-4 . ' ' -fw- ■-■■■;•■ ■ ,*l- '■: !i'-.' ;>;•■':-.'■>■ ^V>'-"'* ■•.•;;■v^<.,^,.>;'^fV^_v;^ ■■■■■■ ■ ■• ■-\;>".;.A .■■ ■ ■*•' — i>r ■■■■^■'■' - -•:■''';■.; :^■■ ■-':■, :■:. :'-. .-V- '^ " •■■'''- ' t'' -■ ■ •*'--^' .''■••■.■•-.■;-■■■'•■,•■ .V.'' ■' ■■.'■>.■.-.'•',•!■",■■.■■;_, ■■ . -J iJi ■ • ;-t-;-.r^^ ■ ♦ T •■ CORNELL UNIVERSITY THE Sflauter Hetertnarji Sltbrary FOUNDED BY^ ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 CORNELL UNIVERSITY LIBRARY 3 1924 104 224 468 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924104224468 CLINICAL HEMATOLOGY DaCOSTA CLINICAL HEMATOLOGY A PRACTICAL GUIDE TO THE EXAMINATION OF THE BLOOD WITH REFERENCE TO DIAGNOSIS. By JOHN C. DaCOSTA, Jr., M.D. ASSISTANT DEMONSTRATOR OF CLINICAL MEDICINE, JEFFERSON MEDICAL COLLEGE HEMATOLOGIST TO THE GERMAN HOSPITAL, ETC. Containing Eight Full-Page Colored Plates, Three Charts, AND Forty-Eight other Illustrations. PHILADELPHIA : P. BLAKISTON'S SON & CO. IOI2 WALNUT STREET 1902 Copyright, 1901, P. Blakiston's Son & Co. TO MY FATHER, JOHN C. DaCOSTA, M.D., THESE PAGES ARE AFFECTIONATELY DEDICATED. PREFACE. This book, designed as a practical guide to the examination of the blood by methods adapted to routine clinical work, repre- sents an endeavor to recount the salient facts of hematology as they are understood at the present time, to cori'elate certain of these facts with familiar pictures of disease, and to apply them to medical and surgical diagnosis. The purpose has been to inter- pret the blood report according to its true value as a clinical sign, neither exploiting it as a panacea for every diagnostic ill, nor belittling it because of its failure consistently to give the sought- for clue in every instance. A minimum amount of theoretical discussion has been intro- duced in the sections dealing with the physiology and pathology of the whole blood and of the cellular elements — only sufficient, in the author's judgment, to add clearness to a number of the mooted points of this science, which in its present transitional stage must still be regarded as one from which more or less hy- pothesis and conjecture are inseparable. Intimate familiarity with technique being an essential qualification for the comprehensive study of the blood, a somewhat lengthy consideration of this sub- ject is given. The methods of examination likely to prove useful in every-day practice have been described in detail, perhaps some- times at the risk of prolixity, in the hope of thus simplifying for the novice the minutiae of blood counting, staining, and other means of investigation. In the discussion of the primaiy anemias and of the anemias peculiar to infancy, prominent clinical features other than those referable to the blood have been briefly men- tioned, in order to add clearness to the differential diagnosis. For convenience in reference, the various diseases included in the sec- tion on general hematology are arranged alphabetically, rather than grouped according to a traditional classification. The greater part of the original data referred to in the text is taken from the records of the Pathological Institute of the Ger- man Hospital, where a systematic account of all blood examina- tions has been kept for the past six years. The remaining data represent the writer's personal examinations in hospital and pri- vate practice and in the army medical service, these sources of statistics together including about four thousand blood reports in various pathological conditions. VIU PREFACE. Hematological literature has been freely consulted in the prep- aration of this volume, special acknowledgment being due to Hayem, Ehrlich and Lazarus, von Limbeck, Rieder, Lowit, Tiirk, Grawitz, Cabot, Stengel, Thayer, Ewing, Taylor, and Coles for the profitable information gleaned from their writings. Due credit in the text has been given to these as well as to the other authors of whose labors use has been made. The colored plates and other histological illustrations, the originals of which were made by Mr. E. F. Faber from fresh and stained specimens, bear evidence of the artist's technical skill cmd faithful attention to structural detail. Mr. S. Trenner has kindly furnished the engravings of several of the special instruments. The author takes pleasure in acknowledging the assistance of his wife and critic in revising the proof of these pages ; in credit- ing Dr. G. P. Miiller for collecting and verifying much statistical matter relating to hospital cases ; and in thanking Dr. J. Chal- mers Da Costa and Dr. T. G. Ashton for helpful suggestions. 313 South Thirteenth Street, Philadelphia, November, 1901. INTRODUCTION. The rapid growth and development of hematology during re- cent years and the practical application of many of its teachings to the diagnosis of various diseases have made this science one which no progressive medical man can afford to disregard. Ex- amination of the blood gives definite clinical information which may be profitable both to the practitioner of internal medicine and to the surgeon, and the procedure is capable of throwing light upon the diagnosis in such a wide range of pathological conditions that it is difficult to single out any disease in which it may not be of some utility, either as positive or as negative evi- dence. In the light of our present knowledge of the subject, clinical information of two different kinds may be derived from hematol- ogy, namely, findings which are pathognomonic of certain dis- eases ; and auxiliary data which, if considered in connection with other clinical manifestations, may prove either essential or helpful in establishing the precise nature of a disease. Pathognomonic blood findings are unfortunately confined to a limited number of diseases : leukemia, the malarial fevers, relaps- ing fever, and filariasis. In pernicious anemia a typical picture is also found, if two conditions capable of exciting identical blood changes are excepted, the profound secondary anemias due to certain intestinal parasites and to nitrobenzol poisoning. The blood examination affords data which, although not pa- thognomonic, are nevertheless essential for the diagnosis of chlo- rosis, Hodgkin's disease, splenic anemia, and secondary ane- mias dependent upon various causes. For example, in chlorosis a definite group of blood changes must exist in order to justify an unconditional diagnosis, although the occurrence of these changes, unassociated with other equally definite clinical signs, is insufficient evidence of this disease. In Hodgkin's disease, a condition indistinguishable from leukemia by an ordinary phys- ical examination, the absence of a leukemic state of the blood at once excludes the latter disease. In the secondary anemias, it is obvious that the blood count alone can give the exact clue to the condition, by determining the degree and character of the blood impoverishment, and by tracing from time to time its progress. INTRODUCTION. In this connection it is important to remember that pallor may go hand in hand with a normal hemoglobin percentage and eryth- rocyte value, and that on the other hand a high color by no means invariably signifies that the individual is not anemic. In addition to the diseases just named, hematology gives informa- tion which is often of great assistance in, although not essential for, the diagnosis of such conditions as enteric fever, sepsis, pneumonia, appendicitis, diabetes, syphilis, malignant disease, trichiniasis, and suppurative processes. Clinical experience has repeatedly illustrated the value of the serum reaction in typhoid and in Malta fevers, of Williamson's test in diabetes mellitus, of eosinophilia in trichiniasis, and of leucocytosis in sepsis, malignant neoplasms, suppui-ative lesions, and many of the acute infections. Negative results from a blood examination also possess diag- nostic value within certain limits, but too great reliance upon evi- dence of this sort more often proves delusive than helpful. In a patient whose waxy, yellowish facies suggests with equal force pernicious anemia, chronic nephritis, and, perhaps, liver cirrhosis, the absence of characteristic blood changes is sufficient to exclude the first-named condition. But failure to detect the malarial para- site does not necessarily exclude malarial fever ; a negative serum test does not absolutely rule out enteric fever ; and an absence of leucocytosis cannot be regarded as an infallible sign that a sup- purative focus does not exist, nor does it always indicate the benignity of a neoplasm. Negative evidence, then, is usually to be considered merely suggestive, the real pertinence of the hint thus obtained depending upon its correlation with other physical signs and symptoms. The significance of positive findings in bacteriological investiga- tions of the blood is patent, and the conclusive value of this means of research in identifying obscure cases of general sepsis, malignant endocarditis, enteric fever, and plague, has been demon- strated in many instances. The conflicting and indifferent results which some investigators have obtained by this procedure were doubtless due largely to faulty technique, but these results promise to become more dependable and certain with the adoption oi more exact technical methods. At the present time the most useful information furnished by hematology has been derived from study of the cellular elements of the blood, but closer familiarity with the chemistry of this tissue, still an undeveloped science, will undoubtedly in the near future afford not only more tangible clues to the etiology and pathology of the blood diseases, but also will bring to light addi- tional facts which may be applied to the diagnosis of these and INTRODUCTION. XI other maladies,. The study of the coagulation time of the blood already promises to be of practical utility in the diagnosis and prognosis of cases of purpura, hemophilia, and jaundice, which are characterized by slow clotting and by a tendency toward hemorrhage. The technique of blood examinations, such as described in the following pages, is neither elaborate nor difficult to master. Necessarily, it must be rigidly exact, but no more so than any other branch of physical diagnosis, if the worker is content only with the best results. To acquire a good working knowledge of hematology takes but a fraction of the time and application that one must spend in familiarizing one's self with the most com- mon heart murmurs or chest signs, and the time thus spent equips the physician with an additional diagnostic agent of the greatest value. If the newly-graduated physician would provide himself with a microscope and a set of blood instruments, and systematically study the blood in the various general diseases which he encounters in practice, many a slip-shod diagnosis might be avoided, and a great stride forward made in popularizing this practical branch of clinical diagnosis. TABLE OF CONTENTS. INTRODUCTION SECTION I. EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Page. General Schema ... . . 19 I. Examination of the Fresh Blood .... ig Obtaining the Specimen . . .... 19 Preparing the Slide . . ..... 21 Microscopical Examination ....... 22 Changes Affecting the Erythrocytes 22 Changes Affecting the Leucocytes ...... 23 Increase of Fibrin, Blood Plaques, and Hemoconia . . 24 Blood Parasites ......... 24 Foreign Bodies . ....... 24 II. Estimation of the Percentage of Hemoglobin ■ • 25 Von Fleischl's Hemometer ....... 25 Oliver's Hemoglobinometer ....... 32 Cowers' Hemoglobinometer ...... 34 Dare's Hemoglobinometer ... ... 36 Tallquist's Method 39 III. Counting the Erythrocytes and the Leucocytes . 40 Methods 40 Diluting Fluids ......... 40 The Thoma-Zeiss Hemocytometer. ..... 42 Counting the Erythrocytes ....... 45 Counting the Leucocytes ....... 49 Cleaning the Pipette 52 Durham's Hemocytometer ....... 52 Gowers' Hemocytometer . . . . . . . 54 Oliver's Hemocytometer ....... 56 IV. Microscopical Examination of the Stained Specimen , 58 Objects of Staining ........ 58 The Aniline Dyes . . 59 Preparing the Films ....... 59 Fixation Methods .... .... 61 Methods of Staining ....... 63 Ehrlich's Triacid Stain 64 Jenner's Stain ........ 65 Prince's Stain 66 xiii XIV TABLE OF CONTENTS. Page. Staining with Eosin and Methylene-blue .... 67 Staining with Eosin and Hematoxylin ..... 68 Staining with Thionin 69 Staining with Polychrome Methylene-blue .... 69 Differential Counting ■ 7° V. Counting the Blood Plaques 7' Determann's Method 72 VI. Estimation of the Relative Volumes of Corpuscles and Plasma 72 Daland's Hematocrit 73 Limitations of the Hematocrit 74 VII. Estimation of the Specific Gravity .... 75 Hammerschlag's Method • 75 VIII. Estimation of the Alkalinity ^^ Engel's Alkalimeter ........ 77 IX. Determination of the Rapidity of Coagulation . 79 Glass Slide Method 80 Wright's Coagulometer ....... 80 X. Spectroscopical Examination 81 The Sorby-Beck Microspectroscope . . • . . 81 XI. Bacteriological Examination 83 Value of Positive Findings ....... 83 Methods 83 Blood Cultures ...... . . 83 Staining Methods ....... 8; XII. Determination of the Serum Reaction ... 86 Widal's Test 86 The Specific Test for Human Blood 88 SECTION II. THE BLOOD AS A WHOLE. I. General Composition . Plasma, Serum, and Cells Salts . Extractives Gases .... II. Color . Normal Variations Density and Opacity Pathological Variations . III. Odor and Viscosity IV. Reaction Reaction in Health 93 93 93 94 94 94 94 94 94 95 95 95 TABLE OF CONTENTS. XV Table of Normal Blood Alkalinity . Physiological Variations Pathological Variations .... V. Specific Gravity Normal Range ..... Pathological Variations .... Relation of Specific Gravity to Hemoglobin Table of Hemoglobin Equivalents . VI. Fibrin and Coagulation . Relation of Fibrin to Coagulation Appearance of Fibrin in Fresh Blood Hyperinosis and Hypinosis . Pathological Variations in Amount of Fibrin VII. Oligemia Definition ... Occurrence . . . . VIII. Plethora Definition ...... Permanent and Transient Polyemia Serous Plethora ..... Cellular Plethora . . . . IX. Hydremia Definition ... Causes . . ... Occurrence . X. Anhydremia Definition ... Causes-. Occurrence XI. LiPEMIA Amount of Fat in Normal Blood Definition ...... Physiological and Pathological Lipemia . Tests for Fat ..... XII. Melanemia . ... Definition . .... Occurrence . XIII. Glycemia Amount of Sugar in Normal Blood Hyperglycemia Test for Sugar . . XIV. Uricacidemia Definition Occurrence ... Test for Uric Acid . . . ■ 96 96 97 98 98 99 99 100 100 100 loi 102 102 103 103 103 104 104 104 104 105 105 105 105 105 106 106 106 106 106 106 107 107 107 107 107 108 108 108 108 108 109 109 109 109 XVI TABLE OF CONTENTS. XV. Cholemia Definition Occurrence Test for Bile XVI. Acetonemia and Lipacidemia Definition Occurrence Tests for Acetone and Fatty Acids XVII. Bacteriemia . . . • Occurrence Latent Infection . Blood Cultures Bacteria Found in the Blood XVIII. Anemia . Definition Pseudo-anemia Classification Pathogenesis Page. no no no no no no no no in 1 1 1 in 112 112 113 113 113 114 115 SECTION III. HEMOGLOBIN, ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. II. III. ■iEMOGLOBIN General Properties . ■ 119 119 Origin ... . . . . Variations in Amount 120 121 Absolute Amount ... . . 122 Color Index .... 122 Hemoglobinemia .... Methemoglobinemia . . . . Carbon Monoxide Hemoglobin . . . . 123 124 125 Erythrocytes . 126 Appearance in Fresh Blood - . . . Histological Structure . . . . . Origin and Life History Size . . . . . . Normal Number . ... 126 127 128 129 129 130 Volume Index . . . . Influence of Physiological Factors on thi ; Eryth- ROCYTES 130 130 131 132 132 132 133 Age and Sex . Pregnancy, Menstruation, and Lactation Constitution and Nutrition Fatigue . . . . . . Digestion and Food . . . . High Altitudes . TABLE OF CONTENTS. XVll IV. Pathological Changes in the Erythrocytes Ameboid Motility Alterations in Isotonicity Hyperviscosity Deformities of Shape and Size Megalocytes . Microcytes Poikilocytes Endoglobular Degeneration Total Necrosis Atypical Staining Reaction Nucleation . Normoblasts Megaloblasts Microblasts . Atypical Erythroblasts Granular Degeneration Oligocythemia Polycythemia V. Blood Plaques Appearance in Fresh Blood Histological Structure . Origin Normal Number . Pathological Variations VI. Hemoconia Appearance in Fresh Blood Histological Characteristics Occurrence . Page s 134 134 13s . 136 136 . 136 137 137 138 139 140 141 141 143 145 146 147 148 149 150 150 ISO 150 151 151 151 151 151 151 SECTION IV. THE LEUCOCYTES. I. General Characteristics • Appearance in Fresh Blood . Ameboid Movement Cell Granules Normal Number ..... II. Classification Number ?ind Percentage of Different Varieties Small Lymphocytes .... Large Lymphocytes Transitional Forms .... Polynuclear Neutrophiles Eosinophiles ... Basophile Cells . ... Myelocytes . . ... Mast Cells 1* iSS 155 156 157 159 159 159 160 161 162 163 165 166 167 168 XVIU TABLE OF CONTENTS. Mononuclear Neutrophiles Neutrophilic Pseudolymphocytes Reizungsformen . Differential Table of the Leucocytes Origin and Development Iodine Reaction . Perinuclear Basophilia . III. Leucocytosis Definition .... Classification of the Leucocytoses Physiological Leucocytosis Character Causal Factors Leucocytosis of the New-bom Digestion Leucocytosis . Leucocytosis of Pregnancy and Parturition Leucocytosis Due ta Thermal and Mechanical Terminal Leucocytosis . Pathological Leucocytosis Occurrence . Degree of Increase Differential Changes Causal Factors Functions Hypoleucocytosis and Hyperleucocytosis Inffammatory and Infectious Leucocytosis Leucocytosis of Malignant Disease Post-hemorrhagic Leucocytosis Toxic Leucocytosis Experimental Leucocytosis IV. Lymphocytosis .... Definition ..... Differential Changes Causal Factors .... Physiological Lymphocytosis Pathological Lymphocytosis . Experimental Lymphocytosis Clinical Significance V. EOSINOPHILIA Definition . ... Causal Factors ..... Physiological Eosinophilia Pathological Eosinophilia Experimental Eosinophilia Diminution in the Number of Eosinophiles Clinical Significance .... Influences Page. 171 171 171 172 173 174 176 176 176 177 177 177 178 178 179 180 181 181 182 182 183 183 184 184 185 187 190 191 192 193 196 196 196 197 197 197 198 198 198 198 199 199 200 201 201 201 VI. Basophilia TABLE OF CONTENTS. XIX Pagb. VII. Myelemia ...... . . 202 Definition ... 202 Occurrence ...... 202 Causal Factors .... 203 VIII. Leucopenia 203 Definition ..... 203 Differential Changes 204 Physiological Leucopenia . 204 Pathological Leucopenia . 205 Experimental Leucopenia 207 SECTION V. DISEASES OF THE BLOOD. Appearance of the Fresh Blood 209 Coagulation . . . . ■ 209 Specific Gravity . . . • 209 Alkalinity . . . . 219 Hemoglobin and Erythrocytes 210 Color Index 210 Deformed and Nucleated Cells 2IO Leucocytes ..... 213 Differential Changes 214 Blood Plaques .... 215 Diagnosis .... 216 Clinical Features .... 2X6 II. Pernicious Anemia 218 Appearance of the Fresh Blood 2l8 Coagulation . . . . ■ 219 Specific Gravity . 219 Alkalinity 220 Hemoglobin and Erythrocytes 220 Color Index 220 The Blood During Remissions 221 Megalocytosis .... 221 Poikilocytosis .... 223 Prevalence of Megaloblasts . 224 Polychromatophila 225 Granular Basophilia 226 Leucocytes ..... 227 Differential Changes 227 Blood Plaques .... 228 Diagnosis ..... 228 Clinical Features .... 229 Pernicious Anemia and Severe Secondary Anemia 230 Pernicious Anemia and Chlorosis . . . • 230 Pernicious Anemia and Bothriocephalus Anemia . 231 Pernicious Anemia and Nitrobenzol Pois oning 231 XX TABLE OF CONTENTS. III. Splenic Anemia. Appearance of the Fresh Blood Hemoglobin and Erythrocytes Color Index .... Deformed and Nucleated Cells Leucocytes . . . • Blood Plaques Diagnosis .... Clinical Features . Splenic Anemia and Spleno-meduUary Leukemia Splenic Anemia and Pernicious Anemia Splenic Anemia and Hodgkin's Disease Splenic Anemia and Splenic Tumors IV. Secondary Anemia .... Appearance of the Fresh Blood Coagulation .... Specific Gravity ...... Alkalinity ..... Hemoglobin and Erythrocytes Color Index .... Deformed and Nucleated Cells Leucocytes .... Differential Changes Blood Plaques .... Diagnosis . .... V. POST-HEMORRHAGIC AnEMIA . Etiology ..... Immediate Effects of Hemorrhage Secondary Effects of Hemorrhage . Degree of Blood Loss Compatible with Life Regeneration of the Blood .... Differential Table ... VI. Leukemia .... . . Varieties ....... Parasitology ....... Spleno-meduUary Leukemia .... Appearance of the Fresh Blood Coagulation ...... Alkalinity ...... Specific Gravity Hemoglobin and Erythrocytes Color Index ...... Relation of Erythrocyte and Leucocyte Counts Nucleated Cells Leucocytes ...... Influence of Arsenic on the Leucocyte Count The Blood During Remissions Differential Changes ; . . . Blood Plaques Pagh. 231 231 231 231 232 233 233 233 234 23s 235 235 235 236 236 236 236 237 237 237 238 238 238 239 239 239 239 240 240 240 241 243 244 244 244 246 246 247 247 247 247 248 248 248 250 250 251 252 256 TABLE OF CONTENTS. XXI Lymphatic Leukemia Appearance of the Fresh Blood Hemoglobin and Erythrocytes Color Index . . . ■ ■ Deformed and Nucleated Cells Leucocytes . . . • • Differential Changes Blood Plaques Acute Leukemia ..... Influence of Intercurrent Infections Diagnosis ...••• Spleno-meduUary and Lymphatic Leukemia Leukemia and Pathological Leucocytosis Leukemia and Lymphocytosis Leukemia and Hodgkin's Disease • Leukemia and Tumors of the Spleen, Kidney, and Pancreas Leukemia and Lymphatic Hyperplasia . VII. Hodgkin's Disease .... Appearance of the Fresh Blood Alkalinity, Specific Gravity, and Coagulation Hemoglobin and Erythrocytes Color Index ...... Nucleated and Deformed Cells Leucocytes ...... Differential Changes Diagnosis ...... Clinical Features ..... Hodgkin's Disease and Tuberculous Adenitis Hodgkin's Disease and Syphilitic Adenitis Hodgkin's Disease and Local Lymphoma Hodgkin's Disease and Lymphatic Sarcoma . Hodgkin's Disease and Lymphatic Carcinoma VIII. The Effect on the Blood of Splenectomy Hemoglobin and Erythrocytes .... Leucocytes ........ Differential Changes ... . . Factors of the Blood Changes Following Splenectomy Differential Table Pagb. 256 256 257 257 257 258 259 260 260 261 263 264 265 265 266 266 266 267 267 267 267 267 , 267 268 268 269 270 271 271 271 271 272 272 272 272 274 274 275 SECTION VL THE ANEMIAS OF INFANCY AND CHILDHOOD I. Characteristics of the Blood in Children Fetal Blood . The Blood at Birth II. Anemia in Children Frequency . General Characteristics 279 279 280 282 282 283 XXll TABLE OF CONTENTS. Classification Primary Anemia . . • • Pernicious Anemia Leukemia Secondary Anemia Mild Anemia . . • • Severe Anemia Anemias with Leucocytosis Etiology of Secondary Anemia Anemia Due to Syphilis . Anemia Due to Rachitis . Anemia Due to Tuberculosis • Anemia Due to Gastro-intestinal Diseases Post-typhoid Anemia Anemia Infantum Pseudoleukemica Bacteriemia in Children Page. 283 284 284 284 287 287 287 287 288 288 288 289 289 289 290 292 SECTION VII. GENERAL HEMATOLOGY. I. Abscess Coagulation, Fibrin, and Iodine Reaction Hemoglobin and Erythrocytes Factors of the Anemia in Abscess Color Index ...... Grade of Anemia in Different Forms of Abscess Cell Deformity and Nucleation Leucocytes ...... Relation of the Leucocyte Count to the Local Lesion Range of the Leucocyte Count in Different Forms of Abscess Differential Changes ....... Diagnosis ......... II. Acromegaly .... III. Actinomycosis IV. Acute Yellow Atrophy of the Liver V. Addison's Disease VI. Anthrax VII. Appendicitis . Factors of the Anemia in Appendicitis .... Grade of Anemia in Catarrhal and Suppurative Cases Hemoglobin and Erythrocytes ..••.. Cell Deformity and Nucleation .... Leucocytes Range of the Leucocyte Count in Different Forms of Appendi cids Differential Changes ....... Diagnosis .... . . 295 29s 295 295 295 296 296 296 297 297 297 297 298 298 298 299 299 300 300 300 300 301 301 302 302 302 TABLE OF CONTENTS. XXUl VIII. Asiatic Cholera . ... IX. Asthma and Emphysema X. Bronchitis . . ... XI. Bubonic Plague Bacteriology . ... Serum Reaction ...... Hemoglobin and Erythrocytes Leucocytes ...... Blood Plaques ..... XII. Cholelithiasis Fibrin and Coagulation .... Bacteriology ...... Hemoglobin and Erythrocytes Leucocytes ...... Diagnosis ....... XIII. Diabetes Mellitus .... Alkalinity, Lipemia, Lipacidemia, and Glycemia Williamson's Test ..... Bremer's Test ...... Hemoglobin and Erythrocytes Leucocytes . . .... Digestion Leucocytosis . ... Iodine Reaction . .... Diagnosis . . .... XIV. Diphtheria Hemoglobin and Erythrocytes Leucocytes ....... Course of the Leucocytosis .... Influence of Antitoxin on the Leucocyte Count Differential Changes ..... Affinity of the Leucocytes for Basic Dyes Diagnosis ....... XV. Enteritis ....... Acute Catarrhal, Chronic Ulcerative, and Phlegmonous Gastro-enteritis ...... Dysentery ....... Effect of Saline Purges .... XVI. Enteric Fever Bacteriology ..... Blood Cultures ...... Spot Cultures ...... Serum Reaction ...... Hemoglobin and Erythrocytes Cell Deformity and Nucleation Leucocytes ....... Differential Changes ..... Effect of Complications .... Page. 304 305 306 306 306 307 307 308 308 308 308 308 309 309 309 309 309 310 311 312 312 312 312 312 312 313 314 314 315 31s 3'6 316 316 316 316 316 3'7 317 317 318 319 326 328 328 330 330 XXIV TABLE OF CONTENTS. Page. Blood Plaques . 331 Diagnosis . 331 XVII. Erysipelas 332 XVIII. Exophthalmic Goitre . • . • • 333 XIX. Fever ... 333 Factors of the Blood Changes 333 Pyrexial Polycythemia 333 Post-febrile Anemia 334 334 Alkalinity 334 XX. FiLARIASIS 334 Occurrence 334 Parasitology 335 The Filaria Nocturna 335 Technique of Examination 340 Staining the Filariae 341 Hemoglobin and Erythrocytes .... 341 Leucocytes ........ 342 342 XXI. Fractures 343 XXII. Gastritis . . . ■ / ■ 343 Acute and Chronic Forms 343 Hyperchlorhydria, Hypochlorhydria, Gastric Achylia, G astric Dilatation, Gastric Neurasthenia 344 Diagnosis 344 XXIII. Gastric Ulcer 345 345 Effects of Hemorrhage and Emesis 345 Leucocytes 345 345 346 XXV. Gonorrhea 346 XXVI. Gout 347 Alkalinity and Fibrin 347 Uric Acid 347 Cellular Elements 347 Perinuclear BasophiUa 348 XXVII. Hemorrhagic Diseases .... 348 Specific Gravity . 348 Bacteriology . 348 348 Coagulation . 349 349 Leucocytes ... 350 Blood Plaques 350 TABLE OF CONTENTS. XXV XXVIII. Hepatic Cirrhosis Anemia in Atrophic Cirrhosis .... Effect of Ascites . Anemia in Hypertrophic Cirrhosis Leucocytes in Atrophic and Hypertrophic Cirrhoses Diagnosis . ...... XXIX. Herpes Zoster . ... XXX. Icterus .... . • Fibrin, Coagulation, Specific Gravity, and Alkalinity Hemoglobin and Erythrocytes .... Leucocytes ........ Diagnosis ...'.. . . XXXI. Influenza XXXII. Insolation XXXIII. Intestinal Helminthiasis Factors of the Blood Changes Hemoglobin and Erythrocytes .... Bothriocephalus Anemia . . . . The Anemia of Ankylostomiasis .... Leucocytes ...... XXXIV. Intestinal Obstruction XXXV. Leprosy XXXVI. Malarial Fever Parasitology . . ..... Developmental Cycle of the Malarial Parasite in Man Developmental Cycle of the Malarial Parasite in the Mosquito Varieties of the Malarial Parasite The Parasite of Tertian Fever Infections with Single and Multiple Groups Anticipation of the Paroxysm Intracellular Hyaline Forms . Intracellular Pigmented Forms Segmenting Forms Extracellular Pigmented Forms Flagellate Forms ... Degenerate Forms . ■ • ■ • The Parasite of Quartan Fever Infections with Single and Multiple Groups Intracellular Hyaline Forms • Intracellular Pigmented Forms Segmenting Forms .... Extracellular Pigmented Forms Flagellate Forms ..... Degenerate Forms .... The Parasite of Estivo-autumnal Fever . Irregularities in Time of Developmental Cycle Disc- and Ring-shaped Forms Page. 35° 351 352 352 353 353 353 353 354 354 3 54 355 356 356 356 356 357 357 357 358 359 359 359 360 361 362 362 362 362 363 364 365 366 367 367 367 368 368 369 370 370 370 370 370 371 XXVI TABLE OF CONTENTS. Pigmented Forms Segmenting Forms Erythropyknosis Spherical, Ovoid, and Crescentic Forms Flagellate Forms . . • • Degenerate Forms Pigmented Leucocytes and Phagocytosis Differential Table of the Malarial Parasites Technique of Examination Hemoglobin and Erythrocytes Causes of Malarial Anemias . Anemia in the Regularly Intermittent Fevers Anemia in Estivo-autumnal Fever Anemia in Malarial Cachexia Types of Post-malarial Anemia Leucocytes . . ... Differential Changes Blood Plaques Diagnosis XXXVII. Malignant Disease . Carcinoma .... Fibrin and Coagulation • Specific Gravity and Alkalinity Glycemia ..... Parasitology . . . • • Hemoglobin and Erythrocytes Color Index ..... Regeneration of the Blood after Operation The Oligocythemia and Polycythemia of Gastric Cancer Deformed and Nucleated Cells .... Leucocytes ........ Frequency of Cancer Leucocytosis .... Causes of Cancer Leucocytosis .... Range of the Leucocytes in Different Forms of Cancer Digestion Leucocytosis in Gastric Cancer Differential Changes ...... Sarcoma ....... General Features of the Blood .... Hemoglobin and Erythrocytes Leucocytes Diagnosis XXXVIII. Malignant Endocarditis Bacteriology .... Hemoglobin and Erythrocytes Leucocytes . Diagnosis XXXIX. Malta Fever XL. Measles XLI. Meningitis 372 372 372 373 374 374 375 376 376 379 379 379 380 381 381 382 383 384 384 384 384 384 385 385 385 38s 386 386 386 387 387 387 388 388 388 389 389 389 389 390 391 392 392 393 393 394 394 395 396 TABLE OF CONTENTS. XLII. Myxedema XLIII. Nephritis Factors of the Blood Changes .... Specific Gravity, Fibrin, Coagulation, and Alkalinity Bacteriology ........ Hemoglobin and Erythrocytes .... Anemia in Acute and Chronic Parenchymatous Nephritis Polycythemia ....... Anemia in Chronic Interstitial Nephritis Leucocytes Uremia . . ..... Diagnosis ..... XLIV. Nervous and Mental Diseases . Neuritis, Beri-beri, Neuralgia, and Brain Tumor Neurasthenia, Hypochondriasis, and Hysteria General Paresis, Dementia, Melancholia, and Mania Convulsions, Apoplectiform Attacks, and Acute Delirium Epilepsy, Chorea, and Tetany XLV. Obesity XLVI. Osteomalacia XLVII. Pericardial Effusion XLVIII. Peritonitis XLIX. Pertussis L. Pleurisy Serous Pleurisy Purulent Pleurisy . Diagnosis LI. Pneumonia General Features of the Blood Bacteriology . . . . ■ Hemoglobin and Erythrocytes Leucocytes . . . . • Relation of Leucocytosis to Intensity of Frequency and Extent of Leucocytosis Effects of Antipyresis . Differential Changes Blood Plaques . . . • Diagnosis LII. Poisoning . . • ■ LIII. Rabies LIV. Relapsing Fever Parasitology . . . • Lowenthal's Reaction . Hemoglobin and Erythrocytes Leucocytes . Diagnosis .... Infection Page, 398 399 399 399 399 400 400 400 401 401 401 401 402 402 402 403 404 405 405 406 406 407 408 409 409 410 411 411 411 411 412 413 413 413 414 415 415 415 416 417 418 418 420 421 421 421 XXVUl TABLE OF CONTENTS. Page. LV. Rheumatic Fever . . • • • 421 Coagulation, Fibrin, and Alkalinity 421 Bacteriology . . ■ • • • 422 Hemoglobin and Erythrocytes .... 422 Leucocytes ...■•••• 423 Diagnosis ....••■• 423 LVI. Scarlet Fever ...... 423 Coagulation, Fibrin, and Specific Gravity 423 Bacteriology 424 Hemoglobin and Erythrocytes 424 Leucocytes 425 Blood Plaques . 426 Diagnosis .....•■■ 426 LVII. Septicemia and Pyemia 427 427 Fibrin ... 427 427 Bacteriology 428 Hemoglobin and Erythrocytes .... 429 Color Index .....-.• 430 Deformed and Nucleated Cells .... 430 Leucocytes 431 Differential Changes 431 Diagnosis .... ... 431 LVIII. Syphilis 432 Hemoglobin and Erythrocytes .... 432 Syphilitic Chlorosis and Pernicious Anemia . 432 432 Justus' Test . . 433 Leucocytes . ... 434 Diagnosis . . . . 434 LIX. Tetanus . . . . . 434 LX. Tonsillitis 434 LXl. Trichiniasis 435 LXII. Tuberculosis .... . . 437 General Features of the Blood 437 Bacteriology ..... 437 Serum Reaction ....... 437 Hemoglobin and Erythrocytes .... 439 Anemia in Pulmonary Tuberculosis 439 440 Anemia in Tuberculous Adenitis, Meningitis, Pericarditi 5, Pleu- risy, and Peritonitis, and in Genito-Urinary Tuberculc )sis • 440 Leucocytes . . 441 441 Iodine Reaction 441 Perinuclear Basophilia 441 Range of the Leucocytes in Pulmonary Tuberculosis 441 TABLE OF CONTENTS. XXIX Page. Range of the Leucocytes in Bone Tuberculosis . . . 441 Range of the Leucocytes in Acute Miliary Tuberculosis, Tuber- culous Adenitis, Pleurisy, Peritonitis, Pericarditis, and Men ingitis, and in Genito -Urinary Tuberculosis . . . 443 Effect of Secondary Septic Infections .... 443 Diagnosis 443 LXIII. Typhus Fever 444 Parasitology ........ 444 Hemoglobin and Erythrocytes 444 Leucocytes 445 Diagnosis .......... 445 LXIV. Vaccination 445 LXV. Valvular Heart Disease 446 Stage of Compensation ........ 446 Acute Rupture of Compensation 446 Effect of Stasis ......... 446 LXVL Varicella 447 LXVII. Variola 448 Fibrin and Parasitology 448 Hemoglobin and Erythrocytes 448 Leucocytes 448 Blood Plaques . 449 Diagnosis . . ....... 449 LXVni. Yellow Fever 449 Fibrin and Coagulation .... . . 449 Bacteriology . 450 Serum Reaction .... . . 450 Hemoglobin and Erythrocytes . .... 450 Degenerative Changes . ..451 Leucocytes .... . . • . 451 Differential Changes . 451 Diagnosis ..... ... 451 LIST OF ILLUSTRATIONS. Plate. I. The Erythrocytes II. The Leucocytes III. Leucocytosis IV. Spleno-medullary Leukemia V. Lymphatic Leukemia VI. The Tertian Malarial Parasite VII. The Quartan Malarial Parasite VIII. The Estivo-autumnal Malarial Parasite Page. ii8 176 246 256 362 366 371 Chart. I. Pernicious Anemia II. III. Spleno-medullary Leukemia Multiple Infections in Malarial Fever 222 250 361 Figure. 1. Blood Lancet ...... 2. Proper Distribution of Cells in a Blood Film . 3. Von Fleischl's Hemometer .... 4. Tinted Wedge of von Fleischl's Hemometer 5. Capillary Pipette of von Fleischl's Hemometer 6. Method of using the von Fleischl Hemometer 7. Light-proof Box for von Fleischl's Hemometer 8. Method of Using Oliver's Hemoglobinometer 9. Gower's Hemoglobinometer .... 10. Dare's Hemoglobinometer .... 11. Horizontal Section of Dare's Hemoglobinometer 12. Method of Filling Blood Chamber 13. Thoma-Zeiss Hemocytometer 14. Thoma-Zeiss Counting Chamber . 15. Ruled Area of Thoma-Zeiss Counting Chamber 16. Ruled Area of Zappert's Counting Chamber . 17. Method of Filling the Hemocytometer . 18. Plan of Counting the Erythrocytes 19. Ocular Diaphragm ..... 20. Expelling Contents of the Erythrocytometer . 21. Cross Section of Durham's Blood Pipette 22. Method of Using Oliver's Hemocytometer 23. Superimposing the charged Cover-glass 24. Drawing Apart the Cover-glasses 25. The Cover-glasses after Separation 26. Oven for Fixing Blood-films 20 21 26 26 26 29 30 33 35 36 37 38 42 43 44 44 45 48 50 52 53 57 59 60 60 61 LIST OF ILLUSTRATIONS. XXXI Figure. Page. 27. Daland's Hematocrit . 73 28. Engel's Alkalimeter . 78 29. Wright's Coagulometer ... 80 30. Sorby-Beck Microspectroscope 81 31. Sorby Tubular Cell ... 82 32. Needle and Tube for Aspirating Blood .... 84 33. Rouleaux Formation and Fibrin in Normal Blood . . loi 34. Hyperinosis ...... 102 35. Blood Spectra ...... 125 36. Degenerative Changes in the Erythrocytes . . . 139 37. Changes in the Erythrocytes in Chlorosis . .210 38. Changes in the Erythrocytes in Pernicious Anemia 221 39. Atypical Myelocytes in Spleno-meduUary Leukemia . . 253 40. Atypical Polynuclear Neutrophiles in Spleno-medullary Leukemia 254 41. Atypical Lymphocytes in Lymphatic Leukemia . . 259 42. Positive Serum Reaction in Enteric Fever . . 319 43. Pseudo-reaction in Enteric Fever ... . . 320 44. Bacillus Typhi Abdominalis ... . 321 45. Fil aria Nocturna in Fresh Blood .... . 336 46. Filaria Nocturna, showing Granular Degeneration 338 47. Filaria Nocturna, showing Changes in Shape 340 48. Spirilla of Relapsing Fever . ... 418 " L'avenir appartient a Vhematolngie. C^ est elle qui nous apportera la solution des grands problimes nosologiques. Elle doit nous apparaitre comnie une vaste science puisant ses matiriaux dans toutes les branches des connais- sanccs biologiques et recueillani les diverses notions de V huinorisme ancien pour les rajeunir et les computer d. la lutniere des dicouvertes modernes en anatomic, en physiologic , en chimie biologique et en pathologic." Georges Hayem. SECTION I. EXAMINATION OF THE BLOOD BY CLINICAL METHODS. SECTION I. EXAMINATION OF THE BLOOD BY CLINICAL METHODS. P A systematic examination of the blood by 5~, clinical methods of established utility includes the following twelve different processes : I. Microscopical examination of the fresh blood. II. Estimation of the percentage of hemoglobin. III. Counting the erythrocytes and the leucocytes. IV. Microscopical examination of the stained specimen. These four procedures, which invariably should be included in every clinical blood-report, furnish the most important informa- tion to be derived from hematological study, and are sufficient for routine clinical work. In certain instances in which more de- tailed investigation of special points is sought, it may be thought advisable to supplement the above plan by employing one or more of these remaining eight procedures : V. Counting the blood-plaques. VI. Estimation of the relative volumes of corpuscles and plasma. VII. Estimation of the specific gravity. VIII. Estimation of the alkalinity. IX. Determination of the rapidity of coagulation. X. Spectroscopical examination. XI. Bacteriological examination. XII. Determination of the serum-reaction. I. EXAMINATION OF THE FRESH BLOOD. The finger-tip or the lobe of the ear is the part Obtaining usually selected from which to obtain the blood, THE by puncture, for examination. The former site Specimen. is preferable in most instances, owing to its con- venient situation and ease of manipulation ; but in nervous individuals and in children the ear-lobe may be chosen, because of its limited sensibility, and on account of the patient's inability to watch the operation. 20 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. The puncture may be made with one of the special blood- lancets devised for this purpose, or, in lieu of such an instrument, a Hagedorn or spear-pointed surgical needle, or a new sharp- pointed steel pen from which one prong has been twisted off, will answer the purpose equally as well. The author is accustomed to use a small steel trocar-blade, mounted on a metal shaft which screws into an outer barrel, by means of a thread. By the use of a threaded locking-nut, any desired length of the trocar may be exposed, so that the depth of the wound may be controlled at will, irrespective of the force used to drive the point of the instrument through the skin. It is not Fig. 1. .,. 1 ^ necessary to sterilize the puncture- ^^~- WlTilfffllfill 1 needle ; wiping it with a towel wet —iPm — -^ with alcohol is all that is required, Blood-lancet. . ,. ... r\r in ordinary examinations. (Ji course, should the patient happen to be a syphilitic, it is safer to pass the blade through an alcohol flame, after having used it. Having chosen, say, the patient's middle or ring-finger, the part is first thoroughly cleansed with alcohol or ether and then with water, and wiped perfectly dry with a clean, lint-free towel, which may be then folded into a pad and slipped behind the fin- ger to isolate it from the neighboring digits, and to serve as a cushion for the back of the hand. The operator, holding the patient's hand in a firm, steady position, makes the puncture with a rapid motion of the wrist, such as one is accustomed to use in percussing the thorax, the depth of the wound being just suffi- cient to cause a free flow of blood in good-sized drops, unaided by the slightest pressure on the finger other than that necessary to start the initial oozing. The needle should be aimed so as to strike a point in the center of the flexor surface of the finger, just back of the extreme tip. The blood-drop to be used for the ex- amination should under no circumstance be squeezed from the finger, for blood secured in this manner is certain to be more or less highly diluted with lymph from the surrounding tissues — a condition which will give rise to erroneous results, especially to lower hemoglobin, specific gravity, and corpuscular estimations than actually exist. In severe anemias, especially in those of the pernicious type, the bloodless condition of the superficial ves- sels is sometimes so marked that it may be impossible to obtain enough blood for the examination by an ordinary puncture, even from the ear-lobe, which as a rule is highly vascular. Relatively deep incisions are unavoidable in such instances. On the con- trary in most cases of leukemia, unless the coexisting anemia is of striking intensity, the blood usually flows very freely, and EXAMINATION OF THE FRESH BLOOD. 21 may even spurt from the wound in a fine jet several inches in height. Most writers on hematology utter an emphatic warning against hemophilics, in whom the slightest prick of a needle may cause troublesome bleeding. The writer has never had the misfortune to^meet with this accident, but recognizes the wisdom of observ- ing the precaution habitually to question every patient concerning an abnormal tendency toward hemorrhage. The observer's attention should be directed to the color and the density of the blood drop as it flows from the puncture, and a note taken of the various macroscopical changes which may oc- cur, such as the pale, hydremic condition of the blood found in severe anemias, and the milky appearance of the drop in leu- kemia and in diabetes. These and other alterations in. the naked- eye appearance of the fresh blood drop have been discussed in another section. The first few drops of blood which follow the Preparing puncture are wiped away, and the site of the in- THE cision freed from every trace of moisture, after Slide. which a perfectly clean cover-glass, held edge- wise between the thumb and forefinger, is lightly touched to the summit of the next drop as it oozes from the puncture, and is then immediately placed, blood side downward, Fig. 2. Proper distribution of the corpuscles in a fresh blood-film prepared for microscopical examination. upon the surface of a clean glass slide. If the cover-glass and the slide are perfectly clean and dry, and if the drop is of the 22 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. proper size, the blood will at once spread out in a thin film con- sisting of a single layer of corpuscles. (Fig. 2.) Gently heat- ing the slide over an alcohol flame just before use will ensure a thin, even spread. If prolonged study of the specimen is intended, it may be advisable to exclude air from the film, by ringing the margins of the cover-glass with a thin layer of cedar-oil, or of vaseline, but ordinarily this precaution is unnecessary. In order to prevent distortion of the corpuscles, pressure must be avoided while adjusting the cover-glass. If the blood does not spread of itself, without the aid of pressure, it is usually owing to the pres- ence of particles of dust or grease between the opposed suifaces of the slide and cover-glass. Absolute cleanliness of the covers and slides is an essential detail to which too great attention cannot be paid, for neglect of this precaution is responsible for the majority of failures to secure good specimens. Perhaps the most useful cleansing agent is the solution popularly known as " acid alcohol " (hydrochloric acid, I part ; absolute alcohol, 29 parts ; water, 70 parts), which quickly and effectually removes all traces of grease and dirt from the glasses, so that their preliminary soaking in soap-suds or in a strong mineral acid, as some recommend, may be dispensed with. The slides and covers may be conveniently kept in closed glass receptacles containing this solution, from which they are removed as the occasion demands, being then dried and polished with a bit of clean linen, or with tissue-paper. Ordinary soft "toilet-paper" is excellent for this purpose. Oblong cover- glasses, measuring f x i;| inches and of " No. i " thickness, are more easily handled without forceps than smaller square or cir- cular slips, and also have a much larger surface than the latter, which is often decidedly advantageous. The use of forceps is unnecessary, if care is observed to hold the cover-glass in the manner already directed, so that only its edges come in contact with the thumb and finger. The specimen, prepared in the manner just Microscopical described, is examined under the microscope Examination, with both low and high powers, a ■!■ or 1 inch dry, and a Jg i"ch oil-immersion objective being the most satisfactory lenses for the purpose. The substage con- denser and diaphragm should be adjusted so that the field is but moderately illuminated, rather than flooded with a glare of white light. Microscopical examination of the fresh blood-film fur- nishes information about the following points : Changes Affecting the Erythrocytes. With a little practice, one soon becomes able to detect with a tolerable degree of accuracy EXAMINATION OF THE FRESH BLOOD. 23 any conspicuous decrease in the number of erythrocytes, by the relatively small number of cells in the field in comparison with their number in a similar field of normal blood. With less con- fidence, it is also possible to decide whether or not the number of erythrocytes is much in excess of the normal standard. Deficiency in hemoglobin produces unmistakable changes in the appearance of the cells, those in which this change is well- defined appearing as pale, washed-out bodies which stand in striking contrast to the darker, yellowish-green color of the nor- mal erythrocytes. Abnormal viscosity of the erythrocytes, their tendency towards rouleaux formation, the presence of deformities of size and of shape, and the occurrence of structural degenerative changes may also be distinguished in the fresh, unstained blood-film. Nucleated erythrocytes are not demonstrable in the fresh speci- men. Changes Affecting the Leucocytes. A glance is usually suffi- cient to determine whether or not the number of leucocytes is markedly in excess of normal, but too great dependence should not be placed on such a method of detecting the presence or absence of a leucocyte-increase, since it is at the best approximate, and sometimes erroneous. As will be explained elsewhere, any marked decrease in the number of erythrocytes, the leucocytes remaining normal, may so increase the ratio of the latter to the former, that the leucocytes may be apparently increased. Having tentatively determined that an increase in the total number of leucocytes is present, it is furthermore possible for one familiar with the morphology of the unstained leucocyte to make a fairly accurate differential count of these cells, and thus to decide whether the increase is due to a pure leucocytosis, or to some form of leukemia. This distinction is not at all diffi- cult in most instances, when one recalls the characteristics of the several forms of leucocytes in the fresh blood-film, viz.: small lymphocytes, large lymphocytes, and transitional forms, appear- ing as cells having a single, spherical, or indented nucleus, and a clear, shining, non-granular protoplasm ; polynuclearneutrophiles, as cells with polymorphous or multiple nuclei, and a protoplasm crowded with very fine, moderately refractive granules ; eosino- philes, as cells with a single, polymorphous nucleus, or multiple nuclei, and a protoplasm containing coarse, spheiical, highly refrac- tive, fat-like granules ; and myelocytes as cells with a single spherical or ovoid nucleus, and a protoplasm crowded with very fine, moderately refractive granules. It is, of course, obviously impossible to distinguish basophile cells in the fresh blood, as 24 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. well as some of the cells containing fine eosinophile granules, but the characteristics noted above are sufficiently plain to justify at least a provisional diagnosis of either of the conditions in ques- tion, which, in every instance, should be verified by a careful ex- amination of the stained specimen. While most of the degenerative changes which affect the leuco- cytes are clearly demonstrable only in the stained specimen, it is still possible to recognize some of the grosser examples of such a process by a study of the fresh film. Vacuolation of both nucleus and protoplasm, extrusion of portions of the cell-substance, and the various stages of nuclear disintegration and of apparent solu- tion of the protoplasm are the alterations most commonly ob- served. In certain specimens " fractured " leucocytes are seen with more or less frequency, a cell thus affected being drawn out into a diffuse, irregularly shaped body with indistinct and ragged margins, about which the cell-granules, which have escaped from the protoplasm, are scattered in the form of a nebulous mass. The eosinophile leucocytes seem especially prone to undergo this disintegration. The exact significance of this phenomenon is not clear, but it probably represents a degenerative change in which the cells have become abnormally vulnerable, and thus highly susceptible to mechanical injury from the pressure of the cover-glass. Ameboid activity of the leucocytes, and pigmentation of these cells are among the other changes to be observed in a histolog- ical examination of the unstained blood-film. Increase in Fibrin, Blood-Plaques, and Hemoconia. The den- sity of the fibrin network and the rapidity with which it forms may be studied as coagulation of the blood-film progresses. Unless the blood-plaques are very greatly increased in number, they are not usually noticeable in the specimen prepared in the ordinary manner. The presence of hemoconia or "blood- dust" is at once rendered conspicuous by the rapid and incessant molecular motion with which these bodies are endowed. Blood Parasites. The hematozoa of the malarial fevers, the spirilla of relapsing fever, and the embryonic forms of the para- site of filarial disease should be studied in the fresh blood when- ever this is possible, rather than in the fixed and stained film, since in the latter the characteristic morphology of these parasites is greatly altered, and their motility lost. Foreign Bodies, such as free fat-droplets, collections of extra- cellular pigment, and, very rarely, the crystalline bodies of Charcot may also be observed in the fresh specimen, during the course of certain diseases. ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 25 Microscopical examination of the fresh specimen should form the initial step taken in every systematic examination of the blood, since it may be the means of determining whether or not a more elaborate investigation is necessary. By this simple pro- cedure an immediate diagnosis may be made in a number of in- stances, while in others the findings, although not pathogno- monic, are of distinct clinical value. Close familiarity with the normal histology of the blood is, of course, essential for the ap- preciation of the various pathological changes which have been outlined above. Fuller reference to these changes has been made in other parts of this book. (See Sections III and IV.) II. ESTIMATION OF THE PERCENTAGE OF HEMO- GLOBIN. No less than half-a-dozen different hemoglo- Methods. binometers, or instruments for estimating the amount of hemoglobin in the blood, are in vogue at the present time, of which the most reliable for general clinical use are the instruments devised by von Fleischl, by Oliver, and by Gowers. The hemometer of von Fleischl has been the general favorite for a number of years, both in this country and on the Continent, but in England it has been sup- planted to some extent, first by Gowers' hemoglobinometer, and in recent years by the hemoglobinometer lately invented by Oliver. All three instruments are based upon a similar principle, that of measuring the depth of color of the diluted blood by a standard color-scale of varying intensity, the gradations of which correspond to different hemoglobin values. With this instrument, which is the one pre- VON ferred by the great majority of clinicians, the color Fleischl's of a fixed volume of blood in an aqueous solu- Hemometer. tion of a definite strength is compared with the color of a movable glass wedge, tinted with Cas- sius' " golden purple." The hemometer consists of the follow- ing parts : (i) A tinted glass wedge, the thickest portion of which is of a deep pink color, and the thinnest portion almost colorless, with every intermediate color gradation between the two extremes. It is mounted in a metal frame provided with a scale, graduated at every five degrees from o to 1 20, the former corresponding to the thinnest, and the latter to the thickest part of the wedge. The metal frame is grooved so that it fits beneath (2) a small 26 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Fig. Stage, in which it may be moved backward and forward by turn- ing a milled wheel. In the center of this stage there is a circu- lar opening through which the light of a candle is reflected by a disc of calcium sulphate, mounted on the pillar supporting the stage, hke the mirror of a micro- scope. Back of this opening there is a small oval slot through which the scale of the underlying tinted wedge is visible, when the latter is adjusted to the stage. (3) A mixing chamber, consisting of a short metal tube closed at the bot- tom by a disc of glass and divided into two equal compartments by a vertical partition, fits accurately over the circular opening in the stage. When properly adjusted to the latter, the vertical partition exactly coincides with the upper edge of the underlying tinted wedge, so that the upper com- partment of the chamber is illuminated by the dull white light from the reflector, while the lower compartment receives the VoN Fleischl's hemometer. (4) A capillary pipette mounted in a Fig. 4. Tinted glass wedge of the von Fleischl hemometer. color of the tinted wedge, short metal handle, used for making the blood dilution. A single pipetteful of nor- mal blood mixed with suffi- cient distilled water to fill exactly one of the compart- ments of the mixing cham- ber gives a solution which matches the color of the tinted wedge opposite the mark 100. (5) A small fine-pointed glass dropper, used for filling with water the compartments. Method of Use. As a preliminaiy step, each compartment of the mixing chamber is filled iH about one-quarter full of distilled water, by Capillary pipette mcans of the glass dropper to one end of which a rubber cap has been fitted. A puncture having been made, as previously directed, a measured volume of blood is collected by bringing one end of the capillary pipette lightly in contact with the blood drop, as it oozes from the wound, so that the tube is instantly filled with blood, by capillary force. No difficulty will be experienced Fig. OF VON Fleischl's hemometer. ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 2/ in quickly filling the tube if it is applied horizontally to the side of the blood drop, rather than vertically to its summit, care being observed not to immerse the end too deeply. It is needless to add that the interior of the tube must be abso- lutely clean and dry, to insure which a very fine needle and thread may be passed through it just before using. As soon as the pipette is filled, every trace of blood must be removed from its outer surface, and the precaution taken to see that the column of blood is exactly flush with the ends of the tube, being neither bulged out nor depressed. The blood is then washed into one of the compartments of the mixing chamber, by forcing a stream of distilled water through the pipette by means of the glass dropper, this rinsing being repeated until it is certain that every trace of blood has been removed. The preceding steps must be carried out quickly, in order to avoid errors arising from coagulation of the blood. The blood and water in the compartment are now thoroughly mixed, by stir- ring with the handle of the pipette, until the color of the so- lution is uniformly diffused, after which water is added, drop by drop, to each compartment until they are both filled ex- actly to their brims. In doing this, no water must be spilled on the thin edge of the vertical partition, for should this occur it may cause an overflow of the liquid from one com- partment to the other, and thus alter the strength of the blood solution. If the latter should appear turbid, or muddy, as it sometimes does with leukemic blood, a few drops of a weak aqueous solution of potassium hydrate may be added to the diluent as a preventive of this change. The addition of a little ether will clear the solution, if the turbidity is due to the presence of fat. Having carried out the preceding steps, the mixing chamber is adjusted over the circular opening in the stage of the instrument, so that the compartment containing the blood solution is upper- most, overlying the semicircle illuminated by the clear, white light; while the compartment filled with water fits over the semi- circle which receives the tint of the underlying glass wedge. The remainder of the test, the comparison of the color of the two compartments, must be completed by artificial light prefer- ably by candle-light. Moderately bright illumination is better than a strong glare, for the latter interferes seriously with the accurate determination of delicate color differences. By means of the milled wheel the tinted glass wedge is moved backward and forward until its color precisely corresponds to that of the diluted blood. When this occurs, the percentage of hemoglobin 28 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. is read off from the scale visible through the oval slot in the stage of the instrument. While making the color comparison the observer should stand facing one end of the glass wedge {not the milled wheel), so that the partition between the two compartments of the mixing cham- ber is in a line with the vertical axis of his eyes, the distance from the latter to the top of the stage of the instrument being about- ten or twelve inches. Gross errors may be avoided if the observa- tion is made with one eye, and if the same eye is habitually used, since the two eyes may differ radically in their sensitiveness to color impressions. It is important to decide the color differences as quickly as possible, for prolonged examination rapidly dulls one's color perception, and creates uncertainty as to the proper reading. It is a good plan first to bring into the field of vision the darkest portions of the wedge between the figures loo and 1 20 of the scale, and then, by short, sudden turns of the milled wheel, to produce abrupt color contrasts of from 5 to 10 de- grees at each turn, until the two tints approximately correspond.^ When this point is reached the eye should be rested for a few moments, and then, by a succession of shorter turns, the wedge is again swept to and fro until the colors appear identical. In the average instance an error of about 5 degrees must be antici- pated, in spite of every precaution to insure accuracy. In cases in which low hemoglobin percentages (30 per cent., or less) are suspected, it is essential to use two or three pipette- fuls of blood in making the dilution, dividing the percentage indi- cated by the instrument by two or three, as the case may be. This precaution effectually removes the objection which has been urged against this instrument on account of its inaccuracies in the determination of low hemoglobin percentages. Another criticism of the von Fleischl instrument has been made on the ground that, since the length of the tinted wedge visible through the compartment of the mixing chamber includes a color range of 20 per cent., it is impossible for one to select a single point in the center of this color for comparison with the even, diffuse tint of the blood solution. This objection may be overcome to a great extent by using a metal diaphragm, provided with a slit one-eighth of an inch in width, which is placed over the glass disc at the bottom of the compartments, to limit the field of vision. Adjusted so that the slit crosses at right angles the par- tition separating the two colors, the use of this device cuts down 1 It is important to bear in mind the fact that the judgment of color differences is much easier if marked contrasts in color value are made, than if a gradual blending of the two tints is attempted, by slowly moving the wedge past the visual field. ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 29 the field of observation to a portion of the glass wedge corre- sponding to about 2.5 degrees on the scale. The hemoglobin percentages indicated by this instrument ap- pear to be low for the blood of the average healthy American, Fig. 6. Method of using the von Fleischl hemometer. Note that the septum between the two halves of the blood compartment is at right angles to the horizontal axis of the observer's eyes. A cylinder of paper may be fitted over the blood compart- ment, to serve as a camera-tube, since it is more common to obtain readings of from go to 95 than of the arbitrary standard 100, in persons in whom there is no good reason to suspect subnormal hemoglobin values. In in- struments of recent manufacture, however, this fault is largely corrected. 30 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. In order to exclude the light of the candle from the field of vision, while making the color comparison, it is customary to use a tube of cardboard or stiff paper, which is slipped over the mixing chamber, and rests upon the platform of the instrument. This sort of a device answers veiy well when the examination is made in a darkened room, as, for example, at a patient's residence. In hospital work, however, the inconvenience, sometimes con- siderable, of being compelled to carry the diluted blood some distance from the bedside to a dark-room may be avoided by the use of a light-proof box, which may be conveniently carried from ward to ward, so that the test may be completed at the bedside. (Fig. 7.) A box of this kind should measure sixteen inches Fig. 7. Light-proof box for the von Fleischl hemometer. The door of the box is closed and the color comparison made through the camera-tube. Note. — Reichert, at the suggestion of Miescher, has recently introduced a modifi- cation of the original von Fleischl hemometer, designed to increase the accuracy of the test, by making it possible, by definite dilution of the blood, to select that part of the tinted wedge which is best adapted for the examination of any particular sam- ple. This innovation was prompted by the discovery that the intermediate portions of the wedge are better adapted for obtaining accurate readings than the terminal parts. The principal modification of the new hemometer consists in the substitution for the original capillary blood pipette of a special mixing pipette, similar to a melangeur, graduated so that the blood may be diluted I : 200, i : 300, and I : 400 times. A table supplied with the instrument translates the combined results of the dilutions and the figures indicated by the scale on the wedge into absolute hemoglobin percentages. The instrument is also supplied with mixing chambers of different depths, and with a diaphragm designed to limit the field of vision. The writer has had no practical experience of the Miescher-Fleischl hemometer, but an examination of the instrument justifies the belief that its elaborateness renders it undesirable for gen- eral clinical work. Its cost (§50.00) is also a bar to many. ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 3 1 in height by twelve inches in length and in width, being fitted with a hinged door which may be fastened shut by a simple catch, and provided with a circular opening through which the milled wheel of the hemometer projects when the door is closed. A metal camera-tube, flanged at the upper extremity for the obser- ver's eye, pierces the top of the box and communicates inside with the mixing chamber of the hemometer. The tube fits loosely in a circular opening in the top of the box, so that it may be readily raised and lowered ; its diameter is a trifle greater than that of the mixing chamber around which it should fit snugly when lowered into position ; and its length is governed by a fixed collar outside the box, which prevents it from slipping and jarring the instrument. Wooden guides, such as are used for securing a microscope in its box, are provided to receive the horseshoe base of the hemometer, holding it firmly in such a position that when the camera-tube is lowered into position, the milled wheel of the instrument projects through the opening in the closed door. The interior of the tube and of the box is painted a dull black. A candle is placed in position on the floor of the box, in a line with the " mirror " of the instrument. In using this device, first the candle within the box is lighted, and the hemometer base is slipped into place between the wooden guides. The blood dilution having been made in the usual man- ner, the mixing chamber is then set upon the platform of the in- strument, and the camera-tube which has been raised to allow this to be done, is lowered until it telescopes around the mixing chamber, and rests firmly upon its collar. The door of the box is now closed, and the two compartments are brought into their proper positions over the glass wedge by turning the camera-tube from the outside of the box, the observer mean- while noting the result by looking through the flanged ex- tremity of the tube. This accomplished, the projecting wheel of the instrument is turned to and fro until the colors of the two compartments are the same, when the door is opened, and the percentage read off" from the scale of the hemometer. Care must be observed to see that the exterior of the mixing chamber is perfectly dry, for if any moisture collects between its outer sur- face and the inner surface of the camera-tube, the contents of the compartments may be disturbed and serious errors re- sult. As the opening in the door of the box is covered by the hand with which the milled wheel is turned, sufficient light to interfere with the test cannot leak in at this situation. With a light-proof box of this sort it is possible accurately to carry on hemoglobin estimations in the brightest daylight. 2,2 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. which may be entirely excluded from the instrument, while the observer's field of vision is limited to the two semicircles illumi- nated by the candle burning within the box. With this instrument the principles of Lovi- Oliver's bond's tintometer are applied to the quantitative Hemoglobin- estimation of hemoglobin, the color of a blood OMETER. solution of a definite strength being compared, by light reflected from a dead white surface, with a series of tinted glass standards which constitute a progressive color scale. Thus, a series of fixed, definite tints is provided, each of which accurately corresponds to the specific color-curve of progressive dilutions of normal blood, this having been de- termined individually, by means of the tintometer. Two sets of color standards have been devised : one for daylight readings, and one for observations by candle light, the latter being prefer- able on account of the greater delicacy of its readings. Oliver's complete apparatus consists of: (i) A capillary blood measure, made of heavy glass tubing, and having a capacity of 5 cubic millimeters. The end to be presented to the blood drop, in filling the measure, is tapered to a blunt point, and highly polished. (2) A mixing pipette, provided with a short rubber tube which fits over the tapered end of the blood measure, while rinsing out the blood from the latter into the (3) standard blood cell, which, when filled exactly to the brim with distilled water in which one measureful of blood has been dissolved, yields a blood solution of approximately one per cent. When filled, the cell is covered with a glass slip provided for this purpose. (4) A standard color scale, consisting of 1 2 tinted glass discs, mounted in two series, and corresponding to hemoglobin percentages ranging from 10 to 1 20. (5) A set of riders, or squares of tinted glass, used for determining the intermediate degrees of color between the deci- mals indicated by the fixed tints of the scale. For ordinary clin- ical work two riders are sufficient, which when laid over the discs of the standard scale read 2.5 and 5 degrees respectively on its upper half, but double this amount on the lower half. For physi- ological observations requiring readings in units a set of nine riders is supplied. (6) A collapsible camera-tube, through which the color comparisons are made. Method of Use. In making hemoglobin estimations with Oliver's apparatus, first the capillary measure is filled with blood by the method directed for filling the pipette of the von Fleischl instrument. The rubber nozzle of the mixing pipette, previously filled with distilled water, is then adjusted over the polished end of the blood measure, and the blood washed into the standard cell by ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 33 forcing through the water, drop by drop. As soon as all of the blood contained in the bore of the measure has been thus washed out into the cell, the rubber nozzle of the pipette is re- moved, and the handle of the measure used as a stirrer to mix the blood solution, more water being added in single drops, from time to time, until the cell is accurately filled. The blue cover- glass is then adjusted, with the result that, if the cell has not been overfilled, a small air bubble forms on the surface of the liquid. The blood cell, filled in this manner with a blood solution of definite strength, is now placed by the side of the standard scale opposite the tinted disc to which it corresponds most closely, the eye readily recognizing its approximate position on the scale. More accurate matching of the two colors is made with the aid of the camera-tube, the cell being moved from disc to disc in an endeavor to match exactly the color of the blood solution by one Fig. 8. Method of using Oliver's hemoglobinometer. of the standard tints of the scale. If this is successful, the hemo- globin percentage indicated by the disc is read off, and the obser- vation is completed. But if it happens that the tint of the blood solution is obviously deeper than a certain disc, but paler than the 3 34 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. one immediately above, the cell is kept alongside the lower of the two, over which a rider is adjusted, in order to deepen its color, while the square of white glass is placed over the cell, so as to compensate for the thickness of the rider. If now the colors correspond, the final reading is ascertained by taking the percent- age of the disc plus the value of the superimposed rider. If the color of the blood solution is darker than that of any one of the standard discs, but paler than the disc plus a rider, the mean average of the two is taken as the final reading ; similarly, if the color of the blood solution is darker than a certain disc plus a rider, but paler than the disc immediately above, the values of the two must be averaged. An error of two per cent, is un- avoidable, even in the hands of a skilful observer. During the observation the candle should be placed three or four inches from the end of the color scale, being adjusted so that the flame is in a line with the opposed sides of the cell and of the scale, thus illuminating both with equal intensity. The positions of the candle and of the apparatus are shown in the accompanying illustration. (Fig. 8.) Small sized candles, such as are used for decorating Christmas trees, furnish a flame of the proper de- gree of brilliancy, the candle of ordinary size giving too intense a light. Total exclusion of daylight is not necessary, so that the observation may be made in the corner of a partly darkened roonl, as, for example, behind a closet door or some other similar shield against direct rays of light. Oliver's hemoglobinometer is a trial to the patience of one who has habitually used the von Fleischl instrument, and it takes some time to become accustomed to it after having worked with the comparatively simple color comparisons of the hemometer. But the results obtained with the newer instrument are so much more accurate than are possible with the older, that no one should hesitate in choosing Oliver's apparatus as the better of the two. This instrument, which for many years has GowERs' been popular in England and is used to some Hemoglobin- extent in this country, consists essentially of two OMETER. small flattened tubes of equal diameter, which when in use are fixed upright and parallel to each other in a small wooden support furnished for this purpose. One tube contains glycerine jelly colored with picrocarmine to cor- respond to the tint of a I in loo solution of normal blood (or, 20 cubic millimeters of blood in 2 cubic centimeters of water), this being taken as the standard with which the blood solution, contained in the second tube, is compared. The second tube is provided with a scale graduated in units from 5 to 1 20, each de- ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 35 Fig GOWERS' HEMOGLOBI- NOMETBR. gree of which equals the volume of blood required for the test. Twenty cubic millimeters of normal blood, dissolved in sufficient distilled water to fill this tube to the loo mark on the scale, give a solution which corresponds to the tint of the standard tube. The special capillary pipette used for measuring the blood is graduated at lo and at 20 cubic millimeters, and fitted with a bit of rub- ber tubing and mouthpiece for filling it by suc- tion. Method of Use. The technique of hemoglobin estimations with Gowers' apparatus is extremely simple. Having made the puncture in the usual manner, the blood is sucked up the caliber of the capillary pipette until the mark 20 is reached, and then immediately blown out into the grad- uated tube, into which a few drops of distilled water previously have been placed, in order to insure instantaneous solution of the measured amount of blood. All traces of blood which may have adhered to the bore of the capil- lary pipette are removed by filling it several times with water, the rinsings being added to the mixture of blood and water in the tube. During the preceding steps the usual precautions must be observed, to wipe all surplus blood from the outside of the pipette before expelling its contents, and to measure the blood rapidly so as to guard against errors arising from rapid clotting. Distilled water is now added, drop by drop, to the mixture in the tube until the color of the blood solution exactly corresponds to that of the picrocarmine standard, the contents of the tube being mixed between each addition, by rapidly reversing it two or three times, with its open end closed by the thumb. The drop or two of liquid adhering to the thumb should be wiped off against the wall of the tube so that it may drain back into the liquid. When the tints of both tubes are precisely similar, the division of the scale to which the diluted blood reaches is read off, to express the per- centage of hemoglobin in the specimen under consideration. In comparing the colors, which is done by daylight, the tubes should be held against a sheet of white paper, or, as suggested by Gowers, between the eye and a window, and viewed at such an angle that their adjoining edges appear to overlap, thus cutting off the vertical streak of white light visible between them, should this precaution be neglected. Owing to the diagonal position in which the two tubes are adjusted in their support, the proper angle to produce this effect may be readily determined. 36 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Fig. 10. The chief drawback to the use of this instrument is the hability to over-dilution of the blood after it has been mixed in the grad- uated tube, the occurrence of this accident necessitating, of course, a repetition of the entire operation. To the novice it is usually- hard to decide just when sufficient water has been added to the blood to bring its color down to that of the standard tint, since one must depend solely upon a gradual weakening of the tint of the blood solution, and this is much more difficult than to com- pare a definite blood color with the sliding scale of the hemom- eter, or with the series of discs of the Oliver apparatus. The instrument may be regarded as accurate within 2 or 3 per cent, for hemoglobin percentages above 10, below which figure it is impossible to distinguish a difference between the tints of the two tubes. This source of error, however, is too remote a possibility to detract from the instru- ment's practical value. Dr. Ar- Dare's thur Dare, Hemoglobin- of the Jef- OMETER. fersonHos- pital Med- ical Clinic, has recently de- vised a new form of hemo- globinometer, by the use of which a thin film of un- diluted blood is brought into direct comparison with a standard semicircularwedge of tinted glass ranging in color from a claret red at the thickest part, to a pale pink at the thinnest. The instrument consists of the following parts : (\) h. cap- illary blood chamber, con- structed of two rectangular plates of polished glass, the opposed surfaces of which are so ground that, when clamped together in a metal bracket, a shallow compartment holding a thin film of blood is formed. One plate is made of transparent, the other of opaque glass, the latter being next to the source of light, in order to soften its glare, when the instrument is in use. The metal Dare's hemoglobinometer. R, milled wheel acting by a friction bearing on the rim of the color disc ; S, case enclosing color disc, and provided with a stage to which the blood chamber is fit- ted ; T, movable wing which is swung outward during the observation, to serve as a screen for the observer's eyes, and which acts as a cover to enclose the color disc, when the instrument is not in use ; U, telescoping camera- tube, in position for examination ; V, aperture admitting light for illumination of the color disc; X, capillary blood chamber adjusted to stage of instrument, the slip of opaque glass, W, being nearest to the source of light ; Y, detachable candle-holder ; Z, rectangular slot through which the hemoglobin scale indicated on the rim of the color disc is read. ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 37 Fig. II. bracket of the blood chamber is adjusted to the stage of the in- strument in such a manner that the blood-film fits over an aper- ture communicating with a camera-tube screwed to the op- posite side of the rubber case. (2) A graduated color standard made of a semicircle of glass tinted with Cassius' "golden- purple," and thinning out like a wedge with various depths of color corresponding to the tints of fresh blood containing dif- ferent percentages of hemoglobin. It is mounted upon a disc ad- justed in the frame of the instrument so that it may be revolved to bring various portions of its surface over an aperture directly alongside of the one through which the blood film is vis- ible. A scale, read from the outside of the instrument, in- dicates in units the hemoglobin percentages from 10 to 120. (3) A hard rubber case encloses the color standard, when the instrument is in use, the disc upon which the standard is mounted being revolved by turning a small milled wheel acting upon the rim of the disc by a friction bearing. To one side of the case a telescopic camera-tube, fitted with an eye-piece, is attached, while on the opposite side a stage furnishes support for the blood chamber, back of which a candle, held between a pair of spring clips, is adjusted. Two apertures of equal diameter, placed side by side on the same level, transmit the light of the candle through the bloo^-film and the color standard to the field of vision enclosed by the camera-tube. By reference to the accompanying diagram (Fig. 1 1 ), it will be seen that the light of the candle, J, equally illu- minates the blood film enclosed be- tween the two rectangular glass plates, O and P, and the edge of the color standard, L, mounted upon the glass disc, K. The differences in the two colors are visible through the two apertures, M and M', communicating with the camera-tube, N. By revolving the disc the tint of the color standard may be altered until it matches that of the blood-film. Method of Use. The instrument is prepared for use by first swinging outward the movable screen which serves as a cover for the case. The two apertures overlying the blood-film and the color scale are thus brought into view, the direct light from the -■'■^ =f-Ti fr— m/" N i 1 Horizontal section of Dare's hemoglobinometer. 38 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. candle being shaded from the observer's eyes by the intervening screen. The camera-tube and the candle-holder are then fitted to their attachments on opposite sides of the instrument, and a candle adjusted so that the surface of its " wick end " is just on a level with the top of the spring clips. The blood chamber is filled by touching its edge to the side of a rather large drop of blood, as the latter flows from the punc- ture, so that the blood Fig. 12. at once flows into and fills, by capillary force, the shallow compart- ment between the pair of glass plates. As soon as this occurs, any excess of fluid which may have ad- hered to the outer sur- face of the blood cham- ber is carefully wiped Manner of filling blood chamber. -^ ^ away, and the latter is slipped into the tongue which holds it in position on the stage of the instrument. The candle having been lighted, the observer holds the instru- ment as a field glass, and compares with one eye the colors of the blood-film and the standard disc which are seen side by side in the field of vision limited by the camera-tube. The disc is made to revolve by making short, quick turns with the milled wheel, until the two colors are identical, and the hemoglobin percentage indicated by the scale is then noted. The color comparisons need not be made in a darkened room, although the observer should avoid facing the direct sunlight, and, in order to exclude reflected light, should hold the instru- ment against a dark surface, such as a black coat sleeve. When the observation is completed, the two glass plates of the blood- chamber are removed from the bracket by loosening the screw which holds them in position. They are then cleaned with water and with acid alcohol, dried, polished, and replaced in the bracket. The various parts of the instrument, when detached, fit into a small leather carrying-case. The chief advantage of Dare's instrument lies in the fact that dilution of the blood is not required, and therefore errors due to incorrect measuring and dilution of the blood, which must be care- fully guarded against in the older hemoglobinometers, are entirely eliminated. A film of whole blood also gives a relatively deep ESTIMATION OF THE PERCENTAGE OF HEMOGLOBIN. 39 and definite color, which may be judged with greater ease and accuracy than the paler and more indefinite tint of a blood solu- tion. It is also obvious that errors due to the turbidity of an aqueous solution of leukemic blood, are avoidable by the use of an undiluted film. Coagulation of the film does not occur with suf- ficient rapidity to constitute a source of error, since the test may be completed within a few seconds after the blood has been drawn. One year's more or less constant use of this instrument in the Jefferson Medical Clinic has shown that its readings closely cor- respond to those of Oliver's hemoglobinometer, and average some- what higher than those of the von Fleischl instrument. The color standard of Dare's apparatus, being wedge-shaped, and therefore gradually blending the tints, is open to- the same criticisms which have been urged against the scale of the hemometer. A simple method of approximately determin- Tallquist's ing hemoglobin percentages without the aid of Method. a special instrument has recently been devised by Tallquist,^ the procedure consisting, in brief, in allowing a drop of blood to soak into a bit of filter-paper and comparing with the naked eye the color strength of the stain with a series of printed standard tints of known value. The lat- ter are arranged as a scale of ten different colors corresponding to the colors of stains produced by bloods having hemoglobin values ranging from loto loo per cent., the latter being regarded as the normal. A lithographed copy of the color standard ac- companies Tallquist's original article. The test is made in the following manner : A drop of blood, large enough to make a stain about five or six millimeters in diameter, is caught in the center of a piece of white filter-paper, care being taken in col- lecting it to apply the paper to the exuding drop in such a man- ner that the blood soaks in very slowly, and thus produces a stain which is evenly colored throughout. Perfectly white filter- paper having a smooth surface, and of a thickness corresponding to about fifty-five leaves to the centimeter, should be used for the test. The blood stain thus made is pressed lightly against a pad of filter-paper, and then compared, by direct daylight, with the series of standard tints, the figure opposite to the tint which the stain most accurately matches being read off, to indicate the per- centage of hemoglobin in the specimen under examination. The comparison must be made immediately after the stain loses its humid gloss, since blood soon changes its color after exposure to the air. This direct method of hemoglobin testing is, of course, only •St. Paul Med. Joum., 1900, vol. ii.,p. 291. 40 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. approximate, at the best, and cannot be expected to furnish re- sults comparable in point of accuracy with those to be obtained by any of the instruments just described. It may, however, be employed to excellent advantage when a hemoglobinometer is not at hand, or in certain cases in which only a rough estimate of the amount of coloring matter of the blood is sought. Tall- quist, who has tested his method under the control of the he- mometer, in his clinic at Helsingfors, claims that the limit of error generally does not exceed ten per cent. III. COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. Of the various instruments used for counting Methods. the blood corpuscles, the hemocytometers de- vised by Thoma and by Gowers are most gener- ally employed at the present time, the former being used almost to the exclusion of the latter everywhere except in England, where Gowers' apparatus has many firm adherents. Durham, by adapting and modifying a number of the details of the older instruments, has succeeded in devising an improved form of hemo- cytometer which possesses many advantages over the original models, being of simple construction, accurate, and comparatively inexpensive. The method of making the estimate, which is es- sentially the same with all three of these instruments, consists, briefly, in first diluting the fresh blood in definite proportions with some indifferent preservative fluid, and in then counting, under the microscope, the number of corpuscles in a drop of the di- luted blood, the latter being contained in a small glass cell on the floor of which is ruled a series of micrometer squares of cer- tain dimensions. The cubic contents of the cell and the degree of the blood dilution being known, the number of corpuscles counted in any given number of these squares may be taken as a basis for calculating the total count of corpuscles to the cubic millimeter of blood. In addition to the method of actually counting the corpuscles in a known volume of diluted blood, Oliver has devised an instru- ment with which the number of erythrocytes may be estimated by means of their optical effect, without the use of the microscope. Diluting fluids for use with the hemocytom- DiLUTiNG eters of Thoma, Gowers, and Durham should Fluids. be of such a composition that when mixed with the fresh blood they preserve unaltered the form of the corpuscles. This requirement being met, the examiner COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 4 1 may choose from the numerous formulae in current use the one which best suits his individual preference. Among the most sat- isfactory solutions used for this purpose the following may be mentioned : Toisson's Solution. Methyl-violet, 5 B 0.025 Sodium chloride i.o Sodium sulphate 8.0 Neutral glycerine 30.0 Distilled water 160.0 Sherrington's Solution. Methylene-blue o.i Sodium chloride 1.2 Neutral potassium oxalate 1.2 Distilled water 300. o For general clinical work no better formulae have ever been suggested than the preceding two. Both solutions act as excel- lent preservative fluids, 'and each contains just sufficient quantity of a basic aniline dye to stain the leucocytes with great distinct- ness, so that they may be readily differentiated from the erythro- cytes, which remain uncolored. Hayem's Solution. Mercuric chloride 0.25 Sodium chloride 0.5 Sodium sulphate 2.5 Distilled water 100.0 Oliver specifies this solution as the diluent invariably to be employed with his instrument, but it may be used also with the other forms of hemocytometers, although with less satisfaction than the formulae first mentioned. Among the simpler diluting fluids, all of which are depend- able, are solutions in distilled water of common salt (0.7 per cent.), of potassium bichromate (2.5 per cent), and of sodium sulphate (5 per cent.), to any of which about 0.5 per cent, of an alco- holic solution of methyl-violet may be added, in order to stain the leucocytes, and thus to facilitate the counting. An aqueous solution of acetic acid, varying in strength from 0.3 to 0.5 per cent., which destroys the erythrocytes and at the same time renders more conspicuous the leucocytes, has been recommended by Thoma as a diluent in counting the latter cells, by means of his special pipette. 42 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Fig. 13. As spores are liable to develop and precipitates to form in all the above-mentioned solutions, they should always be filtered before using, and kept in tightly corked bottles. The Thoma-Zeiss hemocytometer, which is The Thoma- to-day regarded as the standard instrument for Zeiss Hemo- blood counting, consists of two graduated capil- CYTOMETER. lary pipettes, for diluting and mixing the blood, and a counting chamber in which a measured volume of diluted blood is placed for the purpose of counting the corpuscles under the microscope. One of the pipettes is intended for counting the erythrocytes, and, for con- venience's sake, may be termed the erythro- cytometer ; while the other pipette, which is used for counting the leucocytes, may be called the leucoc5;tometer. It is not, however, necessary to purchase both pipettes, as sup- plied with the complete apparatus, since both erythrocytes and leucocytes may be counted accurately with the erythrocytometer. The erythrocytometer consists of a heavy glass capillary tube, the lumen of which is expanded near the upper end into a bulb con- taining a small cubical glass bead, which serves as a stirrer. The lower end of the tube is ground to a blunt point, and to -the upper end is fitted a short bit of rubber tubing capped by a bone mouthpiece, for filling the tube by suc- tion. A scale is enameled into the glass wall of the pipette, the three main divisions of which are indicated by the figures, 0.5, i, and 10 1, jl II the first two gradations being below, and the . I tI latter one above, the bulb ; the lower portion ^ f 11 °^ *^^ ^^^ '^ further graduated in tenths, by cross lines, from o.i to i. If blood is drawn up in the pipette to the mark i, and the diluent added until the mark loi is reached, the blood is thus diluted one hundred times ; or if the blood is drawn up only to the mark 0.5, and the diluent added as before, a two hundred-fold dilution is obtained. The leucocytometer is a capillary tube similar to the former, but having a larger lumen, . so that lowe r dilutions are obtained with it. If blood is drawn up to and the diluent added until the mixture reaches the J" B The Thoma-Zeiss hemocytometer. a, erythrocytometer; B, leucocytometer. the mark i. COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 43 mark 1 1, the blood is diluted ten times ; or if the blood column reaches the mark 0.5, with the same addition of diluent, the dilu- tion thus made is twenty-fold. In the latest model of this pipette the lower end tapers to a fine point, the diameter of the lumen thus gradually decreasing as the extreme tip is approached. The chief object of this modification is to prevent accidental leaking out of the column of blood when the tube is held vertically, while sucking up the diluting fluid — an accident difficult to avoid with the old-style pipette, having a large lumen from tip to bulb. The counting chamber (Fig. 14) consists of a heavy glass slide in the center of which is cemented a square glass plate provided with a circular opening which fits around a ruled disc ; the diameter of the latter being slightly less than that of the opening in the sur- rounding plate, a shallow, narrow gutter is thus formed between the two. The surface of the ruled disc is exactly -^-^ millimeter below the level of the glass plate by which it is enclosed, so Fig. 14. Thoma-Zeiss counting chambek. that a chamber of this depth is formed when both are superim- posed by a cover-glass having an absolutely plane surface, two of which are furnished with each instrument. An ordinary cover- glass should never be used, for owing to the unevenness of its surface a deviation from the standard in the depth of the underly- ing chamber must necessarily result. When an objective having an extremely close " working distance " is employed, the special hollow-cell cover-glass, made by Zeiss, will prove useful. The central part of the disc's surface is divided, by microscopical diamond-rulings, into 400 small squares, each of which has an area of -j^ square millimeter, these small squares being grouped into sets of sixteen, by a series of vertical and horizontal double rul- ings bisecting each fifth column of squares. (Fig. 15.) The cubic contents of each small square, when the cover-glass is ad- justed, is T^-^-^-^ cubic millimeter, since they measure individually 44 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. tV ^y 2V ^y 2V millimeter. In Zappert's modified ruling of the Thoma-Zeiss counting chamber, extra hnes have been added so as Fig. 15. Ruled area of the Thoma-Zeiss counting chamber (ordinary ruling). to increase the ruled area of the disc to nine times its original size. As illustrated in the accompanying diagram (Fig. 16), the Fig. 16. Zappert's modified ruling of the Thoma-Zeiss counting chamber. surface of the disc is thus divided, by heavy cross-ruUngs, into nine large squares each equal in area to the central group of 400 small COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 45 squares, the whole ruled surface therefore equalling an area covered by 3,600 of the latter. To simplify the counting, the peripheral squares ^lre subdivided into four, each of the latter being of the Scune area as i oo of the small central squares. This improved form of counting chamber is an invaluable convenience in leucocyte counting, and should be chosen in preference to the older model. If any difficulty should be experienced in distinguishing the ruled hnes under the microscope, they may be made more con- spicuous by blackening them with a little soft lead pencil dust, placed on the surface of the disc and thoroughly rubbed in with the pulp of the finger, the excess being \\-iped off Emd the disc polished with a bit of lens-paper, or a soft handkerchief. Fig. 17. Method of Fn.xjNG the capillary titbe of the Thoma-Zeiss hehocttometer with blood. Counting the Erythrocytes. Having made the puncture, as already described, the point of the erythrocytometer is plunged into the blood drop, as it flows from the wound, and, by making gentle, uniform suction, a column of blood is drawn up the capil- lary tube exactly to the mark 0.5. The point of the instrument 46 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. is then wiped perfectly dry, and immediately dipped into the diluting fluid, which is drawn up the tube in the same manner until the mixture of blood and diluent reaches the mark loi, above the bulb. While adding the diluent, the pipette should be twisted to and fro between the thumb and forefinger, in order that the blood and diluent may be mixed, by the whipping about of the glass bead, as they fill the bulb ; if this precaution is neglected, a por- tion of the blood will rise in a distinct layer above the diluent, as the latter flows into the bulb, and may be drawn, unmixed, into the capillary constriction beyond. A more thorough mixture of the blood and diluent is now made, the rubber tubing being slipped from the instrument, which is then grasped so that its ends are closed by the thumb and middle finger, and rapidly shaken for about half-a-minute. By the above steps, a mixture is made in which the proportion of blood to diluent is i : 200, a degree of dilution with which it is most convenient to work, in the great majority of instances. For two reasons a i : 200, rather than a i : 100, dilution is to be preferred in routine work : (i) If, as not infrequently happens, the blood column is accidentally drawn up the tube beyond the mark 0.5, in an attempt exactly to reach this gradation, it is a simple matter to correct the error by gently blowing or shaking the blood column down to the proper height ; whereas, in attempting to make a i : 1 00 dilution, should the mark i be exceeded, the blood column will almost surely escape into the bulb whence it cannot be blown back again into the capillary tube, thus necessitating a repetition of the whole operation with a fresh drop, after having cleaned and dried the erythrocytometer. (2) It is easy to count the corpuscles in a I : 200 dilution, since the surface of each ruled square of the counting chamber is, as a rule, occupied by not more than half a dozen cells ; on the contrary, in a i : 100 dilution, except in an occasional instance in which there is a striking paucity of cells, the field may be so overcrowded with corpuscles that their enu- meration is difficult, and often inaccurate. The next step is to place a drop of the diluted blood in the counting chamber, preparatory to counting the corpuscles under the microscope. The unmixed diluting fluid in the lower portion of the capillary tube is first expelled, by blowing out four or five drops, after which the point of the pipette is dried with a soft cloth, and a small drop of the blood mixture is allowed to fall, by force of gravity, exactly in the center of the surface of the ruled disc. The cover-glass is then immediately placed in posi- tion, and the slide left undisturbed for several minutes, so that the corpuscles may settle. The drop placed on the disc should COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 47 be of sufficient size to occupy only its central portion, the object being to use just enough of the blood mixture to cover the ruled area and exactly to fill in the vertical space between the surfaces of the disc and cover-glass when the latter is placed in position. If the drop contains air-bubbles, or if it is so large that it over- flows into the gutter and perhaps finds its way between the cover- glass and the glass plate beneath, errors will result, so that in event of either of these accidents the procedure must be repeated with another drop, after having cleaned and dried the cover-glass and the counting chamber. Water, and not alcohol or xylol, is to be used for this purpose, since the repeated use of chemicals will soon dissolve the cement which fixes the disc to the counting chamber. In repeating the operation the original technique must be rigidly followed, i. e., the erythrocytometer must be briskly shaken for half a minute or so, and the contents of its capillary stem blown out, before placing the new drop in the counting chamber. In a properly prepared slide concentric rings of color — New- ton's rings — may be seen at the points of contact between the cover-glass and the underlying glass plate. If these rings are in- visible, or if they do not appear when pressure is made upon the cover-glass, it is a sign that the contact between the two glass surfaces is not true, this being due to the presence of particles of dust or of moisture beneath the cover-glass. Inasmuch as this may seriously affect the correctness of the count, it is a safe rule invari- ably to reject a slide in which these color rings are not visible. As soon as sufficient time has elapsed for the corpuscles to sink to the bottom of the counting chamber — about five minutes — the slide is transferred to the stage of the microscope, which should not be inclined, for fear of disturbing the uniform distribu- tion of the cells. The field is first brought into focus with a low- power objective (a No. 3 objective of Leitz, for example), and the slide moved across the stage until the extreme upper left-hand comer of the group of small ruled squares is brought into view, when a higher power, to be used in counting, is substituted. For this purpose the writer is accustomed to use a Leitz No. 6 objective and No. 4 ocular, which lenses, with a tube length of 155 mm., cut off a field occupied by a block of 25 small squares. As a basis for the final calculation, the erythrocytes in 400 small squares, or the entire ruled surface of the old-style disc, should be counted, preferably by going over two groups of 200 squares each in two different drops, rather than by taking the en- tire 400 squares in a single specimen. By following this plan the count of one drop may be " controlled " by the count of the other, and any discrepancy between the two discovered, for if the dif- 48 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. ference in the counts is striking, a third group of 200 squares must be examined in an additional drop, and an average taken of the two counts which most closely correspond. In order to simplify the process of counting, some routine method of examining the ruled area, such as the following, should be adopted : Beginning at the upper left-hand corner of the ruled disc, the corpuscles in the first 100 small squares are counted, the slide being moved from above downward, preferably by the aid of a mechanical stage, as the successive groups of squares are covered. By employing the magnification to which reference has just been made, three shifts of the slide are sufficient to bring into the field the requisite number of squares in blocks of 25 each. Examining each small square in succession, proceed from left to right along one row of five, then drop to the next row and Fig. 18. Plan of counting the erythrocytes. The small squares are examined in the order indicated by the arrow, successive blocks of 25 squares being covered until the required number of cells has been counted. count from right to left, and continue in the manner illustrated by the diagram (Fig. i8) until all the erythrocytes in the first group of 100 squares have been counted, the totals of each block of 25 squares being noted as they are completed. To avoid repe- tition in counting it is necessary to include in the total all the cor- puscles which touch the upper and left boundary lines, and to disregard those which touch the lower and right boundaries. A second group of loo squares, not immediately adjacent to the COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 49 first, is then inspected in a similar manner, after which the cover- glass and counting chamber are washed with water and dried, and the operation repeated with a second drop. Thus the 400 squares are covered by examining 1 6 blocks of 2 5 squares each, 8 in the first, and 8 in the second drop of diluted blood. In a i : 200 di- lution of normal blood this involves the counting of approxi- mately from 2,400 to 2,800 eiythrocytes, and gives.results which are accurate within one-and-one-half per cent. To calculate the number of erythrocytes to the cubic milli- meter of blood, the following formula is employed : Number of eryth- Degree of diln- Cubic conte7its Total num- rocytes counted y. tioji {200) x of each square beroferyth- (if.,ood) rocytes per Number of squares counted {406) ^^- "'"'• For example, supposing that in the 400 squares of a i : 200 blood dilution a total of 2,500 erythrocytes is counted, the cal- culation is made thus : 2,500 X 200 X 4,000 , [a) = 5,000,000 erythrocytes per cb. mm. (b) 2,500 X 2,000 = " " " " " Counting the Leucocytes. The leucocytes may be counted by two different methods : («) with the erj'throcytometer, in the same drop of diluted blood in which the erythrocytes are esti- mated ; or {B) with the special leucocytometer, as a separate pro- cedure. Of the two methods the former is greatly to be preferred, since it is fully as accurate, and much more convenient and time- saving than the latter. Furthermore, there is an undoubted ad- vantage in counting both the red and the white corpuscles in the same drop of the blood dilution. {a) If the leucocytes are counted with the erythrocytometer the same technique is followed as in determining the number of erythrocytes, except that a much larger area of the counting chamber must be examined, owing to the comparatively small number of leucocytes contained in the i : 200 blood mixture. It is necessary, for the sake of accuracy, to count the leucocytes in the entire space enclosed by Zappert's ruling, and to repeat the count in a second drop, making an area equal to eighteen times the ruled space of the old-style counting chamber to be examined. If the totals of both counts are approximately the same, their combined figures, representing the coi-puscles found in a space corresponding to 7,200 of the small ruled squares, is taken as a basis for the final estimate ; but if these totals widely differ, a 4 so EXAMINATION OF THE BLOOD BY CLINICAL METHODS. third drop is to be examined in the same manner, and, as in counting the erythrocytes, an average taken of the two totals which are nearest alike. Since in normal blood, in a i : 200 dilution each block of 400 small squares contains from 3 to 6 leucocytes, the examination of the above-mentioned area of the counting chamber involves the counting of approximately from 54 to 108 of these cells — an operation which, practically, is not nearly so laborious as it appears from the description, being easily completed within ten or fifteen minutes, in most cases. ^ As an example of the method of calculating the final estimate, supposing that 90 leucocytes have been counted in the area equal to 7,200 small squares, the blood dilution being i : 200, this for- mula is employed : 90 X 200 X 4,000 -=- 7,200 = 10,000 leucocytes per cb. inm. If the old-style counting chamber is used, the leucocytes in the unruled portion of the disc outside of the central block of 400 squares may be counted with the aid of an eye-piece diaphragm, which when adjusted inside the tube of the ocular cuts off a field exactly the size of 100 small squares (Fig. 19). A black metal or cardboard disc having a central aperture ^ "^- '9 - of the proper size will answer just as well for , this purpose as the more expensive and elab- n orate mechanical eye-piece devised by Ehrlich, I "" ^ i\ which is provided with a diaphragm having a 1 Afcf*'' jjp square opening the size of which is regulated , V» ^/ '^ by a small lever. Having first counted all the leucocytes in the 400 small squares, the cells are Ocular diaphragm. then Counted in 3 2 of the diaphragm-fields out- side the latter, in order to cover an area of the disc corresponding to the entire ruled surface of the Zappert count- ing chamber. This operation having been repeated in a second drop, the totals of both counts are taken as the basis for the final calculation, which is made in the manner already described. If one happens to possess neither an eye-piece diaphragm nor a Zappert counting chamber, the following method of calculat- ing the cubic contents of the portions of the disc outside the ruled area may be adopted, as advised by Stengel.^ Using, for example, a -^-inch objective and a i -inch ocular, the ruled lines are brought into focus, and the tube of the microscope drawn out until one of the parallel lines of the ruled disc exactly coin- cides with either boundary of the field of vision. Assuming that 8 of ' Should the leucocytes be decidedly increased, it is unnecessary to cover such a large number of squares. One hundred cells taken as a basis for the calculation will give an accurate estimate. 2 " Twentieth Century Practice of Medicine," N. Y., 1896, vol. vii., p. 271. COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 5 1 these parallel columns, each ^-^ millimeter in width, are included in the visual field, the diameter of the latter is therefore ^-^ or -| millimeter, and the radius one-half of this figure, -^-^ or -^ milli- meter. The area of the field may then be readily determined by multiplying the square of its radius by 3 . 14 1 6. Its cubic contents are obtained by also multiplying by J-g- millimeter, formula being : i ^ i -^ iV ^ 3-^4^6 =-Oi2e,664., cudic contents o/tke visual Jie/d. Having in this manner ascertained the cubic contents of each field of vision, the final calculation of the number of leucocytes to the cubic millimeter of undiluted blood is made by multiply- ing the total number of these cells found in a definite number of fields (for instance, 50) by the degree of dilution (usually 1:200), and by then dividing by the cubic contents of each field (.0125- 664) multiplied by the number of fields examined. The formula for this calculation is : ( Total number of leucocytes counted x Degree of dilution ) h- {Cubic contents of visual field x Number of fields examined') = Total number of leucocytes per cb. mm. For example, in a i : 200 blood dilution a total of 30 leucocytes are noted in 50 fields, each having a cubic contents of .0125664, since they individually include 8 parallel columns of the ruled disc : ( 30 X 200 ) -i- ( .0125664 X 50 ) = 9,550 leucocytes per cb. mm. (b) If the special leucocytometer is used for counting the leu- cocytes, a 0.3 per cent, aqueous solution of glacial acetic acid must be employed as a diluent, in order to render invisible the erythroc5rtes and at the same time to make the leucocytes appear more conspicuously in the field. A 1:10 dilution is made, by drawing the blood up the capillary tube of the instrument until the mark i is reached, and by then adding the diluent until the mixture reaches the mark 11. The leucocytes are then counted in an area of the counting chamber equal to 800 of the small squares (preferably by examining 400 squares in two separate drops), and the calculation made according to the method pre- viously described. For instance, if in a given case 130 leucocytes were counted in 800 squares, the estimate would be made as follows : 130 X 4,000 X 10 -H 800 = 6,500 leucocytes per cb. mm. The chief objection to this method of leucocyte counting lies in the difficulty in distinguishing the cells, owing to the unavoid- able presence in the field of masses of granular debris resulting from the action of the acetic acid solution upon the erythrocytes. For this reason, if for no other, it seems advisable to dispense 52 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. with the leucocytometer, and to make the count of both red and white corpuscles with the erythrocytometer, in the same drop. Cleaning the Pipette. As soon as the count has been finished the pipette should be carefully cleaned and dried. Having first expelled what remains of the blood dilution, the instrument is rinsed out, first with distilled water and then with a mixture of equal parts of absolute alcohol and ether, the latter being used to remove all traces of the dye, in case either Toisson's or Sherring- ton's solution has been employed as a diluent, as well as to dry the interior of the tube. The pipette, while it may be filled with a fluid by suction, should not be emptied by blowing through it, for if this is done a certain amount of moisture from the breath unavoidably becomes deposited in its lumen. Its contents may be expelled in the form of a fine jet, simply by twisting the rubber suc- tion tube into a tight spiral rope, as shown in the illustration (Fig. 20). When the interior of the instrument is perfectly clean, it Fig. 20. Expelling contents of erythrocytometer. By twisting the rubber suction tube into a tlglit spiral rope, the fluid in the bore of the pipette may be forcibly expelled in a fine jet. is dried by forcing through it a current of air, by means of a rubber atomizer bulb, or an ordinary bicycle pump, until the glass bead no longer clings to the wall of the bulbous expansion, as it will as long as the slightest trace of moisture remains.^ A new form of hemocytometer has been Durham's Hem- recently described by Durham, who has em- OCYTOMETER. bodied in this device the principles of the older instruments, together with the substitution of a self-measuring pipette designed to overcome the sources of error v/hich may occur in making blood dilutions with a suction pipette. Durham's instrument, which appears to be a valuable improve- ^ A o. 1 per cent, solution of pepsin in I per cent, hydrochloric acid is useful for removing any bits of clotted blood which may adhere to the caliber of the instrument. COUNTING THE- ERYTHROCYTES AND THE LEUCOCYTES. 53 ment over other forms of blood-counting apparatus, consists of tile following parts : 1. Several capillary pipettes, of the Oliver type, each mounted in a glass tube, provided with a rubber nipple having a lateral perforation. The capacity of the pipettes is of 5 and 10 cubic millimeters. 2. A number of mixing vessels, each consisting of a small glass test-tube, graduated for i and for 0.5 cubic centimeters of fluid. The tubes holding i cubic centimeter measure 2|- x Jg- inches, and those holding 0.5 cubic centimeter, 2|x|- inches. One or more glass beads are shaken about in the tube, to mix the blood and the diluting fluid. 3. A number of graduated pipettes, for measuring the diluting fluid, of I and 0.5 cubic centimeters capacity, marked at 995 and 990 cubic millimeters, and at 495 and 490 cubic millimeters, respectively. Used with the appropriate capillary pipette, dilu- tions of I in 200, I in 100, and i in 50 may be obtained. 4. A counting chamber of the Thoma-Zeiss pattern. Fig. 21. Cross section of Durham's blood-pipette. T, glass tube ; N, rubber nipple ; p, lateral perforation in nipple ; c, cork in which a capillary pipette is fitted. Method of Use. Having placed in one of the mixing vessels some of the diluting fluid, the quantity of which is measured with one of the graduated pipettes according to the dilution desired, the capillary pipette is filled with blood, by touching it lightly to the blood drop as it flows from the puncture. All traces of blood are then removed from the outside of the pipette, the contents of which are now expelled into the fluid, contained in the mixing vessel. This is accomplished by inserting the pipette into the latter, keep- ing its point about half an inch above the level of the diluting fluid, and by then rotating it between the thumb and forefinger so that the lateral perforation is brought under the pulp of the thumb ; the nipple is now squeezed gently, and, continuing the pressure, the pipette is rotated back so that the perforation is free again. In this manner the blood is forced from the pipette, but is not sucked back. The blood remaining in the pipette is now com- pletely washed away by thrusting its point into the diluting fluid, this at once filling its caliber, by capillarity. Withdrawing the 54 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. pipette from the fluid, the rotation and pressure of the nipple are repeated, the capillary tube being thus rinsed out several times in order to remove completely all the blood clinging to its interior. The blood and the diluting fluid are now mixed by briskly ro- tating the mixing vessel between the opposed hands, so that the tumbling about of the glass beads in the vessel may thoroughly distribute the cellular elements through the fluid. When the mixing is completed, a drop of the fluid is transferred to the counting chamber, and the corpuscles counted under the micro- scope, in the usual manner. Durham's device makes it possible for the unskilled to measure accurately the desired volumes of blood and diluting fluid, and largely eliminates the errors which are likely to occur in sucking up the blood and the diluent with either the Thoma-Zeiss, or the Gowers hemocytometer. The ease and thoroughness with which the capillary blood pipette may be cleaned is also an ad- vantage, this being done by passing through its caliber a piece of darning cotton, dry or soaked in ether, by means of a needle. Comparative observations made with the Thoma-Zeiss hemocy- tometer have shown that the readings of the two instruments are practically identical. In this form of hemocytometer, the blood and GowERs' Hem- the diluting fluid are each measured in a separate OCYTOMETER. pipette, and deposited in a small receptacle in which they are mixed, a small portion of the mix- ture then being placed in a counting chamber and the number of corpuscles counted under the microscope. Gowers prefers to use as a diluent an aqueous solution of sodium sulphate having a specific gravity of 1025, but Toisson's solution, or any of the other diluting fluids previously mentioned will prove satisfactory. The instrument comprises five working parts, as follows : 1. A pipette, graduated to hold a volume of 995 cubic milli- meters, for measuring the diluting fluid. 2. A capillary pipette, graduated to hold a volume of 5 cubic millimeters, for measuring the blood. 3. A small glass mixing jar, in which the dilution of the blood is made. 4. A glass stirring rod, for mixing the blood and the diluent in the jar. 5 . A counting chamber, consisting of a glass slide, mounted on a brass plate, and containing a cell one-fifth of a millimeter in depth, the floor of the cell being divided by cross rulings into squares the sides of which measure one-tenth of a millimeter. When a cover-glass is fitted over this cell, being retained in po- COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 55 sition by means of a pair of clips attached to either end of the brass plate, the cubic contents of the space overlying each square measure 1/500 of a cubic millimeter. Method of Use . In using the instrument, 995 cubic millimeters of the diluting solution are first measured by means of the larger pipette, and blown out into the mixing jar. The latter must be perfectly clean and absolutely free from moisture before it is used, in order to avoid errors in the count. Now, using the capillary blood pipette, 5 cubic millimeters of blood are secured from the puncture, and immediately added to the diluent contained in the jar. The blood and the diluent are then thoroughly mixed, by rapidly stirring the solution with the glass rod. The dilution thus made is in the proportion of i in 200 of blood to diluent. As soon as the mixture is completed, a small drop of the solution is transferred to the center of the cell in the middle of the counting chamber, the small end of the glass rod being used for this pur- pose, after which the cover-glass is gently placed in position, and the clips adjusted so as to hold it in place. The counting cham- ber may then be placed upon the stage of the microscope, and the corpuscles overlying the ruled portion of the cell brought into focus with a low-power objective. It is necessary to use a small drop of the diluted blood, and to place it exactly in the center of the block of ruled squares, otherwise the fluid may flow towards the walls of the cell, alter- ing its volume, and making it necessary to reject the specimen, and to prepare a new drop, after thoroughly cleaning and drying the cell, and again stirring the blood solution. The corpuscles having settled to the bottom of the cell, their number in a given number of squares is noted, and the final calculation made according to the formula : Number of Number of t- ^ / 7 ^ / ■' 1 otaL number of cor- corpusdes x 200 x 500 h- squares = ^ , ^ / ^ ^ J ■' ^ _, J puscles per cb. mm. counted counted ^ ^ In counting the erythrocytes at least 20 squares of the count- ing chamber should be inspected, in different drops, a procedure involving the enumeration of about 1,000 cells, in normal blood. Except in high leucocytoses, the number of leucocytes is usually estimated indirectly, by determining their ratio to the erythro- cytes, and basing their actual number upon this figure. This plan (the necessity for which is a serious drawback to the use of this instrument) is followed so as to dispense with the tedious filling and refilling of the counting chamber, in an endeavor to find a sufficient number of leucocytes to serve as a basis for the S6 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. calculation, should the latter be direct. Ordinarily, not more than two of these cells are contained in an area including 20 squares. Gowers^ claims that the limit of error with his instru- ment is less than 3 per cent. After use, the different parts of the instrument are to be thor- oughly cleaned and dried, in the manner already described. For making rapid numerical estimates of the Oliver's Hem- erythrocytes Oliver has designed an instrument OCYTOMETER. based upon the following principles : When a candle flame is viewed through a flat glass test- tube filled with water, a bright transverse line is visible, com- posed of densely packed, minute images of the flame produced by the longitudinal corrugations of the glass. If for the water a mixture of blood and Hayem's solution '^ is substituted, a more or less opaque fluid results, so that, in low dilutions, this illuminated line is invisible, reappearing only when a definite degree of higher dilution is reached, by the gradual addition of the diluent ; when this point has been obtained, the line is again detected as a bright, delicate streak horizontally crossing the tube. Experiments having proved that the development of such a line, by gradual dilution of the blood with Hayem's fluid, is an accurate gauge of the percentage of erythrocytes in the specimen tested, it remained for Oliver to devise a hemocytometer consisting of the following essential parts : 1 . A capillary pipette, for measuring the blood. 2. A glass dropper, one end of which is capped by a rubber nipple, the other by a short rubber nozzle which fits over the blunt end of the pipette. 3. A standard graduated tube, in which the blood and the diluent are mixed. The four walls of the tube are flattened so that it is rectangular on cross-section, one wall being provided with an etched scale, indicating units from 10 to 1 20. Each of these divisions is equiv- alent to 50,000 erythrocytes, the point marked 100 degrees representing the arbitrary normal number, 5,000,000. Small-sized wax candles, known as " Christmas candles," are to be preferred for the illumination, as they give the small flame requisite to obtain a sharply defined line, but the flame from a gas-jet turned low may also be used with satisfaction. Method of Use. — In making the observation the pipette, which has been previously cleaned and dried, is filled with blood in the usual manner, and any excess of blood on the outside carefully 'Lancet, 1877, vol. ii., p. 797. 2 For formula, see page 41. COUNTING THE ERYTHROCYTES AND THE LEUCOCYTES. 57 removed. The rubber nozzle of the dropper, filled with Hayem's iiuid, is then slipped over the blunt end of the pipette, and the blood washed out into the graduated tube by squeezing the nipple. This preliminary dilution is continued until the column in the tube rises to within lo or 15 degrees below the figure for the hemoglobin percentage of the same blood, this having been pre- viously determined. For instance, if the hemoglobin percentage was found to be 70, the diluting fluid is added in large quan- tities until the mixture in the tube reaches to about the mark 60, after which it is added more cautiously, and in smaller quantities at a time, careful search for the bright line being made after each addition. In cases of chlorosis and of pernicious anemia, in which parallelism between the hemoglobin and corpuscular loss is lacking, it is, of course, impossible to depend upon the hemo- globin percentage as an index to the amount of diluent required, so that in instances of this kind the line must be developed more slowly, by making a smaller primary dilution, and by adding the requisite volume of liquid more deliberately. After the first dropperful of diluent has been added to the con- tents of the tube, the latter are mixed by inverting the tube a number of times with the thumb held over its mouth, the precau- tion being taken also to remove the thumb by drawing it over the mouth of the tube, in order to restore to its contents any liquid which may have ad- hered to the skin. The tube should be thus inverted after each ad- dition of the diluting fluid. The steps of the ob- Method of using Oliver's hemocytometer. Fig. 22, Showing manner in whicli the blood is washed from the capil- lary pipette into the tube containing Hayem's solution. servation succeeding the measuring of the blood and its primary dilution are to be made in a dark-room, free from cross lights, the candle being placed about ten feet distant from the observer. In order to shut out the diffused light of the candle, the tube should be held vertically in the concavity formed between the thumb and forefinger, the tube being kept close to the eye while S8 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. searching for the bright line. Oliver states that the earliest indications of this line are obtained by turning the tube on its axis, when it will become visible at the sides of the tube. It is claimed that tests made with this instrument are accurate within one per cent., and that in many cases it may be used as a substitute for the more laborious method of counting the cor- puscles under the microscope. It is obvious, however, that, apart from the " personal equation," the serious drawback to this test is its failure to indicate the number of leucocytes, this fact alone being sufficient to curtail its use for routine clinical work. It is also apparent that in cases of marked leucocytosis and of leuke- mia the optical principles of the test must necessarily fail because of the enormous number of leucocytes in the blood. Further- more, it is reasonable to infer that the instrument will give false results with blood in which conspicuous deformities of the eryth- rocytes exist, for the reason that the standard tube is corrected for normally shaped corpuscles, so that blood composed largely of microcytes, or megalocytes, or poikilocytes will give different readings from blood in which the cells are of unaltered biconcave shape and of normal size. IV. EXAMINATION OF THE STAINED SPECIMEN. The microscopical study of the dried and Objects of stained blood-film, which should supplement the Staining. methods of investigation just described, is for many reasons the most important step in the clinical examination of the blood. By means of this method of " color analysis " it is possible to differentiate easily and with absolute certainty the various forms of leucocytes and, by differ- ential counting, to calculate the relative percentages of each variety of these cells ; to distinguish the several structural de- generative changes affecting chiefly the erythrocytes, and to a less extent the leucocytes ; and to recognize and classify accord- ing to their histological character the nucleated forms of the eryth- rocytes. To sum up, in the words of Ehrlich,^ to whom we owe this rational means of investigation : " Everything that is to be seen in the fresh specimens — apart from the quite unim- portant rouleaux formation and ameboid movements — can be seen equally well, and indeed much better, in a stained prepara- tion ; and there are several important details which are only made visible in the latter, and never in wet preparations." lEhrlich and Lazaras : "Die Anaemic," Wien, 1900. (Nothnagel's "Spec. Path. u. Therap.," vol. viii., a. 2.) EXAMINATION OF THE STAINED SPECIMEN. 59 According to the classification introduced The Aniline twenty years ago by Ehrlich/ the aniline dyes Dyes. are divided into three different groups : acid, basic, and neutral. Acid dyes, or compounds in which the coloring principle acts or exists as an acid, possess a special affinity for cell protoplasm, and, therefore, are generally employed as plasma stains ; in hematological work acid fuchsin, eosin, and orange G are the principal dyes used for this purpose. Basic dyes, or compounds in which the coloring principle exists chemically as a base in combination with a colorless acid, are especially useful as nuclear stains, since they exhibit a special affinity for chromatin structures ; members of this group of dyes commonly used in blood staining are methylene-blue, tol- uidin-blue, methyl-green, methyl -violet, thionin, and hema- toxylin. Neutral dyes are the coloring principles which re- sult from the mixture of solutions of an acid and a basic dye ; they are used for the demonstration of the so-called neutro- phil granules of the leucocytes, for which they show a selective affinity. For the preparation of the dried blood-films it Preparing is advisable to have at hand at least half a dozen THE Films, perfectly clean, polished cover-glasses, which may be arranged in pairs on a sheet of white paper within convenient reach of the examiner. After having wiped away the blood which immediately follows the puncture, a minute portion of the next drop is collected, by lightly '°' touching the center of one of the cover-glasses to its sum- mit, care being taken to avoid bringing the polished surface of the glass in contact with the skin of the patient's finger. The charged cover-glass is then at once dropped, blood side downward, upon the sur- face of the second glass (Fig. 23), with the result that the blood quickly spreads out in a thin film between the two, and ex- tends to their peripheries, provided that the proper quantity of blood has been used. (Fig. 24.) As soon as the film has reached the margins of both cover-glasses, they are rapidly drawn apart in a horizontal direction, so that the surface of each, when thus separated, is covered with a thin layer of blood (Fig. 25), which ■Zeitschr. f. klin. Med., 1880, vol. i., p. 555. Superimposing the charged cover-glass. 6o EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Fig. 24. 2. Drawing apart the cover-glasses. should be rapidly dried, either by blowing briskly upon its sur- face, or by holding the glass for a few seconds high over the flame of an alcohol lamp. If care is taken to use but a very small drop of blood, to avoid pressure in opposing the sur- faces of the two cover-glasses, and to separate them in their true horizontal planes, the films will consist of a single layer of corpuscles, most of which will be sufficiently iso- lated to allow the study of their individual morphology and other characteristics. The beginner should persistently practice the technique of film making until he is able to obtain a satisfactory percentage of good specimens from every batch of spreads. Thick, uneven spreads, in which the corpuscles are heaped up and glued together in dense masses, are practically of no value for microscopical study ; such specimens should therefore be rejected at once, since it is time wasted ^'°- ^S- to attempt satisfactorily to stain them. The films, after having been dried, may be placed in a pill box, and labelled, to await fixation and stain- ing at the examiner's con- venience. Dried specimens will keep for an indefinite period, if not exposed to dust or to moisture. Unfixed cover-glass specimens of leukemic blood have been stained by the writer, with perfect results, more than three years after they were spread. Many histologists recommend the use of special forceps for hold- ing the cover-glasses while making the spreads, claiming thus to avoid the injurious effects upon the blood corpuscles which may be caused by the moisture of the fingers, if the latter come in contact with the films. The careful worker need have no fear on this score, for if the covers are held in the manner shown in the illustrations this accident will not occur. A pair of light thumb forceps is useful for picking up the cover-glasses from a flat sur- face, but the employment of special spreading forceps is quite superfluous. 3. The cover-glasses after separation. EXAMINATION OF THE STAINED SPECIMEN. 6l Fig. 26. As a step preliminary to staining, the albumi- FiXATiON noid principles of the blood must be fixed, by ex- Methods. posing the dried film either to a high degree of dry heat, or to various chemical hardening agents, the choice between these two methods being determined by the character of the staining solution to be used subsequently. Heat Fixation. This method may be employed with any of the stains described in the following pages ; it must be used with Ehrlich's triple stain, in preference to fixation by chemicals, in order to obtain crisp, clean-cut pictures. The author is accustomed to use an oven, such as is illustrated below (Fig. 26), consisting of a copper box with a heavy bottom, and hinged cover, mounted on an ordinaiy iron burette stand, by means of a thumb-screw. A small " baby" Bunsen lamp placed under- neath the box furnishes the requi- site degree of heat, the temperature being indicated by a thermometer mounted at one end, and resting upon the floor of the oven. By sliding the latter up and down the vertical rod to which it is attached, the desired degree of temperature may be obtained at will. The blood- films are enclosed in the copper box, and the latter fixed at a point eight inches above the summit of the burner, after which the gas is lighted and allowed to burn until the temperature, as indicated by the thermometer, has gradually crept up to 160° C. As soon as this degree of heat has been reached, the gas is extinguished, the cover of the oven thrown back, and, after the temperature has fallen to 30° C, the films removed, being now thoroughly fixed, and ready for staining. Fifteen minutes suffice for the whole operation, from the time the gas is lighted, until the films have been removed and cooled, for staining. A less satisfactoiy method of heat fixation is by the use of a copper plate upon which the films are kept at a temperature of from 100° to 110° C, for from one-half to one hour. The ap- paratus used for this purpose consists of a rectangular plate of sheet copper, about fifteen inches long, by four inches wide, by one-sixth of an inch thick. An alcohol or a Bunsen lamp bums Oven for fixing blood-films. 62 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. under one end of the plate, which is elevated about six inches above the flame, by four metal legs. After having heated the plate for ten or fifteen minutes, until a relatively constant tem- perature becomes established, water is dropped upon its surface, beginning with the end farthest from the flame, until a point is reached at which the water boils. This part of the plate is con- sidered to have a temperature of ioo° C, and at this point the blood-films are placed, " spread" side downward, and heated for the required time. No one with much blood staining to do will choose this method of prolonged heating at a relatively low, ap- proximate temperature in preference to brief heating at a high, definite temperature in an oven. The use of the latter, aside from its convenience as a time-saver, insures constant and cer- tain results, for over- and underheating of the blood-film may be avoided, since the degree of heat is exactly indicated and easily controllable. In triple stained specimens the blood cells are much more brilliantly colored and sharply differentiated when the films are fixed at a temperature of i6o° C, than at a lower degree. Should nothing but a Bunsen or an alcohol lamp be available, the cover-glass film, held with a pair of forceps, may be fixed by passing it rapidly through the flame thirty or forty times and then holding it twelve or fifteen inches above the flame for a minute or so. This makeshift method, which is often sufficient for a hur- ried clinical examination, usually gives fair, and sometimes very good, results, but the fixation is generally uneven, and the speci- men is frequently scorched in some places and underfixed in others. Chemical Fixation. Immersion of the dried films in ether, in absolute alcohol, or in a mixture of equal parts of the two (Niki- foroff's method) gives satisfactory results with specimens stained by any of the single basic dyes, or with the simpler double stains, such as eosin and methylene-blue or hematoxylin. The time of fixation varies from five to fifteen minutes with any of these agents, the specimen then being dried without using heat, and stained without previously washing. If time is an object, the specimens may be boiled for one minute in a test-tube containing absolute alcohol, as advised by Ehrlich.^ Some workers employ one minute's fixation by a one per cent, alcoholic solution of forma- lin (Benario's method), while others prefer to expose the films to the vapors of this chemical for the same length of time. Solley ^ has recently suggested that the film be flooded with a two per cent. 1 Loc. cit. 2 Med. and Surg. Reports of the Presbyterian Hospital, N. Y., 1900, vol. iv., p. 169. EXAMINATION OF THE STAINED SPECIMEN. 63 aqueous solution of chromic acid, which is washed off after exactly thirty seconds, the specimen being then stained, while still wet ; he recommends this procedure as a substitute for heat in fixing specimens for triple staining, but the method, while fairly good, cannot be regarded as entirely satisfactory. In the author's hands a two per cent, aqueous solution of osmic acid has been found to be the best substitute for heat fixation. In hematological, as in other histological work Methods of the choice of a staining method is determined by Staining. the character of the investigation to be undertaken. For general clinical purposes it is advantageous habitually to employ some routine method by means of which the greatest possible number of elements may be demonstrated in a single blood-film, this procedure being known as panoptic staining. Thus, by using a solution containing several of the aniline dyes the stroma of the erythrocytes, the cell-granules, the cell-nuclei, and the various blood parasites may be simultaneously stained each in a characteristic manner, owing to the selective affinity displayed by the different coloring principles of the mix- ture towards these several histological elements. The most useful solutions which have been devised for this purpose are the triacid mixture of Ehrlich,^ which has long served as the standard stain for hematological investigation, and the methylene-blue eosinate solution suggested by Jenner.^ Practically all the information that it is possible to derive from the study of the stained dry blood-film may be obtained with the aid of these two solutions. Combinations of an acid and a basic dye, such as eosin and methylene-blue, eosin and hematoxylin, and orange and hema- toxylin are used by many investigators, chiefly for the purpose of staining the cell-stroma and the nuclear structures ; but, as a general rule, such mixtures are not adapted for clinical work, since with none of them is it possible to differentiate the neutro- phile granules. Solutions of a single dye are but seldom used except for the demonstration of special elements, such, for in- stance, as the staining of the malarial parasite by thionin, the mast-cells by dahlia, and certain bacteria by the basic dyes, such as methylene-blue and gentian-violet. Since by this method of staining only the particular elements toward which the dye reacts are differentiated, the employment of single stains is inadequate for the study of the general morphology of the blood cells. The following formulae will be found sufficient for all purposes of clinical investigation : ' Loc. cit. ^Lancet, 1899, vol. i., p. 370. 64 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Ehrlich's Triacid Stain. This valuable triple stain, con- taining one basic and two acid dyes (methyl-green, orange G, and acid fuchsin), is peculiar in that a chemical combination is formed by its acid and basic components which may be regarded as a neutral coloring principle, serving the purpose of selectively staining the so-called neutrophile elements for which the primary components of the mixture have no affinity. With this stain his- tological structures having an affinity for the acid dyes are stained the color of one of its acid constituents, basic structures the color of its basic dye, and structures having an equal affinity for acid and basic dyes the color of the neutral compound. Saturated aqueous solutions of the three dyes are first pre- pared, and allowed to stand for several days until they have become thoroughly cleared. It is essential that the aniline dyes used for making these " stock " solutions should be chemically pure, to ensure which the products of Griibler, or of the Berlin Aniline Dye Company should invariably be chosen. From these saturated solutions the following mixture is made : Acid fuchsin solution 6- 7 cc. Orange G solution 13-14 cc. Distilled water 15 cc. Absolute alcohol 15 cc. Mix the above thoroughly, and add, drop by drop, with continuous agitation, in the following order : Methyl-green solution 12.5 cc. Absolute alcohol 10 cc. Glycerine 10 cc. The mixture should under no circumstance be filtered, but al- lowed to stand for about twenty-four hours in order that a slight precipitate may form. As soon as this occurs the stain is ready for use, the necessary quantity being pipetted from the super- natant fluid without disturbing the precipitate. Technique of Staining. The heat-fixed film, held preferably with a pair of Stewart's staining forceps, is flooded with the stain, which is washed off in running water after the lapse of from five to eight minutes, the specimen then being dried by gentle heat, and mounted in xylol balsam, or in cedar-oil. In the specimen thus prepared the stroma of the erythrocytes is stained orange, the nuclei of the leucocytes greenish-blue, the neu- trophile granules violet or lavender, and the eosinophil granules copper red. The nuclei of the erythroblasts react with • varying degrees of intensity toward the basic component of the mixture, those of the normoblasts staining deep puiple or black, and those of the megaloblasts pale green or greenish-blue. The basophile EXAMINATION OF THE STAINED SPECIMEN. 65 granules remain unstained, appearing as dull white, coarse, stippled areas in the cell-protoplasm — " negative staining." In order to stain these granules, as well as the basic protoplasm of the lymphocytes, Hewes ^ suggests that the triple stained film, after having been washed, be subjected for from one-half second to ten seconds to Lofider's ^ methylene-blue solution, after which it is again washed, and mounted as above directed. This modifica- tion is of undoubted value, chiefly because it usually enables one to differentiate the larger forms of lymphocytes from the large mononuclear leucocytes. Malarial parasites, and bacteria are also distinctly stained by this method. Unsatisfactory results with the triple stain, provided that the latter is properly made, can almost always be attributed to faulty fixation. As already remarked, heat is the only method of fixa- tion which will insure faultless differentiation in the specimen stained with this mixture. The perfect specimen is of a deep, rich orange tint to the naked eye ; if underheated, the film reacts too strongly toward the acid fuchsin of the mixture, and, conse- quently, is the color of this dye ; if overheated, the plasma stain, orange G, is but feebly displayed, so that the color of the film is pale lemon yellow.^ Jenner's Stain. This solution, made by dissolving in methyl alcohol the neutral precipitate obtained by the addition of methyl- ene-blue to eosin, is especially valuable in the study of the lym- phocytes, and the mast-cells. It should be made according to the following somewhat complicated formula : (a) Eosin (aqueous) 1.25 grm. Distilled water 100 cc. (i) Methylene-blue (medicinal) i.o grm. Distilled water 100 cc. Equal parts of these two solutions, "a" and "b," are mixed together, stirred thoroughly with a glass rod, and set aside for twenty-four hours, so that complete precipitation may occur. In making this mixture, the methylene-blue solution should be added to the eosin solution (never vice-versa) in small quantities at a time, the fluid being constantly stirred during the addition. A burette with the stop-cock regulated so as to deliver the former dye-solution, drop by drop, in rapid succession, will prove useful to insure the slow admixture of the two fluids. The precipitate 1 Boston Med. and Surg. Joum., 1899, vol. cxli., p. 39. 2 Saturated alcoholic solution of methylene-blue, 30 cc; i-io,ooo aqueous solution of potassium hydrate, 100 cc. 3 A reliable triacid stain, made according to Ehrlich's formula, is sold by Messrs. Shinn and Baer, Philadelphia. 5 66 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. is collected by filtration, dried in an incubator, at a temperature of 55° C, and powdered in a mortar. The powder should then be washed, by shaking it up with distilled water, and filtering, after which it is allowed to dry in the air, and kept in a tightly corked bottle. The mixture used for staining is made by dis- solving 0.5 grm. of this powder in 100 cc. of pure methyl alcohol (Merck), and filtering. Technique of Staining. Owing to the methyl alcohol which it contains, Jenner's solution fixes and stains the blood-film simultaneously, so that preliminary fixation may *be omitted. The unfixed specimen is stained for from three to five minutes, washed in water until the film is of a rose -red color, dried in air, and mounted in the usual manner. Since the solution is of an exceedingly volatile character, as much of it should be used as can be placed on the cover-glass without spilling ; if this is not done, the specimen may be ruined, owing to the rapid evapora- tion of the stain around the margins of the cover-glass. Jenner's stain gives the following results : erythrocytes, terra cotta ; nuclei of the leucocytes, pale sea-green ; neutrophile granules, pale pink ; eosinophile granules, deep pink ; fine baso- phile granules, deep blue ; and coarse, mast-cell granules, deep royal purple. The nuclei of the erythroblasts, and the blood parasites stain various shades of bluish-green, and the pseudo- granular protoplasm of the lymphocytes, blue. The chief defect in Jenner's stain is the occasional presence of a coarse, granular precipitate which mars the appearance of an otherwise perfect specimen. If, however, the precaution is ob- served always to filter the stain before use, and to dry the film in air — not by holding it over a Bunsen flame — this accident may generally be avoided. Aside from its obvious value as a panop- tic staining fluid, this solution will often prove of great convenience for the reason that it does not require special fixation of the blood- film. Prince's Stain. This mixture, which consists of an aqueous solution of one basic and two acid dyes, is an excellent stain for the differentiation of both nuclei and granules, and may be em- ployed as a fair substitute for either of the two preceding solutions. It should be made in this manner : Saturated aqueous solution of toluidin-blue 24 cc. Saturated aqueous solution of acid fuchsin i cc. 2 per cent, aqueous solution of eosin 2 cc. These solutions are mixed in the order named, and shaken briskly for several minutes, so as to secure complete precipitation EXAMINATION OF THE STAINED SPECIMEN. 6/ of the basic toluidin-blue by the acid dyes. The solution, which should not be filtered, is ready for use as soon as made. Only the supernatant fluid should be employed, care being taken not to disturb the sediment. Technique of Staining. If a newly made solution is used, the films are stained for from one-half to one minute, after which they are rinsed in water, dried in air, and mounted ; but if the solution has stood for several weeks, its basic constituent becomes less active, so that the specimen requires to be stained for from five to ten minutes. Either chemical or heat fixation of the blood-film may be used with this stain, both methods giving equally sharp dif- ferentiation. Prince's solution colors the erythrocytes rose-red, the nuclei of the leucocytes and erythroblasts blue, the neutrophile granules pink, the eosinophile granules maroon, and the fine and coarse basophile granules blue. Blood parasites are also stained the color of the basic dye. Double Staining with Eosin and Methylene-blue. Crisp, clear pictures of nuclear and stroma structures, of the ma- larial parasites, and of the basophile granules may be obtained be the use of these two dyes, and to investigations of this nature should this staining method be restricted. It is impossible, for example, accurately to distinguish a large lymphocyte from a myelocyte in a specimen stained in this manner, so that for differ- ential counting a more elaborate stain is essential. In films stained by this method the stroma of the erythrocytes and the eosinophile granules react toward the acid dye, staining the color of eosin ; while the nuclei of the leucocytes and erythrocytes, the basophile granules, and all blood parasites show an affinity for the basic dye, being colored various shades of blue. The protoplasm of the polynuclear neutrophiles is either colorless or tinged a deli- cate pink, the granules of these cells remaining unstained. The author has always found the following simple formula de- pendable : Eosin (aqueous), to which sufficient water has been added for solution 0.5 grm. Absolute alcohol 0.5 cc. Saturated aqueous solution of methylene-blue.. 96.0 cc. Technique of Staining. Films are fixed by immersion for ten minutes in absolute alcohol, or in equal parts of absolute alcohol and ether. The cover-glass is flooded with the stain, gently heated for one minute over a Bunsen flame, allowed to stain without heat for two or threeminutes longer, and then thoroughly washed in running water, dried in air, and mounted. OS EXAMINATION OF THE BLOOD BY CLINICAL METHODS. Another method of staining with eosin and methylene-blue, slower than the above, but as a rule giving sharper differentiation, is to stain without heat for five minutes with a 0.5 per cent, solu- tion of eosin in absolute alcohol to which an equal quantity of water is added. Then, after having washed off the eosin solution and dried the film in air, the specimen is counterstained for one minute or less with a saturated aqueous solution of methylene- blue, after which it is rinsed again in water, dried in air, and mounted. Among the many other methods of staining with eosin and methylene-blue, those suggested by Chenzinsky,' by Plehn,^ and by Holmes^ will be found the most useful. Double Staining with Eosin and Hematoxylin. By the em- ployment of these two dyes the erythrocytes and the eosinophile granules are stained the color of eosin, and all nuclei and parasites, the color of hematoxylin. This method, which is decidedly in- ferior to staining with the eosin and methylene-blue mixtures just described, is useful for little else than the study of nuclear struc- tures. It should not be used for differential counting, since in films stained in this manner the neutrophile granules are invisible. Ehrlich^ recommends this formula : Eosin (cryst.) 0.5 grm. Hematoxylin 2.0 grm. Absolute alcohol 100. o cc. Distilled water 100. o cc. Glycerine 100. o cc. Glacial acetic acid lo.o cc. Alum in excess. This mixture must "age" for several weeks before it can be used for staining. Technique of Staining. Specimens, fixed either chemically or by heat, are stained for from one-half hour to two hours, thor- oughly washed in water, dried and mounted. In order to obtain the best results, it is advisable to filter the solution before using, and to wash the films very thoroughly after staining. If time is an object, the following rapid method may be substi- tuted for the above: The film is first stained for about five minutes with a 0.5 per cent, solution of aqueous eosin in fifty per cent, alcohol, washed, and dried in air ; it is then counterstained •Zeitschr. f. wiss. Mik., 1894, vol. xi., p. 260. 2 " Aetiologische und klinische Malaria Studien," Berlin, 1890. 3 Joum. Am. Med. Asso., 1898, vol. xxx., p. 303. *Loc. cit. EXAMINATION OF THE STAINED SPECIMEN. 69 for about one-half minute with Delafield's hematoxylin,' washed for a second time, and mounted in the usual manner. Staining with Thionin. Thionin ( also known as the " violet of Hoyer," and the "violet of Lauth") is an excellent stain for blood parasites in general, being especially useful for the demonstration of the malarial parasites and the filarial embryos. Thionin should not be used as a stain for films in which the gen- eral morphology of the blood cells is to be studied, since the baso- phile granules and the nuclei are the only histological elements for which it displays any decided affinity. Structures reacting toward the dye are stained violet of varying degrees of intensity. The following recipe, suggested by Futcher and Lazear,^ will prove satisfactoiy : — Thionin 0.3 grm. Absolute alcohol..'. 10. o cc. I per cent, solution of carbolic acid q. s. ad loo.o cc. Technique of Staining. Films which have been fixed either chemically or by heat are stained in the above solution for from one to three minutes, being then washed in water, dried, and mounted as usual. The best results are obtained by using the French thionin, made by Cogit et Cie, of Paris. Staining with Polychrome Methylene-blue. Goldhorn's solution of methylene-blue and lithium carbonate affords a rapidly acting stain, superior to all others for the demon- stration of the finer structure of the malarial parasite in every phase of its development. In addition to giving crisp, clear-cut pictures of the chromatin of this organism, the solution also brings out distinctly the granular degeneration of the erythro- cytes, the nuclear characteristics of the erythroblasts and leuco- cytes, the basophile granules, and all ordinary bacteria. Technique of Staining. The films are fixed for fifteen seconds in methyl alcohol, rinsed in water, and then stained, unheated, for from one to two minutes, after which they are thoroughly ' This solution is made by first adding 4 grms. of hematoxylin crystals, dissolved in 25 cc. of alcohol, to 400 cc. of a saturated aqueous solution of ammonia-alum. The mixture is left exposed to the sunlight and air in an uncorked bottle for four days, at the end of which time it is filtered, and mixed with 100 cc. each of methyl alcohol and glycerine. This solution is allowed to stand until it becomes dark- colored, when it is filtered, and placed in a tightly corked bottle, to "age" for at least two months before it can be used successfully for staining. Owing to the com- plicated manner in which Delafield' s hematoxylin must be prepared, it is usually pref- erable to purchase it ready-made, from a dealer in microscopical supplies, Griibler's make being entirely reliable. 2 Johns Hopkins Hosp. Bull., 1899, vol. x., p. 70. JO EXAMINATION OF THE BLOOD BY CLINICAL METHODS. washed in running water, diied without the use of heat, and mounted in balsam. Preliminary staining for ten or fifteen sec- onds with a o. I per cent, aqueous solution of eosin, followed by washing, gives a picture in which the contrast between the plasma and the basic elements of the cells is clearly differentiated. Poly- chrome methylene-blue, prepared according to Goldhorn's for- mula,^ is sold by dealers in laboratory supplies, or it may be made in this manner : — Two grammes of methylene-blue are dissolved in 300 cubic centimeters of warm water and 4 grammes of lithium carbonate are added, with constant agitation. The mixture is poured into an uncovered porcelain capsule, which is heated over a shallow water-bath for ten or fifteen minutes, being frequently stirred with a glass rod. After removal from the water-bath, the fluid is bot- tled, without filtering, and set aside for several days, after which its reaction is corrected by the cautious addition of a 5 per cent, acetic acid solution until the dye is but very faintly alkaline. Should the solution become too alkaline after having been kept for some time, its reaction may be corrected by adding a small quantity of acetic acid, as in the preparation of the original mixture. A differential count of the leucocytes consists in Differential determining, by microscopical examination of the Counting, stained specimen, the relative percentages of the different varieties of these cells, the estimate be- ing based upon a count of several hundred cells, which are clas- sified according to the several forms described in a following section. (Section IV.) This procedure, by means of which qualitative changes affecting the leucocytes may be detected, is obviously a most important step in every blood examination, and one which should not be regarded as of secondary importance to the numerical estimate with the hemocytometer. The technique of differential counting consists simply in exam- ining successive microscopical fields until at least five hundred leucocytes have been counted, the cells in each field of vision being identified as they appear, and jotted down on a piece of paper by the observer under their appropriate class. As soon as the requisite number of cells has been counted, the percentages of the different forms are calculated, to express the final result. For the examination a one-twelfth inch oil-immersion objective is practically indispensable, for to any but the skilled worker it is difficult, if not sometimes impossible, to distinguish the various 1 Johns Hopkins Hosp. Bull., 1899, vol. a., p. 70. Also, N. Y. Univ. Bull, of Med. Sc, 1901, vol. i., p. 57. COUNTING THE BLOOD PLAQUES. /I forms of leucocytes with a lower magnification than this lens provides. In order to be certain that each field is taken in ac- curate succession to its neighbor, the slide should be moved across the visual field by the aid of a mechanical stage ; systematic examination of any given area of the specimen is well nigh an impossibility, if the slide is simply laid on, or clipped to, the stage of the microscope, and pushed across it with the fingers alone. If nucleated erythrocytes are found in the specimen, it is equally important to include them also in the differential count, classifying them in two histological divisions, normoblasts and megaloblasts. In calculating the number of these cells, it is obviously impossible to employ any direct method, so that the estimate must of necessity be more or less approximate, since it is based upon the ratio of erythroblasts to a given number of leucocytes. Having first counted the latter with the hemocytom- eter, the number of nucleated eiythrocytes is noted in an area of the stained specimen in which a fixed number of leucocytes is contained, and having ascertained these data, the estimate is made according to the formula : Number of erythroblasts Number of leucocytes Number of countedin the stained film per cb. mm. erythro- Number of leucocytes ~ blasts per counted m the stained film cb. mm. For example, in a case of pernicious anemia in which the leu- cocytes number 4,000 per cubic millimeter, and a total of 35 erythroblasts are noted while counting 1,000 leucocytes in the stained film, the calculation is as follows : 35 X 4,000 -h- 1,000 = 140 erythroblasts per cb. mm. Whenever erythroblasts are found, it is important to determine their number to the cubic millimeter of blood, and should nor- moblasts and megaloblasts both occur, to estimate the ratio be- tween these two types of cells. V. COUNTING THE BLOOD-PLAQUES. Determann's method^ of indirectly estimating the number of plaques to the cubic millimeter of blood is both simple and ac- curate. It consists, briefly, in first determining the ratio of these elements to the erythrocytes, which are then counted, to furnish the basis for the final calculation. ' Deut. Archiv. f. klin. Med., 1899, vol. Ixi., p. 365. 72 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. In obtaining the blood, a drop of the diluting fluid is placed upon the patient's finger and the puncture made through it, in order that the blood, as it flows from the puncture, will instantly mix with the diluent without coming in contact with the air. The blood and diluent are then thoroughly mixed for a few mo- ments by the aid of a cover-glass, after which a small portion of the mixture is transferred to a Thoma-Zeiss counting chamber, and the ratio of plaques to erythrocytes determined under the microscope. In the healthy adult this ratio, according to Deter- mann, ranges from i to i8 to i to 30, averaging about i to 22. With another drop of blood the erythrocyte count is then made by the usual method, and the actual number of plaques to the cubic millimeter of undiluted blood calculated from the figure thus obtained. For example, in a given specimen of blood in which the ratio of plaques to erythrocytes is found to be i to 25, the count of the latter cells being 5,000,000, the actual number of plaques is therefore 200,000 per cubic millimeter. The diluents for which Determann expresses a preference are either a 9 per cent, aqueous solution of sodium chloride to which a little methyl-violet has been added, or an aqueous solution con- taining one per cent, of sodium chloride and 5 per cent, of potassium bichromate ; but any of the diluting fluids already mentioned are suitable for the purpose. VI. ESTIMATION OF THE RELATIVE VOLUMES OF CORPUSCLES AND PLASMA. The use of centrifugal force for the purpose of determining the relative volumes of blood corpuscles and plasma was first applied in a practical manner by Hedin,^ who embodied the earlier ideas of Blix in an instrument known as the hematocrit. More re- cently Daland,^ by improving the mechanical construction of the original instrument and by simplifying the technique of using it, has made centrifugalization of the blood a method of investigation adapted to general clinical work. By the use of the hematocrit a pair of capillary glass tubes filled with undiluted blood are ro- tated in their horizontal axes at a high rate of speed until, as the result of the centrifugal force thus applied, the corpuscular and liquid portions of the blood become separated, the former being distinguishable in the lumen of the tube as a column the length 1 Skandinavisch. Arch. f. Physiol., 1890, vol. ii., p. 134. 2 University Med. Magazine, 1891, vol. iv., p. 85. Also, Edwards' supplement to Keating's " Cyclopedia of the Diseases of Children," Philadelphia, 1899 ; vol v P- 537- ESTIMATION OF CORPUSCLES AND PLASMA. 73 D Aland's Hematocrit. Fig. of which is dependent upon the volume which the corpuscles con- stitute in relation to the rest of the blood mass. This instrument (Fig. 27) is composed of a set of cog-wheels enclosed in a metal box and geared in such a manner as to cause ten thou- sand revolutions per minute of a vertical spindle, by turning a handle at a definite, uniform rate of speed. A metal frame, which may be securely fastened to the spindle by a modified bayonet-lock, carries a pair of capillary glass tubes, each of which fits into two cup-like, rubber-lined de- pressions, and is adjusted and held in place by a spring. Each tube measures fifty millimeters in length with a lumen of half-a-millimeter, and has engraved upon its outer surface a scale represent- ing one hundred equal divisions, the glass immediately above the scale being moulded so as to form a lens-front, to magnify the column of blood and to facilitate the reading of the divisions. A bit of rubber tubing, fitted with a mouth piece, is used for filling the capil- lary tube, in the same manner in which the blood is measured with the hemocy- tometer. While in use the instrument is securely attached to the projecting edge of a table or shelf, by means of a clamp operated by a thumb-screw. Method of Use. Having cleaned and punctured the pa- tient's finger in the usual manner, the beveled end of one of the capillary tubes is immersed in the drop of blood, which is sucked up the lumen of the tube until it is exactly filled. The forefinger, smeared with a little vaseline, is then applied to the beveled end of the tube, while the rubber suction tube is carefully removed by twisting it free — not by forcibly pull- ing it off, since this may accidentally cause removal of a portion of the blood column, by suction. The tube thus charged with blood is at once adjusted to one arm of the frame, and the empty tube similarly fixed in the other arm, to equalize the balance, this step being completed as rapidly as possible, in order to anticipate coagulation. When the tubes have been thus adjusted, and the frame securely locked in the spindle, the handle of the instrument is turned for three min- Daland's hematocrit. 74 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. utes^ at the rate of seventy-seven revolutions a minute, this rate of speed securing ten thousand rotations per minute of the frame, since the latter revolves one hundred and thirty times with each complete turn of the handle. The centrifugalization having been finished, the charged tube is carefully removed from the frame, and held against a piece of dull white paper, so that the height of the blood column may be easily determined. In order to make the reading with accuracy, it is sometimes necessary to use a small magnifying glass, for the divisions on the scale of the tube are but one-half a millimeter apart — a distance too small to judge easily with the naked eye. On examination, three distinct di- visions of the lumen of the tube containing the centrifugalized blood may be distinguished : first, a dark-colored column con- sisting of erythrocytes, reaching, in normal blood, to a point be- tween the divisions marked 50 and 51 ; second, a thin layer of leucocytes, showing, in blood in which these cells are not largely increased, as an indistinct, milky stratum overlying the erythro- cytes ; and, third, a layer of clear plasma occupying the remainder of the lumen. The normal volume of erythrocytes being arbi- trarily regarded as one hundred per cent., to compute this result the figure of the scale to which these cells rise is multiplied by two. Unless the leucocytes are greatly increased in number, the layer formed by these cells is too delicate and too dully defined to be read with any degree of accuracy ; but in cases of high leucocytosis and of leukemia it is quite possible to estimate roughly the relative proportions of leucocytes to erythrocytes. The capillary tube which has been filled with blood should be cleaned as soon after use as possible, water, followed by alcohol and ether, being used for this purpose. A fine wire should be passed through its lumen, to dislodge any obstruction which may result from drying of the column of closely packed corpuscles. The hematocrit, if its clinical application is limited to the de- termination simply of the relative volumes of the blood corpuscles and plasma, may be relied upon to furnish, on the whole, de- pendable information, the necessary errors attending its use probably being within two per cent. If employed in the role of a hemocytometer, however, its results must needs be highly inac- curate just in those instances in which exact methods of investiga- tion are all important. It is true that in normal blood, in which the size of the corpuscles ranges within the physiological limits, it is correct to consider each percentage volume as representing 1 In a recent personal communication Dr. Daland advises that, in order to insure the most accurate results with his instrument, the centrifugalization be continued for three, instead of for two, minutes, as he formerly recommended. ESTIMATION OF THE SPECIFIC GRAVITY. 75 approximately a count of 100,000 erythrocytes per cubic milli- meter. In blood characterized by any considerable deformity in the size and shape of these cells, as in high-grade anemia or in leukemia, it is perfectly obvious that no such correspondence between the count and the percentage volume can be expected — blood in which microcytosis is pronounced is certain to show a lower percentage volume of erythrocytes than blood in which megalocytosis prevails, or than blood containing normal-sized cells, although the counts of all three may be identical. Similarly, a given number of lymphocytes should indicate a lower percentage volume than an equal number of myelocytes, or even polynuclear neutrophiles. On account of these sources of fallacy, if for no other reason, the hematocrit estimate should never be taken as a basis for calculating the count in pathological conditions, in lieu of the more accurate, if more laborious, method of counting the corpuscles. Capps^ considers that the hematocrit may be used to advan- tage, in conjunction with the hemocytometer, in determining the actual size or volume of the individual erythrocyte, and he regards this method as far more reliable than the use of the micrometer, since with the latter only the transverse diameter of the cells, and not their depth, can be measured. The formula for calculating this "volume index" has been given elsewhere. (See p. 130.) VII. ESTIMATION OF THE SPECIFIC GRAVITY. This method of investigation is used as an indirect means of computing the percentage of hemoglobin, owing to the more or less constant parallelism maintained between it and the specific gravity of the whole blood. The correspondence between the two, together with the sources of error inseparable from the test, has been pointed out in another section. (See page 98.) Hammerschlag's modification^ of Roy's Hammer- method' of determining the specific gravity of schlag's the blood best serves the purpose of those who Method. choose this roundabout means of approximating the hemoglobin percentage. It consists in first making a mixture of benzol and chloroform of such a specific gravity that a small drop of blood deposited in the liquid remains suspended, after which the specific gravity of the mixture is de- termined with a hydrometer, the figure thus obtained representing ijourn. Am. Med. Assn., 1900, vol. xxxvi., p. 464. ''Zeitschr. f. klin. Med., 1892, vol. xx., p. 444. 3 Cited by Devoto : Zeitschr. f. Heilk., 1889, vol. xi., p. 175. 76 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. the density of the blood used in the test. The hemoglobin per- centage corresponding to this figure is then selected from a table giving the various degrees of blood densities and the percentages of hemoglobin to which they are equivalent. The apparatus required for making the test is neither elaborate nor expensive, a hydrometer provided with a scale graduated to 1.070, a hydrometer jar having a wide, substantial base, a glass capillary tube, and a glass stirring rod being the only instruments needed. In most instances an ordinary urinometer may be used instead of a special hydrometer, since specific gravities in excess of 1060 (the highest gradation on the scale of most urinometers) are not often encountered. Either a Thoma-Zeiss leucocytom- eter, or a medicine dropper the free end of which should be heated in a flame and bent into an obtuse angle, will serve as a capillary pipette. Benzol and chloroform are mixed together in the hydrometer jar in such proportions that the specific gravity of the liquid is approximately equal to that of normal blood, 1060. This mix- ture having been made and its specific gravity taken, the point of the capillary pipette, charged with blood, is plunged beneath the surface of the liquid and a small bead of blood gently expelled. If the blood drop rises to the surface of the mixture a few drops of benzol are added, while if it sinks to the bottom of the jar chloroform is used, the addition of the appropriate reagent being continued until the drop neither rises nor sinks, but remains sta- tionary, suspended in the mixture. When this point has been determined the specific gravity of the liquid is taken by means of the hydrometer, this figure obviously representing the specific gravity of the blood drop itself To translate the specific gravity into its hemoglobin equivalent the figure obtained by the above procedure is compared with one of the tables given on page 100. After each addition of benzol or of chloroform the contents of the jar must be thoroughly mixed by stirring with the glass rod, in order to secure uniformity in the density of the liquid. The latter, if it is filtered free from blood and preserved in a tightly stop- pered bottle, may be used again in subsequent tests. In spite of the enthusiasm evinced by certain authors for this method of ascertaining hemoglobin values, considerable experi- ence with the test has convinced the writer that it is both crude and untrustworthy, — it is useful, no doubt when a hemometer cannot be obtained, but in no sense is it an efificient substitute for colorimetric methods. The liability of the blood drop to split up into numerous fine particles, to adhere to the inside of the jar, and to become altered in composition from the influence of the ESTIMATION OF THE ALKALINITY. "JJ reagents, as well as the tedious attempts which must usually be made to add just the proper quantities of benzol and chloroform to secure a mixture in which the drop neither sinks nor rises, are a few of the drawbacks which must make the test unpopular with busy clinicians. VIII. ESTIMATION OF THE ALKALINITY. The most available clinical method of deter- Engel's mining the alkalinity of the blood is by the use Alkalimeter. of Engel's alkalimeter. ( Fig. 28. ) By means of this instrument a measured quantity of fresh blood is diluted with distilled water in the proportion of one to ten, and then titrated with a -^-^ normal solution of tartaric acid until the mixture reacts with lacmoid paper, the total alkalinity being calculated from the amount of the tartaric acid used. The methods of alkalinity estimation devised by Landois ^ by Lieb- reich,^ by Haycraft and Williamson,' by Wright,* and by Kraus,^ are not well adapted to routine blood-work, being either too com- plicated and elaborate for such a purpose, or too inaccurate. The apparatus which Engel has devised consists of the follow- ing parts : a diluting and mixing pipette, resembling a large-sized Thoma-Zeiss erythrocytometer ; a graduated burette by means of which the amount of tartaric acid solution used in the test is meas- ured ; a glass cylinder in which the titration is made ; and a glass stirring rod. The mixing pipette is graduated in three principal divisions marked 0.025, 0.05; and 5.0 respectively, the first two divisions being further scaled in tenths by fine horizontal mark- ings ; otherwise the instrument is modeled like a blood counting pipette. The burette has a capacity of five cubic centimeters, and is provided with a scale indicating one hundred equal divi- sions ; when in use, it is clamped upright, by means of a special at- tachment, to a vertical brass support which screws into a fitting in the box containing the apparatus. Method of Use. The technique of using the alkalimeter is simple and time saving in comparison with that required by other well-known methods of alkalinity testing. Finger-blood, ob- tained by a rather deep puncture so as to afford a good-sized drop, is sucked up in the pipette until it reaches the mark 0.05, imme- diately after which distilled water is similarly drawn up the lumen iReal-Encyclop., 1885, vol. iii., p. 161. ^Berichte d. deutsch. chem. Gesellsch., 1868, vol. i., p. 48. 'Free, of the Roy. See, Edinburgh, 1888, June 18. * Lancet, 1897, vol. ii., p. 719. sZeitschr. f. Heilk., 1889, vol. a., p. 106. 78 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. of the tube until the mixture of blood and water fills the bulbous expansion and reaches the mark 5.0, in the constricted portion beyond. While sucking up the water, the pipette should be rap- idly twisted to and fro between the thumb and forefinger, to insure Fig. 28. EnGEL'S ALKALIMETER, thorough mixing of the blood and water as they together fill the expanded portion of the instrument. As soon as the dilution has been made, the pipette should be shaken for a minute or so, until the mixture becomes of an uniform "laky" tint, which indicates that all the hemoglobin has been dissolved from the corpuscular stroma. The contents of the pipette are blown out into the glass cyhnder, which is placed beneath the faucet of the burette, the latter having been previously filled to the mark o with a -^^ nor- mal solution of tartaric acid. By turning the stop-cock of the burette, the test solution is now added, drop by drop, stirring be- tween each addition, to the measured amount of diluted blood in the cylinder. From time to time a drop of the mixture is re- moved by means of the glass rod and tested with the lacmoid paper, the titration being continued until the reaction, recognized as a bright red halo which forms around the edge of the drop, is 7 8 9 10 II 12 13 14 DETERMINATION OF RAPIDITY OF COAGULATION. 79 obtained. The titration is then stopped, and a note made of the number of drops of the test solution which have been used. In normal blood the writer finds that from 9 to 1 1 drops are re- quired to give the reaction. The estimate of the total alkalin- ity of the blood is made by multiplying by the figure 53.3, the number of drops of the tartaric add solution used, according to the formula, 10: a : : 533.0 : jt, « representing the drops of the reagent.' The result thus obtained is expressed in milligrammes per hundred cubic centimeters of blood. The following table may be useful for reference in determining the various degrees of alkalinity : If 6 drops of the solution are used the alkalinity equals 319 mgrms. NaOH ' " " " " 373 " '. " " " " 436 " " ' " " " " 479 " " ' " " " " 533 " ' " " " " 586 " ' " " " " 639 " " < U (< II 11 6g2 " " " 746 " After use the pipette should be thoroughly washed out with water, alcohol, and ether, and then dried, in the manner already directed for cleaning the Thoma-Zeiss instrument. While, up to the present time, it cannot be claimed that infor- mation of any real diagnostic pertinence has been obtained from the study of the alkalinity of the blood, this procedure should prove of value in the systematic investigation of many cases, espe- cially those of high-grade anemia. As elsewhere mentioned, the degree of normal blood alkalinity vaiies greatly according to the particular method by which this figure is ascertained, so that it follows that the results obtained by means of Engel's apparatus cannot be compared with those based upon different methods of research. IX. DETERMINATION OF THE RAPIDITY OF COAGULATION. This procedure is obviously more useful in experimental labora- tory work than as a means of clinical investigation, yet in some diseases, such as hemophilia, it will prove of real value, since in 1 Assuming that 0.5 cc. of tartaric acid are used to neutralize 0.05 cc. of blood, therefore for every 100 cc. of blood 1,000 cc, or one liter, of a -^j normal solution of tartaric acid are required. As the alkalinity of the blood is not expressed by the amount of acid necessary to saturate it, but in milligrammes of an alkali, sodium hy- drate, the calculation is made thus : as the equivalent weight of tartaric acid is 75, and that of sodium hydrate 40, one liter of water dissolving 75 grammes of the for- mer saturates 40 grammes of the latter, that is, one liter of a -^ normal tartaric acid solution saturates f^ grammes, or, in other words, 533 milligrammes, of sodium hy- drate, this figure being taken by Engel as the degree of normal alkalinity of the blood. 80 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. this condition it is possible thus to determine the influence of remedies administered with a view to promoting coagulation of the blood. In a number of other pathological conditions, char- acterized either by delay or by unusual rapidity of coagulation, this method of research furnishes information which at least adds completeness to the clinical histoiy, if nothing more. The coagulation time may be determined ap- Glass Slide proximately by collecting several individual drops Method. of blood of the same size upon the surface of a perfectly clean, slightly warmed glass slide. At regular intervals of about one minute a straw of a whisk-broom is lightly trailed through each drop in succession, until sooner or later a delicate thread of fibrin may be observed clinging to the straw. The period which has elapsed" between the deposit of the blood on the slide and the appearance of this indication of clotting is expressed in minutes, to represent the coagulation time of the specimen under investigation. Normal blood thus treated coagulates in from two and one-half to five minutes. This instrument (Fig. 29) consists of a tin Wright's Co- water can surrounded by a flannel-lined leather AGULOMETER. jacket provided with nine pockets, one of which is intended to hold a thermometer, and the others a set of glass coagulation tubes. The latter are each about 10 centimeters in length, with a lumen 0.25 millimeter in diameter, and are open at both ends ; they should be con- secutively numbered or lettered, in order that they may be distinguished apart at a later stage of the test. The thermometer is of the same outer di- ameter as that of the tubes, and registers the same degrees of temperature as an ordinary clin- ical thermometer. Method of Use. The central receptacle is first filled with water having a temperature of about 99° F., and the tubes slipped into the pockets sur- rounding it, being allowed to remain in them for a few minutes, so that they may become warmed to the temperature of normal blood. Having then pricked the patient's finger, each tube is about half filled with blood, by aspiration, at suc- cessive intervals of one minute, a tube as soon as it is filled being replaced in its appropriate pocket. An equable temperature of the tubes should be maintained by the addition of hot water to the can, as its contents cool. Within three minutes after filling the first F16. 29. Wright's coagu- LOMETER. SPECTROSCOPICAL EXAMINATION. 8 1 tube, it should be tested, by blowing out its contents upon the surface of a sheet of white filter-paper, the remaining tubes being similarly tested at regular intervals of one minute or less, until after thus trying a variable number, one is found from which the blood cannot be expelled. Coagulation may then be con- sidered to have occurred, the time required for this process being expressed by the number of minutes elapsing between the filling of the tube in question and the evidence of clotting thus demonstrated. With normal blood the coagulation time, as de- termined by this instrument, ranges from about three to five minutes. After use, a fine wire should be forced through the lumen of the tubes, to dislodge the clots, after which the remaining traces of blood are to be removed by thorough washing with distilled water, alcohol, and ether, in the order named. X. SPECTROSCOPICAL EXAMINATION. For clinical work the Sorby-Beck microspectroscope, to be used in connection with the microscope, is an excellent instrument, being both accurate, and, comparatively speaking, easy to manip- ulate. Other very perfect instruments for the spectroscopical examination of the blood, differing but little from the original Sorby model, are Fig- 3°- also made by Zeiss, by Leitz, and by Browning. This instrument (Fig. The 30) when in use fits into Sorby-Beck the tube of the micro- MiCROSPECTRO- scope, like an ordinary SCOPE. ocular, for which it is substituted. Its essen- tial part consists of a tube. A, in which a series of five prisms, two of flint and three of crown glass, is arranged in such a manner that the emergent rays, which are separated by dispersion, leave the prisms in practically the same direction soeby-Beck microspectroscope. as that taken by the entering immer- gent ray. At one side of the tube is fixed a right-angle reflecting prism, so that the spectrum of a solution of normal blood may be thrown alongside that of the specimen under investigation, the two spectra thus being comparable. The adjustment of the spectra is effected by means of the two small screws, B, B' . The 6 82 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. receptacle containing the control solution of blood is clamped to the stage, C, by a spring clip, D, light being reflected through the liquid and into the rectangular aperture, E, by the swinging mirror, F. The width of this aperture is controlled by the screw, G. The receptacle containing the blood solution to be examined is placed upon the stage of the microscope, being brought into focus with a low-power (2^ or i inch) dry objective. Beneath the tube enclosing the series of prisms is mounted an achromatic ocular, below which a narrow slit-like diaphragm is situated, the vertical size of this opening being regulated by a milled screw, not shown in the illustration, and its breadth by the two small levers, /, /'. Both ocular and prisms may be moved simultaneously toward and away from the diaphragm, by a rack-and-pinion mechanism controlled by the Fig. 31. wheel, /, so that any part of the spectrum may be brought into focus. The liquids to be examined should be placed in Sorby's tubular cells, and cover-glasses super- SoRBYTUBULAKCEi-i.. imposcd. Thcsc cells (Fig. 31) are narrow- lumened glass receptacles made of barometer tubing, both ends of which are accurately ground to parallel surfaces, one end being cemented to a small polished glass plate. Method of Examination. The specimen of blood obtained in the usual manner, by puncture, is first diluted with distilled water one hundred times, by means of the Thoma-Zeiss erythrocy- tometer, and sufficient of this laked blood dropped ioto a Sorby cell to fill it exactly to the brim. A cover-glass is then carefully laid over the open end of the cell, the precaution being taken to prevent the formation of air-bubbles upon the surface of the column of liquid thus enclosed. A second cell, to be used as the control, is filled with normal blood, similarly diluted, and both are then adjusted in their respective positions, as already explained. In making the examination, a ray of artificial light (that from a Welsbach incandescent burner being most suitable) is projected by the microscope mirror through the lumen of the cell contain- ing the suspected blood, and the surface of the liquid focused with an ordinary ocular. The latter is then removed from the microscope tube and replaced by the spectroscope ocular, and the second spectrum, that of the normal blood, is brought into proper position alongside that of the first, so that any differences between the two may be contrasted by the observer. The appearance of the spectra of normal and of pathological BACTKRIOLOGICAL EXAMINATION. 83 blood, together with the circumstances under which the latter occur, have been described in another section. (See page 123.) XI. BACTERIOLOGICAL EXAMINATION. The demonstration of bacteria in the circulating blood, pro- vided that faultless technique is employed, furnishes in some instances a diagnostic sign of the greatest importance. The pathological significance of such a finding is much greater than that of a similar result obtained post-mortem, since with the latter there is no means of determining whether the bacterial invasion of the blood current took place during the active stages of the disease, or whether it occurred either as a pre- or a postagonal process. Cultural methods with blood aspirated directly Methods, from a superficial vein should invariably be used whenever such a procedure is practicable, for blood obtained simply by pricking the skin is most likely to be contaminated with various bacteria which have their normal hab- itat in the epidermis and its appendages, notably by the staphyl- ococcus epidermidis albus. Welch,' who first drew attention to this source of error, emphasizes the fact that no diagnostic sig- nificance should be attached to the demonstration of this bac- terium in blood obtained by puncture of the skin. Direct examination of stained cover-glass specimens prepared from finger blood gives either negative or erroneous results in the great majority of instances. In certain overwhelming infections, notably in some of the severer forms of bubonic plague, it may often be possible to detect the specific micro-organism in the stained film, but the method must be regarded as too crude and unreliable to furnish accurate findings, in the average case. Blood Cultures. In order to secure the most reliable informa- tion from blood culturing, the systematic observance of three pre- cautions is essential. First, contamination by the skin bacteria above referred to must be carefully avoided, by the thorough ster- ilization of the patient's skin at and adjacent to the site from which the blood is aspirated. Second, not less than o. 5 cubic centimeter of blood should be used for each culture, since only in rare in- stances^are bacteria so numerous in the peripheral circulation as to be demonstrable in a single drop of blood. Third, fluid, rather than solid, culture media should be used, in sufficiently large quantities to dilute the blood freely — about one hundred parts of media to each part of blood — the object of this precaution being 1 Dennis' System of Surgery, Phila., 1895, vol. i., p. 251. 84 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. to secure attenuation of the bactericidal properties of the blood, which otherwise might prove strong enough to prevent all bac- terial development. For aspirating the blood the author prefers to use the needle- capped glass tube devised for this purpose by James and Tuttle.' (Fig. 32.) This consists of a piece of glass tubing five inches in length and one-quarter of an inch in diameter, having a capacity of about two cubic centimeters ; it is tapered at one end and ground to fit the cap of a number 42 hypodermic needle, while the free end of the tube is plugged with a bit of cotton. The ap- paratus is enclosed in a larger glass tube both open ends of which are also plugged with cotton, and sterilized by dry heat, the aspi- rating tube being removed at the time the blood is to be collected. This instrument is far superior to an antitoxin or a hypodermic syringe for the purpose intended, being simple, inexpensive, easily Fig. 32. Needle and tube for aspirating blood for culturing. sterilized, and readily cleaned after use. It is especially well adapted for making cultures at a distance from a laboratory, where the sterilization of an ordinary piston-syringe is difldcult, if not impossible. At least six hours before the aspiration of the blood, the skin of the patient's arm at and for some distance on all sides of the bend of the elbow should be thoroughly scrubbed for several minutes with either a strong ethereal soap or with tincture of green soap, after which the part is well rinsed with hot sterile water, and finally washed with alcohol and ether. A moist i : 500 bichloride compress is then applied over the site thus cleaned, being left in place until the time of the withdrawal of the blood. As a preliminary to this operation, the dressing is removed, and the part freely douched and scrubbed with hot sterile water, in order to remove eveiy trace of the bichloride. A rubber drain- age tube, previously sterilized, is twisted tightly around the pa- tient's arm above the bend of the elbow so as to cause disten- tion of the superficial veins in this situation, and the point of the needle is then thrust obliquely into the most prominent of these iMed. and Surg. Reports of the Presbyterian Hosp., N. Y., 1898, vol iii p. 46. BACTERIOLOGICAL EXAMINATION. 85 vessels, with the result that the blood immediately begins to flow into the bore of the instrument. If, for any reason, the force of the blood flow should fail to fill the caliber of the tube, sufficient blood may be easily obtained by making gentle suction through a bit of rubber tubing slipped over the cotton-plugged end of the instrument. While introducing the needle it should be held almost parallel to the long axis of the vein, for should it be simply plunged inta the vessel at right angles, there is danger that the point will pass completely through the vessel from wall to wall and penetrate the surrounding tissues — an accident which may explain the cause of many a " dry-tap." The site of the as- piration may be made anesthetic by preliminary freezing with a spray of ethyl chloride, but to most patients the operation is not painful enough to necessitate such a procedure. Having thus collected, say, two cubic centimeters of blood, the contents of the tube are divided equally among four Pasteur flasks each containing at least fifty cubic centimeters of broth or other suitable fluid culture media. The flasks are then shaken for a few moments, in order to mix the blood and media and to dilute thoroughly the former, after which they are placed in an incu- bator. The identity of the growths, should any occur, remains to be determined by secondary culturing and microscopical ex- amination, for descriptions of which the student should consult text-books on bacteriology. Cultures made by this technique, suggested by Adami,^ are much more favorable to the growth of any bacteria which may be in the blood-stream than the older methods of using solid media. Staining Methods. In the limited number of instances to which such methods are applicable the technique described below will be found useful. An attempt should be made to sterilize the skin of the finger from which the blood is obtained, by thoroughly scrubbing the part first with ethereal or green soap, and then with a 1:500 bichloride solution, alcohol and ether, in the order named, this being followed by sponging with sterile water. A deep punc- ture having been made with a needle which has been sterilized by the naked flame, and the first few drops of blood escaping from the wound allowed to drip away, one of the succeeding drops is transferred by means of a sterile platinum needle to the surface of a cover-glass upon which a second cover-glass is at once laid, the two being drawn apart, in order to secure a pair of spreads. The latter are immediately dried by gentle heat and then passed several times through a Bunsen flame. It is ijoum. Am. Med. Assn., 1899, vol. xxxiii., p. 1514. 86 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. needless to add that the cover-glasses used for making the films must be sterilized by heat, and handled by means of a pair of sterile forceps. Films thus prepared may be stained with any of the basic aniline dyes (thionin, methylene-blue, and methyl- or gentian-violet being most useful for this purpose), after which they are washed in water, dried, and mounted in Canada balsam or in cedar-oil. Should a double stained specimen be desired, one of the eosin and methylene-blue solutions referred to previously may be depended upon to give satisfactory results. Gunther's method^ will be found useful, if the object is to de- stroy the color of the erythrocytes, so as to leave a freer field of vision for any bacteria which may be present in the film. Ac- cording to this method, the specimen is first immersed for ten seconds in a five per cent, aqueous solution of acetic acid, until the tint of the hemoglobin has entirely faded away, after which the reagent is removed by briskly blowing upon the surface of the cover-glass ; the latter is then held, face downward, over the open mouth of a bottle containing strong ammonia water, so as to neutralize all remaining traces of the acid. The film is now stained for twenty-four hours with the Ehrlich-Weigert fluid (contained in a covered staining dish), at the end of which time it will be found to be colored a deep blue. It is then decolorized by a few seconds' immersion in a i : 14 aqueous solution of nitric acid, until the color fades to a light green ; rinsed in alcohol ; dried in air ; and mounted in balsam. The Ehrlich-Weigert fluid is prepared by adding from 10 to 15 drops of aniline oil to 6 cubic centimeters of distilled water, held in a test-tube. The fluid is thoroughly mixed by shaking, and then filtered. To the filtrate a few drops of a con- centrated alcoholic solution of methyl- or gentian-violet is added — -just sufficient of the dye to produce a slight turbidity of the liquid, which clears up in a few minutes. The mixture prepared in this manner is employed as the staining agent. XII. DETERMINATION OF THE SERUM REACTION. In 1894 Pfeiffer^ noticed that the vibrios of Widal's Asiatic cholera, if injected into the peritoneal Test. cavity of a guinea-pig immunized against this disease, rapidly lost their characteristic motility, and tended to become granular, broken up, and dissolved, while 'Forschr. d. Med., 1885, vol. iii., p. 775. 2Zeitschr. f. Hyg., 1894, vol. xviii., p. i. Ibid., 1895, vol. xix., p. 75. Also, Centralbl. f. Bakt. u. Parasitenk., 1896, vol. xix., p. 191. Also, Deut. med. Woch., 1896, vol. xxii., p. 97. DETERMINATION OF THE SERUM REACTION. 8/ in the healthy, non-immune animal they developed normally and abundantly, and failed to show'any such changes in their mor- phology. He claimed that this reaction, known as " Pfeiffer's phenomenon," was specific, and emphasized its value as a means of laboratory differentiation. Two years later PfeifiFer and KoUe * found that the same changes occurred in experiments with the bacillus of Eberth and animals rendered immune to enteric fever, and, furthermore, discovered that the test could be conducted in vitro, by mixing in a test-tube typhoid cultures and immune serum. It is of interest to note that results somewhat analogous to those of Pfeiffer had been observed in 1891 by Metchnikoff,^ and in 1889 by Bordet,^ and by Charrin and Roger,^ although none of these workers appeared to recognize the significance of their observations. In 1896 Griiber and Durham' applied the principles of Pfeif- fer's phenomenon to many other motile as well as non-motile bacteria, deduced new facts regarding its utility as a means of differentiating various species of germs, improved the technique of the test, and made the important announcement that aggluti- nation and immobility of typhoid bacillus cultures were pro- duced by the action of blood serum from a patient having re- cently recovered from an attack of enteric fever. It remained, however, for Widal,* in 1896, first to apply the reaction clinically, and to announce that enteric fever could be diagnosed by noting the clumping and immobilization of the typhoid bacillus when mixed in definite proportions with blood serum from a patient suffering from typhoid. This reaction, Widal insisted, was one of infection, and was demonstrable not only during convalescence, but during the incipiency and the height of the disease. The serum reaction is to-day recognized as an important sign in the diagnosis not only of enteric fever, but also of Asiatic cholera, of Malta fever, and of relapsing fever, while its value still remains less certainly established in many other conditions, such as, for example, leprosy, tuberculosis, yellow fever, bubonic plague, and pneumococcus infections. The technique of the test and its diagnostic significance under various circumstances, will iZeitschr. f. Hyg., 1896, vol. xxi., p. 203. Also, Deut. med. Woch., 1896, vol. xxii., p. 735. ^Annal. de I'lnstitut Pasteur, 1891, vol. v., p. 473. Ibid., 1894, vol. viii., p. 714. Ibid., 1895, vol. ix., p. 433. ^Annal. de I'lnstitut Pasteur, 1895, vol. ix., p. 462. Ibid., 1896, vol. x., p. 191. *Compt. rend. Soc. Biol., 1889, 9 s., vol. i., p. 667. ^MUnch. med. Woch., 1896, vol. xUii., p. 285. 6 Bull. m6d., 1896, vol. x., pp. 618 and 766. Sem. m6d., 1896, vol. xvi., p. 259. Ibid., 1897, vol. xvii., p. 69. Lancet, 1896, vol. ii., p. 1371. Munch. med. Woch., 1897, vol. xliv., p. 202. 88 EXAMINATION OF THE BLOOD BY CLINICAL METHODS. be described under the headings of the diseases in which it occurs. (See " General Hematology.") Originally Bordet/ more recently Uhlenmuth,^ The Specific and Wassermann and Schiitze^ have demon- Test for strated the important fact that the blood serum Human Blood of an animal subcutaneously injected with the blood of another animal of a different species rapidly develops the property of agglutinating and dissolving the erythrocytes similar to those injected, but has no effect upon blood derived from any other source. The last-named ob- servers, for example, administered to rabbits, at intervals of two days, several subcutaneous injections of lo cubic centimeters each of defibrinated human blood, the animals being bled to death six days after the last dose, and their shed blood placed upon ice, to effect separation of the serum. Sufficient blood for this purpose may be readily obtained by wet-cupping, leeching, or placental expression. If 0.5 cubic centimeter of this rabbit- serum (or "antiserum" for human blood) is added to a solution of the blood of man, diluted about one hundred-fold with distilled water or with normal salt solution, a distinct cloudy precipitate rapidly occurs at ordinary room temperature, the turbidity be- coming much more dense after brief incubation of the mixture at 37° C. On the contrary, no definite change occurs on the ad- dition of the serum to the diluted blood of other animals, no less than twenty-three different species having failed uniformly to react positively, with the single exception of the monkey, and in this instance the reaction was delayed and incomplete, being in no way comparable to the prompt cloudiness produced by the mixture of human blood with its antiserum. Old dried, and even putrefied blood, diluted i to 100 with normal salt solution, has been found to react typically, while Nuttal and Dinkelspiel * have re- ported characteristically positive results with human blood mixed with an equal volume of the diluted blood of different animals, such as sheep, oxen, horses, and dogs. These workers also found that positive results were obtained with human nasal and lachrymal secretions. Uhlenmuth * discovered that blood specimens could be frozen for two weeks at a temperature of 10° below zero, C, without in any way affecting the sensitiveness of the reaction, and that blood mixed with soapy water, menstrual urine, and other contaminating liquids responds promptly and typically. lAnnal. de I'lnstitut Pasteur, 1898, vol. xii., p. 688. Ibid., 1899, vol. xiii., p. 273. ^Deut. med. Woch., 1901, vol. xxvii., p. 82. SBerl. klin. Woch., 1901, vol. xxxviii., p. 187. ''Brit. Med. Journ., 1901, vol. i., p. 1141. *Loc. cit. DETERMINATION OF THE SERUM REACTION. 89 From a medico-legal standpoint, the value of this test is ob- vious, for, even at this early stage of its development, it has received sufficient corroboration to justify its use as a means of identifying human blood stains, no matter how old and how contaminated they may be. For this purpose, the reaction ap- pears to be entitled to much greater confidence than the spec- troscope, or chemical tests. SECTION II. THE BLOOD AS A WHOLE. SECTION II. THE BLOOD AS A WHOLE. I. GENERAL COMPOSITION. Blood is a tissue consisting of fluid and cor- Plasma, Serum puscular elements, the former constituting about AND Cells, three-fifths, and the latter two-fifths of its total volume. It has been approximated that the total quantity of blood in the normal individual is from -J^ to j^^ of the body-weight, the proportion being somewhat less in the infant than in the adult. The fluid element of the blood, known as the plasma or liquor sanguinis, is an alkaline, yellowish liquid, of a specific gravity ranging from about 1026 to 1030, and con- taining approximately ten per cent, of solid matter, of which three-fourths are proteids ; the latter consist of fibrinogen, serum-albumin, and serum-globulin. Coagulation of the blood results in its separation into a densely reticulated, somewhat granular substance, fihin, and into a clear, straw-colored, alka- line fluid, serum. Fibrin is a sparingly soluble, highly elastic, proteid body, which encloses and imprisons within its multitude of delicate fibrils the corpuscular elements, the whole forming the blood-clot or crassamentum. Serum is a clear, straw-colored, alka- line fluid, having a specific gravity of about 1026 and containing practically the same amount of solids and relative proportion of proteids as are found in the plasma ; its proteid constituents are fibrin-ferment, which replaces the fibrinogen of the plasma, serum- albumin, and serum-globulin. The corpuscular elements of the blood are free cellular bodies suspended in the plasma. They are of two varieties : the eryth- rocytes or red corpuscles, and the leucocytes or white corpuscles. In addition to these cells, two other elements are also found, namely, the blood-plaques, and the hemoconia, although these bodies, while they may be conveniently grouped with the red and white cells, are not to be regarded as definite corpuscular entities. The salts of the blood include sodium chloride, potassium chloride, sodium carbonate, sodium phosphate, magnesium phos- phate, calcium phosphate, and sulphates ; of these salts sodium chloride is the most abundant, constituting from 60 to 90 per cent, of the total amount of mineral matter. 94 THE BLOOD AS A WHOLE. Certain extractives are also found, among which are urea and uric acid, creatine, creatinine, xanthine, hypoxanthine, sugar, fats, soaps, and cholesterine. The gases of the blood consist of oxygen, nitrogen, and carbon dioxide, the former existing chiefly in combination with hem- oglobin in the erythrocytes, and the latter as carbonates ; the nitrogen is held in simple solution. About 60 volumes of gas are contained in each 100 volumes of blood. Arterial blood contains roughly 20 volumes of oxygen, and 40 of carbon dioxide, while venous blood contains less than 10 volumes of oxygen, and almost 50 of carbon dioxide ; the quantity of nitrogen in both arterial and venous blood is from i to 2 volumes. II. COLOR. The distinctive color of the blood is due to Normal the presence of the hemoglobin contained in the Variations, erythrocytes, and alterations in the chemical com- position of this pigment produce corresponding changes in the color of these cells, and, consequently, in the naked-eye appearance of the whole blood. The color of arte- rial blood is bright scarlet, inasmuch as it contains a large amount of oxygen in chemical combination with the hemoglobin ; while venous blood, on the other hand, is of a dark purplish-blue tint, owing to its deficiency in oxygen and to the presence of more or less uneliminated carbon dioxide. This difference in color is so obvious that a cursory glance suffices to distinguish arterial and venous bloods. The presence of immense numbers of hemo- Density globin-containing elements accounts for the vary- AND ing degree of density and opacity which the Opacity. blood possesses, distinguishing it from a mere transparent, colored fluid. If, for any reason, the hemoglobin escapes from the erythrocytes into the surrounding plasma, this characteristic opacity is quickly lost, and the blood becomes transparent, and of a " laky " color. The density and the opacity, and, consequently, the color of the blood increases and diminishes according to the fluctuations which occur in the relative amounts of plasma and erythrocytes, and also according to the cells' richness in hemoglobin, irrespective of their numerical variation. In anemic conditions the blood is usually pale Pathological in color, somewhat transparent, and thin and Variations, watery-looking. This is the case particularly in primary pernicious anemia, in chlorosis, and in leukemia ; in the former disease, it is sometimes difficult to believe REACTION. 95 that the watery, pale fluid which flows from the puncture is any- thing but pure serum ; in leukemia, the blood drop may have a peculiar light, mottled, streaked appearance, or an uniform milky- white tint may predominate over the normal red hue. In cases of dyspnea, arterial blood, because of its inadequate oxygenation, may be dark blue, closely resembling blood from the veins. This similarity has also been noted in cases of poisoning by sulphur- etted hydrogen, in which condition the blood may even be changed to a dark greenish tint. In some cases of diabetes mellitus, the presence of large quantities of free fat in the circulation seem- ingly divides the blood drop into two distinct layers, an upper, light-colored portion, containing supernatant fat-droplets, and a lower, darker layer of pure blood ; at first glance diabetic blood has a somewhat pinkish hue. In poisoning by aniline, nitrobenzol, hydrocyanic acid, and potas- sium chlorate, the blood is chocolate- or dun-colored ; and in poisoning by carbon monoxide, bright cherry-red. In severe icterus a yellowish-red tint of the blood has been observed. III. ODOR AND VISCOSITY. Owing to the presence of certain volatile fatty acids, blood possesses a peculiar and characteristic odor or halitus, which may be intensified by the addition of concentrated sulphuric acid, and which rapidly disappears after the withdrawal of the blood from the body. The slippery feeling of freshly drawn blood is quickly lost after its exposure to the atmosphere, and is replaced by a viscosity or stickiness, as coagulation progresses. IV. REACTION. Under normal conditions, the reaction of the Reaction blood is alkaline, owing chiefly to the presence IN Health, of sodium carbonate and disodium phosphate. Clinically, the degree of alkalinity is determined by ascertaining the amount of sodium hydroxide which is ex- actly neutralized by one hundred cubic centimeters of blood, the result being usually expressed in milligrammes of NaOH per hundred cubic centimeters of blood. The figures given by differ- ent investigators as representing the normal alkalinity range within the widest limits, chiefly in consequence of the many dif- ferent methods by which such data were obtained. In view of these marked discrepancies, the alkalinity figures of different workers are in no sense comparable unless they are based upon precisely similar methods of investigation pursued with identical 96 THE BLOOD AS A WHOLE. technique. The following table, compiled from reliable data, il- lustrates the range of the normal blood alkalinity, as estimated by various observers : Observer. Degree of Alkalinity. Kraus 162-232 mgrms. NaOH per 100 cc. of blood. Burmin!.'.'.'.' 182-218 " " " " ' Rumpff 182-218 " " " " ' Jeffries 200 " " " " ' Freudberg 200-240 " " " " ' Lupine 203 " " " " ' Canard 203-276 " " " " ' Drouin 206 " " " " ' Von Limbeck 218 " " " " ' Zuntz and Lehmann 240 " " " " ' Vonjaksch 260-300 " " " " ' Schultz-Schultzenstein 260-300 " " " " ' Strauss 300-350 " " " " ' Brandenburg 330-370 " " " " ' Lowy 449 " " " " ' Berend 450-5°° " " " " ' Engel 479-533 " " " " ' Mya and Tassinari 516 " " " " ' With the titration method, now generally admitted to furnish fairly accurate results, appreciably higher figures are obtained with laked whole blood than with serum alone, since by the former method the alkalinity of all the plasma and cellular ele- ments is estimated, while by the latter the influence of the cor- puscles is entirely eliminated. The alkalinity of the blood is slightly higher. Physiological as a general rule, in men than in women and Variations, children, and is somewhat influenced by the time of day, being at its minimum during the early morning hours, gradually rising during the afternoon, and falling again during the evening. Some observers maintain that it is increased during the period of digestion, but this fact is dis- puted by others. It is temporarily diminished by the effects of muscular exercise, and by a diet deficient in nitrogenous substances ; on the contrary, richly nitrogenous food eaten during the per- formance of muscular work overcomes the effect of such exertion in lowering the alkalinity. The effects of cold baths are said to increase the alkalinity of the blood. In health, by the perfect mechanism of the emunctory organs of the body, the normal balance of blood alkalinity is constantly maintained, in spite of the entrance of acids into the blood, whether by the ingestion of acid substances, or by their produc- tion within the system, for the excess acidity from such causes is promptly removed from the blood by the action of the kidneys, REACTION. 97 the skin, and the lungs. Thus, the ingestion of acids is quickly- followed by increased acidity of the urine and sweat, while at the same time an increased quantity of carbon dioxide is given off by the lungs. It is also probable that the tendency to acidity is partly neutralized by the ammonium salts generated from proteid foods, and by the action of the liver. Increased alkalinity goes hand in hand with increased antidotal action of the blood against bacterial infection, as experiments have shown that animals whose blood had been artificially rendered highly alkaline, by the administration of sodium salts, showed much greater resistance to the effects of virulent micro-organisms, than untreated animals. Therefore, it is believed that the power of immunity against infections may, to a certain degree, be meas- ured by the alkalinity of the blood, for, in animal experimenta- tion, the fact is evident that the greatest degree of blood alka- linity is found in animals whose immunity is absolute. Unfortunately, the question of alteration in the Pathological alkalinity of the blood in various pathological Variations, conditions is at the present time one about which the opinions of different observers conflict, so that conclusions concerning this subject must be accepted with more or less reserve. It is of interest, however, to note that most observers agree that, as a rule, the alkalinity of the blood is perceptibly lowered in those diseases associated with 2^ febrile movement, but no definite relation between the intensity of the pyrexia and the degree of lessened alkalinity has been established. Subnormal alkalinity figures have also been met with \v\.\}s\& primary 2Xidi secondary ane- mias, with the exception of chlorosis, in which condition the blood alkalinity usually is either normal, or perhaps slightly increased. Desevres * has drawn attention to the fact that in the early stages of acute diseases the alkalinity is either normal or somewhat in- creased, and in the majority of instances it becomes perceptibly diminished during convalescence. In chronic diseases it is usu- ally decreased if the duration of the disease has been of long standing. Drouin ^ found a lessened alkalinity of the blood in enteric fever, in pneumonia, in m,cdarial fever, in diphtheria, in rheumatic fever, in erysipelas, in appendicitis, and in many other acute infec- tions. Cantani ^ maintains that in the algid stage of Asiatic chol- era the reaction of the blood during life in some cases may be ^ Th^se de Lyon, 1897-98. 2 " H^mo-alcalim^trie et H6mo-aci 5) 6. 7. 8, 9, 10, II, 12, 13. Poikilocytes. Many of these cells are highly polychromato- philic, especially 11, 12, and 13. {£osin and Methylene-blue.) Fig. 5. Erythrocytes Showing Degenerative Stroma Changes. Granular basophilia is shown by i and 2 ; extreme decolorization by 3, 4, and 5. The other cells represent various stages of hemoglobin loss and protoplasmic degeneration. Note.— Plates I, II, III, IV, and V are all drawn on the same scale, a Leitz ^-inch oil- immersion objective and 4 ocular, with a Zeiss camera-Iucida. being used. PLATE I. Fig. 1. > Fig. 2. o Q 3 v €) Fig. 3. Fig. 5. Fig. 4. ThK ER^■THROCVTK,S. (Figs. I, 2, 3, and 4, Tr-iacul Stain ; Fig. 5, Eosui and Mfthytc-tu:-blut\) (E. F. FABEK.yVt.) SECTION III. HEMOGLOBIN, ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. I. HEMOGLOBIN. Hemoglobin, which occurs in the circulating General blood in chemical union with oxygen as oxyhem- Properties. oglobin, is an extremely complex ferruginous and albuminoid substance contained within the stroma of the erythrocytes. It constitutes approximately nine-tenths of the latter's total bulk, and a triile less than fourteen per cent, of the whole blood. Hemoglobin displays a striking avidity for combining with oxygen to form a peculiarly unstable, but defi- nite, chemical compound, and a similar facility for yielding up to the tissues much of its oxygen, during its passage through the capillary circulation. Under the influence of deoxidizing agents, oxyhemoglobin may be deprived of its loosely combined oxygen molecule, the resulting oxygen-free constituent being known as reduced hemoglobin. Rhombic crystals of oxyhemoglobin, scarlet or reddish-green in color, are rapidly formed if, for any reason, separation of this substance from the corpuscular stroma takes place. Methemoglobin is an oxygen compound of hemoglobin containing the same quantity of combined oxygen as the latter, but differing from it in holding its oxygen constituent in a more intimate union. The dingy brown color which develops in a so- lution of oxyhemoglobin after prolonged exposure to the atmos- phere, evidences the production of this variety of blood-pigment. (See " Methemoglobinemia," page 124.) The amount of iron (in the form of hemochromogen) which hemoglobin contains is considerable — somewhat in excess of four per cent. It has been shown, clinically, by estimates made with the ferrometer and the hemometer, that no fixed parallelism is maintained between the percentage of hemoglobin and the iron contained in the blood.' Under the action of acids, strong alkalies, or heat, hemoglobin may be readily decomposed into two constituents : hematin, or an iron-containing principle ; and an albuminous residue of un- known character, but somewhat resembling globulin. In combi- 1 Rosin and Jellinek : Zeitschr. f. Win. Med., 1900, vol. xxxix., p. 109. I20 ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. nation with hydrochloric acid hematin forms a crystalline hydro- chloride of hematin, termed hemin, or Teichmann's crystals. Under the microscope these crystals appear as black or dark brown, elongated rhombic prisms belonging to the triclinic sys- tem, which are insoluble in water, alcohol, ether, chloroform, and dilute acids. They may be demonstrated by preparing a slide of blood (or of any dried substance containing blood-pig- ment) to which a small quantity of common salt has been added; a drop of glacial acetic acid is then run beneath the cover-glass so that it mixes with the blood and salt, and the specimen thus prepared is heated to just below the boiling point over a Bunsen ilame. On cooling, Teichmann's crystals may be seen under the microscope with a low-power dry objective. Iron-free hematin, or hematoporphyrin, may be derived from blood by the admixture of concentrated sulphuric acid. This substance is closely related chemically to urobilin, and occurs oc- casionally as a pigment in nature and in normal and pathological urines. Hematoidin, which also is free from iron, occurs in the form of reddish rhombohedral crystals, only in old clots resulting from blood extravasations, such as cerebral hemorrhages, and splenic infarcts. It is derived from hematin, and is probably identical with bilirubin. The chief source of hemoglobin is the iron Origin. contained in various food products, about ten milligrammes daily representing the amount of this metal ingested in an ordinary diet, according to the analyses of Stockman.' In event of a stoppage of this source of an iron supply, the formation of hemoglobin may proceed from the supply of iron stored up in various organs of the body, notably in the Hver. Bunge^ has shown that in the young infant, whose natural food, milk, contains but a slight trace of iron, this source of hemoglobin manufacture is most potent. The recent experiments of Aporti^ regarding the origin of hemoglobin and the erythrocytes have shown that animals sub- jected to repeated bleedings and kept on an iron-free diet, are able up to a certain point to utilize the supply of body iron for hemo- globin manufacture ; but that when such a demand became so severe that this supply was exhausted, the red corpuscles became progressively paler and paler, and the animal finally died. During the course of these experiments, if the animal received injections of iron, a prompt and striking increase in hemoglobin occurred, ijourn. of Physiol., 1897, vol. xxi., p. 55 ; 1895, vol. xviii., p. 484. ^Zeitschr. f. physiol. Chem., 1892, vol. xvi., p. 177. 'Centralbl. f. inn. Med., 1900, vol. xxi., p. 41. HEMOGLOBIN. 121 the gain ranging from 50 to 95 per cent, within a week's time. The injection of arsenic, on the contrary, produced no effect upon the hemoglobin percentage, although it caused a marked and rapid increase in the number of red corpuscles. Similar effects from the administration of these drugs in the treatment of the different anemias may be observed as everyday clinical occurrences. Diminution in the amount of hemoglobin, as Variations indicated by the hemometer, is known as oligo- IN Amount, chromemia, or achroiocythemia. It is a condition usually, but not invariably, associated with a cor- responding decrease in the number of erythrocytes. An ap- parent increase in the hemoglobin percentage may result from the concentration of the blood caused by a reduction in the quantity of blood plasma consequent to excessive drains upon the liquids of the body. By a similar physical mechanism, factors pro- ducing a dilution of the blood are capable of causing an ap- parent diminution in the hemoglobin. Marked oligochromemia is commonly observed in chlorosis, pernicious anemia, and leukemia ; and in the secondary anemias dependent upon such factors as hemorrhage, mineral poisoning, acute and chronic in- fections, malignant neoplasms, and constitutional diseases. The behavior of the hemoglobin under such conditions is more fully alluded to in connection with the lesions in question. Poggi,^ from a series of experiments upon normal women, has shown that the hemoglobin is slightly lowered (10 or 1 5 per cent.) for a few days before menstruation, but with the establishment of the flow the oligochromemia soon disappears. The primary loss he attributes to retarded hemogenesis consequent to the lessened consumption of albumin occurring in menstruating women, while the subse- quent gain he explains by the increased functional activity of the hematopoietic organs. In passing, it may be of interest to compare the degree of hemoglobin loss in the various forms of anemia, as illustrated by the following averages determined by the writer : Average of 50 estimates in pernicious anemia 25.5 per cent. " " " " " chlorosis 43.2 " " " " " " " leukemia 39.4 " " " " " " " secondary anemia 55.2 " " Bierfreund's investigations ^ in Mickulicz's clinic have led to the current impression among surgeons that it is highly dangerous to give a general anesthetic to a patient whose hemoglobin percent- age is below 30 ; some operators regard 40 per cent, as the iPoliclin., Roma, 1899, vol. vi., p. i. 2 Langenbeck's Archiv., 1890-91, vol. xli., p. i. 122 ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. lowest limit of safety, and refuse to employ any but a local anes- thetic in cases with an oligochromemia exceeding this figure, ex- cept under circumstances of imperative necessity. Any one, however, who has attempted to verify the correctness of this gen- eral belief must accept it with a shrug of the shoulders. The writer knows of four patients whose hemoglobin percentages all were below 30, in whom operations under general anesthesia with ether were followed by uneventful recovery ; in one instance (a pan-hysterectomy, lasting more than an hour and a half), the hemoglobin was but 21 per cent., yet no ill effects were observed. Assuming that in the normal adult 14 grammes Absolute represent the average amount of hemoglobin in Amount. 100 grammes of blood, the absolute amount of hemoglobin may be readily calculated, thus : rr , I- , Grammes of hemozlobin Hemoglobin percentage x 14 -h 100 = • ^ i, 1 ^ ^ ° ^ m 100 grammes of blood. For example, in blood in which the percentage of hemoglobin, as determined by the hemometer, is found to be 40, the calcula- tion (40 X .14) gives the absolute amount of hemoglobin as 5.6 grammes. The proportionate amount of hemoglobin con- CoLOR tained in each red blood corpuscle, or its cor- Index. puscular richness in hemoglobin, is known as the color index, or blood quotient, or valeur globulaire. In normal blood the color index is theoretically expressed by the figure I, although, practically, it varies from .95 to 1.05 in men, and from .9 to l in women.' In those anemias in which the decrease in the amount 01 hemoglobin in the blood is coincident with a proportionate de- crease in the number of erythrocytes, the color index remains practically at the normal figure. If, however, the cellular de- crease happens to be relatively greater than the hemoglobin loss, then the index will naturally be found to rise above normal ; thus, in pernicious anemia, in which condition the loss of cells is proportionately much greater than the loss of hemoglobin, high color indices, approaching or even exceeding 1.25, are fre- quently observed. On the contrary, if the hemoglobin loss is rela- tively more excessive than the corpuscular decrease, the color index falls below normal ; for example, in chlorosis, in which, as a rule, the decrease affects the hemoglobin much more strikingly than the erythrocytes, low indices, such as .50 or less are common. To calculate the color index, the percentage of hemoglobin is ' Oliver : Loc. cit. HEMOGLOBIN. 1 23 divided by the percentage of erythrocytes, the result being ex- pressed in decimals. In order to simplify this procedure, 5,000,- 000 erythrocytes per cubic millimeter must be arbitrarily con- sidered ds normal, or 100 per cent. To obtain the percentage of corpuscles, the actual number counted in one cubic millimeter of blood is simply multiplied by two, and two or three decimals pointed off from the left, depending upon whether the count is below or above the normal 5,000,000. The following examples serve to illustrate the calculation in several conditions : Normal Adult. Erythrocytes : 5,000,000 per cb. mm. (100 per cent.). Hemoglobin : 100 per cent. 100 -T- 100 = I : Color index. Secondary Anemia. Erythrocytes : 2,650,000 per cb. mm. (53 percent). Hemoglobin : 40 per cent. 40 -;- 53 = .75 : Color index. Pernicious Anemia. Erythrocytes : 840,000 per cb. mm. (16.8 per cent.). Hemoglobin : 1 8 per cent. 18 -;- 16.8 = 1.07 : Color index. Chlorosis. Erythrocytes : 4,100,000 per cb. mm. (82 per cent.). Hemoglobin : 32 per cent. 32 -H 82 = .39 : Color index. These examples, of course, refer only to the usual blood-find- ings, for the color index is by no means always high in pernicious anemia, nor always low in chlorosis. The color index shows simply the relative relations of the hemoglobin and the corpus- cular percentages. It is only suggestive, not diagnostic of a specific blood disease. The term hemoglobinemia is used to designate Hemoglo- a condition in which the hemoglobin is dissolved BiNEMiA. from the corpuscular stroma, as the result of some pathological factor, and is held in solution by the blood plasma. In extreme instances this condition is sooner or later succeeded by hemoglobinuria. Among the most potent causal factors of hemoglobinemia are certain drugs which act as blood-poisons, when administered in toxic doses, of which the following are examples : arseniuretted hydrogen, sulphuretted hydrogen, potassium chlorate, carbolic acid, hydrochloric acid, sulphuric acid, pyrogallic acid, nitrobenzol, anti- mony sulphide, iodine, naphthol, and many of the coal-tar deriva- tives, such as acetanilid, antipyrin, and phenacetin. A similar 124 ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. liberation of the hemoglobin may be observed as the result of poisoning by certain varieties of mushrooms, by some snake- venoms, by the bite of scorpions, and by a number of vegetable glucosides. Sunstroke, extensive burns, and exposure to excessive cold, are also capable of giving rise to hemoglobinemia. Experi- mentally, hemoglobinemia may be produced by the transfusion of blood from one animal into the circulation of another belonging to a different species. Hemoglobinemia is observed with more or less constancy in a number of acute infectious diseases such as grave cases of septice- mia, diphtheria, malignant jaundice, syphilis, malarial fever, enteric fever, scarlet fever, yellow fever, typhus fever, and variola. It also may occur in scurvy, and in Raynaud's disease, and is a prominent blood-finding in those two obscure conditions known as epidemic hemoglobinuria of the new-born, and paroxysmal hemoglobinuria. Hemoglobinemia may be readily detected by the following method, recommended by von Jaksch : ^ — A small amount of blood, drawn from the patient by means of a cupping-glass, is immediately placed in a refrigerator, in which it is allowed to re- main for twenty-four hours. In normal blood, the serum which separates at the expiration of this period, is of a perfectly clear straw-color, whereas if hemoglobinemia exists the serum is colored a beautiful ruby-red. If this hemoglobinemic serum is examined with the spectroscope, the two characteristic absorption bands of oxyhemoglobin may be observed. If it is coagulated by heat, a deep brown color is imparted to the coagulum. Methemoglobinemia, or the presence in the Methemoglo- circulating erythrocytes of methemoglobin, is BiNEMiA. produced by the action of a number of toxic substances, which, if given in sufficiently massive doses, may seriously or fatally cripple the oxygenating functions of the blood. Among the agencies which cause this conversion of oxyhemoglobin into methemoglobin are potassium chlorate, aniline, iodine, bromine, ether, turpentine , acetanilid, potassium per- manganate, hydrochinon, kairin, thallin, and pyrocatechin. The inhalation of amy I nitrite, and the intravenous injection of sodium nitrite also act in a similar manner. Spectroscopical examination of the blood is essential for the de- tection of methemoglobinemia. The spectrum of methemoglo- bin in alkaline solution shows three absorption bands : one well-marked band between C and D of Fraunhofer's lines and two others of much less distinct appearance, lying between D and E, each immediately adjacent to the lines. In acid and neu- 1 " Clinical Diagnosis," etc., London, 1897, 3d ed. HEMOGLOBIN. 125 tral solutions the spectrum of methemoglobin shows four absorp- tion bands : a decided one between C and D, two between D and E, and one closely adjacent to F. This spectrum, it is true, is identical with that produced by an acid solution of hematin, but it may be easily distinguished from the latter by the fact that the spectrum of methemoglobin, when acted upon by ammonium Fig. 35. Oxyhemoglobin Methemoglobin Reduced Hemoglo- C Hemoglobin Principal blood spectra. sulphide, changes first to that of oxyhemoglobin, and later to that of reduced hemoglobin, while when hematin is thus treated, a spectrum which shows two bands between D and E is produced. Aside from the bright, cherry-red color of the Carbon Mon- blood in coal-gas poisoning, the presence of car- oxiDE Hemo- bon monoxide hemoglobin may be determined by GLOBiN. spectroscopical examination, and by a number of distinctive chemical reactions. Recalling the characteristic spectrum of oxyhemoglobin ( two distinct absorption bands between D and E, the one nearest D being darker, narrower, and more sharply defined), it is found that in the spectrum of carbon monoxide hemoglobin these bands are replaced by two others, also between D and E, but nearer together, and somewhat closer to the violet end of the spectrum. This distinction, which may be so slight as to appear confusing, is at once emphasized by the fact that the addition of ammo- 126 ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. nium sulphide has absolutely no effect upon the carbon monox- ide spectrum, while it transforms the spectrum of oxyhemoglo- bin into that of reduced hemoglobin. Carbon monoxide hemoglobin in the blood is also demonstra- ble by the following simple test devised by Hoppe-Seyler : ' — A small quantity of blood, removed from the patient by means of a wet-cup, is mixed with twice its volume of a ten per cent, solu- tion of potassium hydrate. Thus treated, blood containing car- bon monoxide hemoglobin changes the color of the mixture to a rich cinnabar-red, while with normal blood the solution turns brownish-green. II. THE ERYTHROCYTES. The erythrocytes or red corpuscles are thin. Appearance flattened, biconcave discs, of sharply-defined, IN regular outline, and of smooth, even surface. In Fresh Blood, the blood of the normal individual they do not possess a nucleus. When the corpuscle is ex- amined microscopically as it rests upon its flat surface, its cen- tral concavity is plainly indicated by a dark, central area sur- rounded by a narrower, lighter rim, as the periphery of the cell is brought into sharp focus ; changing to a pale, white center encircled by a darker periphery, as the objective is brought closer to the corpuscle. When viewed in profile the corpuscle is shaped somewhat like a slim dumb-bell, with regularly rounded poles tapering from -either end toward a shallow central concavity on either surface. Their color, when examined singly under the microscope, is a pale greenish-yellow, but when collected to- gether in masses a more or less marked reddish tint becomes ap- parent. The erythrocytes possess a peculiar tendency of collect- ing and adhering together in more or less regularly arranged piles, like rolls of coins stacked up face to face, this being known as rouleaux formation. After withdrawal of the blood from the body various structural changes in the erythrocytes, commonly known as crenation, may be observed. In normal blood, the rapidity with which these changes progress depends upon the quantity of air which leaks in between the slide and the cover-glass, and thus causes de- generation of the corpuscular stroma. The development of one or more small, bright, highly-refractive spots in the body of the corpuscle, or a slight indentation of the cell's periphery are the most conspicuous indications of beginning crenation. As the process goes on, more and more of these hyaline points develop, • Loc. cit. THE ERYTHROCYTES. 12 f until finally the whole surface of the corpuscle- becomes thickly studded with glistening, bead-like spines. As the stroma be- comes drier and drier, its typical biconcavity and sharply-cut out- line are lost, contracting strands of the stroma are seen to extend from point to point among the beaded projections, the periphery of the cell changes to a cogged rim, and finally the cell becomes shrunken and shrivelled up into a small, many-starred asterisk. Some of the erythrocytes become fragmented, and small bits of their stroma are observed to break off and float through the plasma. Others become progressively paler and paler, as the hemoglobin is dissolved out, until complete decoloration occurs. Still others become distorted into designs of every conceivable shape, so that their resemblance to the normal cell becomes most remote. These changes, which never occur in normal blood until the cells have been exposed to prolonged atmospheric in- fluence, must not be confused with similar alterations in the structure of the erythrocytes occurring as the result of patholog- ical states of the blood. The latter changes are described more fully in another place. (See page 141.) The finer structure of the erythrocyte is still a Histological mooted point among different histologists, the Structure, view most generally accepted regarding it as a homogeneous cell composed of an insoluble spongy network, the stroma of Rollet, in the interstices or trabec- ulae of which is embedded a soluble, finely granular substance, the hemoglobin, existing probably as a compound with some un- known constituent of the cell. In lieu of a distinct limiting membrane, the portions of the stroma nearest to the surface of the corpuscle are condensed, to protect it from injury during its movements through the blood stream. The corpuscles are highly elastic and contractile, to permit of the rapid and marked tempo- rary distortions of shape which they constantly undergo in the circulating blood. Other authorities, notably Schafler,^ disagree with this view, inclining rather toward the opinion advocated by the earlier in- vestigators, who considered the eiythrocytes as vesicular masses, consisting of an external envelope enclosing a fluid contents. Thus, Schaffer believes that the cell consists of two distinct por- tions, a colored and a colorless, the former being a solution of hemoglobin, while the latter, or so-called stroma, consists chiefly of lecithin and cholesterin, together with a small amount of cell globulin. Without attempting to discuss the correctness of either of these two views, a single tangible reason for regarding the cor- ■Quain's "Anatomy," Phila., 1891, pt. 2, p. 210. 128 ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. puscle according to RoUet's opinion may be stated, viz : the fact that exposure of blood to destructive temperatures results in fragmentation of the corpuscles into numerous minute portions, each one of which consists of a bit of hemoglobin-containing stroma. This obviously seems to disprove the existence of a limiting membrane, without further investigation. In the human body an active manufacture Origin and of red corpuscles constantly goes on during Life History, health, in order to compensate for the continuous drain on their number by the destruction of those cells which have become incapable of function and useless, their life cycle being run. That this reproduction is the direct answer to a call for new cells is proved by the prompt and rapid increase of corpuscles following the loss of blood from hemorrhage ; that such a manufacture is attempted in severe pathological conditions, although the attempts are sometimes abortive, is evinced by the large numbers of immature and misshapen erythrocytes which appear in the blood in certain of the grave anemias. In the adult it is generally conceded that the erythrocytes are reproduced in the red bone marrow, being developed from their direct antecedents, the nucleated erythrocytes or erythro- blasts, which exist in this tissue in large numbers. The erythro- blasts appear to multiply in the thin-walled capillaries and veins of the red marrow, and having lost their nuclei, become trans- formed into normally developed erythrocytes, which pass from the marrow blood channels into the general circulation. Some authorities have attributed to the spleen and lymphatic glands a share in the formation of the red cells, while others have main- tained that they may be transformed from the leucocytes in the circulating blood, but none of these theories has been associated with convincing evidence, so that it is fair to consider the red bone marrow the chief, if not the only, seat of production, in the light of our present knowledge of the subject. Hayem's ingenious theory, that the red corpuscles arise from the hema- toblasts, does not enjoy the confidence of modem investiga- tors. When finally the erythrocyte, after having executed its function for a certain length of time, becomes useless in its primary office as an oxygen carrier, its death ensues, the destruction of the cell probably taking place largely in the spleen, whence the freed hemoglobin is carried to the liver to be eliminated as bile pigment. Warthin's^ recent studies apparently show that de- struction of the erythrocytes also occurs in the splenolymph 'Jour. Boston Soc. of Med. Sci., 1901, vol. v., p. 414. THE ERYTHROCYTES. 1 29 glands, minute vascular sinuses situated chiefly in the retroperi- toneal and mediastinal tissues, and in the thyroid and thymus regions. The possibility that certain of the partly degenerate red corpuscles also undergo a certain form of repair, first in the spleen and then in the liver, rendering them still capable of func- tion, is an interesting but obviously unproved conjecture. The average diameter of the erythrocyte is about Size. y.$fJL,^ its average thickness being about 1.8 /i. Ac- cording to Gram,^ the diameter appears to vary some- what with the geographical and climatic conditions surrounding the individual, being considerably larger in inhabitants of northern countries than in southerners, as the following average measure- ments of this observer attest : Country. Average Diameter. Italy 7 to 7.5^ France 7.5 to 7.6/i Germany 7.8/i Norway 8.5^ Hayem ^ distinguishes three different sizes : large, averaging 8.5 //in diameter; medium, averaging 7.5 fi in diameter; and small, averaging 6.5 //in diameter. Of these three classes, ap- proximately 75 per cent, are of the medium size, while 12.5 per cent, each, are large and small. The diameter varies within some- what wider limits in the infant and in the young child than in the adult. It is, however, not materially influenced by sex. The pathological increase and decrease in the diameter of the erythro- cytes occurring in certain anemias, are discussed in another place. The normal number of erythrocytes in the Normal healthy male adult may be approximated at Number. 5,000,000 to the cubic millimeter of blood. Higher counts than this are frequently observed, however, especially in healthy, well developed men, so that this figure should be taken to represent a rather low average, subject to an upward fluctuation of half-a-million cells, and occasionally even more. In females a count of about 4,500,000 red cells per cubic millimeter may be regarded as normal. Arterial and venous blood contain practically the same number of corpuscles, the apparent slight increase in favor of the latter, mentioned by some observers, being within the limits of technical error. For a like reason, under normal conditions, peripheral 1 The Greek letter /i is used to represent a micromillimeter, or l/l,ooo of a milli- meter, -which is a standard unit of measurement used in microscopy. "Forschr. d. Med., 1884, vol. ii., p. 33. i! Loc. cit. 130 ERYTHROCYTES, BLOOD PLAQUES, AND HEMOCONIA. blood may be taken as representative of the blood of the entire body. Blood derived from dependent parts of the body contains a diminished proportion of corpuscular elements. Oliver's ' studies of this question have shown that blood from the finger invariably gives a higher count of red cells than blood from the toe, this disparity being explained by the fact that the larger quantity of lymph gravitating to the more dependent parts of the body causes a dilution of the blood in these areas. This term has been applied by Capps ^ to the Volume figure representing the percentage volume of the Index. individual erythrocyte, in contradistinction to the color index, which expresses the amount of hemo- globin in the single cell. It is calculated by dividing the percent- age volume of the erythrocytes as a whole, obtained by centrifu- galization of the blood, by the percentage number of erythrocytes, as determined by the actual count with the hemocytometer, the normal volume index being taken as i.oo. For example, the erythrocyte column, after centrifugalization with the hematocrit, reaches to the mark 40 on the capillary tube, indicating a total volume of 80 per cent.; while the count with the hemocytometer gives 3,000,000 cells per cubic millimeter, or 60 per cent, of the normal number. Then, 80 -f- 60, or 1.33, equals the volume in- dex, a figure which in this instance shows an increase of 33 per cent, in the volume of each corpuscle. As a general rule, it may be stated that the volume index and the color index rise and fall together, although the parallelism between the two is not al- ways closely maintained. The volume index is generally lowered in chlorosis, in leukemia, and in most of the secondary anemias, while in pernicious anemia it tends to rise above the normal standard. III. INFLUENCE OF PHYSIOLOGICAL FACTORS ON THE ERYTHROCYTES. Polycythemia, associated with a proportionately Age AMD Sex. high percentage of hemoglobin, is found in the blood of the new-bom infant immediately after birth, the maximum counts being observed some time during the first twenty-four hours of life, after which period they progres- sively diminish until at the end of about eight or ten days an average of one million cells has been lost. Each period of nursing is generally followed by a prompt temporary decrease in the count, and a similar change has been observed as the effect of 1 Loc. cit. 2 Loc. cit. INFLUENCE OF PHYSIOLOGICAL FACTORS. . I3I premature ligation of the cord, at birth. Hayem ' found an aver- age of 5,368,000 red corpuscles per cubic millimeter in 17 infants at birth, the highest count being 6,262,000, and the lowest 4,340,000. The cause of this polycythemia is attributed to con- centration of the blood from the abstraction of water by the tissues to replace the fluids of the body lost during the first few days of life. As soon as this loss is made up by the ingestion of a sufficient amount of liquids by the child, the normal relation be- tween the liquid and the solid portions of the blood is reestab- lished, so that the polycythemia disappears. During the growth of the adult the average number of eryth- rocytes continues to rise, until the maximum numbet is attained at some time between the third and fifth decades, after which a decrease is observed, usually becoming more marked as the decline of life progresses. Schwinge^ and others have shown that during the period of sexual activity the counts in females are generally lower than in males, but that after the climacteric the number of cells in the two sexes is practically identical. The influence of age and sex upon the number of red corpuscles is well illustrated in the following table prepared by Sorensen : ^ Age. Males. Age. Females. 5 to 8 days 5,769,500 i to 14 days 5,560,800 5 years 4,950,000 2 to 20 years 5,120,000 19.5 to 22 years 5,600,000 15 to 28 years 4,820,000 25 to 30 years 5,340,000 41 to 61 years 5,010,000 50 to 52 years 5,137,000 8 2 years 4, 1 74, 700 There are no conspicuous changes in the num- Pregnancy, ber of erythrocytes in any of these conditions. Menstruation, In primiparse there is often a slight decrease in AND the number of corpuscles, particularly in the Lactation, later months of pregnancy, but in multiparse this change is rarely observed. During men- struation there may be a trifling reduction caused by the physi- ological hemorrhage of the phenomenon, but the loss is rapidly made up in a few days' time. Sfameni * found that a transient polycythemia usually occurs shortly before the establishment of the menstrual flow, and that the average loss of hemoglobin and corpuscles, which takes place in the majority of cases during the ^ Loc. cit. 2Pfluger's Archiv., 1898, vol. Ixxiii., p. 299. 3 Cited by von Limbeck : " Grundriss einer klinischn Patbologie des Blutes," Jena, 1896. ■' ° 13 o 19 20 oo 29 •sag ■'r'MUd e •© ^^. 30 .% •8'...-,;.-:. .■\ 33 s Jl|