often crenate many cells. The writer prefers to use glass slides, finding that beginners are much more successful with the slides than with cover-glasses, that, after very little practice, every specimen can be spread successfully ; that forceps are not required ; that slides^ may be handled and trans- FlQ. 21. Method of making blood smears. ported without fear of breakage ; that they need not be mounted, and, therefore, do not fade like cover-glass specimens, which require mounting in balsam ; that they may be restrained if necessary ; and, above all, that they may safely be fixed in the free flame. After spreading, all specimens should be well dried in the air. They may then be kept for weeks if wrapped in tissue paper and kept from moisture, but it is better to fix them at once. Fixation. 1. Heat. In routine work one may discard all other methods for that of fixation in the free flame of a Bunsen burner. The slide, specimen side up, is passed slowly through the flame untii it is decidedly too hot for the hand to bear. At this temperature, which probably varies between 110° and 150° C, fixation is com- plete in one to two minutes. A little practice will give the confidence necessary to heat the slides hot enough, as one's initial failures from this method almost always result from incomplete fixation and subsequent vacuolization of the red cells. Overheated slides can usually be seen to change TECHNICS. 55 color in the flame, after which the red cells stain yellowish with eosin. The beginner is strongly recommended to perfect himself in this simple method of fixation. Small ovens provided with a thermometer are made for the fixa- tion of blood slides, and may be used when many specimens are in hand, or when one does not care to risk the free flame. Specimens should be exposed five to ten minutes to a temperature of 110° to 120° C. 2. Alcohol. Fixation for ten to thirty minutes in 97 per cent, alcohol, or in equal parts of alcohol and ether, is a very reliabe method in very general use. Specimens may be left in alcohol for twenty-four hours, but do not then stain quite so well. There appears to be no advantage in adding ether to the alcohol, which even without mixture with the more volatile agent must frequently be replaced. Methyl alcohol fixes much more rapidly than ethyl, requiring only one to two minutes. It may be advantageously employed as a routine fixative, and it is used as a combined fixative of blood and solvent of dyes in Jenner's and Goldhorn's stains. Fixation in alcohol is to be specially recommended for the malarial parasite, but is unsatisfactory when Ehrlich's triacid stain is to be used. 3. Fixation by Vapors. Specimens may be fixed by being laid, specimen side down, over a wide-mouthed bottle containing 25 per cent, formalin, to which the exposure is five minutes, or 2 per cent, osmic acid, to which expose two minutes. Both these fluids have to be replaced frequently, they considerably alter the staining relations of the blood cells, and are inferior to other methods of fixation. 4. Fixation without Drying. Jolly and others claim that fixation after drying destroys many of the essential characters of leucocytes, which may be demonstrated in specimens fixed while moist in solu- tions of chromic acid. Flemming's stronger solution gave the best results in Jolly's hands (1 per cent, chromic acid, 15 parts ; 2 per cent, osmic acid, 4 parts ; glacial acetic acid, 1 part). Other fixa- tives recommended for the same purpose are saturated bichloride in 0.6 per cent, salt solution (Hermann's fluid). All these fixatives un- doubtedly give better demonstration of nuclear structures and mitotic figures than can be obtained after fixation with drying. Methods of Staining Dry Blood Specimens. . 1. Eosin and Methylene Blue. The solutions required are : A saturated alcoholic solution of Ehrlich's blood-eosin. A saturated watery solution (1 per cent.) of Ehrlich's rectified methylene blue. The latter should be at least one week old, as fresh solutions lack selective quality and stain the specimen diffusely. After several weeks methylene blue in solution diminishes in staining power, while the alcoholic eosin absorbs water, and becomes less selective and more powerful. In staining flood the specimen with eosin for a few seconds and wash in water. If the stain is not effective add more eosin, but the water on the slide dilutes the alcohol and renders the second applica- tion of eosin much more powerful than the first. Next flood the specimen repeatedly for one minute with methylene blue, wash hastily in water, and dry. 56 GENERAL PHYSIOLOGY AXD PATHOLOGY. This method may be recommended for all ordinary examinations. The blood is stained as shown in Plate II., readily distinguishing the various forms of normal leucocytes. It does not stain neutrophile granules in leucocytes unless the action of eosin has been prolonged, in which case the neutrophile leucocytes can be distinguished from the eosinophile only by the size of the granules. Its chief advantage is the clear differentiation of basophilic leucocytes and of nuclear structures. It clearly demonstrates the malarial parasite, but in this field is greatly inferior to Nocht's method. Its chief disadvan- tage is the danger of overstating with eosin, which prevents the full action of methylene blue. 2. Ehrlich's Triacid Mixture. This fluid has the following com- position : Saturated watery solution of orange G .... 120-135 c.c. " " " acid fuchsine ... . . 80-165 " " " " methyl green . . 125 " To these add : Aqua . .... 300 " Absolute alcohol .... .... 200 " Glycerin . . . 100 " The attempt to prepare this mixture is not always successful. The smaller quantities of orange G and acid fuchsine are best employed, and the solution of methyl green, well seasoned, should be added slowly, with stirring, to the mixture of the other dyes. The water should be added next, then the alcohol, and, finally, the glycerin, with constant stirring. After standing one week the mixture is ready for use. Griibler's preparation of this mixture is in the market and is reliable. In staining it is only necessary to flood the specimen with the dye for one to two minutes, and wash hastily in water. It cannot over- stain, but overheated specimens are usually faint, and the red cells are yellowish. It stains neutrophile and eosinophile granules deep red, the latter being distinguished by their size. It is, therefore, indispensable in the diagnosis of leukemia. It is a poor nuclear stain, fails to demonstrate the structure of normal mononuclear leucocytes, and does not stain the malarial parasite. On account of the uniformity of its results many prefer it to eosin and methylene blue as a routine method. 3. Jenner's Stain. Jenner's method of fixing and staining blood has now withstood sufficient trial to warrant its acceptance as one of the most important recent methods in blood technics. The speci- mens are fixed and stained in the same solution, which is prepared as follows : equal parts of 1.2 per cent, to 1.25 per cent, of watery solution of Griibler's yellow water-soluble eosin and of 1 per cent, watery solution of Griibler's medicinal methylene blue are mixed together in an open basin, thoroughly stirred, and allowed to stand twenty-four hours. The mixture is then filtered, dried in the air, or oven, at 55° C, the filtrate powdered, shaken up with distilled water, and washed on a second filter. It is again dried, powdered, and stored in bottles for use. The stain is prepared by dissolving 0.5 TECHNICS. 57 gramme of the powder in 100 c.c. pure methyl alcohol (Merck's " for analytical purposes "). Very thin smears of blood, made on thoroughly clean slides, are dried in the air. The dye is poured on the specimen, and staining is complete in one to three minutes. The specimens are washed, preferably in distilled water, until of a pink color, which usually appears in ten seconds. All the cells, their nuclei, and the various granules, are well differentiated, while the malarial parasite is densely stained and only in the larger parasites does the chromatin fail to appear deeply red stained. For this last purpose the method is inferior to Nocht's. The powder or fluid dye may be obtained from New York dealers. 4. Demonstration of " Mast-cells." The large basophilic granules of these cells retain basic dyes with tenacity, and may be demon- strated by a mixture of one of these dyes, with a strong decolorizer, which removes the stain from most other basophilic structures. Ehrlich's dahlia solution is adapted to this purpose : Absolute alcohol 50.0 c.c. Glacial acetic acid 12.5 " Distilled water 100.0 " Add dahlia to saturation. Stain several hours, wash in water, decolorize in alcohol or more rapidly in 20 per cent. Ac, until the nuclei fade, and wash in water. The nuclei of leucocytes are then very pale blue, the mast-cell granules very dark blue or black. Recent experience has shown that mast-cell granules are very sol- uble in water and that their reliable demonstration requires fixation and staining by alcoholic solutions. Michaelis recommends a solution of thionin or kresyl violet in 50 per cent, alcohol. The author prefers to use Goldhorn's solution in strong methyl alcohol of eosin and polychrome methylene blue. ESTIMATION OF THE SPECIFIC GRAVITY OF THE BLOOD. Hammerschlag's method is the most practical of the various indirect procedures devised for this purpose. A small urinometer of suitable dimensions is partly filled with a mixture of chloroform (s. g. 1526) and benzine (s. g. 0.889) of a gravity of about 1060. By means of a pipette, such as the red-cell mixer of Thoma, a drop of blood, expressed with the usual precautions, is transferred to the fluid. In expelling the blood the tip of the pipette should be sub- merged and no air should be allowed to pass out with the blood. The drop should not be very minute in size and should float on the fluid. If it is allowed to sink it will often be lost by spreading out on the bottom of the vessel. By adding chloroform or benzine drop by drop, as required, and carefully mixing by inverting the urin- ometer closed by the palm, a mixture is secured in which the drop neither rises nor sinks, but which is of exactly the same density as the blood. The specific gravity of the mixture and of the blood 58 GENERAL PHYSIOLOGY AND PATHOLOGY. may then be taken as with urine. The urinometer should be gradu- ated up to 1065, and should be tested in distilled water at 60° F. The apparatus should be clean and perfectly dry. The mixture may be filtered and used repeatedly. Great accuracy can hardly be expected from this method, yet it is sufficiently reliable for clinical purposes. Errors arise from changes in the blood drop during and after its transfer to the mixture, from evaporation, from the escape of gases, from the inclosure or adher- ence of minute air bubbles, and perhaps also from the possible action of chloroform and benzine upon the blood, which is at present an unknown factor. Accordingly, Hammerschlag's method gives a uniformly higher gravity than is obtained by more accurate direct estimates. Practically, in performing the test one finds that the behavior of the drops varies, for reasons which are not clear, some rising and others falling in the same mixture. A large drop should be secured and followed in the test. Since the specific gravity of the blood in simple anemia varies principally with the Hb, the percentage of Hb may in many cases be calculated with considerable accuracy from the specific gravity. Hammerschlag has prepared the following table showing the rela- tion of Hb to the specific gravity as determined by his method : Specific gravity. Hb. 1033-1035 .... . . ... 25-30 per cent. 1035-1038 30-35 " 1038-1040 .... 35-40 1040-1045 . 40-45 " 1045-1048 .... . 45-55 1048-1050 . 55-60 " 1050-1053 . ... . .... 65-70 1053-1055 . 70-75 1055-1057 75-85 1057-1060 85-95 The suggestion that Fleischl's hemoglobinometer be discarded for this indirect method of estimating Hb has not found favor. The changes in the plasma in severe anemia, leukemia, dropsy, and diarrheal diseases, render this practice always unscientific and fre- quently very unreliable (cf. Siegel, and Stintzing and Gumprecht). Bs. 22. Capillary glass tube adapted to various details of blood analysis. Schmaltz 's Method. A thin-walled capillary glass tube is pre- pared, about 12 mm. in length, and of a calibre of about § mm. at the ends and 1J mm. at the middle. This is thoroughly cleaned, dried, and weighed before and after filling with distilled water. After drying with ether the tube is filled with blood and again weighed. The weight of the blood divided by that of the water gives the specific gravity of the former. This is the best of the methods requiring a small quantity of blood, and in experienced hands gives more accurate results than TECHNICS. 59 Hammerschlag's method. It is to be especially recommended in experimental and laboratory work, but usually requires too much blood for its adoption as a routine clinical method, although the quantity required, two drops, is not great. The chief sources of error are in the difficulty of thoroughly cleaning the tube and inac- curacy in the use of the scales. Schmaltz found by controlling his results by the use of salt solutions of known density that the error did not exceed 0.003 in the computed gravities. Comparing the percentage of Hb obtained by Fleischl's method with the specific gravity obtained by his own method, Schmaltz con- structed the following table : Specific gravity. Hb. Specific gravity. Hb. 1030 . . 20 per cent. + 1049 . . 60 per cent 1035 . . 30 " 1051 . . 65 1038 . 35 " 1052 . . 70 1041 . . 40 " 1053.5 . . 75 1042.5 . . 45 " 1056 . . 80 1045.5 . . 50 " 1057.5 . . 90 1048 . . 55 " 1059 . . 100 ESTIMATION OF THE ALKALESCENCE OF THE BLOOD. The determination of the alkalescence of the blood is attended with very great practical difficulties. This alkalescence being refer- able to the presence of carbonates, bicarbonates, and of albumins which are retained in solution by acid phosphates, it is always diffi- cult to judge of the changes in these principles and the consequent variations in reaction produced by the procedures required in alka- limetry. If serum alone is titrated, the alkaline principles of the clot are left out of account, and if " laked " blood is employed there is an uncertain factor in the chemical changes produced, especially in the delicately balanced albumins and phosphates, during the solu- tion of red cells. Nevertheless it appears from a considerable number of painstaking studies by Landois, v. Jaksch, Kraus, Tausczk, Lowy, Schultz- Schultzenstein, Limbeck, and Rigler, that in blood and in serum there is a fairly constant group of alkaline principles which may be rather accurately measured and which have a distinct and important relation to disease (cf. Blood in Fever). Four of the many methods employed may be recommended as most reliable. 1. Lowy's Method. In a 50 c.c. flask containing 45 c.c. of 2 per cent, solution of ammonium oxalate, 5 c.c. or less of fresh blood are accurately measured and dissolved. Of the solution 5 c.c. are titrated by means of a -^ normal solu- tion of tartaric acid, using litmus paper as an indicator. The latter may be prepared by soaking prepared paper in an alcoholic solution of litmus to which dilute HC1 has been added until a violet color appears. The end reaction is obtained by adding a drop of blood solution to the paper and closely inspecting the color of the outer 60 GENERAL PHYSIOLOGY AND PATHOLOGY. zone into which the fluid diffuses. The result is not affected by changes in temperatures. Engel's Alkalimeter. Engel has devised an apparatus for the clinical estimation of alkalinity according to Lowy's method. A large drop of blood is drawn into a special pipette up to the mark 0.05 and diluted with distilled water to the mark 5.0. After shaking the dissolved blood is discharged into a glass cylinder and titrated by ^g- normal solution of tartaric acid (ac. tartar., 1 gr.; aq. dest., 1 litre). The acid is added drop by drop until a distinct red zone appears, when a drop of blood is allowed to diffuse through litmus paper. In normal blood about ten drops of acid bring the end reaction. Computation. If 0.4 c.c. of acid is required to neutralize 0.05 c.c. of blood, 8 c.c. of acid will be required to neutralize 1 c.c. of blood. One cubic centimetre of -^ normal tartaric acid neutral- FlG. 23. Engel's alkalimeter. izes 0.533 mg. NaOH (Engel), so that 8 c.c. of acid solution indicate the presence in 1 c.c. of blood of 4.264 mg. of NaOH, to which terms the alkalinity of the blood is usually reduced. 2. Method of Schultz-Schultzenstein. By means of the pipette of the Fleischl hemoglobinometer, 5 or 7.5 mg. of blood are meas- ured, which is dissolved in 12 c.c. of distilled water. This is acidi- fied by adding 1.5 c.c. -^ normal H 2 S0 4 . After careful mixing a drop or two of ethereal solution of erythrosin is added as an indicator and the solution titrated, with frequent stirring, by -^§-$ normal solu- tion of KOH. The end reaction is shown by the first appearance of a red color in the supernatant ether. The test must be performed speedily to avoid obscuring the end reaction by a layer of fibrin pre- cipitated by the ether. The requirement of a very small quantity of blood renders this method specially suitable for clinical purposes. TECHNICS. 61 3. v. Limbeck's Method. To about 200 c.c. of boiled distilled water are added 5 c.c. decinormal HC1 solution, and drop by drop, with stirring, 5 c.c. of serum spontaneously expressed from a clot. The stirring rod should be covered with black gutta-percha. The resulting clear and slightly opalescent fluid is now titrated with decinormal solution of NaOH. After adding a few drops a precipi- tate forms which soon dissolves. The end reaction is reached when the abundant precipitate (of albumin) no longer dissolves, which is best determined by finding a flocculent precipitate persisting on the black rod. The same quantity (5 c.c.) of fresh blood may be titrated by this method and the total alkalinity of the blood determined. In adding blood, however, it must be dropped very carefully into the hot water to avoid coagulation. The chief technical difficulty with this method lies in detecting the end reaction. In case of doubt the precipitate may be dissolved by adding 1 to 2 c.c. of acid, as above, and titrating as before. There is also the objection that it takes no account of the alkalinity referable to albumins. Limbeck doubts, however, if the capacity of albumins to neutralize acids ever comes into action in the body, and argues that his method estimates exactly, without regard to the quantity of albumin, the alkalinity of the salts present in the blood. 4. Rigler's Method. The blood flows from the vein through a canula into two vessels: (1) 3 to 4 c.c. in a tube for centrifuging ; (2) a similar quantity into a 50 c.c. flask containing 10 c.c. of 96 per cent, alcohol. The serum is obtained by centrifuging, and is transferred to a flask containing 10 c.c. of 96 per cent, alcohol. Both flasks are weighed both before and after the addition of blood or serum to determine the weight of blood. After shaking the specimens stand for half an hour, and then to each is added 10 c.c. or neutral distilled water. The alkalinity is determined by titration with ^ normal H 2 S0 4 , using red and blue litmus paper as indicators. 5. Wright has devised and extensively used one of the most practical clinical methods of estimating the alkalinity of the blood, for the full explanation of which the reader should consult the original description. Determination of the Coagulability of the Blood. Vierordt and Wright have employed methods of determining the coagulability of the blood the results from which do not appear to be sufficiently accurate even for clinical purposes (Limbeck). Estimation of the Osmotic Tension of the Plasma. Although comparatively little attention is usually paid to the osmotic tension of the plasma, it is evident from the frequent occur- rence of hemoglobinemia in various forms of severe anemia, malarial 6 2 GENERAL PHYSIOL OGY AND PA THOL G Y. and especially hemoglobinuria fever, jaundice, acute poisonings, etc., that the condition of the blood in this respect is of prime importance in the clinical and pathological study of these diseases. In Ham- burger's method we possess ready and very exact means of investi- gating this problem. In a series of test tubes are poured small quantities, accurately meas- ured, of the serum to be examined, and to each is added an increas- ing quantity of distilled water. The surface of the mixed fluids in each tube is then touched with a pipette holding a very little normal blood, and the tubes are allowed to stand twelve hours. By that time the cells have settled to the bottom in some tubes, while in others the supernatant fluid is tinged with dissolved Hb, and one notes that tube in which the first traces of Hb appear. Normal red blood cells begin to lose their Hb in solutions of salt containing any less than 0.46 per cent, of XaCl. Estimated in terms of NaCl that tube of the series which shows the first faint traces of dissolved Hb, therefore shows the same osmotic tension as a 0.46 per cent, solution of salt. The dilution being known the tension of the original serum can be computed as follows : Suppose that to 1 c.c. of serum 0.9 c.c. of water added, caused the solution of Hb. Then the tension of the original serum is equivalent to 1 -f- 0.9 X 0.46 = 0.874 per cent, of XaCl. Hamburger* points out that all red cells do not dissolve with equal rapidity in the same salt solution, and he defines as minimum resistance that strength of salt solution in which the weakest cells begin to dissolve, and as maximum resistance that solution in which the strongest cells begin to dissolve. In order to obtain comparable results he recommends for universal use a series of simple tubes in which is secured a dilution of blood in proportion of 1 : 40 of salt solution. He determines the minimum resistance by adding 0.05 c.c. of blood to each of several salt solutions, allows the specimens to stand for two hours, and centrifuges in order to determine the first appearance of dis- solved Hb. The apparatus with twelve tubes can be obtained from W. J. Weller, Kerk Stratt, Utrecht. CRIOSCOPY. In 1898 Koranyi pointed out that certain important clinical deduc- tions could be drawn from the determination of the freezing point of the blood and urine. The method is based upon the fact that the presence of elements in solution in a fluid increases the osmotic ten- sion of the fluid and lowers its freezing point, both effects following invariable physical laws. The freezing point of normal blood he found to be 0.56° lower than that of water — a fact which is commonly indicated by the formula J = 0.56°. A series of clinical studies showed that in uncompensated heart disease the lowering of the freezing point of the blood, -J, is increased, reaching as much as 0.67°, although the chlorides of the blood are diminished. This result he shows is owing to the retention of C0 2 in the system. In nephritis A was shown to be much increased, reaching 0.71° in a case of uremia. Various characteristic changes were noted in the TECHNICS. 63 blood in anemia and in fevers, where in the absence of cyanosis A was less than 0.56°, and in hemoglobinemia, in which A rose to 0.70°. Various contributions regarding the clinical value of the test have appeared. Koeppe noted variations in healthy subjects between 0.558° and 0.570°, and in hysteria, menstruation, gastritis, cancer of pylorus, diabetes, nephritis, neurasthenia, pleurisy and pneumonia, variations between 0.508° and 0.634°. Lindemann finds that in disease of the kidneys the freezing point of the serum is unaffected as long as uremic symptoms are absent, while in uremia A rises to 0.70°. Kummel, in ten cases of nephritis, cystic kidney, and anuria after operation, always found A increased to 0.60° to 0.65°, and once to 0.71°. Ogston reports various conditions in which the value of the test appears very uncertain. Ceconi and Micheli could not distin- guish by the method the stage of an inflammatory exudate, nor between exudates and transudates, but found that the freezing point of the cerebrospinal fluid was not higher than normal (0.56°), while in uremic seizures simulating meningitis A was increased. In pneumonia A was always increased to 0.60°, while in typhoid fever several observers have demonstrated no change. Carrara finds crioscopy of the blood serum the most reliable test for the medico- legal diagnosis of death by submersion. The absorption of fresh water through the lungs, gastro-intestinal tract, and skin, raises the freezing point of blood serum, while salt-water lowers it, in each instance more markedly in the left than in the right heart. His results were as follows : Dog, drowned in fresh water, serum of right heart . . . A = 0.42° " " " " " left heart . . . A = 0.29 Man, drowned in sea water, serum of right heart . . . A = 1-04 ■■ " " " " left heart . . A = 1-18 In the determination of the freezing point of blood and blood serum, Beckmann's apparatus is employed, and preferably the modi- fication of Lindemann. About 10 c.c. of blood is required, and may be drawn from the basilic vein. Coagulation makes no difference in the freezing point (Ogston). Beckmann's Apparatus. Description of Apparatus (Fig. 24). The large vessel (C) for holding the mixture of salt and ice is fitted with a perforated iron lid through which passes the handle of a wire stirrer, while in the centi'e is an opening for the tube (B). Another tube (A) containing the liquid to be tested, the thermometer (D), and a small wire or glass stirrer (E) fits in the mouth of the large tube. The thermometer is of peculiar construction, graduated in y^ C, and permitting very accurate readings over a scale of 10°. It con- tains the usual main reservoir at the bulb and also an accessory reser- voir at the top of the column (F), by means of which one may add to or take from the column of mercury at will. A thermometer with " fixed zero " and without an accessory reservoir may be obtained from the makers of Beckmann's apparatus as modified by Linde- 64 GENERAL PHYSIOLOGY AND PATHOLOGY. mann, but even with this thermometer the exact freezing point of water on the instrument varying from day to day must first be deter- mined as described later. Lindemann's ap- Fia. 24. paratus has the advantage of requiring not more than 5 c.c. of serum. Application of the Test. The large jar is half filled with finely crushed ice and salt. The thermometer must first be "set" so that the top of the column of mercury at freezing point falls about the middle of the graduated scale. This may be done by im- mersing the bulb in warm water until the mercury in the column joins that in the accessory reservoir. Then fill the inner tube with distilled water and cool to about 10° C, immersing an ordinary thermometer in the water. Now place the thermometer of the instrument in the cool water, and when it is thoroughly cool break the column of mercury in the reservoir by tapping it sharply with the finger. The distilled water is now frozen with the thermometer immersed, and the exact point to which the broken column of the mercury falls is taken as the zero of the instrument. It will be found that the moment that the water congeals the mer- cury falls slightly below the true freezing point, shortly to rise and remain steady at the exact point. With Lindemann's thermometer the breakage of the column is not required, but the exact freezing point of water on the thermometer must be determined. Having determined the zero of the instru- ment, the blood or serum to be tested is placed in the inner tube, cooled to near the freezing point, and the thermometer im- mersed in it. As the fluid cools it must be stirred by the glass stirrer. When the fluid congeals the mercury drops a fraction of a degree, but soon rises and becomes stationary at the freezing point of the fluid. The difference between the freezing point of water and that of the fluid, J, may then be determined by the read- ing, which should be made as soon as the column becomes stationary. Bremer's Specific Reaction of Diabetic Blood. Saturated watery solutions of eosin (watery ?) and of methylene blue are mixed in about equal proportions so that a neutral reaction is obtained, and the mixture produces little or no stain on litmus Beckmami's apparatus. TECHNICS. 65 paper. A precipitate forms, soluble in alcohol, insoluble in water, which is filtered, washed, dried, and powdered. To twenty-four parts of this powder are added six of powdered methylene blue and one of eosin Of this mixed reddish brown powder 0.025 to 0.05 grm. are dissolved in 10 c.c. of 33 per cent, alcohol, in which solu- tion the specimen is stained for four minutes. The specimens are prepared by smearing the blood on glass slides or covers, and fixed by boiling in equal parts of alcohol and ether for four minutes. This may be accomplished by placing the bottle of alcohol and ether in hot water at 60° C. After washing the stained specimens in water, diabetic blood has a greenish tint, while normal blood is reddish violet, and on micro- scopic examination the erythrocytes of diabetic blood are found to be greenish, while those of normal blood are red. Bremer found the reaction in 50 out of 51 cases of diabetes. The negative result was obtained in a well-established case, a boy of six- teen, in whom glycosuria began after an electric shock. He found that the reaction persisted in the absence of glycosuria ; that normal blood floated on diabetic urine for fifteen minutes gave the reaction ; that blood treated with solutions of glucose failed to give the reac- tion ; that the blood in glycosuria artificially produced in animals by phloroglucin gave the reaction, while in that produced by phloridzin it did not. The nature of the reaction is not understood. While some observers have convinced themselves that the presence of glucose is not sufficient to bring about the altered staining qualities of the blood, Hartwig concluded that the glucose first causes a change in the Hb which shows itself in the altered reaction of the red cells to aniline dyes. Other reducing agents besides glucose are probably of importance in this relation (R. Muller). Some have attempted to explain the reaction by reference to the changes in alkalescence of the blood. Hartwig found the reaction to disappear after the addi- tion of alkali to the blood. Although Lowy found a pronounced reaction in cases with relatively high alkalescence, Schneider and others have shown that the reaction runs parallel with the quantity of abnormal acids in the blood. Various modifications of the above most approved method have been employed successfully by Bremer and others. One of these, employed by Bremer, is as follows : 1 per cent, solutions of Congo red or of methylene blue stain diabetic blood very slightly, while 1 per cent, solution of Biebrich scarlet stains it intensely. A directly opposite relation holds with normal blood. Rather thick smears of blood should be used with this procedure, and the colors compared by the naked eye. The value of Bremer's test has been confirmed by Le Goff, Eichner and Folkel, Lepine and Lyonnet, James, Jeanselme, Badger, and Hartwig, but similar reactions have been found in normal blood in leukemia, Hodgkin's disease, exophthalmic goitre, and multiple neuritis. A partial reaction has been obtained in cachectic condi- tions, and Bremer failed to find it in a case of glycosuria of neurotic 5 66 GENERAL PHYSIOL OGY AND PA THOL OGY. origin. Yet in most conditions, other than diabetes, the reaction has been found, when present, to be inconstant, and to occur in a very small proportion of cases (Lepine, Eichner, Hartwig). The technical difficulties in carrying out the test are considerable, while a slight variation in technique appears to vitiate the result, as is indicated by the failures reported by Patella and Mori, after both Bremer's and Williamson's methods. Williamson finds that diabetic blood decolorizes solutions of methylene blue, while normal blood does not. His test is performed as follows : 20 c.mm. (2 drops) of blood are dissolved in 40 c.mm. of water, and to the solution is added 1 c.c. of methylene blue (1 : 6000 solution) and 40 c.mm. of liquor potassce (s. g. 1.058)._ The vessel is then placed in boiling water for four minutes; diabetic blood Pronounced lipemia. Specimen treated with osmic acid. Lower half shows extracellular fafglobules, upper half having been cleared by oil of turpentine. (Gumprecht.) decolorizes the solution, normal blood leaves it deep blue. Diabetic urine has the same effect. Williamson found this reaction in 6 diabetics and failed to find it in 160 cases of other diseases, including one of leukemia. Demonstration of Glycogen in Blood. Gabritschewsky's method may be employed. The blood smears, thoroughly dried in the air, are stained for several minutes in : iodum pur., 1 ; KI, 3 ; aq., 100 ; acacia pulv. in excess. The presence of glycogen is indicated by the appearance of mahogany-brown granules of variable size, in leucocytes and plasma. (See Plate XII.) TECHNICS. 67 Czerny claimed that this method demonstrates the presence in the blood not of glycogen but of a carbohydrate more nearly related to amyloid. Huppert's later studies on the blood of animals support the belief that the substance thus demonstrated is really glycogen. It is probable that many colorless globules visible in leucocytes treated by ordinary staining methods are referable to the former presence of glycogen, which is soluble in water and of which the reactions are largely destroyed by heat. Demonstration of Fat in the Blood. The blood smear is fixed in 1 per cent, osmic acid for twenty-four hours and counterstained with eosin. The fat particles are then stained black. Since all that blackens under osmic acid is not fat, a control preparation should be fixed twenty -four hours in alcohol and ether, then in 1 per cent, osmic acid for twenty-four hours, counterstained with eosin, and the extraction of the fat by ether demonstrated by the absence of black particles in cells and plasma. BACTERIOLOGICAL EXAMINATION OF BLOOD. Apparatus. Any aspirating syringe of the capacity of 10 c.c. which can be thoroughly sterilized may be used for drawing the blood, but the difficulty of sterilizing the ordinary syringe renders it advisable Fig. 26. Blood aspirator. (Half size.) to employ a special apparatus without joints and permitting the adjustment of the needle on a ground-glass orifice (Fig. 26). The other end of the tube, considerably narrowed and bulbed, is plugged with cotton, and over it a rubber tube is slipped, through which the blood is sucked by the operator. The glass tube and needle are inclosed in a glass case, sterilized by heat, and kept ready for use. Such an apparatus — a modified form of one devised by Dr. James — has been made for the writer by Stohlmann, Pfarre & Co., New York City. 68 GENERAL PHYSIOLOGY AND PATHOLOGY. Operation. The blood should be drawn from the median basilic vein. The skin of the arm must be thoroughly washed with soap and water, cleaned with alcohol, and sterilized with bichloride solu- tion 1 : 500, or formalin 5 per cent. In most female subjects an Esmarch bandage must be applied at the axilla to render the basilic vein prominent, but in many male subjects this is unnecessary. It is better to incise the skin over the vein before inserting the needle, but this precaution is not essential under ordinary circumstances. When the needle enters the vein obliquely the blood should flow into the tube, and the flow may be hastened by suction through the rubber tube. At least 10 c.c. should be drawn, a quantity which is usually of no consequence to the patient. The elbow may be snugly bandaged after the operation. Preparation of Cultures. Before clotting the blood should be distributed in culture media. The medium chosen depends upon the particular organism which one expects to demonstrate. For the usual pathogenic species, staphylococcus, streptococcus, typhosus, etc., ordinary bouillon suffices, but for the gonococcus, ascitic broth should be preferred. The dilution of the blood should be considerable, and for 10 c.c. of blood it is advisable to employ at least 300 c.c. of broth. Unusual care should be employed at every step of the process to prevent contamination. Bibliography. Badger. Cited by Cabot. Baumgarten. Johns Hopkins Bull., vol. 13, p. 176. Becker. Arch. kl. Med., Bd. 70, p. 17. Bergmann. Cited by Limbeck, Inaug. Dis., Munchen, 1884. Biernacki. Zeit. f. Physiol. Chem., Bd. 19, p. 179. Zeit. f. kl. Med., Bd. 24, p. 460. Bleibtreu. Pfliiger's Archiv, Bd. 51, p. 151. Blix, Hedin. Scand. Arch. f. Physiol., 1890, p. 134. Bordet. Annal. Inst. Pasteur, T. 12-14. Brandenburg. Munch, med. Woch., 1900, p. 183. Bremer. N. Y. Med. Jour., vol. 63, p. 301. Ibid., vol. 66, p. 808. Cent. f. inn. Med., 1897, p. 521. Buntzen. Virchow-Hirsch, Jahresb., 1879. Carrara. Arch. Ital. de Biol., T. 35, p. 349. Ceconi, Micheli. Rif. Med., 1901. Cheron. Compt. Rend. Acad. Sci., 1895, vol. 121, p. 314. Cohnheim. "Vbrles. u. Allg. Pathol., Berlin, 1882, Bd. 1, p. 397. Cohnstein, Zuntz. Pfliiger's Archiv, 1888, Bd. 42. Corin. Viertelj. f. ger. Med., Bd. 23, p. 61. Czerny. Archiv. f. exp. Path. u. Pharm., Bd. 34, p. 268. Ibid., Bd. 31, p. 190 Daland. Fort. d. med., 1891, No. 21. Deutsch. Cent. f. Bact., Bd 29, p. 661. Dieudonne. Munch, med. Woch., 1901, p. 14. Ekgren. Deut. med. Woch., 1902, p. 519. Elzholz. Wien. klin. Woch., 1894, No. 32. Engel. Berl. klin. Woch., 1898, p. 308. Eichner, Folkel. Wien. klin Woch., 1897, p. 1103. Eyckman. Arch. f. ges. Physiol., 1895, Bd. 40, p. 340. Gabritschewsky. Arch. f. exper. Path., Bd. 28, p. 272 Gartner. Allg. Wien. med. Zeit., 1892, p. 513. Gartner, Romer. Wien. klin. Woch., 1892, No 2. TECHNICS. 69 Graham-Smith, Sanger. Jour, of Hyg., vol. 3, p 258. Grawitz. Zeit. f. klin. Med., Bd. 21, 22. Methodik d. kl. Blutuntersuch, Berlin, 1899. Haldane. Jour, of Physiol., vol 26, p. 497. Haldane, Smith. Jour." of Physiol., vol. 22, p. 232; vol. 25, p. 331. Hamburger. Die physiol. Kochsalzlos. u. d. Yolumbestimmung d. korperl Elemente im Blute. Cent. f. Physiol., 1893, 1894, p. 656. Virchow's Archiv, Bd. 141, p. 230. 4 Jour, de Physiol, et de Path, gen., 1900, p. 889. Hammerschlag. Wien. klin. Woch., 1890, p. 1018. Hartwig. Deut. Arch. klin. Med., Bd. 62, p. 287. Hedin. Arch. f. ges. Physiol., Bd. 40, p. 360. Heidenhain. Versuch. zur Lehre von der Lymphbildung, Pfluger's Archiv, 1891, Bd. 49, p. 209. Heissler. Arbeit, aus d. path. Tnstit. zu Munchen, 1886, p. 322. Huppert. Zeit. f. Physiol. Chem., Bd. IS, p. 144. Jacobi, M. P. N. Y. Med. Record, 1898, I., p. 933. v. Jaksch. Zeit. f. klin. Med., Bd. 13. Jenner. Lancet, 1899, I., p. 370. Jolly. Arch, de Med. exper., T. 14, p. 73. Klein. Volkmann's Vortrage, No. 29. Knopfelmacher Wien. klin. Woch., 1893, pp. 810, 886 Koeppe, Phvsikal. Chem. in d. Med., Wien, 1900. Koranyi. Zeit. f. kl. Med., Bd. 33, 34. Berl. kl. Woch., 1901, p 424 Kraus. Arch. f. exper. Path., Bd. 26, p. 186 Kummel. Munch, med. Woch., 1900, p. 1525. Landois. Text-book of Physiol., 1893. Le Goff. React, chrom. du sang diabet, Paris, 1897. Leclainche, Yallee. Semaine Med., 1901, No. 4. Lepine, Lyonnet. Lyon Med., T. 82, p. 187. Leichtenstern. Unters. u. d. Hemoglobingehalt d. Blutes, Leipzig, 1878, p. 57. Lenossier, Lemoine. Semaine Med., 1902, No 13. v. Lesser. Sitzungsber. d. k. Sachs. Gesell. d. Wissenschaften, 1873, p. 573; 1874, p. 153. Du Bois-Raymond's Archiv, 1878, p. 41. v. Limbeck. Grundriss einer klin. Path. d. Blutes, Jena, 1897, p. 68. Lindemann. Arch f. klin. Med., Bd. 65, p. 1. Pfluger's Archiv, Bd. 56, p. 579. Lloyd Jones. Journal of Physiol., 1887, viii Loewy. Pfluger's Archiv, Bd. 58, p. 462. Cent. f. med. Wissen., 1894, p. 785 Fort. d. Med., 1898, p. 171. Loioit Studien z. Phys. u. Path. d. Blutes, etc., Jena, 1892. Ludwig. Cited by Lukjanow. Lukjanow. Grundzuge ein. allg. Path, des Gefass-systems, Leipzig, 1894 Lyon, Thoma. Virchow's Archiv, Bd. 84, p. 131. Michaelis. Munch, med. Woch., 1902, p. 225 Mitchell. Amer. Jour. Med. Sci., vol. 107, p. 502. Muller, R. Munch med. Woch., 1899, p. 820. Muller, Rieder. Deut Arch. klin. Med., Bd. 48, p. 96. Nuttall, Dinkelspiel Brit. Med. Jour., 1901, I., p. 1141. Jour, of Hyg., 1901 Ogston. Lancet, 1901, II., p. 1253. Oertel. Klinische Zeit. u. Streitfragen, Wien, 1889. Oliver. Lancet, 1896, I., p. 1778. Patella. Mori. Gaz. deg. Osped., 1896, II., p. 1441. Pepper, Stengel Cong. f. inn. Med., 1896, p. 631. Pfeiffer. Ibid., 1895, No. 4. v. Recklinghausen. Handb. der allg. Path. d. Kreislaufs u. der Ernahrung, Stuttgart, 1883, p. 179. Reinert. Zahlung der Blutkorp, Leipzig, 1891 Rigler. Cent. f. Bact,, 1901, Bd 29. Rostocki. Munch, med. Woch., 1902, p. 740. Scamiell. Boston Med. Surg. Jour., vol. 142, p 160. Schmaltz. Arch. f. klin. Med., Bd. 47, p. 145. Schneider. Munch, med. Woch., 1899, p. 817. Schultz-Schtdtzenstein. Cent. f. med. Wissen., 1894, p. 801 Siegel. Wien. klin. Woch., 1891, No. 33. Stem. Deut. med. Woch., 1901, p. 135. Stintzing, Gumprecht. Deut. Arch. f. klin. Med., Bd. 53, p. 265. 70 GENERAL PHYSIOL OGY AND PA THOL OGY. Stoenesco. Annal. d'Hyg. Pub., T. 48, p. 211. Strube. Deut. med. Woch., 1902, p. 24. Tallquist. Arch. Gen. de Med., 1900, I. p. 421. Tausczk. Ungar. Arch. f. Med., 1895, p. 359. Thayer. Johns Hopkins Hosp. Bulletin, 1893, p. 37. Thibault. Bull. Acad, de Med., Bruxelles, 1894, p. 112. Uhlenhuth. Deut. med. Woch., 1901, pp. 82, 260. Valentin. Reporter, f. Anat. u. Physiol., 1838, Bd. 3, p. 281. Vierordt. Arch. f. Heilk., 1878, Bd. 19, p. 193. Wagner's Handb. d. Phvsiol., 1842, Bd. 1, p. 84. Wasserman. Cong. f. inn. Med., 1900. Wasserman, Schutze. Deut. med. Woch., 1901, 1902, 1903. Wendelstadt, Bleibtreu. Zeit. f. klin. Med., Bd. 25, p. 204. Wick. Wien. med. Zeit., 1887. Winternitz. Cent. klin. Med., 1893, Bd. 14, pp. 177, 1017. Worm-Muller. Transfusion u. Plethora, Christiania, 1875. Wright. Lancet, 1897, II., pp. 719, 814, 833. Lancet, 1892, I., pp. 457, 515. Zappert. Zeit. f. klin. Med., Bd. 23, p. 227. Ziegler. Cent. f. Path., 1902, p. 545. Ziemke. Deut. med. Woch., 1901, pp. 424, 731. CHAPTEE II. CHEMISTRY OF THE BLOOD. THE CHEMICAL COMPOSITION OF RED BLOOD CELLS. In the analyses of Schmidt, Hoppe-Seyler, and Judell the red cells were separated from the serum by the addition of salts (Na 2 S0 4 , NaCl), a procedure which considerably alters the chemical composition of cells and plasma. The accuracy of their results, which are the best available, is therefore only approximate. The specific gravity of red cells is usually about 1088. They contain about 90 per cent, of oxyhemoglobin and a small proportion of a globulin-like albumin (nucleoproteid) coagulating at 75° C. There are also traces of lecithin and cholesterin. The principal salts are phosphates of Na, K, Ca, and Mg, and chloride of K, whereas in the serum the chief salt is NaCl. In cholera, dysentery, and dropsy, Schmidt found the red cells to be of increased specific gravity in proportion to the duration of the exudative process, while their chemical analysis showed that they participate in the changes which first affect the serum, losing first water, then salts, and finally albumin. More recently v. Jaksch, 1 after establishing the normal content in N of the red cells, followed the variations in this principle in various diseases. He found very marked and irregular variations both in health and disease, which indicate that the method used (determination of total N) was unre- liable. Biernacki, estimating the dry residue of red cells settled after the addition of sodium oxalate, found a normal residue of 29.28 to 30 per cent. ; in carcinoma of the esophagus, 27.9 per cent. ; in tabes with anemia, 25.51 per cent. ; in chlorosis (Hb 25 per cent.), 22.24 per cent. ; in chronic nephritis (Hb 20 per cent.), 22.88 per cent. Among the important observations of Biernacki's are the increased content in water of the red cells in hydremia, the general parallelism between P 2 5 , Fe, and K, and between water and NaCl in the red cells. Hemoglobin (Hb) and its Derivatives. Hemoglobin belongs to the group of proteids, containing about 96 per cent, of albumin and 4 per cent, of an iron-holding pigment, hemoehromogen. Hb exists in the red cells in combination probably with the nucleoproteid of the stroma. The nature of this union is not certainly understood, but it renders Hb comparatively insoluble, greatly concentrated, and capable of actively forming unstable com- pounds with oxygen (Hoppe-Seyler, Gamgee). 72 GENERAL PHYSIOL OGY AND PA THOL OGY. Its chemical composition is very complex and apparently variable, bat its spectroscopic relations are constant. In the circulation it exists principally as reduced Hb in the veins, and in molecular union with oxygen as oxyhemoglobin, in the arteries. One gramme of satu- rated oxyhemoglobin yields about 1.16 c.c. of oxygen, but in the cir- culation the degree of saturation with oxygen varies, and in health is never complete. Meyer and Biarnes found in the arterial blood of a dog 76 per cent, of saturation with oxygen ; after a large hemor- rhage it rose to 85 per cent, of saturation, while after inducing CO poisoning it rose to 90 per cent. Limbeck found 97 per cent, of saturation with oxygen in the blood of a dog poisoned with potassium chlorate. It thus appears that with a relative loss of functionating Hb the demands of the system may cause a more complete saturation of the blood with oxygen than exists in health. Oxyhemoglobin is bright red in color, and forms with difficulty yellowish red rhombic crystals. These crystals are very soluble in water and in very dilute solutions of alkaline carbonates, but when treated with strong alcohol they are modified and become insoluble (parahemoglobin of Nencki). They are insoluble in ether, chloroform, benzol, or carbon disulphide. Oxyhemoglobin is absolutely non- diffusible (Gamgee). Hemoglobin (reduced Hb) is dichroitic. In thick layers or in thin layers of concentrated solutions it presents a dark cherry-red color, while very dilute solutions exhibit a green tint (Gamgee). This dichroism is characteristic of the blood of simple asphyxia. Reduced Hb is more soluble, but less easily crystallizable than oxyhemoglobin. Methemoglobin is brownish red in color, readily soluble in water, and crystallizes in brownish red needles, prisms, and hexagonal plates. It contains the same proportion of oxygen as oxyhemoglobin, but in much firmer and probably in chemical union. Methemoglobin is found in the blood chiefly in cases of poisoning by a variety of substances, the so-called " blood poisons." Tschirkoff claims to have found it in Addison's disease, and Ruyter has recognized a- very similar but not identical coloring matter in a case of malignant edema. Hemoglobinemia, a solution of Hb in the plasma, a normal condi- tion in the blood of some vertebrates, is in man always pathological, and results from lessened resistance on the part of the red cells and from abnormal states of the plasma. The former condition is prob- ably concerned in cases of paroxysmal hemoglobinemia and in the destruction of blood which follows general burns (Lichtheim, Murri, Chvostek, Silberman), while the latter condition exists in the hemo- globinemia of acute poisonings and in cases referable to increased globulicidal power of the serum. Carbonic oxide Hb is a firm compound of Hb and CO, and imparts a bluish red or rose-red color to the blood. Its crystals are similar to those of oxyhemoglobin, but more bluish and less soluble. In cases of poisoning by the inhalation of fire-damp or illuminating gas, CO-Hb is formed in considerable proportion and the respiratory capacity of the blood is largely destroyed. CO-Hb persists in the CHEMISTRY OF THE BLOOD. 73 blood for several days, in cases that recover, and for a much longer period in fatal cases. Hematin, one of the advanced decomposition products of Hb, is a dark brown or blackish, non-crystalline solid, decomposed at 180° C. It is insoluble in water, dilute acids, alcohol, ether, and chloroform, but dissolves in acidified alcohol and ether, and readily in dilute alkalies. It is found in old blood transudates, in the feces where digestive fluids have acted upon blood, and in the urine in cases of arsenical poisoning. Crystals of hemin, or the hydrochlorate of hematin, are formed in Teichmann's test. Hematoidin is a crystalline derivative of Hb, found in old blood extravasations. It crystallizes in orange-colored rhombic plates, is soluble in chloroform, ether, carbon disulphide, and ammonium sul- phide, contains no iron, and gives no absorption bands, although absorbing most of the violet end of the spectrum. It is generally regarded as identical with bilirubin. Hemosiderin is an amorphous iron-holding product of the decom- position of Hb, and is abundantly found in the viscera in disease accompanied by much destruction of blood. It is probable that when red cells are disintegrated hemosiderin is formed by the action of living cells upon Hb, while hematoidin originates apart from any cellular activities (Perls, Thoma, Ziegler). Hemosiderin is black- ened by ammonium sulphide and turned blue by acidified potassium ferrocyanide. Melanin is a yellowish brown or black pigment produced by the action of the malarial parasite upon Hb. It is insoluble in water, alcohol, ether, chloroform, carbon disulphide, and acids in moderate strength, but is destroyed by heat, and is soluble in strong alkalies and in ammonium sulphide. It fails to yield the microchemical reactions of iron, and probably contains no iron. The term " melanin " is also loosely applied to a variety of black pigments occurring in the body, some of which contain iron but whose origin is uncertain. From "antialbumid," formed by heating egg-albumin with 3 per cent. H 2 S0 4 for ten hours at 100° C. , and from hemipeptone, Chittenden and Albro produced black melanin by digesting these substances with 10 per cent. H 2 S0 4 and boiling for seventy-nine hours. The change they believe to consist in a process of hydrolytic cleavage. Sulphur and fatty acids were thrown off in the process, and the melanin was found to contain C, H, N, and S. THE CHEMICAL COMPOSITION OF LEUCOCYTES. Attempts to determine the chemical composition of leucocytes have been made by analyses of leukemic blood, of pus, and of fresh lymphoid tissue. In all of these it cannot be doubted that abnormal products were constantly present, so that the chemical composition of normal leucocytes cannot be learned by such methods. Probably Lilienfeld's analyses 1 of lymphoid tissue furnish the most reliable data. 74 QENERA L PH YSIOL 00 Y AND PA THOL OGY. In cadaveric leukemic blood various fatty acids have been isolated, and lecithin, a normal constituent of blood, has been found in ex- cess. Glycogen has been repeatedly isolated in this and other con- ditions. In fresh pus, Miescher found five different forms of albumin, an abundance of lecithin, and cholesterin, phosphates of Na, K, Ca, Mg, Fe, and chloride of Na. In analyses of the thymus gland in calves Kossel and Lilienfeld isolated various albumins, principally nucleins, lecithin, and choles- terin, besides about 3 per cent, of inorganic principles. In older cells they believe that secondary products develop in the form of glycogen, protagon, and fats. The nuclein of Kossel is a combination of the organic nucleinic acid which contains phosphorus, with albumin, and in the leucocytes is found in com- bination with another albuminous body, histon, with which it forms nucleo- histon. Under certain conditions Kossel and Lilienfeld believe that nucle- inic acid becomes free in the cells, and exerts bactericidal action before and during phagocytosis. The nucleins are bodies obtained from animal (or vegetable) cells after digestion with pepsin to which they are resistant. They are rather insoluble in water, alcohol, and ether, give the biuret and Millon's reactions, and acting as strong acids and uniting with bases, they may be identified from the basic albumins by their tinctorial qualities. Boiled with dilute acids they yield " nuclein bases " or xanthin bodies; or treated with alkali they yield albumin and nucleinic acid. They are rich in phosphorus and iron. Nucleinic acid is of variable composition, but that from the calf 's thymus is a combination of a xanthin body with a complex phosphoric acid (Hammarsten). Milroy and Malcolm have demonstrated some peculiar reactions of the granules of leucocytes. They found that weak watery solutions of sodium carbonate or of acids remove the pseudoeosinophile granules. Injections of nu- cleinic acid reduce the number of pseudoeosinophile and eosinophile granules and cause them to become basophile. This change they explain on the theory that the granules are composed of a nucleoproteid and an albumin, the latter being removed by the nucleinic acid. The change being most marked in the pseudoeosinophile granules, they conclude that in these granules the albumin is less firmly bound than in eosinophile. The complete solution of the baso- phile residue of the granules seemed to be accompanied by an increase of excretion of P„Oc. THE CHEMICAL COMPOSITION OF BLOOD PLATES. Since the true nature of blood plates has been at least partly shown the chief inquiry concerning them has related to their chemical com- position. Lowit has strongly maintained that they are composed of globulin, a claim which is probably true of some of the bodies which appear in coagulating shed blood and which must be classed with the blood plates of different origins. CHEMISTRY OF THE BLOOD. 75 Lilienfeld 2 regards the substance of blood plates as belonging to the nucleo-albumins, and identifies them with the remnants of the nuclei of leucocytes, basing this opinion on their content in phos- phorus, their resistance to digestion by pepsin, and their micro- chemical reactions. These observations may be regarded as definitely settling the question of their chemical composition, but do not prove their exclusive origin from leucocytes. The Serum. The Albumins of the Serum. Blood serum contains two albu- minous bodies, serum albumin and serum globulin, which together form 7.62 per cent. (Hammarsten) or 8.26 per cent. (Schmidt) of the weight of normal serum. The quantitative changes in these principles in disease are not usually very marked, owing to their relatively slight diffusibility. From the studies of Becquerel and Rodier, Schmidt, v. Jaksch, Limbeck and Pick, it has been shown that the albumins of the serum are considerably diminished in severe anemias, and in nephritis or endocarditis with dropsy, but that in most severe infectious dis- eases they are but slightly reduced and bear a fairly constant rela- tion to the other solids of the serum. If temporarily reduced by some exudative lesion, as in diarrhea, dysentery, etc., they are soon replaced if the disease continues. Becquerel and Rodier, however, found the albumins much reduced in puerperal septicemia, and Schmidt found a marked increase in the concentrated blood of cholera, v. Jaksch, 2 determining the total N of the serum, found slight variations in many acute and chronic diseases, but a well-marked loss in leukemia, pernicious and secondary anemia, and chlorosis. Limbeck, using the same method, found less marked changes in cases of anemia. The ratio of serum albumin to serum globulin, on the other hand, varies much more than does the total quantity of albumin in the serum. The normal limits are placed by Limbeck and Pick, for globulin, 16.9 to 38.3 per cent., for albumin 61.7 to 83.1 per cent, of the total albumin of the serum. When such wide limits may be found in normal subjects it is difficult to attach much importance to very considerable changes demonstrated in disease, as reported by Estelle, Hoffman, Halliburton, and Mya and Viglezio. In a considerable series of observations in various diseases Limbeck and Pick succeeded in showing only that serum globulin, the less diffusible principle, is less subject to change than is serum albumin, but they were unable to establish any other general rules. The inorganic salts of the serum include phosphates, chlorides, carbonates, and sulphates, but in what proportion or form the alkalies and earths are combined with these acids is not definitely known. Sodium and potassium are combined especially as neutral chlorides, partly as phosphates and carbonates. Pathological variations in the phosphates of the serum are of slight degree and importance, so far 76 GENERAL PHYSIOLOGY AND PATHOLOGY. as is known, probably on account of their occurrence in scant quantity and their relative lack of diffusibility. A retention of phosphates in the blood of pathological grade has not been demonstrated. The variations in the chlorides of the serum investigated by Schmidt, Biernacki, and Limbeck have not been found extreme, nor of notable pathological import, although this principle is chiefly responsible for the isotonic relations of cells and serum. A high percentage of chlo- rides is usual in anemias. The specific gravity of the serum in disease varies slightly from the normal limits, 1025 to 1030 (Hammerschlag, Limbeck). While Becquerel and Eodier and Hammerschlag found the specific gravity of the serum normal in chlorosis but reduced in secondary anemia, Limbeck found it low in both (1021 to 1023). In infectious diseases Becquerel and Rodier found the serum of normal gravity. The color of human serum is yellowish with a slight greenish fluorescence, but after a hearty meal the increased quantity of fat may yield a cloudy or whitish color. The coloring matter of normal human serum belongs to the group of luteins or lipochromes (Ham- marsten). It is extracted by alcohol and by ether, yields a blue color with iodine and sulphuric acid, and on spectroscopic analysis causes an absorption of the violet and part of the blue, which is unaffected by reducing or oxidizing agents. Icteric serum causes more complete absorption, beginning sharply in the blue (Lim- beck). When, from lessened resistance of the red cells or abnormal con- ditions of the plasma, red cells are dissolved the serum contains Hb in solution (hemoglobinemia), as occurs principally in malaria, par- oxysmal hemoglobinuria, septicemia, and with the blood poisons. In jaundice the serum has a characteristic orange-yellow color and bilirubin is readily detected. Hemolytic Properties of Human Blood Serum. Maragliano (1892) first investigated the hemolytic power of pathological blood serum upon the red cells of human subjects (isolysis), finding that the serum in many cases of primary anemia, leukemia, purpura, pneumonia, malaria, typhoid fever, cirrhosis, etc., possessed in vary- ing degree the power of dissolving healthy red cells. In the solution . he obtained the spectrum of hematoidin. Heating to 50° to 53° C. did not destroy this hemolytic property. Various later writers have confirmed and extended these observations, and suggested various explanations of the toxic effects of blood serum, but the systematic study of the subject has been possible only since the contributions of Bordet and Ehrlich on the nature of hemolysis. Many of the earlier studies probably deal with other forms of hemolysis than those dis- cussed by Bordet and Ehrlich. Halpern (1902) reviews the subject to date, finding considerable but rather inconstant variations in the hemolytic power of blood serum in disease, both upon human and upon rabbit cells. In typhoid fever hemolysis was usually increased, and in septicemia it was decreased ; but in most other conditions it varied slightly from that of normal blood. (See section on Hemolysis.) CHEMISTRY OF THE BLOOD. 77 THE WHOLE BLOOD. Albumins. The albuminous principles of normal circulating blood include hemoglobin, serum albumin, serum globulin, and fibrinogen. In shed blood a nucleoproteid exists which is probably derived from the nuclei of leucocytes, probably also from red cells, which combines with calcium salts to form the fibrin ferment, and which is called "prothrombin." Traces of albumose have been found in the blood in various diseases, and other nitrogenous princi- ples, occasionally present and commonly reckoned with albumins, are urea, uric acid, and xanthin bodies. Varying considerably with the method of determination, the proportion in which these elements exist in the blood has been placed for the entire group by v. Jaksch 2 at 22.62 per cent. ; by Limbeck at about 25 per cent. ; by Schmidt at 10.82 to 16.63 per cent. A relative increase of total albumin is seen, according to v. Jaksch, in diseases accompanied by marked loss of fluids, as in cholera and severe diarrhea, but an absolute increase has not been demonstrated. In infectious diseases the albumins are moderately reduced, even when the number of red cells remains normal (typhoid fever). In nephritis there is usually but not always a reduction. In endocarditis there is little change. In chlorosis, pernicious anemia, and leukemia the total albumins of the blood are constantly diminished, but in secondary anemia the diminution usually noted sometimes fails. v. Jaksch found his lowest proportion, 8.46 per cent., in a case of gastric cancer. Peptone has been demonstrated in the blood of leukemia by Ludwig, v. Jaksch, 3 and Freund and Obermayer, and deutero-albumose by Matthes. These observations were made on blood from the cadaver. Devoto and Wagner, who examined blood obtained during life, both failed to find peptone. The suspicion that the peptone previously demonstrated was of postmortem formation was followed up by v. Limbeck in a case of myelogenous leukemia. In the fresh blood no peptone was found, but in a specimen that had stood at room temperature for forty-eight hours albumose was demonstrated, but its exact character was not determined. The patient died three weeks later and deutero-albumose was demonstrated in the blood of the cadaver, v. Jaksch states that peptone (albumose ?) is found in leukemic blood only when eosinophile cells are abundant. Freund's observation that peptone is found in the blood in cases of sarcoma but not in cases of carcinoma has not yet been confirmed. The addition of small quantities of peptone to plasma in vitro reduces its content of C0 2 and its coagulability, and it seems prob- able that if peptone (or albumose) exists in the circulating blood a similar influence is exerted, intra vitam, by its presence. Quantitative estimation of total albumin of the blood may be accomplished by one of two methods. 1. By precipitating the albumins by excess of alcohol, and weigh- ing the dried precipitate. 78 GENERAL PHYSIOLOG Y AND PATHOLOQ Y. 2. By estimation of total N by Kjeldahl's method and multiplying the result by 6.25 (v. Jaksch). By the first method an uncertain quantity of inorganic matter is carried down which cannot be thoroughly removed by washing, and which therefore disturbs the result. In the second method N is derived from other principles besides albumins, and, as Limbeck and Pick have shown, there may be a difference of 10 to 20 per cent, in the results of the two methods. Most of the nitrogenous principles other than albumins are soluble in alcohol, and some of the sources of error in this method are there- fore removed by estimating the total N of the alcoholic precipitate, v. Jaksch has determined that by multiplying the percentage of N by 6.25 the average percentage of weight of albumin is obtained. Limbeck and Pick, after investigating the ratio between N and the weight of albumin from which it was derived, concluded that v. Jaksch's figure is sufficiently reliable in the great majority of cases, but that considerable inaccuracies may result in the cases of venous stasis, nephritis, and especially uremia, in which nitrogenous principles in the blood other than albumins are increased. The determination of total N by Kjeldahl's method is generally employed in estimating the albumins of the blood. The method may be found fully described by Halliburton, Chem. Phys. and Path., and Sutton, Volumetric Analysis. Inorganic Principles of the Blood. The Blood Ash. After incineration of two specimens of normal blood Schmidt found 0.84 per cent, and 0.91 per cent, of ash. This quantity was increased to 1 per cent, in a case of cholera, but the relation of ash to dry residue remained normal, 3.1 to 3.5 per cent. In the watery blood of nephritis, however, the ash was relatively much increased (6.5 per cent.). Chemical analysis of the blood ash in health and disease have shown that pathological importance attaches principally to variations in the chlorides, phosphates, and the iron of the blood. With regard to the chlorides (principally NaCl) the law has been established that the larger the proportion of plasma the greater is the percentage of chlorides in the blood. In pneumonia the chlorides are low, probably owing to diminished ingestion and the effects of an exudative process which drains the blood of a considerable quantity of salts. In typhoid fever and erysipelas a reduction usually exists which is neither so marked nor so constant. In nearly all forms of anemia the proportion of chlorides is high, following the rule above stated. Yet Limbeck found normal proportions in two cases of chlorosis, which he refers to diminished salts in the red cells, and Biernacki refers to cases of severe anemia with normal chlorides, which he also explains from the loss of red cells. Becquerel and Rodier found considerable variations, but a normal average in chlo- rides in six cases of chlorosis. From the known influence upon the urinary chlorides of dimin- CHEMISTRY OF THE BLOOD. 79 ished ingestion of food, vomiting, diarrhea, and exudation, it appears probable that these factors may to some extent affect the chlorides of the blood, for in many of the available analyses of the blood ash in general diseases there are numerous contradictory results (tubercu- losis, syphilis, cancer). The phosphates exist as neutral or alkaline salts of Na, Ca, Mg, in the plasma and in various combinations (lecithin, nuclein) in the red and white cells. Few facts of importance have been established in regard to the variations in these principles of the blood. Biernacki demonstrated a considerable diminution of P 2 5 in anemia, and at the same time a certain parallelism between P 2 5 and K in these condi- tions. In leukemia, Freund and Obermayer found an increase in P 2 6 and Na, while the K was diminished. The alkalies Na and K are principally combined with CI in the blood, but Na is united in considerable proportions as phosphates and carbonates. The sodium salts, being found principally in the plasma, are subject to marked quantitative variations, being usually increased in watery blood. Potassium being located chiefly in the red cells is diminished in most hydremic conditions, and not alone in scurvy as suggested by Garrod. Sodium carbonate is probably the next most abundant salt of the plasma after sodium chloride, and to this salt mainly the plasma owes its alkalinity and its power to absorb C0 2 . Iron. The iron of the blood is principally found in the hemoglo- bin, a compound of albumin and iron containing, in human blood, about 0.42 per cent, of iron. It also occurs in traces in the plasma, and is found in relatively large proportion in nuclein (Hammar- sten). The percentage of iron in normal blood is placed by Limbeck, from the results of several analyses, between 0.056 and 0.058. Jolles found variations between 0.0413 and 0.0559 per cent., and Hladik an average of 0.0425 per cent., using the ferrometer devised by the former. While the principal depot of iron is in the Hb of the red cells, and the quantity of iron is closely proportionate to the percentage of Hb, the ratio between the two is often disturbed, because all iron of the blood does not form colored compounds. A further discrepancy between the Hb per cent, (after Fleischl) and the iron content of the blood results from the presence of iron-free pigments, as the lutein of Thudicum or the hydrobilirubin of Maly. Thus Biernacki found that direct quantitative estimation always yields more iron than the computation from the percentage of Hb, after Fleischl. Some- times twice as much iron was found as was to be expected from the Hb per- centage. Similar results have been obtained by Jolles and Jellinek. It seems probable that the general introduction of a practical clinical method of esti- mating the iron of the blood may develop some new facts of interest in the pathology of the blood. Thus, Jellinek found in a case of purpura hemor- rhagica that iron-free pigment was apparently absorbed from extravasations, since the Hb registered 50 per cent, after Fleischl, but only 38 per cent, as computed from the iron-content. Also in a case of malaria immediately after a paroxysm the Hb sank 10 per cent, while the iron remained con- stant. 80 GENERAL PHYSIOLOG Y AND PATHOLOG Y. Estimation of the Inorganic Principles of the Blood. It falls outside the scope of the present volume to consider the details of inorganic quantitative analysis of the blood. For the estimation of Fe, however, reference should be made to the clinical method and results of Jolles. This method permits the es- timation of Fe within fifteen minutes, and with considerable accuracy. The apparatus is offered by Keichert's agents in this country and is accompanied by full directions for use. Mackie has described clinical methods of estimating Fe and phos- phoric acid in a drop of blood, to which the reader is referred. ("Iron," Lancet, 1898, vol. i. p. 219; "Phosphoric Acid," Lancet, 1899, vol. ii. p. 484.) Urea in the Blood. Urea occurs in traces in normal blood (Picard) and in increased quantity in fevers (Gescheidlen), and when, as in nephritis, its excre- tion by the kidneys is imperfect. In uremia Munzer found 0.4 per cent, of urea in the blood, but the amount is regarded as insufficient to cause the toxic symptoms of this condition. Demonstration of Urea in Blood, v. Jaksch recommends the following procedure : 200 to 300 c.c. of blood are precipitated with three to four times as much alcohol, and after twenty-four hours the precipitate is repeatedly washed in alcohol. The alcohol is then evaporated, the residue treated with nitric acid, and the crystalline mass, secured after some hours, is dried between filter paper, dissolved in water, treated with barium carbonate till CO, ceases to form, dried on a water-bath, and extracted with hot alcohol. On evapora- tion urea is found in slender rhombic prisms. If secured in sufficient quantity the crystals may be treated with nitric acid and evaporated, when character- istic crystals of urea nitrate form. Or the biuret test may be employed, dis- solving the crystals in a little caustic potash, and adding a drop of dilute cupric sulphate. Various other methods may be found described in text-books of physiologi- cal chemistry — e. g., Gamgee. Uric Acid in the Blood. Scanty traces of uric acid have been demonstrated in normal blood (Picard, Abeles). Garrod found 0.025 to 0.145 per cent, during acute attacks of gout, and Salomon also found an increase in acute gout, v. Jaksch regards their methods as inexact. A moderate increase (0.08 per cent.) has been demonstrated in pneumonia and anemia (Salomon, 1 v. Jaksch 3 ), in cardiac and other forms of dyspnea, and in nephritis (v. Jaksch). Klemperer 1 and Weintraud noted moderate increase in leukemia (0.09 per cent.), in nephritis (0.06 per cent.), and in uremia (0.19 per cent.), but failed to find any trace in three healthy subjects and in one case of pneumonia. Estimation of Uric Acid.— Garrod took 10 c.c. of serum from 30 c.c. to 35 c.c. of coagulated blood, added 10 per cent, of dilute acetic acid, and obtained crys- tals of uric acid on a thread placed in the fluid. In blood containing not less than 0.025 per 1000 of uric acid the thread was covered with crystals in twenty-four to forty-eight hours. These crystals should be submitted to the murexide test. CHEMISTRY OF THE BLOOD. 81 v. Jaksch recommends the qualitative and quantitative estimation of uric acid by means of the Ludwig-Salkowski method employed in urinary analysis. The blood is prepared for this method as follows : 100 c.c. to 300 c.c. of blood are diluted three to four times with water, heated on the water-bath until coagu- lation begins, when it is feebly acidified by acetic acid (s. g. 1.0335). After fifteen to twenty minutes it is filtered, the precipitate washed with hot water, boiled again after feebly acidifying, cooled and filtered. A little sodium phosphate is now added to the filtrate, which is then submitted to the Ludwig- Salkowski method. For a full description of this method, see Simon's Clinical Diagnosis. Glucose in the Blood. Normal blood always contains traces of glucose, which may be increased by diet rich in carbohydrates, and is diminished by mus- cular exercise and hunger (Seegen, Chauveau and Cavazanni, v. Mering). Limbeck found in the blood of two healthy subjects, five hours after eating, 0.075 per cent, and 0.089 per cent, of glucose, Freund and Trinkier find that glucose is very considerably increased in the blood in cases of carcinoma, readily reducing cupric oxide when freed from albumins, which cannot be accomplished with the blood of any other disease except diabetes. In one case of cancerous cachexia Trinkier found 0.3 per cent, of glucose, which reaches the limit stated by Claude Bernard to be capable of producing diuresis. In diabetes the quantity of glucose in the blood is subject to' great variations, according to the character and progress of the disease. Hoppe-Seyler found in one case 0.9 per cent. Estimation of Glucose in the Blood. A weighed quantity of blood is freed from albumin by boiling with an equal quantity of sodium sulphate and filtering. The precipitate is well washed and the presence and quantity of glucose in the filtrate determined by Fehling's solution or by polarimetry. A certain amount of sugar is carried away by the precipitated albumins, and unless the blood is fresh, the action of the glycolytic ferment may cause serious error in the result. Glycogen in the Blood. The long discussion regarding the feasibility of demonstrating glycogen in the blood by chemical methods has led to many contrary opinions, but in recent years positive results have been more constant (Salomon, Frerichs, Cramer, Lepine and Barral, Huppert, Czerny). The microscopic test on specimens dried in the air and stained by iodine, after Ehrlich's suggestion, has given more uniform results. Gabritschewsky, using this method, found intracellular and extra- cellular glycogen in the blood of both healthy and diseased persons. Extracellular glycogen, in the form of fine or coarse granules (1/i to 6/i) he found to be the only form usually present in normal blood ; and it was increased in diseases in which intracellular glycogen was abun- dantly present. He states that extracellular glycogen is derived from the disintegration of leucocytes, but offers no evidence to prove this origin. The glycogen was found in the bodies of neutrophile leucocytes in cases of diabetes and leukemia, and in the plasma in a considerable variety of other diseases. The results of his experi- . ments, injecting sugar and peptone into the blood of animals, indicate 82 GENERAL PHYSIOLOG Y AND PATHOLOG Y. that the leucocytes are capable of transforming both sugar and pep- tone into glycogen. Livierato also found extracellular glycogen in normal blood, but failed to discover any in the leucocytes in diabetes. From his clinical observations he concludes that the glycogen of the blood is increased in febrile cases with an active exudative lesion and with leucocytosis. In typhoid fever he found only extracellular glycogen. Czerny's studies added considerably to the knowledge of the subject. He found the increase of glycogen in exudative processes with leucocytosis. In the blood of children with cachectic leucocytosis he found an almost equal quantity. In two or three dogs exposed to prolonged cold he found that glycogen appeared in the leucocytes after twenty-four hours, and persisted for several days. From the severe disturbances of respiration following section of both vagi, or pneumothorax artificially introduced, as well as in severe anemia from hemorrhage, he found a marked increase in glycogen. An inter- esting inquiry of Czerny's related to the exact chemical nature of the brown staining granules demonstrated by iodine, a reaction common to both gly- cogen and amyloid. That these brown granules are glycogen is indicated by (1) their reaction to iodine; (2) by the disappearance of their brownish stain on beating (Barfurth), and (3) by their complete digestion by saliva (Czerny). On the other hand, Czerny points out that pure glycogen is soluble in water, while the glycogen of leucocytes is insoluble in water (as the writer, also, has found), that iodine* with dilute sulphuric acid colors these granules violet, which is the reaction of amyloid ; and, finally, that in dogs suffering from prolonged suppuration whose blood continually shows abundance of " glycogen," the viscera, on the other hand, develop extensive amyloid changes. These observations indicate that the brownish staining granules are not pure glycogen, but a comparatively insoluble compound of glycogen with some other substances, or else an intermediate product between glycogen and amyloid. Caminer failed to find any glycogen in normal blood, and in disease very rarely found it in the plasma. He distinguished three stages of the deposit in leucocytes: (I) the presence of a light, diffuse mahogany stain; (2) the presence of a few isolated globules; (3) the complete transformation of the body of the cell into glycogen. In cases of extreme sepsis, all stages were seen ; in pneumonia glycogen was usually present, but never in the third stage of its formation ; in phthisis only the first stage was found ; and in rheumatism it was absent. In cases of carcinoma it was found only when suppuration occurred. In four diabetics none was found, but in a case of diabetic coma it was abundant. It was absent in most cases of carcinoma, in chlorosis, in two cases of leukemia, and in secondary anemia from hemor- rhage. From experimental studies, he finds that three factors are concerned in the glycogenic degeneration of leucocytes : (1) fever, (2) leucocytosis, (3) toxemia, of which the last is most potent. T. Dunham found the glycogenic degeneration of leucocytes a valuable aid in the diagnosis of suppuration, and introduced the term " iodophilia." Locke has contributed extensive clinical observations on the occurrence of glycogen in leucocytes. He found an extracellular reaction, especially in the blood plates, in all cases, and regards this occurrence of no clinical importance. In septicemia an intracellular reaction was marked and constant. In abscess it- was present in 22 of 25 cases, but disappeared in forty-eight hours after drain- age of the pus. The three negative cases were very small abscesses without fever or leucocytosis. In appendicitis, peritonitis, and empyema it was abun- dant when pus was forming, but otherwise absent or slight. In serous pleurisy no reaction occurred even with high fever. In 49 cases of pneumonia it was constant and persistent. In malaria 22 of 29 cases gave a positive reaction. In 17 of 42 cases of typhoid fever the reaction was present, but referable to inflammatory complications. After perforation the reaction increased within six hours. In tuberculosis it was absent or faint if there were no suppurative complications. In diabetes a positive reaction occurred only with coma or gangrene. In pernicious anemia a very faint reaction was occasionally seen. CHEMISTRY OF THE BLOOD. 83 [Numerous other reports on the occurrence of glycogen in leuco- cytes have appeared, showing that the subject is of considerable clinical interest and sometimes of value in diagnosis. Regarding the microscopic demonstration of extracellular glycogen, it should be mentioned that other principles, such as myelin, lecithin, and amyloid, stain brown with iodine. Lipemia. The occurrence of free fat (palmitin, stearin, olein) in the blood, both in health and in disease, has repeatedly been observed. While usually present in such small quantity as to be recovered in very small amounts from the ethereal extract, and recognized with diffi- culty by the microscope, it is sometimes so abundant as to give the blood a milky appearance (Gumprecht). Its physiological variations probably exceed the pathological, as it has been found very much in- creased in healthy individuals after a hearty meal. In disease its occurrence appears to follow no general rule, so that its real patho- logical significance remains doubtful, v. Jaksch, extracting the blood with ether and thereby including fat, lecithin, cholesterin, and a trace of nitrogenous compounds, found in three cases of diabetes 0.05 to 0.16 per cent., in nephritis 0.1 to 0.5 per cent., in typhoid fever 0.16 per cent., and in pneumonia 0.15 per cent. It has also been found in increased quantity in phthisis, poisoning by carbonic acid, and in fat embolism after traumatism. Englehart found considerable variations in the ether extract of human blood in healthy subjects, viz., 0.101 to 0.273 per cent., average 0.194 per cent., while in cachectic subjects, although no greater variations were found, the average was lower than 0.174 per cent. In fasting dogs, Cohnstein and Michaelis found an average of 0.121 per cent, in seven analyses, while after fatty diet the average rose to 0.153 per cent. Acetonemia. Lipacidemia. Deichmuller and v. Jaksch, by extracting the blood with ether and by distillation, have isolated a principle which gives the reaction of acetone, and which they found increased in many processes, espe- cially in fevers. Fatty acids have been found in the blood by v. Jaksch, in diabetic coma, leukemia, acute yellow atrophy of liver, and acute infectious diseases : ; 3-oxybutyric acid, in the cadaveric blood of diabetes, by Hougounenq ; and sarcolactic acid, in normal blood, by Gaglio, Spiro, and Berlinerblau. Limbeck doubts the reliability of these results, believing that fatty acids may develop from lecithin during the technical procedures followed in isolating these principles. Cholemia. The poisonous symptoms developed in cholemia have been referred by most authorities to the presence of biliary acids. Flint's stale- 84 GENERAL PHYSIOLOGY AND PATHOLOGY. ment that cholesterin is the poisonous agent has not been accepted, although it has been found in considerable traces in icteric blood. To the naked eye icteric blood may appear of yellowish red color, while in the serum or its foam small quantities of bile pigment are readily detected by the peculiar yellowish tinge. On repeated heat- ing the yellowish red bilirubin changes to the green biliverdin. Diminished isotonic tension and increased resistance of the red cells are a peculiar character of the blood in jaundice. Limbeck found the tension of the cells reduced 0.4, 0.38, and 0.32 per cent. NaCl, and that of the serum 0.76 and 0.864 per cent. NaCl. The well-attested fact that in intense jaundice red cells are frequently dissolved by biliary principles, can, with some difficulty, be reconciled with this markedly hyperisotonic quality of the serum. Limbeck believes that bile acids affect the union of Hb with the stroma of the red cells, rendering the Hb more easily soluble, and thinks that the solution of red cells in jaundice, as well as in other conditions, depends on other than simple osmotic factors. Other characters of icteric blood are, according to Limbeck's analyses, an increase of nitrogenous bodies (3.29 to 3.52 per cent.) ; diminution of chlorides of both blood and serum, which he refers to their displacement by biliary acids ; and a well-marked increase in the volume of red cells (Bleibtreu's method). Detection of Biliary Principles in the Blood. Well-marked cholemia may be detected by naked-eye inspection of serum or its foam. On heating to 50° C. bilirubin may be changed to biliverdin. v. Jaksch has been able to demonstrate bilirubin in the blood when none was to be found in the urine, or when only urobilin existed in the urine, by the following procedure : A little blood obtained by a wet-cup is allowed to coagulate, and after one to two hours the serum is drawn off and forced by aspiration through an asbestos filter. The froth of the filtrate is yellow if any bilirubin is present, and this yellow tinge becomes green on heating two or three hours at 35° C, if any minute traces of bile pigment are present. Or the blood may be coagulated slowly at 78° to 80° C. when the serum becomes greenish in the presence of minute traces of bile. Biliary acids may be demonstrated by Pettenkofer's method on serum prepared as follows : Albumins are removed by boiling or by alcohol, and the filtrate is treated "with lead acetate, and with ammonia, which precipitates the acids with the lead compounds. The acids are then recovered, by washing the precipitate on a filter, boiling in alcohol, filtering, and decomposing the lead salts by carbonate of soda. The solution is again filtered, evaporated to dryness, and the acids extracted by boiling in absolute alcohol. Finally, on evaporating the alcoholic extract, biliary acids crystallize out, or an amorphous substance remains, from which the crystals may be obtained by extracting with ether. The Specific Gravity of the Blood. The specific gravity of the blood changes with the content of water, its most variable constituent, the proportion of salts, which are less variable, and the percentage of albumins, which are the last principle affected by pathological processes. CHEMISTRY OF THE BLOOD. 85 The normal limits have been placed by Becquerel and Rodier between 1.058 and 1.062 for men, 1.054 and 1.060, including both sexes; by Hammerschlag, between 1.056 and 1.063, including both sexes; by Lloyd Jones, between 1.045 and 1 .066. Some of these discrepancies are doubtless referable to the differences between the methods employed. There are considerable ■physiological variations in gravity. Accord- ing to Lloyd Jones the blood of newborn infants shows the highest gravity, averaging 1.066; after the second week of life up to the second year the gravity sinks, 1.048 to 1.050 ; rising with men between thirty-five to forty-five years to 1.058, with women after the climac- teric, to 1.054 ; in old age the blood of both sexes approaches the initial high gravity of infancy. Limbeck finds an explanation of these progressive changes in the decreasing capacity of the tissues to absorb water. From forty-four estimations on his own blood Schmaltz found minimal variations at different hours of the day, from 1.061 from 7 to 8 a.m., to 1.058, from 2 to 8 p.m. Muntz found a marked increase, 1.038 to 1.058, in the gravity of the blood in overfed sheep. Muscular activity, if accompanied by sweating, slightly decreases, sleep slightly increases the gravity (Schmaltz, Jones). Schmaltz found that menstruation is followed by a slight increase of gravity, while the slightly lower gravity of pregnant and parturient women has been frequently observed. For somewhat obscure reasons the gravity of the blood is increased by residence in high altitudes and a considerable difference (0.015) has been observed in the blood of animals pastured on mountain tops and those grazing in the valleys (Muntz, Viault, Glogner). In pathological conditions lowered specific gravity is a constant character of the blood in anemia. In chlorosis the change is usually referable to and proportionate with the loss of Hb, but Stintzing and Gumprecht and Siegel have shown that this parallelism is not invariable. In -pernicious anemia the specific gravity and dry residue suffer more than the Hb, owing to loss of albumins from the serum. Extremely low gravity, in comparison with the Hb-content, is characteristic of this condition. In leukemia the gravity is reduced as in simple anemia, but extreme reductions are seldom observed, owing to the increase of white cells and the presence of abnormal products soluble in the plasma. In secondary anemias there are numerous exceptions to the parallelism between specific gravity and Hb-content. These are found especially in diseases in which an exudative process drains the blood of albu- mins (dysentery), or when from edema there is relative hydremia of serum or red cells. In the infectious diseases the specific gravity of the blood depends upon associated conditions, such as profuse perspiration, diarrhea, exudation, etc., more than upon any specific property of bacteria to increase the water of the blood. That the power to impoverish the blood varies, however, with different infectious agents is strongly indicated by clinical observation. Grawitz's claim that tuberculin and the diphtheria toxin tend to increase the gravity of the blood, while the toxins of streptococcus and staphylococcus pyogenes tend to 86 GENERAL PHYSIOLOG Y AND PATHOLOG Y. diminish it, is frequently confirmed by comparison of the blood in cases of miliary tuberculosis and uncomplicated diphtheria with that of septicemia. Although acute stasis is usually followed by marked increase in the gravity of the blood, yet in chronic endocarditis the variations observed are very irregular. General edema is usually associated with lowered gravity. The same observation applies to the blood of nephritis, normal gravity being observed in many cases of chronic interstitial nephritis (Hammerschlag), while anemia and low gravity of the blood and edema of the tissues are nearly constantly associated with the large white kidney. Numerous studies of the blood of pulmonary tuberculosis, including that of Grawitz, have failed to bring to light any uniform variations in gravity peculiar to the blood in this disease. As a rule, extreme reductions are not observed. In the cachexia of malignant neoplasms some of the lowest gravi- ties on record have been observed (1.030, 1.032, Lyonnet), especially in ulcerating and bleeding tumors of the stomach and uterus. In certain shin diseases (pemphigus, eczema, psoriasis, prurigo, morbus maculosus) an increased gravity of the blood has been noted by Schlesinger. In cases of general burns, Tappeiner, Baraduc, and Schlesinger have found very high gravities, 1.065 to 1.073, which, in cases that recovered, fell to normal in twenty-four hours. Finally, various drugs appear to exert a moderate but in no sense peculiar influence on the gravity of the blood. Purges, diaphoretics, and diuretics remove water from the system, and when this loss is not immediately replaced from the tissues the blood is temporarily concentrated. The action of mercury is somewhat uncertain, but in syphilis it appears to diminish the gravity after a short initial period of increase (Schlesinger). To recapitulate, it has been found that considerable variations in the water of the blood may under ordinary conditions be promptly equal- ized by the action of the tissues on the one hand replacing a loss, or of the kidneys and skin removing an excess, so that the change in the blood is very transitory. Only when there is interference with these processes or when the demands upon them are excessive does a more or less permanent alteration in the gravity of the blood follow. On the other hand, when the albumins of the blood are affected, more marked and permanent changes are produced. In general, the content of the tissues in water and that of the blood are very closely interdependent. The Ferments of the Blood. In recent years the metabolic processes in the cells, both catalytic and synthetic, have been referred more and more to the agency of ferment-like bodies, many of which have been demonstrated in the blood. It falls outside the scope of this work to consider the rela- tions of the fibrin ferment and of the probable ferment action of the CHEMISTRY OF THE BLOOD. 87 toxophore group of Ehrlich's complement, but some other special fermentative processes inherent in the blood or its cells deserve brief mention. Oxidase. The presence of an oxidizing ferment in the blood was first demonstrated by Schonbein, who located the ferment in the stroma principally of the red cells. Its importance in physiology is now fully recognized. Lillie has applied an interesting method of demonstration of an oxidase in leucocytes and red cells. When blood is mixed with an alkaline solution of a-naphthol or paradiamidobenzene, in equi- molecular proportions, the solution, at first colorless, becomes deeply tinted from oxidation, and many stained granules of oxidation prod- ucts are deposited about the nuclei of leucocytes and in the red cells. Lipase. The presence of a fat-splitting ferment in the blood has been claimed by Hanriot and others, but its existence is not fully proven (Jacoby). Proteolytic Ferment. Ascoli and Moreschi report that the washed sediment of sterile pus from empyema contains a proteolytic ferment. To the action of such a ferment in the leucocytes has been attributed the dissolution of the pneumonic exudate, in which, of course, the leucocytes are mingled with other cells and are dying. Glycolytic Ferment. The presence of a glycolytic property in normal blood has been long known. Lepine found that it depends on the red cells more than upon the plasma, and he elaborated the theory that diabetes results from the diminution or absence of this glycolytic ferment. It has been shown, however, that the glycolytic property of diabetic blood is not distinctly inferior to that of normal blood (Minkowski, Kraus). The experiments of Seegen and Arthus indicate that this property is not exerted during life, but is a cadaveric phenomenon, and that the extent of its action depends upon the time consumed in the examination of the specimen. This objection applies to present views regarding nearly all of the cell ferments and is not generally regarded as valid (Jacoby). Spitzer, in an exhaustive study, finds that a glycolytic ferment is present not only in red and white blood cells, but in all tissue cells ; that it is precipitated in active form by alcohol, and that its action is possible only in the presence of oxygen as contained in oxy-Hb. Diastatic Ferment. The study of the property of fresh blood to digest starch has lately been extended to the blood in various diseases. This property may be demonstrated by adding 1 c.c. of blood to 50 c.c. of a solution of starch, allowing the mixture to stand a few hours in the thermostat, when glucose may be demonstrated by Fehling's solution. Castellino and Pracca found that 2 c.c. of normal human blood added to 50 c.c. of starch solution produced 0.07 per cent, of sugar in twenty-four hours, at 30° C. Arterial blood is more active than venous, and the fermenta- tion most active between 30° and 38° C, is inhibited at 75° C, and diminished by slight changes in reaction (Oavazanni). Oavazanni and Pracca found con- siderably increased fermentative power of the blood in anemia, chlorosis, leukemia, pneumonia, malaria, nephritis, cirrhosis, and carcinoma, while in other cases of these and other diseases it was diminished. 88 GENERAL PHYSIOLOG Y AND PATHOLOGY. The principal source of the diastatic ferment is placed in the red cells by Tiegel and Plosz, in the serum of Bial, and in the leucocytes by Castellino and Pracca. The last-named observers find that the diastatic property is closely related to the globulicidal and coagulative power of the blood ; that it increases after the blood is shed ; that it is inhibited by the addition of nuclein, and increased by sodium sul- phate and chloride. Tschereskoff was able to precipitate from blood by alcohol small amounts of a diastatic ferment which remained active forty-five days. It was more abundant in the serum than in the clot, and the presence of sodium oxalate did not destroy it. The Osmotic Relations of the Blood. When a drop of blood is placed in distilled water the red cells are promptly dissolved, but when blood is placed in a solution of salt of a certain concentration the red cells retain their Hb, and sink to the bottom of the fluid. The solution of the cells results from the law of osmosis, that when two solutions of different concentration are separated by an animal membrane the solutions pass through the membrane until the quantity of salt in each is equal. The force which leads to this interchange is called " osmotic tension," and two fluids with equal content of salt are said to be " isotonic," or of equal " isotonic tension." Fluids are likewise said to be hyperisotonic or hypisotonic when they contain greater or lesser quantities of diffusible salts than other fluids, and are capable of drawing water from or yielding it to such fluids, according to the laws of osmosis. A solution containing 0.46 per cent, of NaCl is just sufficient to prevent the solution of red cells in the average specimen of normal human blood (Limbeck), and the isotonic tension of human red cells may, therefore, be said to be 0.4-6 per cent. NaCl. Yet when red cells are placed in a 0.46 per cent, solution of salt they absorb water and swell, although they do not yield up Hb, and when placed in strong solutions of salt, red cells shrink, yielding water to the fluid. Ham- burger finds that a 0.9 per cent, solution of salt causes neither swelling nor shrinkage of the red cells. This solution, therefore, represents the isotonic tension of the blood plasma, and is properly called, the " normal salt solution." Any considerable lowering of the osmotic tension of the plasma must therefore lead to swelling of the red cells and eventually to their solution. The hyperisotonic quality of the plasma with refer- ence to the red cells is a physiological necessity, otherwise the inges- tion of a considerable quantity of water would cause the solution of many red cells. The exact limits of osmotic tension between which the red cells suffer no alterations of volume are not known, but it is certain that they are often exceeded in pathological conditions. Changes in osmotic tension which affect the volume of the red cells may occur not only in the plasma but also in the red cells. Thus, if the red cells in chlorosis are for some developmental anomaly deficient in salts they would shrink in plasma of normal (0.9 per CHEMISTRY OF THE BLOOD. 89 cent.) osmotic tension, while normal cells would swell in the watery plasma supplied after hypodermoclysis for hemorrhage. The isotonic relations of the blood do not apparently depend entirely upon the presence of salts, but are affected also by the pres- ence of other diffusible principles, as the albumins. Limbeck finds only 0.2 per cent, of salts in red cells, yet their isotonic tension is equivalent to at least 0.46 per cent. NaCl. Hamburger finds that albumins, phosphates, and chlorides behave differ- ently under changing osmotic conditions. When a little acid is added to blood, albumins and phosphates pass from red cells to serum, while chlorides pass from serum to cells, but when alkali is added the opposite transfer is induced. Similar physical effects are produced by the passage of oxygen and carbonic acid through the blood, and Hamburger suggests that these factors take important part in the metabolic exchanges in the capillaries. One of the chief physiological relations of the isotonic tension of the blood is its influence in confining Hb to the red cells. Hamburger, who was one of the first to study this subject, regarded the fixation of Hb as the result solely of osmosis on the fluid Hb lying within the membrane of the red cell. Yet the opinion of Hoppe-Seyler that Hb enters into chemical or molecular union with the stroma of the red cell, and the fact that the existence of a membrane about the erythrocyte has not been satisfactorily proven (Limbeck) render this belief uncertain. Limbeck offers evidence to show that there are other than purely physical influences con- cerned with this important function, viz., the chemical union of Hb with other elements of the red cells, and the influence of albumins oh osmosis. Physiological variations in isotonic tension of the blood are numerous. That of venous is slightly higher than that of arterial blood (0.02 per cent.). The addition of CO, C0 2 , hydrogen, nitrogen, arsenic, or a trace of acid increases isotonic tension, while oxygen and traces of alkali diminish it (Limbeck). In pathological conditions, from a series of fifty-four observations on blood from venesections, Limbeck concludes : During acute infections, especially typhoid fever, erysipelas, and pneumonia, the isotonic tension of the blood is frequently much increased, but not always or constantly so. In general disturbances of nutrition the tension of the blood is very variable ; in diabetes and osteomalacia it was normal ; in leukemia it was much increased ; in jaundice it was low, while in chlorosis it was low, and in severe secondary anemia higher than normal. In pregnancy and lactation Vicarelli found a distinct increase in osmotic tension — i. e., a diminished resistance of the red cells to water (0.6 to 0.66 per cent. NaCl). While Limbeck and Oastellino found the red cells less resistant in typhoid fever, Bianchi and Mariotti found that experimental injections of B. typhosus lowered the isotonic tension of the blood, although filtered cul- tures of this and other bacteria had the opposite effect. Cavazanni found that injections and inunctions of mercury slightly increased the resistance of the red cells. The tension of the serum has been investigated by Hamburger, using another method, who found no change after bleeding. Viola and Jona, during seven hours following venesection, found a moderate diminution, while Limbeck, in three portions of blood, taken at intervals during the exsanguination of a dog, found nearly constant 90 GENERAL PHYSIOLOG Y AND PATHOLOG Y. conditions, and Adler found little variation in the tension of the serum in various diseases. It will thus be seen that the knowledge of this subject is yet in a very rudimentary condition, although its importance in hematology invites further investigation. Alkalescence of the Blood. It is an established principle of physiology that the capacity of the blood to absorb CC) 2 depends on its alkalescence. When one compares the results of direct alkalimetry obtained by any of the recognized methods with the proportion of C0 2 obtained after the dissociation of carbonates by strong acids, marked discrep- ancies are observed. The volume of C0 2 differs greatly from the degree of alkalinity obtained by direct titration. To explain this fact it must be supposed that certain basic properties of the blood are brought into action by alkalimetric methods which are not active during life, or else that under changing conditions of metabolism the blood is required to absorb varying quantities of C0 2 , which are by no means necessarily proportional to the capacity of the blood to absorb this element. There are good physiological grounds for supposing that both of these con- ditions actually exist. It is probable that the existing methods of titration are sensible to the alkaline carbonates and phosphates, which are principally concerned in the alkalinity of the blood, and, as well, to some acid-neutral- izing albuminous principles that are liberated from plasma and red cells during alkalimetric procedures (Limbeck and Steindler). To what extent the phosphates and albumins are concerned in the physiological functions centred in the alkalinity of the blood is not known, but it has seemed to the writer that the opponents of the titration method have failed to consider the possibility that other important functions besides that of absorbing C0 2 may depend on the alkalinity of the blood {e.g., fibrin formation), and that the results of the titration method, therefore, deserve recognition in the study of the pathology of the blood. On the other hand, the attempt to measure the alkalescence of the blood by the volume of C0 2 recovered after addition of acids involves a needless confusion of the problem, as such estimations include both the C0 2 loosely combined with Hb and that more firmly united with the alkalies. Yet the physiological significance of each of these combinations is probably very different, the former representing the respiratory activity of the blood and the metabolic activity of the tissues, while the latter is related to other less definitely known functions. Moreover, it by no means follows, as has been said, that the volume of C0 2 recovered from the blood represents the total quantity that the blood is capable of absorbing, or is any indication of its acid neutralizing power. Bunge calculates that after allowing for the amount of sodium required to saturate the only strong mineral acid of the plasma (HOI), there is enough sodium left to fix 63 volumes per cent, of C0 2 as car- bonate and an equal additional amount as bicarbonate, which is far more than the amount of C0 2 actually present in the blood. Again, the attempt to estimate the alkalescence of the blood, considered from either the biological or chemical standpoint, by the content in 0O 2 involves several probable errors. There may, first, be a diminished produc- tion of 00 2 in the tissues in pathological conditions. There may be a dimin- ished absorption of C0 2 by the blood, owing to chemical changes in the tissues or mechanical impediments in the circulation. Finally, there may be simple absorption of C0 2 uncombined with acid neutralizing principles (Schafer). CHEMISTS F OF THE BLOOD. 91 Of the actual existence or importance of these factors, it is. with the present knowledge of physiology, difficult to judge, but since they stand as uncertain quantities, it seems unwise to rely upon any such indirect method of judging of the alkalinity of the blood. It would seem, therefore, that both alkalimetry and the estimation of CO, furnish important information in regard to the state of the blood, but there are no good a priori reasons to suppose that both measure the same property of the blood, while the practical results obtained positively disprove such a view. When laked blood is titrated a high degree of alkalescence is obtained, as this method takes account of all acid-neutralizing principles, carbonates, phosphates, and albumins of plasma, and red cells. When serum is titrated, the acid neutralizing principles of red cells are ignored, and lower grades of alkalinity are obtained. When the CO, is estimated account is taken only of the carbonates, but albumins and phosphates are ignored, and the presence of any dissolved CO s disturbs the computation, while the possibility still remains of accidental variations in the ratio between the CO, actually present and total capacity of the blood to absorb this acid. Brandenburg distinguishes between the total alkalescence of the blood and its "alkaline tension," the latter signifying the diffusible alkalies, the former those in non-diffusible combination with albumins. The percentage of diffu- sible alkali increases as the albumins of the blood diminish. In normal blood he found about 20 per cent, of the alkali diffusible, in chlorosis about 33 per cent. The freezing point of blood was considerably influenced by the diffusible alkalies, but very little by those combined with albumin. (See Diabetes.) The stiulies of the CO i -content of the blood have given the follow- ing chief results, as summarized by Limbeck : Venous blood is always richer than arterial in CO,, and both are subject to minor physiological variations, 33.37 to 45.3 volumes per cent. (Schafer, Kraus). Febrile processes are generally accompanied by diminution of C0 2 in the blood, often in proportion to the height of the fever, 34.18 to 20.9 volumes per cent. (Geppert). This condition, according to Geppert and Minkowski, is referable to the abnormal production of acid metabolic principles of the blood, a view which is supported by Kraus, who, in fevers, along with a diminution of CO, (10 to 20 volumes per eent.\. found an increase in the acid principles of the blood. In the cachexia of carcinoma Limbeck and F. Kleniperer found marked diminution of the C0 2 of the blood (9.67 to 20.5 volumes per cent.), but did not estimate the acid principles. Similar results fur- nished by other observers indicate that in diabetic coma the blood is frequently very deficient in CO., (Minkowski, Stadelman, Kulz, Hallervorden, Kraus). Yet Kraus in one case found a normal quantity. A simultaneous increase in acid principles has been demonstrated in one case of diabetic coma by Kraus. and on these grounds it has been concluded by various authors that in diabetes there exists an acid intoxication probably from oxybutyric and diacetic acids. Limbeck accepts this view and concludes from the various studies that in acute febrile infectious diseases, in cancerous cachexia, and in diabetic coma, a diminution in C0 2 and an increase of acid principles of the blood have been fully demonstrated, pointing in all probability to the existence of an acid-intoxication in these diseases. In leukemia Kraus 2 found a slight deficiency of C0 2 (20.29 volumes per cent.). 92 GENERAL PHYSIOLOG Y AND PATHOLOG Y. From the direct alkalimetry of the blood or serum much less uniform results have been obtained. A fairly constant normal alka- linity has been established with titration methods, v. Jaksch 5 placed the alkalinity of normal blood at 0.26 to 0.30 per cent. NaOH; Kraus, at 0.226 per cent. ; Jeffries, at 0.2 per cent. ; Drouin, at 0.206 per cent. ; Freudberg, at 0.2 to 0.24 per cent. ; Limbeck, 0.220 to 0.256 per cent. In laked blood it has been found much higher by Loewy, 0.449 per cent.; by Berend, 0.45 to 0.5 per cent. Peiper found slightly greater alkalinity in the blood of women than in that of children, and in the blood of men over that of women. Berend and Preisich, using Tausczk's method, found the alkalinity of the blood to be very high at birth and for the first six months, after which it diminishes rapidly, reaching its lowest point in the second year. After the third year it increases until at the sixteenth year it reaches the normal degree of adult life. A constantly diminished alkalinity in the blood during fevers has been reported by the above authors, by Rumpf, and others, and in carcinoma (Rumpf), anemia (v. Jaksch, Peiper), leu- kemia (Peiper, Rumpf), uremia, cirrhosis, and osteomalacia (v. Jaksch). On the other hand, the results obtained by Loewy and by Limbeck and Steindler, using their particular methods, were extremely varia- ble, and these variations were about equal in health and disease. From the observations of Fodor, Behring, Zagari, Calabrese, Cantani and Rigler it has been shown that during infectious diseases the alkalinity diminishes as the disease progresses unfavorably, reaches its lowest point just before death, and that the alkalinity of the blood is essentially connected with the natural or acquired immunity of the animal organism against bacterial infection. Fodor found that immunization of animals against anthrax, cholera, and swine plague increased the alkalinity of the blood. No increase followed injec- tions of bacillus typhosus or tuberculosis. Burmin found that the alkalescence of the blood in anemia varies directly with the red cells. Using his own method Rigler has recently contributed an elaborate study, with the following main conclusions : The alkalinity of normal rabbit blood does not vary more than 6 per cent. After infection by various pathogenic bacteria, including typhosus and tuber- culosis, especially if fatal, there is uniform and usually marked diminution in the alkalinity of the blood. As recovery from the infection follows the alkalinity increases. Similar results followed injections of various bacterial toxins, of phosphorus, potassium chlorate, picric acid, biliary acids, pilo- carpin, and atropin. He found, as did Cantani and Fodor, that injections of diphtheria anti- toxin, anthrax vaccine, etc., causes a marked but temporary increase of alkalinity. In rabbits treated by injections of serum of various animals considerable variations in the alkalinity of the blood were observed. In twenty-three patients suffering mostly from infectious diseases Eigler found similar variations with those observed in animals. The Acidity or Basic Capacity of the Blood. 3> There are certain unsaturated salts in the blood, NaHCO NaH 2 P0 4 , and probably Na 2 HP0 4 (Limbeck), which although alka line to litmus are acid to phenolphthalein, and are capable of uniting CHEMISTRY OF THE BLOOD. 93 with bases. While fresh blood is alkaline, serum reacts as acid to phenolphthalein. The capacity of the blood salts to neutralize bases has been called its basic capacity by Kraus, 2 who devised a delicate and somewhat difficult method of measuring this capacity. In normal venous blood Kraus found a basic capacity of 0.162 to 0.232 per cent. NaOH, which increased in febrile conditions to 0.209 to 0.272 per cent. NaOH. His demonstration of a marked increase of basic capacity (0.347 per cent.) in diabetes has been regarded as strong evidence of an acid intoxication in this disease. Bibliography. Chemistry. Abeles. Wien. med. Jahrbucher, 1887, p. 479. Adler. Cited bv Limbeck, p. 165. Arthus. Arch, de Physiol., T. 24, p. 337. Ascoli, Moreschi. Deut. med. Woeh., 1901, V. B., p. 295. Baraduc. Union Med., May, 1863. Barfurth. Arch. f. micr. Anat., Bd. 25, p. 305. Beal. Pfliiger's Archiv., Bd. 54, p. 72. Becquerel, Rodier. X T ntersuch. u. d. Zusammenset d. Blutes in gesund. u. krank. Zustande, Erlangen, 1845. Behring. Cent. f. kl. Med., 1888, No. 36. Berend. Zeit. f. Heilk., 1896, p. 351. Berlinerblau. Arch. f. exper. Path., Bd. 23, p. 333. Bianchi. Mariotti. Wien. med. Presse, 1894, No. 36. Biernacki. Zeit. klin. Med., Bd. 24, p. 460. Brandenburg. Zeit. f. kl. Med., Bd. 45, p 157. Buchner. XI. Cong. inn. Med., 1892. Bunge. Cited by Schafer, Text-book of Physiol., 1898, p. 157. Burmin. Zeit. f. kl. Med., Bd. 39, p. 365. Calabrese. Giorn. inter, della sci. med., 1892. Caminer. Deut. med. Woch., 1899, No. 15. Cantani. Cent. f. Bact., Bd. 20, p. 566. Castellino. 1 II Morgagni, Ann. 37, May- June, 1895. 2 Gaz. d. Osped., 1891, No. 20. Castellino, Pracca. II Morgagni, Aug., 1894. Cavazanni. Arch. p. 1. sci. med., T. 17, No. 6. Chauveau, Cavazanni. Cent. f. Physiol., VIII., p. 22. Chittenden, Albro. Amer. Jour. Physiol., 1899, p. 291. Chvostek. U. d. Wesen d. parox. Hemoglobinaemia, 1894. Cohnstein, Michaelis. Arch. f. d. Physiol., Bd. 69. Cramer. Zeit. f Biol., 1888, p. 97. Czerny. Arch. f. exper. Path., 1893, Bd. 31, p. 190. Deichmuller. Cited by v. Jaksch, Clinical Diagnosis. Devoto. Revista Clin., T. 30. Drouin. These de Paris, 1892. Dunham. Bost. Med. and Surg. Jour., vol. 144, p. 580. Ehrlich. Zeit. f. klin. Med., 1883, p. 33. Englehart. Arch. kl. Med., Bd. 70, p. 182. Estelle. Rev. mensuelle, 1880, p. 704. v. Frerichs. Zeit. f. klin. Med., 1885, p. 33. Freund. Wien. med. Blatter, 1885, No. 9. Freund, Obermeyer. Zeit. f. physiol. Chem., Bd. 15, p. 310. Freudberg. Virchow's Archiv, Bd. 125, p. 566. Fodor. Cent. f. Bact., Bd. 17, p. 225. Cabritschewsky. Arch. f. exper. Path., Bd. 28, p. 272. Gaglio. Arch. f. Physiol., 1886, p. 400. Gamgee. Text-book of Physiol. (Schafer), 1898, I., p. 185. Carrod. Med.-Chir. Transac, 1854, p. 49. Oeppert. Zeit. f. klin. Med., Bd. 2, p. 364. 94 GENERAL PHYSIOLOGY AND PATHOLOG Y. Gescheidlen. Stud. u. d. Ursprung d Harnstoffs im Thierkorp, Leipzig, 1871. Glogner. Virchow's Archiv, Bd. 126. Grawitz. Deut. med. Woch , 1893, No. 51. Gumprecht. Deut. med. Woch., 1894, p. 756. Hallervorden. Archiv. f. exper. Path., Bd. 12^ p. 237. Halliburton. Text-book of Phvsiol. Chem., 1893. Hal-pern. Berl. kl. Woch., 1902, p. 1121. Hamburger ' Arch. f. Anat. u. Physiol. Phys. Abt., 1886, p. 476; 1887, p. 31. Zeit. f . phys. Chem., 1890, VI., p. 319. Virchow's Archiv, Bd. 140, p 503. 2 Cent, f. Phys., Bd. 9, No. 6. Hammarsten. Physiol. Chemie. Hammerschlag. l Zeit. f. klin. Med., Bd. 21, p. 472. 2 Wien. klin. Woch., 1890, p. 1018. Zeit. f. klin. Med., Bd. 20. Hanriot. Compt. Rend. Soc. Biol., 1896, p. 925. Hayem. Compt. Rend. Soc. Biol., 1894, p. 227. Hladik. Wien. klin. Woch., 1898, No. 4. Hoffman. Arch. f. exper. Path., Bd. 16, p. 133. Hoppe-Seyler. Physiol. Chemie, 1881. Hougouneng. Maly's Jahresber., 1888, XVII., p. 430. Huppert. Zeit. f. phys. Chem., Bd. 15, pp. 335, 546. v. Jaksch. ' Zeit. f. klin. Med., Bd. 24, p. 429. 2 Ibid., Bd. 23, p. 187. 3 Zeit. f. physiol. Chem., Bd. 16, p. 243, and Clinical Diagnosis. 4 Zeit. f. klin Med., Bd. 11, p. 307. 5 Ibid., Bd. 13, p. 353. Jeffries. Boston Med. and Surg. Jour., 1889. Jellinek. Wien. klin. Woch., 1897, No. 47. Jolles. Deut. med. Woch., 1897, No. 10. Judell. Pfliiger's Archiv, Bd. 6-7. P. Klemperer. Charite Annalen, Bd. 15, p. 151. Klemperer. l Deut. med. Woch., 1895, No. 40. Kossel. Zeit. f. physiol. Chem., Bd. 22, p. 74. Kraus. 1 Zeit. f . klin. Med., Bd. 21, p. 315. 2 Zeit. f. Heilk., Bd. 10, p. 106. Arch. f. exper. Path., Bd. 26. Kulz. Zeit. f. Biol., Bd. 20, p. 165. Lepine. Lyon Med., 1890, No. 3. Lepine, Barral. Compt. Rend. Soc. Biol., 1891. Lichtheim. Vollfmann's Vortrage, No. 134. Lilienfeld. 1 Zeit. f. Phys. Chem., Bd. 18, 473. 2 Ibid., Bd. 20, p. 155. Lillie. Amer. Jour, of Physiol., vol. 7, p. 412. Limbeck, Pick. Prag. med. Woch., 1893, Nos. 12-14. Limbeck, Steindler. Cent. f. inn. Med., 1895, No. 27. Livierato. Arch. f. klin. Med., Bd. 53, p. 303. Lloyd Jones. Jour, of Physiol., vol. 8, p. 1. Locke. Bost. Med. and Surg. Jour., vol. 147, p. 289. Loewy. Arch. f. d. ges. Physiol., Bd. 58, p. 498. Cent. f. med. Wissen., 1894, p. 785 Lowit. Studien u. Phys. u. Path. d. Blutes, Jena, 1892 Ludwig. Wien. med. Woch., 1881, p. 122. Lyonnet. De la densite du Sang., Paris, 1892. Maragliano. XI. Cong. inn. Med., 1892. Berl. klin. Woch., 1892, No. 3L Matthes. Berl. klin. Woch., 1894, Nos. 23, 24. Mering. Pfliiger's Archiv, Bd. 37, p. 348; Bd. 39, p. 21. Meyer, Biarnes. Compt. Rend. Soc. Biol., 1893, p. 821. Miescher. Med. Chem. Untersuch. (Hoppe-Seyler), p. 411. Milroy, Malcolm. Jour, of Physiol., vol. 25, p. 105. Minkowski. l Berl. klin. Woch., 1892, No. 5. 2 Arch. f. exper. Path., Bd. 19, p. 233. 3 Ibid., Bd. 18, p. 33. Muntz. Compt. Rend. Acad. Sci., T. 112, p. 298. Munzer. 3 Cited by v. Jaksch. Murri. Klin. Zeit! u. Streitfragen, Bd. 8, H. 7. Mya, Viglezio. Rivista clin., 1888. Nencki, Sieber. Ber. d. Deut. chem. Gesell , 1885, Bd. 18, p. 392. Peiper. Virchow's Archiv, Bd. 116, p. 337. Picard. Virchow's Archiv, Bd. 11, p. 189. Rigler. Cent. f. Bact., Bd. 30, 1901. Rumpf. Cent. f. inn. Med., 1891, p. 441. de Ruyter. Arch. f. klin. Chir., Bd. 37, p. 766. CHEMISTRY OF THE BLOOD. 95 Salomon. 1 Zeit. f. phys. Chem., Bd. 2, p. 65. Charity Annalen, 1880, V., p. 137. 2 Deut. med. Woch., 1877, pp. 92, 421. Schafer. Sitzb. kais. Acad. Wissen., Wien, Bd. 41, Abt. 3, p. 589. Schlesinger. Arch. klin. Med., Bd. 50, p. 407. Schmaltz. Deut. Arch. klin. Med., Bd. 47, p. 145. Deut. med. Woch., 1891, No. 16. Schmidt. Zur. Charact. d. epidem. Cholera, Leipzig, 1850. Seegen. l Pfluger's Archiv, 1884, 1885, 1886. 2 Wien. klin. Woch., 1892, Nos. 14-15. Siegl. Prag. med. Woch., 1892, No. 20. Wien. klin. Woch., 1891, No. 33. Silberman. Virchow's Archiv, Bd. 119, p. 88. Spiro. Zeit. f. phvsiol. Chem., 1887, I., p. 110. Spitzer. Berl. kl. Woch., 1894, p. 949. Stadelman. Arch. f. exper. Path., Bd. 16, p. 419. Stintzing, Gumprecht. Deut. Arch. klin. Med., Bd. 53, p. 265. Tappeiner. Cent. f. med. Wissen., 1881, Nos. 21-22. Tiegel, Plosz. Pfluger's Archiv, Bd. 6, p. 249; Bd. 7, p. 391. Tschereskoff. Arch. d. Physiol., 1895, p. 628. Tschirkoff. Zeit. klin. Med., Bd. 19, Suppl., p. 87. Trinkler. Cent. f. med. Wissen., 1890, p. 498. Viault. Compt. Rend. Acad. Sci., T. Ill, p. 97 Yicarelli. Cited by Limbeck, p. 165. Viola, Jona. Arch, d Physiol., VIII., 1, p. 37. Wagner. Cited by v. Jaksch. 3 Weintraud. Deut. med. Woch., 1895, V. B., p. 185. Zagari. Giorn. inter, della sci. med., 1892. CHAPTER III. MORPHOLOGY AND PHYSIOLOGY OF THE RED CELLS. In fresh normal blood the red cells appear as homogeneous bicon- cave disk-shaped bodies, with opaque yellowish rims and nearly trans- parent centres. In the capillary circulation they exhibit remarkable elasticity, folding, indenting, and greatly elongating, without rupture. After early infancy they are invariably non-nucleated. They show a strong tendency to cohere to one another by their flat surfaces, forming long rows (rouleaux). This property has been referred to the presence of a fatty material surrounding the red cells. Brunton placed cork disks covered with soap in acidulated water, and found that they formed rouleaux, probably with the liberation of fatty acids. Rou- leaux formation by red cells, he suggests, is probably due to the liberation of fatty acids under the influence of C0 2 . Peskind has shown that agglutination of red cells is produced by various acids and acid salts through alteration in the alkalinity, and combination with the nucleoproteid, of the cell envelope. In dry specimens when thinly spread and rapidly dried the cells are circular in outline, their concavities are obliterated, and they stain evenly throughout. When rather thickly spread the rouleaux are retained, the concavities persist, and the centres may be nearly trans- parent while the thicker rims are densely stained. Neither membrane nor reticulum, have been fully demonstrated in the human red blood cells, although both, from analogy, have been supposed to exist. It is more probable that the hemoglobin is held in compact form principally by a chemical union with other albu- minous constituents of the cell (Schafer). Peskind's studies indicate that the red cells are enveloped by a hemoglobin-free layer composed of lecithin, cholesterin, and a nucleo- proteid. Staining Reactions. The living red cell is achromatic, but when fixed is markedly acidophile in quality, a property probably residing exclusively in the Hb. In the centre of the cell there is an achromatic constituent which fails to stain with acid dyes, but under some circumstances this central substance may become partially separated or even extruded from the cell, when -it stains lightly with methylene blue, and yields the specific reaction of chromatin (Nocht's method). Two views regarding the structure of the red blood cell have been main- tained. According to the first, the red blood cell is vesicular, consisting of colored semifluid contents, hemoglobin, surrounded by a membrane and sup- ported by a stroma composed of various substances, principally lecithin, cholesterin, and cell-globulin (Schafer). According to the second view,' the red cell is not. vesicular, but a viscous solid mass, consisting of a colorless PLATE I. v.; .v:. •**:•• .:fvW£B£% 5. ■.v.... 3* :'&l Myelogenous Leukemia. Triaeid Stain. Figs. 1. Normal-sized red cells deficient in Hb. Figs. 2. Pear-shaped poikilocyte. Fig. 3. Normoblast. Fig. 4. Myelocytes (Ehrlich's). Fig. 5. Myelocyte (Condi's). Fig. 6. Myelocyte (eosinophile) . Fig. 7. Eosinophile leucocyte (normal). Fig. 8. Blood plates. Fig. 9. Lymphocyte. Fig. 10. Polynuclear leucocyte, nucleus subdivided. LEUKEMIA. 241 Later the estimates are untrustworthy, but the Hb-index probably rises. The Leucocytes. The white cells are usually so much increased as to leave no doubt, from their numbers alone, as to the nature of the disease. In cases of very moderate severity there are usually from 100,000 to 200,000 leucocytes ; Cabot's average of first examinations in 39 cases was 438,000. Rarely 1,000,000 or more white cells are present in the cubic millimetre of expressed blood, but it appears doubtful if any such excessive number actually exists throughout the circulation. As a rule, the severity of the general condition is proportionate to the increase of leucocytes, 'except in acute leukemia, when the number of leucocytes may not exceed that of inflammatory leucocytosis. Variations in the number of leucocytes may be observed at differ- ent periods of the day, as shown by Hayem, who found 122,500 at 10 a.m., and 235,000 at 4 p.m. of the same day. These rapid changes must be referred largely to vasomotor disturbances leading to unequal distribution of cells, or, possibly, to a sudden but temporary increase of the leucocytes discharged from the marrow. Other variations in number of leucocytes will be considered under the course of the disease. Morphology of Leucocytes in Myelemia. 1. Neutrophils Myelo- cytes. These are large mononuclear cells with neutrophile granules. It is of some moment to distinguish two varieties of neutrophile myelocytes in leukemia. (a) Cells of about the same size as normal polynuclear leucocytes, with well-staining, central nucleus, as described by Ehrlich and Utheman. These cells are abundantly present in leukemic blood and are generally the only form of myelocyte seen in secondary anemia. (Plate VIII., Fig. 4.) (b) Cornil and H. F. Miiller have called special attention to the large myelocyte with pale eccentric nucleus. This cell is not usually seen except in leukemia, but in some other conditions, especially in the secondary anemia of children, it may appear in moderate numbers. These cells have frequently been seen in mitotic division. (Plate VIII., Fig. 5.) As first shown by Mosler, and abundantly verified by later observers, the presence of large numbers of neutrophile myelocytes is pathognomonic of leukemia. In 28 cases (chronic ?) Cabot found that between 20 to 60 per cent, (average 35 per cent.) of all leuco- cytes present were myelocytes. With the extreme leucocytosis of chronic leukemia there are nearly always enough myelocytes present to establish the diagnosis as against any other condition that has yet been observed. In acute myelocytic leukemia, however, this rule may fail and both the numbers and proportions of myelocytes may not exceed those seen by Mngel in fatal diphtheria. Of recent cases of acute leukemia in which the type of the disease was satis- factorily determined, Frankel (1895) could fiud only three of myelocytic type, the great majority being of the lymphatic variety. In 1897 the writer saw 16 242 SPECIAL PATHOLOGY OF THE BLOOD. three cases of rapidly fatal leukemia, verified by autopsies, and all of the mye- locytic type. They occurred in the services of Drs. Thomson and Delafield, at Roosevelt Hospital. In two of these the changes in the blood were from the first observation typical of the condition and the marrow was puriform, while the spleen and lymph nodes were but slightly affected. In the third case, on the first examination, with a leucocytosis of ordinary inflammatory grade, 5 per cent, of the cells were with difficulty recognized as myelocytes. Eosinophile myelocytes and normoblasts were absent, and there was then not sufficient ground on which the diagnosis of leukemia could rest. Later, the leucocytosis and the proportion of myelocytes increased and the diagnosis of acute leukemia, made with reserve just before death, was fully verified at autopsy. In this case, on account of the small numbers and indistinctness of the granules in the myelocytes, as well as the moderate grade of leucocytosis, the writer did not feel certain of the diagnosis until after the microscopic exam- ination of the bone-marrow. Degenerative Changes in Myelocytes. The myelocytes of leukemia are usually deficient, and may be entirely lacking, in neutrophile granules. In the latter case they are indistinguishable from large lymphocytes, except by the great pallor of their nuclei (triacid stain). Such forms occur especially in acute leukemia. The nuclei of degenerating myelocytes may undergo hydropic degeneration. It is then practically impossible to determine the origin of these altered cells. (Plate X., Figs. 1 and 2.) Some very small mononuclear cells with neutrophile granules seen in leukemic blood Ehrlich regards as the result of subdivision of polynuclear leucocytes (neutrophile pseudolymphocy tes) . 2. Polynuclear neutrophile leucocytes are excessively" numerous in myelocythemia, but are commonly found in diminish- ing proportions, though in increasing numbers, as the percentage of myelocytes increases. Cabot, using the triacid stain, found between 17 and 72 per cent, (average 46 per cent.) of polynuclear leucocytes. Even when the myelocytes are relatively few their presence seems always to be at the expense of polynuclear cells, a fact which indi- cates a slower progress to full development as well as increased production of these cells in the marrow. The most successful demon- stration of nuclear figures in these cells (e. g., by x^ocht's stain) show very numerous transitional forms between the spheroidal and the multilobate nucleus, while after the triacid stain it is usually impos- sible to distinguish between many single and polymorphous nuclei in neutrophile cells. In lymphatic leukemia polynuclear leucocytes are usually scarce and may not be found at all. Degenerative changes in the polynuclear leucocytes are very common and very marked. Their cohesiveness is increased and they appear in large inseparable groups with myelocytes Their nuclei are usually pale (karyolysis) and may become excessively faint. Gumprecht suggests that this pallor of the nuclei of neutrophile cells indicates the transformation of the nucleins into xanthin bodies ; or the lobes may become more numerous, entirely separate, very compact, and densely staining (pyknomorphous), while the granular cytoplasm is replaced by homogeneous, highly refractive material (Plate X., Figs. PLATE X. Degenerating Leucocytes in Myelogenous Leukemia. (Triacid Stain. ) Fig. 1. Myelocyte deficient, in neutrophile granules. Fig. 2. Necrotic myelocyte, complete knryolvsiw; hydropic degeneration; lows of neutrophile granules. Fig. 3. Degenerating myelocyte. Hydrops of nucleus. Fig. 4. Polvnuelear leucocyte deficient in neutrophile granules; hydrops of nucleus. Figs. - r », 6, Polynuclear leucocytes. Loss of neutrophile granules. Advanced subdivision of nucleus. Fig. 7. Myelocyte in mitotic division. (Eosin and methylene blue,) Fig. S. Eosinophil leucocyte. Some granules basophilic. (Eosin ami methylene blue.) LEUKEMIA. 243 5 and 6) ; or the nuclei may undergo hydropic degeneration. (Plate X., Fig. 4.) All grades of deficiency of granules may be observed, but the writer has been unable to demonstrate fat in the neutrophile cells of leukemia. Considerable variations in the size of these cells may be noted in some cases of leukemia. 3. Eosinophile cells are usually much increased in number in myelocythemia, but their proportions to other forms of leucocytes commonly vary within normal limits. In some cases, however, the proportions are increased, but never so much as in cases of pem- phigus, etc. Their proportions are not pathognomonic of the disease, but in most cases their total numbers greatly exceed those found in any other condition. Ehrlich places their numbers between 3,000 to 100,000 per c.mm., thereby practically demonstrating, as he originally claimed, that the excess of eosinophiles is among the pathognomonic signs of the disease. In some specimens of blood the eosinophile cells vary greatly in size, some of them being very small, while their neighbors are hypertrophic. A few large basophilic granules may be found in the polynuclear as in the mononuclear eosinophiles of leukemia. These cells are actively ameboid. In acute leukemia of both types and in chronic lymphocythemia eosinophiles are scarce or absent. Eosinophile myelocytes are mononuclear cells with eosinophile granules. In some of these cells the granules are of uniform size and staining quality, or there may be some basophile granules among the eosino- phile, or the granules may vary greatly in size and in density of stain. Eosinophile myelocytes with granules of very unequal size and density of stain are, in the writer's experience, pathognomonic of myelo- cythemia. (Plate VIII., Fig. 6.) Bignami, however, speaks of the occurrence in pernicious malaria of eosinophile myelocytes " such as are seen in leukemia." In the majority of cases of myelocythemia eosinophile myelocytes constitute a large proportion of all eosinophile cells present. In lymphatic leukemia they are almost always absent, and in acute leukemia they are scarce or absent. 4. Lymphocytes. The numbers and proportions of lymphocytes in myelocythemia vary in different cases and at different periods in the same case, and one must be prepared to find marked variations in the proportions of lymphocytes without being able to attach much significance to such changes. As a rule, the lymphocytes are much increased in numbers, while diminished in relative proportions to the neutrophile cells. The increase of lymphocytes appears to have no uniform relationship to the stage or character of the disease, as small numbers are found at early stages of some cases, and large numbers in the late stages of others, and vice versa. Cabot found an average of 10.6 per cent., but when at their lowest (2 per cent.) they were still more numerous than in normal blood. The proportions of large and small lymphocytes also vary without apparent relation to the other features of the disease. Usually but not always the large cells outnumber the small. It is probable that many myelocytes 244 SPECIAL PATHOLOGY OF THE BLOOD. deficient in neutrophile granules are commonly enumerated among large lymphocytes, especially in acute myelocythemia. Turk's " Reizungsformen," mononuclear cells with opaque, densely staining cytoplasm, are also commonly included among the large lymphocytes. They are probably ancestral forms of megaloblasts. The access to the blood stream in myelocythemia of large numbers of true lympho- cytes, small and large, may be referred to a secondary, but, neverthe- less, continuous hyperplasia of these cells in the marrow and lymphoid tissues, and to accidental and temporary factors which excite their proliferation and attract them to the blood. Their presence, even in considerable numbers, cannot at present be regarded as an indica- tion that the disease is of mixed type. 5. Large mononuclear leucocytes, with very faint cytoretic- ulum and vesicular nucleus, are seen in considerable numbers in most cases of leukemia, but they appear to lack special significance. They are very apt to suffer damage in the smearing process and to appear in the dry specimen as large, coarsely reticular nuclei, without demonstrable cell body. (Plate IX., Fig. 3.) These cells may con- tain granules giving the reaction of glycogen. The larger forms may show horseshoe-shaped nuclei. In the cases of acute leukemia described by Frankel and others, the majority of cells were large mononuclear leucocytes. Degenerative changes ure noted in the small and often in the large lymphocytes of leukemia. The nuclei of the small cells, instead of remaining compact, may become incurved and finally bilobed or trilobed, while the cell body remains basophilic (Rieder, Ehrlich). Litten 2 describes, in two acute cases, very large cells in which the nucleus was obscured by many globules of fat blackened by osmic acid. There are some other reports of fatty degeneration of leuco- cytes in leukemia, but apparently none from very recent literature. Mitotic figures in lymphocytes of lymphemia are reported by Wertheim. 6. Mast-cells. These cells are very constantly increased in chronic myelocythemia, but in some cases a prolonged search is required for their discovery. Although occasionally seen in other conditions, in leukemia they are usually so markedly increased as to constitute a very reliable diagnostic feature of the blood. They are at times more abundant than the eosinophile cells. They are usually absent in lymphemia, and the writer has failed to find them in acute myelo- cythemia. Strauss found 5 per cent, of mast-cells among the leucocytes in blister fluid from a case of myelocytic leukemia, and Milchner reports 23.9 per cent, of mast-cells in the sediment from ascitic fluid in a similar case. In the blood of one case Taylor reports the remarkable number of 140,000 mast-cells. Special Characters of the Blood of Lymphatic Leukemia. In this type of the disease the lymphocytes are the only form of white cell appearing in the blood in increased numbers. In cases in which a positive clinical diagnosis is possible, the nature of the condition is at once evident from the great abundance of these cells, but owing to PLATE IX. Lymphatic Leukemia. (Eosin and Methylene Blue.) Figs. 1. Small lymphocytes. Figs. 2. Medium-sized and large lymphocytes. Fig. 3. Degenerating basket-shaped nucleus of large lymphocyte, without cytoplasm. Fig. 4. Polynuclear leucocyte. Nodal points of cytoreticulum. Fig. 5. .Red cell. Polyehromasia of Maragliano. LEUKEMIA. 245 the numerous other causes of chronic lymphocytosis it is at present impossible to state what is the lowest proportion of such cells seen in the blood of genuine lymphemia. In well-marked cases they are quite as numerous as the leucocytes of myelocythemia, while some of the highest counts on record nave been reported in this type of the disease. Usually the lymphocytes are of small size and normal structure, and the writer has specimens of one verv marked case, in which almost all the leucocytes were small mononuclears. Their percent- age commonly runs between 80 and 90. In rather rare cases reported by Frankel, Grawitz, Cabot, and others, and in one child observed by the writer, the great majority of the cells were unusually large and their cytoreticulum faintly staining. Between the two extremes are cases showing various pro- portions of small, medium-sized, and large lymphocytes. The small lymphocytes are usually more abundant in chronic cases and in adults, the larger cells tending to become prominent in acute cases and in children. Gerhardt refers to a case in which large lympho- cytes, abundant in the early acute stage, were replaced during a chronic course of six months, by small lymphocytes. Frankel believed that these cells could be regarded as pathognomonic of acute lymphatic leukemia, but his claims have been fully disproved. Large mononuclear cells with hyaline bodies were common in the writer's cases of acute myelocythemia, and reasons have been given to show that some of these cells may result from loss of neutrophile granules of myelocytes. Askanazy insists that the large cells of Frankel are not lymphocytes, but myelocytes deficient in granules, a view to which the writer is inclined from the study of three cases of acute leukemia. Hirschlaff has recently reported two acute cases in which most of the leucocytes were large mononuclear cells without granules. In a moderate proportion of them neutrophile granules and in a larger number eosinophile granules were noted. This variation in size of lymphocytes may have an anatomical basis in the structure of the hyperplastic lymph nodes, as in some cases these nodes con- tain mostly small lymphocytes, at other times large mononuclear cells. (Birch-Hirschfeld, Benda ) These and other considerations oppose the interesting view recently ex- pressed by Eosenfeld, that when the lesion of lymphemia is in the marrow many large lymphocytes appear in the blood, anemia rapidly develops, and the disease ruDS a severe course, while if the lesion affects only spleen and lymph nodes large lymphocytes are absent from the blood and anemia is of slow development. Myelocytes, both neutrophile and eosinophile, and mast-cells are usually absent or extremely scarce in lymphemia, but are occasionally seen in scant numbers (Miiller). Polynuclear leucocytes, both neu- trophile and eosinophile, are also comparatively scarce. The red cells suffer the same changes as are seen in myelocy- themia, but nucleated red cells are usually very scanty and may not be found at all. Da Costa, however, found 10,678 nucleated red cells in one case, the great majority normoblasts. Variations in the Blood Changes in Leukemia. Both the numbers and the proportions of leucocytes in leukemia are subject to considerable variations from many causes. 246 SPECIAL PATHOLOGY OF THE BLOOD. 1. Intercurrent Diseases. A considerable number of cases have been reported showing that intercurrent infections may greatly alter the appearance of the blood in leukemia. These cases have been col- lected by Frankel and Marischler, Froelich, Cabot, Kormoczi, and McCrae. Some of the reports refer to terminal septicemia which has resulted usually in a marked and rapid decrease of leucocytes, usually with an increase in the proportion of polynuclear cells. Frankel, who saw the leucocytes fall from 220,000 to 1200, refers this result to pure leucocytolysis. Such cases have been observed after typhoid fever, pneumonia, empyema, erysipelas, septicemia, tuberculosis, car- cinoma, and influenza (Kovacz). Beitzke, from observations in six- teen cases, concludes that all diseases which cause polynuclear leuco- cytosis tend to increase the proportion of these cells in leukemia. Distinct changes in leukemic blood do not invariably result from such intercurrent diseases. In cases where the infection is localized (empyema, erysipelas) the norrUal activities of the marrow seem to be stimulated, and while the leucocytes diminish the proportion of polynuclear cells increases (Freudenstein, Kovacz, Miiller'). Kraus has recently reported a reduction of from 393,000 to 4000 leucocytes in a few days as the result of double pneumonia and empyema. The viscera (marrow, liver, spleen) showed no evidence of leukemic infil- tration, and Kraus suggests that the suppurative process had not only transformed the blood, but had resolved the essential visceral lesions as well. Usually intercurrent infections diminish the volume of spleen and lymph nodes even when, as in Midler's case, the leuco- cytes are increased. After the subsidence of the infection the blood soon resumes its original condition (Grawitz), or even before the infection subsides the first effect may pass off, as occurred in Eisen- lohr's case, within fourteen days. Although no permanent improvement resulted in any cases suffering from intercurrent infections, several attempts have been made to favorably influ- ence the course of leukemia by artificial leucocytosis. Jacob reduced the leucocytes from 850,000 to 282,000 in a few days by repeated injections of glycerin extract of spleen, but could not report any improvement in the patient. The same result was obtained by Richter using spermin. Richter and Spiro claim to have increased the leucocytes in leukemia from 170,000 to 560,000 within three hours after injection of cinnamic acid, followed by prompt return to the previous condition. Heuck 2 has also reduced the leucocytosis of leukemia by injection of tuberculin. In mild inflammatory processes there may be no effect upon the leucocytes of leukemia, as indicated by the cases of Richter and Heuck. 1 Antemortem leuoocytosis of considerable degree (172,000) was observed by Thorsch in a case complicated by pneumonia. In a case of lymphatic leukemia dying of septicemia, Miiller found, four days before death, 400,000 leucocytes when there had previously been but 180,000. A differential count was not made, but Miiller regarded the increase as referable to a polynuclear leucocytosis. Chronic infections have much less effect in altering the character of leukemic blood. Quincke and Stintzing reported a general improvement of the leukemic process during acute miliary tuber- culosis and during an exacerbation of chronic phthisis. LEUKEMIA. 247 In a case of lymphatic leukemia dying with carcinoma of the kidney Marischler found a decrease of leucocytes from 96,000 to 48,000, with marked increase in the proportion of polynuclear cells. 2. Spontaneous Changes in the Blood of Leukemia. A few instances are recorded in which the disease, as indicated by the blood changes, appeared to become transformed from one type into another. The first of these cases was that of Fleischer and Penzoldt, who observed splenic leukemia pass into lymphatic. It seems possible that this case may be placed with others described by Frankel 2 and Gerhardt, in which lymphatic leukemia began acutely with large lymphocytes, but progressed more slowly with small lymphocytes. Wey observed a case of myelocythemia in which, within ten weeks, the polynuclear cells fell from 33.5 per cent, to 3.7 per cent., the mononuclears rose from 66.5 to 96.3 per cent., among which were neutrophile myelocytes, large hyaline cells, and a few lymphocytes. This and Reiman's case represent a series of transformations sometimes observed, in which the disease begins with a considerable proportion of polynuclear neutrophile cells, passes through a stage in which myelocytes exist in high proportions, and ends in more or less fulminant form, with many large cells deficient or entirely lacking in neutrophile granules. Gerhardt observed the blood of a case of leukemia of marked grade pass into that of pernicious anemia, the excess of leucocytes disap- pearing in three days. The possible transformation of pernicious anemia into leukemia has already been discussed. Pseudoleukemia has been described as changing into leukemia in several cases. (See Pseudoleukemia.) In view of the occasional reports of cases of leukemia in which the diagnosis rested upon the study of cadaveric blood, reference may be given, but with some caution, to the report by Seelig, of a case in which during life many myelocytes but few lymphocytes were found, while in the blood of the cadaver only lymphocytes were encountered. Chemistry. Specific Gravity. The specific gravity of the blood is usually reduced, owing principally to the loss of Hb. But leu- kemia is one condition in which Hb is often replaced by other albumins, so that the gravity of the blood is relatively high in com- parison to the Hb-content, and in exact proportion to the increase of leucocytes. Thus Diebella reports a case with 2,600,000 red cells, 750,000 leucocytes, and specific gravity of 1.060. The lowest observations are placed by Grawitz at 1.036. The low figures are usually seen in cases complicated by hemorrhages or other causes of secondary anemia. The specific gravity of the serum has been found by Grawitz and by Taylor to fall within normal limits, 1.023 to 1.030. Alkalescence. The alkalescence of leukemic blood has been found by v. Jaksch, Peiper, and others to be much diminished. Early observers found the blood distinctly acid soon after death (Scherer), probably owing to the postmortem formation of acids, v. Noorden referred the diminished alkalinity to the development of acids during life. Lactic and formic acids have been isolated from the fresh 248 SPECIAL PATHOLOGY OF THE BLOOD. blood by Scherer, Mosler, and others, and acetic acid, after death only, by Mosler. Albumins. Fibrin has been found in excessive quantity, 5.7 per cent, (normal 0.25 per cent.), by Parkes, but in chronic cases with severe anemia it is diminished (Robin). Peptone. Considerable interest attaches to the demonstration, first by Bockendahl and Landwehr, of considerable traces of peptone in the splenic pulp and in the blood of leukemia. This observation has lately been verified by Mathes, who, however, has shown that the principle in question is not Kuhne's peptone, but deutero-albumose. Freund believes that the retarded coagulation of leukemic blood is . largely referable to the presence of albumose. The products of the excessive destruction of leucocytes have been traced in the presence of their various derivations in blood and urine. Nucleo-albumin and deutero-albumose, but not peptone, have been isolated from the serum by Mathes ; mucin or a closely allied sub- stance by Scherer and others ; and a principle resembling gluten by Salkowski. Xanthin bodies, which represent further decomposition products of leucocytes, have been found in the spleen and blood by Scherer, and many others The xanthin bodies of Kossel are more abundant and more easily recognized in leukemic than in normal blood. Uric acid has been found in traces by Mosler, Fowlwarczny, Klem- perer, and Weintraud. An excessive excretion of uric acid in the urine of leukemia has often been observed, and Magnus-Levy reported as high as eight grains daily, but since the most abundant appearance of leucocytes need not indicate the time of their greatest destruction, there is no parallel between the excretion of this prin- ciple and the excess of white cells in leukemia (Minkowski, Wey). Gumprecht 2 finds that in those cases of leukemia in which uric acid excretion is normal there is an increase of " alloxurkorper," which consists of alloxan, urea, uric acid, and other principles of similar significance with uric acid. Glycogen. An increased quantity of glycogen has been extracted by Salomon and by Gabritschewsky. The fat-extract has been found distinctly increased by Robertson, Isambert (0.72 per cent.), and Freund and Obermayer. Tyrosin was isolated by Fowlwarczny, and lecithin and cholesterin from the blood of a hematoma by Freund and Obermayer. Of inorganic principles the iron has uniformly been found dimin- ished (2.24 to 2.97 per cent, of ash), usually in proportion to the loss of Hb (Strecker, Scherer, Freund, and Obermayer). Phosphorus, sulphur, and sodium were much increased and potas- sium and chlorine much diminished in Freund and Obermayer' s 2 case, while the total salts were moderately increased. Charcot-Leyden Crystals. Robiu first observed these peculiar crystals in the tissues of a leukemic cadaver, and they were described by Charcot and Robin in 1853. Zenker later (1855) claimed priority in their discovery. They were found in all parts of the circulation in a case examined after death by Wallace in 1855. Later they PLATE XI. Mast Cells. (Ehrlieh's Dahlia-stain.) LEUKEMIA. 249 were carefully studied by Charcot and Vulpian, who described them as colorless, refractive, elongated octahedra, 0.016 X 0.005 mm., or occasionally of much larger dimensions, insoluble in cold water, alcohol, ether, or glycerin, soluble in hot water and in most acids and alkalies. After death they appear in the blood, exudates, and especially in the spleen, and their numbers increase with post- mortem changes in the tissues. Like most other crystals of post- mortem formation, they are often seen in or on the leucocytes. Their occurrence in sputum and relation to eosinophile cells was pointed out by Charcot and Leyden. According to Neumann, 1 they are found in leukemia only when the blood contains many large cells with abundant cytoplasm and large nuclei, and hence are not seen in lymphatic leukemia. Chemically they were regarded by Charcot as albuminates, by Salkowski as mucinous. Lately Pohl has shown that they are identical with Bottcher's spermin crystals, which are a product of the destruction of the nucleins of disintegrating cells, and probably consist of spermin phosphate. Scherer also con- cluded that they are composed of phosphoric acid and an organic base. They were obtained in the blood drawn during life by Neumann and later by many others. They are not peculiar to leukemia, but appear in the sputum of asthma, in feces and mucus surrounding intestinal parasites, and have been found in the fetal blood in simple anemia (Growers). Lewy states that eosinophile granules may be thrown down in the form of Charcot-Leyden crystals by fixation of tissue containing eosinophile leuco- cytes in magnesium sulphate, 1 part; water, 7 parts; or sodium sulphate,! part; water, 10 parts. The tissues thus fixed should be thoroughly washed in water and hardened in alcohol. "The Diagnosis of Leukemia. In no other department is the examination of the blood to be interpreted with greater certainty in one case or greater reserve in another than in its application to the various types of leukemia. 1. The changes in the blood may yield positive diagnostic signs of leukemia. This result is obtained in the great majority of cases of both types, whether acute or chronic. These signs are briefly : In Myehcythemia. An excessive leucocytosis (150,000 to 1,000,000). A large number and considerable proportion of neutrophile mye- locytes (20 to 60 per cent.). A large number of eosinophile cells (3000 to 100,000) of which many are mononuclear and exhibit very large densely staining granules. An excessive number of polynuclear neutrophile cells. Charac- teristic changes in the red cells. To these may be added : The presence of many mast-cells, many nucleated red cells, mitotic nuclei in leucocytes, extreme and pecu- liar degenerative changes in leucocytes. 250 SPECIAL PATHOLOGY OF THE BLOOD. In Chronie Lymphoeythemia. An excessive leucocytosis (150,000 to 1,000,000). The presence of 85 to 99 per cent, of lymphocytes. Scanty numbers of myelocytes, eosinophiles, nucleated red cells, and mast-cells. For the positive diagnosis of either type of the disease the essen- tial point is the excessive leucocytosis, at least 150,000 cells, of which a considerable proportion are myelocytes or a still higher proportion are lymphocytes. No other condition thus far observed yields such characters in the blood in the presence of which the diagnosis of leukemia is estab- lished beyond doubt. 2. The changes in the blood may justify only a probable diagnosis of leukemia. This situation is encountered under several condi- tions. (a) In myelocythemia the effect of intercurrent infections may so reduce the number of leucocytes, and especially the proportion of myelocytes, that the blood does not differ from that of some cases of acute inflammatory leucocytosis with 5 to 16 per cent, of myelo- cytes. (b) In the less marked stages of lymphoeythemia, especially of the acute form, the number and proportion of lymphocytes may be far from characteristic of leukemia. When there are less than 150,000 white cells and less than 90 per cent, of lymphocytes the condition of the blood does not differ from that seen in some forms of inflam- matory leucocytosis, or of lymphocytosis in the secondary anemia of children, or of the obscure condition called " v. Jaksch's anemia." In inflammatory lymphocytosis, however, there are always a fair proportion of polynuclear leucocytes, which are very scanty in lymphemia, and the lymphocytosis is usually transient. In secondary anemia the lymphocytosis is seldom excessive, the proportion of lymphocytes is usually lower, and a few myelocytes, eosinophiles, and nucleated red cells are commonly present. In "v. Jaksch's anemia" the lymphocytes are usually less numer- ous, there are more large hyaline mononuclear leucocytes than are usually seen in lymphemia of equal chronicity, and myelocytes, eosinophiles, and many nucleated red cells are usually present. Frankel has classed as acute lymphemia certain obscure cases attended with moderate hyperplasia of lymph nodes and the presence in the blood of a considerable number of cells regarded by some as large lymphocytes. Frankel's cases have not been fully accepted by his countrymen, and Grawitz especially has shown that the above condition of the blood is not pathognomonic of lymphemia, while Benda has paved the way for the division of these cases into other categories, including acute myelocythemia. Frankel's cases may belong in the class of lymphoeythemia, but the condition of the blood is not characteristic, and the result of his autopsies is inconclusive. ^ 3. In some stages of leukemia the blood may fail to furnish indi- cations of the nature of the disease, which may then be overlooked. (a) Intercurrent infections have been shown to temporarily trans- form the blood of leukemia into that of inflammatory leucocytosis. LEUKEMIA. 251 (6) In a case of acute myelocythemia, with the disease fully estab- lished, the writer found on first examination, 5 per cent, of myelo- cytes with a leucocytosis of ordinary inflammatory grade. Later the myelocytes increased to the lower limits of leukemia, but a positive diagnosis was not established until the marrow was examined micro- scopically. (e) Most writers agree that the early stages of leukemia often escape detection, and that many spurious cases have appeared in the literature. BlBLIOGEAPHY. Leukemia. Ambros. Inaug. Diss., Munchen, 1893. Arnold. Virchow's Archiv, Bd. 97, p. 107. Askanazy. Virchow's Archiv, Bd. 137, p. 1. Audeod. Traits de mal. de l'enfance (Grancher), T. ii. p. 112. Bard. Lyon med., 1888, p. 239. Becquerel, Rodier. Recherches sur le Sang., 1845. Beitzke. Inaug. Diss., Kiel, 1899. Benda. XV. Cong. f. inn. Med., 1897, p. 371. Bennett. Edin. Med. Jour., 1845, vol. lxiv., p. 413. Monthly Jour. Med. Sci., 1851-52, XII., XIII., XIV. Biesiadecki. Wien. med. Jahrb., 1876, p. 233. Bignami. Twentieth Cent. Pract., Article " Malaria,'' 1900. Birch-Hirschfeld. XV. Cong. f. inn. Med. Bizzozero. Virchow's Archiv, Bd. 79, p. 378. Bockendahl, Landwehr. Virchow's Archiv, Bd. 84, p. 561 Bollinger. Virchow's Archiv, Bd. 59, p. 341. Bonardi. Rev. gen. ital. di clin. med., 1889, No. 5. Bottger. 1 Virchow's Archiv, 1858, Bd. 14, p. 483. 3 Ibid., 1866, Bd. 36, p. 342. Bramwell. Cited by Pawlowsky. Cadiot, Roger. Path, du Sang. Traits de m6d. (Charcot), Paris, 1892. Cameron. Cited by Stengel, Twentieth Cent. Practice, vol. ii. Casali. Rev. clin. Bologna, 1872, vol. ii. p. 118. Charcot, Robin. Compt. Rend. Soc. Biol., 1853, p. 44. Charcot, Vulpian. Gaz. Hebdom., 1860, p. 756. Cornil. Archiv d. phys. norm, et path., 1887, T. x. p. 46. Cornil, Ranvier. Histol. path. Craigie. Edin. Med. Jour., 1845, vol. lxiv. p. 400. Denning. Munch, med. Woch., 1901, p. 140. Dock. Amer. Jour. Med. Sci., vol. cvi. p. 152. Donne. Cited by Gowers, Lancet, 1878, vol. i. p. 550. Eberth. Virchow's Archiv, 1868, Bd. 43, p. 8. Ebstein. Deut. Archiv klin. Med., Bd. 44, p. 343. Ehrlich. Gesam. Mittheil., 1891. Eichhorst. 'Virchow's Archiv, Bd. 130, p. 365. 2 Ibid., Bd. 113. Eisenlohr. Virchow's Archiv, 1878, Bd. 73, p. 56. Engel. Deut. med. Woch., 1897, pp. 118, 137. Erb. Virchow's Archiv, 1865, Bd. 34, p. 138. Ewing. N. Y. Med. Jour., 1895, vol. ii. p. 161. Fermi. Cited by Litten. Fleischer, Penzoldt. Deut. Archiv klin. Med., Bd. 26, p. 368. Fowlwarczny. Deut. Zeit. f. prak. Med., 1875. Frankel. J Deut. med. Woch., 1895, p. 666. 2 XV. Cong. f. inn. Med. Freudenstein. Inaug. Diss. Berlin, 1895. Freund, Obermayer. 1 Zeit. f . physiol. Chem., Bd. 17, p. 318. 2 Ibid., Bd. 15, p. 310. Friedrich. Virchow's Archiv, Bd. 12, p. 37. Froelich. Wien. med. Woch., 1893, p. 285. Fuller. Lancet, 1846, vol. ii. p. 43. Gabritschewsky. Archiv f. exper. Path., Bd. 28, p. 272. 252 SPECIAL PATHOLOGY OF THE BLOOD. Gerhardt. XV. Cong. f. inn. Med., p. 382. Gilbert. Path, du Sang. Traite de Med. (Charcot), Paris, 1892, vol. ii. p. 457. Gowers. Reynolds' Syst. Med., 1879, vol. v. p. 216. Grawitz. Klin. Path, des Blutes, 1902. Greene. N. Y. Med. Jour., vol. xlvii. p. 144. Greime. Berl. klin. Woch., 1892, No. 33. Gumprecht. 1 Deut. Archiv klin. Med., Bd. 57, p. 523. 2 Cent, f . Path., 1896, No. 20. Guttmann. Berl. klin. Woch., 1891, p. 1109. Hajek. Wien. klin. Woch., 1897, Xo. 20. Hayem. Du Sang., pp. 382, 856, 857. Herzfeld. N. Y. Polyclinic, 1894. Heuck. 1 Virchow's Archiv, Bd. 78, p. 475. 2 Deut. med. Woch., 1891, p. 747. Hinterberger. Deut. Archiv klin. Med., Bd. 48, p. 324. Hintze. Arch. klin. Med., Bd. 53, p. 377. Hirschlaff. Deut. Archiv klin. Med., Bd. 62, p. 314. Isambert. Cited by Mosler. Jacob. Cited by Grawitz, p. 127. Jaderholm. Cited by Litten (Nothnagel, Spec. Path.), p. 148. v. Jaksch. Zeit. f. klin. Med., Bd. 23, p. 187, also Klin. Diagnostik. Jolly. Arch, de med. exper., 1902, p. 73. Kelsch, Vaillard. Annal. de l'Institut Pasteur, 1890. Klebs. 1 Virchow's Archiv, 1867, Bd. 38, p 190. 2 Eulenberg's Realencyc, Bd. 1, p. 357. Klein. Cited by Pawlowsky. Klemperer, Weintraud. Deut. med. Woch., 1895, Xo. 40, V. B. Kormoczi. Deut. med. Woch., 1899, pp. 238, 775. Kottman. Inaug. Diss. Bern., 1871. Kottnitz. Berl. klin. Woch., 1890, No. 35. Kovacz. Wien. klin. Woch., 1893, p. 701. Kraus. Prag. med. Woch., 1899, Xo. 41. Lannois, Begaud. Arch. d. med. exper., 1895. Lauenberg. Archiv f. Gyn., Bd. 40, p. 419. Leube, Fleischer. Virchow's Archiv, Bd. 83, p. 124. Lewy. Zeit. f. klin. Med., Bd. 40, p. 59. Limbeck. Prag. med. Woch., 1893, Nos. 12-14. Litten. 1 Berl. klin. Woch., 1877, p. 256, also Krankh. d. Milz. (Nothnagel, Spec. Path.), p. 133, etc. 2 XI. Cong. f. inn. Med., p. 159. Lowit. 1 Sitzungsber. kais Acad. Wien., 1885, also ibid., 1887, Bd. 95, III. Abt., p. 22. 2 Die Leukaemie als Protozoeninfec. Also, XVIII. Cong. inn. Med., pp. 251, 322. Magnus, Levy. Cited by Litten. Mannaberg. XD7. Cong. f. inn. Med., p. 252. Marischler. Wien klin. Woch., 1896, p. 686. Masius, Francotte. Cited by Ebstein. Mathes. Berl. klin. AVoch., 1894, pp. 531, 556. Mayet. 1 Compt. Rend. Acad. Sci., 1888, p 762. 2 Soc. med. de Lyon, 1888, T. xxviii. p. 47. McCrae. Brit. Med. Journ., 1900, vol. i. p. 760. McGillavry. Schmidt's Jahrb., Bd. 192, p. 19. Michaelis. Zeit. f. klin. Med., Bd. 46, p. 87. Milchner. Zeit. f. klin. Med., Bd. 37, p. 194. Minkowski. XVII. Cong. f. inn. Med., 1899 Mosler. 1 Berl. klin. Woch., 1864. 2 Ibid., 1876, pp. 701, 720. 3 Virchow's Archiv, Bd. 114, p. 461. Mosler, Westphal. Semaine m6d., 1889, p. 372. Muller. * Deut. Archiv klin. Med., Bd. 48, p. 51. 2 Ibid., Bd. 50, p. 47. 4 Cent, f. Path., 1894, p. 628. Muller, Rieder. Deut. Archiv klin. Med., Bd. 48, p. 96. Mursick. Amer. Jour. Med. Sci., vol. lxix. p. 449. Musser. Trans. Phila. Co. Med. Soc, 1885. Neumann. 1 Archiv f. micr. Anat., Bd. 2, p. 507. 2 Cent, f . Med. Wissen., 1868. p. 689. 3 Ibid., 1869, p. 292. 4 Archiv f. Heilk., 1874, p. 441. 5 Berl. klin. Woch, 1878, p. 607. v. Noorden. Path, des Stoffwechsels, 1893, p. 342. Obrastzow. Deut. med. Woch., 1890, *p. 1150. LEUKEMIA. 253 Osterwald. Archiv f. Ophthal., Bd. 27, p. 224. Palma Deut. med. Woch., 1892, p. 784. Pappenheim. Zeit. f. klin. Med., Bd. 39, p. 171. Parkes. Med. Times, 1851. Pawlowsky. Deut. med. Woch., 1892, p. 641. Pfeiffer. Cited by Litten (Nothnagel's Path.). Pohl. Deut. med. Woch., 1895, p. 475. Ponfick. Virchow's Archiv, Bd. 67, p. 368. Quincke. Munch, med. Woch., 1890, p. 19. Reed. Amer. Jour. Med. Sci., 1902, vol. cxxiv. p. 653. Renaut. Archiv de Physiol., 1881, T. xiii. p. 649. Richter. Cited by Grawitz. Richter, Spiro. Archiv f. exper. Path., Bd. 34, p. 289. Rieder. "Leucocytose." Rindfleisch. Lehrbuch d. path. Gewebel. Robertson. Cited by Mosler. Roux. La Prov. Med., 1890, p. 280. Salomon. Charite-Annalen, 1878, vol. v. p. 139. Salkowski. Virchow's Archiv, Bd. 81, p. 166, also Bd. 50. Sanger. Cited by Stengel, Twentieth Cent. Practice, vol. ii. Schede, Stahl. Mitiheil. a. d. Chir. Abt., Friedrichshain Hosp. Berlin, Leipzig, ] 878, cited by Ebstein. Scherer. Annal. de Chimie et de Pharm., T. cxliv. Schwarze. Ehrlich's Gesamt. Mittheil. Seelig. Arch. f. klin. Med., Bd. 54, p. 537. Senator. Berl. klin. Woch., 1882, p. 533. Sittmann. Deut. Archiv klin. Med., Bd. 53, p. 323. Steinbrugge. Zeit. f. Ohrenheilk., 1886, p. 238. Stintzing. Munch, med. Woch., 1890, p. 19. Stintzing, Oumprecht. Deut. Archiv klin. Med., Bd. 53, p. 264. Strauss. Charite-Annalen, Bd. 23. Thomson, Ewing. N. Y. Med. Record, 1898, vol. i. p. 333. Thorsch. Wien. klin. Woch., 1896, p. 395. Troje. Berl. klin. Woch., 1892, p. 285. Turk. x Wien. klin. Woch., 1900, p. 293. J XVIII. Cong. f. inn. Med., pp. 251, 325. "Zeigler's Beit., Bd. 30, p. 371. Utheman. Ehrlich's Gesamt. Mittheil. Vehsemeyer. Munch, med. Woch., 1893, p. 564. Virchow. Froriep's Notizen, 1845, No. 780. Med. Zeitung, Berlin, 1846, p. 157. Virchow's Archiv, 1847, Bd. 1, p. 563. Vogel. Virchow's Archiv, 1851, Bd. 3, p. 570. Waldeyer. Virchow's Archiv, 1871, Bd. 52, p. 305. Waldstein. Virchow's Archiv, Bd. 91, p. 12. Wallace. Glasgow Med. Jour., 1855, vol. iii. p. 1. Walz. Cent. f. Path., 1894, p. 553. Wende. Amer. Jour. Med. Sci., vol. cxxii. p. 836. Weiss, J. Haematolog. Untersuch. Wertheim. Zeit. f. Heilk., 1891, Bd. 12. Wey. Deut. Archiv klin. Med., Bd. 57, p. 300. Ziegler. Lehrbuch d. Allg. u. Spec. Path. Anat. Zenker. Deut. Archiv klin. Med., 1876, p. 125. CHAPTEE IX. PSEUDOLEUKEMIA . Definition. Pseudoleukemia is a primary disease of the lymph nodes and lymphatic structures, characterized by progressive enlarge- ment of various chains of lymph nodes, and by secondary multiple growths of lymphoid tissue throughout the lymphatic system. It is of toxic and usually of infectious origin, and its essential feature is an extreme hyperplasia of lymphocytes, which approaches or reaches the grade of a neoplasm. Historical. The first attempt to demonstrate the specific character of the lymphadenopathy of the disease appeared in 1828, when Craigie called attention to the difference between certain firm tumors of lymph nodes and the caseating, scrofulous, and the cancerous enlargements. In 1832 Hodgkin described several cases observed chiefly at Guy's Hospital, some of which were cancerous, tubercu- lous, or syphilitic, but two of which were undoubtedly genuine examples of pseudoleukemia. He distinguished the " organized " nodes from the caseous and cancerous ones. Before the discovery of leukemia, therefore, it was known that there are enlargements of the lymph nodes apart from cancer and tuberculosis. In 1839 Velpeau drew attention to the occurrence of hypertrophied lymph nodes apart from scrofula. In 1856 Wilkes described several cases, which he regarded as a special form of dis- ease of lymph nodes, but did not fully distinguish the lesions from those of tuberculosis. The first complete description of the malady appears to have been that of "Wunderlich, 1858, who noted an idiopathic origin of the dis- ease, and the cellular character of the new-growths in both nodes and spleen, mentioned the severe anemia, and called attention to the absence of the extreme leucocytosis which characterizes leukemia. In the same year, 1858, Billroth described the histological structure of the enlarged nodes, noting the limitation within the capsule, the obliteration of follicles and of lymph sinuses and vessels, the pro- liferation of cells by nuclear division, and concluding that the hyperplasia is closely related to and may pass into sarcomatosis. Recognizing in the condition a tumor of lymph nodes of fatal ten- dencies, but differing from sarcoma, he employed the term " malig- nant lymphoma" as specially applicable to this condition. In 1864 the general pathological features were described by Virchow, under the term "lymphosarcoma." Cohnheim in 1867 described a case, and, noting especially the absence of leucocytosis, suggested the term " pseudoleukemia." The nearly constant affection of the spleen was noted by the earlier observers, Wilks, Woillez, Griesinger, Miiller, and Striimpell, and PSEUDOLEUKEMIA. 255 the prominence of this symptom in some cases led Griesinger to employ the term " splenic anemia " for such forms of the malady. In France, Bonfils described a case in 1858, using the designa- tion " cachexie sans leukemia," on account of the absence of leuco- cytosis. A few other examples of the disease were reported before 1865, when Trousseau described in detail the clinical characters and pro- posed the term " adenia." French writers from Trousseau to Gilbert have regarded the condition as very closely related to or identical with leukemia, employing the term " lymphadenie aleukemique." In 1870 a full description of the disease, clinical and pathological, with historical notes to date, was published by Murchison and San- derson. Cornil and Ranvier, in 1867, very fully described the histological structure of the nodes, regarding the process as a true lymphadenoma. In 1887 Ebstein 1 described an acute form of the malady. Later writers have described similar cases, possibly includ- ing among them some conditions not related to pseudoleukemia (Band, Potain, Bruhl). The peculiar localization of the lesions to different structures has been illustrated in many reported cases. Bonfils and Trousseau claimed that the spleen need not participate at all in the lesion. A special affection of the tonsillar ring was described by Demange, and of the intestinal mucosa by Gilly, and later by many others. A testicular form has been described by Monod and Terillon. A case of Reineberg's indicates that the lesion may be limited to the marrow. Many have regarded the anemia infantum pseudoleukemia of v. Jaksch as the splenic form of Hodgkin's disease occurring in an infant (Luzet, Gilbert). The dermal type of the disease has been described by many writers, including Biesiadecki, Kaposi, Hochsinger and Schiff, and Joseph, but appears to have been first described in France by Gillot. Anatomical Characters. Lymph Nodes. The essential lesion in the disease is an extreme hyperplasia of lymphocytes. In some cases, especially in the early or active febrile stages, there are promi- nent signs indicating that the hyperplasia is inflammatory, but in other cases the lesions are those of a simple hyperplasia approaching the neoplastic grade, and the inflammatory changes are in the back- ground. Gowers has described in different nodes of the same case various stages between simple inflammatory and neoplastic hyperplasia of lymphocytes. The essential lesion may, therefore, be inflammatory hyperplasia or lymphadenoma. In the typical chronic cases one lymph node in a chain is affected, followed by the other members of the chain, or the entire chain is affected simultane- ously. The disease seldom spreads by gradual extensions from one chain to another, but suddenly involves a new chain on the same or opposite sides of the body. Sarcoma of lymph nodes affects the intervening tissues between •chains of nodes. For a long period the swollen nodes are retained in their capsules, but late in progressive cases the members of the chain are variously fused and their capsules largely obliterated. This fusion may result either from rupture of the capsules or from periadenitis. 256 SPECIAL PATHOLOGY OF THE BLOOD. It is regarded by some as an important distinction of Hodgkin's disease that secondary growths arise only in pre-existing lymphoid structures, while in lymphosarcoma the secondary growths are indiscriminate and more dif- fuse. Microscopic examination shows the new tissue to be composed of lymphocytes supported by reticular tissue. There are considerable variations in the character, both of the cells and of the reticular tissue. The cells are usually lymphocytes of small and medium size, among which are a few polynuclear leucocytes, exfoliated endothelial cells, and a few small giant cells. Eosinophile cells are frequently but not always found in considerable abundance (Goldman, Kanter). This local eosinophilia is regarded by Dietrich and Fischer as of diagnostic importance. Reed found it in six of eight cases. Its presence is regarded by most pathologists as an argument favoring the belief in a tuberculous origin of such growths, as eosinophile cells are so frequently found in atypical tuberculous lesions. The lympho- cytes are either diffusely distributed or the normal follicles may be preserved for a long period. In the early stages or in less active growths the lymph paths are preserved, but later are obliterated. The reticular tissue is frequently less abundant than in the normal node ; later a diffuse fibrosis occurs and the proportion of cells diminishes. The varying proportions of cells and connective tissue yield soft and cellular or hard and fibrous growths. The latter is probably a later stage of the former condition, but all stages are com- monly seen in different nodes of the same subject, and dense nodes are said to have been replaced by softer ones (Gowers). There is little tendency in these structures toward caseation, or suppuration, or hyaline changes of chronic tuberculosis. The lymph nodes of pseudoleukemia may not differ in microscopic structure from those of lymphatic leukemia in any known particular. Nevertheless, they commonly show more of the signs of chronic inflammation and less of the distinctly neoplastic features. In mye- locytic leukemia the lymph nodes commonly show a diffuse growth of larger mononuclear cells. According to Birch-Hirschfeld 1 the nodes of leukemia can be injected through afferent vessels and the fluid will pass through into the efferent vessels, while in pseudo- leukemia the injection is imperfect and fluid fails to pass through. Attempts to demonstrate lymph vessels about pseudoleukemia nodes have often failed (Billroth), but sometimes they are found distended with lymph. The occlusion of lymph paths may prevent the afflux into the general circulation of cells from lymph nodes, but it still remains an obscure fact that the very abundant and unusually diffuse lymphoid deposits in the viscera fail to cause lymphocytosis. Relation to Lymphosarcoma. In many cases reported as pseudo- leukemia, especially of more acute type, the lymph nodes in the group are fused together, capsules and surrounding structures are infiltrated and destroyed, different chains are united by a continuous growth, and the process has all the gross characters of a malignant tumor. Such nodes show no traces of sinuses or follicles, but are com- posed of a diffuse growth of cells usually larger than lymphocytes, PSEUDOLEUKEMIA. 257 often containing giant cells and sometimes masses of fusiform cells. In some cases with more or less involvement of pre-existing lymph nodes multiple secondary growths of very wide distribution occur in situations where distinct collections of lymphoid cells do not normally exist. Thus the. serous membranes, pleura, and peritoneum may be found studded with myriads of fine nodules of lymphoid tissue. In such cases the local original growth may or may not exhibit distinpt sarcomatous characters. There appear to be all transitional forms between the chronic lympho- mata and the rapidly growing, infiltrating, giant-celled lymphosarco- mata. In at least one case it has been possible to observe the transformation of pseudoleukemia into sarcoma of lymph nodes (Eisen- menger). Moreover, of the cases of pseudoleukemia which have passed into leukemia, some have shown the histological structure of giant-celled lymphosarcoma. There remains as a separate class of sarcoma of lymph nodes the spindle-celled sarcomata or endothelio- mata, which arise from the reticular stroma. Two views are possible regarding the classification of such growths. One may enlarge the scope of the term pseudoleukemia to include lymphosarcoma, or may throw out such cases of lymphosarcoma, and limit the application of the term to the lymphadenomata. In the present state of our knowledge it seems unwise to rob the terms Hodgkin's disease and pseudoleukemia of their specific meaning by including with the cases which resemble lymphatic leukemia those which possess all the characters of malignant tumors. It is admis- sible and convenient to limit the term pseudoleukemia, as is com- monly done, to cases exhibiting the following characteristics : general similarity to lymphemia without leucocytosis, considerable involve- ment of various chains of lymph nodes, in which the separate nodes long retain their identity, absence of involvement of intervening tissue between affected chains, development of secondary growths principally in pre-existing lymphoid structures, histological structure indicating slight malignancy of an inflammatory origin. Spleen. The spleen is always enlarged, usually to a very consid- erable size. Microscopically the lesion is found to consist in hyper- plasia of the lymphocytes in the Malpighian bodies with more or less obliteration of their outlines, in some increase in the cells of the pulp cords, and in hyperplasia and exfoliation of the endothelial cells resulting from chronic congestion and inflammation. While in some instances the enlargement of the Malpighian bodies produces mul- tiple tumor-like growths, more often these bodies are but slightly enlarged, and are not prominent. The early cellular stages of the splenic lesion may later be replaced by a diffuse deposit of fine fibrils of connective tissue running out from the Malpighian bodies, and involving the pulp cords. Marrow. The hyperplasia of the lymphocytes in the marrow is usually not marked. The changes usually consist in moderate excess of lymphocytes, swelling, hyperplasia, and exfoliation of endothelial cells, and corresponding loss of the normal cells of the marrow. In some cases there are added the changes of secondary pernicious anemia . 17 258 SPECIAL PATHOLOGY OF THE BLOOD. Acute Pseudoleukemia. Various acute and fatal processes of somewhat uncertain nature have been classed with pseudoleukemia. 1. That pseudoleukemia may run an acute course appears from the reports of Cohnheim, Eberth, and Falkenthal, in which the dis- ease lasted from eleven days to four and one-half months, and in which microscopic examination showed the presence of lymphadeno- matous hyperplasia of the nodes and metastatic growths in the viscera. These cases occurred in children under fifteen years. 2. Chronic pseudoleukemia may suddenly assume a grave char- acter and prove rapidly fatal from the ulceration of enlarged lymph follicles in the intestines, as first shown by Berthenson's cases, and now not infrequently verified at the autopsy table. Usually such fulminant features in the disease are seen in the early stages of cases, which later subside and pursue a chronic course ; or in latent processes which have long existed without attracting attention ; while in other cases the acute course is referable to secondary infections grafted on the original process. 3. Acute lymphosarcoma may run a rapidly fatal course and lead to changes in the viscera, which are very similar to those of pseudo- leukemia. Fagge collected several cases to illustrate this fact, but their real nature is not always apparent, and the microscopic reports were meagre. They were all attended with purpuric symptoms. As already stated, these cases cannot properly be included in the class of pseudoleukemia. The most rapid case of lymphosarcoma that the writer has seen lasted four months. The nodes showed a diffuse growth of mononuclear cells much larger than lymphocytes and a few giant cells. There was prompt recurrence of the tumor in loco, rapid metastases, marked anemia, and no leucocytosis. Much more rapidly fatal cases have been observed, while Birch-Hirschfeld's case of Hodgkin's disease following typhoid fever was fatal in six weeks. Relation of Pseudoleukemia to Leukemia. The similarity of the process in the lymphoid tissues in pseudoleukemia and leukemia, is strongly indicated by the histological features already described. The process in pseudoleukemia differs from that in tymphemia in its constant origin in the lymph nodes, whereas lymphemia originates in or constantly involves the marrow. In pseudoleukemia the lymph paths are occluded and the growth is more circumscribed, although usually producing larger tumors, while the signs of an inflammatory process are often distinct in one, while commonly lacking in the other. Pseudoleukemia may lead to the same changes in the marrow as are seen in lymphatic leukemia (Perrin, Schulz, Dyrenfuth, Kelch, Ponfick, 1 Schmuziger, Baumgarten). The transformation of pseudoleukemia into leukemia has been re- ported in several cases Most of these on examination prove to be of uncertain nature, or were undoubtedly instances of terminal or moderate temporary leucocytosis in the course of some disease of the lymph nodes. Mosler reports an acute case in a child, aged fourteen years, whose blood a few weeks after the onset of the disease showed no leucocytosis, while about three weeks before death the white cells were as numerous as the red. The PSEUDOLEUKEMIA. 259. varieties of cells were not stated. Senator has reported a case of transforma- tion of v. Jaksch's anemia into leukemia. The case of Fleischer and Pen- zoldt is the most significant. Their patient was a male, aged forty years, who suffered from enlargement of many lymph nodes for sixteen months before death. Four months after the beginning of the illness there was no leucocy- tosis, but after twelve months there was one lymphocyte to nine red cells.. The spleen was moderately enlarged, the lymph nodes extremely large ; there was diffuse lymphoid infiltration of the liver, but the marrow of the femur was normal. The microscopic structure of the nodes was not reported, while the absence of a characteristic lesion in the marrow speaks against leu- kemia. Wende reports a case of pseudoleukemia beginning with multiple tumors of skin and lymph nodes with terminal lymphocytosis of 45,000 cells, 96 per cent, of lymphocytes appearing after the fourth month of the disease and one month before death. The day before death the leucocytes fell to 600. The marrow was unchanged. It thus appears that the statement that pseudoleukemia may pass into leukemia rests upon rather uncertain observations. The more recent case of Posselt is an apparently genuine example of the same transformation, but the clinical history is meagre. The remarkable case of Lucke's of lymphosarcoma with rupture into a vein and development of lymphatic leukemia is also of interest in this con- nection. One must, therefore, accept with considerable reserve the opinion that some forms of pseudoleukemia represent an aleukemic preliminary stage of leukemia. The vast majority of cases of pseudo- leukemia pursue a chronic course showing no tendency to develop leukemia, and recent experience does not support Rothe's belief, that early pleurisy and pneumonia carry off many cases of pseudoleuk- emia before the leukemic stage has had time to appear. Eisen- menger reports a case of pseudoleukemia which, after pursuing the ordinary course for four years, developed malignant characters infil- trating surrounding tissues and perforating the larynx. The pres- ence of 90,000 leucocytes in this case would have led many observers to suggest the presence of lymphatic leukemia as well. Relation of Pseudoleukemia to Pernicious Anemia. Euneberg reported a case showing pernicious anemia, which he regarded as an example of purely myelogenous variety of pseudoleukemia, on account of the lymphoid changes in the marrow. The transforma- tion of pernicious anemia into pseudoleukemia was claimed to have occurred in a case described by Laache, and there can be no doubt that the marrow in some cases of pernicious anemia greatly resembles that of pseudoleukemia, as first noted by Scheby-Buch and Pepper. On evidence of this sort, Sevestre suggested that all cases of perni- cious anemia fall in the class of pseudoleukemia, but this view cannot be supported on either clinical or anatomical grounds. Relation of Pseudoleukemia to Tuberculosis. That cases of tuberculous lymphadenitis cannot always be distinguished clinically from pseudoleukemia was early recognized, and so commonly ob- served that the scope of the disease was by some widened to include such cases. Many of the cases of so-called chronic intermittent fever with swelling of lymph nodes described by Pel, Ebstein, 1 Renvers, and many others, probably belong in this class, as shown by Combe- male and Musser. 260 SPECIAL PATHOLOGY OF THE BLOOD. The exact significance of these cases appears not to have been sus- pected until Askanazy, in 1888, demonstrated tubercle bacilli in the swollen nodes of such a case. In most of the cases in which micro- scopic examination of the nodes was reported, there were distinct evidences of tuberculous inflammation, in the presence of miliary tubercles, or caseous foci, or at least abundant hyaline degeneration, and often there was a general tuberculosis (Delafield), but Waetzoldt's case furnished some nodes, showing pure lymphoid hyperplasia, with only very scanty and minute foci of hyaline material, although con- taining many tubercle bacilli in sections. Finally, Brentano and Tangl have described a chronic case in which, with tuberculous lesions in other regions, the lymph nodes showed no recognizable evidences of tuberculosis, not even minute hyaline areas, nor bacilli in section. Inoculation experiments, however, demonstrated the tuber- culous nature of the process. Numerous observers have recently reported similar cases, while Sternberg demonstrated the tuberculous nature of fifteen of eighteen routine cases. On this evidence, of which further confirmation appears desirable, it is necessary to admit either that tuberculosis of lymph nodes may follow the most typical course of acute or chronic pseudoleukemia or that tuberculosis and pseudoleukemia may coexist. It is evident, also that microscopic examination of lymph nodes is not a sufficient test of the tuberculous nature of lymphoma ta, and that the entire subject requires readjustment on the lines indicated above. That all cases of pseudoleukemia are not tuberculous, however, is indicated by the negative results of inoculation obtained by West- phal, Sciola and Carta, Fischer, Reed, and others, but it must be remembered that the inoculation of moderate quantities of tubercu- lous material in guinea-pigs is not always followed by tuberculosis. Miscellaneous Infectious Origin of Pseudoleukemia. Acute or chronic, non-tuberculous, inflammatory hyperplasia of lymph nodes may reach such extreme degree as to resemble the condition found in pseudoleukemia. The reported cases of this character are numer- ous, and have arisen from a great variety of infections, as from carious teeth (Ebstein, 2 Stengel), quinsy (Ponfick 2 ), ulcerative pharyn- gitis (Chvostek), typhoid fever (Birch-Hirschfeld 2 ). Other cases have been referred to otitis media, chancroid, eczema, etc. The Writer has seen more extensive lymphoid hyperplasia in the intestine in typhoid fever than existed in two cases of pseudoleukemia dying with intestinal ulcerations. Microscopic examination, where reported, has shown that the hyperplasia of nodes is inflammatory in character and not lympho- matous, as in the cases of Lannois and Courmont, while metastatic growths are wanting. Many of these cases have resembled purpura hemorrhagica, as pointed out by Kossler and regarded by him as evidence of infectious character. Lannois, Courmont, and Guiller- met isolated pyogenic cocci in their cases. The occurrence of these febrile forms of the disease and its fre- quent development after many forms of suppurative inflammation, especially of the mucous membranes, early suggested the idea that PSEUDOLEUKEMIA. 261 pseudoleukemia is, in all instances, of infectious origin. This view was reached by Westphal, after a full review of the subject in 1893, and has since been maintained by Barbier, Verdelli, and others. Ver- delli collected fifteen cases of pseudoleukemia, in which the presence of pyogenic organisms was demonstrated in the blood or lymph nodes, viz., staphylococcus pyogenes aureus, seven times ; streptococcus pyogenes, three times ; pneumococcus of Fraenkel, once ; unidentified cocci or bacilli, four times. Barbier refers the failure of suppuration in these cases and the negative results of some inoculations to a diminished virulence of the germ or to rela- tive insusceptibility of the individual. The accumulating evidence points to the possibility of separating from true pseudoleukemia a considerable group of inflammatory hyperplasias of bacterial origin. At the same time it must be admitted that the micro-organisms in some of the above cases were prob- ably the cause of secondary infections only, and there are opposed to these positive results of culture a number of negative reports. That tertiary syphilis of the lymph nodes may occasionally pursue a clinical course indistinguishable from that of chronic pseudoleu- kemia has long been accepted by surgeons, and the existence of a syphilitic element in some cases which yielded to mercury has been pointed out by Lannois and Lemoine and others. Lowenbach's study of these cases indicates that the lesion closely resembles that of the tuberculous variety, with rather greater tendency to fibrosis. "With few exceptions the syphilitic lymphomata have been of moderate size. It may be added that Lowit claims to have found the hemameba leukemice magna in a case of pseudoleukemia. Summary of Etiology and Pathogenesis. It thus appears that the term pseudoleukemia applies to a group of cases showing chronic enlargement of the lymph nodes usually originated by or associated with an infectious agent, but in which the histological examination reveals a multiplication of cells, apparently beyond the limits of inflammatory hyperplasia, in some cases developing into lymphosarcoma, and possibly in rare instances into lymphemia. That the clinical course of the disease may be perfectly simulated by tuberculosis, and that the more or less specific histological changes in the nodes are in the majority of cases induced by the tubercle bacillus, there is no longer good reason to doubt. Such overgrowth of lymphocytes in pronounced degree is exhibited in some other manifestations of tuberculosis, so that no new char- acter of this micro-organism is required to account for its connection with Hodgkin's disease. It seems almost equally certain that other micro-organisms, the common pyogenic bacteria, and syphilis may give rise to the same lesions and history. The essential feature appears to be not a par- ticular micro-organism, but a particular clinical course and histolog- ical lesion. This lesion is an extreme hyperplasia of lymphocytes, which lies in the well-recognized border-land between inflammatory and neoplastic processes. In most cases the inflammatory features are distinct, and a tendency to fibrosis is evident. In others the 262 SPECIAL PATHOLOGY OF THE BLOOD. grade of hyperplasia progresses until it seems to fall within the group of neoplasms. That the lymphatic hyperplasia of typical chronic Hodgkin's disease ever does fall distinctly into the group of true neoplasms may still be doubted. It is possible that the advent of a true neoplastic process affecting lymphocytes is revealed at once by the clinical features of lympho- sarcoma or lymphemia, in which case Hodgkin's disease is resolved into lymphosarcoma, on the one hand, and inflammatory hyperplasia, usually tuberculous, on the other. An essential feature, as pointed out by Grawitz, is the general systemic involvement giving rise to multiple lesions throughout the lymphatic system. It also appears that the peculiar clinical condition is not, as claimed by Grawitz, without a peculiar pathological basis, but is, on the other hand, char- acterized by a most striking cellular reaction, which has numerous counterparts within the domain of cellular pathology. It may even be claimed that the histological process is more distinctly limitable than is the clinical picture, especially in the cases of lymphosarcoma, which may resemble pseudoleukemia at a time when the histology of the nodes clearly indicates a malignant process. It remains to consider the significance of those cases in which dis- tinct tuberculous lesions are added to the lymphatic hyperplasia. Of these it may be said that the essential feature of pseudoleukemia is present, viz., the excessive hyperplasia of lymphocytes, and that the mere additional presence of the ordinary lesions of tuberculosis is insufficient ground for eliminating these cases from the category of pseudoleukemia, so long as they exhibit the usual clinical and anatomical features of the disease. Nor is it necessary to suppose that tuberculosis is here grafted upon some other process. Usually in such cases, the ordinary tuberculous lesions, if present at all, are coextensive with the simple hyperplastic changes, which is very unlikely to follow a secondary infection by tuberculosis. The existence of numerous transitional forms between frank tuber- culosis of lymph nodes and pseudoleukemia, between pseudoleukemia and lymphosarcoma, and between pseudoleukemia and lymphemia, as well as the increasing number of observations of the coexistence of tuberculosis and lymphemia, suggest that all of these conditions may at times have a common origin. The Changes in the Blood. Red Cells. It is a uniform observation that in the early stages of pseudoleukemia the anemia may be very slight. The red cells frequently number 5,000,000 or more when the nodes are distinctly swollen. With the progress of the lesion there is usually a progres- sive loss of red cells, which, however, is less marked than in corre- sponding stages of 1 eukemia. In fatal cases there Tnarybe-surprismgly little anemia, usually the cells fall below 3,000,000, and occasionally the condition of the blood resembles secondary pernicious anemia. In acute cases the anemia may rapidly increase, and the disease resembles a septic infection or a malignant new-growth. PSEUDOLEUKEMIA. 263 In morphology the red cells usually show the changes of .simple secondary anemia of chronic course. Laache has called attention to the very uniform size of the red cells in the average case. In some of the writer's cases the cells have been very uniformly undersized. Later megalocytes deficient in Hb may appear, but they are seldom numerous, and do not lead to confusion with pernicious anemia. Laache also reports an obscure case regarded as showing the trans- formation of pernicious anemia into pseudoleukemia. Nucleated red cells are usually very scarce, even in late stages, and when present are usually of moderate dimensions. Jawein reports an obscure case of splenic type and afebrile course, in which there were 3840 to 5914 normoblasts. The condition of the blood resembled that of v. Jaksch's anemia, but the patient was an adult. The Hb is in all cases diminished. A low Hb-index is commonly seen in early stages or with slight diminution of red cells, while in advanced cases with marked reduction of red cells the Hb-index is usually higher. These characteristics are common to most secondary anemias. The anemia of the typical case of Hodgkin's disease stands in an intermediate position between simple secondary and pernicious anemia. It is usually much less marked than in corresponding stages of leukemia, and almost never approaches the type of perni- cious anemia. On the other hand, it is usually more marked than the general condition of the patient would lead one to suspect, thus differing from most forms of secondary anemia. As Grawitz has pointed out, the character of the blood changes offers little encour- agement of the tendency to find in this disease a condition related to either pernicious anemia or leukemia. Leucocytes. The number of leucocytes in the blood is consider- ably influenced by the character of the process in the lymph nodes. In the majority of cases the leucocytes are normal or diminished in number, and there is a tendency toward relative lymphocytosis. In many cases, however, the leucocytes are continuously increased, with periods of well-marked leucocytosis. The increase of white cells may be considerable, but does not pass beyond the limits of inflam- matory leucocytosis, 50,000 to 60,000. Exceptions to this rule must be allowed in the cases of pseudoleukemia which appear to have passed into leukemia, while in Eisenmenger's case, which terminated with sarcoma, there were 90,000 leucocytes. A few eosinophiles are commonly seen in afebrile cases, while an occasional myelocyte has sometimes been encountered. The cases showing few white cells the writer has found to be usually the chronic ones of slow progress and without fever, while a high proportion of polynuclear cells, with or without absolute in- crease, usually belongs to the more rapid or the febrile cases. Lim- beck finds that when the polynuclear cells are increased the nodes usually show inflammatory changes, but when lymphocytosis is found the nodes approach more to the sarcomatous type. In lymphosar- coma, also, not simulating pseudoleukemia, the small lymphocytes may be very numerous. The writer has been unable to find any 1264 SPECIAL PATHOLOGY OF THE BLOOD. features in the blood distinguishing between pseudoleukemia and frank chronic tuberculosis of lymph nodes. Diagnosis of Pseudoleukemia. 1. From myelocytic or pro- nounced lymphatic leukemia the diagnosis is readily accomplished by the examination of the blood, demonstrating the absence of char- acteristic leucocytosis. 2. From less pronounced cases of lymphatic leukemia the diag- nosis may not in every instance be possible. Usually the excess of leucocytes in pseudoleukemia affects the polynuclear type, but as the histological structure of the nodes approaches sarcoma the lympho- cytes may be greatly increased. 3. In tuberculosis of lymph nodes the diagnosis may require microscopic examination of the nodes. If tuberculous, these may show (a) distinct tuberculous lesions, miliary tubercles, and cheesy areas ; (6) small areas of hyaline material associated with demon- strable tubercle bacilli ; (c) lymphoid hyperplasia, without any trace of distinct inflammatory changes and without bacilli in demonstrable numbers. Such nodes should be tested by ino.culation. 4. Non-tuberculous inflammatory hyperplasia of lymph nodes usually causes irregular fever, polynuclear leucocytosis, and suppura- tion of the nodes. It is probable that some pyogenic infections of lymph nodes may run the course of subacute pseudoleukemia. Chronic Splenic Anemia. This term is commonly applied to a group of cases characterized by idiopathic enlargement of the spleen and anemia, but without enlargement of the lymph nodes. While the clinical aspects of these cases are rather characteristic, the under- lying pathological conditions have been found so diverse or obscure that many deny that any peculiar condition exists to which such a term can be properly applied. This state of affairs is largely owing to the incomplete investigation of the clinical history and the patho- logical lesion in the spleen. The writer believes that after all recog- nizable and well-understood conditions have been separated from this group there remains a set of cases of somewhat peculiar clinical characteristics and with somewhat specific lesion in the spleen to which the name chronic splenic anemia may be given. To reach this conclusion one must separate from the group of cases of large spleen with anemia the following conditions, all of which are clearly distinct from the others. Syphilis of the spleen. Gummata. Large round-celled sarcoma of the spleen. Chronic splenitis of the type of Gaucher (epitheliome primitive). Chronic malarial splenitis. Splenomegaly of anemia infantum (v. Jaksch). Splenomegaly secondary to cirrhosis of the liver. Many writers include under splenic anemia the anemia infantum of v. Jaksch — a practice which is entirely unwarranted seeing that the histology of the viscera, both liver and spleen, and of the blood is quite different in these conditions. Others include under splenic anemia the peculiar condition of the spleen described by Gaucher as epitheliome primitive, although the PSEUDOLEUKEMIA. 265 splenic lesion indicates positively that this condition must stand by itself. There remain for consideration the splenomegaly of Banti's disease, that of marasmic infants suffering from chronic catarrhal gastro- enteritis, and that of certain cases in which the lesion of the spleen is that of chronic hyperplastic splenitis, but with which there is neither gastroenteritis nor cirrhosis. The writer has studied the histological lesion in the spleen in each of these three conditions without finding any essential differences. All show phases of chronic hyperplastic splenitis, with variations only in proportion of cells and diffuse connective tissue While the splenic lesions in these cases do not differ essentially from some stages of the lesion of pseudoleukemia, yet it appears unwise to regard any of these cases as examples of splenic pseudo- leukemia, because none of them develop the lesions in the lymph nodes of that disease while the spleen is often larger than that of pseudoleukemia. The question remains, therefore, to determine the relation between the chronic splenic anemia of adults, young or old, that of Banti's disease, and that of marasmic infants suffering from chronic gastro- enteritis. That all of these conditions are referable to intestinal toxemia has been repeatedly claimed ; and it may be that all represent different stages of essentially the same process. The majority of continental writers accept this view. Until this etiological relation is established and until it is proven that a marasmic infant with splenomegaly may pass through adoles- cence as a case of chronic splenic anemia and die later with the lesion of Banti's disease, it may be just as well to apply the term, of splenic anemia to the supposed intermediate stage of the process. The typical case of splepic anemia does not suffer from gastro- enteritis and seldom gives such a history, while the disease com- monly persists for years without developing the cirrhosis of Banti. A description of the changes in the blood of this particular group of cases is rendered difficult because current reports usually refer to cases of uncertain nature, and include, especially, examples of v. Jaksch's anemia and Banti's disease. In the more limited group described above, the changes in the blood are those of a slowly pro- gressive secondary anemia without leucocytosis and with relative lymphocytosis. The red cells commonly number from 3,000,000 to 4,000,000, sometimes less, very rarely falling to the grade of pernicious anemia. They usually exhibit slight changes in size and moderate poikilocy- tosis. Nucleated red cells are rare, and if present are usually of normal size. The Hb-index is low. Leucocytes are usually dimin- ished in numbers, with relative lymphocytosis, and mast-cells are very scanty. The anemia usually continues for many years without aggravation, and most of the patients die of other diseases, although some develop pernicious anemia, and in some instances the condition is probably the forerunner of Banti's disease. 266 SPECIAL PA T30L OGT OF THE BL OB. This entire subject, especially in its clinical aspects, has been reviewed by Osier, through whose writings an introduction to the extensive literature may be secured. Bibliography. Pseudoleukemia. Askanazy. Ziegler's Beitrage, 1888, Bd. 3, p. 411. Banti. De 1' Anemia splenica, Florence, 1882. Barbier. Gaz. hebdom., 1894, No. 5. Baumgarten. Arbeit, a. d. Path. Instit. Tubingen, 1899. Berthenson. St. Petersburg med. Woch., 1879, p. 101 Biesiadecki. Wien. med. Jahrb., 1876. Billroth. Beitrage z. path. Hist., 1858, p. 158. Birch-Hirschfeld. 'Lehrbuch d. Path., 1895, p. 191. 2 Ziemssen's Handb., Bd. 13, p. 100. Bonfils. Soc. Med. d'Observ., Paris, 1856. Brentano, Tangl. Deut. med. Woch., 1891, p. 588. Bruhl. Arehiv. gen. de Med., 1891, I., II. Chvostek. Cited by Stengel. Cohnheim. Virchow's Arehiv, Bd. 33, p. 451. Combemale. Revue de Med., 1892, p. 540. Cornil, Ranvier. Man. d. Hist. Path., p. 251. Courmont. Arehiv. de M6d. Exper., 1892, No. l/"p. 115. Craigie. Path. Anat., 1828, p. 250. Delafield. N. Y. Med. Record, 1887, I., 425. Demange. These de Paris, 1874. Dietrich. Zeit. f. kl. Chir., Bd. 16, p. 376. Dreschfeld. Deut. med. Woch., 1891, p. 1175. Dyrenfuth. Diss. Breslau, 1882, cited by Grawitz. Eberth. Virchow's Arehiv, Bd. 49, p. 63. Ebstein. 'Berl. klin. Woch., 1887, No. 31. 2 Deut. Arehiv klin. Med., Bd. 44, p. 389. Eisenmenger. Wien. klin. Woch, 1895, p. 505. Falkenthal. Diss. Halle, 1884, cited by Ebstein. Fischer. Arch. f. kl. Chir., Bd. 55, p. 487. Fleischer, Penzoldt. Deut. Arehiv klin. Med., Bd. 26, p. 368. Gaucher. These de Paris, 1882. Gilbert. Traite de Med. (Charcot), II., p. 528. Gillot. These de Paris, 1869. Gilly. These de Paris, 1886. Goldmann. Cent. f. Path., 1892, p. 665. Gowers. Pseudoleukemia (Reynolds' Svstem of Medicine). Griesinger. Berl. klin. Woch., 1866, p." 212. GuiUermet. These, Lyon, 1890. Hochsinger, Schiff. Yiertelj. f. Dermat. u. Syph., 1877. Hodgkin. Med. Chir. Trans., 1832, p. 69. Jawein. Berl. klin. Woch., 1897, No. 33. Joseph. Deut. med. Woch., 1889, Xo. 46. Ranter. Cent. f. Path., 1894, p. 299. Kaposi. Oester. med Jahrb., 1885, p. 129. Kelsch. Bull, de Soc. Anat., 1873. Kossler. Jahrb. d. Wien. Kxankenanstalten, 1893, p. 565. Kundrat. Wien. klin. Woch., 1893, pp. 211, 234. Laache. Die Anaemie, 1883. Lannois. Lyon M6d., 1890, No. 34. Lannois, Lemoine. Rev. de Med., 1887, vol. ii. p. 257. Lowenbach. Arch. f. Derm. u. Syph., Bd. 48, p. 71. Lucke. Virchow's Arehiv, Bd. 35, p. 524. Luzet. These de Paris, 1891. Monod, Terillon. Arehiv. gen. de Med., 1879, vol. ii. pp. 34, 325. Mosler. Virchow's Arehiv, Bd. 114, p. 461. Muller. Berl. klin. Woch., 1861. PSEUDOLEUKEMIA. 267 Murchison. Trans. Path. Soc. London, 1870, p. 372. Musser. Trans. Assoc. Amer. Phys., 1901, p. 638. Osier. Amer. Jour Med. Sci., vol. cxxiv. p. 751. Pel. Berl. klin. Woch., 1885. No. 1; 1887, No. 35. Pepper. Amer. Jour. Med. Sci., 1875, vol. lxx. p. 313. Perrin. Bull, de Soc. Anat., 1861, p. 247. Ponfick. 'Virchow's Archiv, Bd. 50, p. 550. a Cited by Stengel. Posselt. Wien. klin. Woch., 1895, p. 407. Potain. Semaine Med., 1887. Ramond, Picou. Archiv. de M6d. Exper., 1896, p. 168. Reed. Johns Hopk. Hosp. Rep., vol. x. Nos. 3-5. Renvers. Deut. med. Woch., 1888, No. 37. Rothe. Inaug. Diss. Berlin, 1880. Runeberg. Deut. Archiv klin. Med., Bd. 33. Sabrazes. Soc. Anat. et Physiol, de Bordeaux, Feb. 8, 1892. Scheby-Buch. Deut. Archiv klin. Med., Bd. 17. Schulz. Archiv f. Heilkunde, 1874, p. 200. Schmuziger. Archiv f. Heilk., 1876, p. 279. Sciola, Carta. Gaz. d. Osped., No. 5, 1894. Senator. Berl. klin. Woch., 1882, p. 533. Sevestre. Prog. Med., 1877, No. 34. Sharp. Jour, of Anat. and Physiol., vol. xxx. p. 59. Stengel. Twentieth Cent. Pract., vol. ii. p. 445. Sternberg. Zeit. f. Heilk., Bd. 19, p. 21. Strumpet. Archiv f. Heilk., 1876, Bd. 17, p. 547. Trousseau. Clinique Med. Velpeau. Lecons Orales de Clinique, T. III. Verdelli. Archiv. Ital. de klin. Med., 1894, T. 32, 4, p 595 Virchow. Krank. Geschwulste, II. Waetzoldt. Cent. f. inn. Med., 1890, No. 45. Wagner. Deut. Archiv klin. Med., Bd. 38. Wende. Amer. Jour. Med. Sci., vol. cxxii. p. 836. Westphal. Deut. Archiv klin. Med., Bd. 51, p. 83. Wilkes. Guy's Hosp. Rep., 1856, p. 114. Woillcz. Soc. med. d. Hop., 1856. Wunderlich. Archiv f. Physiol. Heilk., 1858, p. 123. CHAPTEE X. ANEMIA INFANTUM PSEUDOLEUKEMIA. SPLENECTOMY. ANEMIA INFANTUM (V. JAKSCH). Historical. In 1889-90 v. Jaksch described a form of infantile anemia clinically resembling leukemia, but failing at autopsy to show the visceral lesions of leukemia. The disease was said to be characterized by grave anemia, high and persistent leucocytosis, marked enlargement of the spleen, slight swelling of the liver and occasionally of the lymph nodes, and was to be distinguished from leukemia by the disproportionate size of the spleen as compared with that of the liver, by the more moderate leucocytosis, by the more favorable prognosis, and by the absence of leukemic infiltration of the viscera. Peculiar forms of grave anemia in infants had previously been described by Italian observers, Cardarelli, Somma and Fede, under the term " infective splenic anemia." Mosler and Senator also had long before recognized and described numerous cases of infantile anemia which they placed in an intermediate position between leukemia and pseudoleukemia. Shortly after v. Jaksch's first reference to this condition Hayem described a case in which the blood contained numerous nucleated red cells, many showing mitotic figures. The leucocytes were chiefly mononuclear, but the eosinophiles were increased. In Luzet's case nucleated red cells were not very numerous, but eosinophile cells were increased and some mitotic leucocytes were seen. Thereafter reports of cases multiplied rapidly, so that in 1892 Monti and Berggrun were able to collect sixteen cases, not including those of Hausse and Loos, and added four of their own. In recent years the knowledge of the condition has not been greatly extended, and opinions regarding its significance are still at variance. Etiology. The typical condition is usually limited to infants and children between one-half and four years of age. The majority of the reported cases have occurred in girls. Rachitis is of undoubted importance in the disease, since it was prominent in sixteen of 'the twenty cases of Monti and Berggrun. In the cases of severe rachitis reported by Hock and Schlesinger 1 the changes in the blood resem- bled those of v. Jaksch's anemia, even when the spleen was not enlarged. Syphilis, chronic intestinal catarrh, and chronic tubercu- losis were found in other cases, collected by Fischl. An infectious origin was held for very similar cases as early as 1880 by Cardarelli, and in 1887 by Somma and Fede. Later Mya ANEMIA INFANTUM PSEUDOLEUKEMIA. 269 and Trambusti, Toeplitz, and Gianturco and Pianese, again main- tained the infectious nature of the malady. Recently Lowit reports finding the " hemameba leukemics magna " in one case. Pathological Anatomy. The changes in the viscera have been described principally by v. Jaksch (Eppinger), Luzet, Baginsky, Audeoud, and Rotch. The spleen is much enlarged, and usually rather firm. Histologi- cally the changes are those of simple hyperplasia of ■ all elements, while the sinuses contain an excessive number of leucocytes. Luzet found some mitotic normoblasts, but in his cases nucleated red cells were not numerous in the blood. Baginsky found many eosinophile cells in the spleen. Audeoud described extensive proliferative changes in the splenic follicles and pulp, gorging of sinuses with leucocytes, and occasional extravasations of blood. The liver, in the majority of cases, has been found moderately enlarged, usually less so than the spleen, and of uniform color and normal consistence. Luzet could not verify the claim that the liver is less affected than in leukemia, while in a case of the writer's the liver was quite as large as in fatal cases of leukemia in young chil- dren. Histologically there is an absence of leukemic infiltration, but Luzet found between the liver cords a considerable number of large cells (15/j. to 25/i in diameter) which he regarded as progenitors of the red blood corpuscles. In a case of the writer's, examined in 1896, within the liver capillaries there were small collections of nucleated red cells and leucocytes, of which some of both were found in mitotic division, but the characters of leukemic infiltration were entirely wanting. The infiltration with these groups of small cells gave much the same appearance as the late fetal liver. In the fetal liver, however, these foci are composed almost exclusively of nucleated red cells. In one of Rotch's cases also a similar condition was described. The lymph nodes were moderately enlarged in twelve of twenty cases, but in no degree comparable to the changes of leukemia. The- marrow was described by Luzet as diffusely reddened and moist, and as showing evidence of excessive multiplication of red cells. The Changes in the Blood. The red cells are always markedly diminished, v. Jaksch's case,, in which they numbered 820,000, showed exceptionally severe anemia, the majority of cases having 1,500,000 to 3,500,000 red cells. The ordinary changes of grave secondary anemia are to be noted in the red cells. Alt and Weiss found poikilocytosis very prominent, but this condition, together with megalocytes and micro- cytes deficient in Hb, are common to other anemias. An excessive abundance of nucleated red cells has been shown to- be one of the most characteristic features of the blood of this condi- tion, in which they may be even more numerous than in leukemia. Yet they are not always present, in which case the disease becomes difficult to distinguish from pseudoleukemia. Luzet, Alt, and "Weiss, 270 SPECIAL PATHOLOGY OF THE BLOOD. and others, have noticed that an unusual number of these cells are found in mitotic division. In well-marked cases the nucleated red cells may all be of normal size for this age, but in the graver stages of anemia megaloblasts reach a considerable proportion or a majority. The usual degenerative changes in the red cells of severe anemia have been described in several of these cases. The leucocytosis is an important characteristic. Usually the white cells number between 20,000 and 50,000, but in an apparently genuine case of Baginsky they varied between 122,000 and 40,000. In more typical instances the uniform persistence of the leucocytosis without marked variations has been somewhat peculiar. Some cases have recovered with diminishing leucocytosis. In most instances in which differential estimates are reported the mononuclear cells have been slightly in the majority. In apparently genuine cases (Rotch, Hock and Schlesinger 2 ) the mononuclear cells, large and small, formed 80 per cent., 84 per cent., and 75 per cent, of a greatly increased number of leucocytes (in one case 116,000). Id other cases the polynuclear cells have been the more numerous. The proportion of eosinophile cells varies. Although considerably increased, up to 6 per cent. (Vickery), they do not reach either the proportions or numbers seen in leukemia. Myelocytes have been noted by Klein and in the more recent reports, but only in small numbers. In one of Vickery's cases 10 per cent, of myelocytes are reported among 22,000 white cells. Usually these cells are not so abundant as to suggest leukemia. Great variations in the size of the neutrophile leucocytes have been described, but cannot be regarded as a special character. The great variety of degenerative changes seen in all the blood cells, espe- cially in the leucocytes, has been very fully described and depicted by Engel. Significance of "v. Jaksch's Anemia." The attempt to deter- mine the true nature of the cases described under this term must be guided by the known characteristics of the blood of infants. The more important of these characters are : (a) The relative lymphocy- tosis ; (b) the more active leucocytosis excited by chemotactic influences ; (c) the tendency to enlargement of the spleen in all chronic anemias of infants; (d) the hyperemia of the marrow associated with rachitis; (e) the facility with which blood formation in infants partially regresses toward the embryonal type. With these disturbing factors in view, it would appear that some of the reported cases of anemia pseudoleukemica could better be ■classed as pernicious anemia by referring the peculiar leucocytosis to the special tendencies of infants' blood. Such a case is that of v. •Jaksch, in which there were 820,000 red cells and 54,660 leucocytes of undetermined varieties^ Other cases, like that of Rotch, with 1,311,500 red cells, 116,500 leucocytes, 80 per cent, lymphocytes, enlarged lymph nodes, no autopsy, etc., might perhaps better stand as lymphatic leukemia with unusual proportion of nucleated red cells. Yet the present tendency is to accept too uncertain evidence in the diagnosis of lymphatic ANEMIA INFANTUM PSEUDOLEUKEMICA. 271 leukemia, and the writer agrees with Rotch in his classification of this case. One of Senator's cases also showed enlargement of lymph nodes and a ratio of 1 to 10 between leucocytes and red cells. Likewise Vickery's case, with 22,000 leucocytes, 35.8 per cent, lymphocytes, 10 per cent, myelocytes, and 6.2 per cent, eosinophiles, and no autopsy, may have been an early stage of myelogenous leu- kemia, but myelemia in infants, even if acute, ought to give more than 22,000 leucocytes and 10 per cent, of myelocytes, while if chronic its characters are usually unequivocal. The real nature of these cases must remain obscure in the absence of repeated and very complete examinations of the blood, and microscopic study of the viscera. Finally, it is possible that many of the cases are to be regarded as grave anemia with leucocytosis of peculiar character. This view is supported by the occasional record of recoveries and of gradual trans- formations into grave anemia with ordinary leucocytosis (Monti, Berggrun). Nevertheless, in spite of the resemblance of many cases to perni- cious anemia, leukemia, or grave anemia with leucocytosis, there appears to be, in the clinical history, in the morphology of the blood, and especially in the condition of the viscera, sufficient ground on which to separate, at least for the present, certain peculiar forms of chronic anemia in children from any of the above conditions. In the clinical history these signs are, chiefly, the chronic course, the frequent association with rachitis, syphilis, or chronic intestinal catarrh, the pronounced enlargement of the spleen, and the usual absence of distinct features of pernicious anemia, leukemia, or of a cause of leucocytosis. In the blood the changes are rarely such as to cause confusion with pernicious anemia, though the condition appears at times to precede pernicious anemia. While the acute cases with many lymphocytes or myelocytes may be indistinguishable from leukemia (one case of Luzet, XXV., having passed slowly into leukemia), in other more numerous instances the peculiar condition of the blood persists unchanged for months and shows no tendency to declare itself as leukemia. In the average case of grave secondary anemia the leucocytosis fails to show the peculiar characters of this condition. The excessive numbers of nucleated red cells and the very active multiplication of these cells and of leucocytes, as indi- cated by the large proportion of mitotic nuclei, and the abundance of myelocytes and eosinophile cells, or of large lymphocytes, are signs not necessarily connected with grave anemia in children, but here constitute a striking and distinctive blood picture. Considering the changes in the viscera, it is found that while the other features of the disease simulate pernicious anemia or leukemia, there are neither the leukemic infiltrations and hyperplasias nor the excessive iron-content of the liver, nor the megaloblastic changes in the marrow, of pernicious anemia. The writer is inclined to believe that the peculiar groups of mitotic red cells and leucocytes found in the hepatic capillaries of the single case which he has had opportunity to examine may indicate 272 SPECIAL PATHOLOGY OF THE BLOOD. the essential nature of the condition. The presence of these cells shows that the liver had resumed or retained its fetal function of blood-cell formation. Similar groups of cells resembling leucocytes have been described by Hotch and similarly interpreted, but without mention of Hb-content or mitotic figures in the cells. Luzet also mentioned the presence of peculiar groups of cells in the liver. If these observations can be verified a specific anatomical condition will have been established for v. Jaksch's anemia, viz., the resumption of the liver under the. influence of grave anemia or toxemia of its fetal function of developing red and possibly also white blood cells. The conditions would then differ from pernicious anemia in the absence of extreme megaloblastic changes in the marrow, and in the extension, rather, of megaloblastic red-cell formation beyond the marrow and into the next most available tissue, which is the liver. On this anatomical basis it would still be impossible to explain the frequent presence of many lymphocytes or myelocytes which, how- ever, may be referred to the coincident affection of lymphoid tissues, especially of the marrow, a tissue which in this disease has as yet received inadequate attention. In a case observed by the writer in 1896 the patient was a markedly rachitic infant of twenty months. The spleen extended three inches below the costal border. The liver was considerably enlarged. The lymph nodes were not enlarged. The red cells numbered 1,820,000, about 10 per cent, of which were pale megalocytes. Nucleated red cells were extremely abundant and usually of normal size. Hb not taken. The leucocytes numbered 48,000, of which 22 per cent, were small lymphocytes, 34 per cent, large lymphocytes, 3 per cent, myelocytes, 38 per cent, polynuclears, 3 per cent, eosinophiles. The spleen was the seat of uniform hyperplasia of pulp cells, while the follicles were much reduced in size and number. Throughout the pulp there were numerous small collections of small cells with compact or mitotic nuclei. Many of these were nucleated red cells. Mitotic leucocytes were not identified. There was beginning increase of connective tissue and the sinuses were often obliterated. Eosinophile cells were very abundant. The liver exhibited numer- ous small intracapillary foci of cells, many of which contained mitotic nuclei. Of these the majority were nucleated red cells, others were much larger and granular. Occasionally they appeared fused together in one cell mass, resem- bling the multinuclear masses of Luzet. These groups of cells resembled the foci of nucleated red cells of the embryonal liver, but were much less numerous. The liver cells were not fatty. The marrow of the femoral shaft was cellular throughout, no fat cells being found in this situation, which, in normal subjects of this age is at least partly fatty. There was general hyperplasia of cellular elements, but the cells were not densely packed as in leukemia. The islands of nucleated red cells were very numerous, these cells being distinctly iu excess. Many mitotic leucocytes with granular protoplasm were identified. The sinuses were obliterated. Eosinophile cells were not overabundant. The condition of the spleen, liver, and marrow indicated that excessive demands were being made upon the blood- forming organs, and that these demands were being met by the marrow, the liver, and probably also by the spleen. Bibliography. Anemia Infantum. Alt, Weiss. Cent. f. med. Wissen., 1892, Nos. 24-25. Audeoud. Revue med. de la Suisse Rom., 1894, p. 507. Baginsky. Archiv f. Kinderheilk., 1892, Bd. 13, p. 304. SPLENECTOMY. 273 Cardarelli. Atti d. primo Cong. Pediatr., Naples, 1891. Engel. Virchow's Arohiv, Bd. 135, p. 369. Fede. Atti d. primo Cong. Pediatr., Naples, 1891. Fischl. Zeit. f. Heilk., 1892, Bd. 13, 277. Gianturco, Pianese. Gaz. d. Clin., Naples, 1892, III., p. 305. Hausse. Diss. Miinchen, 1890. Hayem. Gaz. des. Hop., 1889, No. 30. Hock, Schlesinger. l Beitrage zur Kinderheilk., Wien., 1892. 2 Cent, f klin. Med., 1891. v. Jaksch. Wien. klin. Woch., 1889, Nos. 22-23. Prager med. Woch., 1890, No. 22. Klein. Berl. klin. Woch., 1890, No. 31. Loos. Wien. klin. Woch., 1891, p 26. Lowit. Die Leukaemie als Protozoeninfec. Wiesbaden 1900. Luzet. Archiv. gen de Med., 1891, 1., p. 579. These de Paris, 1891. Monti, Berggrun. Die chron. Anaemie, etc., Leipzig, 1892. Mosler. Cited by Senator. My a, Trambusti. Lo Sperimentale, 1892. Rotch. Pediatrics, Philadelphia, 1897, p 359. Senator. Berl. klin. Woch, 1882, p. 533. Sornma. Allg. Wien. med. Zeit., 1891, Nos. 4-11. Toeplitz. Jahrbuch f . Kinderheilk., Bd. 33, p. 367. Vickery. Med. News, 1897, vol. lxxi. p. 731. SPLENECTOMY. The effects of splenectomy upon man are usually the combined results of severe hemorrhage, or pre-existing anemia, of the loss of the function of the organ, and very often of intravenous infusion prac- tised immediately after the operation. How much of these effects are referable solely to the loss of the organ can better be determined by comparing the blood changes following splenectomy in animals with those observed in the human subject. Effects of Splenectomy in Animals. Mosler was one of the first to study the effects of splenectomy upon healthy dogs. He found a diminution of red cells, persisting for several months, but no changes in the leucocytes. The blood-forming function, then regarded as inherent in thespleen, he believed to have been transferred to the marrow, in which tissue he found changes comparable to those of leukemia. The chief permanent chemical alteration he found to be a loss of iron. Malassez found the oligocythemia to persist only one month, while the Hb remained deficient much longer. Zesas found in rabbits a marked leucocytosis, reaching a maxi- mum in the tenth week, when the red cells begin to diminish. The blood was restored to the normal six months after the operation. Winogradoff found that the red cells in dogs diminish for 200 days, and many megalocytes appear. In the second year smaller cells reappear, and the total number gradually be- comes normal. In two of three dogs there was considerable leucocytosis. Diminution of specific gravity and of Hb was constantly noted. The lymph nodes of the animals were found to be smaller, and the marrow hyperemic. Gibson found the maximum diminution of red cells sixty days after splenec- tomy (three dogs). Pronounced leucocytosis was observed the day after the operation. The restoration of the blood required six months. More recently Kourloff followed the course of the leucocytosis of splenec- tomy, finding in the first year following the operation varying grades of lym- phocytosis, the proportion of these cells rising from 30 up to 60 per cent. The proportion of granular leucocytes fell from 40 or 50 per cent, to 20 per cent., or less. The number of large mononuclear cells did not change, indicating that the spleen cannot be considered as their place of origin. During the second 18 274 SPECIAL PATHOLOGY OF THE BLOOD. year a prominent alteration of the blood was a marked eosinophilia, while in this period the lymphocytes fell to the normal proportion. Emilianoff demonstrated in splenectomized dogs a slight diminution of red cells and an extreme leucocytosis marked by an initial loss of small cells, and a sudden increase of larger (polynuclear ) cells. Vulpius also observed in several rabbits a moderate leucocytosis lasting not longer than nine weeks, and a loss of 20 per cent, in the number of red cells, which were restored within five to six weeks. It has therefore been shown, by the above observers among others, that splenectomy in animals is followed by moderate reduction in red cells lasting from one to twelve months, by relatively greater loss of Hb more slowly restored, and in some cases by the appearance during the first year of megalocytes. Leucocytosis follows the operation, but its extent and duration are very variable. A polynuclear leucocytosis is observed during the first days or weeks, followed by relative or absolute lymphocytosis during the first year, while during the second year distinct eosino- philia may be observed. With these changes in the blood are associated a marked cellular hyperplasia of the marrow, approaching at times that of leukemia, and often also affecting the lymph nodes. In the swollen nodes an excessive number of nucleated red cells have been found by Wino- gradoff, Tizzoni, Gibson, Kourloff, and Grunberg. Splenectomy in Man. The most marked changes in the blood following splenectomy in man are seen in those cases in which the organ has been removed for rupture or idiopathic enlargement. Successful cases are recorded by Czerny, 1 Regnier, Hartley, McBurney, and others. In Czerny's case of idiopathic enlargement (1500 grm.) the Hb fell to 56 per cent., while the red cells were very slightly reduced and reached over 5,000,000 within a month ; but four years later the patient, for reasons not stated, was found to have only 3,300,000 red cells with 85 per cent, of Hb. The leucocytosis was very marked, reaching 70,000 within a week and persisting for at least eight weeks. In Regnier' s case of rupture of spleen with severe hemorrhage the Hb fell to 20 per cent., rising gradually to 80 per cent, in the eighth week. The red cells, falling to 2,500,000, rose to 4,700,000 by the eighth week. An acute leucocytosis, 25,000, appeared soon after the operation. One month later the polynuclear cells had been largely replaced by an equal number of lymphocytes. Nucleated red cells and eosinophiles were scarce. Hartley's case (examined by the writer) was complicated by infu- sion, and possibly by pre-existing malaria. The anemia on the fourth day was profound ; the leucocytosis was extreme, estimated roughly at 75,000 cells, of which 77 per cent, were polynuclear. Many " spleno- cytes " and a moderate number of myelocytes with deficient neutro- phile granules, and a few normoblasts, were present, so that the blood strikingly resembled that of leukemia. At the end of three weeks the young patient's blood had greatly improved, and the SPLENECTOMY. 275 leucocytes were normal. After three months the only abnormality was the presence of a few moderate-sized megalocytes and an apparent deficiency of neutrophile granules. Later examinations, extending over three years, failed to show at any time an absolute in- crease of either lymphocytes or eosinophiles or any persistent anemia. In McBurney's case the writer found the anemia and the resem- blance to leukemic blood even more pronounced. The same prompt improvement followed, and six months later the blood was practically normal. After splenectomy for various other general indications, not includ- ing malaria and leukemia, rather variable results have been noted. In the cases collected by Vulpius, Hartley, and Litten, and reviewed by various authors, the writer finds a general resemblance of the blood changes when reported to those described after experimental splenectomy. The grade of anemia and the period required for the restoration of the blood seem to vary with the general condition of the patient and the circumstances of the operation. Dominici describes a case in a tuberculous subject in whose blood, twelve days after the operation, a very large number of nucleated red cells began to appear, 9800 per c.mm. having been counted at one time. After three weeks they disappeared. In the majority of the cases of splenectomy for wandering spleen and other conditions not seriously affecting the patient's health, the blood was described as normal before and after the operation, but in some the usual reduction of red cells and leucocytosis have been observed. The slight changes normally noted are probably referable to the effects of the laparotomy, as Hartmann and Vaquez found that after every such operation there is slight anemia and leucocytosis. The malarial spleen has been excised by many surgeons with favorable results. Jonnesco, who reports a series of cases, found a prompt increase of red cells after a temporary diminution (500,000 to 2,000,000) and a somewhat persistent leucocytosis (15,000 to 30,000). The operation effected a prompt cure of long-persistent cachexia. Similar favorable results were obtained by Hartley, Vulpius, and others. In leukemia Hagen collected forty-two extirpations of the spleen with four recoveries from the operation, and to these must be added one case of Richardson, reported by Warren, in which the patient recovered while the effect on the blood remained sub judice. One case survived thirteen days, another eight months, and a third, in which the diagnosis must be doubted (Franzolini, 1882), was reported cured. In two genuine cases the operation was followed by steady diminution of red cells and increase of leucocytes (Bardenhauer, Burckhardt). Visceral Changes following Splenectomy. Enlargement of lymph nodes has been observed after splenectomy in man by Czerny, 2 Kocher, Lennander, and Regnier. In Regnier's case the enlarge- ment of the nodes was associated with lymphocytosis. The marrow was examined by Regnier four weeks after the splenectomy and found very hyperemic. There had been marked anemia in this case. 276 SPECIAL PATHOLOGY OF THE BLOOD. Resume. In comparatively healthy subjects splenectomy has often been performed without affecting the blood more than does any other laparotomy. In many graver cases the loss of blood and the shock of the opera- tion give rise to a considerable grade of secondary anemia. The red cells are, in favorable cases, restored in one to three months, but in less favorable cases there may be more persistent anemia. The restoration of Hb seems in some cases to fall behind the improve- ment in cells rather more than in most secondary anemias. The operation is usually followed by considerable polynuclear leucocytosis (15,000 to 50,000), which commonly persists for two to six weeks, but may continue for months, in which case the polynuclear cells may be largely replaced by lymphocytes. Eosinophilia has been observed in a few cases during the second and third years. In traumatic cases suffering from large hemorrhages, splenectomy, especially when complicated by infusion, may lead to very profound anemia, marked by extreme loss of red cells, the presence of many very large, pale, sometimes polychromatic, and dissolving red cells, nucleated red cells, and to a high grade of leucocytosis. Among the leucocytes there may be a considerable proportion of large, pale, mononuclear cells and myelocytes, so that the blood resembles that of acute leukemia. This condition, however, is transitory and the blood may improve rapidly. Leukemia and the amyloid spleen are con- traindications to splenectomy. In other conditions the choice of operation may depend entirely upon the general condition of the patient. Beyond a moderate persistent leucocytosis or lymphocytosis, and possibly a slight delay in the restoration of Hb, there are no specific effects known to follow splenectomy in man. Bibliography. Splenectomy. Bardenhauer. Deut. Zeit. f. Chir., Bd. 45, p. 181. Burckhardt. Archiv f. klin. Chir., Bd. 43, p. 446. Czerny. 1 Cited by Vulpius. 2 Wien. med. Woch., 1879, p. 333. Dominici. Compt. Rend. Soc. Biol., 1898, p. 1193. Emilianoff. Archiv des Sci. Biol., St. Petersburg, Bd. 2, No. 2. Franzolini. Gaz. med. Ital., 1882, Guil. Gibson. Jour, of Anat. and Physiol., 1886, vol. xx. p. 674. Grunberg. Diss. Dorpat, 1891. Hagen. Archiv f . klin. Chir., Bd. 62, p. 188. Hartley. Med. News, 1898, vol. lxxii. p. 417. Hartman, Vaquez. Compt. Rend. Soc. Biol., 1897, p. 126. Jonnesco. Archiv f. sci. Med. (Bucharest), 1897, p. 301. Kocher. Cent. f. Chir., 1889, p. 14. Kourloff. Wratch, 1889, 1892, cited by Ehrlich, Die Anaemie. Lennander. Wien. klin. Woch., 1893, No. 30. Malassez. Gaz. med. de Paris, 1878, p. 317. McBurney. N. Y. Med. Record, vol. liii. p. 601. M osier. Leukemia, 1872. Regnier. Berl. klin. Woch., 1893, p. 181. Tizzoni. Archiv. Ital. de Biol., 1882, T. 1. Vulpius. Beitrage zur klin. Chir., 1894, Bd. 11. Warren. Annals of Surgery, vol. xxxiii. p. 513. Winogiadoff. Cited by Laudenbach, Archiv. de Physiol., 1896, p. 724. Zesas. Langenbeck's Archiv, Bd. 28, p. 157. PART III. THE ACUTE INFECTIOUS DISEASES. INTRODUCTORY SECTION. THE BLOOD IN FEVER. While all observers have agreed that the blood in fever suffers a reduction in red cells, it still remains a matter of doubt whether a febrile process alone actually destroys red cells or only causes them to be unequally distributed in the body. The theory of unequal distribution of red cells in the acute fevers is supported by a variety of observations. Maragliano demonstrated a contraction of arterioles during the height of a febrile process, fol- lowed by dilatation during defervescence, and he was able to verify these results by watching the effects of antipyretics. Breitenstein found an excess of red cells in the livers of heated animals coincident with a deficiency in peripheral vessels, while Naunyn under the same conditions could find no evidence of destruction of red cells. Increase in specific gravity of the blood was demonstrated by Stein, with rising temperature, followed by lowering of gravity in defer- vescence. Reinert believes that excessive loss of fluids diminishes the volume of blood during the height of the fever, while retention of fluids results from lower blood pressure and heart weakness after defervescence. Tumas believes that the blood in fever may be re- duced in bulk as well as in proportion of red cells. In favor of the view that the red cells are destroyed in fever is the demonstration of an excess of potassium (Salkowski) and of hydrobilirubin (Gerhardt, Hoppe-Seyler) in the urine. Yet Bunge has shown that the red cells, which are very rich in potassium, may take up and discharge large amounts of this element without them- selves being destroyed. In pure types of experimental pyrexia Naunyn and others have found no solution of Hb in the. blood serum and no evidences of destruction of red cells. Werhowsky, however, exposed rabbits to a temperature of 38.5° to 40° C. for two to twenty-nine days and found a steady diminution, reaching 30 per cent, of Hb, followed by progressive loss of red cells, moderate leuco- cytosis, and deposits of hemosiderin in the marrow and spleen. Similar indications were obtained long ago by Mobitz in septic animals whose blood at first showed from day to day considerable variations in the red cells, but eventually a permanent loss. 278 THE ACUTE INFECTIOUS DISEASES. These results leave no doubt of the capacity of prolonged fever to destroy red cells, but the length of time required furnishes opportunity for factors other than pyrexia to intervene. Consequently Lowit 1 con- cludes that the real nature of the oligocythemia in the early febrile process is still doubtful. Fever is usually accompanied by marked disturbance of the coagu- lation of the blood, which has sometimes been found increased, at others diminished or entirely inhibited. From the studies of Schmidt and his pupils it has been shown that in septic fever coagulation is diminished at an early stage and increased at another later stage. The variations depend, according to Bojanus, upon changes in the quantity either of fibrinoplastic substances or of fibrin ferment, and are connected with the presence of dissolved Hb in the plasma. In non-febrile pyrexia, insolation, the coagulability of the blood is lost. Changes in Chemical Composition. A progressive loss of albu- mins of the blood in febrile diseases has been noted by many observers, but it is not yet known whether the loss is referable to the pyrexia or to other associated factors. Rather favorable clinical conditions for determining the effects of fever were found by Stejskal in a case of intermittent Ebstein's disease, in which during the febrile intervals he demonstrated a decrease of albumins and diminution of ash in the whole blood, in the serum, and in the red cells. While there is good reason to believe that the less diffusible globulin should suffer less than the albumin, as Gottschalk claims to have found in some instances, yet Limbeck and Pick found that no general rule could apply to variations in the globulin of the blood in infectious diseases. A contrary conclusion also was reached by Emmerich and Tsuboi, who found the globulin of the blood to diminish when rabbits are being immunized against hog-cholera. Resistance of Red Cells in Fever. A diminished resistance of the red cells and increased isotonic tension of the plasma were demonstrated by Maragliano in severe anemia, and by Celli and Guarnieri in various fevers, while the morphological characters which indicate this change in the red cells have been described by Maragliano, Gabritschewsky, and Grawitz. Yet the resistance of the red cells is not found diminished in all fevers and may, as Limbeck 1 has shown, be at times increased. While Hamburger has shown that the resistance of the red cells depends principally upon the osmotic tension of the plasma, Demoor refers the low resistance in fever to the presence in the blood of bacterial toxins and of excess of C0 2 , while Lowit 2 connects it more closely with changes in the vessel walls. Febrile Hydremia. A marked diminution of the albumins of the plasma has been demonstrated in many infectious diseases by Ham- marschlag, Limbeck, 2 Biernacki, 1 Wendelstadt, and others, and has been referred not to increase in the total bulk of water in the blood, but to destruction of albumins in the febrile process. Herz believes further that the febrile hydremia is characterized also by swelling of the red cells with corresponding oligoplasmia, and Limbeck and Steindler found the volume of the serum in three healthy subjects INTB OB UCTOR Y SECTION. 279 to average 72.6 per cent., while in eight acute febrile cases it fell to an average of 54.8 per cent. On the other hand, Pfeiffer denies that the red cells swell in febrile diseases, finding rather that they shrink, and Biernacki denies the existence of any uniform febrile hydremia. The conclusions of each of the above observers appear to be justified by their results, and their conflicting opinions must be referred to technical errors or to particular conditions existing in the subjects of their study. Alkalescence. Abundant sources of acid metabolic products are believed to exist in fever as a result of imperfect oxidation of albu- mins and the formation thereby of fatty and lactic acids, as well as in the development of acids as a result of bacterial growth. Numerous observations, also, by Senator, v. Jaksch, Kraus, Klemperer, and others, have apparently shown by different methods that febrile processes are regularly marked by diminished alkalescence of the blood. Yet Minkowski has from the first pointed out that at the height of the fever the loss of alkalescence is not proportional to the pyrexia. Limbeck and Steindler in a large series of febrile subjects found in both blood and serum variations in alkalescence quite within normal limits. On the other hand, Lowy and Richter, using a special method which they claim to be more reliable than others, find that in a cer- tain period of febrile processes corresponding to the stage of hypo- leucocytosis the blood shows an increase and not a decrease of alka- lescence. The cause of this condition is not yet explained, but Lowy's results have been verified by Lowit, Biernacki, 2 and Strauss. Important contributions to the subject were made by Fodor and Rigler. These observers found that the serum of rabbits infected with anthrax at first shows an increase of alkalescence, but after twenty-four hours a rapid and marked diminution. With rabies the alkalescence diminished from the first. Between the quantity of diphtheria toxin injected and the final loss of alkalinity therefrom they found a parallel. Injections of vaccine increased the alkales- cence for seven to eight days, of diphtheria antitoxin for only forty- eight hours. Since the alkalinity of the serum-ash did not vary with that of the serum they concluded that the property depends on the presence of organic substances. Other important studies of the alkalescence of the blood by Cantani, Calabrese, and others have shown that the important variations in the quality of the blood are dependent less upon fever than upon other obscure chemical processes. Lowift concludes that the alkalescence of the blood may be increased at one time and diminished at another period of an infectious disease, that this property is not dependent in any large measure upon the leuco- cytes, and that its significance is still unexplained. Action of Bacteria and Their Products on the Blood. Many of the phenomena classed among the effects of fever on the blood can be directly traced in part to the action of bacteria and bacterial products which are the exciting causes of the febrile process. Bouchard has demonstrated for the bodies of bacillus pyocyaneus, and Gley and Charrin for their filtered products, a vasoconstricting 280 THE ACUTE INFECTIOUS DISEASES. action, and therefore a tendency to concentrate the blood. An increased flow of lymph and probably a similar concentrating effect upon the blood has been shown to follow the injection into animals of peptone and animal extracts, by Heidenhain ; of tuberculin, pyocyanin, pneumotoxin, by Gartner and Romer. Rapid variations in the gravity of the blood have, in Grawitz's 1 hands, followed the injection of cultures of cholera, diphtheria, and pyogenic organisms. Small doses of toxins of various bacteria were found by Bianchi and Mariotti to increase the isotonic tension of the blood quite beyond normal limits, while large doses of many germs or even small injec- tions of bacillus typhosus had the opposite effect. Specific bacterial products, toxalbumins, bacterioproteins, and ptomains are believed by most observers to be the active agent in the destruction of the albumins of blood and tissues in infectious diseases, acting at times without the accompaniment of fever (Miiller). As a specific effect of the chemotactic action of bacteria and their products upon the white blood cells there is the whole series of phenomena of leucocytosis, the significance of which is considered in another section. Conclusions Regarding the Blood in Fever. From the fore- going brief review it will be seen that the changes in the blood in fever form an extremely complex subject, about which our knowledge is still rather fragmentary. The same phenomena have been encoun- tered in different lines of investigation and attributed to different single factors, though probably referable to many. While some main facts are rather distinctly apparent, other questions must remain undecided until fully adequate technical methods are devised. Decrease or relative increase in the proportion of red cells, but ending always in a loss in their total numbers, must be accepted as accompanying all cases of pyrexia, although requiring some time to become clearly apparent. Coagulability varies in different stages of febrile diseases, but is not clearly connected with the pyrexia as such. The progressive loss of albumin of the blood is probably essentially connected with the febrile process, but occurs in increased degree when the fever is of infectious origin. Febrile hydremia is an accidental condition which may or may not occur as a result of the loss of albumins of the blood. Diminished resistance of red celts occurs in the majority of fevers, and depends on a variety of factors. Variations in alkalinity are frequent and con- siderable in fever, but are not proportional either to the height of the temperature or to the toxic condition of the blood. BlBLIOGKAPHY. Blood in Fever. Bianchi, Mariotti. Wien. med. Presse, 1894, No 36. Biernacki. 1 Zeit. f. klin Med., Bd. 24. 2 Ibid., Bd. 31, p 312. Bojanus. Inaug. Diss. Dorpat, 1881. Bouchard. Lecons sur les Autointoxications, Paris, 1887 Breitenstein. Arohiv f. exper. Path., Bd. 18, p 42 Bunge. Zeit. f. Biol., Bd. 9, p. 129. INTR OD UCTOR Y SECTION. 281 Calabrese. Policlinico, 1896, fasc. 1, 2. Cantani. Cent. f. Bact., Bd. 20, p. 556. Celli, Guarnieri. Fort. d. Med., 1889, No. 14. Demoor. Jour. med. de Bruxelles, 1896, No. 36. Emmerich, Tsuboi. XL Cong. f. inn. Med., 1892, p. 202. Fodor, Rigler. Cent. f. Baot., Bd. 21, p. 134. Gabritschewsky. Archiv f. exper Path., Bd. 28, p. 83. Gartner, Romer. Wien. klin. Woch., 1892, No. 2. Gerhardt. Ueber Hydrobilirubin, Diss. Berlin, 1889. Gley, Charrin. Cent. f. Bact., 1894, p. 688. Gottschalk. Zeit. f . physiol. Chem., Bd. 12, cited by Limbeck. Grawitz. 1 Zeit. f. klin. Med., Bd. 22, p. 411. Hamburger. Virchow's Archiv, Bd. 140, p. 503. Hammarschlag. Zeit. f. klin. Med., Bd. 21, p. 475. Herz. Virchow's Archiv, Bd. 133. Hoppe-Seyler. Virchow's Archiv, Bd. 124, p. 30. v. Jaksch. Zeit. f. klin. Med., Bd. 13. Kraus. Zeit. f. Heilk., Bd. 10. Limbeck. l Klin. Path, des Blutes, 1896, p. 158. 2 Prager med. Woch., 1893, Nos. 12-14. Limbeck, Steindler. Cent. f. inn. Med., 1895, p. 649. Lowit. ' Die Lehre v. Fieber. 2 Ibid., p. 151. 3 Ibid., p. 164. Lowy, Richter. Deut. med. Woch., 1895, No. 33. Maragliano. Berl. klin. Woch., 1887, No. 43. Minkowski. Archiv f. exper. Path., Bd. 19, p. 215. Mobitz. Inaug. Diss. Dorpat, 1883, cited bv L6wit. Muller. Zeit. f. klin. Med., Bd. 16, p. 496. " Naunyn- Archiv f. exper. Path., Bd. 18, p. 81. Pfeiffer. Cent. f. inn. Med., 1895, No. 4. Reinert. Zahlung d. Blutkorp, p. 174. Schmidt. Pfluger's Archiv, Bd. 11, pp. 291, 515. Senator. Untersuch. u. d. fieberhaften Process, Berlin, 1873. Stein. Cent. f. klin. Med., 1892, No. 23. Stejskal. Zeit. f. klin Med., Bd. 42, p. 309. Strav^s. Zeit. f. klin. Med., 1896, Bd. 30, p. 315. Tumas. Deut. Archiv klin. Med., Bd. 41, p. 323. Wendelstadt. Zeit. f. klin. Med., Bd. 25. Werhowsky. Cong. f. inn. Med., 1895, p. 345. CHAPTEK XI. PNEUMONIA. DIPHTHERIA. PNEUMONIA. The gross characters of the blood in pneumonia were accurately- described by Piorry, who noted the prompt and firm clotting due to excess of fibrin, while the distinct orusta phlogistica, most marked about the seventh or eighth days, was the earliest recorded observa- tion concerning the leucocytosis of this disease. Fibrin has since been shown to be abundant in nearly all cases of pneumonia, and very much increased in the great majority. The excess often continues beyond defervescence. In very severe cases without leucocytosis, fibrin is usually deficient (Turk 1 ). The total volume of blood is probably somewhat reduced in severe cases of pneumonia, owing to the concentration which occurs in fever, from cyanosis, and from the loss of cells and plasma in the exudate. Evidences of this concentration have accumulated from several sources, among which are : the increase in specific gravity during the height of the process (Monti, Berggrun), and the persistence of a high pro- portion of red cells and Hb which usually continues until the crisis, after which there is a rapid decline in the quality of the blood. Large exudations into the lungs are believed by Bollinger to reduce the quantity of blood to such an extent that oligemia may usually be noted at autopsy. In 65 per cent, of his cases marked diminution of blood was thus noted at the postmortem examination, a loss which was fully explained by the extent of the exudate, which averaged over 1000 grm. in weight. The Red Cells. . Although destruction of red cells undoubtedly occurs in the disease, as shown by the increased excretion of hydro- bilirubin, and the occasional appearance of jaundice, yet the propor- tion of red cells remains high during the active febrile period, when this destruction is taking place, to diminish only when the tempera- ture begins to fall. This result can be referred only to the concen- trating effects of fever, exudation, and local vasomotor phenomena, and is to be seen in other infectious fevers. In spite of these complicating factors the red cells usually show a slight but steady decline during the course of the fever, as shown by the reports of Sorensen, Boeckman, Halla, Tumas, Sadler, and others. Slight polycythemia (maximum 7,000,000) frequently appears in the reports of Sadler, while Sorensen observed an increase of red cells during the febrile period in one case. The decrease of red cells often occurs suddenly with the crisis, Tumas reporting one case with a fall of 600,000 on the day of defervescence. The diminution con- PNEUMONIA. 283 tinues for a very variable period depending upon somewhat complex factors, but commonly ceases within ten to fourteen days, or in mild cases much sooner. The grade of anemia established is usually not very marked. In many recorded estimates the loss of cells did not exceed the limit of error. A loss of 500,000 to 1,000,000 is not uncommon, while a re- duction of 2,000,000 cells is recorded by several observers. Such changes in the red cells, however, must be interpreted with great caution. There was, for instance, a loss of 2,000,000 cells in one of Sadler's cases, yet no anemia was established, as the blood had been concentrated and the lowest count was 5,100,000. Morphological changes in the red cells are usually slight. Poly- chromasia is seen in the severe cases only. Nucleated red cells were present in seven of Turk's eighteen cases. These were usually normoblasts, occasionally megaloblasts. The cases were all very severe, but only one of the seven was fatal. A similar phenomenon may occur in other severe infections. The Hb suffers considerably more than the red cells, being almost invariably reduced after defervescence, but in the absence of compli- cations seldom falling below 65 per cent. Chemistry. The specific gravity was found to remain normal or to distinctly increase during the febrile period in nine children studied by Monti and Berggrun. 1 The albumins of the blood were slightly diminished, while those of the serum were normal or increased in seven cases reported by v. Jaksch. 1 The toxicity of the blood serum was found much increased in pneu- monia and other infectious diseases by Albu, who locates the poisonous principle in the albumins of the serum. Leucocytes. Leucocytosis appears in the great majority of cases of pneumonia, failing only in very mild attacks and in very severe infections with feeble reaction and bad prognosis, and when some pre- existing conditions have already excited leucocytosis or debilitated the system. In leukemia a complicating pneumonia reduces the leucocytes, while the onset of pneumonia in the course of some infec- tious diseases is not always traceable in the blood. Although the leucocytosis of pneumonia was noted by Virchow, Nasse, and other early writers, its closer study was begun by Sorensen in 1876, and continued by Boeokman, Halla, Tumas, Hayem, and Limbeck. 1 Even Boeckman (1881) gathered from the literature that most febrile diseases are accompanied by leucocytosis, and that typhoid fever and malaria are ex- ceptions to the rule. Halla noted that the leucocytosis is not proportional to the fever, having encountered three cases without leucocytosis, all fatal. Hayem and Limbeck followed the course of the leucocytosis over considerable periods, pointed out relations between the grade of leucocytosis and the severity of the disease, discussed the time and manner of its disappearance, and reported other fatal cases without leucocytosis. Limbeck's study was especially minute and he was able to detect a fall in the leucocytosis some hours before the crisis, and to note the absence of any change during pseudocrises. He drew the important general deduction that leucocytosis precedes and is a part of the inflammatory exudate, and that non- exudative diseases are unaccompanied by leucocytosis. Eieder, in 1892, was 284 THE ACUTE INFECTIOUS DISEASES. probably the first to demonstrate that the leucocytosis of pneumonia is not determined by the height of the fever or the extent of the exudate, but de- pends upon the intensity of the infection and the degree of resistance of the subject, and this view was fully supported shortly afterward in the writer's series of cases. Since Limbeck's study the leucocytosis of pneumonia has been a favorite field of investigation, so that an extensive literature in many languages has accumulated, and many interesting and important details have been added to the knowledge of the subject. Course of the Leucocytosis. Leucocytosis appears very early in the course of pneumonia, simultaneously with the chill, according to Klein ; preceding the exudation, according to Limbeck, and having repeatedly been found on the first day of the disease. In one of the writer's cases 25,000 cells were counted within four hours after the beginning of the first symptom, a sharp chill, while Rieder and Laehr found a marked increase within six hours of the chill. Theoretically the leucocytosis should be preceded by a period of hypoleucocytosis, but this period has never been observed clinically, except in fatal cases with prolonged hypoleucocytosis. The maximum increase is reached usually just before the crisis (Hay em, Klein, Bieganski), but has been observed on the first day of the disease or on succeeding days. When the leucocytes increase slowly they usually diminish slowly, and the disease defervesces by lysis. Peculiar cases of marked severity in which there was no dis- tinct leucocytosis until the temperature began to fall are reported by Bieganski and Turk. During the high febrile movement there is usually little alteration in the leucocytosis, but extension of the lesion to other lobes, or to adjoining serous membranes, may cause irregular rises in the count. Yet in a case in which bronchial breathing passed successively up one side of the chest and down the other, the writer found rather uniform and high leucocytosis. In six cases Lambert found the leucocytes doubled in numbers with the advent of empyema, which sometimes failed to raise the temperature or give physical signs. Empyema caused a greater increase of leucocytes than did extension of the lesion to new lobes, or resolution. A few hours before or after crisis the leucocytes begin to diminish rapidly, sometimes falling from a high to a normal figure within twenty-four hours, after which there are commonly some slight oscilla- tions. During lysis the reduction of leucocytes usually keeps pace with the temperature, but Limbeck's, Laehr' s, and Billings' 1 charts show some marked upward curves of the leucocytes after lysis. Such rises are most often due to complications (Kuhnau 1 ). Several of Lambert's cases showed very pronounced increase of leucocytes (maximum 100 per cent.) as resolution began. In fatal cases there is often a continuous increase (Pick), but some- times the leucocytes are high at first, but steadily diminish as the patient grows worse (Bieganski, Rieder), while in many fatal cases there is no leucocytosis. Hayem believed that in fatal cases without leucocytosis the lesion is " less exudative," but subsequent reports have shown this view to be inapplicable to the majority of cases. PNEUMONIA. 285 Alcoholism, virulent infection, and old age are more important factors. In four such cases of the writer the lesion was of the usual type. In one of these, although the exudate was considerable, the marrow of the ribs and vertebras failed to show the usual hyper- plasia. Pseudocrises usually have no effect upon the leucocytosis (Limbeck, Klein, Laehr, Bieganski), but Turk and others have shown that this is not an invariable rule. Fib. 33. 17000 106° 1 1 I 1 ,, 1 _r ft f lsdoo 1 i \ n A \ n n 1 1 IT 1 1 15000 i I 1 , t \ / I if t \ / ft n t \ \ H t V , / 1 11000 Tj V 103° , T f T ■ i A \ J ' \ 13000 ^r l \i \ 102° |. \ i ,, v T l ' 12000 i" 1 101° fj ii i t^ 1 1 i 1 . ^A i 1 _i i 1 11000 i 1 100° i 1 1 . i jl 1 1 11 ii 1 l 10000 . r ii i 99° t ii t i. i <• M \ 9000 98° *, A \ / J \ 1 \- \ 8000 \ \ 97' \ > \ \ / \ / I / \ 7000 - t l-H 90° Temperature. Leucocytosis. Course of leucocytosis in pneumonia. (After Laehr.) The grade of leucocytosis is usually considerable, and is affected by several factors. Hay em found in mild cases 6000 to 12,000 leuco- cytes, in moderately severe cases 18,000 to 20,000, and in very severe cases a maximum of 36,000. In children the increase is usually greater than in adults (Monti, Berggrun, Felsenthal). Severe un- complicated cases often show 50,000 cells, while some writers have found purulent complications with many of the high leucocytoses, especially when the temperature is relatively low (Smith). The highest figures appear to be 115,000, recorded by Laehr, in a peculiar 286 THE ACUTE INFECTIOUS DISEASES. case with delayed resolution. The fever and the leucocytosis often run parallel, but there are numerous individual exceptions to this general rule, the most significant of which are the fatal cases, show- ing normal or diminished leucocytes. The extent of the exudate has considerably more influence upon the leucocytes, as first shown by Limbeck. Although contrary conclusions have been reached by many writers reviewing short series of cases, it nevertheless remains true that the leucocytosis in general bears a rather close relation to the extent of the exudate. In the writer's cases, especially in those which came to autopsy, this rule was readily verified, although its application to individual cases proved unsafe. Thus, in sixty-three cases in which one lobe was affected the average number of leuco- cytes was 20,000 ; in twenty-four cases with two lobes involved, 22,700 ; in twelve cases with three lobes, 25,000 ; in one case with four lobes, 27,000, and in one in which there was bronchial breath- ing over the entire back of chest, 32,000. In ten cases with lesions extending to the pleura (empyema), pericardium, and peritoneum the average was 17,000 — a tendency toward hypoleucocytosis beginning to appear. The degree of systemic reaction to the disease chiefly determines the grade of leucocytosis. This view, first clearly stated by Bieder, has been fully verified by many observers, and embodies the true mean- ing of leucocytosis in infectious diseases. In forty-seven cases marked by vigorous systemic reaction (temperature 105°, full pulse, sthenic condition), the writer found an average of 30,000 leucocytes ; in twenty-seven moderate cases (temperature below 105°, symptoms less severe), the average was 20,000 ; in twenty-seven cases with deficient reaction, 9000, while in twelve asthenic cases the leucocytes were subnormal. The significance of hypoleucocytosis, observed in many fatal cases, has been demonstrated in various experimental infections like those studied by Tschis- towitch, who found that rabbits die without leucocytosis when inoculated with virulent cultures of the pneumococcus, but with attenuated cultures there is leucocytosis and the animals usually recover. Types of Leucocytes in Pneumonia. At the height of nearly all well-marked leucocytoses in pneumonia the polynuclear neutrophile cells form 80 to 95 per cent, of the cells. Turk counted as high as 96.5 per cent, of these cells, and they are frequently above 90 per cent. This high proportion may be seen when the leucocytosis is slight or absent, but is usually most marked when the white cells are very numerous. At the same time there is a marked reduction, relative or often absolute, of lymphocytes to 2 to 4 per cent. The large mononuclear cells usually persist in considerable numbers (Turk), may sometimes be distinctly increased (Klein), and are never entirely absent (Jez). The eosinophile cells at the height of the leucocytosis are always very scanty and frequently cannot be found after very prolonged search (Zappert, Turk). Becker, being unable to find any eosino- phile cells in fatal cases, concluded that their complete disappearance is an unfavorable sign. PNEUMONIA. 287 Instead of polynuclear leucocytosis there may be well-marked lymphocytosis, as in a case reported by Cabot, in which in a child aged six years there were 66 per cent, of lymphocytes among 94,600 white cells. Stienon refers to similar cases, and the writer and others have observed such inflammatory lymphocytoses in diphtheria. During defervescence the polynuclear cells diminish rapidly, usually to a high normal proportion, at which they are apt to remain several days. The lymphocytes at the same time increase in proportion, and often in numbers, reaching their normal figures also after a few days. Fig. 34. l'-2 13 1- 15 1 If li i 1 ) a ) % sk j a i 4 1 ; 1 1 i \ -f \ \ ; i , f- / \ ... \ / ] 1 \ \ 1 ■ < - 1 A 1 fl \y /A / V A V \ -f / 1(1"° v \ / i -\ v -\ ,' \ T 1 ■ i ' A l\ /\ \ / A \ / \ A \{ \ \ Vi '-4 — }x v 1 / v — f \ I ■ t \ \/ \ u V \ • V i \ N N Temperature. Leucocytosis. Precrltical leucocytosis in pneumonia. (After Limbeck.) The large mononuclear leucocytes are usually overabundant at this stage, reaching 1 6 per cent, in one of Turk's cases, and constituting the " large-celled lymphocytosis " described by Klein at this period. Eosinophile cells usually reappear in scanty numbers on the day before defervescence, or rarely a day earlier (Turk). Distinct post- critical eosinophilia occurs in a moderate proportion of cases (Zappert, Bieganski), but not in all (Turk). In severe cases without leucocytosis the polynuclear cells are usually relatively increased (Rieder), but may be normal (Billings). 288 THE ACUTE INFECTIOUS DISEASES. Very often in such cases there is a moderate leucocytosis during or after defervescence (Laehr). Degenerative Changes in Leucocytes in Pneumonia. Klein described numerous " leucocyte shadows" in severe pneumonia. They are seen in other infectious diseases, especially diphtheria, and are to be classed with the degenerative changes in tissue cells which mark the status infectiosus. Glycogen granules in the leucocytes have been found in considerable abundance by Livierato and others, usually in proportion to the severity of the disease and height of the leucocytosis. Myelocytes were found by Turk in nearly all cases. They were most abundant about the time of defervescence, and once reached a proportion of 11.9 per cent, among 8800 cells. Peculiar cells (" reizungsformen ") of large or small size, with single or double nuclei, staining densely and with a brownish tinge by the triacid mixture, are described by Turk as of frequent occurrence, and as resulting from abnormal stimulation of blood-forming tissues. Applications in Diagnosis and Prognosis. The very numerous situa- tions in which the examination of the blood is of great value in the diagnosis of pneumonia have been pointed out by numerous writers. It is especially in such acute uncomplicated infections that the results of examination of the blood are to be relied upon, but even here a word of warning is needed against too implicit confidence in this or any other isolated clinical sign, while it is especially unwise to transfer to other fields in blood analysis the rather positive rules which may usually be applied to the changes in the leucocytes in pneumonia. Barring mild cases, and those fatal ones in which there is no increase of white cells, leucocytosis is an invariable accompaniment of pneumonia, and its absence is, therefore, very strong negative evidence against pneumonia. When present, leucocytosis excludes, with somewhat less certainty, a considerable class of diseases which are not usually accompanied by leucocytosis. These are principally typhoid and typhus fevers, malaria, acute tuberculous pneumonia, uncomplicated influenza, and the catarrhal pneumonia of influenza (Rieder). In prognosis a slight leucocytosis indicates a mild case, while in severe cases a low proportion of leucocytes is an extremely unfavor- able prognostic sign. This fact was first pointed out by Kikodse and v. Jaksch, although such cases had previously been reported by Halla and Hayem. Yet absence of leucocytosis appears not to be so unfavorable as was at first supposed. Out of fifty-seven severe cases without leucocytosis, reported by Halla, Hayem, 2 Laehr, Ewing, Sadler, Bieganski, Zappert, Turk, Billings, Cabot, only forty-four died, although all of them were unusually severe cases. Lambert points out that some severe cases may show absence of leucocytosis in the first examination, but later a well-marked increase, whereas a continuously low or decreasing number of white cells, as reported in three of his patients, is probably seen only in fatal cases. The persistence of leucocytosis may be of value in distinguishing pseudocrises, but Bieganski, Turk, and others have seen the leuco- cytes fall during a pseudocrisis. Continuous or increasing leucocy- PLATE XII. Glycogenic Degeneration of Leucocytes. (Iodine and Potassium Iodide in Mucilage of Acacia.) Fig. 1, Polynuclear leucocyte showing early stage of iodine reaction. Fig. 2. Mononuclear and polynuclear cells with circumscribed areas containing glycogen. Fig. 3. Polynuclear leucocyte in advanced stage of glycogenic degeneration. From a case of purulent peritonitis. Fig. 4. Large isolated granules of glycogen in a polynuclear leucocyte. Fig. 5. Forms of extracellular glycogen. PNEUMONIA. 289 tosis after defervescence often indicates a relapse or complication. Ine reappearance of eosinophile cells indicates that the lesion has passed its acme. Bacteriology of the Blood in Pneumonia. That infection with the pneumococcus may often take the form of a general blood infec- tion is indicated by the clinical course of virulent infections in man and animals and by the frequent occurrence of the micro-organism in the blood of the cadaver. Similar indications are found in the report by Netter and Levy of finding the pneumococcus in the blood of a dead fetus whose mother had died of septicemia, and by the discovery of the same germ in the milk of a nursing woman who was attacked by pneumonia (Bozzolo). Numerous bacteriological studies of the blood during life have shown also that in a moderate proportion of severe cases the pneu- mococcus may be obtained in culture by the usual procedure. Sittmann obtained the largest proportion of positive results, 4 out of 16 cases examined, three of which were fatal and gave signs during life of general sep- ticemia. Similar cases of pneumococcus septicemia are reported by Belfanti, Netter, Marchiafava and Bignami, Goldscheider, Grawitz, Bacciochi, and others, and it has become evident that in this group of infections the pneu- mococcus is usually to be found in the blood during life. Other successful re- sults were obtained only a few hours before death from pneumonia, and the pres- ence of the pneumococcus in the blood in these cases must be regarded as an ante-mortem invasion (Boulay). As regards the bacteriological condition of the blood in ordinary uncomplicated lobar pneumonia evidence has steadily increased which indicates that this disease is often a form of bacteremia, but the question cannot yet be regarded as settled. Most of the early observers were unable to isolate the pneumococcus from the circu- lating blood in any considerable proportion of cases. The following table represents the more important work in this field : Bacteriological Studies of Blood in Pneumonia. • Year. No. of cases. Positive results. Negative results. Author. Total. Fatal. Total. Fatal. Sittmann 1894 16 4 3 12 S Kraus, Kuhnau .... 1897 21 2 2 19 Kohn . . . 1897 32 9 7 23 James, Tuttle . 1898 12 12 10 Sello . . . . 1898 48 12 10 27 9 White 1899 19 16 i« 3 Beco .... 1899 49 7 5 42 15 Silvestrini, Sertoli 1900 16 15 1 Prochaska . 1900 50 50 12 Pieraecini . 1900 28 11 17 Landi, Cionini . 1900 27 25 2 Cole ... 1902 30 9 9 21 4 19 290 THE ACUTE INFECTIOUS DISEASES. Prochaska's results were remarkable, as he claims to have found the pneumococcus in all of a series of fifty cases, twelve fatal. He sowed 10 c.c. of blood in distinctly alkaline broth. Sometimes, especially in cases with metastatic inflammations, staphylococcus aureus or streptococcus were found associated with the pneumococcus. Some of his examinations occurred as early as the second day. He naturally claims that bacteremia is invariable in the disease, but he found no relation between the leucocytosis and the grade of bac- teremia and no relation to prognosis. It appears premature to draw positive conclusions from these varying results. Fraenkel claims to have verified Prochaska's con- clusions, but Cole, who sowed more blood than Prochaska, secured rather few positive cultures. None of the recent observers mention the difficulty of distinguishing the pneumococcus from the strepto- coccus. From these studies it may be concluded that when pneumonia leads to metastatic inflammations the pneumococcus is frequently found in the blood ; that in some cases the blood is invaded just before death ; and that in uncomplicated pneumonia the pneumo- coccus is rarely found in the blood during the progress of the lesion, and that when it is present the disease is usually fatal. DIPHTHERIA. Red Cells. The concentration of the blood demonstrated to a slight degree in pneumonia is even more distinct in the highly febrile stages of diphtheria. Grawitz refers the tendency to a lymphogogic action of diphtheria toxin which he has observed clinically and experi- mentally to cause an increase in the specific gravity of the blood. Although Bouchut found an average of 4,300,000 cells in ninety- three cases, a distinct reduction, yet many of his observations were made late in the disease, and individual cases often showed slight polycythemia. Cuffer reported extreme polycythemia, 7,200,000 to 7,800,000 in three cases. Later observers, especially Morse and Billings, have closely followed the changes in the red cells, finding high normal proportions, 5,100,000 to 5,600,000 during the first week. In the second week Morse found even higher numbers, once 6,800,000, while Billings, although finding an average loss of 500,000 cells, encountered some cases with distinct polycythemia. With the fall in temperature slight anemia appears in many cases, but not in all. Bouchut and Billings observed losses of 2,000,000 cells in several cases. Morphological changes in the red cells are not marked, but when anemia develops there is slight polychromasia and the usual deficiency of Hb. Nucleated red cells were noted by Engel. The Hb is slightly diminished in the average case, but may be considerably reduced in cases which have lost many red cells. Billings found an average loss of 10 per cent, in patients not receiving antitoxin. The Leucocytes. The early observers, Bouchut, Cuffer, Pee, Halla, Limbeck, and Rieder, fully established the presence of leuco- DIPHTHERIA. 291 cytosis in diphtheria, but only Bouchut attempted to deduce any rules governing its occurrence. He observed an increase in all cases, usually proportionate to the severity of the disease, but in septicemic cases the increase was less marked and the prognosis fatal. In view of the recent observations of many myelocytes or an extreme propor- tion of lymphocytes in diphtheria, Bouchut's statement that the blood of diphtheria may be " leukemic " is not far from the truth. More detailed studies were contributed later by Gabritschewsky, Morse, Ewing, 2 Billings, 2 Engel, Schlesinger, and File. The leucocytosis of diphtheria resembles in most respects that of pneumonia. It begins very early when the disease develops sud- denly, and reaches its highest point within one or two days, or begins more slowly and increases steadily for several days or until death. It probably begins earlier and reaches its acme sooner in more refrac- tory individuals, with whom the prognosis is comparatively good. The writer observed two cases without leucocytosis until the fourth and sixth days. In one of these the condition was probably refera- ble to prolonged toxic hypoleucocytosis, and the patient died later. Not infrequently the white cells begin to rise only after deferves- cence, while long persistent leucocytosis is also of rather common occurrence. In favorable cases the leucocytes usually diminish steadily after the first few days. In fatal cases the leucocytes may steadily increase or may steadily decrease during the days before the fatal issue, or there may be no leucocytosis. The height of the leucocytosis is usually considerable, 25,000 to 30,000 cells being frequently present in severe cases. The majority of fatal cases at some period show between 25,000 to 50,000 cells, hence the conclusion of Gabritschewsky that a high and progressive leucocytosis is a bad prognostic sign. Mild attacks, especially in adults, may not show any increase. The writer found 72,000 cells in one case with lymphocytosis, while in a peculiar case marked by hemorrhagic eruption Felsenthal counted 148,000 — possibly an agonal hyperleucocytosis. The grade of leucocytosis is little affected by the fever, but in many cases is proportional to the extent and depth of the membrane. It may be distinctly affected by pneumonia, but the writer failed to find any uniform lymphocytosis when the cervical nodes were greatly enlarged, nor did the character of the infection, whether pure or mixed, appear to influence the leucocytes (cf. File). In general, the leucocytosis of diphtheria is less marked than that of pneumonia with equal temperature, and less than that of non-diphtheritic angina of equal extent (File). Types of Leucocytes in Diphtheria. With well-marked leucocytosis the polynuclear neutrophile cells are usually much increased in num- bers and proportions, but they very seldom reach the very high ratios seen in pneumonia, probably on account of the greater tendency of chil- dren to lymphocytosis. Lymphocytes, probably for the same reason, are relatively more abundant. A striking lymphocytosis occurred at the height of the disease in two of the writer's cases, in one of which the lymphocytes, large and small, formed 60 per cent, of 72,000 leucocytes, and in another 62 per cent, of 22,500 cells. In 292 THE ACUTE INFECTIOUS DISEASES. neither was there marked enlargement of cervical lymph nodes. Of quite different significance is the relative lymphocytosis without increase in total numbers which may be seen in the earlier stages of the disease, and was noted by Billings in a fatal case without leuco- cytosis, and by Bieder and File during convalescence. Eosinophils cells are usually reduced in proportions, often persist in low normal ratio, and frequently cannot be found at all. They are relatively more numerous than in pneumonia. Engel made the interesting observation that myelocytes, both neutrophile and eosinophile, are frequently present in the blood of diphtheria, and that when these cells form over 2 per cent, the prog- nosis is very unfavorable. In cases which recovered the myelocytes never formed over 1.5 per cent., while in seven fatal cases they ran from 3.6 per cent, to 14.6 per cent. It should be added that the numbers of myelocytes varied greatly from day to day, and that eight of thirty-two patients died with few or no myelocytes in the blood. Degenerative changes in the leucocytes in diphtheria are often well marked. The ' ' leucocyte shadows " of Klein are seen in all severe infections and may become very numerous in fatal septic cases. Even before the cells become fragmented many of them, principally the polynuclear forms, show deficiency of chromatin and of neutro- phile granules. The writer, examining the blood in 0.6 per cent, salt solution tinged with gentian violet, found a striking deficiency of chromatin in a case in which the leucocytosis disappeared before death. In the other septic and fatal cases also it was plainly appar- ent, although not demonstrable in less severe infections. This change in the leucocytes, which may be seen in other infectious diseases, and especially in acute leukemia, must be classed with the general cellular degenerations which mark the status infectiosus. Its significance in diphtheria has been fully discussed by Gabritschewsky, while File believes that these pale-staining cells are necrotic. The writer believes that, with careful technique, variations in the staining capacity of leucocytes may be made of value in prognosis. An increased acidophile tendency of the neutrophile granules appeared to be demonstrable in some of the writer's fatal cases. Kanthack has also noted a similar change in the staining tendency of the pseudoeosinophile granules in rabbits which had received small injections of microbic poisons. Effects of Antitoxin upon the Blood in Diphtheria. The red cells show no distinct or uniform effects from the injections of anti- toxin, although in some healthy subjects there is a moderate reduc- tion lasting a few hours. On the other hand, the use of antitoxin, by limiting the progress of the infection, tends to prevent further destruction of blood cells. "Within half an hour after the injection of antitoxin, the leucocytes, especially the polynuclear forms, if previously abundant, show a marked diminution, and in most cases, although the leucocytosis returns after twenty-four to forty-eight hours, it seldom reaches its previous grade. DIPHTHERIA. 293 The writer noted the reduction of leucocytes after antitoxin in all but two fatal cases, while Schlesinger found it in all of his exami- nations. It has also been noted by File\ In the writer's cases the loss was from 4000 to 15,000, but Schlesinger observed a loss of 20,000 within seven hours after the injection. In some fatal cases the use of antitoxin is followed immediately by distinct hyperleuco- cytosis (the writer, Gundobin). The most favorable cases show a steady diminution of white cells after the injection, but in many fatal cases this reduction is interrupted after the third or fourth day. In critical cases with many pale-staining leucocytes the writer found an improvement in the staining quality of many polynuclear cells shortly after the injection of antitoxin. In some fatal cases this change could not be detected. CHAPTER XII. THE EXANTHEMATA. VARIOLA. Red Cells. During the febrile period ITayem found very little change in the red cells, while in the stage of suppuration polycy- themia was sometimes to be noted. Immediately after defervescence, when the destruction of blood cells through the septic process was no longer masked by the concentrating influence of fever, the red cells showed a sudden reduction. In a case of confluent smallpox there was a loss of 2,000,000 cells. In a fatal case before death there were 4,600,000 cells, while in another, markedly hemorrhagic, there were only 2,000,000. Pick, from his study of forty-two cases, found very little change in the red cells throughout the entire course of the disease. He failed to find any severe anemia, although many of his cases were hemorrhagic and confluent. In severe cases which recovered the red cells remained normal before the eruption, almost invariably increased during the suppurative stage, even in hemorrhagic cases, and after defervescence or before death were rarely found much below their original figure. The usual condition of the blood after smallpox appears to be, therefore, a mild form of chlorotic anemia, while under some rather unusual conditions this anemia may become severe. Leucocytes. Although leucocytosis was demonstrated in the eruptive stage of variola by Brouardel, Hayem, Halla, and Pee, the detailed knowledge of the subject has been contributed principally in the studies of Pick, and of Courmont and Montagard. Mild cases of varioloid with scanty eruption ran their course, under Pick's observation, with normal or subnormal numbers of leucocytes, but when suppuration occurred the leucocytes were increased, reach- ing in one case 20,000. In nearly all of Pick's cases of variola there was distinct leucocy- tosis beginning with the appearance of vesicles, increasing as the exudate became purulent, and reaching its height when suppuration became most active. In cases which recovered the leucocytes returned slowly to the normal as suppuration subsided. Abscesses frequently interrupted recovery and caused marked exacerbations of the leucocytosis. In seven of eight cases dying of pneumonia there was a rapid and steady decrease of leucocytes, although suppuration continued. The grade of leucocytosis was usually proportionate to the severity of the septic process. THE EXANTHEMATA. 295 Failing to find leukocytosis in the high febrile periods of the early stages of severe cases, Pick concluded that variola uncomplicated does not tend to produce leucocytosis, but that the usual leucocytosis is referable to secondary infection. This conclusion must be doubted. Courmont and Montagard have also contributed an extensive study of the blood of variola, and their results require a modification in some respects of the conclusions of Pick. They observed a mixed leucocytosis with excess of lymphocytes in nearly all cases, beginning in the early stages of the eruption, reaching its acme in the pustular stage, and in uncomplicated cases slowly declining. In uncompli- cated cases the leucocytosis was usually slight, and varied between 10,000 to 20,000, but once reached 33,000 before the development of any pustules. In confluent and fatal cases there were usually more than 20,000 leucocytes. In hemorrhagic cases the leucocytosis was usually more marked, and in two cases reached 39,000 and 41,000 on the fourth day, the day of death. In some fatal cases the leucocytes tended to diminish markedly toward the end of the dis- ease, from 19,700 on the ninth day to 2500 four hours before death. In subjects who had never been vaccinated the leucocytosis was distinctly greater than in vaccinated subjects. The increase affected principally the lymphocytes, the polynuclear cells, with few exceptions, being below normal in percentage, though usually increased in actual numbers. They properly distinguish between pustulation and true secondary suppurative complications, such as boils and abscesses. The former condition did not increase the percentage of polynuclear cells, although the pustules constantly yielded streptococci in cultures. Boils and abscesses, however, usually ran the percentage of poly- nuclear cells above normal. They conclude from these observations that pustulation is not the result of secondary infection of the skin, but is a part of the true variolous process. In a detailed study of the varieties of leucocytes occurring in variola, it appears that the majority of new cells are medium- sized lymphocytes which constitute usually 35 to 45 per cent, of the white cells. The authors attribute great importance to the presence of 5 to 10 per cent, of very large mononuclear non-granular leucocytes in the blood of variola. Neutrophile myelocytes were never absent, occurring in an average of 3 per cent, of all cells, with extremes of 2 to 16 per cent. Marked eosinophilia was observed in one hemor- rhagic case which recovered under serum therapy. On the second day in a fatal case 15,000 white cells were counted, of which 3 per cent, were mast-cells. A few nucleated red cells were always to be found, except in infants, most numerously in hemorrhagic cases. As the diagnostic features of the blood in variola, much reliance was placed upon the presence of lymphocytosis with excess of very large cells and associated with neutrophile myelocytes in moderate proportion. Very similar results were obtained by Weil, who found slight leucocytosis, 6000 to 10,000, in six cases; 6000 to 15,000 in thir- teen cases; 15,000 to 20,000 in nine; 20,000 to 25,000 in three ; 296 THE ACUTE INFECTIOUS DISEASES. 25,000 to 30,000 in three ; 30,000 to 35,000 in one ; above 35,000 in one. The leucocytosis was most marked at the period of vesicula- tion, while with pustnlation the leucocytes increased or diminished or remained stationary. In cases which were fatal without complica- tions there was usually a diminution of leucocytes before death. The increase of large mononuclear cells and myelocytes was characteristic, especially in cases ending in streptococcus septicemia. In the pus- tules he found 10 to 20 per cent, of mononuclear cells and myelo- cytes, and 30 to 60 per cent, of polynuclear cells. While claiming that the pustulation is not due to secondary infection he found that the pustules always contain streptococci, which make their appear- ance very suddenly, as on one day he would find none and on the next enormous numbers. The writer examined the blood in twelve cases of variola at various stages. Distinct leucocytosis was found in only six of the cases, but the low proportion of polynuclear cells was always observed if complications were absent, and once at the height of pustulation only 21 per cent, of these cells were present. A few myelocytes, however, were seen in only five of the cases. It was found, as did Courmont and Montagard, that the low proportion of polynuclear cells exists in the blood when the cutaneous lesions contain pus and streptococci. Until the contagium of variola is discovered and until the relation of the streptococcus to the disease is determined it will probably remain undecided whether the leucocytosis of the disease is referable to the true virus or to the streptococcus. It is certain that leuco- cytosis is absent in the very early stages in many cases of ordinary severity, but it is also true that the streptococcus is often multiply- ing in the cutaneous lesions before any pronounced leucocytosis appears in the blood, and when the peculiar increase of mononuclear cells is prominent. In the marrow of variola Roger, Josue, and Weil found very little reaction in adult cases, corresponding to slight leucocytosis observed clinically. In children with bronchopneumonia leucocytosis was marked and the hyperplasia of the marrow was distinct. In the adult marrow they found an excess of mononuclear cells and diminu- tion of polynuclears. Biological Examinations of the Blood in Variola. In 1887 G. Pfeiffer called attention to the presence in vaccine lymph of small unicellular ameboid bodies, and the following year he found these same bodies in human lymph. These observations were soon confirmed by Eieck, and later by Ogata, who classed the bodies as protozoa, order gregarinidce. Guarnieri, in 1892, believed that he had cultivated the same bodies in the epithelial cells of the rabbit's cornea, and although Ferroni and Massari claimed that Guarnieri's parasites were degenerating nuclear products, some but not all subsequent observers have succeeded in verifying Guarnieri's conclusions. In 1894 Pfeiffer reported the discovery of amebse in the blood of smallpox patients, describing them as free in the plasma, about one-fourth the size of red cells, possessing one or more nuclei, and projecting pseudopodia. Weber and Doehle have described in the blood of measles, scarlet fever, and smallpox, several forms of ameboid bodies which they believe to be develop- mental stages of a parasitic protozoon. THE EXANTHEMATA. 297 In 1897 Eeed found pigmented ameboid bodies in the blood of vaccinated monkeys and children, and in the blood of variola, but he found the same bodies in normal subjects, and he could not demonstrate any nucleus. Kruse, reviewing the evidence in 1897, ascribed great significance to Guar- nieri's bodies in the epithelial cells of the cornea, believing that they represent the first stage in the solution of the etiology of variola, but he did not grant equal importance to the bodies found in the blood. Widal and Bezancon are reported to have found streptococci in the blood of six cases of variola, and Arnaud in two primary hemorrhagic cases. The writer 3 sowed in broth 10 c.c. of blood in five cases of variola of ordinary severity and found all the cultures sterile. Only one of the cases died. Serum therapy in variola has been attempted by Courmont and Montagard by the use of calf serum drawn fifteen days after vaccina- tion. The treatment seemed to lessen complications, while the injections usually caused a diminution of the leucocytes in cases showing leucocytosis. VACCINIA. Billings, in fourteen infants, noted a moderate but distinct poly- nuclear leucocytosis, usually about 15,000, maximum 20,000. Sobotka examined the blood of forty-three children after vaccina- tion, making the following observations : The red cells and Hb showed no constant variations, and usually remained normal. Vaccination always caused a leucocytosis, beginning usually on the third or fourth day after inoculation and gradually falling until the seventh or eighth day, when the leucocytes were frequently sub- normal. From the tenth or twelfth day a secondary leuoocytosis regularly appeared, lasting two to six days, and showing close rela- tion to the activity of the virus and the number of the pustules. The height of the first leucocytosis varied between 12,000 and 23,000; that of the second between 10,000 and 17,500, while in the intermediate period the cells fell as low as 3500. In uncompli- cated cases the lowest figures usually corresponded to the highest temperature, the leucocytosis preceding by several days the local and general manifestations of vaccinia. Similar changes are noted in infants by Enriquez and Sicard, but they failed to detect the intermediate decline observed by Sobotka. In these cases the mononuclear cells numbered 60 to 70 per cent., the polynuclear 30 to 40 per cent., and once there were 3 per cent, of myelocytes. Of twelve cases in adults they found leucocytosis, 10,000 to 12,000, in only four. In seven rabbits vaccination, which proved fatal in two to three weeks, caused well-marked large-celled lymphocytosis. VARICELLA. Engel 2 found 67 per cent, of polynuclear cells, with absence of eosinophiles, in a child at the height of the pustular stage. Three days later, when most of the pustules were healed, the polynuclear cells had fallen to 47 per cent., and the eosinophiles risen to 16 per 298 THE ACUTE INFECTIOUS DISEASES. cent. The total numbers of leucocytes were not stated, but the above proportions indicate a moderate leucocytosis. Nobecourt and Merklen, in fifteen cases, found no change in the red cells and little in the leucocytes. In seven cases there was an excess of large mononuclear cells, in six slight polynuclear leucocy- tosis, maximum 4500. The eosinophiles were nearly always dimin- ished. In five cases there were 1 to 12.5 per cent, of myelocytes. Weil and Descos, from a study of twenty cases, concluded that varicella sometimes yields a slight polynuclear leucocytosis, maximum 13,500, while in thirteen of their cases the leucocytes were within normal limits. They state that the absence of large mononuclear cells and myelocytes will serve to distinguish varicella from variola. Stengel and White, in four cases, found distinct leucocytosis but once, and in a patient suffering from bronchitis. From these somewhat discrepant results it will be seen that the blood in varicella may not differ in any particular from that of mild cases of variola. SCARLET FEVER. Hayem gave the first systematic account of the blood changes in scarlatina, finding a loss of about 1,000,000 red cells after deferves- cence, and a moderate leucocytosis in average cases, much increased by severe angina or by suppurative processes. Somewhat isolated observations were reported by many of the earlier blood analysts, Halla, Pee, Pick, Reinert, Sadler, while considerable series of cases were studied by Rieder and Kotschetkoff. The whole blood suffers in a slight degree the usual effects of fever. The formation of fibrin is usually increased, especially when there are extensive angina or suppurative complications (Hayem). Heubner noted hemoglobinemia in one case, representing the septicemic type of the disease. Red Cells. The gradual loss of red cells noted by Hayem was fully verified by Kotschetkoff, who found a reduction to 3,000,000 or lower in nearly all cases. The regeneration of the blood was slow and complete only after six weeks or longer. On the other hand, Zappert found less than 4,000,000 cells in only one of six cases, and very slight anemia was noted in a considerable number of cases examined at varying periods by Leichenstern, Arnheim, Pee, Pick, Reiner, Sadler, Felsenthal, and Vandenberg. When complications supervene, however, as nephritis or endocarditis, very severe anemia may rapidly develop. In such cases Vandenberg reports as low as 25 per cent, of Hb, 2,000,000 red cells, and specific gravity 1031. In the late stages of fatal cases this severe anemia may be referred to the widespread occurrence of the streptococcus. Leucocytes. Kotschetkoff classified the leucocytoses in three groups, the mild cases, showing between 10,000 and 20,000 white cells, the moderately severe cases, with 20,000 to 30,000 cells, and very severe and usually fatal cases with a leucocytosis of 30,000 to 40,000 cells, while in some rapidly fatal cases over 40,000 leucocytes were found. Yet Rieder's ten observations never gave more than 25,000 cells, THE EXANTHEMATA. 299 and usually less than 20,000, although some of his cases were com- plicated with pneumonia and croupous pharyngitis, and were fatal. Felsenthal's six cases in children were of moderate severity and showed 18,000 to 30,000 cells. The leucocytosis begins one to two days before the appearance of the rash, reaches its height with or shortly after the full development of the eruption, and while in some cases rapidly falling with the erup- tion, usually continues for four to five days longer, and very often persists for days or weeks after the temperature has become normal. Mackie, who found leucocytosis in twenty-five cases, places the period of greatest leucocytosis in mild cases at the fourth day after the appearance of the rash. The grade of leucocytosis seems in general to correspond with the severity of the disease, especially of the angina, but not with the height of the temperature. Complications such as lymphadenitis, otitis, nephritis, usually have little effect on the leucocytes (Kotschet- koff), but Pee observed two cases in which the leucocytosis increased when the lymph nodes began to swell late in the disease. Pneumonia caused but slight increase in some of Rieder's cases. Mackie counted 95,300 cells in a case of septic angina, and noted a steady decrease of white cells in a fatal case. Types of Leucocytes. The percentage of polynuclear cells is in all cases much increased, varying from 85 to 98 per cent., according to the intensity of the disease, reaching the highest point on the second day of the exanthem, and thereafter slowly declining. In fatal cases the proportion of polynuclear cells falls but slightly, or soon regains or passes the original figure. The eosinophils cells may show characteristic variations. In all but very severe eases they are normal or subnormal at first, steadily increase after two to three days, reaching a maximum of 8 to 15 per cent, in the second or third weeks, and thereafter, declining slowly, reach the normal figure about the sixth week. In ten cases Reckzeh found that they began to increase on the first day, reached an average of about 6 per cent, on the seventh to eighth days, thereafter gradually diminishing until the fourteenth day. In • fatal cases they may disappear early in the disease. The lymphocytes are at first dimin- ished, but later rise to normal proportions. The above rules deduced by Kotschetkoff, while probably repre- senting the average case, are not always applicable. Thus Weiss found no eosinophiles in one case at the height of the exanthem. Rille observed marked eosinophilia in a fatal case ; Bensaude observed as high as 20 per cent, of eosinophiles in one instance ; Klein reports lymphocytosis during convalescence; and many writers (Rieder, Turk) have described a high persistent leucocytosis, especially in those cases followed by nephritis or other complications. Turk has called attention to the remarkable change which the leucocytes undergo about the fifth day of the disease, when the polynuclear cells rapidly diminish and eosinophiles and lymphocytes rapidly increase. This " secondary leucocytosis " he likens to the somewhat similar phenomenon seen in variola. 300 THE ACUTE INFECTIOUS DISEASES. Bacteriology. Although the specific contagium of scarlet fever has not been demonstrated, practically all observers who have dealt with the cadavers of such cases have found a coccus widely distributed in the viscera and blood. Many have found in this coccus certain characters which they believe could distinguish their particular micro- organisms from streptococcus pyogenes, while others, dealing appar- ently with the same germ, have been content to class it with the polymorphous streptococcus. Very few of these observers have attempted to demonstrate the presence of the germ in the blood dur- ing life, nearly all reports being concerned with its occurrence in the blood after death. Charlton, however, claims to have isolated a streptococcus from the circulating blood of all those cases, in a series of twenty-five, in which the examination was made during the first five days of the disease. Baginsky and Sommerfeld have collected about all the available evidence which indicates that the only micro-organism constantly present is the streptococcus pyogenes, and that this germ is the specific contagium. Kurtb, d'Espine and Marignac, and Class, Gradwohl, and Page, have endeavored to maintain an independent position for certain cocci which they succeeded in isolating from the throat, skin, and cadaver, occasionally from the circulating blood. Raskin found streptococcus pyogenes in the circulating blood in two of sixty-four cases, while all other cultures were sterile. Negative results were obtained in two cases by Sittmann and in four cases by Kraus. At present it is impossible to decide what is the relation between the different cocci described, what is their relation to the disease, or how frequently they are to be found in the circulating blood. MEASLES. The red cells have been found in the great majority of cases to suffer little or no change, but a loss of Hb is usually demonstrable after defervescence. Reckzeh mentions the occurrence, in very severe cases, of anemia with unusual variations in the size of the cells, with microcytes, macrocytes, and normoblasts. In adults uncomplicated measles seldom causes leucocytosis, but is characterized rather by hypoleucocytosis, reaching in one of Rieder's cases 2700 cells. From 4000 to 6000 cells are commonly seen. This fact was first noted by Pee, and has been confirmed by Pick, Rieder, Rille, Felsenthal, and others. Considerable variations in the behavior of the leucocytes in the first days of measles are apparent in the numerous reports collected by Reckzeh. Normal or slightly reduced numbers of white cells are usually present at the onset of the disease. Combe finds that the decrease begins during the last two days of the period of invasion and becomes more marked during the stage of eruption. Sobotka and Cazal, however, observed hyperleucocytosis before the eruption appeared. THE EXANTHEMATA. 301 At the height of the exanthem they are usually at their lowest figure (Pee, Turk), and return to the normal within a few days or a week after defervescence. When the bronchitis is severe there may be a moderate leucocytosis, Hayem finding 10,000 to 14,000 cells in such cases occurring in children. Rieder observed slight leucocytosis in a case complicated by catarrhal pneumonia. Cabot observed 9000 cells in two cases, one hemorrhagic. The writer found no leucocy- tosis in three cases occurring in malarious subjects. The malarial parasites reappeared, with chills, during convalescence. The proportions of the various leucocytes show no distinctly abnormal changes. Turk found a rather high percentage of poly- nuclear cells during the fever, with diminution of small lymphocytes, and Reckzeh's tables also show a high proportion of polynuclear cells, 62 to 88 per cent., on the third and fourth days. Pee, Klein, Turk, and Combe noted an excess of large mononuclear cells appear- ing with the eruption. The eosinophiles are usually in low normal proportions during the early febrile period, and tend to diminish as the eruption declines. Yet Turk found nearly 5 per cent, during the second week of the disease. Bacteriological examination of the blood was negative in ten cases examined by Barbier. Weber claims to have found in the blood of measles a protozoon, which he has also seen in variola. Applications in Diagnosis. Typical cases of measles and scarlet fever may sometimes be distinguished from each other in their early stages by the examination of the blood. Yet, as Turk says, the blood in measles strongly resembles that of a mild scarlatina, as both show a nearly normal number of leucocytes and normal proportions of eosinophiles. Yet equally severe constitutional disturbance should give, on the second to third days, leucocytosis, if scarlatinal; normal or diminished leucocytes, if from measles. In German measles there was no leucocytosis in two cases men- tioned by Cabot. CHAPTER XIII. TYPHOID FEVER. The whole blood commonly suffers concentration in the early stages of the disease as a result of the febrile process, while at any period profuse diarrhea and repeated hemorrhages considerably reduce the total bulk. This concentration is so marked and constant that the deep-red appearance of the blood drop in typhoid fever is a very characteristic differential sign between this disease and perni- cious malaria. Fibrin formation is usually deficient and may not be demonstrable at all, but with inflammatory complications this element may reap- pear and become abundant (Hayem, Turk). Red Cells. During the febrile period the red cells usually show a slight and gradual decline. Yet the initial concentration of the blood often yields moderate polycythemia during the first two weeks or longer, so that the slight anemia is not to be noticed until the fever and diarrhea subside (Sadler, Felsenthal). Many observers have found over 5,000,000 red cells in individual cases at varying periods of the fever, while Thayer reports nearly 7,000,000 in the first, second, and third weeks. At such times a considerable grade of pre-existing anemia may be obscured by the febrile concentration of the blood. Even when distinct polycythemia does not result the same factors tend to keep up the proportion of red cells, so that in the vast majority of cases of typhoid fever the red cells number more than 4,000,000, and in order to demonstrate an anemia referable to the disease it is necessary to follow the case from the first. That the typhoid toxemia tends nevertheless to produce anemia is clearly shown in the large series of cases reported by Thayer and Da Costa, in which there is each week a progressive loss of red cells and Hb, the weekly loss averaging for the first five weeks 100,000 to 500,000 red cells and 5 to 6 per cent, of Hb. The Hb suffers to a great extent, and in spite of the concentration of the blood most febrile cases register between 60 and 80 per cent, of Hb. Severe diarrhea or large hemorrhages may at any time cause marked oscillations in the red cells and Hb. After and often before defervescence the blood may show distinct anemia, which, with rapid defervescence, may appear suddenly (Arnheim, Zaslein, Boeckman, Laache). During convalescence the anemia slowly disappears, the Hb being restored much later than the red cells. The grade of anemia observed after typhoid fever varies greatly with the character of the disease. Uncomplicated cases may show no distinct reduction of cells and very slight loss of Hb. In a con- siderable proportion of severe cases the red cells fall below 4,000,000 TYPHOID FEVER. 303 and the Hb below 70 per cent. Kohler, in twenty-nine cases, found an average loss of 19.4 per cent, of Hb in men and 20.4 per cent, in women, while the average of red cells for men was 4,030,000 and for women 3,580,000. Thayer's average for the fourth week was 4,200,000 and Da Costa's 3,500,000. Hayem, Thayer, Henry, and others have reported severe grades of pernicious anemia as sequelae of typhoid fever. In one of Henry's cases there were only 804,000 red cells. Morphological changes are usually not marked, but in severe cases Turk found megalocytes, small microcytes, and polychromasia in comparative abundance. Nucleated red cells may appear after hemorrhage. Leucocytes. Numbers. In the first week uncomplicated cases nearly always show a normal number of leucocytes, but severe angina, bronchitis, enteritis, etc., may yield a moderate polynuclear leucocy- tosis. Klein and Aporti regarded this initial leucocytosis as of fre- quent occurrence, but most other observers have failed to encounter it. The writer has seen so many examples of moderate polynuclear leucocytosis in the first week of typhoid fever that he would warn the observer that the behavior of the leucocytes at this time is quite variable, and that complications can seldom be discovered to account for the leucocytosis. In the second week the leucocytes usually show a distinct reduc- tion, especially of the polynuclear forms, but the number of leuco- cytes found at this time is by no means constant. In some cases the lowest figures of the disease are reached in the second week (Turk, Chetagurow), while in others there is no distinct reduction at this time (v. Jaksch, 2 Sadler). In the third and fourth weeks the leucocytes usually continue to diminish until the acme of the disease is reached (Turk), after which they slowly increase. In many cases the lowest figures are reached at this period (Rieder, Turk, Thayer), but sometimes the reduction continues until after defervescence (Klein). The average course of leucocytosis is shown in Thayer's collection of 826 examinations : Examinations. Leucocytes. First week Second " .... Third " Fourth " Fifth " Sixth ■' . . . Seventh " . . Eighth " Ninth " . . Tenth " A relation between the leucocytes and the fever seldom exists, nor does the size of the spleen appear to influence the number or type of the leucocytes (Turk). That the reduction of the white cells is dependent on the action of the typhoid toxin is indicated by the further losses which commonly go with unfavorable turns in the dis- 119 6442 25S 6251 200 5528 117 5431 70 5510 25 5690 14 6132 14 6614 7 5057 2 5000 304 THE ACUTE INFECTIOUS DISEASES. ease (Rieder, Jez, Turk, Nagaeli). It is therefore a general rule that the more severe the typhoid intoxication the lower is the count of leuco- cytes. The reduction is seldom below 2000 cells, but 1000 to 2000 were reported by Hayem, Limbeck and Rieder, Cabot, and Kohler. The majority of cases at some periods show between 4000 and 6000 cells, and many fall below 4000. Leucocytosis during the later course of typhoid fever is by no means uncommon. Da Costa claims that it is present at some period in about 10 per cent, of all cases. Usually a severe diarrhea from extensive ulcers, or hemorrhage, or pneumonia, or other exudative or suppurative complications, may be found to explain the increase. Yet several reported cases with leucocytosis have shown no such complications (Aporti), and a short experience at the bedside seldom fails to bring to light some moderate leucocytoses without apparent cause. Courmont and Barbaroux observed polynuclear leucocytosis (89 per cent.) throughout an uncomplicated fatal relapse, and in several cases at some time during the continuous pyrexia, usually at its acme, there was an increase of polynuclear cells. In grave cases they frequently found a series of elevations of the polynuclear cells, and conclude that the typical course of the leucocytes can be expected only in cases of moderate severity. On the other hand, leucocytosis may fail in the presence of pneumonia or other markedly exudative complications (Turk, the writer, Cabot). Kohler found 6200 and 4200 leucocytes in cases complicated by severe bronchitis. After hemorrhage in one case the white cells remained at 2600 ; in two others there was an increase of about 4000 cells. In eleven cases of hemorrhage Thayer observed a reduction of red cells, and after twenty-four hours a leucocytosis, maximum 24,800, in only half the cases. Complicating pneumonia once reduced the leucocytes from 2600 to 1000; at another time they remained at 6300, and twice 11,200 and 11,800 cells were counted (Kohler). Regarding the effects of the numerous other inflammatory compli- cations of the disease it appears that the occurrence or failure of leucocytosis is quite as uncertain as after pneumonia. Cold baths have long been known to cause temporary massing of leucocytes in peripheral capillaries (Winternitz). In twenty cases Thayer 2 found an average increase of 5346 cells, and a maximum increase of 17,000. With the return of peripheral circulation the excess of cells disappears. Perforation usually causes polynuclear leucocytosis, but there may be no effect upon the leucocytes, or the percentage of polynuclear cells may rise without increase in their total number. One may refer the failure of leucocytosis after perforation to the low condition of the patient or to the discharge into the peritoneum of much septic material, or to violent infection by the streptococcus, as in three cases reported by Thayer. As Russell found, leucocy- tosis in these cases may be due not so much to the perforation as to the subsequent peritonitis, and may, therefore, be delayed until after other signs have become urgent. The leucocytosis when present TYPHOID FEVEB. 305 may be very evanescent, and its presence may in general be regarded as more favorable than its absence. Types of Leucocytes. During the first week of the disease the persistence of a normal proportion of lymphocytes without noticeable increase of neutrophile cells is a very characteristic feature of typhoid blood. Barring the occasional occurrence of initial polynuclear leucocytosis, from the end of the first week there is a progressive increase of the lymphocytes and diminution of the neutrophile cells. At first the lymphocytes do not pass beyond high normal limits, but during the third, fourth, and fifth weeks, or later, they usually occur in distinct excess. While mononuclear cells seldom fall below 25 per cent, at any stage of the disease, a proportion of 40 to 60 per cent, is often seen. Among these cells the proportion of small lymphocytes is often striking, but usually the medium-sized leuco- cytes are most abundant. In some cases there is a very marked absolute lymphocytosis. In one of the writer's cases at Montauk the blood resembled that of lymphatic leukemia, a resemblance rendered still more striking by the enormous size of the mesenteric nodes at autopsy. With the increase of mononuclear cells the num- bers of polynuclear cells gradually fall, reaching 60 to 65 per cent, in most cases and occasionally a much lower figure (35 per cent., Jez ; 20 per cent., Klein). The excess of lymphocytes usually per- sists during the first weeks of convalescence, and may not reach its acme until this time, while Ouskow found that, normal relations were not restored until the tenth or eleventh week. Nagaeli describes the development during convalescence, especially in children, of a well-marked lymphocytosis with a moderate increase of neutrophile cells. In children this lymphocytosis was most marked two or three months after the fever, while in adults it was less marked and dis- appeared by the end of the second month. Eosinophile cells are usually absent or very scarce during the febrile period, but reappear shortly before (Nagaeli), during, or after defervescence. Aporti found as high as 18 per cent, during the intermittent pyrexia at the end of the disease, but they are usually much less abundant. Degenerative changes in the leucocytes occur as usual, Jez reporting large numbers of pale leucocyte shadows in severe cases. Glycogen was found in the leucocytes in increased quantity between the twelfth and twentieth days by Livierato. Applications in Diagnosis. The morphological examination of the blood is often of great assistance in the diagnosis of typhoid fever and the diseases which simulate it. The writer knows of no clearer illustrations of this fact than those which he reported 1 in 1893, and which have been paralleled from numerous later observations and by many writers both before and since. Suppurative processes, if active, may usually, but not always, be distinguished from typhoid fever by the presence of polynuclear leucocytosis. Yet it should be remembered that slow suppuration, or the mere presence of pus the secretion of which has ceased, are frequently unaccompanied by leucocytosis or are even marked by 20 306 THE ACUTE INFECTIOUS DISEASES. relative lymphocytosis. Thus the writer found 50 per cent, of lymphocytes among 11,000 cells in a case of large abscess of the liver with mild typhoidal symptoms, and 45 per cent, of lymphocytes among 7000 cells with the chest full of pus. "With increasing experience, therefore, the writer believes that most careful observers will recognize the partial justice of Grawitz's claim that in difficult cases the blood examination here often fails to be of service. Among the conditions which, by the almost invariable presence of leucocytosis, may be distinguished from typhoid fever, are bacterial endocarditis, suppurative appendicitis, and pneumonia. Malaria of sufficient gravity to simulate typhoid fever can nearly always be distinguished by the anemic appearance of the blood drop as com- pared with the deep red, concentrated blood of typhoid fever. An astonishing change in the blood was often noted at Montauk when typhoid fever developed in subjects of malarial cachexia, and in no instance were malarial parasites found in such concentrated blood. In acute paroxysms the discovery of the parasite is usually possible, but chronic malaria without parasites in the blood may simulate typhoid fever. Between miliary tuberculosis, typhoid fever, and some forms of meningitis, Widal's test is required and the enumeration of leuco- cytes is of little value. From an extensive study of the leucocytes in typhoid fever Nagaeli draws the following conclusions regarding 'prognosis. The prognosis is favorable : (1) When eosinophiles are present at the height of the disease, or reappear in the second or third stages of the febrile period. (2) When lymphocytes begin to increase after the severest toxemia is past. (3) The diminution of neutrophile cells is slight only in more favorable cases. Unfavorable signs are : (1) Very small numbers of all varieties of leucocytes. (2) Failure of leucocytosis with complications. Bacteriological. In the hope of developing a method of early diagnosis of typhoid fever many biological studies of the blood have been undertaken. Aspiration of the spleen has given successful results in the majority of cases (Chan tem esse and Widal, Redtenbacher, Lucatello, Neisser, Bruschettini), but this dangerous procedure cannot be generally adopted. The results of Silvestrini,who found the typhoid bacillus in the aspirated blood of the spleen in four cases in which there were no characteristic intestinal lesions, are most suggestive. In blood drawn from the rose spots, while early attempts were rather unsuccessful, Neuhaus and later observers have been able to isolate the typhoid bacillus in nearly all cases often before the appearance of Widal's reaction. Scholz and Krause recommend that the test be made as soon as possible after the spots appear, since they found that the bacillus disappeared after three to five days. Richardson plants the material curetted from five or six incised spots, as well as the blood which oozes from the incisions. Freezing by ethyl chloride diminishes the pain of the operation. Bacteriological studies of blood in typhoid fever usually gave nega- tive results until Kuhnau reported eleven positive cultures in forty- TYPHOID FEVER. 307 one cases, examining blood drawn from the basilic vein at the acme of the disease. In 1900 Auerbach and Unger pointed out that large dilutions in broth are necessary to annul the bactericidal action of the blood, and they secured positive results in seven of ten cases by planting ten to thirty drops in 300 c.c. of broth. The growth was at first often scanty and the bacilli non-motile and thread-like, but grew abun- dantly in subcultures. Later it was shown that the bacillus is very constantly present in the blood in early stages of the disease, but may disappear in the third week or with defervescence. Thus, Schotmuller was successful in forty of fifty cases ; Castellani, in twelve of fourteen cases ; Cole, in eleven of fifteen, and Hewlett, in twenty of twenty-four ; while Courmont, 1 who was successful in nine consecutive cases, claims that the bacillus can always be isolated from the blood before the decline of the disease, although more than one culture may be required. Not only have these recent studies demonstrated that typhoid fever is probably always a form of bacteremia, but it has been shown that the characters of the bacterial strains concerned in typhoid fever vary considerably, each producing highly specific agglutinins in the blood (Achard and Bensaude, Widal, Gwyn, Cushing, Schotmuller, Kurth, and Buxton). That the disease may assume the character of a mixed septicemia is indicated by a considerable number of reports of the presence in the blood of other bacteria, especially staphylococcus aureus (Loison, Sittmann, Kraus, Pennato). Serum Therapy in Typhoid Fever. For a history of this sub- ject and a consideration of its problems and the various attempts that have been made to meet them, the reader is referred to the author's review, 4 which includes literature up to 1902. The most noteworthy results in serum treatment of typhoid fever are those of Chantemesse. This investigator began in 1896 to inject horses with very toxic cultures of bacillus typhosus, secured and handled anaerobically. In 1901 he treated 100 cases with the serum thus obtained which had very high agglutinative properties and strong preventive powers in animals. The mortality was 6 per cent., the chief value of the treatment proving to be in the relief of symptoms and in shortening the course of the disease. Three of the fatal cases died of perforation, against which the treatment was of little avail. Two of the fatal cases were critically ill of pneumonia when treatment was begun. • The serum caused rapid defervescence and improvement in all symptoms. Occasionally the temperature rose again after ten days of improvement, but the relapse, though often severe, was promptly controlled by the serum. The injections caused distinct leucocytosis for twenty-four to forty-eight hours, after which the neutrophile cells diminished and eosinophile cells reappeared. 308 THE ACUTE INFECTIOUS DISEASES. Bibliography. Pneumonia, Diphtheria, Typhoid Fever, Exaxthems. Achard, Bensaude. Bulletin Soc. d'Hop., Nov. 27, 1896. AVm. Virchow's Archiv, Bd. 149, p. 405. Aporti. Eleventh Internat. Cong., Rome, III., p. 381. Arnaud. Rev. de Med., 1900, p. 303. Amheim. Jahrbuch f. Kinderheilk., Bd. 13, p. 293. Auerbach, Unger. Deut. med. Woch., 1900, p. 796. Bacciochi. Lo Sperimentale, 1893, Nos. 16-17. Baginsky, Sommerfeld. Berl. klin. Woch., 1900, pp. 588-618. Barbier. Semaine Med., 1897, p. 37. Becker. Archiv klin. Med., Bd. 26, p. 558. Beco. Rev. de Med., 1899. Belfanti. La Rif. Med., 1890, 1., p. 338. Bensaude. Cited by Besaneon, Archiv. Gen., 1900, p. 491. Bieganski. Deut. Archiv. klin Med., Bd. 53, p. 433. Billings. 1 Johns Hopkins Bulletin, 1S94, p. 105. 2 N. Y. Med. Record, vol. xlix. p. 577. Billings. Med. News, vol. lxxiii. p. 301. Block. Johns Hopkins Bulletin, 1897, p. 119. Boeckman. Deut. Archiv klin. Med., Bd. 29. p. 4S1. Bollinger. Munch, med. Woch., 1895, No. 32. Bouchut. Gaz. d. Hop., 1879, p. 153. Boulay. Des. infec. a pneumococ, Paris, 1891. Bozzolo. La Rif. Med., 1889, No. 45. Brouardel. Gaz. med. de Paris, 1S74, p. 141. Bruschettini. La Rif. Med., 1892, No. 34. Buxton. Jour, of Med. Research, 1902, vol. viii. p. 201. Castellani. La Rif. Med., 1900, I., pp. 63, 76. Cazal. Gaz. d. Hop., 1899, No. 94. Chantemesse. Bulletin Soc. des H6p., 1901, p. 1133. Chantemesse, Widal. Gaz. hebdom., 1887, p. 146. Charlton. Montreal Med. Jour., Oct., 1902. Chetagurow. Abstr. Virchow's Archiv, Bd. 126, p. 187. Class. N. Y. Med. Jour., vol. lvi. p. 330- Jour. Amer. Med. Assoc, vol. xxv. p. 799. Cole. Johns Hopkins Bulletin, 1901, p. 203; 1902, p. 136. Combe, Archiv. de Med. des Enf., T. 2, p. 345. Courmont. Jour. d'Physiol. et de Path, gen., 1902, p. 165. Courmont. Rev. de Med., T. 20, pp. 317-483. Courmont, Barbaroux. Jour, de Phys. et de Path, gen., 1900, p. 577. Courmont, Montagard. Jour, de Phys. et de Path, gen., 1900, pp. 557-781- Cuffer. Rev. mens, de Med., 1878, p. 519. Cushing. Johns Hopkins Bulletin, 1900, p. 156. Doehle. Cent. f. Path., 1892, p. 150. Engel. 'Deut. med. Woch., 1897, pp. 118, 137. fifteenth Cong. f. inn. Med. Enriquez, Sicard. Compt. Rend. Soc. Biol., 1900, p. 1011. Ettlinger. Cited by Bloch. d'Espine, Marigna'c. Archiv. de Med. Exper., 1900, T. 13 Ewing. 1 N. Y. Med. Jour., vol. lviii. 1893, p. 713. 2 Ibid., 1895, vol. lxii. p. 161. 3 Trans. N. Y. Path. Soc, 1902, p. 73. 4 Medical News, 1902, vol. lxxx. p. 577. Felsenthal. Archiv f. Kinderheilk., Bd. 15, p. 78. Ferroni, Massari. La Rif. Med., 1893, II., p. 602. File. Lo Sperimentale, 1896, p. 284. Fraenkel. Deut. med. Woch., 1886, No. 1; 1901, V. B., p. 298. Cabritschewsky. Annal. de l'Institut Pasteur, 1894, p. 673. Goldscheider. Deut. med. Woch., 1892, No. 14. Gradwohl. Phila. Med. Jour., 1900, V., p. 683. Gundobin. St. Petersburg med. Woch., 1897, Lit. No. 7, p. 37. Gwyn. Johns Hopkins Bulletin, 1898, p. 54. Holla. Zeit. f. Heilk., 1883, p. 198. Hayem. 2 Archiv. gen. de Med., Mar., 1884 Henry. Cited by Cabot. TYPHOID FEVER. 309 Heubner. Deut. Archiv klin. Med., Bd. 23. Hewlett. Med. Rec, vol. lx. p. 849. v. Jaksch. l Zeit. f. klin. Med., Bd. 23, p. 207. ' Prag. med Woch., 1890. James, Tuttle. Presbyterian Hospital Reports, III., p. 44. Janowski. Cent. f. Bact., Bd. 5, No. 20. Jez. Cited by Turk. Kanthack, Hardy. Jour, of Physiol., vol. xvii. p. 91. Kikodse. Cited, Cent. f. Path., 1893, No. 3. Klein. Cited, Baumgarten's Jahresb., 1894, p. 266. 1 Volkman's Vortrage, 1893, No. 87. Kohler. Deut. Archiv klin. Med., Bd. 60, p. 221. Kohn. Deut. med. Woch., 1897, p. 136. Kotschetkoff. Ref. Cent. f. Path., 1892, No. 11. Kraus. Zeit. f. Heilk., Bd. 17, pp. 117, 138. Kruse. Pfliigge, Microorganismen, 1897. Kuhnau. 1 Zeit. f . klin. Med., Bd. 28, p. 534. 2 Zeit. f. Hygiene, Bd. 25, p. 544. Kunth. Arbeit, a. d. Kais. Gesund., Bd. 7, p. 389, 1891. Kurth. Deut. med. Woch., 1901, pp. 502, 519. Laehr. Berl. klin. Woch., 1893, Nos. 36, 37. Lambert. St. Paul Med. Jour., Dec, 1902. Landi, Cionini. Deut. med. Woch., 1901, V. B., p. 296. Limbeck. 1 Zeit. f. Heilk., Bd. 10, p. 392. Livierato. Deut. Archiv klin. Med., Bd. 53, p. 303. Loison. Rev. de Med., 1893, p. 257. Lucatello. Baumgarten's Jahresb., 1886, p. 176. Marchiafava, Bignami. La Rif Med., 1891, Nos. 251, 252. Menas. Cited by Kuhnau. Menzer. Diss. Berlin, 1892. Merkel. Cent. f. klin. Med., 1887, No. 22. Monti, Berggrun. Archiv f. Kinderheilk., Bd. 17, p. 1. Morse. Boston Med. Surg. Jour., vol. cxxxii. p. 228. Nagaeli. Deut. Archiv klin. Med., Bd. 67, p. 279. Neisser. Zeit. f. klin. Med., Bd. 23, p. 93. Netter, Levy. Cited bv Sittmann. Neuhaus. 1 Berl. klin. Woch., 1886, Nos. 6, 24. 2 Zeit. f. Hygiene, Bd. 30, p. 498. Nobecourt, Merklen. Jour, de Physiol, et de Path., 1901, p. 439. Ogata. Mitt. a. d. Kaiserl. Japanish Universitat, Tokio, 1892, p. 403. Ouskow. Cited by Turk. Page. Jour. Boston Soc. Med. Sci., vol. iii. p. 344. Pasquale. Baumgarten's Jahresb., 1891, p. 249 Pee. Diss. Berlin, 1890. Pennato. Cent. f. Bact., Bd. 24, p. 583. Pfeiffer. Monatshefte f. prak. Dermat., 1887, No. 10. Handb. d. spec. Thera- pie, 1894, p. 229. Pick. Prag. med. Woch., 1890, No. 24. Pieraccini. Cent. f. Path., 1900, p. 460. Piorry. Hematopathol., Leipzig, 1839. Prochaska. Archiv f. klin. Med., Bd. 70, p. 559. R. Pick. Archiv f . Derm. u. Syph., 1893, p. 63. Raskin. Ref. Cent. f. Bact., 1889, p. 286. Reckzeh. Zeit. f. klin. Med., Bd. 45, p. 107. Redtenbacher. Zeit. f . klin. Med., Bd. 19, p. 305. Reed. Jour, of Exper. Med., 1897, p. 515. Richardson. Phila. Med. Jour., 1900, vol. v. p. 514. Rieck. Rundschau a. d. Gebiet. d. Thiermedecin, 1887, p. 77. Rieder. Beitrage zur Kenntniss d. Leucocytose, Leipzig, 1892. RilU. Archiv f. Derm. u. Syph., 1892, p. 1028. Roger, Josue, Weil. Archiv. de Med. Exper., T. 12, p. 545. Russell. Boston Med. and Surg. Jour., 144, p 374. Rutimeyer. Cent. f. klin. Med., 1887, No. 9. Sadler. Fort. d. Med., 1892, Suppl. Heft. Schlesinger. Archiv f. Kinderheilk., Bd. 19, p. 378. Scholz, Krause. Zeit. f. klin. Med., Bd. 41, p. 405. Schotmuller. Deut. med. Woch., 1900, p. 511. Seitz. Stud. u. Typhusaetiol., Miinchen, 1886. 310 THE ACUTE INFECTIOUS DISEASES. Sello. Zeit. f. klin. Med., Bd. 36. p. 112. Silvestrini. Settimana Med., 1897, Nos. 45, 46. Silvestrini, Sertoli Rif. Med., 1899, No. 116. Sittmann. Deut. Archiv klin. Med., Bd. 53, p. 323. Smith. N. Y. Med. Jour., vol. xlix. p. 649. Sobotka. Zeit. f. Heilk., Bd. 14, p. 411. Sorensen. Cited by Reinert, Diss. Copenhagen, 1876. Soudakojf. Cited by Kuhnau. Stengel, White. Univ. of Pennsylvania Bulletin, vol. xiv. p. 310. Stern. Cent. f. inn. Med., 1896, p. 1249. Stienon. Jour, de Med. (Brussels) Annales, 1895. Thayer. 1 Johns Hopkins Hospital Reports, vol. iv. p. 82. 2 Johns Hopkins Bulletin, 1893, p. 37. Thiemich.i& Bsbumgaxten's Jahresb., 1894, p. 266. Tschistowitsch. Annal. de l'Institut Pasteur, 1891. Archiv des Sci. Biol., St. Petersburg, 1893. Tumas. Deut. Archiv f. klin. Med., Bd. 41, p. 323. Turk. 1 Klin. Blutuntersuch. Wien., 1898, p. 96. Urban. Wien. med. Woch., 1897, Nos. 32-35. Weber. Cent. f. Baot., Bd. 21, p. 286. Weil. Compt. Rend. Soc. Biol., 1900, p. 615. Weil, Descos. Jour, de Physiol, et de Path., 1902, p. 504. Weiss. Jahrb. f. Kinderheilk., Bd. 35. White Jour, of Exper. Med., vol. iv. p. 425. Widal. Semaine Med., Aug. 4, 1897. Widal, Bezancon. Cent. f. Path., 1896, p. 569. Wiltschour. Cited, Cent. f. Bact., 1890, p. 279. Winternitz. Cent. f. klin. Med., Bd. 14, p. 177. Zappert. Zeit. f. klin. Med., Bd. 23. Zaslein. Diss. Basel, 1881. CHAPTEE XIV. WIDAL'S TEST. The history of the development of Widal's test from the discovery of the phenomenon of Pfeiffer in 1894 through its first application to the diagnosis of typhoid fever by Pfeiffer and Kolle, and Gruber and Durham, in 1896, up to its more practical modification by Widal, has now been very fully reviewed in numerous monographs and treatises on bacteriology and need not here be repeated. It may only be said that while the scientific basis of the test had been firmly laid by previous studies, up to the. time of Widal's communication there had been no indication that the specific reaction could be obtained from small quantities of serum and at the beginning of the disease, while the use of dried blood and the introduction of the test into municipal laboratories, thus rendered possible, was first accom- plished by Johnston, of Montreal. Description of the Reaction. When to a few cubic centimetres of blood serum of a patient suffering from typhoid fever is added a few drops of an actively motile culture of bacillus typhosus, the bac- teria are precipitated in flakes, leaving the supernatant fluid clear. When the sediment is examined microscopically it is found that the bacilli have lost their motility and are agglutinated iu masses, while with very intense reactions they may be broken up into granules or even completely dissolved. When the test is performed with a drop of dissolved blood inoculated by means of a platinum loop with a small portion of broth culture, all stages of the reaction may be fol- lowed under the microscope. With very intense reactions the agglutination is found to be com- plete as soon as a cover-glass can be adjusted over the drop and the specimen placed under focus. The bacilli are motionless, they are nearly all gathered in characteristic clumps, which stretch in a net- work irregularly through the field, and very soon, in some cases, evidences of subdivision or solution of the bacteria may be detected. If the reaction is less complete, the clumps are less compact and a few motile bacilli may be found between the masses. With high dilutions of the blood, all stages of the reaction may be followed. The first change noted is a partial loss of motility. The bacilli cling to one another after struggling to free themselves, but gradually entangling their neighbors. In the course of ten or fifteen minutes the process results in the gradual clumping of all bacilli, or, accord- ing to the strength of the reaction, a variable number of actively motile bacilli may remain between the loose masses. When the reaction is present in traces only, much longer time is required for the appearance of small clumps, but these reactions 312 THE A G UTE INFECTIO US DISEASES. must be discarded as unreliable. All experienced observers have insisted that distinct islands of clumped bacilli must be present to constitute a positive reaction, and Fischer and others maintain that all bacilli must be rendered quiescent before the result can be regarded as positive. Wilson describes as a partial reaction one in which well-defined clumps appear in every field, but composed of motile bacilli, while the edges of the hanging drop are not free from bacilli. He found such reactions in thirteen different diseases. Pseudoreactions. While partial reactions occur under many conditions when the technique has been faultless, certain effects simulating the true agglutination may appear in specimens contain- ing not a trace of the specific principle on which the test depends. In old cultures many loose clumps of bacteria may be transferred by the loop from the culture tube and be mistaken for an effect pro- duced by the serum. Normal blood or serum, in somewhat concen- trated form, exerts a marked influence upon the motile typhoid bacillus, causing it to lose its motility after a time, but failing to develop typical clumps. If the specimen has been allowed to dry many bacteria adhere to slide or cover-glass or gather at the thicken- ing edges of the specimen, and sometimes these collect in motionless groups. Such clumps always lack the reticular arrangement seen in the true reactions. A considerable variety of confusing appear- ances will meet the beginner, resulting from the formation of fibrin and the incomplete solution of masses of red cells. Technique : Methods of Obtaining the Body Fluid. Although specific reactions have been obtained from many secretions, excre- tions, and other fluids of the body, only the blood, its serum expressed from the clot, or the fluid gathering in blisters, yield sufficiently constant results to be available in clinical diagnosis. The Whole Blood. When the examiner can reach the bedside there is no better method than to employ the whole blood diluted in known proportion by distilled water. In order to obtain an accurate dilution a medicine dropper may be graduated in one and ten or twenty drop marks, as suggested by Cabot, but such dilution cannot be very accurate. The writer has long used for this purpose the leucocyte pipette of the Thoma-Zeiss hematocytometer, which gives an exact dilution of 1 : 20 or 1 : 50, or more, and which is itself a very convenient instrument for handling the blood. Levy and many others have devised special tubes adapted to this purpose. The chief objection to this method is the failure of the blood to com- pletely dissolve in dilution of 1 : 20 or 1 : 50 of water, but no other method can furnish more exact dilution, which is essential in accurate work. This difficulty may be avoided by laying the pipette on the side until the corpuscles settle. Blood dried on a glass slide and dissolved by mixing with water is now used in probably 90 per cent, of all examinations. One or two drops are touched to a clean glass slide and dried in the air. Such a specimen protected from moisture may be kept for days, transported for long distances, and being easily obtained, even by the patient WIDAL'S TEST. 313 himself, furnishes by far the most practical method of obtaining the necessary material for the test. Its great disadvantage is the impossi- bility of securing an exact dilution of the blood when it is redissolved. This difficulty, while theoretically insurmountable, proves to be of comparatively little moment in practice, since an experienced worker can learn to control the dilution by the color of the drop. Also, when imperfectly dissolved the detritus of red cells may obscure the field, although it should not fail to be distinguished from masses of bacilli Some observers have found less powerful and constant effects from dried blood than from serum (Widal, Delepine, Johnston), but for diagnostic purposes Park finds the two methods of nearly equal value. Nevertheless, for accurate studies of variations in the reac- tion, the use of dried blood is inapplicable, although it will probably always remain in extensive employment for simple diagnostic pur- poses. Serum. 1. The Bulbed Tube. A capillary tube with central bulb is filled by capillary attraction from the expressed drop, the ends are fused, and when the blood coagulates a few drops of serum are exuded. A great many modifications suited to individual pur- poses have been suggested for this method. It offers considerable technical difficulties, but furnishes serum without delay. 2. The Blister. Serum may be obtained by means of a Spanish fly blister in from six to eighteen hours, and in quantity sufficient for extended examination without involving much pain or incon- venience. The fluid may be drawn in a sterile test tube and after- ward divided in measured quantities, or it may be sealed in a capil- lary tube. For detailed study blister serum is superior to all other fluids. For measuring serum the " mixer " of the hematocytometer is well adapted, and the test may be performed in the hanging drop or in the test tube. The Culture. Considerable difference in the response of different cultures of bacilli have been demonstrated by Kolle, Achard and Bensaude, Durham, v. de Velde, and Ziemke. Although Foerster found very little difference in a considerable series of cultures, most observers agree that attenuated cultures are more readily agglutinated. Miiller succeeded in greatly diminishing the agglutinability of a given species and at the same time increasing its virulence by passing it through animals. It appears that for each culture there is a limit beyond which normal serum is positively inert, while at the same dilution typhoid serum exerts a specific action (Foerster). While any pure culture may be used, Miiller and others have shown that a freshly isolated culture is not suitable for diagnostic use, and must first be transplanted many times in artificial media. The best results have been obtained in New York City by a so-called " Pfeiffer specimen" imported by Park. A stock culture of this particular growth is kept in sealed tubes of nutrient agar in the ice chest and replanted every few months. From this growth the specimen employed in testing is obtained in broth culture grown for twenty- four hours at a temperature of 37° C. In such broth cultures the 314 THE ACUTE INFECTIOUS DISEASES. bacilli are completely isolated and actively motile, so that the slightest change in motilhy'or tendency to clump can be detected by com- parison with a control specimen. Dead Bacilli. Widal first observed that cultures of typhosus which had been killed by heat (57° to 60° C, thirty to forty-five minutes) had lost little or none of their capacity to agglutinate, and this fact has been abundantly attested by others. Many have there- fore preferred to use a dead culture killed by heat, or by 10 per cent, formalin, or by thymol, or by corrosive sublimate, 1000 to 5000 (Park), thereby dispensing with the trouble of preparing a fresh culture. Such an expedient, however, can hardly meet with general adoption, on account of the difficulty of keeping the dead bacilli well isolated, and because the gradual loss of motility is a desirable and reassuring feature of the test in mild reactions, which are just the ones in which uncertain clinical signs render a positive test more desirable. Attenuated cultures have been recommended by Johnston, who found that in using dried blood pseudoreactions were not infre- quent with active virulent cultures, while broth cultures planted from old agar stock were less susceptible and did not respond to healthy or non-typhoid sera. Paratyphoid Strains. From the observations of Achard and Bensaude, "Widal, Gwyn, Cushing, Schotmuller, Kurth, Coleman and Buxton, and others, it has been shown that the bacteria in typhoid fever may show certain characters, such as the fermentation of sugars with the production of gas and acid, which gives them an intermediate position between typhosus and bacillus coli communis. Buxton has shown that these intermediate forms, isolated by him- self and obtained from the above observers, may be still further classified into three rather distinct groups. It appears, also, that the agglutinins developed by these strains are highly specific, that of one group usually having little effect on other groups. Buxton found a certain grade of interaction between the members of his groups. These considerations suggest that where the blood is negative to typhosus it should be tested with various paratyphoid strains. The writer attempted to do this in a short series of cases of suspected typhoid fever, but where the reaction was negative to typhosus it was negative also to the paratyphoid cultures. Degree of Dilution of Serum and Time Required. Although it has been believed that the serum reaction in typhoid fever depends on the presence in the blood of a peculiar substance which exerts a specific action on the typhoid bacillus, yet it has been found that in a considerable proportion of cases healthy serum, or that of patients - suffering from other diseases, contains substances which exert a very similar influence upon typhoid cultures. In typhoid fever, however, the serum may usually be diluted twenty to fifty times, or often very much further (1000 to 2000, Widal 2 ) without destroying the reaction, while in other diseases the reactions usually disappear with dilutions of 1 : 10, or almost certainly with dilutions of 1 :@0. The element of time is also of great importance in distinguishing the specific typhoid reaction from that produced by other sera. WIDAL'S TEST. 315 While a distinct reaction often occurs instantly with typhoid serum, blood of other diseases almost never acts immediately, but requires one-half to one hour or longer to disclose its effects. Consequently, in order to demonstrate the specific typhoid reaction, it is necessary to increase the dilution and to limit the time. " The results obtained in the New York City Health Department laboratories and elsewhere have shown that in a certain proportion of cases of typhoid there occurs a delayed moderate reaction in a 1 : 10 dilution of serum or blood ; but very rarely, if ever, excepting in typhoid fever, does a complete reaction with this dilution occur within five minutes. When dried blood is used the slight tendency of non- typhoid blood in 1 : 10 dilution to produce agglutination is increased by the presence of fibrinous clumps and perhaps by other substances derived from disintegrating red cells. From many cases examined by Fraenkel, Stein, Foerster, Scholz, ourselves, and others, it has been found that in dilution of 1 : W or mare,, a decided quick reaction is never produced by any febrile disease other than typhoid infection, in which it often occurs in dilution of 1 : 50" (Park). Yet even this dilution appears insufficient to eliminate all sources of error, and there has been a constant tendency among experienced workers to increase the grade of dilution. Grunbaum placed it at 1 : 30 ; Simon, at 1 : 30 or more ; Stern, after a large experience, at 1 : 40 ; and Mewius, at 1 : 60. From these authoritative opinions it becomes necessary to prescribe a dilution of at least 1 : 20, better 1 : 30, while the limit of time should not be greatly extended. Even these limits will be found insufficient to avoid all possible error, and in doubtful cases one should use a dilution of 1 : 40 or 1 : 60, increase the time to one or two hours, and require a distinct result. Occurrence of the Reaction. Statistics vary considerably regarding the dates of occurrence and the constancy of the reaction in typhoid fever. Cabot collected over 3000 cases of supposed typhoid fever, of which 95 per cent, gave the reaction at some period of their course, while in 2500 control cases, not typhoid, 2 per cent, gave positive reactions. Park reports positive results in a large series : During the first week, in " second " " third " " fourth " 1 ' second month, in 20 per cent, of the cases. 60 80 90 75 In 88 per cent, of hospital cases in which repeated examinations could be made, a definite reaction was obtained at some period of the disease. Wilson reports further on the work of the New York City Department of Health, as follows : In 256 examinations the test was positive in 8 cases not diagnosed as typhoid, while 3 were malaria, and 1 puerperal sepsis with tuber- culous intestinal ulcers. Of 1115 cases 117 which gave no reaction proved to be typhoid. 316 THE ACUTE INFECTIOUS DISEASES. The earliest date of appearance of a positive reaction has not been and cannot- well be determined in the human subject, but in animals inoculated with dead typhoid bacilli the reaction appears between the third and eighth days. In man a positive reaction has been found by Johnston and Taggart, and by Fraenkel, on the second day, but the reactions were not sharp. Levy found the first distinct reac- tion on the sixth day in the human subject inoculated with dead typhoid bacilli. It may be found before the appearance of rose spots, splenic tumor, or the diazo reaction in the urine. During the course of the disease the reaction may continue without interruption, or may be absent one day while present the next. It may be absent entirely in mild cases, may appear only in a relapse (Lichtheim, Breuer, Thoinet), may disappear entirely after a few days (Elsberg), or may persist for months or even a year. Incomplete reactions have been reported after many years (Stewart). It not infrequently appears for the first time during convalescence or may exhibit a sudden increase at this time. According to Widal, in the majority of cases the reaction disappears by the fifteenth to thirtieth day of convalescence, but Stewart believes that it persists at least one year in 50 per cent, of the cases, for two years in 25 per cent., and for ten years in 5 per cent. His conclusions can only apply to very indis- tinct reactions. In the blood of the fetus delivered during the course of typhoid fever, several observers have failed to find any agglutinative power (Mahnt, Stahelin), but Schumacher 2 claims that agglutinins are always present in the fetus during the last month of pregnancy, but that their persistence is of short duration. Relation of the Reaction to Other Features of the Disease. In some cases the reaction is most intense at the height of the dis- ease, as in cases observed by Jemma. Widal and Sicard, who found that in the active stage a dilution of 1 : 60 or 1 : 80 does not usually prevent the reaction, noted a marked weakening as convales- cence proceeded, some cases failing to react with dilution of 1 : 10. They noted a reaction in one case with a dilution of 1000 or 12,000. Foerster, who found a reaction in dilutions varying from 1 : 60, 1000, or 5000, could not detect any relation between the intensity of the reaction and the severity of the disease. Pfeiffer and Kolle, and Foerster have shown that the agglutinating power has no relation to the bactericidal activity of the serum. Courmont 2 has contributed an elaborate study of the course of the agglutinative power of the blood in typhoid fever, reaching some important conclusions regarding prognosis and offering evidence that the agglutinative power of the blood is closely connected with the defensive reaction of the organism. His conclusions are as follows : In the great majority of cases of typhoid fever of ordinary or moderate severity the power of agglutination reaches a maximum at the period of defervescence, or during the first days of apyrexia, and falls, often rapidly, with the fever, or during convalescence. Agglu- tination curves that are continuously low (less than 1 : 200) are rarely seen in cases of moderate severity, but frequently in very WIDAL'S TEST. 317 toxic forms of the disease which are prolonged or fatal. Very irreg- ular agglutination curves with premature fall are rare in moderate cases, very frequent in toxic and fatal cases. In some prolonged cases the curve of agglutination rises rapidly shortly before defervescence. In relapsing cases the agglutination curve varies, but usually fol- lows the same types as in single attacks. It is usually higher in the relapse than in the initial attack if the patient recovers and lower if he dies. In mild and irregular types of the disease the power of agglutination is very variable and usually low or evanescent. Effect of Typhoid Serum upon the Colon Bacillus. Although Fraenkel in a large series of cases reported that typhoid serum has no marked power to agglutinate the colon bacillus, and recommended the use of this serum in the separation of colon from typhoid bacilli, this claim has not been verified. Stern and Biberstein found, very shortly, five cases of typhoid fever of which the blood serum agglu- tinated the colon bacillus even more powerfully than the typhoid, and many other observers, including Park, Rodet, Courmont, Ustvedt, Bensaude, and Kubnau, have had similar experiences in many cases. Baumgarten, therefore, concludes that this method can no longer be accepted as yielding positive proof of the identity of bacillus coli communis. It nevertheless remains true that a bacillus which on cultural characters falls between the typhoid and colon groups, is almost certainly coli communis, if it is not agglutinated by a well-tested and active typhoid serum. The possibility that infec- tion by the colon bacillus is intermingled with that of bacillus typhosus in enteric fever has been abundantly considered by several writers. The safest method of identifying the colon bacillus is not by the serum of a case of typhoid fever, but by the serum of an animal which has been inoculated with a pure culture of bacillus typhosus. Reactions in Conditions Other than Typhoid Fever. In view of the fact that positive reactions have been obtained both with healthy serum and in diseases other than typhoid fever when a dilution of 1 : 10 was used, many observers have recommended that higher dilutions only be employed. Thus Schultz obtained com- plete reactions in eleven of 100 cases of various febrile diseases with a dilution of 1 : 10 ; in seven cases with a dilution of 1:15, and in three with 1 : 20, while a faint response followed a dilution of 1 : 25 in a single instance. The time limit, however, was one to two hours. In single cases complete reactions with dilutions of 1 : 10 or more have now been reported in so many cases that it is no longer possible to refer the results to faulty technique. Some of these conditions are as follows : septicemia (Ferrand) ; malaria (Bloch, Yilliers, Catrin, and the writer) ; pneumonia (Kasel and Mann) ; tuberculosis (Wesbrook, Jez, d'Espine) ; otitis (Stern) ; influenza (Wesbrook) ; typhus (Park) ; meningitis (v. Oordt) ; nor- mal serum (Kuhnau). In many of these cases the dilutions were from 1 : 10 to 1 : 30, or more, and the clumping was prompt and complete. In several cases of chlorosis Kohler claims to have secured a reac- tion in a dilution of 1 : 40, and in a case of severe anemia at 1 : 50. 318 THE ACUTE INFECTIOUS DISEASES. A large percentage of cases of jaundice have been found to show high agglutinative power over bacillus typhosus, which Greenbaum refers to the presence of taurocholic acid. In Weil's disease Eckhart obtained positive reactions in two cases, and Zupnik, in four of six cases of Weil's disease, two of cholelith- iasis, one of cholangitis, and one of carcinoma of the liver, all the subjects being jaundiced. Stewart, using dry blood in 538 cases, found typhoid lesions at autopsy in five which failed at three periods during the disease to give a positive reaction. Thompson also reports six negative results in 163 cases, while a slight reaction was obtained in 12 per cent, of various conditions other than typhoid fever. Ustvedt encountered a peculiar epidemic of typhoid fever among soldiers in which four of fifteen cases failed to yield a reaction. Total failure of reaction, under full precautions, is also reported in single cases by Artaud, Haushalter, Schumacher, Fischer, and others. The significance of these negative results have been made apparent by the work of various observers, who have shown that a consider- able number of bacterial strains, each developing highly specific agglutinins, may give rise to the symptoms of typhoid fever. Value in Diagnosis. The difficulty in determining the value of Widal's test in diagnosis arises principally from the divergent opin- ions regarding the essential features of a complete reaction. Although the belief in the specific quality of Widal's reaction has been aban- doned, the test remains an extremely valuable diagnostic procedure under several conditions. It must be granted at once that absolute certainty cannot be ascribed to any test unless the dilution has been very high, certainly not less than 1 : 60, with a time limit of not more than fifteen to thirty minutes. A positive result with much less dilution (1 : 30) must stand as almost certain evidence that the disease is typhoid fever. Such results are usually obtainable, however, only after the disease is well estab- lished and its clinical symptoms distinct. Accordingly the chief value of the test lies in its capacity to distinguish, during the height of the illness, certain conditions, such as acute gastritis, tuberculosis, meningitis, and pneumonia, which may simulate typhoid fever. The application of the test in the early diagnosis of typhoid fever has, in the experience of most observers, proved disappointing. It is rarely to be found until other signs render the diagnosis very prob- able, when the blood test may often be added, as any other isolated clinical symptom, to the evidence for or against typhoid fever. The writer believes, however, that the combination of an indistinct serum reaction, diminution of fibrin, absence of leucocytosis, and presence of relative or absolute lymphocytosis, can very rarely be demonstrated in the early stages of any obscure febrile disease except typhoid fever. The morphological examination of the blood is, therefore, a valuable adjunct and control in the application of Widal's test, and the writer believes that in doubtful cases it should never be omitted. WIDAL'S TEST. 319 Negative results are of very moderate import until the third or fourth week, and unless often repeated, while some reported cases, often fatal, have failed at any time to yield the reaction. In case of a negative result in a case strongly resembling typhoid fever, one should if possible test the blood with various paratyphoid strains. Summary. The most reliable method for clinical purposes is the use of fresh blood diluted with water in a graduated pipette, and inoculated as a hanging drop preparation. Blood or blister serum should be employed for more extended study, while the use of dried blood, sometimes necessary, gives very slightly less reliable results. The culture should be grown in broth at 37° C, twenty-four hours old, actively motile, and of moderate virulence. A recognized brand should be obtained if possible, and the behavior of all newly isolated cultures should be tested before use, and replanted until they become readily agglutinable. A positive reaction consists in the complete immobilization of all bacilli and the compact clumping of the great majority. A time limit of five minutes is all that is necessary, with dilutions of 1 : 20, in the great majority of positive reactions. With higher dilution (1 : 40) the time may be extended to thirty minutes, and with dilution of 1 : 60, to two hours (Mewius). A dilution of at least 1 : 20 is required in all positive reactions, with which, however, occasional errors will occur. Dilutions of 1 : 30 or 1 : 60 are to be strongly recommended, for the higher the dilution the more certain are all positive results. A negative result is of little import unless obtained during the height of the disease (third to fifth weeks) and on repeated examina- tions. In the great majority of cases the reaction disappears within a few weeks after convalescence, and several authentic cases, usually fatal, have not given a positive reaction at any time. The agglutinative power of typhoid serum is usually slight and transient in mild cases, marked and persistent in cases of average severity, low or variable in toxic cases, but bears no constant rela- tion to the gravity of the disease, and gives no certain prognostic indications. Bibliography. Widal's Test. Achard, Bensaude. Compt. Rend. Soc. Biol., 1896, p. 940. Artaud. Presse Med., 1898, No. 18, p. 106. Bensaude. L 'Agglutination des Microbes, Paris, 1897. Biberstein. Zeit. f. Hygiene, Bd. 27, p. 347. Biggs, Park. Amer. Jour. Med. Sci., vol. cxiii. p. 274. Bloch. Jour. Amer. Med. Assoc., 1897, vol. xxix. p. 7. Breuer. Berl. klin. Woch., 1896, p. 1037. Catrin. Soc. des H6p., 1896, p. 698. Courmont. Compt. Rend. Soc. Biol., 1898, p. 756. Also, Sero-prognostic d. 1. fiev. typh., Paris, 1897. Dele-pine. Brit. Med. Jour., 1897, I., p. 1894. Durham. Lancet, 1896, II., p. 1746. Eckhart. Munch, med. Woch., 1892, No. 27. Elsberg. N. Y. Med. Record, vol. li. p. 510. d'Espine. Rev. med. de la Suisse Romande, 1898, p. 113. 320 THE ACUTE INFECTIOUS DISEASES. Ferrand. Bull. Soc. d. Hop , 1897, p. 104. Fischer. Zeit. f. Hygiene, Bd. 32, p. 407. Foerster. Zeit. f. Hygiene, Bd. 24, p. 500. Fraenkel. Munch, med. Woch., 1897, p. 107. Gruber, Durham. Munch, med. Woch., 1896, pp. 206, 285. Grunbaum. Brit. Med. Jour., 1897, II., p. 1852. Haushalter. Presse Med., 1896, p. 505. Jemma. Cent. f. inn. Med., 1897, p. 65. Jez. Wien. med. Woch., 1897, p. 98; 1898, p. 890. Johnston. N. Y. Med. Jour., 1896, vol. lxiv. p. 573. Johnston, Taggart. Brit. Med. Jour., 1896, II., p. 1629. Kohler. Cent. f. Bact., Bd. 29, p. 683. Kolle. Deut. med. Woch., 1897, p. 132. Kasel, Mann. Munch, med. Woch., 1899, p. 581. Kuhnau. Berl. klin. Woch., 1897, p. 397. Levy. Munch, med. Woch., 1897, p. 1435. Lichtheim. Cited by Foerster. Mahnt Cent. f. Bact., Bd. 30, p. 675. Mewius. Zeit. f. Hygiene, Bd. 32, p. 422. Midler. Munch, med. Woch., 1903, p. 56. v. Oordt. Munch, med. Woch., 1897, p. 327. Pfeiffer. Zeit. f. Hygiene, Bd. 18, p. 1. Pfeiffer, Kolle. Deut. med. Woch., 1896, p. 185. Rodet. Compt. Rend. Soc Biol., 1898, p. 756. Scholtz. Hyg. Rundschau, Bd. 8, p. 417. Schumacher. Zeit. f. Hygiene, 1899, Bd. 30, p. 364; Bd. 37, p. 323. Simon. Cited, Baumgarten's Jahresber., 1898, p. 336. Stahelin. Corresp. Schweizer Aerzte, 1898, p. 161. Stern. Cent. f. Bact., 1898, p. 673. Stewart. Amer. Public Health Assoc, vol. xxiii. p. 151. Thoinet. Semaine Med., 1896, p. 504. Thompson. Brit. Med. Jour., 1897, II., p. 1775. Ustvedt. Cited, Baumgarten's Jahresber., 1898, p. 342. v. d. Velde. Acad. med. de Belgique, 1897, Mar. 27. Villiers. Presse Med., 1896, p. 54. Wesbrook. Ref. Cent. f. Bact., 1898, p. 713. Widal. 1 Soc. med. des Hop., 1896, p. 561. 2 Presse Med., 1897, I., p. c. Wilson. Medical News, 1901, vol. Ixxix. p. 81. Ziemke. Deut. med. Woch., 1897, p. 234. Zupnik. Munch, med. Woch., 1902, p. 1305. CHAPTEE XV. MISCELLANEOUS INFECTIOUS DISEASES. SEPTICEMIA, PYEMIA, OSTEOMYELITIS. Red Cells. In no other disease do the red cells suffer destruction so constantly and to such an extent as in the toxemia of diffuse inflammation caused by the common pyogenic bacteria. This fact was very early noted, Braidwood finding shrinkage of the red cells and absence of rouleaux, Mannassein remarking on their reduced diameter, and Quincke and Patrigeon observing extreme loss of Hb in cases of pyemia. Hayem and Toenissen placed the average loss of red cells in ordinary septic fever at 200,000 to 1,000,000 per week, while a con- tinuous diminution was found to persist as long as suppuration con- tinued. That the loss of red cells is often very much more rapid than Hayem supposed is shown by Grawitz's remarkable case, in which fatal puerperal sepsis combined with considerable hemorrhage reduced the red cells in about twenty-four hours to 300,000. Various forms of acute septicemia not infrequently reduce the red cells below 2,000,000, but none appear to act more violently than does puerperal or uterine sepsis. Hayem 2 found only 1,450,000 cells, 20 per cent. Hb, in a recent case of puerperal sepsis. Cabot reports 1,800,000 cells from a "suppurating fibroid." The writer found 1,600,000 cells and 20 per cent. Hb in a septic form of endo- metritis, not puerperal. When the suppurative process is localized the red cells are usually found to be only slightly affected, but with the first appearance of the " septic " condition the reduction of red cells promptly begins, Roscher finding evidences of diminished concentration of the blood within the first few hours. In empyema, suppuration in wounds, pelvic abscesses, appendicitis, peritonitis, etc., the red cells are seldom markedly reduced, while the usual effects of fever are present and polycythemia is often found. The Hb is, as usual, more susceptible than the red cells, and its loss is usually out of proportion to that of the red cells, the Hb-index being invariably low. Timofjewsky found that nucleated red cells appear promptly in the blood of dogs after moderate injections of pyogenic bacteria, in one instance reaching the enormous proportion of 25,698 per c.mm. Turk found considerable numbers of nucleated red cells in two cases of septicemia from cystitis. In cases of intense puerperal sepsis the writer has usually been able to find a few nucleated red cells, but they have never been very numerous. 21 322 THE ACUTE INFECTIOUS DISEASES. In chronic suppuration the red cells tend to diminish as long as the discharge continues. In a case of chronic empyema of one year's duration the writer found 1,800,000 cells and 25 per cent, of Hb, but an old pelvic abscess discharging a small quantity of pus for two years had induced very slight anemia. Morphological changes in the red cells in septicemia are usually present in moderate degree. Most cases show a pure type of sec- ondary chlorotic anemia with marked loss of Hb. The reduced diameter of the anemic cells, previously noted, was distinct in the writer's case of septic endometritis, but did not approach the grade seen in some forms of primary disease of the blood. In severe cases many cells show granular degeneration, but polychromasia has, in the writer's experience, not been prominent. Marked differences in the size and shape of the cells do not appear, as a rule, until after the second or third week of a severe septic process. Leucocytes. A considerable number of cases of asthenic septi- cemia run their course without leucocytosis, or with distinct reduc- tion of white cells. Such cases have been reported by Limbeck, Krebs, Cabot, Turk, and, usually proving fatal, their significance is similar to cases of pneumonia, diphtheria, etc., with hypoleucocytosis. In Turk's case, and in one observed by the writer, while the leuco- cytes were subnormal, the proportion of polynuclear cells was very high. Da Costa reports absence of leucocytosis in twelve of twenty-one cases of severe septic processes, and the writer has observed a pro- longed puerperal case with severe chills in which leucocytosis failed to appear. The great majority of cases, however, show pronounced leucocy- tosis, which is usually in proportion to the severity of the disease. Among the reported cases showing no leucocytosis are doubtless some in which the examination of the blood was made during the antemortem decline in the number of leucocytes. Regarding the more minute factors determining the grade of leuco- cytosis little is definitely known, and the examination of the blood must be interpreted according to the circumstances in each case. While the rule that suppuration induces leucocytosis is almost invari- able, it must be remembered that leucocytosis may promptly disappear when the exudation ceases, and that suppurations involving mucous sur- faces may induce very slight leucocytosis. The polynuclear leucocytes are almost always in high proportion in cases of sepsis, but Klein has recently described a case of hemor- rhagic septicemia 1 with 76 per cent, of eosinophiles in the pleural exudate and 40 per cent, in the blood. Chemistry. The loss of Hb has already been mentioned as one of the chief alterations in the blood of septicemia. Its solution in the plasma and the occurrence of hemoglobinemia is observed in severe cases with rapid destruction of blood. In cases of puer- peral sepsis, or pyemia in infants, the solution of Hb may reach a very extreme grade. In a puerperal case the writer found the viscera discolored, and so great was the deposit of pigment that MISCELLANEOUS INFECTIOUS DISEASES. 323 the liver and spleen closely resembled, in gross and microscopic appearance, the organs of pernicious malaria. The increased globulicidal action of the serum in these cases, to which the solu- tion of red cells must be referred, has been described by Maragliano and others. An increased tendency toward crystallization of the Hb has been noted by Bond, who, in examining fresh specimens, found a rich deposit of such crystals about the edge of the cover-glass. Rosoher noted in severe cases a very rapid lowering of the specific gravity of the blood, beginning a few hours after the initial symp- toms. In fatal cases the dry residue of the whole blood fell to 15 per cent., while in favorable cases the loss of solids was usually much less. This rapid and extreme loss of albumins Roscher and Grawitz regard as a valuable prognostic sign. The serum also usually showed a pronounced loss of albumin, proportionate to the severity of the septic process, its dry residue falling from 10.5 per cent, to a mini- mum of 6.25 per cent. Yet in a very severe case the dry residue of the serum was 13.1 per cent, of its weight, which Grawitz explains as resulting from the solution of Hb. In various forms of pyogenic infection Livierato found an excess of glycogen in the blood, even when the temperature was normal. Goldberger and Weiss also describe in the leucocytes during abscess- formation a reaction to iodine either in the form of a diffuse stain or a granular deposit. They also find many brownish staining extra- cellular granules after fractures, and believe that the somewhat obscure " iodine reaction " of the blood may be made of value in the diagnosis of abscess, fractures, etc. Bacteriology. The frequency with which specific bacteria can be isolated from the circulating blood in cases of septic infection, in spite of the vast amount of study devoted to the subject, still remains a matter of doubt. There are, on the one hand, a large number of studies of the blood in pyemia, septicemia, puerperal fever, osteomyelitis, etc., reporting the discovery of bacteria in a large proportion of cases, while in many others, reported by competent observers, very uniformly nega- tive results were obtained. On reviewing many of these reports it appears that the majority of them must be set aside as unreliable. Rosenbach, Garrod, Raskin, Brunner, Blum, Czerniewski, Eisel- berg, Cantu, Roux and Lannois, Bommers, Stern and Hirschler, and others, all used blood squeezed from the finger, and when they attempted to demonstrate the same germ in the viscera after death, were not always successful. Many of their successful cultures were made only a few hours before death. When accurate details are given, as by Czerniewsky, it appears that contaminations by clearly non-pathogenic germs frequently occurred. On the other hand, Brieger, drawing blood from a vein by a sterilized syringe, obtained negative results in five cases of puerperal sepsis. Sanger, by the same method, in three cases of osteomyelitis found staphylococcus aureus or albus in the blood, but cultures from 324 THE ACUTE INFECTIOUS DISEASES. an inflamed joint gave only streptococcus pyogenes. Likewise Kraus reports six positive results in ten cases of puerperal sepsis, but one was examined postmortem, and two showed only staphylococcus albus. Cannon, who secured a number of successful cultures from the blood in puerperal sepsis, osteomyelitis, wound infection, etc., took some of his specimens from a puncture of the skin, and Petruschky, who usually obtained some bacteria, drew the blood through the " disin- fected " skin by means of a wet cup. Sittmann, accepting as genuine the results of previous investigators, and adding nine positive cases of his own, concludes that in septicopyemia the bacterial agent is always to be found in the blood, and that its presence does not insure a fatal issue. Somewhat striking conclusions may be drawn from the reports of Bertelsman, who secured fifty-four negative and forty-seven positive results from examinations in 101 cases of various forms of suppura- tion. In twenty-seven cases the streptococcus was obtained, and in fifteen staphylococci. Important features of his study were the uni- formly positive cultures in osteomyelitis (six cases), the demonstration of bacteremia in panaritium, and in five of nine cases of urethral fever, in phlegmonous erysipelas, and in three cases of cryptogenic strepto- coccus septicemia which recovered, while in several instances it was shown that bacteria were present in the circulating blood for short periods and in rapidly decreasing numbers. In the absence of much greater detail than accompanies the report, such results cannot be accepted without confirmation. Old observers, as Kuhnau, do not accept the conclusions of Sitt- mann and do not admit much significance in the isolation from the blood of staphylococcus albus or the colon bacillus. Kuhnau examined the blood repeatedly in twenty-three cases of septicopyemia, nineteen of which were fatal. In two streptococcus pyogenes was obtained, in one staphylococcus aureus. Several cultures gave non-pathogenic germs, among them staphylococcus albus, which was obtained from the blood, but not from the abscesses after death. The only success- ful cultures were made shortly before death, or in one case during a chill. White also found streptococcus pyogenes only three times and staphylococcus aureus once in eighteen fatal cases of sepsis. The results of Kuhnau's study cannot fail to raise the suspicion that manyjnvestigators have been too lax in technique, or have not fully identified their species. From the review of the above studies the writer draws the follow- ing conclusions : 1. The only reliable method of obtaining blood for bacteriological examination during life is to draw it from a vein, in quantity not less than 5 c.c, through the thoroughly sterilized, or, better, the incised skin, by means of a sterile syringe or aspirator. 2. In the great majority of cases of local or general septic infec- tion, septicopyemia^ septicemia, pyemia, diffuse suppuration, osteo- myelitis, etc., bacteria are present in the circulating blood only for short periods and at infrequent intervals, most frequently during chills. MISCELLANEO US INFECTIO US DISEASES. 325 3. A few hours before death from septic infection, various bac- teria, some of which may not be active in the original process, make their way into the circulation. 4. In a very moderate number of cases of septic infection, espe- cially those which are not attended by local abscess formation, the bacterial agent may be isolated from the blood during the progress of the disease. APPENDICITIS. Red Cells. From the observations of Da Costa in 139 cases it appears that anemia of moderate grade exists in nearly all cases of appendicitis. It is least marked in catarrhal cases, in thirty-eight of which Da Costa found an average of 4,180,000 cells, with some exceptions showing much greater loss, while the Hb commonly registered 20 to-40 per cent, below normal. Anemia is most marked in cases with abscess of long standing, in which the toxemia fre- quently causes a loss of 25 to 45 per cent, or more of Hb and a re- duction of the red cells below 4,000,000. In Da Costa's series a reduction to 2,000,000 red cells was twice observed, once with a catarrhal and once with a suppurative lesion, while more than 5,000,000 cells were found in twenty cases. Leucocytes. The behavior of the leucocytes is extremely vari- able. While catarrhal or simple exudative appendicitis usually fails to show leucocytosis, Da Costa found over 15,000 cells in five of forty- five such cases, average 8987, and once 17,100. This leucocytosis he attributes to local peritonitis or to conditions apart from the appendicitis. With necrotic lesions, abscesses, gangrene, and general peritonitis there is well-marked leucocytosis in nearly all cases, except in the fulminant and asthenic type of the disease, in which the leucocytosis may fail. The majority of all cases fall in this category, as did thirty-three of seventy-two reported by Cabot, and ninety-two of 139 by Da Costa. While most of these cases show more than 1 5,000 white cells, and many more than 20,000, a considerable number fall below 15,000 within the limit frequently seen in simple exudative processes. As in pneumonia, etc., very marked or very slight leucocytosis may be found in grave cases calling urgently for operation, while the sudden transformation which follows perforation from a necrotic process of small extent commonly fails to give warning by a disturb- ance of the leucocytes. Cabot reports four cases of general periton- itis without leucocytosis and two with slight increase (14,800, 16,000). In following the course of appendicitis the daily examination of the blood may bring to light some important considerations. A steadily increasing leucocytosis usually indicates an extending local process. After an abscess has been formed the leucocytes commonly fluctuate between distinctly high limits, rising abruptly if peritonitis develops. As the abscess becomes walled off the leucocytes slowly diminish, and when pus is evacuated a favorable course is indicated by a rapid decrease of leucocytes. 326 THE ACUTE INFECTIOUS DISEASES. In chronic cases without active formation of pus the leucocytes are usually subnormal (Bloodgood). It, therefore, appears that the interpretation to be placed upon the examination of the blood in appendicitis, especially in its early stages, is extremely variable. With leucocytosis ranging between 8000 and 16,000 cells, the condition may rather frequently be : (1) simple exudative appendicitis ; (2) necrotic inflammation with perforation and abscess ; (3) a large abscess ; (4) general peritonitis. The blood changes follow no definite rule in those dangerous forms of the disease which pursue a mild course for a time, but suddenly develop peritonitis from perforation. Nor can the question of opera- tion in frank cases be decided from the examination of the blood alone, which indicates only what damage actually exists, while the operation is intended to meet the conditions which may later arise. One of Cabot's cases with 33,000 white cells recovered without inter- ference. After the period of invasion is passed and the character of the lesion established, the indications of the blood have proved much more reliable. In differential diagnosis a positive conclusion seems to be warranted only in those cases which show well-marked leucocytosis, from the presence of which it is possible to rule out nearly all forms of typhoid fever, most but not all cases of fecal impaction, and nearly all forms of abdominal neuralgia. Hubbard, Da Costa, and Cabot have offered some very practical comments on the limitations of blood analyses in this field. ABSCESS FORMATION. Active suppuration in a confined space is almost invariably accom- panied by distinct leucocytosis. Cabot has remarked on the high leucocytosis usually observed with very limited suppurative foci in subcutaneous, submucous, or interstitial connective tissues, and reports very considerable increase of white cells, with furuncle, car- buncle, and abscess of vulva, vagina, middle ear, uterus, Fallopian tube, ovary, lung, brain, parotid gland, neck, and in pectoral, psoas, and perinephritic regions. The writer's experience in a considerable variety of such conditions fully accords with these reports, leucocy- tosis having been present in the active stages of all such processes, but subsiding slowly or rapidly after operation or after the exudative process had ceased. An important exception to the rule was found in abscess of the liver with mucopurulent exudate. (See Liver.) Glycogenic degeneration of the leucocytes, according to Locke's observations, occurs in considerable degree with all large abscesses, but is not a reliable indication of the presence of pus, since it may be absent with small abscesses, and disappears in forty- eight hours after the abscess is drained. ERYSIPELAS. A slight diminution of red cells and Hb was observed in severe cases by Malassez, Hayem, and Reiuert. In mild cases the fibrin MISCELLANEOUS INFECTIOUS DISEASES. 327 is not affected, but in severe forms of the infection its formation is much increased (Turk). Leucocytosis occurs in nearly all cases, but Rieder reported one instance of facial erysipelas with a temperature of 39° C. and 6800 white cells. Turk observed one case of considerable extent with temperature 38.5° to 40.2° and the leucocytes between 7000 and 8900. Later the temperature rose to 40.5° and the leucocytes to 10,100. Zappert, also, observed two febrile cases with subnormal numbers of white cells (5500 to 6500). ITayem found between 7000 to 8000 leucocytes in very mild cases, but 12,000 to 20,000 when the rash was extensive. Most of the cases of Pee, Rieder, Ehrlich, Limbeck, and Cabot gave leucocytosis of moderate grade, but in proportion to the severity of the disease. Higher leucocytosis was observed by Halla in uncom- plicated facial erysipelas, and in a case complicated by pneumonia (23,000). Suppuration raised the count to 39,600 in a case of Rein- ert, and to 59,400 in one of Epstein. Chantemesse and Rey found that the leucocytes and the temperature vary as a rule together, although in some cases high fever was unaccompanied by leucocy- tosis. "When the total increase is considerable the polynuclear cells are much increased in proportion, but with slight increase the polynuclear cells are usually unaffected. Eosinophiles are usually diminished or absent (Zappert, Turk). Scanty myelocytes may also be found dur- ing the active stages, 5 per cent, of these cells having been recorded by Turk. Negative bacteriological examinations of the blood in erysipelas are reported by Petruschky in three cases and by Kraus in one case. Bertelsman had negative results in three severe cases, and positive cultures in two fatal cases of phlegmonous type. ACUTE RHEUMATISM. A much increased formation of fibrin has been noted by many observers, Halla, Hayem, Berggrun, Turk. Garrod failed to find any uric acid in the blood of acute rheumatism, and Salmon demon- strated the absence of lactic acid. Red Cells. It is a general clinical rule that patients who pass through an attack of acute rheumatism become distinctly anemic. In many cases of ordinary severity the loss of red cells is slight, while the Hb falls to 80 or 65 per cent. In more severe cases Hayem seldom found more than 4,000,000 cells. Sorensen found an average of 4,160,000 in eight cases. In prolonged and relapsing cases the red cells not infrequently fall slightly below 4,000,000. Yet in few of the reported studies are the examinations sufficiently extensive to show that such anemia is referable to the acute disease. Turk, while admitting the usual presence of anemia even during the febrile period, saw a distinct increase in red cells beginning with defervescence, and in Cabot's cases it does not appear either that there was any uniform loss of red 328 THE ACUTE INFECTIOUS DISEASES. cells or that the cases of long standing were invariably the more anemic. It seems, therefore, that the severity of post-rheumatic anemia has been overestimated. The Hb suffers more severely and much more constantly than the red cells. Cabot's average in thirty-one cases was 67 per cent., and a few cases fell below 60 per cent., while Turk found between 60 and 80 per cent. Prompt use of salicylates prevented the loss of Hb in a case of Lichtenstern's, and apparently also in one of Cabot's. Da Costa reports two very anemic cases with 26 and 30 per cent, of Hb, and 1,240,000 and 1,600,000 red cells. In convalescence the restoration of Hb remains considerably behind that of the red cells. Leucocytes. In mild cases without exudation there is usually no distinct leucocytosis (Pee, Rieder). When fever and swelling of joints exist there is almost always an increase to 10,000 or 1 5,000 cells. Turk insists that when the leucocytes reach 20,000 or more there are nearly always complications, such as pleurisy, pericarditis, or pneumonia. Hayem also found 17,000 to 18,000 cells in moder- ately severe attacks, and as high as 25,000 only in extremely severe and cerebral types of the disease. Cabot reports 21,000 to 31,000 cells in six cases, one complicated by acute endocarditis, while in the others no complication was mentioned. The writer, from the exami- nation of forty cases, in 1893, can support Turk's statement, having found signs of pneumonia, or pericarditis, or hyperpyrexia, whenever the leucocytes rose above 20,000. The pneumonic signs, however, were not always those of complete consolidation. With defervescence the leucocytes promptly fall to normal, and in relapses are much less affected than by initial attacks (Turk). Types of Leucocytes. When the white cells do not greatly exceed 10,000, the proportions of the various forms are not much disturbed. With distinct leucocytosis the proportion of polynuclear cells rises to a considerable height. Eosinophile cells are absent only in the early stages ; later, in spite of fever and exudation, they are always present in moderate numbers, "while after defervescence most cases show a distinct tendency toward eosinophilia (Turk, Achard, Loeffler). In one of Turk's cases there were 13.8 per cent, of eosino- philes, and in another 8.33 per cent, shortly after defervescence. This observer believes that a high proportion of eosinophile cells during the -febrile period is a good prognostic sign, and occurs principally in self-limiting cases. Bacteriology. In several studies, reviewed by Sittmann, various pathogenic bacteria were obtained from the circulating blood, but these cases have later been classed as examples of septicemia and not of articular rheumatism. The principal bacteriological studies of the blood of acute rheumatism are those of Sittmann, Singer, Kraus, and Kuhnau. Sittmann obtained negative results from repeated cultures in five cases, and Kraus in twelve cases. Singer conducted an elab- orate study of the blood and urine in sixty cases, but while he found staphylococcus alhus in several instances, this germ was probably a contamination from the skin. In one case streptococcus pyogenes was MISCELLANEOUS INFECTIOUS DISEASES. 329 isolated, but the history shows the patient to have been suffering from hemorrhagic septicemia and endocarditis. Even more conclusive were the totally negative results obtained by Kuhnau in sixty-seven eases representing all types of the disease and many complications. In the etiology of rheumatism several theories are at present actively maintained. Singer urgently claims that the disease is a mitigated form of pyemia from infection by streptococcus or staphylo- coccus pyogenes. A considerable number of observers, including Leyden, Triboulet, Wasserman and Meyer, and Poynton and Paine, have isolated from the blood and tissues a diplococcus which repro- duces the disease in rabbits more or less perfectly and which they regard as the etiological agent. Singer thinks that this diplococcus is the streptococcus pyogenes, but it decolorizes by Gram's method. Achalme and various other French observers have endeavored to prove the importance of an anaerobic spore-bearing bacillus obtained in several cases from the heart's blood and tissues. Others regard this bacillus as a contamination from the media. Predetchensky, who found the streptococcus in the blood in two of five cases, failed to secure any trace of Achalme's bacillus in large quantities of blood with liquid media and complete anaerobiosis. It is generally believed that bacterial invasion is a secondary phenomenon, and that while the disease may be of bacterial origin the circulating blood i« nearly always sterile. At present it is impossible to decide between the claims of Singer and the results of Kuhnau, and those of Poynton and Paine, Triboulet and Coyon, or Achalme. TONSILLITIS. Follicular tonsillitis usually causes moderate leucocytosis, seldom rising above 15,000 (Halla, Pick, Pee), yet in mild cases it may be absent (Cabot). In phlegmonous tonsillitis more marked leuco- cytosis, reaching 20,000 cells or more, usually occurs (Pee, Rieder). The leucocytosis is generally more marked than in diphtheria with equal constitutional disturbances (Pee). WHOOPING-COUGH. Meunier has observed, in thirty cases of pertussis in children of various ages, a pronounced or extreme leucocytosis which "far exceeds the increase found in any other afebrile disease of the respira- tory passages." The grade of leucocytosis varies with the age of the patient, being most marked in children under four years, and usually reaching a slightly lower figure in children from four to seven years. It appears in the catarrhal stage before the characteristic cough, when its demon- stration may be made of diagnostic value, and disappears slowly with the improvement of the disease. It is little influenced by complica- tions. The average leucocytosis was 27,800, but in several cases 40,000 cells were found, and in one 51,150. The lowest figure 330 THE ACUTE INFECTIOUS DISEASES. obtained during an active period was 15,500. _ The proportion of lymphocytes was always high, averaging 53.8 in a portion of the cases, while the polynuclear cells maintained a low average (39 per cent.), and the eosinophiles were scarce or normal in number. Meunier refers the lymphocytosis to hyperplasia of bronchial lymph nodes. De Amicis and Pacchioni also report marked leucocytosis in whooping-cough, beginning in the first days of the disease, reaching its height in the spasmodic stage, and being sometimes prolonged after the cessation of the typical cough. Cabot found 12,600 white cells and 78 per cent. Hb in a girl aged six years. In four cases in children between the ages of two and six years Stengel and White noted leucocytosis— 12,145 to 34,667 — of which the polynuclear cells constituted 30 to 40 per cent. INFLAMMATIONS OF SEROUS MEMBRANES. Pleurisy, Pericarditis, Peritonitis. Serous inflammations of the large serous membranes usually cause a slight increase of leucocytes in the blood during the acute febrile stages. Thus Hayem found between 7500 and 12,000 leucocytes in acute pleurisy ; Eieder 11,000 to 13,000 ; and some of Cabot's early cases registered as high as 15,000. Higher leucocytosis belongs to the more severe inflammations, with temperatures reaching 101° to 104° F. In two cases of serofibrinous pleurisy Limbeck found, with a temperature of 38.8° C, 18,000 to 19,000 leucocytes, and with a temperature of 41.5° C, 22,000 cells. The grade of leuco- cytosis excited by fibrinous pleurisy, pericarditis, etc., has apparently not been determined. The inflammation stands in an intermediate position in point of intensity. With purulent processes the leucocytosis is usually much higher. In 1893 the writer found no difference in the leucocytosis of pneu- monia from that of idiopathic empyema and purulent pericarditis. All the cases of pericarditis were complicated by pneumonia or rheu- matism and gave very high counts (maximum 60,000). The writer has seen one case of very rapidly fatal empyema, yielding streptococcus pyogenes in cultures, in which there was scanty purulent exudate and slight and diminishing leucocytosis. In purulent peritonitis, also, leucocytosis is not infrequently absent. After the exudative process has ceased the leucocytosis subsides, very rapidly in the case of serous exudates and usually more slowly with purulent processes. The majority of serous effusions are there- fore encountered, as in Halla's and Cabot's cases, when the leucocytes are normal in number ; nor is it rare to find extensive empyema with no excess of white cells in the blood. Tuberculous inflammations of serous membranes, when pure and uncomplicated, are usually not accompanied by leucocytosis. In serous pleurisy it is seldom possible to distinguish the quiescent stages of simple serous effusions from beginning tuberculous exudates, MISCELLANEOUS INFECTIOUS DISEASES. 331 and the examination of the blood cannot prove of much value in diagnosis. Further considerations of the changes in the blood in inflammations of serous membranes is to be found in the sections dealing with appendicitis, rheumatism, tuberculosis, etc. GONORRHEA. In acute gonorrheal urethritis the red cells remain unaffected. A moderate leucocytosis is usually observed in severe febrile cases, and is markedly increased by many complications, including cystitis, epididymitis, orchitis, etc. The polynuclear cells are in the usual excess, while eosinophiles, although sometimes very abundant in the urethral discharge (Bett- man), usually remain within moderate limits (0.5 to 11.5 per cent.) in the blood (Vorbach). Bettman, however, while finding that there is no relation between the eosinophile cells of the discharge and those in the blood, believes eosinophilia is usually present in gonor- rhea, especially in posterior urethritis. He found 25 per cent, in the blood in a case of gonorrheal epididymitis. In gonorrheal rheumatism the changes in the blood are similar to those of the idiopathic joint affections. The gonococcus has several times been isolated from the blood in gonorrheal endocarditis (q. v.). Panichi, in two cases of gonorrheal rheumatism without endocarditis, reports the isolation of the gono- coccus from blood sowed on human blood serum. He believes that the culture of the blood in order to be successful must be made at the time of invasion of the rheumatic symptoms. YELLOW FEVER. From Jones' interesting observations about yellow fever, it would appear that anemia is infrequent, that fibrin formation is deficient, that the globulicidal action of the serum is very greatly increased, and that cholemia is responsible for some of the changes observed in the red cells before and after shedding. " When a drop of blood from a yellow-fever patient falls upon blotting-paper, a dark, brownish ring due to diffusion of bile-stained plasma spreads about the central mass of blood cells " (Jones). Pothier examined in some detail the blood of 154 cases. The red cells never fell below 4,280,000, and in one fatal case were normal. The Hb suffered a considerable loss during the course of the disease, registering between 50 and 72 per cent. In the above fatal cases 90 per cent, of Hb was found. The restoration of the Hb was slow. Morphological changes in the red cells were usually absent, hut the presence of normoblasts was noted in a few specimens. The leucocytes fell between 4660 and 20,000. The polynuclear cells were found in high proportions in some of the slides from these cases examined by Cabot, while in others their proportions were normal. Eosinophiles were very scarce and myelocytes were found 332 THE ACUTE INFECTIOUS DISEASES. in two cases. The writer examined dry specimens of the blood of two rapidly fatal cases of yellow fever brought from Rio Janeiro by Dr. J. M. Masury, and noted slight hemoglobinemia and hypoleuco- cytosis. Pothier tested the action of the serum of some of his patients on cultures of Sanarelli's bacillus, but the results did not indicate the presence of any specific power in the blood. Arohinard and Woodson claim to have found the blood in yellow fever to agglutinate the bacillus of Saranelli in 75 per cent, of the cases. Reed and Carroll 1 have shown, however, that while this reaction occurs in a small proportion of cases (34 per cent.), it is in those same cases exhibited also with the bacillus of hog-cholera, with which they identify the bacillus of Sanarelli. Through the labors of Reed, Carroll, Agramonte, and Lazear, the importance of Sanarelli's bacillus in yellow fever has been set aside, and it has been shown that the specific virus of the disease is present in the circulating blood and is transferred from one subject to another by means of the mosquito, cule.v fasciatus. An interval of about twelve days or more after contamination appears to be necessary before the mosquito is capable of carrying the infection. They succeeded in producing yellow fever in a series of non-immune subjects through the agency of mosquitoes under con- ditions precluding the possibility of error. They also transferred the disease by injections of blood taken during the first and second days of illness. 3 The blood itself was found sterile. In three cases blood heated for ten minutes at 55° C. failed to cause any symptoms when injected in doses of 1.5 c.c. in non-immune subjects. Of three subjects receiving injections of 1.5 c.c. of yellow-fever serum passed through a Berkfeld filter two developed the disease. From one of these two cases serum was obtained and injected into a non-immune subject who also developed the disease. Reed and Carroll, therefore, conclude that the contagium of yellow fever is a very minute organ- ism capable of passing through a Berkfeld filter. TYPHUS FEVER. In four cases the writer found between 5000 and 9000 leucocytes. The patients were adults, the examinations were made during the high fever of the early period of the disease, and at least two of the patients died. Tumas followed one fatal case during a three weeks' course, observ- ing a steady reduction of red cells from 4,400,000 to 3,100,000, of Hb from 80 to 50 per cent., and hypoleucocytosis 9600 to 1600. EPIDEMIC INFLUENZA. The scanty reports of the condition of the blood in epidemic influ- enza indicate that the disease in uncomplicated form fails to cause leucocytosis. This fact, while in accordance with the catarrhal character of the essential lesions, is somewhat difficult to reconcile MISCELLANEO US INFECTIO US DISEASES. 333 with the acute infectious nature of the disease. Grippe appears to be the only bacterial disease, beginning acutely with marked chill, which fails to induce leucocytosis. The absence of leucocytosis in grippe was first demonstrated by Rieder, 2 who, in seven cases, found that the white cells at the acme of the disease were reduced in number (7000 to 2800). In the catarrhal pneumonia which complicates the disease and which is distinguished by signs of incomplete consolidation, he found little or no leucocy- tosis (maximum, 13,000). In lobar pneumonia following grippe the usual leucocytosis was observed. In 1893 the writer found no differ- ence in the leucocytosis of lobar pneumonia following grippe from that of primary pneumonia. Cabot reports the examination of the blood in sixty-seven cases, most of which showed normal or reduced numbers of white cells, and when there was leucocytosis (maximum, 14,000) complications were usually present. Bacteriological Examination. In a considerable proportion of cases Canon, Klein, 2 Hirschfeldt, Bruschettini, and others have obtained from the blood cultures of what they believed to be Pfeiffer's bacillus. Successful cultures are sometimes found to have been secured from blood squeezed from the finger-tip, as by Slawyk. Pfeiffer and Kruse and others were unable to isolate the bacillus of influenza from the blood, and denied the identity of some of the germs obtained from the blood by others. Recently Kuhnau has added twelve negative results, and there seems little reason to doubt that the bacillus of influenza has no special capacity to invade the blood stream. BABIES. Friedrich, 2 in 1869, observed an increase of white blood cells eighteen hours before death. Courmont and Lesieur have recently followed the leucocytes in the disease in botli man and animals. Rabies in man is apparently unaccompanied by leucocytosis until a few hours before death, when a terminal increase, principally of polynuclear cells, appears. In two cases one hour before death 21,000 and 24,000 cells were counted, while in three cases the antemortem proportion of poly- nuclear cells was 83 to 88 per cent. In experimental rabies no leucocytosis developed until nervous symptoms appeared. In both man and animals, however, an excess of polynuclear cells was found before any distinct increase in their numbers. In spon- taneous rabies in dogs (five cases) there was leucocytosis, with 88 to 98 per cent, of polynuclear cells, twenty-four to forty-eight hours before death. TETANUS. Cabot reports 70 per cent, of Hb, 11,900 leucocytes, and persist- ence of eosinophile cells, in a fatal case of tetanus treated by antitoxin, and 19,600 in a second case. 334 THE ACUTE INFECTIOUS DISEASES. BUBONIC PLAGUE. Coagulation of the blood failed entirely in ten of twelve fatal cases in which the blood was collected in sealed tubes by Corthorn, while in two other fatal cases the clot was imperfect. The Austrian Pest Commission found 65 to 80 per cent. Hb in the majorty of cases. Leucocytosis was always present, but not in excessive degree, the numbers varying between 12,000 and 28,000. Aoyaina, in 1895, examined the blood of many cases, reporting four in full. The red cells varied from 4,400,000 to 8,100,000, the latter in a case which recovered, and in the report of which no cause of polycythemia is apparent. The Hb was normal. The leucocy- tosis also was reported to have reached a very high grade. Poly- nuclear cells made up the bulk of the increase, but large and small lymphocytes were numerous, and eosinophiles scarce. Zabolotny studied the reaction of the blood serum on cultures of the bacillus. During the first week no effect was observed even with a dilution of 1:6; in the second week reactions were obtained with dilutions of 1 : 10 ; in the third week with 1 : 25 ; in the fourth week with 1 : 50. The reaction was most marked in the severer cases, and the blood serum of the cadaver was inert. Kitasato, in 1894, reported the demonstration of bacillus pedis in' twenty-five of twenty-eight cases in smears of blood squeezed from the finger-tip, but this method gives no reliable indication of the presence of the organism in the circulating blood. Many later observers, relying on the same method, have drawn erroneous con- clusions regarding the occurrence of this bacillus in the blood. Thus, Rees found it in the smears of the blood when cultures of the blood drawn aseptically were sterile. The Austrian Commission and others came to the conclusion that the bacteriological examina- tion of blood smears in plague is not only unreliable, but fallacious and mischievous. They placed little diagnostic value even on the proper bacteriological examinations of the blood. MALTA FEVER. The hemorrhages which mark the severer forms of this malady commonly give rise to severe anemia. Bruce places the average number of red cells at 3,500,000, and reports the absence of leuco- cytosis and occasional presence of free pigment in the blood. In a prolonged case without hemorrhages Musser and Sailer found 5,050,000 red cells, 60 per cent. Hb, 11,564 leucocytes, all varieties being in normal proportions. Wright and Smith found that the blood serum of cases of Malta fever agglutinates cultures of baeillus melitensvs, and diagnoses of the disease have been made by this method by Musser and Sailer, Cox, and others. A dilution of serum 1 : 50 should give a prompt reac- tion, while typhoid serum has no effect. MISCELLANEOUS INFECTIOUS DISEASES. 335 ACTINOMYCOSIS. The writer found 21,500 leucocytes in a case of pulmonary actino- mycosis, clinically resembling acute phthisis, and Cabot reports a case of actinomycosis of liver with 3700 white cells. The markedly purulent character of the exudate excited by the ray fungus explains the grade and character of the leucocytosis. Bierfreund found marked chlorotic anemia in actinomycosis, the Hb registering 30 to 50 per cent. GLANDERS. Cabot refers to a case of acute glanders in which the fibrin was increased, the Hb normal, and the leucocytes varied between 11,600 and 13,600, 86 per cent, of which were polynuclear. Coleman and the writer reported an acute case with extensive pulmonary lesions, in which the leucocytes steadily numbered 13,000. Bacillus mallei was isolated from the blood three days before death. Duval isolated the bacillus of glanders from the blood during life, and Noniewitch reports that in fatal cases in horses the bacilli can be found in the circulating blood, usually within the leucocytes. ANTHRAX. Anthrax in some lower animals frequently develops into a pro- nounced bacteremia early in the disease. Man, however, usually exhibits only a moderate grade of susceptibility to the infection and the majority of cases recover after excision of local foci of infection. Although it is probable that in the severe septicemic forms of the disease anthrax bacilli may multiply in the general blood stream early in the disease, the literature appears to contain very few re- ports of competent bacteriological analyses of the blood in such con- ditions. The rather frequent impression that the diagnosis of anthrax can be made by morphological examination of a drop of blood from the general circulation is almost entirely without foundation, as the great majority of the cultures of the blood, even in severe cases, have been found sterile. Only in the late stages of some septicemic cases in man do the bacteria invade the circulation and yield positive cul- tures of the blood. Such a case is that of Blumer and Young, who report successful cultures, and claim to have identified the germ in blood smears during life, but the time before death was not stated. Antemortem invasion of the blood by anthrax in man is apparently not much if any more abundant than with some other infections, and even postmortem cultures are frequently negative. Detailed clinical examinations of the red cells and leucocytes in the blood of anthrax are still wanting. 336 THE ACUTE INFECTIOUS DISEASES. Bibliography. Miscellaneous Infectious Diseases. Achard, Loefler. Compt. Rend. Soo. Biol, 1900, p. 1029. Aoyama. Mittheil. a. d. kaiserl. Japanisch. Univers., 1895, Bd. 3, No. 2. Bein. Zeit. f. klin. Med., Bd. 17, p. 545. Bertelsman. Arch. f. kl. Chir., Bd. 67, p. 940. Bettman. Archiv f. Derm, und Syph., Bd. 49, p. 227. Bierfreund. Archiv f. Chirurg., Bd. 41. Blum. Munch, med. Woch., 1893, p. 297. Blumer, Young. Johns Hopkins Bulletin, 1895, p. 12't Bommers. Deut. med. Woch., 1893, p. 552. Bond. Lancet, 1887, II., pp. 509, 557. Braidwood. On Pyemia, etc., London, 1868. Brieger. Charit6-Annalen, 1886, p. 198. Bruce. Lancet, 1892, II., p. 1101. Annal. de l'lnstitut Pasteur, 1893. Brunner. Wien. klin. Woch., 1891, p. 391. Bruschettini. Rif. Med., 1893, No. 186. Cannon. Deut. Zeit. f. Chirurg., Bd. 33, p. 571. Canon. Virehow's Archiv, Bd. 131, p. 401. Cantu. Rif. Med., 1892, II., p. 243. Coleman, Ewing. Jour of Med. Research, vol. ix., p. 223. Corthorn. Brit. Med. Jour., 1902, I., p. 1143. Courmont, Lesieur. Jour, de Phys. et de Path., 1901, p. 599. Cox. Philadelphia Med. Jour., vol. iv. p. 491. Czerniewsky. Archiv f. Gynecol., 1888, Bd. 33, p. 73. De Amicis, Pacchioni. Ref. in Amer. Jour. Med. Sci., vol. cxix. p. 599. Duval. Archiv. de Med. exper., 1896, p. 361. Ehrlich. Charite-Annalen , Bd. 12, p. 288. Eiselberg. Wien. klin. Woch., 1890, p. 731. Epstein. Allg. Wien. med. Zeit., 1889, No. 43. Friedrich. ' Arbeit, a. d. kaiserl. Gesundheit., 1890, p. 250. 2 Schmidt's Jam-., Bd. 144, p. 245. Carrod. Fort. d. Med., 1885, p. 165. Oarrod. Reynolds' System of Medicine. Coldberger, Weiss. Wien. klin. Woch., 1897, p. 601. Holla. Zeit. f. Heilk., Bd. 4. Hayem. 1 Du Sang. 2 Med. Moderne, 1897. Hirschfeldt. Baumgarten's Jahresber., 1893, p. 206. Hubbard. Boston Med. and Surg. Jour., vol. cxlii. p. 409. Jones. Jour. Amer. Med. Assoc, vol. xxiv. p. 402. Klein. * Cent. f. inn. Med., 1899, p. 97. 2 Baumgarten's Jahresber., 1893, p. 206. Kraus. Zeit. f. Heilk., Bd. 17, p. 117. Krebs. Diss. Berlin, 1893. Kruse. Cent. f. Bact., Bd. 7, p. 657. Kuhnau. Zeit. f. Hygiene, Bd. 25, p. 492. Livierato. Deut. Archiv f. kl. Med., Bd. 53,' p. 303. Locke. Boston Med. and Surg. Jour., vol. cxlvii. p. 289. Malassez. Archiv de Physiol., 1874, p. 32. Manassein. U. d. Verand. in d. Dimens. d. rot. Blutkorp., Tubingen, 1872. Maragliano. Cong. f. inn. Med., 1892. Meunier. Compt. Rend. Soc. Biol., 1898, p. 103. Musser, Sailer. Philadelphia Med. Jour., 1898, p. 1408. Noniewitch. Baumgarten's Jahresber., 1897, p. 415. Panichi. Settimana Med., Aug. 26, 1899. Patrigeon. Recherch. s. 1. nombre d. glob, rouges., Paris, 1877. Pee. Diss. Berlin, 1890. Petruschky. Zeit. f. Hygiene, Bd. 17, p. 59. Pfeiffer. Deut. med. Woch., 1893, p. 816. Pothier. Jour. Amer. Med. Assoc, vol. xxx. p. 884. Poynton, Paine. Brit. Med. Jour., 1901, II., p. 779; Lancet, 1900, II., pp. 861. 932. Predetschensky . Vratch, 1901. MISCELLANEOUS INFECTIOUS DISEASES. 337 Quincke. Virchow's Archiv, Bd. 54, p. 537. Raskin. Cent. f. Bact., 1889, pp. 286, 433. Reed. Med. Record, Aug. 10, 1901. Reed, Carroll. 1 Jour. Exper. Med., vol. v. p. 216. 2 Med. Record, 1901, vol. lx., p. 641. 3 Amer. Med., 1902, vol. iii., p. 301. Reed, Carroll, Agramonte. Jour. Amer. Med. Assoc, 1901, vol. xxxvi. p. 431. Rees. Brit. Med. Jour., 1900, II., 1236. Lancet, 1894, II., 428. Reinert. Zahlung d. Blutkorp, etc. Rieder. ' Leucocytose. 2 Munch, med. Woch., 1892, p. 511. Roscher. Blut. bei septisch. Fieber, Diss. Berlin, 1894. Rosenbach. Microorg. bei Wundinfection, Wiesbaden, 1884. Roux, Lannois. Rev. de Med., 1890,-p. 1011. Salmon. Jahresbr. f. Thierchemie, Bd. 10. Sanger. Deut. med. Woch., 1889, p. 148. Singer. Aetiol. d. acut. Gelenkrheumat. Singer. Wien. klin. Woch., 1901, p. 482. Sittmann. Deut. Archiv f. klin. Med., Bd. 53, p. 323. Spronck. Semaine Med., 1898, p. 393. Stengel, White. Univ. of Penna. Bull., vol. xiv. p, 310. Stern, Hirschler. Wien. med. Presse, 1888, p. 1102. Timofjewsky. Cent. f. Path., 1895, p. 108. Toenissen. Inaug. Diss. Erlangen, 1881. Triboulet, Coyon. Le Rheum, artic. aigu en Bacteriol., Paris, 1900. f.Turk. Klin. Blutuntersuchungen, Wien, 1897. Vorbach. Diss. Wurzburg, 1895. Wasserman. Berl. klin. Woch., 1899, p. 638. White. Jour, of Exper. Med., vol. iv. p. 425. Wright, Smith. Lancet, 1897, I., p. 656. Zabolotny. Compt. Rend. Soc. Biol., 1897, p. 520. 22 CHAPTEE XVI. SYPHILIS, TUBERCULOSIS, LEPROSY. SYPHILIS. Very accurate conclusions regarding the state of the blood in syphilis were reached by Becquerel and Rodier, who stated that a moderate grade of anemia were to be found in the great majority of cases, that as long as the disease progressed without complications the blood maintained a high or normal standard, and that if the course of the disease were prolonged there was a loss of red cells. They noted also that the abuse of mercury might lead to the same changes. The resemblance of the anemia of syphilis to that of chlo- rosis was also early recognized, especially by Eicord, and this belief was later supported by analyses of the blood by Grassi, Wilbouche- witch, Keyes, Laache, Malassez, 1 Gaillard, and others, who found in the secondary and tertiary stages of most cases a loss of red cells and Hb. Keyes was the first to point out that mercury in small doses, while curing the disease, increases the red cells. The grade of anemia observed was usually moderate, the majority of cases showing slight loss of red cells, which, however, sometimes fell to 3,000,000 to 4,000,000. The Hb was usually found dimin- ished more than the cells, especially by Lezius, who claimed that the only essential lesion of the blood in syphilis is a loss of Hb. Other patients appeared to have normal blood, and Sorensen in twenty cases failed to detect any distinct anemia. On the other hand, Miiller reported cases in which all the typical lesions of pernicious anemia were present, and in one of his cases the red cells fell to 428,100. Other such cases are reported by Ponfick, Kjerner, Klein, Laache, Fisischella (cited by Dominici). It was thus established that syphilis in some stages and in some patients may entirely fail to reduce the red cells, while in others grave pernicious anemia must be charged to its action. The later studies have been concerned with the more detailed course of the blood changes in the disease, and have been contributed principally by Anc, Lezius, Bieganski, Konried, Rille, Loos, Justus, and Riess. From these contributions it appears that the blood suffers in a somewhat uniform degree in the several stages of uncomplicated syphilis, but unfavorable conditions and abuse of mercury may greatly aggravate the changes observed. Primary Stage. During the first four to seven weeks after infec- tion the red cells do not diminish perceptibly in number (Lezius, Konried, Oppenheim and Lowenbach) unless there is fever or some other disturbing factor, when a moderate decrease may be noted from SYPHILIS, TUBERCULOSIS, LEPROSY. 339 the first (Stoukovenkoff). No doubt, as Hayem says, these disturb- ing factors are frequently present, and it still remains uncertain whether syphilis alone affects the number of red cells during the first few weeks. The majority of observations indicate that it does not, but Riess came to a contrary conclusion, while Bieganski found uniform polycythemia in the early stages of syphilis. Recently Oppenheim and Lowenbach in thirty-four cases failed to find any distinct reduction in red cells. The Hb is almost invariably diminished from the first, a loss of 15 to 30 per cent, being commonly noted before the appearance of secondaries (Konried). Secondary Stage. There is uniform agreement among very numerous observers that with the outbreak of secondary symptoms the red cells begin to fall rapidly, reaching in untreated cases as low as 2,000,000 or less (Konried). In ten cases Wilbouehewitch found an average decrease of 229,000 cells daily. At the same time the Hb continues to diminish and may fall to 55 or 25 per cent, within a few weeks or months. Riess denies that the Hb suffers particu- larly at the outbreak of the eruption. With the appearance of fever and new eruptions a further and more marked loss of red cells and Hb has been observed (Stoukovenkoff). Under favorable hygienic con- ditions and in feeble and especially in young subjects the anemia in this stage may be unusually severe. In untreated cases the disap- pearance of the eruption is not followed by any immediate improve- ment in the blood. Tertiary Stage. In untreated cases there can be no doubt that the anemia may progress until the pernicious type is established, but treatment usually limits the impoverishment of the blood, so that only moderate grades of anemia are commonly observed. Konried found an average of 4,000,000 red cells in ten cases, and from 50 to 80 per cent, of Hb in twenty-two cases suffering from gummatous lesions. In several cases showing advanced tertiary lesions (gummata) at autopsy the writer has found different grades and types of secondary chlorotic and pernicious anemia. In one the spleen was much enlarged and contained gummata, while the blood showed the lesion of secondary pernicious anemia, with a tendency toward the micro- cytic type, and with low Hb-index. In other cases the abundant megalocytes with increased Hb closely resembled those of primary pernicious anemia. It is especially in infants that syphilis induces the grave types of anemia. Effects of Mercury upon the Blood in Syphilis. The effect of proper doses of mercury in arresting the progress of the anemia of syphilis was clearly stated by Wilbouehewitch, who reported an average gain of 102,000 cells daily in ten cases, while Keyes and many later observers have fully verified these results. That pro- longed use of mercury may of itself lead to marked anemia resem- bling that of syphilis was early noted by Becquerel and Rodier, and was also demonstrated both in man and animals by Wilbouehewitch, and later by Bieganski, Hayem, Lezius, Anc, Schlesinger, and 340 THE ACUTE INFECTIOUS DISEASES. Jelleneff. The extent to which mercurial treatment may be carried without diminishing the red cells or Hb has been placed at twenty- four days, by Gaillard ; at twenty-five to thirty-five inunctions, by Konried ; at sixteen injections (grammes 0.5, 1 per cent. Hg-benzoate) by Jelleneff; and at 140 to 150 milligrammes of bichloride or 77 milligrammes of benzoate, injected in increasing doses, by Lindstrom. During this period most observers agree that both red cells and Hb increase from the first, and many instances of moderate poly- cythemia are recorded. At the same time the eruption commonly disappears. If treatment is continued beyond this point, especially if the patient is salivated, the cells and Hb steadily and sometimes rapidly decline and well-marked mercurial anemia is established. Jawein, however, failed to observe any anemia during prolonged courses of inunctions. The Leucocytes in Syphilis. Leucocytosis was early observed in syphilis and its usual connection with the hyperplasia of lymph nodes was one of the facts that led Virchow to locate the origin of the lymphocytes in these structures. More detailed study of the behavior of the leucocytes was made by Wilbouchewitch, Bieganski, Jelleneff, Konried, and Rille, who have shown that the leucocytosis of syphilis is connected principally with the eruptions and the anemia of the disease. In the primary stage, before the appearance of the eruption, the leucocytes are usually normal (Rille), but Jelleneff found that an increase of white cells usually precedes the development of anemia, and Konried's cases showed a slight leucocytosis (maximum, 16,400) in the first weeks of the primary stage. In the secondary stage, with the appearance of eruptions, anemia, and hyperplasia of lymph nodes, the leucocytes are nearly always increased. In Konried's cases of untreated secondary syphilis the white cells were never under 10,000, while the maximum" figure was 17,500. Riess, however, found leucocytosis absent in many cases, but observed an increase to 20,000 in some instances, while the excess of lymphocytes was usually very distinct (maximum, 68 per cent.). The administration of mercury commonly reduces the white cells, which become normal with the disappearance of the eruption and the anemia. Jelleneff found the leucocytosis to be more nearly proportionate to the extent of the eruption than to the size of the lymph nodes, and noted leucocytosis in the absence of external signs of lymphoid hyperplasia. The increase affects principally the small and large lymphocytes, but the eosinophile cells may also be increased, especially in those cases with marked papular exanthems (Rille). Zappert found a slight increase of eosinophiles, 4.91 per cent., in one early case, but normal numbers in seven others. The proportion of lymphocytes diminishes as the patient improves, falling after each inunction (Riess). In very severe cases there may be progressive polynuclear leucocytosis. Myelocytes have been found by Rille and others in both secondary and tertiary syphilis. In tertiary stages with gummatous lesions the leucocytosis usuallv SYPHILIS, TUBERCULOSIS, LEPROSY. 341 persists, but lymphocytosis, though sometimes distinct, is less con- stant. Konried found 8500 to 17,710 leucocytes in nine cases. Justus' Test in the Blood of Syphilis. By a series of observa- tions in over 300 cases Justus came to the conclusion that in the blood of florid syphilis after injections of moderate doses of various preparations of mercury, or after inunctions, there is a period of a few hours or days during which the Hb is considerably reduced (10 to 20 per cent.). After a certain period, varying with the general condition of the patient and the severity of the symptoms, the Hb Fia. 35. XI XII 1 3 10 1 ■ 18 10 2 21 22 23 U •S, 2 27 28 20 30 1 2 3 i 5 7 8 10 u 12 13 M 15 2 VII / / / / VII 8 / / / ,/ / / 105 I III . 1 V V VI / 1 4 j / / / / / 1 » 2 / 1 ' / / 1 1 / 1 / 1 .100 1 , 1 1 \ / 1 1 \ / / 1 8 / 1 1 / ■1 | 1 \ ' / OS f .1 3 1 2 | 1 HB.jJOO Course of changes in Hb in Justus' test. (Roman numerals indicate intravenous injections of sublimate.) begins to increase. In some cases the diminution continued only one day and in others it was repeated after each of three or four in- jections. The reaction was noted in all stages of the disease after the primary swelling of lymph nodes, and in hereditary syphilis, but could not be obtained " during or shortly before the symptoms of the disease begin to disappear." In the serum, within two minutes after the intravenous injection of bichloride, Justus found the spectrum of oxyhemoglobin. In blood smears immediately after the injection many pale degenerating 342 THE AOUTE INFECTIOUS DISEASES. red cells were found, which disappeared within a few minutes. It thus appeared that the mercury had caused an immediate solution of red cells in the plasma. The specific gravity of the blood was_ not studied except in one case in which it showed irregular fluctuations. Justus' test has been applied in 121 cases, collected from the reports of Cabot, Jones, Christian and Foerster, Brown and Dale, Huger, Oppenheim and Lowenbach, and Tucker. In active syphilis it was positive in 46 cases, nega- tive in 15, and the Hb rose instead of falling in 13. In cases of chancre there are 11 positive and 20 negative results, and in chancroid 6 positive and 3 neg- ative tests. Three negative results with herpes, 2 positive results in genito- urinary tuberculosis, and 1 positive result in chlorosis and in sexual neuras- thenia complete the observations of the above writers. With only 62 per cent, of positive results in active syphilis, 30 per cent, in chancre, and 67 per cent, in chancroid, it is evident that Justus' test can have only a limited value in the diagnosis of syphilis. Lately, however, Justus has reiterated his belief that the test is a specific sign of florid syphilis. He explains the negative results of other observers as due to the use of too little mercury or the absence of lesions of the lymph nodes as a necessary sign of a florid stage of the disease. Only inunctions of ung. hydrarg. he now finds suitable for the test, and three grammes must be used with adults or one gramme with children. Further studies are required before the value of Justus' test can be shown, but on general grounds it would seem that the test would prove unreliable in many forms of anemia, when the globulicidal action of mercury might dissolve red cells. Congenital Syphilis. Syphilitic infants invariably suffer from anemia. In the mildest cases Loos found over 5,000,000 red cells and 65 to 75 per cent, of Hb. Schiff also found the usual polycythemia of infants in mild cases of congenital syphilis. In the majority of cases there is con- siderable loss of red cells and the Hb falls to a low figure, minimum 21 per cent. (Loos). In the severer forms the blood may show the changes of secondary or progressive pernicious anemia, examples of which have been described by Loos, Luzet, Monti, Berggrun, and others. Whatever the grade of anemia, the changes are influenced by the special tendencies of infants' blood. Marked differences in the size of the cells are early established, nucleated red cells are frequently seen in abundance, and white cells, especially lymphocytes, are often present in greatly increased numbers. Loos noted an extreme degree of polychromasia in the large nucleated red cells in congenital syphilis. The leucocytes did not fall below 12,000 in any of Loos' sixteen cases, and in one fatal case rose to 58,000. Baginsky, and Monti and Berggrun have also reported excessive leucocytosis in similar cases, while the former author speaks of a distinct leukemic tendency to be found in the blood of congenital syphilis. The lymphocytes usually fall within normal limits for this age. Eosinophile cells are sometimes increased, especially when the eruption SYPHILIS, TUBERCULOSIS, LEPROSY. 343 is extensive (Loos). The presence of a few myelocytes in the severe cases makes the resemblance of these cases of syphilitic anemia to v. Jaksch's anemia often very close, while the excess of large lympho- cytes may lead to confusion of the disease with lymphatic leukemia of Fraenkel's type. Congenital syphilis is recognized by Demelin as one of the common causes of melena neonatorum. In such a case, dying four days after birth from abdominal hemorrhage, the writer found in the blood the typical characters of idiopathic pernicious anemia, especially in the large number of megaloblasts. The marrow was everywhere lymphoid, and showed pronounced megaloblastic changes. TUBERCULOSIS. The fact that the blood of consumptives may often fail to show any changes comparable with the pallor of the skin and the emacia- tion of these subjects was noted in the first studies of the blood by Andral and Gavarret, Becquerel and Rodier, and others. The dis- crepancy between blood and facial appearance was fully recognized by Laache, who, finding an average of 4,400,000 cells in fourteen cases, stated that phthisis of itself usually does not tend toward marked anemia. Similar results in the hands of Sorensen, Oppen- heimer, Gnezda, Barbacci, and Keinert, confirmed this opinion in a considerable group of cases, although well-marked chlorotic anemia was occasionally encountered. In the Massachusetts General Hos- pital series Cabot found forty-one out of sixty patients with more than 4,000,000 red cells, and sixty-nine of eighty cases with more than 50 per cent, of Hb. Yet Malassez had in 1874 reported well-marked anemia in some early cases, had seen the red cells fall over half a million in the course of one week, and found less than a million cells in an advanced case with diarrhea. In nearly all of their series of cases Laker and Fenoglio found low percentages of Hb, and the specific gravity was found markedly reduced by Schmaltz and Peiper. In 1893 current views were presented by v. Noorden, who stated that in pulmonary tuberculosis a loss of more than 20 per cent, of red cells or Hb is seldom observed unless from complications such as hemorrhage, suppuration, or amyloid degeneration. The subject was at this time thoroughly reviewed by Grawitz 1 and Strauer, who found three distinct periods in the changes of the blood in phthisis : 1. "With beginning apical lesions, they generally found marked chlorotic anemia with loss of red cells, irregular leucocytosis, and reduction in dry residue and specific gravity. In robust subjects, however, the blood during this stage was sometimes normal. 2. In pale, emaciated subjects with chronic phthisis and cavities, but without much fever, the blood commonly did not vary from the normal standard, the red cells numbering 5,000,000 or more, the leucocytes 5000 to 10,000, the dry residue of blood and serum being 344 THE ACUTE INFECTIOUS DISEASES. moderately low, but the specific gravity higher than the dry residue would indicate. 3'. "When hectic fever supervened, usually from suppuration in cavities, and the patient began to suffer from septicemia, the blood became rapidly impoverished, the red cells were then much reduced, the leucocytes frequently much increased, and the dry residue and specific gravity falling to a low point. The intense septicemia of rapidly advancing phthisis was found to yield some very severe grades of anemia, in one of which the red cells fell to 700,000. Similar cases have been reported by Malassez 2 and Limbeck, 1 both of which suffered from marked diarrhea. In Limbeck's case there was also tuberculous peritonitis, and the blood showed marked poikilocytosis and increased Hb-index. In the attempt to correlate his own and Kobert's divergent results, Dehio described two different types of the disease, the marantic and the anemic cases. In the former group the patients showed a marked tendency to emaciate while their blood retained a good standard, while in the others all grades of chlorotic anemia might be observed. It would seem that the classifications of both Dehio and Grawitz are based on accurate clinical study. The following conclusions may be drawn regarding the general course of blood changes in tuber- culosis : 1. The -primary anemia of tuberculosis is seen not only in diseases of the lungs, but quite as frequently in chronic tuberculosis of other tissues, especially of the lymph nodes and bones, constituting the very numerous group of cases of " lymphatic anemia." Moreover, it has recently been placed beyond doubt that the chronic anemia of Hodgkin's disease is in a large proportion of cases the result of tuber- culosis of lymph nodes. The peculiar character of these cases of secondary tuberculous anemia has been described by Laker, JSTeubert, Wiskeman, Vierordt, Bierfreund, Brown, Dane, and many others, and they constitute one of the largest groups of cases encountered in routine blood examina- tions. In appearance the patients clearly resemble cases of chlorosis and the blood shows a slightly or considerably reduced number of red cells, more marked loss of Hb, and usually slight relative or absolute lymphocytosis. Yet this description does not apply to all cases of early tuberculosis or phthisis, as some of them from the first appear anemic and yet show normal cells and Hb. 2. The second group in lohich the blood is but slightly altered in quality, although cavities have formed in the lung and the patient is pale and emaciated, includes the majority of cases of chronic phthisis. One of the most striking examples seen by the writer occurred in a patient suffering from subacute tuberculous empyema from which the pus contained very large numbers of tubercle bacilli. But the same quality of blood is sometimes seen with early apical lesions and moderate emaciation, while considerable anemia may exist in moder- ately advanced cases of phthisis who are not suffering from distinct hectic fever. SYPHILIS, TUBERCULOSIS, LEPROSY. 345 The cause of the concentration of the blood, or oligemia, in the average phthisical subject, for such must be the condition, is probably to be found, as indicated by Heidenhain, Gartner and Romer, and Grawitz, 2 in the specific lymphogogic action of the toxins of the tubercle bacillus, by which there is established a continuous excess in the balance of fluids which leave the tissues through the lymphatics. This view is strongly supported by the resemblance which exists between the tuberculous and the typhoidal processes, both showing a peculiar relation to lymphatic structures and both tending to concentrate the blood. Identical effects upon the blood follow also the experimental injection of tuberculin and typho- toxin. By this means the considerable destruction of blood which results from the chronic toxemia, hemorrhages, and malnutrition of the dis- ease is obscured, and only becomes visible at autopsies on these sub- jects when the shrunken appearance of the tissues finds a parallel in the diminished total volume of blood which is often apparent to the naked eye. In the majority of cases of well-advanced phthisis, there- fore, approximately normal blood indicates considerable absorption of the toxins of the tubercle bacillus. Prolonged and profuse night-sweats, and severe diarrhea, doubtless have a similar effect, which, however, is usually overbalanced by the destruction of blood which results from the associated toxemia. Normal blood is found in many phthisical patients who do not sweat or suffer from diarrhea. Yet in some extreme cases one or both of these factors may very well prove to be the chief influence in the concentration of the blood. 3. The third group of cases includes those who are anemic from the first or who become anemic in the terminal stage of the disease, in either instance from the destruction of blood which occurs in all severe toxemias. Since the severe hectic fever of phthisis is largely referable to mixed infection, the condition established is not very different from the ordinary type of pyogenic sepsis. The destruction of cells may be quite rapid. Malassez observed losses of 730,000 cells in one month, and 760,000 in three weeks in cases without hemorrhage. Here belong the acute cases with grave anemia described by Malassez, Limbeck, and Grawitz, from which it appears that the blood in tuberculosis may develop much the same characters as in pernicious anemia. Hills reports a chronic case of general tubercu- losis with melancholia, in which the red cells fell to 155,000. Morphological changes in the red cells are rather less marked than in most other types of secondary anemia, owing probably to the con- servative effect of a plasma of high gravity. When anemia exists it is usually of the simple chlorotic type with relatively high Hb-index. In his case of grave anemia Limbeck described extreme poikilocy- tosis, but nucleated red cells were apparently absent. In two very anemic acute cases lasting four and five weeks, the writer found under 2,000,000 red cells, but there were no distinct megalocytes and no nucleated red cells. Cabot finds that nucleated red cells are usually absent in tuberculosis, even after hemorrhage, and contrasts this fact with their abundance in carcinoma. Degenerative changes in the red 346 THE ACUTE INFECTIOUS DISEASES. cells in tuberculosis are not marked. In thirteen cases of uncompli- cated phthisis Grawitz 3 found no signs of granular degeneration. Effects of Complications. Hemoptysis causes impoverishment of the blood in proportion to the extent of the hemorrhage. Malassez observed a reduction of 940,000 cells from small hemorrhages in a period of eight days. Amyloid degeneration is seen with advanced and anemic cases, but its effects on the blood have not been specially studied. Stenosis of the larynx and diabetes have been found with concentrated blood (Grawitz). Regeneration of the Blood in Phthisis. Prompt regeneration of the blood after hemoptyses has been observed by Malassez, but such improvement must depend largely upon the general condition of the patient. Laker came to the conclusion that if the Hb did not steadily improve after operation on tuberculous foci, it might safely be con- cluded that all of the disease had not been removed. In seven of Bierfreund's cases and in two of Brown's this rule held good, and three times a steady decline in Hb preceded for several weeks the development of symptoms of general tuberculosis (vide infra). In phthisis as well as in other tuberculous processes great caution must be used in judging of the patient's improvement from an increase in red cells or Hb. The writer has seen the Hb and red cells increase while the -patient was rapidly losing flesh, the lesions advancing, and the total quantity of blood doubtless falling. In several of Bierfreund's cases the Hb steadily increased while the patient was developing general tuberculosis. The Leucocytes in Tuberculosis. In the majority of cases of uncomplicated tuberculosis the leucocytes remain within normal limits or are distinctly deficient in number. This rule has been established principally by later observers, since the earlier studies cited by Rieder, Reinert, and others indicated that tuberculous in- flammation usually excites leucocytosis. Yet Halla noted that leucocytosis was usually found only in advanced cases with fever, and Rieder distinguished the fresh cases from chronic febrile tuberculosis by the absence of any increase of white cells in the former. It is perhaps a still more uniform rule that leucocytosis is found almost exclusively in the febrile and anemic cases, but it is absent also in many febrile periods. In pulmonary tuberculosis an increase of leucocytes is usually referable to suppurating cavities, advancing pneumonia, severe anemia, or hemoptysis. Suppurating cavities, when of recent formation, usually raise the leucocytes distinctly, 15,000 cells being frequently seen, and when the lung is softening the excess may be much greater. The leucocytosis usually persists while the fever continues. With old cavities the expectoration may be abundant, but the leucocytes usually fall to normal. In the pneumonia of various types which complicates phthisis the leucocytes are usually much increased, but considerable areas of diffuse tuberculous pneumonia may be found with acute miliary tuberculosis, which have failed to increase the leucocytes during life. A lobar pneumonia causes the usual leucocytosis, but the writer has SYPHILIS, TUBERCULOSIS, LEPROSY. 347 seen both lungs consolidated and riddled with small cavities, in a case lasting five weeks, yet the leucocytes were never found above 12,000. The absence of leucocytosis in these cases of acute phthisis which resemble pneumonia may often be of value in diagnosis. Simi- larly in a case of subacute empyema in which the tubercle bacillus was largely concerned, the leucocytes were found not to exceed 14,000 during an acute febrile period. The leucocytosis of tuberculous cachexia is moderate, usually between 10,000 and 15,000. After hemoptysis the leucocytes are increased in proportion to the hemorrhage, but the increase is tran- sient. Acute general miliary tuberculosis offers the best illustration of the failure of pure tuberculous inflammation to induce leucocytosis. Rieder, Limbeck, Warthin, and Cabot find rather a diminution of leucocytes, often to an extreme degree, and, as Warthin has shown, during a prolonged period. Minor suppurative complications seem to have no capacity to raise the white cells, but in some reported cases a high percentage of polynuclear cells was noted among the scanty leucocytes (Warthin). Tuberculous inflammations of serous membranes, when uncomplicated by pyogenic infection, follow the type of pure tuberculous inflamma- tion elsewhere and fail to increase the leucocytes. In many cases, however, there appears to be mixed infection, and the effusion, instead of remaining serous or serosanguinolent, becomes seropurulent and purulent. In such cases of pleurisy with seropurulent or puru- lent exudate the writer has found a moderate increase of leucocytes, usually in proportion to the height of the temperature. While it is undoubtedly true that the tubercle bacillus may of itself produce pus, the fact remains that it is seldom or never seen alone in purulent exudates. The slight leucocytosis that accompanies cases of purulent tuberculous inflammations of serous membranes is perhaps a further indication that these cases represent mixed infections. Tuberculous Meningitis. In the majority of reported cases of this important localization of the disease leucocytosis has been absent, as seen by Limbeck, 2 Pick, Rieder, Sorensen. Yet in five of seven cases reported by Cabot and in one by Ziemke there was distinct leucocytosis, from 14,700 to 34,300, while in one of Rieder's cases there were 14,400 white cells. It is difficult to reconcile these con- flicting results. Some of the leucocytoses may have been ante-mortem phenomena, but not all. Ziemke's case was one of advanced general tuberculosis. The writer has seen cases with leucocytosis, but com- plicating terminal pneumonia was in each instance found at autopsy. Morphology of Leucocytes in Tuberculosis. In most cases of chronic tuberculosis, when the leucocytes are normal or diminished, the mononuclear cells are relatively in excess, but in some instances the usual rule is reversed, as in Warthin's case, and the polynuclear cells are in excess. Distinct lymphocytosis is seen in many chronic cases, especially those with large lymph nodes (lymphatic anemia). Lymph- atic leukemia sometimes develops in tuberculous subjects, but the relation of the two diseases is uncertain. When distinct leucocytosis 348 THE ACUTE INFECTIOUS DISEASES. occurs, excess of polynuclear cells is usual, and pyogenic infections, etc., are indicated. Neusser believes that eosinophilia indicates relative immunity to tuberculo- sis. General experience seems to show that eosinophiles are frequently present, usually in normal numbers, in the blood in chronic tuberculosis, and some- times absent in the acute cases, or when there is fever and leucocytosis. Zappert found none in 2 of 5 cases of febrile tuberculosis. They are commonly increased, sometimes to an extreme degree, in the febrile period following in- jections of tuberculin. (Zappert.) Tuberculosis of Bones and Joints. Valuable studies in this field have been contributed by Brown in seventy-two and Dane in forty- one cases of tuberculosis of hip and spine. From these studies it appears that the above conditions are very frequently accompanied by moderate or marked leucocytosis (maxi- mum, 41,000), which is usually referable to secondary infection and the formation of pus. Yet Brown observed several cases with leuco- cytosis but without formation of abscess, and believes that leucocy- tosis may be referable alone to increased activity of the tuberculous process. A frequent cause of leucocytosis, often of marked degree, was secondary infection of the sinus after operation. The leucocy- tosis was usually but not always associated with fever. Both authors agreed that when leucocytosis was absent in cases with abscess sec- ondary infection had not occurred, since the pus was sterile. They did not apparently consider the possibility that a micro-organism once present may disappear, or that leucocytosis may disappear when pus ceases to form. Myelocytes have been reported in scanty numbers in a few eases. Chemistry. Special chemical alterations of the blood in tubercu- losis have not been demonstrated, but the usual changes of secondary anemia have been reported in a few cases. Hammarschlag found a low gravity only in cachectic cases. Moderate reductions only were observed by Devoto and Scholkoff, but Schmaltz found a gravity of 1.036 in an advanced case of phthisis, and Grawitz's case of grave anemia gave 1.032. The gravity is much lower, in proportion to the loss of cells, than in pernicious anemia. Dieballa found a density of 1.039 in a case of phthisis with 2,400,000 cells, and in one of pernicious anemia with only 840,000 cells. Mircoli found the serum actively hemolytic in the early stages of some cases. Freund found in the blood of tuberculous subjects a body which he regarded as cellulose, which Nischimura has likewise identified, while others have found the same substance in tuberculous tissues. It is probably derived from the bodies of the tubercle bacilli. Bacteriological Examination of the Blood in Tuberculosis. Even before the discovery of the tubercle bacillus Weigert had pointed out the considerable involvement which the vessels may show in acute miliary tuberculosis. Weichselbaum, in 1884, was able with prolonged searching to find numbers of tubercle bacilli in stained specimens of the clotted blood of the great vessels in three cases of acute general tuberculosis. In the same year Meisels and Lustig SYPHILIS, TUBERCULOSIS, LEPROSY. 349 both succeeded in finding tubercle bacilli in the blood intra vitam, the_ specimens having been squeezed from the finger-tip during a period of rising pyrexia. In the blood of the spleen the bacilli were much more numerous and Meisels recommended aspiration of the spleen as an aid in the diagnosis of obscure cases. Rutimeyer also succeeded in finding many bacilli in the aspirated splenic blood of one of two cases examined shortly before death. In a case which eventually recovered Sticker, after considerable search, found a few bacilli in the finger blood taken on the tenth day of the illness. With the introduction of tuberculin, Liebman claimed to have found tubercle bacilli in 56 of 141 blood specimens, most abundantly about twenty-four hours after the injection of tuberculin. Grave doubt was, however, thrown upon all previous work in this field by the negative results obtained by Guttmann, and by Kossel, who called attention to the danger of contamination in such speci- mens. This method of diagnosis has not been followed up in later years, although Kronig, in 1894, recommended the staining of centrifuged laked blood in doubtful cases of miliary tuberculosis. In the blood of tuberculous cadavers various micro-organisms have been isolated by Pasquale, Petruschky, Canon, Welch and jN T uttall, and others. Cultures of the blood during life have been made, by reliable methods, by Sittmann, Kraus, Kuhnau, Hewelke, White, Michaelis and Meyer, and Hirschlaff . Of their seventy-nine cases of advanced and usually febrile phthisis, there were twenty-three positive and fifty-six negative results. Staphylococcus pyogenes aureus or albus was isolated in twenty cases ; streptococcus pyogenes and a diplococcus in one case each, and the tubercle bacillus was once obtained from inoculation by Kuhnau. The pyogenic germs were sometimes found together. Jakowsky and Petruschky were rather more successful, drawing the blood through the skin, but this method is unreliable. There can be little doubt, therefore, that the blood in the late stages of phthisis suffers bacterial invasion as in other forms of septi- cemia. Kuhnau's one positive result from twelve inoculations of the blood of severe febrile cases does not encourage further search for the tubercle bacillus in the blood of tuberculous subjects. Lasker also, in sixty-eight cases of hectic tuberculosis, found bacteria in the blood, streptococci but once. LEPROSY. Winiarski, in 1892, studied the blood in seventeen cases illustrat- ing various phases of this disease. In some cases he found polycy- themia, 6,380,000 to 6,090,000 red cells, which may perhaps be referred to local stasis, as the hands appeared cyanosed. Few of the patients showed any marked loss of cells, the average being 5,050,000 for nine men, and 4,300,000 for eight women. Winiarski, there- fore, concluded that the milder cases of one or two years' standing do not suffer from any distinct anemia. Yet in two advanced cases with extensive lesions the red cells numbered 2,300,000 and 1,900,000, and the latter patient presented many signs of pernicious anemia. 350 THE A C UTE INFECTIO US DISEASES. In these cases the anemia was referred to extensive ulceration. The Hb suffered even less than the cells, usually registering 80 to 118 per cent, in all but the two very anemic cases. The Hb-index was uniformly high, and in one case with 1,900,000 cells and 64 per cent. Hb the index reached 1.7. An increased diameter of the red cells was noted in this and other cases. The leucocytes were normal or subnormal in number, and the lymphocytes were in rela- tive excess (maximum, 47 per cent.), except when suppuration occurred. Brown, more recently, reported his observations in sixteen cases, eight of which appeared to be quiescent and presented normal blood, while only one showed severe anemia. He claimed to have found the bacillus in the leucocytes of the circulating blood in nine cases, eight of which showed the tubercular type of lesion. Streker also examined the blood of five " very anemic " cases of leprosy and found bacilli both free in the plasma and inclosed in leucocytes. The blood was drawn with antiseptic precautions from a deep incision through normal skin. These observations are quite in accord with the reports of Joseph regarding the large deposits of bacillus leprce in the spleen. Spronck claims that the serum of leprous subjects, in dilutions between 1 : 60 and 1 : 1000, agglutinates fresh living cultures of the bacillus of Hansen, and he recommends the use of this test in diagnosis. Bibliography. Syphilis, Tuberculosis, Leprosy. Anc. Monatshefte f . Prak. Dermat., Bd. 12, p. 266. Virchow-Hirseh. Jahres- ber., 1892, II., p. 537. Andral, Gavarret. Annal. de Chem. et de Physiol., 1840. Arloing, Courmont. Zeit. f. Tuberc, 1900, p. 11. Deut. med. Woch, 1900, p. 766. Gaz. des Hop., 1900, Dec. 1. Baginsky. Lehrb. d. Kinderkrank., Berlin, 1889, p. 287. Barbacci. Cent. f. med. Wissen., 1887, No. 35. Beck, Rabinowitch. Deut. med. Woch., 1900, p. 400; 1901, p. 145. Becquerel, Rodier Cited by Wilbouchewitch, Archiv. de physiol. norm, et path. , 1874, p. 509. Also, Memoir Acad, des Sci., 1844. Bendix. Deut. med. Woch., 1900, p. 244. Bieganski. Archiv f. Dermat. u. Syph., 1892, p. 43. Bierfreund. Langenbeck's Archiv, Bd. 41, p. 1. Brown. Occidental Med. Times, 1897, p. 537. Also, Trans. California Med. Soc, 1897, p. 168. Brown, Dale. Bost. Med. and Surg. Jour., vol. cxl. p. 323. Buard. These de Bordeaux, 1900. Cabot. Boston Med. and Surg. Jour., 1899, vol. cxl. p. 323. Canon. Deut. Zeit. f. Chirurg., Bd. 37, p. 571. Christian, Foerster. Univ. Med. Mag., May, 1900. Dane. Boston Med. and Surg. Jour., 1896, vol. cxxxiv. p. 559. Dehio. St. Petersburg med. Woch., 1891, p. 1, cited by Limbeck. Demelin. Mai. de l'Enfance (Grancher), II., p. 135. Devoto. Zeit. f. Heilk., Bd. 11, p. 176. Dieballa. Deut. Archiv klin. Med., Bd. 59, p. 308. Dominici. Presse Med., 1898, I., p. 468. Ewing. N. Y. Med. Jour., 1893, vol. lviii. p. 713. Fenoglio. Oesterreich med. Jahrb., 1882, p. 635. Fischl. Zeit. f. Heilk., Bd. 13, p. 291. SYPHILIS, TUBERCULOSIS, LEPROSY. 351 Fraenkel. Hyg. Rund., 1900, p. 630. Freund. Wien. med. Jahrb., Bd. 1, p. 335. Gaillard. Gaz. des Hop., 1885, Xo. 74. Gartner, Romer. Wien. klin. Woch., 1892, p. 23. Gnezda. Inaug. Diss. Berlin, 1886. Grassi. L 'Union Med., 1859. Grairitz. ] Deut, med. Woch., 1893, p. 1347. 2 Zeit. f. klin. Med., Bd. 21, p. 459. 3 Berl. klin. "Woch., 1900. Guttmann. Cited by Kossel. Hammarschlag. Zeit. f. klin. Med., Bd. 21, p. 475. Hayem. Du Sang., p. 920. Heidenhain. Pfliiger's Archiv, Bd. 49, p. 209. Hewelke. (Abstr.) Cent. f. Bact., Bd. 19, p. 563. Hills. Bost. Med. and Surg. Jour., vol. cxxxix. p. 542. Hirschlaff. Deut. med. "Woch., 1897, p. 766. Huger. Philada. Med. Jour., 1902, I., p. 849. Jakowsky. Cited by Sittmann. Jaioein. Cited by Lindstrom. Jellenef. Annal. de Dermat., 1892, p. 924. Jones. X. Y. Med. Jour., 1900, vol. lxxi. p. 513. Joseph. Archiv f. Dermat. u. Svph., Bd. 43, p. 359. Justus. Virchow's Archiv, Bd. 140, p. 91. Ibid., Bd. 140, p. 533. Deut. Arch. kl. Med., Bd. 75, p. 1. Keyes. Amer. Jour Med. Sci., 1876, vol. lxxi. p. 17. Kobert. St. Petersburg med. Woch., 1889, Xo. 32. Konried. Wien. klin. Woch., 1893, p. 341. Kossel. Berl. klin. Woch., 1891, p. 302. Kraus. Zeit. f. Heilk., Bd. 17, p. 117. Kronig. Deut. med. Woch., 1894, V. B., p. 42. Kuhnau. Zeit. f. Hygiene, Bd. 25, p. 492. Laache. Die Anaemie, 1883, p. 63 Laker. Wien. med. Woch., 1886, Xo. 18. Lasher. Cited by Stadelman, Deut. med. Woch., 1901, p. 411' Lezius. Blutver. bei Svphilitisehen, Diss. Dorpat, 1889. Li-ebman. Berl. klin. Woch., 1891, p. 393. Limbeck. l Grundriss, etc., p. 336. 2 Cited bv Rieder, Leucocvtose. Lindstrom. Presse Med., 1898, T. 13, p. 267." Loos. Wien. klin. Woch., 1892, p. 291. Lustig. Wien. med. Woch., 1884, p. 11S7. Malassez. 'Archiv. de physiol. norm, et path., 1S86. 2 Prog. Med., 1S74, p. 562. Meiseh. Wien. med. Woch., 1SS4, p. 11S7. Miehaelis, Meyer. Charit£-Annalen, Bd. 22, p. 150. Mircoli. Gaz. d. Osped., 1901, Nov. 3. Mongour. Compt. Rend. Soc. Biol., 1899, p. 564, 656. Midler. Charite-Annalen, Bd. 14, p. 253. Xischimura. Archiv f. Hygiene, Bd. 21, p. 52. Oppenheim, Lowenbach. Arch. kl. Med., Bd. 71, p. 425. Oppenheimer. Deut. med. Woch., 18S9, p. 859. Pasquale. Ziegler's Beitrage, Bd. 12. Peiper. Cent. f. klin. Med., 1891, p. 217. Petruschky. Zeit. f. Hygiene, Bd. 17, p. 59. Pick. Cited bv Rieder. Reinert. Zahlimg d. Blutkorp, p. 189. Ricord. Schmidt's Jahrb., Bd. 45, p. 45 Rieder. Leucocvtose, p. 126. Rille. Wien. klin. Woch., 1893, p. 155. Rothamel. These de Bordeaux, 1900. Rutimeyer. Cent. f. klin. Med., 1SS5, p. 353. Schiff. Pest. med. chir. Presse, 1S92, p. 48. Schlesinger. Archiv f. exper. Path., Bd. 13. Schmaltz. Deut. Archiv klin. Med., Bd. 47, p. 145; Scholkoff. Diss. Bern, 1892. Sittmann. Deut. Archiv klin. Med., Bd. 53, p. 323. Sorensen. Cited bv Reinert. Sticker. Cent. f. klin. Med., 1SS5, p. 441. 352 THE ACUTE INFECTIOUS DISEASES. Stoukovenkoff. Annal. de Dermat., 1892, p. 924. Strauer. Zeit. f. klin. Med., Bd. 24, p. 295. Streker. Munch, med Woch., 1897, p. 1103. Tucker. Philada. Med. Jour., 1902, L, p. 846. Vierordt. Cited by Reinert. Warthin. Medical News, 1896, vol. lxviii. p. 89. Weichselhaum. Wien. med. Woch., 1884, p. 364. Welch, Nuttall. Hyg. Rundschau, Bd. 2, p. 927. White. Jour, of Exper. Med., vol. iv. p. 425. Winiarski. St. Petersburg med. Woch., 1892, p. 365. Wiskeman. Zeit. f. Biol., 1873, p. 434. Zappert. Zeit. f. klin. Med., Bd. 23, p. 227. Ziemke. Deut. med. Woch , Apr. 8, 1897, cited by Cabot. PART IV. CONSTITUTIONAL DISEASES. CHAPTEE XVII. HEMORRHAGIC DISEASES, THE HEMORRHAGIC DIATHESIS, AND INTOXICATIONS. Hemorrhages of greater or less extent occur in many general diseases, in which the loss of blood is not referable to any specific predisposition on the part of the individual, but to peculiar condi- tions arising in the course of the disease. Such hemorrhages are regarded as symptomatic. The chief causes of symptomatic hemorrhages are : 1. Infections, as in any severe infectious diseases, such as septi- cemia, scarlatina, smallpox, measles, etc. 2. Mechanical, as in partly asphyxiated infants, or other condi- tions of marked venous stasis. 3. Toxic and autotoxic, as in poisoning by the so-called blood- poisons, or in jaundice, anemia, etc. 4. Nervous, as in rare forms of hysteria. In the hemorrhagic diathesis probably all the above factors are at times unusually active, but there is believed to be in addition a specific, pre-existing, often hereditary change in the blood which largely determines the various manifestations of this diathesis. Modern bacteriological and chemical research has now considerably narrowed the scope of the hemorrhagic diathesis, and at present it is impossible to claim that any peculiar predisposition to hemorrhage exists except in cases of hemophilia. Yet it remains convenient to describe the various idiopathic pur- puras, scurvy, and hemophilia as closely related conditions. PURPURA HEMORRHAGICA. (WERLHOFF'S DISEASE.) The etiology of this disease still remains obscure, although the evidence points more and more toward some form of infection as the essential element in the majority of cases. The prominence of hemor- rhage in the symptoms of many infectious diseases has prepared the way for an acceptance of this view. In adults the extent of the hemorrhage attending well-identified forms of infection is rarely so great as in pronounced cases of purpura hemorrhagica, in which often 23 354 CONSTITUTIONAL DISEASES. no bacterial origin has been demonstrable. In infants, however, infections by the ordinary pyogenic bacteria have become recognized as a frequent source of fatal hemorrhages constituting for that age the typical picture of Werlhoff 's disease. There arennw manycasesof purpuraheraorrhagica on record in which bacter- iological examination of the blood or viscera showed the presence of bacteria, which were regarded as the cause of the disease. In some cases bacilli were isolated, which were not fully identified. Letzerich's bacillus purpurea in many respects resembled bacillus anthracis, and produced in animals, and possibly in the investigator's own person, a hemorrhagic disease resembling Werlhoff's. Streptococcus pyogenes has been isolated in typical cases by Hanoi and Luzet, Widal and Therese, Guarnieri, and others. In the first of these the disease was transmitted from mother tofetus, the latter dying and yielding a pure cul- ture from the blood. In infants, rapidly fatal hemorrhagic infections of this nature are not rare. Staphylococcus pyogenes aureus has been isolated by Lebreton, Litten, Fischl and Adler, Lewis, Silvestrini, and others. Fischl and Adler claim to have produced a fatal anemia in animals by inoculation with their coccus. Kolb obtained sterile cultures of the blood in five cases, but from the viscera of three of these, three to four hours after death, he isolated a diplobacillus which caused hemorrhagic septicemia in various animals. Auche obtained both the staphylococcus and streptococcus in a fatal case, and Levi obtained in another both streptococcus pyogenes and the pneumococcus of Fraenkel. The pneumococcus lanceolatus was isolated postmortem by Claisse and by Claude. Bacillus pyocyaneus was obtained postmortem from a case of melena neonatorum by Neumann, and bacillus coli communis by Dansac, Legendre, and others. Hamilton and Yates report a rapidly fatal case showing, ten hours postmortem, bacillus aerogenes capsulatus. McLeod observed a case closely following Malta fever, and conditions described as purpura hemorrhagica have been reported as following pertussis, tuberculosis, congenital syphilis, etc. Negative results are reported in a fatal case by Denys ; in a purpuric complication of angina, by Legendre; and in chronic cases by Marfan, and by Millard. In the majority of the above cases the bacterium was obtained postmortem and from the viscera, or during life, from the lesions in the skin, and was rarely demonstrated in the circulating blood. Nevertheless, there can be but little doubt that a numerous group of cases of purpura hemorrhagica exists which is referable to a variety of bacteria, as represented in the above reports. It seems necessary, therefore, to provisionally separate the cases of purpura hemorrhagica of probable infectious origin from those which in no respect resemble an infectious disease, and are, therefore, probably acute manifestations of hemophilia. Changes in the Blood. In most of the febrile infectious cases of purpura hemorrhagica the anemia is not excessive and appears to be secondary to the infection and the loss of blood. In mild cases the red cells are slightly diminished in number, but exhibit no other alterations. Carriere and Gilbert report cases with 3,350,000 and 3,900,000 red cells, many of which were microcytes. In more severe cases the red cells may be greatly diminished, in proportion to the extent of hemorrhage, but a microcytic type of red cell is usually prominent. The Hb-index is subnormal. Nucleated red cells occur when hemorrhages are large or frequent. In a fatal case in an infant, yielding a pure culture of streptococcus pyogenes at postmortem, the writer found the blood to show all the HEMOBBHAGIC DISEASES AND INTOXICATIONS. 355 characteristics of primary pernicious anemia. In four other cases of fatal hemorrhage in newborn infants at Sloane Maternity Hospital, the subjects had bled to death in the course of three to five days, but the blood showed only the characteristics of secondary anemia with leucocytosis. Leucocytosis has been noted by the majority of observers. In one case Carriere and Gilbert found 126,000 leuco- cytes, 90 per cent, polynuclear, and 5.8 per cent, eosinophile. In the writer's cases in infants the leucocytosis reached as high as 56,000, but the eosiuophiles were scanty. A very marked degree of poiychromatophilia was observed in two cases by Spietschka. Hayem 1 and Bensaude have described a peculiarity in the blood of purpura hemorrhagica which they claim to be pathognomonic. On allowing the blood to clot iu a vessel it was found that after twenty-four hours the retraction of the coagulum is very feeble and fails to express the serum, as happens in normal blood. Associated with this feeble coagulation there is a marked reduction of blood plates (200,000 to 50,000). These two features of the blood were noted in sixteen cases of purpura hemorrhagica, but in 152 examples of other diseases, some with purpura, the feeble clotting and loss of blood plates were never found to be combined. Non-infectious Idiopathic Purpura Hemorrhagica. In addition to the cases of purpura which arise in the course of infectious diseases or after various infectious processes, in which hemorrhage constitutes the chief symptom of a cryptogenic infection, there are other cases of rapidly fatal anemia attended with severe and repeated losses of blood for which a different etiology is indicated. Some of these cases are difficult to distinguish from pernicious anemia, but differ from that disease in the absence of megaloblastic changes in the blood or marrow, the great predominance of micro- cytes in the blood, and the prominence of hemorrhage among the symptoms. They differ from the infectious purpura in the more persistent hemorrhage, the absence of leucocytosis, and the absence of general symptoms of an infectious disease. Such cases have been reported by Ehrlich, 1 Engel, Muir, and others as pernicious anemia without characteristic changes in the blood or marrow. The writer can find no evidence on which to claim that pernicious anemia can exist without such changes in the marrow, and believes that these cases belong to the diseases referable to the hemorrhagic diathesis. This view is supported not only by the great dissimilarity in the blood changes of the two conditions, but by the absence of any marked deposits of iron in the viscera (Zalesky). In Muir's case the marrow of the rib consisted almost entirely of fat tissue with very few normoblasts, while the shafts of the bones were decidedly thicker than normal. Here the condition was apparently one of congenital agenesis of the marrow. The viscera contained considerable deposits of iron. Of the various etiological factors to which fatal purpura has been attributed the non-infectious variety may be referred to any or all 356 CONSTITUTIONAL DISEASES. except the presence of bacteria. The underlying condition is probably identical with that of hemophilia of more typical course. Changes in the Blood. In the writer's case the dotting appeared to be abnormally rapid, as in the course of an hour the specimen in the hematocytometer diluted 1 : 100 with 0.6 per cent, salt solution became jelly-like. Grawitz has also noticed an increased coagula- bility of the blood in these cases after repeated hemorrhages. The red cells are rapidly diminished in number, reaching before death an extremely low figure. In a case of the writer persistent epis- taxis reduced them in three weeks to 456,000. In a case reported by Billings they numbered 483,000. In Engel's case there were over 2,000,000. Contrary to the rule in secondary anemia following hemorrhage, nucleated red cells are extremely scarce or absent. The majority of the red cells are undersized and many are of oval shape. The leucocytes are normal or reduced in number, and of those remain- ing a large proportion (90 per cent., Engel ; 80 per cent., Ehrlich ; 75 per cent., Billings) are lymphocytes. Eosinophile cells are scarce or absent. HEMOPHILIA. Hemophilia is an extremely hereditary constitutional anomaly which predisposes the subject to persistent and fatal hemorrhages of traumatic or spontaneous origin. The peculiar law of heredity which transmits the condition to males only through the female parents who are themselves usually exempt, as fully set forth by Grandidier and Stahel, not only marks this malady as the clearest possible illus- tration of the hereditary transmission of disease, but completely baffles the attempts to prove its dependence on any infectious agent. The essential lesion has been held by many to lie in an unnatural thinness and narrowness of the arteries (Virchow). In somewhat altered form this theory is maintained by Immerman and Oertel, who hold that there is a disproportion between the bulk of blood and, the capacity of the blood system, referable in part to hypoplasia of vessels (Immerman) or to hydremic plethora. Proceeding on this theory Cohen claims to have considerably lessened the tendency to hemorrhages in a pronounced bleeder by a prolonged course of diure- sis and diaphoresis. In partial support of this mechanical theory are cited a few cases in which the heart was hypertrophied, and the vessels of small size, and showing fatty, hyaline, or granular degen- eration. A local origin is perhaps indicated by the case cited by Stengel, in which the hemorrhages were observed to occur from cuts above the neck, but never from those below. Recklinghausen holds that a disorder of the nervous system is the essential cause of the hemorrhages, and Henoch believes that the local condition is one of paralytic dilatation of vessels, followed by diapedesis. The condition has been seen in some very remarkable forms in neurotic and hysterical subjects (Stengel). Little success has followed numerous attempts to demonstrate essential changes in the blood. Albertoni claims to have found HEMORRHAGIC DISEASES AND INTOXICATIONS. 357 diminished resistance in the red cells. The remarkable therapeutic influence of calcium chloride, described by Brunton, and reported by Wallis, supports the view that the chief alteration in the blood is in the content of salts. Wright finds that the bleeding in hemophilia is diminished by administration of calcium chloride, by inhalation of C0 2 , and by local application of the nucleo-albumin of the testicle, thyroid, and gastric mucosa. In morphology there is uniform agree- ment that the blood presents nothing peculiar. Decreased coagula- bility was held to be constant, by older writers (Grandidier, Lossen), and has later been observed by Schmidt and Manteuffel, who found that the blood in hemophilia requires longer than usual to clot, while its coagulation may be greatly hastened by adding a zymoplastic substance (fibrin ferment). Zavialoff claims to have found evidence that the red cells in hemophilia contain a cytoglobin, not present in normal blood, which prevents coagulation when liberated by the cells on shedding of the blood. The fibrin itself is apparently not greatly diminished, 5 per cent, having been found by Heyland ; 2.6 per cent., by Gavoy and Ritter, and 4.3 per cent, by Otto (cited by Litten). Studies in pathological anatomy have also failed to show any constant lesion of essential importance. In most of the viscera there are the lesions which follow acute or chronic anemia. Buhl described an unusually rich network of capillaries in the skin of one case, but the tissue was the seat of a chronic inflammatory process. Birch- Hirschfeld found normal vessels, slightly fatty heart muscle, and slight hyperplasia of the splenic stroma. Litten noted areas of granular degeneration in capillar}' endothelium, and irregularity in outline or widening of intercellular spaces between these cells, but suspected these changes to be artificial. In a series of cases hypo- plasia of the heart furnished the basis of Virchow's theory. The blood changes are determined by the extent of the hemor- rhages, but leucocytosis has been absent in the few cases reported. In general, the blood resembles that of non-infectious purpura hemor- rhagica. SCURVY. Etiology. The most important fact in the etiology of scurvy is its intimate relation to improper diet. Aside from the almost exclu- sive occurrence of the disease in subjects whose food has been re- stricted in variety, it has been repeatedly demonstrated that the malady may be promptly cured by supplying some element previously deficient in the diet. Even a change in water supply has eradicated epidemics in garrisons. Scorbutic infants rapidly recover from grave stages of the disease after such apparently trivial assistance as a change in the milk. Yet it has never been fully determined just what chemical substance is concerned in the causation and cure of the disease. It has even been claimed that diet has no relation to the malady, but all such reports have thus far, on investigation, failed to be supported, although they have demonstrated that the 358 CONSTITUTIONAL DISEASES. dietetic error may be very trivial. Thus Seeland, regarding a some- what limited army ration as full mixed diet, came to regard the cases of scurvy under his care as of miasmatic origin. Accumulating evidence has shown that the most frequent source of the disease is a too exclusive diet of meat, especially if salted, and the absence of fresh vegetables, especially of potatoes, which are very rich in potas- sium. Guided by these observations various theories of origin of scurvy have been elaborated. Among these is the belief that the symptoms were caused by excess of NaCl in the blood, a condition long since shown to characterize most forms of anemia. That the essential element in the disease consists in a defi- ciency of potassium in the blood was held by Garrod,Liebig, and Hirsch.but a loss of potassium from the blood, with excess in the urine, while not constant in scurvy, is observed in other conditions. Ralfe and Cantani hold that a deficiency of salts of vegetable organic acids and corresponding diminution in the alkalinity of the blood is the important factor. Wright also maintains that scurvy is a form of acid intoxication arising from a diet rich in mineral acids and poor in bases. In seven advanced cases of scurvy from the beleaguered garrison of Ladysmith he found, by his alkalimetric method, that the blood /N N N\ was very deficient in alkalescence ( c7i_ 306 epitheliome primitive of, 264 extirpation of, in leukemia, 275 in malaria, 275 hyperplasia of, in rachitis, 380 sarcoma of, 264 syphilis of, 264 Splenectomy, 273 in animals, 273 in man, 274 Splenic anemia, 264 Splenocyte, 126, 184 Spotted fever of Montana, 481 Staining methods, 55 dahlia, for mast-cells, 57 eosinophiles, methylene blue, 55 Jenner's, 56 triacid mixture, 56 Nocht's, 434 Stippling of red cells, 450 Stomach, carcinoma of, 209, 399, 425 digestion leucocs'tosis, 146, 401 dilatation of, 396 diseases of, 395 ulcer of, 398 Stomatitis, a cause of leukemia, 231 Suppuration, 326 Surgical procedures, effects on blood, 110 Surra, 480 Syphilis, 339 a cause of leukemia, 231 congenital, 342 lymphocytosis in, 171, 342 myelocytes in, 343 eosinophiles in, 165 in pernicious anemia, 207 of spleen, 264 TECHNICS, 23 in malaria, 431 Tenia, 473 Tests for blood, qualitative, 23 guaiacum, 24 serum, 24 spectroscopic, 28 Teichmann's hemin, 24 Tetanus, 333 Tetany, 387 Thoma's hematocytometer, 35 Thyroid chlorosis, 387 therapy in myxedema, 381 Toadstools, poisoning by, 364 Tonsillitis, 329 Toxicity of serum, in pneumonia, 283 Toxins, 143 Transfusion, effects of, 117, 364 Trichinosis, 470 Trypanosomiasis, 480 in sleeping-sickness, 485 Tuberculin, 171, 246 Tuberculosis, 343 bacteria in, 348 INDEX. 495 Tuberculosis of bones, 348 chemistry of, 348 meningitis, 347 relation to pseudoleukemia, 259 Tumors, eosinophiles in blood, 163, 425 428 Typhoid fever, 302, aspiration of spleen in, 306 bacteria in, 306 bilious, 468 a cause of pernicious anemia, 208 cold baths in, 304 lymphocytosis in, 305 serum diagnosis of, 311 Typhus fever, 332 Tyrosin in leukemia, 248 UREA, occurrence and estimation of, 80, 417 Uremia, 417 alkalescence in, 418 chemistry of, 417 Uric acid, in blood of gout, 326 in leukemia, 248 occurrence and estimation of, 80 " thread test," 377 VACCINIA, 297 blood in, in children, 297 Varicella, 297 Variola, 294 protozoa in, 296 Varioloid, 294 Vermiculus, of malaria, 452 Volume of red cells and plasma, 31 of blood, total, 31 WANDERING cells, primary, 180 Werlhoff's disease, 353 Whole blood, chemistry of, 77 quantitative estimation of albumins of, 77 Whooping-cough, 329 Widal's test, 311 VANTHIN bodies in leukemia, 248 "yELLOW fever, 331 r/APPERT chamber, 38