COLUMBIA LIBRARIES OFFSITE HEALTH SCIENCES S I ANDARD ^ 'HXe^ 128334 RC641 .Eh82 1910 Anaemia, Pt I vol RECAP Columbia Mnibersiitpx'^ jdj mtf)eCitj>ofi5elD|9orfe " COLLEGE OF PHYSICIANS AND SURGEONS Reference Library Given by OAK^Vjr -^O "i • << Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/anaemiaeOOehrl ANEMIA ALSO NOW READY AN ATLAS OF HEMATOLOGY With a Description of the Technique of Blood Examination. By Dr. Karl Schleip, Scientific Assistant in the Medical Clinic, University of Freiburg i/B. English Adaptation of Text by Frederic E. Sondern, M.D., Professor of Clinical Pathology, New York Post-Graduate Medical School and Hospital, etc. Large Crown 4to, with 71 Coloured Illustrations, remarkable for exceptional accuracy and beauty of detail. \ Leather, price $10*00 net. New York: REBMAN COMPANY, Publishers A N it] M I A BY GEH. OBERMEDIZINALRAT PROFESSOR Dr. p. EHRLICH DIRECTOU OF THK KONIGI.. INSIITUT I'UK EXI'KRF MENTKI.I.K THKKAI'IK, I'RANKFURT A.-.M. AND Dr. a. LAZARUS PNOKESSOR OF THE UNIVEHSI'IV OK DEKI.IN-CHARI.OTTIiNBUKO PART I. VOLUME 1. NORMALand PATHOLOGICAL HISTOLOGY OF THE BLOOD SECOND EDITION {enlarged and to a great extent rewritten) Dr. a. LAZARUS ,,, Dr. O. NAEGELI AND PROFESSOR (bERLIn) PRIVAT-D02ENT (zURICh) TRANSLATED FROM THE GERMAN BY H. W. ARMIT, M.R.C.S., L.R.C.P.(London) WITH 5 ILLUSTRATIONS IN THE TEXT AND 5 COLOURED PLATES ■^^^ NEW YORK REBMAN COMPANY 1123 BROADWAY Ail Kiirhts Eeserved PREFACE TO THE SECOND GERMAN EDITION It is now more than ten years since I, together with my pupil, A. Lazarus, published the First Edition of tJiis part of Ancemia. Apart from a critical description of general clinical methods of examination, it dealt with the position at that time of the normal and pathological histology of the blood. It was, moreover, intended that this book should be especially devoted to a resume of the various works published by me and my pupils, as well as the hitherto unpublished results of my investigations and the views which I had adopted on the basis of these results. A glance over the hematological literature of the last ten years — a literature which has assumed almost immeasurable dimensions — shows that our Ancemia has stimulated investigation widely and has influenced it. The large majority of the com- munications have dealt with the problems which have been discussed in our book, and while some of these works support the views set forth, others oppose them. Histologists as well as clinicians have taken a very lively part in this criticism. It has been of the greatest satisfaction to me that my seed has fallen on such fruitful soil ; but it is especially pleasant for me to find that, after the completion of these ten years, the views which I have from the first defended as the foundation of modern cellular hfematology, in spite of the opposition of many renowned observers, have been more and more acknowledged, and I am convinced that within a very short space of time absolute unanimity with regard to these questions will reign among haematologists, as far as the principles are concerned, in concord with my doctrines. It is true that more recent work has removed V VI PREFACE many a stone from the building which I had erected, but, on the other hand, this same work has materially assisted in the completion of >the structure. The foundation, however, has not been touched in any important detail. This refers more especially to my doctrine of the anaemic conditions, which I have grouped according to the form of reaction of the bone marrow and to the doctrine of dualism of the white blood cells, which I have held from the very first. It is just with regard to this subject that many of my earlier pieces of evidence have been disproved by recent researches, and in many important details this question has assumed another aspect to that which it presented ten years ago. It is, however, with great satisfaction that I note from the recent literature, and particularly from the Transactions of the " Meeting of Scientists and Physic- ians," held in Cologne in 1908, that unanimity has been arrived at with regard to the chief points, and that the " unitarians " seem to have withdrawn from the fight. It appears to be generally accepted now that the various granules of the cells must be regarded as products of specific metabolism. This has been demonstrated more especially in the more recent publications. In this connection the observations, for example, of Kollmann are very important. He showed that in the lower animals {e.g. the crab), a disappearance of the granules can be obtained by starvation. This observation may be able to throw much light on phenomena of human pathology in which the neutrophile cells have lost their granules either completely or in part. I have described this in a case of anaemia. Similar conditions have been met with in leukaemia, and the appearances of the blood in these cases have in many instances led to erroneous suppositions with regard to the genesis of the white blood corpuscles. If I might express a wish for the future, it would be that physiological chemistry should attempt to clear up the chemical nature of the granules, since it is possible that the substances involved might turn out to be of great interest for clinical, therapeutic purposes. During the last five years we have been pressed both by our PREIACK vii publishers and by our leadeiH to edit a new edition of Ancemia, but in view of the large number of questioriB which were still being discussed with energy, this was postponed — much, it must be admitted, against our desires. Pressing obligations have prevented me from taking part in the preparation of this Second Edition, and for this reason I have requested Privat-Dozent Dr. 0. Naegeli of Zurich to utilize his well-known capabilities in assisting my original co-operator, — A. Lazarus, — to undertake the work. P. EHRLICH. Frankfurt-am-Main, April 1909. PREFACE TO THE ENGLISH EDITION The name of Ehrlich conveys two ideas directly to the mind of the ordinary student of medicine in England : blood and immunity. Of these two ideas, that of blood is the clearer in the mind of the average practitioner, because the teaching of the great German savant has reached him with more facility and in greater detail with regard to the histology of the blood than with regard to the intricacies of the mechanisms of side chains and antibodies. The name of Ehrlich is one which the English htematologist and the English research student has learned to value, and one which he cannot regard as Teutonic ; he claims it as an inter- national name. In introducing a work which has emanated from Ehrlich's school, and which in its first edition was in part written by Ehrlich himself, to the medical profession in England, no need exists for any recommendation of the author's. The value of the teaching of the greatest medico-biologist must be recognised by everyone. The task of transferring the ideas of the authors into a readable form of English has been one which the translator has willingly attempted, because he holds the opinion that it will be of use to the medical profession in this country to have at its disposal a work which aims chiefly at defending the manifold doctrines which Ehrlich has introduced in htematology. Some of the views put forward may not be as widely accepted as the one author (Dr. 0. Naegeli) would have the reader believe, but no attempt has been made to introduce any outside opinions into the work. It has been left entirely Ehrlichian. The extraordinary application of intra-cellular chemistry ix X PREFACE which characterises Ehrhch's blood work is well set forth in the pages of this book, and much of the material dealt with must be regarded as the, result of genius working against the disadvan- tages of almost complete ignorance. It is the earnest wish of the translator that his attempts to present Ehrlich's and Lazarus' Aoicemia in a readable form to the British medical practitioner has succeeded, and that the English Edition may find the favour which the original edition undoubtedly enjoys. H. W. ARMIT. LoNDOK', June 1910. TABLE OF CONTENTS Peepace to Second German Edition, by P. Ehklicu Peeface to the English Edition (The Teanslatoe) Definition- chapter I Introduction -Clinical Methods of the Examination of Blood Ev A. LAZARUS Tlie quantity of blood The number of red bloixl corpuscles Relative size of red blood cori^uscles Haemoglobin content of the blood . Colour index Specific gravitj^ of blooil . Hygrsemometry Total volume of red blood corpuscles Reaction of blood . Coagulability of blood Separation of serum Resistance of red blood corpuscles Cryoscopy .... 2 5 12 13 17 19 22 22 24 26 27 28 29 CHAPTER II The Morphology of the Blood Bv A. LAZARUS (A) Methods of Examination Making dry films . Fixing dry films Staining dry films . Theory of staining Combination staining Triacid solution Otlier dj'^e solutions Vital staining . Recognition of glycogen in blood xi 30 30 33 36 39 40 41 43 44 51 52 Xll CONTENTS Microscopical test for the distribution of alkali in blood Bremer's reaction ..... (B) NoRMAX AND Pathological Histology of the Blood The red blood corpuscles . Structure .... Diminution of haemoglobin content Polychromatojihilia Punctated erythrocytes Poikilocytes Nucleated red blood corpuscles Normoblasts and niegaloblasts Physiological occurrence The fate of the nucleus of the erythrocytes The clinical differences of erythroblasts Bibliography .... PAGE 53 54 56 56 57 58 58 61 67 68 69 71 72 74 79 CHAPTER III The White Blood Corpuscles By O. NAEGELI . I, Normal Histology and Classification op the White Blood Corpuscles The lymphocytes The large mononuclear leucocytes The transition forms . The polymorpho-nuclear neutroj^hile leucocytes The eosinophile cells . The mast cells .... Pathological forms of white blood coiyuscles The neutrophile myelocytes . The eosinophile myelocytes The mast myelocytes . The myeloblasts The " stimulation " forms The plasma cells Arneth's method II. On the Origin of the White Blood Corpuscles (a) The spleen .... (h) The Ij'mphatic glands . (c) The bone marrow . 87 90 90 94 95 95 97 98 99 99 100 101 101 104 106 107 109 111 116 124 CONTEN'IS xiii PAO> III, On THIO DlCMONHTHATlON AND SlONIFKJA.NCK OF CkIJ- OkANULKS 133 HisLory of givuiule researclies since Ehrlicli'H diwcovery Metliods of (liMiionstratioii Vital .sUiiiiiiig of granules .... Altmann's Ijioljlast tlieoiy .... Granules as metabolic products oi tlic cells (Eiulicli) Secretory phenomena in granulated cells IV. Thk DuALisTic Theory . , . , . V. Leucocytosis ...... The biological significance of leucocytosis The morj)liology of leucocytosis (a) Polyinorpho-nuclear neutrophil leucocyto.sis Definition .... Clinical occurrence Origin ..... (h) Polymorpho-nuclear eosinophil leucocytosis Definition .... Clinical occurrence Origin ..... (c) Mast cell leucocytosis. . . ' . VI. Leuk.«jiia (Mixed Leucocytosis) Lymphatic leukaemia ..... Myeloid leukremia ..... Mori^hological characters .... Origin 133 136 137 141 142 14.5 146 151 1.52 159 160 160 161 164 164 164 165 170 175 176 178 180 181 192 BiBLIOGRArHY . . . . . . ,193 CHAPTER lY The Blood Platelets : The H^moconia By A. LAZARUS . . .202 Bibliography. ...... 208 ANEMIA CHAPTER I INTRODUCTION DEFINITION— CLINICAL METHODS OF EXAMINING THE BLOOD The term " aureiiiia " as it is applied in clinical medicine does not possess exactly the same meaning as the limitations which scientific investigation has imposed upon it would suggest. In the former certain prominent external symptoms are regarded as the characteristics of anaemic conditions : pallor of the skin and, as compared with the normal condition, a slighter degree of redness of the mucous membranes of the eyes, lips, oral cavity, and fauces. Not only is the existence of an anaemia deduced from the presence of these signs, but even the degree of the affection is measured by the extent of the symptoms. It is obvious that a definition which is formulated on such a common and elementary symptom complex must include conditions which do not belong to it at all, while other conditions may perhaps be excluded which on account of their nature should be grouped with it. This fact is responsible for a number of uncertainties and contradictions. It is therefore the first problem of a scientific consideration of anaemic conditions to carefully define their extent. The external symptoms mentioned above will be found to be little suitable for such a task, although it must be admitted that when applied in the proper place they are of practical importance. The word anaemia in its etymological sense deals with a blood the content of which is smaller than in health. This abnormality 2 INTRODUCTION may be general and affect the whole organism, or it may be local and affect only a limited area or a single organ. The latter forms, the local anae-mias, do not enter into the scope of the present work. The blood content of an organism can clearly differ from that of a healthy individual in two ways : quantitatively and qualitat- ively. A diminution of the total quantity of blood, without any alteration of its composition, may be present; this is called oligcemia. On the other hand, the diminution of the quality of the blood may be absolutely independent of the total quantity, and must be recognised primarily by the diminution of those com- ponents of the blood which are physiologically most important. Accordingly the principal types of qualitative diminution of the blood, which are recognised, are the diminution of the haemo- globin content (oligochromcemia), and the diminution of the red blood corpuscles (oligocythcemia). All conditions in which a diminution of the haemoglobin content is ascertainable must be regarded as anaemic. In the majority of cases, even if this is not quite constant, oligaemia and oligocythsemia exist simultaneously in varying degrees. The most important methods of clinical haematology depend directly or indirectly on the recognition of these changes. Up to the present no clinically applicable method for the determination of the total quantity of blood has been introduced. It is possible to a certain extent to utilise the observations mentioned above of the symptoms of redness or pallor of the skin and mucous membranes for this purpose. These symptoms are, however, dependent to a great extent on the composition of the blood, and not solely on the amount of blood contained in the peripheral vessels. In order, therefore, to utilise this means as a measure of the total quantity of blood, it is advisable to have regard to those isolated vessels which are visible to the naked eye, e.g. in the sclera. The most useful way, however, is to observe the size of the vessels in the fundus of the eye by means of the ophthalmoscope. Eaehlmann has shown that in 60 per cent, of cases of chronic anaemia, in which the skin and rNTRODUCTION 3 mucous membranes arc pule, hyperfcmia of tlio retina exists. This proves that the blood circulating in tlie vesHcls is paler but not sparser than normal. The character of tlie pulse also may give an ini])()rtaiit indication in cases in which the (HniiMu- tion of the (Quantity of blood is considerable ; in marked oliga^mia a very small and soft pulse is always found. The wa,y hi which fresh wounds bleed may serve as a criterion of the quantity of blood ; this, however, can only be utilised within certain limitations, depending to a large extent on the coagul- ability of the blood. Those who have had occasion to examine the blood of anaemic persons frequently will have experienced that this behaviour is subject to great variations. In certain cases scarcely a drop of blood can be obtained in the ordinary way, while in other cases the blood flows freely. There is little risk of mistake, if an absolute diminution of the total quantity of blood is assumed in the former case. The degree of fulness of the peripheral vessels is, however, only an index of relative value, since the blood content of the internal organs may be quite different. The task of determining the exact quantity of blood in a body in figures has always been one of utmost importance. The solution would mark a very great advance in hsematology. Of the methods which have hitherto been suggested for use in clinical medicine, that emanating from Tarchanoff deserves mention. Tarchanoff proposed that by determining the loss of water during profuse sweating, and by comparative red blood cell counts both before and after the sweating, an estimate of the quantity of blood could be arrived at. This method, apart from many theoretical difficulties, is much too complicated to be applicable to practice. Quincke attempted to determine the quantity of blood by means of calculations, while carrying out therapeutic trans- fusion of blood. The quantity of blood in the person into whose vessels blood is being transfused can be calculated by means of a simple mathematical formula, from the number of red cells in his blood before and after the transfusion, and from 4 INTRODUCTION the quantity of the transfused blood and the number of cor- puscles contained therein. But even this method is only applicable in certain cases, and is open to some theoretical objections. In the first place, it is dependent on the relative content of red corpuscles of the blood, inasmuch as the transfusion of normal blood into normal blood would not effect any change in the number. This suffices to show that this method is only of use in a few special cases. It has been demonstrated that an increase of red corpuscles in each c.mm. takes place in an individual with a very low red blood cell count, into whom normal blood has been injected, but it is extremely risky to attempt to determine the amount of the pre-existing blood from this calculation, since there is no doubt that the act of transfusion directly produces compensating currents of fluid and changes in the distribution of the blood. The same objection may be offered to the suggestions recently made by Kottmann and Plesch. These observers injected physio- logical saline solution intravenously in large quantities, and after five minutes determined the total volume of red blood corpuscles (Kottmann) or haemoglobin (Plesch) and compared the values gained with those derived from similar counts before the injections. Apart from the objections mentioned above, there are objections raised from the medical point of view against the introduction of such injections for the purpose of clinical examination. The injections are often followed by a considerable rise of temperature. The method employed by Morawitz of determining the quantity of blood in an arm by means of a plethysmograph and of calculating from this the total quantity in the body seems to be quite worthless, in view of the local and temporary variations in the distribution of the blood, which cannot be ascertained but which are certainly considerable. Haldane and Lorrain-Smith have attacked the problem from another standpoint and have used a comparatively simple method, which, however, is not quite free from objection on the point of safety. They caused their animals to breathe in a definite amount INTRODUCTTON 5 of CO, then ;i,l)slra,c,t(;(l siiiiill ([uiiiilili(!K of blfjcKl ii,)i(l jiHcertained without (h'Cficiilty tlio (JO codIohI. (if the tot;iJ i|U.'uitity of hlood. The resultH obtained in this way did not a, 0-5 grms. 5-0 J5 10 JJ 200-0 J> Hydrarg-perchloridi . . . Sodii sulphatis . . . . . Sodii chlorat ...... Aquae dest. ....... The results obtained by these methods of counting are sufficiently exact for practical purposes, since, according to the researches of E. Thoma and I. P. Lyon, which have been con- firmed by numerous observers, the experimental error is low. When 200 cells are counted it is 5 per cent., when 1250 cells are counted it is 2 per cent., with 5000 cells it is 1 per cent., and with 20,000 cells 0-5 per cent.). As far as the practical application of the method is concerned, further considerations must be taken into account which exercise an unfavourable influence on the accuracy of the values. Cohnheim and Zuntz, inter alia, have shown that the blood in the larger vessels reveals a con^ant composition, but that in the smaller vessels and capillaries the corpuscular elements are subjected to considerable variations as to number, even in other- wise normal blood. For example, samples of capillary blood taken from both sides of persons suffering from hemiplegia do not contain the same number of corpuscles; while marked hypersemia, cold, etc., increase the number of red cells locally. It is therefore necessary that blood taken for the purpose of making counts should be derived only from those portions of the body which are not subjected to marked variations, that all IN'nU)I)(JCTI()N 7 procedures should bo avoided wliich could alter the eajjillaiy circulation, such as violent rubbing, massage, and the like, and that the examination should be undertaken at a time of day when the number of blood corpuscles is not artificially influenced by the taking in of food or by medicaments. It is usual to take the blood from the tip of the finger, and only when this is rendered inadvisable, e.g. when there is oedema of the finger, to select other situations, such as the lobule of the ear, the great toe (especially in children), etc. It is inexpedient to make a prick with a sharp needle or with a specially constructed open or hidden lancet ; instead of all the complicated apparatus, the best instrument for the purpose is a new steel nib, one point of which is broken off, or a Sonnecken's vaccination lancet. The nib or lancet can be readily sterilised by heating in the open flame, and by their means a more suitable cut, rather than a prick, is obtained, from which the blood flows freely without the aid of marked pressure. The material from which the countings of red blood corpuscles in healthy persons has been determined and published seems to be too extensive to deal with. According to the exact compilations of Keinert and von Limbeck, the following values (calculated for cubic millimetres and expressed in round figures) may be regarded as physiological : — Males. 1 Maximum. Minimum. Average. 7,000,000 4,000,000 5,000,000 Females. Maximum. Minimum. AA'erage. 5,250,000 4,500,000 4,500,000 8 INTRODUCTION The difference between the two sexes only exists from the time of puberty in women. Up to the time of the onset of the first menstruation the number of red blood corpuscles is even a little larger (Stierlin). The only other variation in the number of red blood corpuscles due to the age of the individual is found in the case of newly born infants, in whom polycythsemia is always present (up to 8 1 millions during the first few days of life, E. Schiff). From the first taking in of food, however, the number decreases gradually, albeit in stages, until the normal is reached, which takes place in about ten to fourteen days. The oligocythgemia, which is occasionally observed during advanced age, is, according to Schmaltz, not a regular phenomenon, and should therefore not be regarded as a physiological peculiarity of senility, but must be ascribed to the manifold active circumstances which affect this age. , The influence which the intake of food tends to exercise on the number of red blood corpuscles must be ascribed in the main to the addition of water, and is so insignificant that the varia- tions lie as a rule within the limits of error of the method. Other physiological processes : menstruation (i.e. a single period), pregnancy, lactation — do not alter the number of blood cells to any appreciable extent. Nor are there any dijfferences between the numbers in arterial and venous blood. All the fluctuations in the number of blood corpuscles which lie within physiological limits are dependent on vasomotor influences (according to Cohnstein and Zuntz). Stimuli which cause a contraction of peripheral vessels lessen the number of red blood corpuscles m situ; stimulation of vaso- dilators, on the other hand, produces a reverse effect. This means that the physiological variations in the number in any given area is only an expression of an altered distribution of the red elements within the blood channels, and is quite independent of new formation and destruction of the cells. Climatic conditions appear to have a great influence on the number of blood corpuscles. This matter is of equal importance INTRODUCTION 9 to ])liy8i()](),] been witnessed in the othor depiirlnicnt.s oi' noiniul und paLliologicul histology ; improvement in techni([ue must first Vjo achieved before material advances in knowledge can be luiived ;it. I'"or this reason it is difficult to understand how certain observ(;rs still recommend the old methods, and claim that a diagnosis can be made in all cases from the examination of fresh blood specimens. It may be admitted tliat this wcjuld not' 1)0 surprising, since the most important points have already been explained by means of the new methods. ]>ut the recognition of the more difficult cases, c.ij. certain rare forms of antemia, and for the recognition of definite kinds of cells, such as myelocytes, mast cells, etc., stained preparations are indispensal)le, as every experienced hiematologist knows. The object of the examination, moreover, is not to facilitate a rapid diagnosis, but rather to enable an exact study of the details characterising the blood to be carried out, which cannot be ascertained from fresh specimens. It may with safety be claimed that at present it is possible to see all the characteristics of the blood, save those of the formation of rouleaux and of the amoeboid movements of the white corpuscles, in stained dry preparations as well, if not better, than in the fresh specimens ; while it must be admitted that there are many important details which can only be rendered visible by means of staining fixed preparations, and which remain invisible in fresh specimens. Examination with the assistance of the so-called " dark field illumination " forms a valuable extension of the methods of studying the blood, especially in its fresh condition. This was first employed for this purpose by Dietrich. It has become possible with its assistance to demonstrate certain morphological details, e.g. shadows in the corpuscles, more clearly than had been done previously, while even some biological processes, such as haemolysis, can be rendered directly visible by means of the dark field illumination. The special morphology of the blood cell, however, has not been advanced by this method, nor by the method of examining with the aid of nltra-violet rays (Grawitz and Griineberg). As far as the purely technical or practical aspect of the 32 ANEMIA question is concerned, the examination of stained dry films is undoubtedly much more convenient than that of fresh specimens. The former enables the observer to be independent with regard to place and time. Fixed specimens may be put aside for months without any special precautions, and then studied closely under the microscope. The examination of one specimen can be con- tinued for any length of time, and can be repeated at any future date. On the other hand, the examination of the blood in the fresh condition is only possible at the bedside, and must be completed rapidly, since the blood changes quickly, by clotting, by destruction of the white cells, etc., so that an ex- haustive study cannot be undertaken at all. An additional advantage of the former consists in the fact that the making and staining of dry blood films may be regarded as one of the easiest and most convenient of all the methods of clinical histology. It is therefore advisable to describe the technique in detail in this place, in order to awaken wide interest for this mode of examination. It is further found advisable to describe in this place the application of stained dry specimens for the determination of the important numerical relation between the red and white blood corpuscles, and of the proportional percentages of the various forms of white corpuscles. It is absolutely necessary that the investigator should be able to make a perfect, uniform film. Quadratic ocular diaphragms (Ehrlich-Zeiss) are essential.^ These either represent a complete series, so that the sides of the square having measurements in the ratio of 1:2:3 . . . 10, would give segments of the field in the ratio of 1 : 4 : 9 . . . : 100, or take the form of the more handy eye-piece, devised by Ehrlich and constructed by Leitz, which possesses a neat mechanical appliance by means of which a centrally situated, square segment of the field of a desired size can be interposed. The eye-piece is used in the following manner. A normal blood preparation is examined by first counting the white blood corpuscles as seen in the field when a No. 10 dia- ^ These square eye-pieces are not frequent in this country. — (The Translator). phra<^ni (or the Beginont TOO of tlio eye-piece) is interposed. Next the cliM,Y)hragm No. 1 is interposed so that only one-hnndredth part of the (ield is exposed, and in tliis fifild the nunihei- of red blood cells are counted. This is done witliout shifting the speci- men. Next, another part of the specimen is chosen at random, and the count repeated ; in each case only one hundredth or one twenty-fifth of the field employed for the white cell count is used for the red cell count. About 100 such counts are made in each specimen. The number of red cells is then multiplied by 100, and compared to the number of white cells counted. When the white cells are very numerous and the counting in a large segment becomes difficult, a smaller diaphragm is employed, such as 81, 64, 49, and so on. The important determination of the percentage proportions of the various forms of leucocytes is carried out by noting the numbers of each kind in a series of several hundred cells. This can be done by an experienced individual in less than a quarter of an hour. (a) Making a Dry Specimen. For the purpose of making perfect dry films, it is of especial importance to use cover-glasses of a particular kind. The cover- glasses should not be thicker than 0'08 to O'lO mm. ; the glass should not be brittle, it should have no defects, and it must be of such a quality that it will bend to a considerable extent without breaking. The slightest roughness of the glass renders it useless for the purpose. The cover-glasses must be subjected to a scrupulous cleansing, and must be absolutely freed from all traces of fat. For ordinary purposes it is sufficient to immerse the glasses in aether for thirty minutes, without allowing them to overlap, and then to wipe them with an old soft linen or cambric cloth. They are then dipped into alcohol for a few minutes, and are again dried in the same manner as they were after immersion in aether. They should then be placed in a dust-tight glass bottle or box until required. The fact that these cover-glasses are cut out of a large cylinder and not out of a flat plate of glass shows 3 34 ANAEMIA that they are the only kind which would allow of the formation of a capillary space between them when superimposed on one an- other, in which the blood can spread out spontaneously. The slightest unevenness or brittleness of the glass would render it impossible for the curve in the one to correspond suJEiiciently exactly to the curve in the second. It is only when the glasses are of this quality that it becomes possible to slide the one from the other without using such force as would destroy the film. The cover-glasses must be held by means of forceps ^ in order to avoid any soiling, and especially any contamination of the blood by moisture of the finger. The lower glass is best held in a pair of forceps pro- vided with a catch and broad fiat points (a). The inside of these points can be lined to ^i»- 1- the extent of about 3 cms. from the tips with leather or English blotting- paper. The other cover-glass is best held in a pair of forceps (&) with a good spring and smooth but very sharp points. These forceps will enable the operator to catch hold of the cover-glass even when it is lying on a perfectly smooth surface. The lower cover is seized with the clamp forceps, fixed and held in the left hand. The second glass is taken up in the forceps (b) with the right hand, and applied to the drop of blood issuing from the prick in the finger. The cover-glass must pick up the blood without touching the finger itself. Next the cover-glass in the right- hand pair of forceps is carried rapidly to the other and allowed to fall gently on it. The blood then distributes itself in an absol- utely uniform capillary layer, without the application of any pressure, provided that the cover-glasses are suitable. The upper ^ Klonne and Miiller, Berlin, make the forceps, as devised by Ehrlich. THE MORPITOr.OGY OF TFTE BLOOD .',5 glass is then Hoiz(3(l, cither with two iingei's ol' th(; lif^lit h.'ind, oi' better still with the forceps (b), and is carefully slid oil" tlio lowor cover-glass, which is still held by the clamped forcejjs, but without pressing or raising it (sec Fig. 1). v\s a rule only the lower glass yields a perfectly uniform smear, but at tiuK^s both arc; utilisable. During the process of drying in the air, which takes about ten to thirty seconds, it is of course necessary to protect the cover-glasses from moisture, such as that derived from the breatli of persons in the vicinity. The size of the film on the cover-glass depends on the size of the drop which has been picked up. The smaller this has been, the smaller will be the surface over which the blood will distribute itself. Large drops are absolutely useless, if they cause the one cover-glass to swim on the other instead of merely sticking them together. The directions for this method may appear to be somewhat complicated, but a little practice will show that the technique can be easily acquired. The details have been described minutely, because the author frequently sees specimens which he considers to be quite unsuited for the purpose, although they have been made by men who have given special attention to the study of hsematology.^ Jancso and Eosenberger have published the details of another ^ Translator's Note. — A considerable number of English investigators dispense with the use of cover-glasses altogether. Slides of the best quality are employed and cleaned with utmost care. Boiling in sulphuric acid or nitric acid, subsequent rinsing in distilled water, washing in alcohol and storing in mixtures of alcohol and aether yield good results. Wright advises polishing with very fine emery cloth, stretched on a wooden block before use. The drop of blood is applied to tlie slide, and is spread over the whole length by means of a second slide. Before this is done it is necessary to select a second slide, which "dances," i.e. which shows a just perceptible concavity of one of its ends. The concave surface is then gently and lightly pushed up tlie slide bearing the drop of blood until the blood runs toward the edge by capillary attraction. The slide is then gently drawn downwards, when it will be found that the blood follows it. Under no circumstances may the slide be drawn over the blood. AV right performs this drawing in short regular jerks, so that the smear appears as a series of thicker and thinner transverse lines of blood right down the lower slide. Some workers have also used a thread of silk for the purpose of drawing the blood down the slide. The advantage of dispensing with the cover-glass is obvious. In the first place, it is difficult to secure cover-glasses which are reliable ; next, they are much more 36 ANiEMIA method of making fixed blood smears. This method has been employed by a number of investigators. The drop of blood is picked up on the edge of one cover-glass from the finger, and the edge of this cover-glass is then drawn gently over the full length of a second cover-glass. With practice very nice thin smears can be obtained in this way, which dry rapidly in the air. After the specimens have dried thoroughly they may be stored between layers of filter paper in glass vessels provided with properly fitting stoppers. For important cases, when it is desirable to preserve the smears for a considerable time, it may be wise to protect the smear from the damaging effect of the atmospheric air by covering it with a layer of hard paraffin. Before the films can be fixed and stained it is then necessary to remove the wax by dissolving it in toluol or xylol. It is, of course, essential to keep the films in the dark. The procedure may be modified in a number of ways according to the object of the examination. For example, for the detection of blood parasites it is advisable, according to Eobert Koch, to use very large drops of blood. The blood cells, which are of minor importance in these cases, may be dissolved, and the detection of the parasites may be rendered much more easy by the use of a considerable quantity of blood. (b) Fixing a Dry Specimen. All methods of staining blood cells require a preliminary fixation of the albuminous substances in the blood. General directions for fixation cannot be given, since the intensity of the same must depend on the choice of the method of staining. flifficult to clean than slides, and are at the best of times liable to break. Further, tlie slide yields a much larger surface for examination, which may be of great im- portance, especially when it is difficult or impossible to secure another specimen of blood. The slides keep well, and are examined without any cover-glass, the cedar-wood oil being applied directly to the film and the objective immersed in the oil. The oil can be removed from the smear without damaging the latter by means of xylol or other solvent, if applied with care. This should be done after the examination is completed, and not left until a future occasion. THE MORPHOLOGY OF THE HLOOI) 37 Comparatively f-nuill (logrees of hardening sulHce wIkmi the staining is caniod out with watery sohitions, sucii as the triacid solution. This may he a(;hieved hy allowing various agents to act for a short time, and in not too intense a manner. Other methods, which inchule the use of strong acids or which are carried out with sohitions containing free alkalies, ref[uire that the structure should he fixed by a much stronger action of the fixatives. It is just as important to avoid over as under fixation. It is quite easy to determine the optimum fixation for each of the few staiidng solutions which are in use. The following means of fixation may be employed : — 1. DRY HEAT. For this purpose a simple copper plate placed on a stand is heated at one end by means of a Bunsen burner. After the flame has been burning for some little time it may be assumed that the plate has acquired a certain degree of con- stancy of temperature. It will, of course, be hottest at the burner end, and least hot at the other end. By allowing water, toluol, xylol, and other fluids to drop on the plate, the observer can easily ascertain which portion of the plate has approximately the temperature at which the various fluids boil. Victor Meyer's apparatus, which is much used by chemists, is more suitable for this purpose. A modification adapted to the fixing of specimens takes the form of a small copper kettle, the cover of which is a thin copper plate having only one opening for the transmission of the steam pipe. If a small quantity of toluol is allowed to boil in the kettle for a few minutes, it may be assumed that the temperature of the copper lid is also between 107° and 110° C. It is sufficient for specimens which are to be stained by the ordinary watery solutions to expose the cover-glasses to a temperature of about 110° for a half to two minutes. When differential staining (such as the eosin-aurautia-nigrosin mixture) 38 ANEMIA is to be employed, it may be necessary to expose them for longer periods or to higher temperatures. 2. CHEMICAL MEANS. (a) Nikiforoff advised fixing the smears in mixtures of equal parts of absolute alcohol and aether for two hours, in order to obtain good triacid staining. Specimens fixed in this manner, however, are not so fine as those fixed by heat. (5) Absolute alcohol fixes dry smears within five minutes sujfficiently for subsequent staining by Chenzinsky's solution or by hsematoxylin-eosin solution. In some cases, when it is desirable to examine the specimen quickly, it may be advisable to boil the dried cover-glass for one minute in a test tube with absolute alcohol. (c) For Griemsa staining it is advisable to fix in absolute methyl alcohol. If this is undertaken immediately after the drying the fixation takes from three to five minutes, while if it is carried out on the following day it only takes two minutes. (d) Formol was first employed by Benario in 1 per cent, alcoholic solutions for the fixation of blood specimens. The fixing is completed in one minute, and may be used for the demonstration of granulations. Benario recommends it especially for staining with hsematoxylin-eosin. Schiiffner obtained beautiful results by fixing his blood smears in 1 per cent, formol solution to which from 5 to 10 per cent, of glycerine had been added. For certain forms of staining the fixation is carried out simultaneously with the staining (see below). It must be understood that these methods are described as the most suitable for blood examination in general. For special purposes, e.g. the demonstration of mitosis, of blood platelets, etc., the fixation methods generally employed in other branches of histology may be used with advantage. These include perchloride of mercury, osmic acid, and Fleming's solution ioiter alia. THE MOUPIIOLOGY OF TTTK IJLOOT) .39 (c) staining a Dry Specimen. Staining methods may be classified according to the purpose for which they are used. In the first place, stains are employed for tin; purpose of obtaining rapid information of a general character. This may be attained by solutions which stain both tlie haemoglobin and the nuclei (hasmatoxylin-eosin, ha^matoxylin-orange). In the next place, it is at times desirable to have a staining which only afi'ects one special form of cell in a characteristic manner, such as the eosinophile cells, the mast cells, or bacteria. This is termed " single staining," and is carried out in accordance with the principle of maximal decolorisation (see. E. Westphal). In the last place, there is the so-called panoptic staining, i.e. staining which affects as many elements as possible, and which makes use of the greatest variety of colours. These methods are naturally of considerable interest for exhaustive examinations. It is necessary when they have been employed to utilise high magnification for the study of the specimens, but apart from this fact the methods yield more information with regard to the condition of the blood than any other. In order to obtain the greatest possible degree of differentiation, it will be found that double staining is usually not sufficient, but that it is necessary to use three colours which contrast from one another as much as possible. Formerly, the various stains were applied successively for this purpose. But, as every one who has employed these methods knows, it is exceedingly difficult to obtain constant results in this way, for even w^hen the directions with regard to the length of time of staining and the con- centration of the solutions are followed with the most minute care, it is impossible to rely on the results. On the other hand, the methods of simultaneous or combined staining offer undoubted technical advantages. Improvements in technique are of considerable importance for the development of the histology of the blood. Since it appears that there is some want of clearness in the mind of some observers with 40 ANEMIA regard to the principles, a short description of the theory of differential simultaneous staining may be of use. For this purpose a simple example will be selected. This is the employment of picro-carmine, i.e. of a mixture of neutral ammonium-carmine and a salt of picric acid. If a tissue which is rich in protoplasm be stained with carmine, the stain appears to be fairly diffuse, even though the nuclei become prominent. But if picrate of ammonium of the same concentration be added to the solution, the staining gains greatly in definition, by some portions appearing pure yellow and others pure red. The best known example of this is the staining of muscle by picro-carmine. In this case the muscle substance is stained pure yellow, while the nuclei take on a red colour. Now, if, instead of adding the picrate of ammonium, the experimenter uses a dye containing more nitro groups, such as the ammonium salt of hexa-nitro- diphenylamin, the carmine staining will be prevented altogether. All the elements in this case take on the pure colour of aurantia, no matter how long the stain is allowed to act. The explanation is very simple. Myosin possesses a greater affinity for picrate of ammonium than for carmine, and therefore combines with the yellow dye contained in the mixture of both stains. This combination removes the possibility of it taking up any carmine. The nuclei, however, possess a greater affinity for carmine, and therefore stain red in this process. But if a nitro dye be added to the carmine solution, which possesses a greater chemical affinity for all the elements of the tissue, and even for the nuclei than the carmine itself, the sphere of action of the carmine will be more and more limited, until when a very strongly acting nitro stain — the liexanitro compound — is used, it will be prevented altogether. Connective tissue, bone substance, and similar tissues, however, behave in a different manner toward the picro-carmine mixture. In this case the diffuse staining is solely dependent on the concentration of the carmine, and is not influenced by the employment of a chemical antagonist. It therefore is only possible to obtain a limitation of this staining by diluting the stain, and no addition of a dye stuff" possessed of opposite THE MORrH()I.()(iV OF TIIK IM.OOI) 11 characters will make any difference. Tliis example of tissue staining may be regarded as a mechanical attraction of the colour by the tissues, and not as a chemical combination. It may therefore be stated that a chemical staining may be recognised by the fact that it reacts to chemical antagonists, and tliat a meclianical staining reacts to physical modifications. This statement, however, is true only as long as pure neutral solutions of stains are employed. All additions, such as those of acids and alkalies, which could alter the chemical behaviour of the tissue, or which could diminish or increase the affinity of the tissues to the dyes, would also interfere with this test. From this view it may be deduced that all double staining methods, which can be employed by means of successive stainmg, can be advantageously substituted by combination staining, provided that it can be proved that the staining depends on a chemical combination. And, conversely, all those methods of double staining which can only be obtained by successive staining must be dependent on mechanical processes. Only pure chemical staining processes are employed for the purpose of staining dry blood films, and the application of polychromatic combination staining is therefore possible in all cases. The following combinations are available for blood preparations : — 1. Combination Staining with Acid Dyes. — The best- known example of this is the eosin-aurantia-uigrosin mixture, with which the hiemoglobiu stains orange, the nuclei black, and the acidophile granules red. 2. Mixtures of Basic Dyes. — It is a simple matter to prepare mixtures of two dye bases. The most suitable of these are fuchsin, methyl-green, methyl-violet, methyleue-blue, and pyronin. On the other hand, it is somewhat difficult to form a mixture of three of these substances, and this can only be done successfully by paying minute attention to the quantitative relationships. The following may be employed for this purpose : — fuchsin, Bismarck-brown, chromic-green. 42 ANJEMIA 3. Neutral Mixtures. — These mixtures were first introduced by Ehrlich into use for the histology of the blood, and for general histology. They have been found to be of considerable importance and claim careful consideration. Neutral staining depends on the fact that nearly all basic dyes (i.e. the salts of dye bases, e.g. rosanilin acetate) enter into combination with acid dyes (i.e. salts of dye acids, e.g. picrate of ammonium) to form what is known as neutral dyes (e.g. rosanilin picrate). Their use is, however, rendered difficult by the fact that they are very little soluble in water. It was only after Ehrlich had shown that certain series of the neutral dyes are freely soluble in the presence of an excess of acid dyes that their use was rendered practically possible; in this way stable solutions of varying concentration of these dyes can be prepared. The most suitable basic dyes for this purpose are those which contain a so-called ammonium group, and especially methyl-green, methylene-blue, amethyst-violet ^ (tetra-ethyl-safranin-chloride), and, under certain conditions, pyronin and rhodamin. In contradistinction to these, those dyes which form the members of the triphenyl-methane series are on the whole but little suitable for this purpose, with the exception of methyl-green. These include fuchsin, methyl-violet, Bismarck-brown, phosphin, and indacine. The most suitable acid dyes for the purpose of forming the neutral dyes are more especially those highly soluble salts of the polysulphonic acids. The salts of the carboxylic acids and the other phenol dyes are less suitable, while the nitro dyes are the least suitable. The following members of the acid dyes may be enumerated, as being used for the purpose of forming neutral dyes : orange G, acid fuchsin, narcein (a freely soluble yellow dye, the sodium sulphanilate of hydrazo ;S-naphthol). If a solution of an acid dye, such as orange G, be dropped slowly into a solution of methyl-green a coarse precipitate at first takes place, which is completely redissolved on the addition of more orange G solution. The solution is prepared so that the ^ Kalle & Co., " Badische Anilin und Sodafabrik." THE MORPHOLOGY OF TIIK liLOOI) 43 quantity of orange G i'k jiisL .sullicieiit to (IIkhoIvo all the preuiintutc. A. solution ])i'(;])ai'('(l in this manner m a typical example of a simple neutnd dye Bolution. The example given may be explained chemically. All the three basic groups of the methyl-green in this mixture are combined with Uw, acid dye, so that a triacid compound of methyl-green results. Simple neutral mixtures which have one component in common may be combined with one another without further difficulty. This is a very important fact for triple staining, which has proved itself of utmost value. It can only be achieved by mixing together two simple neutral mixtures, i.e. twc mixtures which consist of two components each. Chemical dissociation does not take place under these conditions. The important group of staining mixtures containing three or more dyes are obtained in this way. Theoretically, there are two main possibilities for such combinations. 1. Dye mixtures, composed of one acid and two basic dyes. For example : — Orange-amethyst-nietliyl-green. JSTarcein-pyronin-inethyl-green. JSTarceiii-pyronin-methylene-blue. 2. Dye mixtures, composed of two acid dyes and one basic dyes. For example : — Orange G-acid fuchsin-methyl-green, Narcein-acid fuchsin-methyl-green, and also the corresponding combinations of methylene-blue and amethyst-violet. The first of these mixtures will be described in greater detail later. The importance of these neutral dye solutions depends on the fact that the mixtures colour certain structures separately which cannot be demonstrated by any of the constituents alone, and which are therefore called neutrophik. Elements which, as is the case with the nucleiu substances, possess an affinity to the neutral dyes take on the colour of the 44 ANiEMIA basic dyes from such neutral mixtures ; while the acidophile elements are coloured by one of the two acid dyes of the mixture. Those portions of the tissues which, owing to the presence of definite groups, have equal affinity to the acid and basic dyes, attract the combined neutral dyes to themselves, and are therefore stained the colour of the mixture. Among the many dye combinations which microscopists, and especially hsematologists have tried, the mixtures of methylene-blue and eosin have attracted especial attention. The extraordinarily beautiful colour contrast between these two substances which is not likely to occur frequently is to a large extent responsible for this. Not only have the details of a large number of very active and handy methods of staining with methylene-blue and eosin in two stages been published, and some of these have been found to yield good results in practice, but several dozen formulae for the preparation of eosin-methylene- blue mixtures for simultaneous staining have also been described. It is quite impossible to enumerate all these mixtures and methods in this place, and it is presumed that it will be found impossible for any one student to test the adequacy of each of them thoroughly. Mention will therefore only be made of the more important of these, and of those which have proved them- selves in the hands of the authors to be specially efficient. Three different groups of these mixtures can be described. The first of these includes those formulae which have aimed at the preparation of the most favourable proportions in the mixture of the two dyes, without any attention having been given to the reciprocal chemical action when applied to the specimen {e.g. Chenzinsky's solution); the second and third are based on a fact, which was first discovered by Eomanowsky, that when mixed in certain proportions these two dyes in solution form a new chemical substance. This possesses in its nascent condition specific tinctorial characteristics which are not possessed by either of the dyes in their original solutions. In the course of his studies of the malaria parasite, Eomanowsky THE MORPHOLOGY OF THE BEOOD 45 was able to obtain exceptionally good chifjiiialin slainin^r in this way, and less regularly good neutrophile granule staining. The discovery of the " red in inotliylone-ldue," i.e. luetliylene-aznre, was made from this observation, and it also bid to tiie lecogni- tion of a stable " eosinate " of methylene-bliu! wbicli dciiKJiiKirates the neutrophile elements with certainty. On repeating Eomanowsky's method of cliromatiu staining, other observers failed to obtain regular results. Zicmann, however, was able to show how constant results could be obtained after he had tried all the commercial preparations of methylene-blue, and had further employed borax in addition. Nocht was the first to recognise that the active substance in this staining was a con- stituent of commercial methylene-blue, which he termed " red in methylene-blue." It was left to L. Michaelis, however, to explain this behaviour chemically, thereby materially advancing the practical solution of the difificulty. He was able to prove that active solutions of this kind, among which Unna's polychromic methylene-blue must be classified, contain methylene-azure beside unaltered methylene-blue. This substance has been described by Bernthsen, who regarded it as a sulphonic dye ; but Kehrmann was able to show that this was not the case, but that it was a simple dimethyl-thionin. These researches have rendered it easy to prepare the active dye synthetically, and it can now be obtained in a pure condition, — for example, from the " Badische Anilin- und Sodafabrik." The most advanced use of this methylene-azure staining for haematological purposes takes the shape of " Giemsa " staining, which is justly much in vogue at the present time (see below). A number of investigators, including Eosin and Michaelis, have contributed towards the gaining of a constant eosinate of methylene-blue, on which the htematologist can depend. It must, however, be mentioned that Jenner described, as long ago as in 1899, the most perfectly active product. This compound was obtained by an extremely simple method. His stain yields a true panoptic staining; the oxyphilic, basophilic, and neutrophilic elements of the normal blood are characterised sufficiently sharply, 46 ANEMIA and all the most important pathological changes are demonstrated by its means. The differentiation of the neutrophile from the eosinophile granules may not be quite sharp enough for an inexperienced observer, and in this case it might appear that a control with triacid staining would be advantageous. In this way scientific research has given the practitioner a number of methods which, while they can be applied with absolute ease, guarantee the results with precision. These methods have been arrived at as the result of endless ingenuity and diligence, by means of which great difficulties have been overcome. For practical use, apart from the solution of iodine and iodine eosin (which will be described on pp. 52, 53), the following claim attention : — 1. Haematoxylin Solutions with Eosin or Orange G — E. Eosin (cryst.) 0*5 grm. Hsematoxylin 2 Alcohol abs. Aquae dest. Glycerini . aa 100 grm. Acid acetic glac. . 10 „ Alum, in excess. The solution must ripen for some weeks. Films fixed by a short exposure to heat, or in absolute alcohol, stain in from a half to two hours. The haemoglobin and the acidophile granules stain red, while the nuclei take on the colour of the hsematoxylin. The stain must be rinsed off very carefully. 2. For the practical employment of the triacid solution, it is especially necessary that the stains employed should be chemically pure. Heidenhain first pointed this out.^ The advantage of solutions made with such dyes is particularly well exemplified by the following observation. Formerly what was regarded to be basophile granules were ^ At M. Heidenliain's request the " Aktiengesellschaft fiir Anilinfarbstoffe," Berlin, have prepared the thi'ee dyes in a crystalline condition. THE MORPMOT.OCrY OF Tm^". P»TX)OD 47 frequently seen iji white blood corpuscleH, especially in tlio neighbourhood of the nucleus. Even Huch nu experienced observer as Neusser did not regard these granules as artifices. They were therefore described as perinuclear structures, and were looked upon as being true cellular elements. Since IJio iiiLioduf.iiou of pure solutions of the dyes, these so-called basopliile granules are not often seen. Saturated aqueous solutions of the three dyes are first made up and allowed to stand until clear. They are then mixed in the following proportions : — 13-14 c.c. of orange G solution. 6-7 acid fuchsin solution. 15 distilled water. 15 alcohol. 12-5 methyl-green solution. 10 alcohol. 10 glycerine. These ingredients must be measured out in the same measure glass and added in the order given. After the methyl-green has been added the mixture must be well shaken up. The solution is ready for use at once, and will keep for a long time. Staining blood films with triacid need only be preceded by slight fixation. The staining itself is complete within five minutes. The nuclei then appear greenish the red blood corpuscles orange, the acidophile granules copper colour, and the neutro- phile granules violet. Mast cells are seen as peculiarly pale, almost colourless cells, with pale green nuclear substance. This behaviour is spoken of as negative staining. It will thus be seen that triacid staining is, technically spea'king, quite simple. It can be recommended for the purposes of gaining a general conception of the changes in a given specimen, and must be regarded as being indispensable in all cases in which the neutrophile granules have to be studied. 3. Double basic Staining.— A saturated aqueous solution 48 ANiEMIA of methyl-green is mixed with a small quantity of an alcoholic solution of fuchsin. The fixation for this method need only be slight, and the staining itself is complete within a few minutes. The nuclei appear green, the red corpuscles red and the protoplasm of the lymphocytes take on the colour of fuchsin. This method is therefore particularly suitable for films demonstrating the changes in lymphatic leuksemia. 3a. Pappenheim's double basic staining with Pyronin Methyl-green. — The directions, according to Grawitz, are as follows. The two following solutions are prepared : — 1. Acid carbol. liq. 0-25 Aquse dest. . 100 Methyl-green (pure) . 1-0 2. Acid carbol. liq. 0-25 Aquse dest. . 100-0 Pyronin . 1-0 15 parts of No. 1 are mixed with 35 parts of No. 2, shaken up, filtered, and allowed to stain for a few seconds. The fixation must be carried out with heat. The solution can be obtained ready for use from Griibler. 4. Eosin-methylene-blue Mixtures. (a) Chenzinsky's solution. Concent, watery solution of methylene-blue . . 40 c.c. Half per cent, solution of eosin in 70 per cent, alcohol 20 „ Distilled water . . . . . . . 40 ,, The solution is fairly stable, but should nevertheless always be filtered before use. The films need only be fixed by immersion in alcohol for five minutes. The staining takes from six to twenty- four hours, and is carried out in the incubator in air-tight block glass pots. The nuclei and the mast cell granules stain an intense blue, malaria plasmodia stain a delicate sky-blue, the red blood THE MORrnor.OGY OF TflP: BI.OOD 49 corpuscles and the oosinophile granulen tuko on a l)eautifu] rod colour. This solution is therefore especially suitable for the study of nuclear structure and of baso])hile and eosino])hile f^ranulation. It is largely used for ana'niic and lymphatic Icukjcniic blood. (&) Von JlluUem's Successive Staininfj. — This method has been described as follows by Tlirk, who recommends it wariDly : — (a) Pure French eosin, | per cent, in 70 per cent, alcoliol. (/;) Methylene-blue (B. pat.) I per cent, in water. 1. Fixation for three minutes in methyl-alcohol. 2. The films are transferred directly to the eosin solution, in which they stain for from three to five minutes. o. They are then rinsed with distilled water and dried between layers of blotting-paper. 4. They are then placed in a mixture of 20 drops of the methylene-blue solution and 10 drops of the eosin solution for from a half to at most one minute. The proportions for this mixture must be exactly measured, and it must be prepared fresh for each staining. 5. Eapid rinsing with distilled water, and rapid drying between layers of blotting-paper. Mounting in Canada balsam (c) Ten c.c. of a 1 per cent, aqueous solution of eosm, 8 c.c. of methylal, and 10 c.c. of a saturated aqueous solution of methylcnc-Uue (medicinal) are mixed together and used at once. The staining is continued for one or at most two minutes. The staining is only characteristic if the films have been thoroughly fixed by heat. The mast cell granulations are coloured pure blue, the eosinophile granules red, and the neutro- phile granules the same colour as the mixture. (d) Zicmanns solution, which is specially adapted for malaria specimens and for the demonstration of lymph cells. (a) One. part of Hochst's medical methylene-blue in 100 of distilled water and 2 to 4 parts of borax. ih) Hochst's eosin A. G., 0"1 per cent, in watery solution. These solutions are mixed in proportion of 1:4. The film is 4 50 ANAEMIA fixed in alcohol and is stained for five minutes. A metallic skim which forms on the solution should be removed with blotting paper, to prevent it from coming into contact with the film. The film is then rinsed well in water, immersed several times in very dilute acetic acid, and dried. (e) In the next place, there are the solutions which actually contain " eosinate of methylene-blue " ( Jenner, May-Griinwald). Methyl-alcohol is employed as the solvent, so that the fixation and staining take place at the same time. The authors re- commend the hsematologist to obtain either the eosinate of methylene-blue or the solution ready for use directly from Griibler of Leipzig. Burroughs, Wellcome & Co. put up the dye in small so-called " soloids." A half to 1 per cent, solution of eosinate of methylene-blue in methyl-alcohol is prepared, and the films, after having dried in the air, but without any previous fixation, are immersed in the solution for about five minutes. They are then thoroughly rinsed off with distilled water, during which process they are decolorised to a certain extent. They are then dried and mouated in Canada balsam. When stained in this manner, the red blood corpuscles and the eosinophile granules appear bright red, the neutrophile granules a paler red, the nuclei and the mast cell granules blue. The cytoplasm of the malaria plasmodium also appears pale blue. Granulated erythrocytes can be demonstrated well by this method. (/) Giemsa's Staining. — The preparation of methylene-azure solutions is still very complicated, and the results in the hands of inexperienced workers are uncertain. It is therefore wiser to buy Giemsa's stain solution ready for use from Griibler of Leipzig, or Klonne & Mliller of Berlin. The fixation is performed for ten to twenty minutes in absolute alcohol, or from two to five minutes in methyl-alcohol. For staining blood films, 1 drop of Giemsa's stain is added to 1 c.c. of distilled water.^ This is allowed to act for from ten ^ The translator prefers a stronger solution of Giemsa for blood films (2 drops per c.c. ) to act for three to six minutes. For parasites a weaker solution is, however, preferable. THE MORPHOLOGY OF THE 15LOOD 51 to thirty minut(3K. Tfui lilniH uro tlicii liiiscd willi distillrid water und dried. The erythrocytciH appear ])Jile red, Llie iiueh.'i of the inonoiiU(d(;ii.r ond ])olynucl(,'Jir' loucocyteH brij^Iit refl nnd violet respectively, parasites and tixi plasma of the lympho- cytes, blue, neutrophile granules violet-rtid, the acidophils granules a brownish-red, the mast cell granules a mauve colour, and the granulation of the erythrocytes blue or at times red. What is termed "vital staining" requires to Ije dealt with separately. This term is a very unsuitable one. Ehrlich first employed it for the staining of nerve tissue in the living animal, and in this connection it is descriptive, since it actually conveys a correct meaning. But blood leaves off being a living tissue when it leaves the body, and begins to die from the moment it is abstracted from the vessels, even though under favourable conditions it is possible to retain the form of the elements unaltered for a surprisingly long time. It must, however, be admitted that some of the authors who employed the term {e.g. Eosin and Bibergeil) realised that it was not a satisfactory one. It would, however, have been wiser if they had avoided its use altogether and substituted for it such a term as " post-vital " or " prae-mortal " staining. But whatever name is given to this method, the author is of opinion that it has not served any purpose which could not be served in a much more convenient manner by stained dry films. It is necessary to accept with the utmost caution and reserve all that is claimed as new, since all sorts of uncontrollable phenomena may be produced in the process of dyeing when this staining is employed. It must even awaken suspicion, if this method of examination reveals appearances which cannot be demonstrated in dry stained preparations. These remarks, of course, are not intended to apply to the study of the phenomena of movement of the blood, which cannot be achieved in any other manner. The methods of vital staining depend either on the solution of the still fluid blood in the solution of the dyes (Arnold, Pappenheim), or on the principle 52 ANEMIA of applying the blood to cover-glasses on which an alcoholic solution of the dyes has been previously allowed to dry (Nakanischi, Unna, Eosin, and Bibergeil) ; or, lastly, on the principle elaborated by Deetjens, in which the examination is undertaken in a medium on which the blood can be preserved for a considerable time, and in which the dyes have been impregnated (A. Wolff). Two further important methods for which dry blood films are utilised without any previous fixation must be briefly described before this subject is left and the subject of the histology of the blood proper is entered upon. These are : (1) The detection of glycogen in the blood, and (2) the microscopical demonstra- tion of the distribution of alkali in the blood. To these may be added (3) the so-called diabetes reaction. 1. Eecognition of Glycogen in the Blood. This can be carried out in two ways. The original method consisted in placing the blood in a drop of a thick, cleared solution of iodine rubber under the microscope, in accordance with the glycogen test devised some time ago by Ehrlich. The following method, however, is a better one. The blood is placed in a closed vessel containing iodine crystals. Within a few minutes it takes on a dark brown colour. It is then embedded in a saturated solution of Isevulose, which, as is well known, has a very high refraction index. It is necessary, in order to preserve such specimens, to use a cover-glass cement. When either of these methods has been applied, the red corpuscles, having taken on the iodine stain, become prominent, without showing any morphological changes. The white cells are only stained to a slight extent. But all the structures which contain glycogen, be they in the white blood corpuscle, be they in the blood platelets or extracellularly in the debris, appear very markedly characterised by a beautiful mahogany-brown colour. The second modification of the method possesses this advantage, that on account of the strikingly refractive action of THE MORPHOT.OCY OF TTTK lU.OOD 53 the Isevulose syrup the colour can be seen very clearly, while with the lodiKed rubber solution small quantities of glycogen in the cells niiiy be obscured, ])artly by tb(; o])iUjue iiatiii-c of tbo rubber ii,ud piirlly by the colour of LIk! solution itself. The se.eond, sharjjer method may therefore be recommended for more extended use in the examination of diabetes cases and of other diseases.^ The result is slightly diri'erent if the blood smear is exposed to iodine vapour while still moist (Zollikofer). When treated in this manner normal white blood corpuscles are found to be impregnated by a large number of granules stained deep brown, while the erythrocytes are stained much more intensely than by the dry method. 2. The Microscopical Test for the Distribution of Alkali in Blood. This method is based on the test for alkali in glass, which Mylius has worked out. Iodide of eosin is a red compound which is very soluble in water but insoluble in aether, chloroform, and toluol. On the other hand, the free acid of the dye, as precipitated from the salt by acidifying the solution, is very little soluble in water, but is readily soluble in the organic solvents. On shaking it up thoroughly in an aethereal solvent the precipitate forms a yellow solution. If such a solution is applied to the surface of glass, on which alkali has been de- posited as a result of the decomposition of the glass, the deposit takes on a brilliant red coloration, as a result of the formation of the highly coloured salt compound. In applying this method to blood it is of course necessary to remove any deposition of alkaline salt from the vessels or cover- slips employed in the process by means of acid. The freshly prepared dry film is thrown into a glass vessel containing a solution of free acid iodide of eosin in chloroform, or chloroform ^ This method can be warmly recommended also for the detection of glycogen in the secretions, e.g. in gonorrhoea] pns, which always shows a marked glycogen reaction of the cells ; the same reaction is further found iu cells derived from tumours, either free in exudations or obtained directly from the gi-owths themselves. 54 ANAEMIA and toluol. The film soon becomes dark red in this solution. It is then rapidly transferred to another vessel containing pure chloroform, and the chloroform is changed once. The film is then mounted while still wet in Canada balsam. The mor photic elements in the film will be found to be well preserved. The plasma takes on a distinctly red colour, while the red blood corpuscles do not take up any stain. The white corpuscles show a red staining of the protoplasm, in which the nucleus, being unstained, appears as a vacuole (negative nuclear staining). The debris show intense red coloration, as does also any fibrin which may have been formed. These stainings are extremely in- structive, and frequently demonstrate minutiae which are not visible in preparations made by other methods, which aim more at obtaining elegant specimens. The study of these specimens is of the utmost value, because they show up all the artificial ap- pearances and technical mistakes in a most reliable manner, and thus act as a sort of control to technique. The scientific value of the method consists in giving information with regard to the distribution of the alkali in the various elements of the blood. It appears that free alkali which reacts to iodide of eosin is not present in the nuclei ; these structures must therefore possess a neutral or acid reaction. On the other hand, the protoplasm of the leucocytes is always alkaline, and it is found that the protoplasm of the lymphocytes contains the greatest amount of alkali. It is also necessary to call attention to the marked alkalinity of the blood platelets. 3. Bremer's Diabetes Reaction. Bremer described in 1894 a peculiar colour reaction of the blood of diabetics. At first he employed a somewhat com- plicated procedure, but later described the following method. A fairly thick smear of the blood to be tested is made on a cover-slip, and this is fixed at 125° C. and stained at once in a 2 per cent, solution of methylene-blue. Diabetic blood when examined under the microscope is found to be stained a yellowish- THE MOUrTIOLOCiV OK TIIK lU.OOI) .'J green, while normia blood lakoH on lln; colour ol' iho .sl,:iiii employed. Williamson's test may also he mc.td.ionod: 'ZU o.f,. oF hlo..d (best taken by means of the i.ipotU-, of (Jower's ha^mojrlohiiio- meter), 1 c.c. of a 1 in GOUO aqueous solution of methylene-ljlue, and 40 c.c. of a 6 per cent, solution of caustic potash are mixed in a test tube and boiled in the water bath for three or four minutes. Diabetic blood is either decolorised or tinted faintly yellow in this test, while normal blood solutions retain their colour. The test, as will be seen, is nothing more or less than a simple reduction test. These facts have been confirmed generally by a number of observers (J. Loewy, le Goff, Hartwig, Dannner, and others), but at the same time it was found that the reaction is not specific for diabetes, but occurs at times in other conditions. There are considerable differences of opinion with regard to the interpreta- tion of the phenomenon, and at present it is not possible to consider the matter settled. There is no doubt that the re- action in the blood of diabetics is produced by the presence of grape sugar. Loewy has proved that in Bremer's test the blood corpuscles cause the decolorisation, while in Williamson's test it is the plasma. It is interesting to note that both Williamson and Loewy have still obtained the reaction in diabetics, after the glycosuria has disappeared. According to Ehrlich, these phenomena may be explained on the assumption that a chemical combination between the glucose and some other constituent of the blood, probably the haemo- globin, diminishes the power of taking up the methylene-blue. This view is supported by the fact that normal blood which has been treated with grape sugar gives a Bremer's reaction. There is no doubt that this reaction occurs in other diseases in which there is no increase of the sugar of the blood. 56 AA^iEMIA 5.=— NORMAL AND PATHOLOGICAL HISTOLOGY OF THE BLOOD. The Red Blood Corpuscles. During the past ten years new views have been put forward with regard to the structure of the red blood corpuscles, which to a certain extent stand at variance with the old generally- accepted ideas. Special mention must be made in this con- nection of the investigations of Weidenreich on the shape of the erythrocytes. As a result of extensive comparative anatomical studies he comes to the conclusion that the red blood corpuscles are not discs provided with symmetrical delled concavities on both sides, which when viewed in transverse section would show the so-called dumb-bell shape. He believes that they have the shape of a bowl or bell glass, or, as he very expressively explains it, are like a rubber ball from which some of the air has escaped and in which a dent has been made. These views with regard to the shape of the red cells are of purely academic interest, and do not convey any influence on physiology or pathology. The various hypotheses with regard to the structure may be of importance. The older views of oikoid and zooid (Briicke), of discoplasm (Ehrlich), of a stroma in the meshes of which the haemoglobin is distributed (Eollet, Hayem), were to a great extent based on the demonstration of a framework in the protoplasm of the cells by Arnold and his pupils, E. Bloch, Eosin, Bibergeil, and others. Weidenreich and Grawitz, however, contend that the protoplasm of the erythro- cytes has a perfectly homogeneous structure. The latter bases his opinion on the experiments which he carried out with Grriineberg with ultra-violet rays. The observers named, as well as Hamburger and others, recognise a membrane which forms an external limit of the blood discs. Weidenreich believes that this membrane possesses basophile characteristics, and that it plays a part in the development of punctation and of blood platelets. The existence of a membrane has been accepted by Koppe, THE MORPIIOLOCiY OF ^ITTE IJLOOD 57 and by Albrecht and Hedingor, hh the j'ChuII of fuiUier ui(;lliud8 of examination. The membrane belongs to the lipoid subfitauces. It contains lecithin and cholenterine, and can be diHHolved by definite phyeical and chemical mean.s. Tliriso views, as will be shown subsequently, are of great im])ortance for tlir; dociiin*' of the pathogenesis of various forms of an.'cmia. The nucleoid theory opposes the doctrine of tlu; liomogeneity of the red blood corpuscles. 'J'his theory assumes the remains of nuclear substance in the interior of the blood discs, which it claims to demonstrate by special forms of staining. It has been supported by Pappenheini, Arnold, Hirschfeld, Maximow, and others, and has received remarkable confirmation by Lowit's observations with fresh blood specimens (quoted by Pappenheim), and by the dark-field illumination studies of Pappenheim. The latter describes the nucleoid substance as " molecularly motile," and as of " tenacious fluid consistence." The views with regard to the red blood corpuscles which are of the greatest importance for clinical medicine, however, are not based on these special anatomical and physiological examinations, but have been formed as the result of the clinical methods described above, and especially those dealing with dry films. A technically well made dry film shows the red blood corpuscles in their natural size and shape, and the cupping or delling can be distinctly seen. They present the form of isolated, round, homogeneous structures of a diameter of about 7*5 to 8*5 jO; (maximum 9-0 ^O/, minimum 6"5 ///). They show^ the most intense staining in the peripheral zone, and the least in the dell or centre. The stroma does not take up any of the stains mentioned above. The dyes only affect the haemoglobin, so that an experienced observer can gain some idea of the haemoglobin content of the individual cell by the intensity of the staining, which is much more reliable than the natural colour of the haemoglobin in fresh unstained preparations. Blood corpuscles which are poor in hasmoglobin are readily recognised by their pale staining, and especially by the lightness of the central zone. When this is very marked they appear as structures which 58 " ANEMIA Litteii has appropriately called " pessary " forms, on account of the fact that only the periphery stains at all. The slight power of staining cannot be explained, as Grawitz believes, by a smaller affinity of the haemoglobin to the dye. Qualitative alterations of the hajmoglobin, which would modify its behaviour toward the dye, do not exist, even in ansemic blood. The fact that anaemic blood stains more faintly than normal blood depends entirely on its smaller haemoglobin content. A diminution of the haemoglobin content may be ascertained in this manner in all the anaemic conditions, and especially in posb- haemorrhagic, secondary, and chlorotic anaemias. In contrast to this, however, as Laache first pointed out, there is an increase in the amount of haemoglobin in a large number of erythrocytes in pernicious anaemia. In order to form a correct conception of pathological changes in blood, it is necessary to bear in mind that the individual red blood corpuscles are by no means equal even in normal blood. Under physiological conditions some of the cells are constantly being used up and replaced by new ones. Each drop of blood therefore contains erythrocytes of varying age. It will be readily understood that damaging influences, provided that they are not too great, need not affect all the red blood corpuscles equally. The least resistant of the elements, i.e. the oldest cells, will be destroyed by these damaging influences, while the more robust only react in a more purposeful manner toward the same agencies. The anaemic composition of blood as such undoubtedly constitutes a moderately intense stimulus of this kind. The action may be studied with advantage in cases of acute post- haemorrhagic anaemia. Certain characteristic changes are observed in the blood discs in all anaemic conditions. A. Polychromatophilia. — This term is employed for a condition, which was first described by Ehrlich, in which the red blood corpuscles show a modification in staining, consisting in taking on a mixed colour, instead of a pure haemoglobin tint, THE MORPHC^T.OGY OF THE 1>,L()01) o9 as in iiormiil blood. For example, Uk; vcA l)louOOI) r>\ certain that ])olychroiriatophilia riia,y jiJho be a HJgn of young forms. Thore is absolutely no reason why both views should not be accepted. There are other examples in histology, and especially in hiEmatology, of young and dying elements having similar characteristics ; mononuclear neutrophile cells must be regarded as the precursors of the polynuclear cells, and also without any doubt as a degeneration form of the same cells. ^ The genesis of this phenomenon is of course different for the instance when it represents a degeneration form to that when it represents an early developmental stage. In the former case it is usually dependent on acquired pathological charac- teristics of the circulating blood, produced by the influence of the surrounding blood plasma (see above). The staining peculi- arities may also be produced, according to Engel, by the action of bacteria on the bone marrow. With regard to the demonstration of polychromatophilia, suffice it to mention that Tiirk and Naegeli have found that methylene-blue alone, or LofHer's solution of methylene-blue, are to be recommended as the most suitable stains. Very beautiful results can be obtained with Chenzinsky's solution. JB. The Punctated Erythrocytes. — In various conditions, punctate and at times coarse deposits are met with in the protoplasm of the red blood corpuscles. These deposits cannot be seen in fresh specimens, but may be demonstrated in dry films by means of almost any of the nuclear stains. At times these deposits are so minute and so closely placed that the whole cell appears to be stained with the nuclear dye. For example, when stained with methylene-blue the cell offers an appearance of a homogeneous blue structure. In other cases the deposits are coarse, and look like little heaps of stain. In one and the same cell the deposit may be in part extremely fine and in part very coarse. Again, it may be distributed equally over the whole cell, or it may be limited to a part of it. Such deposits were first found by Ehrlich in 1878, and mentioned in a dissertation, compiled under the supervision of 62 ANEMIA von Noorden. S. Askanazy described these elements minutely in a case" of ansemia in 1893, and in 1894 Schaumann mentioned them in his monograph. A communication made by the author, in which he reported that he had found these elements in more than twenty cases of pernicious ansemia and in leukaemia, appears to have awakened general interest in these bodies. The author in his communication suggested the term of punctate erythrocytes for this condition of the red cells, since this term did not presuppose any special significance. It is essential to avoid the employment of any term which could suggest the slightest similarity between the punctate deposits and the granules of the leucocytes. Granules are structures which possess definite functions of considerable physiological and biological importance, while the punctiform deposits of the erythrocytes cannot be said to serve such a purpose. For this reason it would seem to be advisable to avoid using such designations as " basophile granulated erythrocytes," which might be easily confused with the description of mast cells by inexperienced observers. An extraordinarily large number of communications followed the publication by the author, dealing with further characteristics of these bodies, while others discussed their whole significance (Klein, Zenoni, Lenoble, Litten, Borchardt, E. Bloch, Engel, Bourret, Sabrazes, Strauss and Eohnstein, A. Plehn, Grawitz and his pupils, P. Schmidt, Naegeli and his pupils, S. Askanazy and others). These communications revealed the fact that punctate erythrocytes may be found in all forms of anaemia. It appears to be undoubted that this form of cell is especially marked in those anaemic conditions which either depend on toxic causes or in which these factors play a large part in the causation. Accordingly they are found almost invariably in all cases of progressive pernicious anaemia, carcinoma, etc. The extremely frequent occurrence in lead poisoning has awakened special interest, after Borchardt first called attention to the fact. It has, however, been proved that the phenomenon is not necessarily associated with toxic processes, since it has been seen repeatedly THE MORPirOLOGY OF THE IUX)()I) 03 in cases of ])m-c ]M).si-luemorrhagic auajrnia, ovom if Uu'h in not the rule. This i.s Tiot only true for hseniorrhageH which Uiko place into one of the body cavities, as Grawitz believed, under which conditions he concludcul tliat a toxic action was produced by the abKor[)ti()n of the l)li)()(l wliicb liiul csciajjcd IVoiii the vessels, but applies equally to those forms of lia^rnftrrhage in which the bleeding takes place externally, and which are un- doubtedly cases of pure acute post-lucmorrhagic aUcX-niia (IJloch, Schmidt). Mention must be made of the following facts. The punctate erythrocytes have been found as the only ascertainable blood change in the stage of complete remission in prot^ressive pernicious ana?mia (Lazarus) ; their presence may form the first sign of lead poisoning, before any other symptoms have made their appearance ; and lastly, according to Strauss and liohnstcin, they have been observed in a single instance, in a perfectly healthy young medical man, in which case the examination was carried out with extreme care. Two questions have been thrown up by all the authors who have busied themselves with the subject, since these elements have been known to exist. Firstly, what is the general clinical significance of these structures ; and, secondly, what is their histological explanation. The answers to these tw^o questions naturally stand in close association to one another. No one will dispute that the punctate elements are at all events in part derived from nuclear structures. This becomes quite evident when series of cells are examined, such as those depicted in Part II. of Ancemia, Plate 2, Fig. 5. In these cells an unbroken series of fragments from the largest portions of the nucleus, down to the very finest punctiform dust, is plainly visible. But this by no means proves that all such deposits are derived from nuclei. According to Grawitz, a certain difference in the staining speaks against the view that the deposits are derivatives of the nuclei. This objection, however, is just as little convincing as it would be if it were assumed that basophilic staining is direct evidence of a nucleogenous nature ; fragments of nuclei, as Meyer and Speroui have appropriately pointed out, 64 ANJEMIA need not behave chemically in the same way as intact nuclei. A further objection consists in the statement that this punctate deposit has never been discovered in bone marrow. Pappenheim, however, has shown that it is quite easy to demonstrate it in the medulla if the method of staining be but slightly modified. Schur and Loewy and Bloch have found it in the bone medulla. Naegeli was able to demonstrate the presence of punctate deposits in bone marrow, even in cases in which they were absent in the blood. In opposition to the assumption of the nuclear origin of the punctate deposits, Grawitz alleged that he had found innumerable punctate erythrocytes in the blood of many patients, without having come across a single erythroblast. Quite apart from the fact that this could not be accepted as evidence in favour of Grawitz's views, Meyer and Speroni have found, more especially in lead poisoning, that when the examination is undertaken regularly, erythroblasts are present in every case which shows punctate erythrocytes. The third argument against the view is that punctate deposits are found in erythroblasts with absolutely intact nuclei, and in cells which show mitosis. But it must be pointed out that it is not un- reasonable to suppose that the punctate deposits could have been derived from the disintegration of a second nucleus, or that they may have been formed during the process of division (Schmidt). Meyer and Speroni have recently called attention to a highly important fact. In animal experiment, punctiform deposits can only be produced in those warm-blooded animals whose red blood corpuscles normally are not nucleated. If the phenomenon depended on some damage to the protoplasm, it would be difficult to understand why it should be absent in those warm-blooded animals whose red blood corpuscles contain nuclei. The punctiform deposits only occur when denucleisation takes place as a result of dissociation of nuclei under physiological conditions. It may be deduced from the foregoing that some of the basophile punctiform deposits undoubtedly owe their origin to the nuclei, while it cannot be proved that they stand in any THE MOKPITOLOCiY OF TJIK ]MA)()\) 05 causal relation to ilio protoplasm of the coll. Under UicHe circumstances it seems unnecessary to attempt to explain one morphological phenomenon in two ways. Since the close relationship bctwe(;n the punctifoirn deposits and the nuclei suggests that this ])he)n)menon is more of iiie nature of a regenerative than a degenerative process, it is interest- ing to note that further facts have been In'ought to light which support this view. The most striking evidence has been obtained by experiment. Sabraz^s, and Naegeli and Lutoslawski, whr^se work fully confirms the work of the first named, have shown that punctate erythrocytes can be readily produced in chronic lead poisoning, but that they disappear as soon as the dose of poison has exceeded a certain point. This result can only be explained on the assumption that the punctate ery- throcytes indicate that the bone medulla reacts actively, while when large doses are employed the medulla is paralysed. Analogous conditions are known to exist, e.g. in experimental arsenical poisoning (Bettmann). Schmidt has obtained results which tally well with this theory in his experiments with various other poisons. As Naegeli has tritely pointed out, the appearance of this phenomenon in erythrocytes showing mitosis speaks in favour of it being a regenerative process. It would be absolutely inconsistent to suppose that a degenerative process could take place in the same cell at a time when the most energetic regenerative process is going on. Finally, it may be stated that the fact that basophilic punctiform deposits have been found in embryonal blood, that is, under conditions when all degenerative processes may be regarded as excluded, clinches the evidence in favour of this theory. This does not only occur in the embryonal blood of mice in which it was first observed by Engel, but, according to recent observation of Naegeli, it also takes place in the embryonal blood of all animals, including man. In his last contribution S. Askanazy has admitted ihat these bodies are a sisjn of regeneration. But in contradiction to his earlier views he now considers that the punctiform deposits are 5 G6 ANEMIA a variation of polychromasia, which he attributed to a peculiar abnoriiiahty of the plasma. His arguments are based on the alleged condition, which has been disproved in the preceding paragraph, that the punctate erythrocytes have never been found in bone marrow, and also on the assumption that this phenomenon appears side by side with polychromasia. This is certainly not the case as far as the individual erythrocytes are concerned. A large number of orthochromatic erythrocytes containing punctiform deposits are met with. And even if this assumption holds good for blood as a whole, it must be pointed out that this is not surprising, since the phenomenon of polychromasia is observed in nearly every case of anaemia. In view of all these facts and theoretical considerations it appears that, although this may be to some extent at variance with the views expressed formerly by the author, the following conclusions are justified. The punctiform deposits are the de- rivatives of nuclear substance, and the process must be regarded biologically as a transformation of the nuclear substance, modified by pathological conditions, and perhaps as an altered form of denucleisation. The whole phenomenon therefore bears in its clinical significance the characters of a pathological regeneration. Schleip found in a case of progressive pernicious anaemia that the blood, when stained by Leishman's and Giemsa's methods, contained in the normal or polychromatophilic erythrocytes, small rings or much twisted loops composed of extremely delicate threads. In the larger erythrocytes there were two or three rings, and at times rings were found lying free in the blood plasma, in which case remnants of a red blood corpuscle could be discerned adhering to them. Schleip was able to demon- strate these changes in a few further cases of progressive pernicious ansemia, in severe secondary anaemia, in chronic lead poisoning, in one case of acute leukaemia, and in one case of pseudo-leukaemia. He regarded these structures as remains of the nuclei, and possibly of nuclear membrane, and attributed the condition to an abnormally increased new formation. Schleip THE MORPHOIXJGY OF THE BI.OOD G7 has stated that Ca})ot dcsoribcd the Haiiie condition uh early as 1903, and Gabriel conHrnied Schleip's observation in a single ease of progressive pernicious aniemia. Naegeli (private (•oiiiiniiDi- eation) has found very numerous ring bodies in a number of cases of infantile pseudo-leukajmic anemia, and also in acute lymphatic and acute myeloid leukaemia, as well as in progressive pernicious anaemia (see Plate III.). C. — A third change which is found in the red blood corpuscles in ansemia is known as poikilocytosis (Quincke). This term implies a change in the microscopical appearance of the blood, which is characterised by the presence of large, small, and minute red elements, in addition to a greater or smaller number of normal sized red blood corpuscles, " anisocytosis " (Strauss). As Laache first pointed out, and as has subsequently been confirmed by other hamatologists, extremely large cells, which are rich in hsemoglobin, are found in Biermer's anaemia. In all other severe and moderately severe forms of anaemia the volume and hccmo- globin content of the red blood corpuscles are, as a rule, diminished. This apparent contradiction could not be explained by Laache, who first called attention to it, but has been satisfactorily accounted for by the results of Ehrlich's observations on the nucleated precursors of the megaloeytes and normocytes (see below). The appearance of anaemic blood becomes more complicated by the fact that the smaller cells do not retain their normal shape, but take on well-known irregular forms: pear shape, balloon shape, boat shape, and dumb-bell shape. At the same time, the central cupping can still be recognised even in the smallest forms in well-prepared dry films. The so-called microcytes form an exception to this rule. These are small globular bodies, which used in the early days of microscopical haematology to be regarded as being of prognostic importance in severe forms of autemia. It has, however, been shown that these bodies are merely contraction forms of poikilocy tes ; or, in other words, the microcytes stand in the same relation to the poikilocytes as the crenated forms stand to the normal erythrocytes. Accordingly microcytes are 68 ANEMIA but rarely seen in dry films, whereas they may be seen in fresh specimens after prolonged examination. It is further of importance to know that in fresh blood the poikilocytes show certain movements ; this has given rise to wrong conceptions in many instances. They were regarded as the causal organisms of malaria in the early days of hsematology ; while the larger forms were looked on, at a later date, as amoebae and similar organisms by Klebs and Perles. Hayem from the first described these forms as " pseudo-parasites," and warned the observer against assuming that they possessed a parasitic char- acter. Formerly the origin of poikilocytes was the subject of consider- able discussion, but it is now generally accepted that the explana- tion which Ehrlich has given is the correct one. The fact that poikilocytosis can be produced in any specimen of blood by careful warming has led to the deduction that these forms are products of a fragmentation of the red blood corpuscles (" schistocytes," Ehrlich). This view is further supported by the fact that even the smallest fragments in dry films show a definite delling. They contain the specific protoplasm of the blood disc, the discoplasma, which " possesses the tendency of assuming the delled form in the stage of quiescence." No other qualitative alterations of the protoplasm of the poikilocytes can be demonstrated even by staining. They may therefore be regarded as possessing full functions, and their presence may be ascribed to a purposeful reaction to counteract the diminution in the number of blood corpuscles. The respira- tory surface is considerably increased by the disintegration of a large blood corpuscle into a number of homologous smaller cells. D. — A fourth change in the morphological condition which is seen in blood in the severer degrees of ansemia is the presence of nucleated red blood corpuscles. While the question of the origin of the elements of the blood cannot be followed up in this place in any degree of minuteness, it may be wise to describe in a few words the present position of the doctrine of the nucleated red blood corpuscles. THE MORPITOT.OCxY OF TTTE BLOOD 09 Nucleated cxjUk have been generally accepted as tlu; youiij,' forms of the normal red blood corpnHcle« ever since Neumann and Bizznzero publisluul their Htandard works (October anrl November 186(S). Jlayem's Uicory, on tlie other hand, according to which the blood platelets arc tlic origin oi" tlu; (nythrocytes, may be regarded as having been disputed by evoryoii.i save this author and his pupils. Ehrlich called attention to the clinical significance of the nucleated red blood corpuscles in 1880, by pointing out that cells of normal size — normoblasts — occurred in the so-called secondary antemias and in leukaemia, and very large elements — megaloblasts, gigantoblasts — occurred in Biermer's ansemia. Ehrlich at the same time emphasised the fact that the megalo- blasts play an important part in the embryonal formation of blood. In 1883, Hayem sought to classify the nucleated red corpuscles into — (1) The " globules nuclees geantes," which were only to be found in the embryonal condition ; and (2) the " globules nuclees de taille moyenne," which are j)resent in the late stages of em- bryonal life and always in adults. W. H. Howell (1890) recog- nised two kinds of erythrocytes in cat embryos : (1) A very large blood cell, similar to the blood cells of reptiles and amphibia (" ancestor corpuscles ") ; and (2) a blood cell of the usual size of mammalian blood corpuscles. Three forms of nucleated red blood corpuscles may be dis- tinguished by the following characteristics : — 1. Normoblasts. — These are red blood corpuscles of the size of the ordinary non-nucleated discs ; the protoplasm shows, as a rule, pure haemoglobin coloration ; they have one nucleus as a rule and at times there are from two to four. The nucleus is pycnotic, has well-defined edges, and apparently no differentiated structure. It is usually concentrically situated, and occupies the greater part of the cell. It is characterised by the capability of taking on a more intense degree of staining with the nuclear dyes than do the nuclei of the leucocytes, or indeed of all other cells. This property is so characteristic that, even when free nuclei are met with around which no or exceedingly little haemoglobin can be 70 ANiEMIA distinguished, as is the case at times in anaemias, and especially in leukaemia, they can be readily recognised as normoblast nuclei. 2. Megaloblasts. — These cells are from two to four times the size of normal red blood corpuscles. Their haemoglobin occupies by far the greater part of the cell, and frequently shows more or less marked degrees of anaemic degeneration. The nucleus is larger than that of the normoblasts, but does not occupy so large a portion of the cell as the latter does. It is frequently ill defined, and has a rounded shape. The nuclei of the megalo- blasts may be distinguished from those of the normoblasts by their finely differentiated structure and by their small capability of taking up nuclear stains, which may be so limited that an inexperienced observer may have difficulty in recognising that the cells have a nucleus at all. At times cells corresponding to the form described above, but of a considerably larger size, are met with. These cells are termed gigantoblasts, but are regarded as being of the same type as megaloblasts. 3. Microblasts. — These forms occur at times, as, for example, in traumatic anaemias, but must be regarded as extremely rare cells. They have not attracted the especial attention of the haematologist up to the present. Practically every haematologist of repute has followed Ehrlich's propositions in the morphological classification of the nucleated red blood corpuscles. Marked differences of opinion have only been expressed with regard to the significance of the two chief forms. There is no doubt that this difference of opinion depends in part on the fact that some of these investigators, in using the classification introduced by Ehrlich, have not adhered strictly to his definitions. It is not unreasonable to expect that when a name has been suggested for a definite matter, this name should not be employed to signify some other phenomenon or structure. This claim has not been respected in modern haematology. When an investigator considers that the suggested term is unsuitable in any respect, he can propose a new, better term, but he should not THE MOlMMIOr.OCiY OF IIIK liLOOI) 71 create confusion by ji, rnisc. us(; of Uk; oi'igiiial one. iMariy of the disagr(;onicntB with ro<,rar(l to J^^hrlicli's doctrine conceniing the normoblasts and nicgaloblasts would not have occurred if all the authors had respected this regulation. In order to gain a perfectly clear conception of the physiological and pathological import of the crythroblasts, it is absolutely necessary to consider, in the first place, only typical examples of both forms. There are, as would be expected, a large number of cells which cannot be classified either as normoblasts or as megaloblasts, on account of the fact that some of the characteristics which Ehrlich des- cribed for these cells are wanting. It would, however, be purpose- less and would not contribute towards success if atypical inter- mediate forms were utilised for the purpose of gaining a clear insight into the significance of the definite and typical forms which can be accepted as standards. Turning first to the physiological occurrence of normoblasts and megaloblasts, everyone is agreed that only the latter variety is met with in the earlier embryonal stages. This variety is gradually replaced by the former, so that the blood of the fully developed foetus does not contain any megaloblasts, although the bone marrow still does so. The same remark applies at times to the first two years of life. No one has ever claimed to have found megaloblasts in the blood of healthy adults. In spite of very extensive observations, neither Ehrlich, Bloch, nor the author have ever found megaloblasts in the bone marrow of adults; while Dominici, Naegeli, Grawitz, Engel, and others claim to have seen megaloblasts as rare, and even very rare, constituents of normal bone marrow. These exceptional finds, however, suffice to impel Grawitz to regard the megaloblasts as "a type of normal blood formation." He seeks to establish this view by setting up a hypothesis of a development of megaloblasts as a result of an increased water content of the blood. While this assumption appears to be untenable in view of the function of the megaloblasts in the embryonal blood formation, and of the high hemoglobin content of the cells, some recent experiments of Georgopulos have deprived it of 72 ANiEMIA all foundation. This observer showed that in hydremic con- ditions ■ absolutely no swelling of the erythrocytes takes place, and he was even able to demonstrate that on the appearance of disturbance of compensation of the heart in persons suffering from renal affections, and from the consequent increase in the concentration of the blood, the diameter of the erythrocytes is increased on the average. All other hsematologists are agreed that the normoblasts of the bone marrow alone are the physiological precursors of normal red blood corpuscles. It is at present impossible to prophesy what explanation will be found for the contradictory observations of Ehrlich on the one hand, who failed to find megaloblasts in the marrow, and Dominici and others who found them on a few occasions. It is necessary to consider the fate of the nuclei of the erythroblasts separately, in spite of the fact that this landmark is no longer regarded as important as it formerly was for the differentiation of the two forms of cells. For a long time two views with regard to the transformation of the nucleated erythroblasts into the non-nucleated erythrocytes were opposed to one another. Eindfleisch, who was the chief exponent of the one view, taught that the nucleus of the erythroblast issued from the cell, leaving it in the form of an erythrocyte, and that the nucleus itself took up, by means of a trace of proto- plasm, which still adhered to it, fresh substances out of the plasma, imbibed haemoglobin, and thus shaped itself into an erythroblast again. The other doctrine, which was directly irreconcilable to the former, assumed that the erythroblasts were transformed into non-nucleated discs by the disintegration of the nucleus within itself (" karyorrhexis, karyolysis "). KoUiker and E. Neumann may be particularly mentioned as having championed this view, and as having regarded it as the only way in which the erythrocytes are formed. Eindfleisch arrived at his theory by direct observation of the phenomenon, which he described. He saw this take place in the blood of guinea-pig embryos and in teased bone marrow THE MORPriOT.OCiY OF TTTE lU.OOD 73 preparations, both of whicli lia,(l l.(!(^n lic.;itr'. Enqbl, C. S. — " Ueber enibryonale und paUiologisclie rote Blutkorperchon," Berlin Medical Society TravMictionn, 1899, vol. i. " Ueber einern Fall von pernizioser Anaemie," Zeituchr. f. klin. MedAzin^ vol. xl. ; Leitfaden zur Idin. Untersuchunfj des Bluten, 3rd Edition, Berlin, 1908. Eykmann. — " Blutuntersucliungen in den Tropen," Virchoiv's Archiv, vol. cxxvi. i>. 113. Fano (quoted by Limbeck). FoA, C. — " I mutamenti del sangue sull' alta montagna," Abstract in F(jlia Haem., 1904, p. 344. Friedenthal.^ — Arbeit, aus dem Gehiete der ex2Jerimentellen Patholotjie, Jena, 1908. Gabriel. — " Ueber Ringkorper im Blut Anaemischer," Deutsches Arch. f. hlin. Medizin, 1908, vol. xcii. Gabritschewsky. — " Kliniscli-liaematologische Notizen," Arch. f. expcri- mentelle Pathologie und Pharmacie, 1891, vol. xxviii. Gartner, G. — " Ueber eine Verbesserung des Haematokrit,"' Berl. hlin. Wochenschr., 1892, No. 36. Georgopulos. — " Ueber den Einfluss des "Wassergebaltes des Blutes auf die Dimensionen der roten Blutkorperchen," Zeitschr. f. klin. Medizin, 1906, vol. Iviii. Giemsa. — " Farbungsmethode fiir Malariaparasiten," Zentrulhl. f. BakterioL, 1902, vol. xxxi. Glogner. — " Ueber das spezifisclie Gewicht des Blutes des in den Troj^en lebenden Europiiers,'' Virchoid's Archiv, vol. cxxvi. p. 109. Le Gopp. — " These de Paris," 1897 (quoted by Bezangon and Labbe). GoTTSTEiN. — " Ueber Blutkorperchen und Luftdruck," Berl. klin. TFochenschr., 1898, No. 20. Grawitz, E. — " Ueber die Einwirkung des Hohenklimas auf die Zusam- mensetzung des Blutes," Berl. klin. TFochenschr., 1895, Nos. 33 and 34. Klinische Pathologie des Blutes, 3rd Edition, Leipzig, 1906. " Kliniscli-experimentelle Blutuntersuchungeu," Zeitschr. f. klin. Medizin, 1891, vols. xxi. and xxii. " Ueber kornige Degeneration der roten Blutzellen," Deutsche med. TFochenschr., 1899, No. 36. Grawitz, E., and GrOneberg. — " Die Zellen des menschlichen Blutes im ultravioletten Lichte," Leipzig, 1906. Haldane and Lorrain-Smith. — "The Mass and Oxygen Cajjacity of the Blood in Man," Journ. of Physiol., 1900, vol. xxv. Hammerschlag. — " Ueber das Verhalten des sj)ezifischen Ge-wichtes des Blutes in Krankheiten," Zentralbl. f. klin. Medizin, 1891, No. 44. "Ueber Hydraemie," Zeitschr. f. klin. Medizin, 1892, No. 21. "Ueber Blutbefund bei Chlorose," TT-ien. med. Presse, 1894, No. 27. Hartwig. — " Ueber die Farbenreaktion des Blutes bei Diabetes mellitus," Deutsclies Arch.f. klin. Medizin, 1899, vol. Ixii. 82 AN.EMIA Hayem. — Du sang, Paris, 1889. " Dii caillot non retractile. Supression de la formation du serum sanguin dans quelques etats pathologiques," Acad, des sciences, 1896, November 23 (Sem. me'dic). " Des globules rouges a noj^au dans le sang de I'adulte," Arch, de Phys. normale et Pathologie, 3rd Series, 1883, vol. i. Heidenhain, M. — " Neue Untersuchungen liber die Zentralkorper," u.s.w., Archiv f. miJcrosJc. Anatomie, 1894, vol. xliii. p. 515. Helly. — Die hcoematopoetischen Organe, Vienna, 1906. Herz, Max. — " Blutkrankbeiten," Virchow's Archiv, vol. cxxxiii. Hoppe-Seyler. — " Verbesserte Methode der kolorimetrischen Bestimmungen des BlutfarbstofFgehaltes im Blute und in anderen Fliissigkeiten," Zeitschr. f. phys. Ghemie, vol. xvi. Howell. — " The Life History of the Formed Elements of the Blood," etc. (quoted by H. F, Miiller). Israel and Pappenheim. — " Ueber die Entkernung der Saugetiererythro- blasten," Virchow's Archiv, vol. cxliii. VON Jaksch. — " Ueber die Zusammensetzung des Blutes gesunder und kranker Menschen," Zeitschr. f. Min. Medizin, 1893, vol. xxiii. Jakcso and Rosenberger. — " Blutuntersuchungen bei Malaria," Deutsches Arch. f. Min. Medizin, vol. Ivii. p. 449. Jaquet and Suter. — " Ueber die Veranderungen des Blutes im Hoch- gebirge," Korrespondenzbl. f. Schweizer Aerzte, 1898. VON Jaruntowsky and E. Schroder. — " Ueber Blutveranderungen im Gebirge," Milnchen. med. Wochenschr., 1894, No. 48. Jenner. — "A New Preparation for Rapidly Fixing and Staining Blood," iancee,.1899, i. p. 370. JiJNGER. — " Ueber kernhaltige rote Blutkorperchen im stromenden mensch- lichen Blute," Deutsches Arch.f. Min. Medizin, vol. Ixvii. Klebs. — Vide XI. Medical Congress. Discussion. Klein. — " Die Regenerationfahigkeit des Organismus bei den verschiedenen Varietaten der Anaemic," Wien. med. Presse, 1896, No. 28. KoLLiKER. — " Entkernung der Erythrozyten," Zeitschr. f. rationelle Medizin (quoted by E. Neumann). KoEPPE. — " Ueber Blutuntersuchungen im Gebirge," Medical Congress Transactions, 1893, vol. xii. " Ueber Blutuntersuchungen in Reibolds- griin," Munchen. med. Wochenschr., 1895. " Ueber den Quellungsgrad der toten Blutseheiben durch aquimolekulare Salzlosungen und iiber den osmotischen Druck des Blutplasmas," Arch. f. Anatom. u. Physiol., Physiol. Part, 1895, p. 154. KtJNDiG. — " Ueber die Veranderungen des Blutes im Hochgebirge bei Gesunden und Lungenkranken," Korrespondenzbl. f. Schweiz. Aerzte, 1897, Nos. 1 and 2. Laache. — Die Anaemic, Christiania, 1883. Laker.- — " Ueber eine neue klinische Blutuntersuchungsmethode. (Spezi- fische Resistenz der roten Blutkorperchen.) " Wien. med. Presse, 1890, No. 35. Landois, L.— Lehrbuch der Physiologic des Menschen, Vienna and Leipzig, 1887. Lazarus, A. — "Blutbefund bei pernizioser Anaemic," " Transactions of the THE MORPITOI.OGY OF THE Br.OOD 83 Berlin Medical Society," Deutsche med Wochenschr., No. 23, 1896. " Klinik der Anaemien," Ncdhnayel's Handbuch, vol. viii. 2, Vienna, 1900. LENOBi;ifl. — Garadhrcs smii/iolw/iques du caillot et du s&um, PariH (Steirilieil) 1898. .LlKUMi5EiU)KU. — "Eoiiraj^ ziii' IJi.'liaii'lliiii^ drr Aiik>](<.sUjiriia.si.saiia(:iiiii; iumI der Tropenanaeniien," y^f!'/-/. /cdi'/i, IFochevsrhr., lOOr*, No. 14. V. LiMiJKCK. — Ch-undriss cincr kliniachcn I'athobxjie dea JHiUch, 2iid Edition, ,Jena, 18!iG. " Teljerdie durcli (jrallc.ii.staiiurig bewirktfji Voriin- derungen des Blntos," Zcnlralbl.f. innere Medizin, 189G, No. 33. LiTTEN.— " Ueber einige Veriinderungeu roter Blutkorperchen," Berl. hlin. Wochenschr., 1877, No. 1. " Ueber basophile Kornungen in roten Blutkorperchen," Deutsche med. Wochenschr., 1899, No. 44. LoKWY, A.—" Ueber Veriinderungen des Blutes durch thermische Einfliisse," Berl. Iditi. Wochenschr., 1890, No. 4. "Die Wirkung des Hohen- und Seeklinias auf den Menschen," Berl. Med. Society (1903) Transactions, vol. xxiii. LOEWY, A., J. LoEWY, L. ZuNTZ.— " Ueber den Einfiuss der verdiinnten Luft und des Hohenklimas auf den Mensclien,'Mrc/i. /. d. ges. Physiol, 1897, vol. Ixvi. LoEWY, J. — " Ueber das Verhalten des diabetischen Blutes v.w den Anilin- farbstoffen," Fortschr. d. Medizin, 1898, vol. xvi. Maragliano.— "Beitrag zur Patliologie des Blutes," XI. Medical Congress, 1892. Massloff.— " Einige Bemerkungen zur Morphologic und Entwicklung der Blutelemente," Arckiv f. mikrosk Anat., 1898, vol. li. May and Grunwald.— " Ueber Blutfjirbungen," Zentmlhl. f. innere Medizin, 1902, No. 11. Mayer, Karl Hermann.— " Die Fehlerquellen der Haemometerunter- suchung (v. Eleischl)," Deutsches Arch.f. Min. Medizin, vol. Ivii. pp. 1, 2 (full Bibliography). Meissen and Schroder.—" Eine vom Luftdrucke unabhangige Zahlkammer fiir Blutkorperchen," Miinchen. med. Wochenschr., 1898, No. 4. Mercier. — " Des modifications de nombre et de volume que subissent les erythrocytes sous I'influence de I'altitude," Archives de 2}hysiol, 5th Ser., 1894, vol. vi. p. 769. Meyer and Heineke.—" Ueber den Farbeindex der roten Blutkorperchen," Miinchen. med. Wochenschr., 1906, No. 17. Meyer and Speroni.—" Ueber punktierte Erythrocyten," Miinchen. med. Wochenschr., 1906, No. 17. MiCHAELis, L.— Berl. Medical Society, 1899-1900, p. 49 of Transactions. " Eine Universalfarbemethode fiir Blutprtiparate," Deutsche med. Wochenschr., 1899, No. 30. "Das Methylenblau und seine Zersetzungsprodukte," Zentralhl. f. BaUeriol., 1901, vol. xxix. MiESCHER. — " Ueber die Beziehungen zwischen ]\Ieereshohe und Beschaffen- heit des Blutes," Korrespondenzbl.f. Srhweizcr Aerzte, 1892, xxiii. p. 809. MoRAWiTZ. — "Klinische Untersuchungen ueber Blutverteilung und Blut- menge bei Gesunden und Kranken," Volkmann''s Clinical Lectures, No. 462, 1907. 84 ANEMIA Naegeli.— "Ueber die Entstehimg der basophil gekornten roten Bliitkor- perchen," Munchen. med. JVochenschr., 1904, No. 5. "Ueber basopliile Granulation ■ der Erytlirocyten bei Embryonen," Fol. haemat, 1908, p. 525. BluthranMieiten und Blutdiagnostik, Leipzig, 1907. Neumann, E.— " Ueber Blutregeneration und Blutbildung," Zeiischr. f. Uin. Medizin, 1881, vol. iii. Neusser. — "Ueber einen besonderen Blutbefund bei uratischer Diathese,'' Wien. Jdin., 1892, Nos. 3 and 4. V, NooRDEN. — " Untersuchungen liber scbwere Anaemie," Annals of the Berl. Charite, 1889, vol. xvi. Orum. — " Quantitative Blutuntersuchuugen," Deutsches Arch. f. klin. Medizin, 1908, vol. xciii. Pappenheim. — " Die Bildung der roten Blutscheiben," Inaugural Dissertation, Berlin, 1895 (full Bibliography). "Ueber Entwicklung und Aus- bildung der Erythroblasten," Archiv f. patholog. Anat., vol. cxv. " Dunkelfeldbeleuchtung," Fol. haem., 1908, vol. vi. S. 190. "Ver- gleichende Untersuchungen liber die elementare Zusammensetzung des roten Knochenmarkes einiger Saugetiere," Virchow's Archiv, 1899, vol. clvii. Perles. — " Beobachtungen liber perniziose Anaemie," Berl. Uin. Wochenschr., 1893, No. 40. Ppeiffer, Th. — " Ueber die Bleibtreusche Methode zur Bestimmung des Volumens der korperlichen Elemente im Blute und die Anwendbarkeit derselben auf das Blut gesunder und kranker (insbesondere fiebernder) Menschen," Zentralhl.f. innere Medizin, 1895, No. 4. Plehn, a. — "Ueber Tropenanaemie," u.s.w., Deutsche med. JVochenschr., 1899, Nos. 28-30. Plesch. — " Chromophotonieter," u.s.w., Zeitschr. f. Idin. Medizin, 1907, vol. Ixii. Quincke. — " Weitere Beobachtungen liber perniziose Anaemie," Deutsches Arch. f. klin. Medizin, vol. xx. " Zur Physiologic und Pathologie des Blutes," Deutsches Arch. f. klin. Medizin, vol. xx. " Ueber Eisentherapie," Volkmann^s Collection of Clinical Lectures, New Series, p. 129. Rahlmann. — " Ueber einige Beziehungen der Netzhautzirkulation zu allgemeinen Storungen des Blutkreislaufes," Virchoid's Archiv, vol. cii. Reinert. — Die Zcihlung der roten Blutkorperchen, Leipzig, 1891. RiNDFLEiscH. — " Ueber Kuochenmark und Blutbildung," Archiv f. mikroskop. Anat., 1880, xvii. p. 1. " Ueber die Fehler der Blutkorperchenbildung bei der perniziosen Anaemie," Virchow's Archiv, 1890, vol. cxxi. p. 176. RoMANOWSKY. — " Zur Frage der Parasitologic und Therapie der Malaria," Petersburger med. JVochenschr., 1891. Rosin.- — Berl. Medical Society Transactions, 1899-1900, p. 49. Rosin and Bibergeil. — " Ueber vitale Blutfarbung," u.s.w., Zeitschr. f. klin. Medizin, 1902, vol. liv. (Bibliogra]3hy). Sabrazes (quoted by Naegeli). Sachs, H. — " Ueber DifFerenzen der Blutbeschaffenheit in verschiedenen Lebensaltern," Zentralbl. f. Bakteriol., 1903, vol, xxxiv. THE MORrnOLOGY OF THE lU>OOI) 85 Sahli. — Klinische UntersuchumjHmethoden, 4tli Edition, 1000. "Jjeitriige zur klinischcn Gescliichte der Anaeinie der Gotthardf,iiniiel-Arbf;iter," Deutsches Archivf. hlin. Medizin., 1883, vol, xxxii. ScHAUMAN. — Zur Keniiinit der sogenannteii, Bolhrioceph(ilii,ii-Anacmie, ]3erlin, 1894. ScHAUMAN and Ro.sknquist. — " Ucb(!r die Natur der J'hitveriinderunj^en im Holienklinia" (IJiljliography), Zeiinrhr. f. Jcliv.. Medizin, 1898, vol. xxxv. "Wie ist die Blutkorpercli(;nvernielirung im Geliirgi; zii erkluren?" Therap. Monatsheftc, 1900, No. 1. SCHIFF. — "Ueber das quantitative Verhalten der Bhitkorperchen und des Haemoglobins bei neugeborenen Kindern und Sauglingen unter normalen und pathologischen Verhiiltnissen," Zeitschr. f. Heilkunde, 1890, vol. xi. ScHLEiP. — "Ueber Ringkorper im Blute Anaemi.sclier," Deutsches Archivf. Mill. Med., 1907, vol. xli. Schmaltz. — " Die Untersuchung des spezifischen Gewichtes des menschlichen Blutes," Deutsches Archiv f. klin. Medizin, 1891, vol. xlvii. " Spezifische-s Gewicht und Haemoglobingehalt," Deutsche med. JVochenschr., 1891, No. 16. Die Pathologic des Blutes und die BlutJcrankheiten, Leipzig* 1896. Schmidt, P. — " Ein Beitrag zur Frage der Blutregeneration," MUnchen. med. JVochenschr., 1903, No. 13. Experimentelle Beitrdge zur Pathologic des Blutes, Jena, 1902. " Zur Frage der Entstehung der basophilen Kornchen in den roten Bhitkorperchen," Deutsche med. JVochenschr., 1902, No. 44. ScHUPFNER. — "Beitrag zur Kenntnis der Malavia," Deutsches Archiv f. klin. Medizin, vol. Ixiv. SCHUMBURG and N. Zuntz. — "Zur Kenntnis der Einwirkungen des Hoch- gebirges auf den menschlichen Organismus," Pfliiger's Archiv, 1896, vol. Ixiii. ScHUR and H. Loewy. — "Ueber das Verhalten des Knockenmarkes in Krankheiten," u.s.w., Zeitschr. f. klin. Medizin, 1900, vol. xl. Sellier. — " Contribution a I'etude de Tinfluence de la tension de I'oxygene sur I'hematopoiese, " etc., These de Bordeaux, 1895 (quoted by Schaumau and Rosenquist). Lorrain-Smith and M'Kisack. — Transactions of the Pathol. Soc. London, 1900 (quoted by Parkes-Weber). Stierlin. — " Blutkorperchenzahlung und Haemoglobinbestimmung bei Kindern," Deutsches Archiv f. klin. Medizin, 1889, vol. xlv. Stintzing and Gumprecht. — " Wassergehalt und Trockensubstanz des Blutes beim gesunden und kranken Menschen," Deutsches Archiv f. kbn. Medizin, 1894, vol. xliii. Strauss and Rohnstein. — Blutzusammemetzung hei verschiedemn Anaemien, Berlin, 1908. Tarchanoff, J. R. — "Die Bestimmung der Blutmenge am lebenden • Menschen," Pfliiger's Archiv, \o\s. xxiii. and xxiv. Thoma and Lyon. — " Ueber die Methode der Blutzahluug," Virclioic's Archiv^ vol. Ixxxiv. Turk. — Klinische Haematologie, Vienna, 1904, vol. i. 86 ANJEMIA ViAULT. — " Sur raugmentation considerable du nombre des globules rouge, dans 1-e sang cliez des habitants des hauts-plateaux de I'Amerique du Sud," Compt. rend.- de VAcad. des scienc, vol. cxi. p. 917. Walker. — The Journ. of Boston Soc, November 1899 (quoted by Grawitz). Parkes-Weber. — " Die Zunahme der gesamten Blutmenge bei Polycytliaemie, U.S.W., Fol. haem., June 1908. Weidenreich. — " Studien liber das Blut," u.s.w., Archiv f. mikroslcop. Anat. und Entwichlungsgesch., 1903, vol. Ixi. Wbndelstadt, H., and L. Bleibtreu.- — " Bestimmung des Volumens und des Stickstoffgehaltes des einzelnen roten Blutkorpercliens im Pferde- und Schweineblut," Pjiiiger's Archiv^ vol. Hi. Westphal. — " Ueber Mastzellen," Inaugural Dissertation, Berlin, 1880 (cf. Ehrlich, Farhenanalytische Untersuchungen, etc.). Williamson. — "A Simple Method of Distinguishing Diabetic from Non- diabetic Blood," Brit. Med. Journ., 1896, September 19. Wolff, F., and Koppe. — Ueber Blutuntersuchungen in Eeiboldsgriin," Miinchen. mediz. Wochenschr., 1893, No. 11. WoLOWNiK. — " Ueber das Verhalten der Knoclienniarkzellen bei verschiedenen Krankheiten," Zeitschr. f. Idin. Medizin, 1905, vol. Ivi. Wright. — "Remarks on Methods of Increasing and Diminishing the Coagulability of the Blood," Brit. Med. Journ., 1894, July 14. Zangemeister. — "Ein Ap-pa.Ya.t fiir kolorimetrische Messungen," Zeitschr. f. Biologie, 1896, vol. xxiii. Zenoni. — "Ueber gas Auftreten kernhaltiger roter Blutkorperchen im zirkulierenden Blute," VircJioiv's Archiv, 1895, vol. cxxxix. "Delle Alterazioni degenerative degli Eritroblasti," Policlinico, 1898. ZiEMANN. — " Die Methode der Doppelfarbung bei Flagellaten," u.s.w., Zentralhl. f. Balderiol., 1898, vol. xxiv. ZoLLiKOFER — Inaugural Dissertation, Bern, 1899 (quoted by Naegeli). CHAPTER III THE WIHTi: BLOOD COIil»USCLES The biological siguificiince of the while blood corpuHcles is so varied that these cells represent the most interesting chapter in hsBmatology. They are motile elenjents, which reveal con- siderable changes in response to comparatively slight stimuli. Fig. 2. — Fe.'VYIng of the Peotoplasmal Investment of Lymphocytes; SEPARATION OF THE FREE PlASMA ELEMENTS (PlASMOLYSIS). (After a Photograph of a Fihii from a Case of Chronic Lj'mphatic Leukj)l;LKiii is visible at one side. The nucleus stains intensely with iill the basic dyes, and reveals a dense chromatin network. Wlien suitably stained (the best for this purpose is ^lyronin methyl-green) a distinct nucleolus is rendered visible, with a relatively broad highly coloured membrane ; rarely two such nucleoli are seen. The nucleus often presents a notch, and in older types is not round, but rather of a long oval shape. This cell never shows that peculiar polymorpho - nuclear structure which is met with in other leucocytes, but possesses a more or less round nucleus throughout its whole existence. Only under very rare patho- logical conditions does the lymphocyte have peculiar lobulated nuclear structures, and it is then known as the " Eieder's cell " of lymphatic leukaemia. The protoplasm is usually very narrowly developed. In the larger specimens it is broad, and then reveals a basophile reticulum, in which the crossings of the meshes are so prominent that Ehrlich at first regarded them as granules. When ex- amined under a high magnifying power it will be seen that there are no isolated nodules. A pale area is seen between the nucleus and the protoplasm, in which the protoplasmal reticulum is only very faintly out- lined. In this area in every lymphocyte the fuchsinophile granules, which were discovered by Schridde, are placed. They are demonstrable by means of Altmann-Schridde's staining. Since this method of staining has not yet been included in Helly's treatise, it should be described in this place. (a) INIethod op preparing the Film — 1. The blood is smeared on to the cover-sHp in a thin layer. 2. The cover-glasses are tlieu placed for one to two hours iu formol-Miiller (1 : 9). 3. They are then rinsed first for several minutes with tap water, and then with distilled water 92 ANEMIA 4. They are then immersed for half an hour in the dark in 1 per cent, osmic acid. ' 5. IS^ext, they are rinsed for a short time. 6. They are then stained with Altmann's anihn acid fuchsin solution (100 c.c. of cold saturated filtered solution of anilin in distilled water with 20 grms. of acid fuchsin. — Filtration). This solution is poured on to the cover-glass, which is then warmed five or six times over the flame until the solution begins to steam, and then put aside until cool. 7. After the dried stain on the edges of the film have been removed with filter paper the films are diff"er- entiated with alcoholic picric acid (saturated solution of picric acid in alcohol, 1 part in 7 parts of 20 per cent, alcohol). This solution is dropped on to the film several times, until it appears yellowish or pale yellow. 8. The specimen is then rinsed rapidly with absolute alcohol. 9. It is then passed through toluol or xylol, and 10. Mounted in Canada balsam. The eosinophile granules are dark red, the neutrophile (amphophile), granules pale brownish red, the basophile granules colourless, like vacuoles. The lymphocytes show perinuclear granules or rodlets of a yellow crimson-red colour. {b) Method of demonstrating the Cell Granulation in Sections (including lymphocyte granules). — The tissues, while warm and fresh, are fixed in formalin-Muller (1 : 9) for twenty-four hours at 36° C. They are then washed for twenty-four hours. Next, they are passed through alcohol, 60, 70, 85, 96 per cent., and absolute alcohol, toluol (in each one hour), paraffin (altogether one and a half to two hours). The sections should be 1 or 2 /x, in thickness. 1. The sections are floated on 1 per cent, osmic acid solution for one hour in the dark. 2. JS^ext they are rinsed in distilled water. 3. ISText they are stained in Altmann's solution (see above). 4. They are then diff'erentiated with alcoholic picric acid solution, as described above. THE WHITE HT.OOn CORiniSCr.ES 03 5. They are then taken through alcoliol, 96 por cent, absolute alcohol, toluol, and Canada balsam. The sections should appear yellowish, wiili a trace of red to the naked eye. Microscopically, the cell nuclei are palo brown, the protoplasm yellowish, the granules red (the kind of red varying in different cells). (c) ScIIRIDDE's AZUIIE II -KOSIN-ACETONK MlCTIIOD FOK StAINING Sections. — The fixation may be any of the ordinary methods, e.g. formol-Miiller (formalin 1 part, Midler 9 parts). Staining with Giemsa (2 drops to each 1 c.c. of distilled water) for twenty minutes. Careful washing, drying with blotting paper, and then treatment for one minute in pure acid-free acetone (Kahlbaum). The sections are then passed through acid-free xylol or toluol. They are then mounted in Canada balsam, and kept in the dark. The neutrophile granules are violet-red, the eosinophile granules red, the mast cells dark blue, the erythrocytes grass green. The myeloblasts show a greyish-blue protoplasm without any granules. Besides the fuchsiuophile granules, some of the lymphocytes possess azurophile granules, whicli can be rendered visible by means of Giemsa-Eomanowski's staining. These granules are met with exclusively in the larger cells. They are at times sparse and coarse and at times numerous, fine granules of a bright red colour. Inequalities and fraying of the contour of the cells are always the artificial results of pressure, and many of the apparently large lymphocytes in the smears are merely the results of squeezing. The protoplasm possesses no affinity to the neutral and acid dyes save in the case of the larger cells, vrhen the affinity is very slight. It is markedly hasophile, and with Giemsa staining it takes on a pure pale blue colour. If the cells have been crushed the protoplasm may remain unstained. The azure granules then appear against an almost white background. Large lymphocytes occur in the blood of children, but very rarely in the blood of adults. Very large elements are always pathological, and are met with in leukemia. (Troje erroneously called them " marrow cells " in this connection). 94 ANiEMTA In the blood of adults from 20 to 25 per cent, of the white blood corpuscles are lymphocytes; in the blood of children the number is much greater, and may be as high as 70 per cent. An increase in the number of lymphocytes is not frequently met with. When it occurs it is spoken of as lymphocytosis or lymphaemia. 2. Large Mononuclear Leucocytes. — These cells are relatively very large, being usually twice or three times the size of the erythrocytes. They possess a fairly large oval nucleus, which stains much less intensely with a basic dye than the nucleus of the lymphocyte does. With a suitable dye (hsematoxylin, Giemsa) they reveal a very delicate slender network of chromatin. The nucleus generally shows a marked tendency to assume a polymorpho-nuclear structure. The protoplasm is very broad, possesses a close, delicate basophile reticulum, which is extended equally right up to the nucleus, and when stained by Giemsa takes on a dusky greyish- blue (slate-grey) colour. In the meshes an extremely fine neutro- phile granulation is seen when the cells are properly stained by the triacid or Giemsa method. This granulation is distributed over several areas of the cells, but not uniformly over the whole cell (young, beginning granulation). In some situations the fine granules are so closely packed that in a well-prepared film some of the edges have a diffuse pink appearance when stained by Giemsa (see Plate II.). These cells are quite different from the lymphocytes, even if the neutrophile granulation is not taken into account. They belong to the " transition " forms, which will be described later and can only be distinguished from the latter by the nucleus, which scarcely presents any polymorphous character. When they are really well stained with Giemsa the relationship is so clear that it is practically impossible to divide the two classes from one another sharply. Large and crushed lymphocytes might be mistaken for these cells, if the staining is too pale or the neutrophile granulations are not sufficiently coloured to be distinguishable as such. But THE WIIITK P>I/)()I) (ORiniSCLKS 95 scarcely 1 per cent, of ccIIh is met with wliif-b )iii<^lit ))e coiifuHed with the lar5 > / ^ ^ < i V N \_ \ s, ^ \ \ \ ?5 \ \ ' 1 >^ ^ \ Tf 1 ^ / \ V_ i §; i ^ a W o fer ra ft c3 o >> s .a .o '^ S H H H S fl S .5 p ^ 2. Polymorpho- nuclear eosino- phile cells, about 1 per cent. 3. Mgrosino- phile cells, anal- ogous to the eosinophile cells, but the granules of which take on the colour of nigrosin from the eosin - nigrosin mixture in pre- ference to the red. They take on a darker colour when stained by triacid. 4. Cells with vacuoles, 15 to 20 per cent. 5. Lympho- cytes, 30 to 35 per cent. Kurloff, in his extremely careful and laborious ex- aminations, deter- mined the total S "f . number of leuco- g § =9 cytes and then the percentages of the absolute number of pseudo-eosinophile, neutrophile, eosino- phile, and vacuol- &o 'S rs .s ^ O H H pR TIIK WIIITK IJLOOI) COIIJMISCLKS 11.'} ated cells, as well as of the lynipliocytos. llo was tliiis ahlr; to prove that in xiiiconiplicated cases of icinoval of ilic. s])I(!(;n, in which all inflammatory processes wliich involvi; an imiciisc of iJic ])ol_ynuclcar noutrophilo corpnscles were cxcJiiilctl, a gradual increase limited to the lymphocytes up to twice or three times the original value is noticed, while the numhers of all the other elements remain ahsolutely unulterefl. The increase in the nunil)er of lyinphocytos occurs (Ini'in^r the course of the first year after the removal of the spleen. This increase must be regarded as the expression of a hy][)erplasia of the lymphatic glands, and especially of the mesenteric glands. The loss of the splenic function is thus in part compensated by the lymphatic system. The pseudo-eosinophile cells show a temporary increase after the operation, but no marked variations have been noticed in the transition forms. In the second year after the operation a very considerable increase in the number of eosinophiles is constantly observed. Kurloff's experiments thus prove that the spleen of the guinea-pig only plays a minor part in the formation of white blood corpuscles, and that after splenectomy, compensatory functions are assumed during the first year by the lymphatic glands. In the second year a considerable increase of the eosino- phile cells takes place. It is necessary again to point out that the spleen has nothing to do with the formation of the pseudo- eosinophile polynuclear cells, wdiich are the analogies of the polynuclear neutrophile cells of human beings. It is necessary in the next place to inquire how observations on human beings compare with Kurloff's observations, which after all might be regarded as peculiarities of the species' of animal. Absolutely analogous conditions can be found in those cases of healthy persons who, as the result of trauma, have been sub- jected to splenectomy. Unfortunately, such cases are extremely rare, but it would be of great value if the changes in the blood could be studied systematically for several years in such cases. The observations made up to the present have led to the 114 ANEMIA following results. A lymphocytosis lias been observed after the operation, which at times was of considerable duration and at times merely temporary. In the latter case the increase was probably only an after-effect of the operation, and corresponds to the experience that the diminished formation of lymphocytes after operative interference is overcompensated during convalescence. In a few cases a slight increase of the eosinophiles has been observed. This too could be regarded as a post-infective or post-toxic process, and cannot be regarded as justifying the conclusion, at present at all events, that the bone marrow in human beings takes on a vicarious function. It is not an infrequent find that a considerable increase in the eosinophiles is present in splenic tumours, but the cause of this is probably to be sought in the disease itself and not in the loss of splenic function. It is, however, essential that further careful observations in human pathology are needed to clear up this question. In the meantime, direct histological examination has become applicable for the study of the participation of the spleen in the formation of the blood. By means of modern section staining a perfectly clear insight into the conditions is rendered possible by these methods, at all events as far as the majority of the points under discussion are concerned. These examinations show that the normal human spleen does not contain any nucleated red blood corpuscles. Only a few authors claim to have seen a few such cells. It can therefore be definitely stated that the human spleen during adult life takes no part in the production of erythrocytes, or at all events no material part. The same applies to the occurrence of myelocytes, the precur- sors of the polymorpho-nuclear blood cells. These cells are not met with in stained sections, although* a few authors state that single examples of these types have been seen on rare occasions. The spleen has therefore nothing to do with the normal formation of the polymorpho-nuclear leucocytes ; this takes place exclusively in bone marrow. THE WHITE P,LOOI) COIMM ^SCLKS 115 On the other hand, one origin of tlio lymphocytes in found in the M)ili)igliiiui bodios, iind there (;aii bo no doubt that a certain proportion of the lyini)hocytes of the blood proceed from the spleen. The spleen would, aceordiug to Ibis jxiiiit of vinvv, belong to the lymphatic system. The cells of the splenic pulp, however, must still be taken into consideration. Up to the present the significance and char- acter of these cells are quite unlcnown, and information on this point can only be obtained by means of the most dolicatci methods of cell analysis. The normal functions of the spleen must be regarded as including a process whereby a portion of the used-up white and red blood cells are completely disintegrated and the utilisable material is used for the reconstruction of new cells. Spodogenous tumours of the spleen are consequently met with in many diseases (from a'TTo'hoc = fragments). Comparative anatomy and histology, however, teach that in some of the lower vertebrates and in many mammalians, e.g. mice and rabbits, the spleen fulfils a much more important part in the formation of blood. Nucleated red blood corpuscles may be found in nests, and the same applied with regard to the myelocytes, so that as far as these animal species are concerned there is no doubt that this organ possesses a blood-forming activity. With regard to human beings, observations of this kind have only been made under embryonal or pathological conditions. These observations have been made quite recently, although some finds of an unconvincing nature have been reported during the past two decennia. It is now known that the human spleen in the early stages of embryonal life at first exercises an erythro- poietic and myeloid activity exclusively (Naegeli, Schridde). In a foetus of from 10 J to llf inches length the spleen is composed of almost pure myeloid tissue, so that even an experienced histo- logist would diagnose bone marrow at first sight from a smear. Later on this function diminishes little by little, until the lymphatic structures are developed in the Malpighiau bodies, 116 ANAEMIA and at the time of birth the spleen has lost nearly all traces of its former myeloid-erythropoietic functions. Under pathological conditions, however, the human spleen may again harbour erythroblasts and myelocytes, as if it had reverted to its embryonal habits. Under these conditions the cells mentioned are not merely washed into the organ. Actual formations and even quite extensive transformations can at times be discerned, in which the lymphatic tissue of the follicles is reduced or even stifled and substituted. Observations of this nature have been reported in such great numbers during the past few years that it is impossible even to name the individual authors responsible for them. Suffice it, therefore, to mention that this form of transformation in the structure and function of the spleen actually takes place. The conditions under which this occurs include the infective diseases, severe ansemias of various origins, malignant tumours, provided that they have led to anaemia or destruction of the bone-marrow tissue (carcinoma of the medulla of bone), and especially leukaemia and the allied conditions. Experimental research did not have any great difficulty in discovering some absolutely analogous histological appearances. A complete transformation of the spleen was produced in experi- mental anaemia caused by blood poisons, b}'' artificial infections, and by exposure to X-rays (K. Ziegler). It is proposed to discuss how such a surprising phenomenon may be brought about later on. Consequently it is necessary to adhere to the view, which Ehrlich formulated with considerable precision some years ago, that the normal human spleen does not participate in the forma- tion of the red blood corpuscles and of myeloid tissue ; but, in opposition to the older views, this formation takes place in the spleen frequently and at times extensively under pathological conditions, like a reflection of embryonal times. (b) The Lymphatic Glands Since it is impossible experimentally to eliminate all the lymphatic glands from taking part in the formation of blood, it THE WHITE IJLOOD CORPUSCLES 117 is necosKiU'y io (lo[)(!ii(l eiitiroly on clinical and liiHloltjgical observiitions i'or the [)urpoHO of obtaiiiiiif^ irifoi'inatiori on this subject. Since Virchovv dolnied lynijjliocyteH, the identity of the lymphocytes of the bl(Kjd and tlie lyni])liocyte8 of the lymphatic glands and those of othcu' forms of lymphatic tissue, be they l;i,rg(! or small types, iiiis not been questioned. This identity is proved by the complete correspondence of the general morphological characters, and of the tinctorial peculiarities both of the protoplasm and of the nucleus. With regard to the granules in the lymphocytes, the proof of the identity of the blood lymphocytes and those of the lymphatic glands can be further clinched by the demonstration of Schridde-Altmann's fuchsinophile perinuclear granulation, which is possible in every lymphocyte in lymphatic glands. In the same way, some of the lymph cells show azure granules. The identity is therefore complete in this respect also, and since both these forms of granulation do not occur in any other cells save the lymphocytes, the correspondence may be regarded as absolutely proved. Now, since similar cells (myeloblasts) occur in the parenchyma of the bone marrow, which, however, do not show these specific characters (Schridde, Naegeli), it follows that the bone marrow does not produce any lymphocytes normally. The differential distinction between the two cells is shown in this way. Only a few isolated lymphocytes are met with in the sheaths of the vessels of the medulla. It is characteristic of the advance in hsematology due to Ehrlich's teaching that a direct proof of identity and new formation of cells is now forthcoming, while formerly, owing to the absence of good section staining, the observer was forced to rely on indirect methods which were far less certain. Ehrlich based his doctrine of the origin of the lymphocytes from the lymphatic glands chiefly on biological grounds. He pointed out that when extensive areas of lymphatic tissue were eliminated by new growths and similar changes, the number of lymphocytes was sensibly diminished. This fact has since 118 ANEMIA been confirmed by a number of authors. For example, Eeinbacli described several cases of malignant tumours, especi- ally sarcomata, in which the percentage of lymphocytes, which is usually about 25, was very materially diminished : in one case of lympho-sarcoma of the neck these cells only represented 0'6 per cent, of the total number. The author observed a case (published in the J. D. Chotimsky, Zurich, 1906) of general enlargement of the lymphatic glands, in which, during the course of two years, the absolute value of the lymphocytes varied in a large number of counts between 300 and 500, as compared with the normal 2000. In spite of the extraordinary generalisation of the process, which suggested an aleuksemic early stage of a lymphatic leuksemia, this diagnosis could be definitely excluded on account of the biological- functional phenomena, and a process of destruction of the active lymphocyte-producing tissue had to be assumed. The post- mortem examination and subsequent histological investigation showed that the case was one of a tuberculosis, having the course of a pseudo-leuksemia and leading to complete induration and scarring of the glandular tissue. These appearances can be explained quite readily and naturally on the assumption of the elimination of the lymphatic glands. It is difficult to say how the supporters of the view that the lymphocytes are the precursors of all white blood corpuscles can explain these facts. In accordance with this view, it would have to be assumed that the small number of lymphocytes in such cases would be accounted for by supposing that an unusually rapid transition into the elder forms, the poly nuclear elements, had occurred, or, to adopt Uskoff's vernacular, that a premature getting old of the lymphocytes had taken place. Further proof that the blood lymphocytes are derived from the lymphatic glands can be obtained from those cases in which an increase of lymphocytes in the blood is found. These lymphocytoses are of rare occurrence as compared with other forms of leucocytosis. In the first place, it can be seen that certain conditions, in which a hyperplasia of the lymphatic THE WJIITK iJLooi) c ()iiiM;sc:Li:s llj gland apparatuH occurs, an: aissociaLed vviLli ;iii iiicicasc oi lymphocytes in the l^lood. I^>liilicli iiiid l\;M(;\\Hki f;xaiiiincd a long series of typicaJ cases of ]yiiipli<»iii;i, niali.^iniin (l/licy did not publish tlieir icsulls). 'I'lu'.y noted ;i, r("j,id;ii- lympliocytosiK which was veiy considei'al)l(! in souk; of Hk; c.jises ;M,d li;id almost a leulvtx'niic character. On the bases of tlicsn ivsnlts, I'llulicli ;i,iid Wasscrinaim (^Dermafolor/isrhe Zcr/Kclir///, J804, vol. i.) foinKid tjic diagnosis of malignant lympliomu during life in a fa.'^n of a rarn foim of .skin affection. The lilood showed an absohite increase, wliicli was limited to the lymphocytes. No swelling of the lymjiliatic glands was ascertained by })alpation. The post-mortem examination revcilcd that the retroperitoneal lymphatic glands were swollen to the size of a fist. In cases of this kind there is a marked increase of pro- duction of lymphocytes in the whole lymphatic apparatus, as can be proved by histological preparations. Some parts of the apparatus no doubt are but slightly affected, but the proliferation is well marked in others. This means that there is a system affection of the lymphatic apparatus, which in view of its nature is termed lymphocytomatosis. This affection may last for several years and may pass on to a true lymphatic lenkiemia, from which it differs oidy in point of extension. It is therefore possible to gain information with regard to the natirre of certain affections of the lymphatic glands on biofunctional considerations, and at the same time to determine essential differences between the various forms, even when the clinical appearances do not serve to clear np the matter. It must, of course, be realised that these considerations can only be regarded as correct and utilisable if the anatomic histological premise corresponds to fact, namely, that the lym- phatic glands are the sites of origin of the lymphocytes. Proof has recently beeir adduced that in the earliest embryonal stages, before the lymphatic apparatus has been developed, no lymphocytes are found in the blood, and that the blood then contains cells of the mveloid series exclusivelv. 120 ANAEMIA ■ It is, as would be expected, extremely difficult to say how large a proportion of the lymphocytes is derived from the lymphatic glands. The lymphatic follicles of the intestinal tract and, as has already been mentioned, the. spleen un- doubtedly supply the blood with true lymphocytes. But the clinical experiences made in cases of destruction of lymphatic glands, to which allusion has been made, goes to show that the preponderance of these cells originate in the glands. If this were not so it would be extremely difficult to explain the very low values which have been observed and which persist for years. A marked diminution of the lymphocytic value is met with frequently and with considerable regularity in acute diseases, and especially in the early stages of the infective processes. This diminution is resolved in the later stages by an increase in the absolute numbers which may attain a quite considerable degree during convalescence. Under these con- ditions, even if it cannot be said that histological changes are not present, the changes must depend to a large extent on functional factors, such as the toxic inhibition of the cyto- genesis and a consequent hyperactivity, according to general biological laws. It is just the late increase which cannot possibly be explained otherwise than as a biofunctional process. Consequently the phenomena of hypo- and hyper-lympho- cytosis must always be judged with caution, and it is essential in all cases to think of the possibility of functional changes rather than of gross anatomical lesions, although even when the former are active the latter need not be excluded. An example of this may be quoted in the later stages of pertussis and of enteric fever, when enlargement of the lymphatic glands is met with. This enlargement should be regarded as the anatomical substratum of the existing increase of lymphocytes. Chemical substances induce a preliminary diminution in the number of lymphocytes in the blood as the result of a functional process (toxic inhibition of the cytogenesis), while, as is well known, the myeloid system usually reacts to a stimulation of the THE WHITE ]}L()()1) COllPUSCLES 121 funciioiiH of tli(! organs l»y a inarkfid IciicDcytDsis. An inf,roa80 of tlie lympliocylcB only lakn.s placo lat(!r as an af'tor-oircct, in accordance with biologic-al laws. This r'(^ac,liv(; increase conlinuoB beyond Uh; noi'inal niveau as ilio function recovers itself. Hitiioi'to only one substance lias been mentioned in literature which is stated to l.)e capal)l(; by itself of ])roducing a lym- phocytosis. Waldstein reports that he lias succer^dcd in inducifig a lymplia3mia by injecting pilocarpine. On increasing the number of injections he ol)tained a progressive chaiucter of the changes. Observations of this kind do not jjrove much, since an aleukicmic lymphocytosis may at any time pass over to its leukfcmic stage and then assume a progressive character. It appears to be exceedingly doubtful whether pilocarpine can induce a primary and not secondary functional lymphocytosis on an unprepared soil, and would have to be proved by repeated careful investigation before it could be accepted. The production of a lymphocytosis therefore depends on absolutely different causes to those which act in producing the ordinary leucocytoais in which an increase of the noutrophile elements is found. As Ehrlich pointed out long ago, the chief difference is found in the fact that chemotactic functions play a principal part in the production of leucocytosis, and that this exercises a distant action on the bone marrow. In lymphocytosis this chemotaxis is not present, or is only present to a very slight extent. A primary increase of lymphocytes is therefore unknown. On the other hand, it can be said at present that secondary lymphocytosis, which is seen in the later stages, cannot be regarded solely as the result of an increase in the lymph circulation, which would mechanically cause a larger number of the elements to be washed out of the lymphatic glands. Clinical observation has taught that a functionally augmented activity sets in during recovery after the stage of diminished function, which is usually a sign of toxic inhibition, and that the hyperlymphocytosis is then the expression of a true increase of 122 ANEMIA function. It is therefore necessary to add the functional explanation to the mechanical explanation formulated above. In the same way, in severe pathological affections involving an actual proliferation of the lymphatic tissue, as in lymphocyto- matosis and lymphatic leukaemia, a marked increase of activity of the tissue takes place and not a mere mechanical washing out. The lymphocytes do not play any part, as a rule, in inflammatory processes, and are not met with in the in- flammatory foci. This corresponds to the absence of a chemotactic attraction. Neumann described many years ago a highly interesting experiment bearing on this question. He produced an abscess in a patient who was suffering from lymphatic leukaemia, and whose blood contained a very small number of polynuclear cells. The pus was found on examination to consist exclusively of polynuclear leucocytes ; not a single lymphocyte was found in the discharge, although the blood was full of these cells. The same results have been obtained each time this experiment has been repeated. If, in spite of this, lymphocytes leave their vessels actively, and this has been observed several times (Schridde, Helly, and others), quite a different cause must prevail to that wdiich prevails in ordinary leucocytosis. Histological examination of nearly all fresh inflammatory processes in which the polynuclear elements alone are found in the inflammatory tissue also yields results conforming to this view. Under exceptional conditions lymphocytes may pass out of the vessels in the earliest stages of fresh inflammations. There must in this case be special, undoubtedly different attrac- tions to those which act on the leucocytes. In the case of migration of lymphocytes a local action of the vascular wall and the tissue in its immediate environment must take place, and not a distant action, which reaches as far as the blood- forming organs. It is well known that in the later stages of THE WHITE HLOOI) (OllIMISCLKS 12:5 in(liUiiTn)i,t/i()ii Kiuall ('-(ill in(ill,i'!il,ioii !i,|)])e;u'H, wliicli cojisIkLh appiii'ciiLly (jF lyiiiphocytu.s. J5uL Ll)i,s does iioIj provo ilial thfise lympliocytes liavo migratxid from tli(Mi' vcHseln io the nite of iiillaiuiiialion. IL i.s uiiiiedessary in this |)l;i,('(; to enter into a discussion of the controversy which has h(;eii eiiga^^iiig tlie attention of a number of lia'matologists with regard to this question. It will be sullicient for the present to mention that all tlic investigators have cousidcied, in the (iist place, the possibility of a new formation of the colls in aitu. Evidence of this occurrence has been forthcoming in the examination of the blood in tubercular pleurisy. In spite of the fact that a lymphocytosis exists from the earliest stages in the exudation, no increase of the lymphocytes in the blood is seen. In every chemotactic increase the rule is that increase in numbers of a certain species of cell finds a corresponding increase of the same cells in the blood. It therefore follows from clinical and morphological ex- aminations, and also from the results of investigations of inflam- matory processes, that the lymphocytes do not stand in any correlation to the polynuclear leucocytes. The same result will be arrived at in a different way in the following chapter. Erythropoesis and formation of myelocytes have within recent times been observed under pathological conditions in the lymphatic glands, just as they have been seen in the spleen. The analogy with the conditions obtaining in connection with the spleen is a perfect one. During the embryonal period the myeloid tissue at first claims a place in the lymphatic glands as well, and only disappear gradually as the bone marrow develops. In post-embryonal periods the central portions of the glands undergo a myeloid transformation, in severe antemias, in infec- tions, and intoxication. This takes place more especially when the organism calls forth new fields for the production of the vitally essential red blood corpuscles and myeloid leucocytes, in compensation for defective function of bone marrow. 124 ANEMIA (c) The Bone Marrow It was fermerly thought that the spleen and lymphatic glands were the only organs of production of the blood corpuscles, but general attention was attracted to the bone marrow by the investigations of Neumann and a little later of Bizzozero, in which it was shown that the precursors of the red blood corpuscles are formed in these organs. This discovery was rapidly recognised, and was soon turned to practical use in pathology by Cohnheim and others. In this connection, especially valuable information was adduced in the fact that after severe htemorrhage the medulla of the long bones was reconverted into red marrow, which shows that the regenerative function of the bone marrow may meet an increased demand. No other site of production of red blood corpuscles in man under normal conditions is known. In other mammalians, as has already been mentioned (see p. 115), the spleen may partici- pate to a certain extent in the production of erythrocytes. The type according to which the normal production of blood is carried out in adults, and the variations from this type which are met with in pernicious anaemia, have been discussed in detail in the chapter on the red blood corpuscles, and Ehrlich's views were accorded their proper significance, according to which the production of blood in Biermer's ansemia follows quite a different type, and one which is analogous to the embryonal type. It is therefore only necessary in this chapter to consider the white blood corpuscles and their relation to the bone marrow. In man, as well as in a number of other animals {e.g. monkey, guinea-pig, rabbit, pigeon, etc.), the bone marrow shows a peculiarity in that the cells which it produces contain specific and easily demonstrable granulations. This is sharply contrasted to the lymphatic system, the granules of which are quite difterent, and which differ among themselves. Some of the latter, such as the azure granules and Schridde's fuchsinophile granules, were not recognised for a very long time. THE WHITE BLOOD CORPUSCLES 125 The azuro /.i many thou«aii(l leucocytes, luid in t.lie r)r<4-;i,iis also not one cf^nld be found. A temporary myelocytosis of tli(; l)lof)(l ta,k(!H place frequently in the early stages of lymphaemia as a result of stimulation of the bone marrow. But a continuous decrease in the numljers of the myelocytes in the blood can be seen later, keeping pace with the replacement of the tissue of the medulla. The bone marrow has, as has recently been determined, another extremely important function besides that of forming cells. This function is the power of producing antitoxins (Wassermann, Pfeiffer, and Marx). It therefore must be re- garded as the organ par excellence which has to decide the teruiination of an acute infection. The red blood corpuscles are further disintegrated in the bone marrow, and the available material is utilised again for the re- construction of new cells. III.— ON THE DEMONSTRATION AND SIGNIFICANCE OF CELL GEANULES. In recent times, histological, biological, and also clinical in- vestigation has aimed, to an ever-increasing extent and with most promising results, at the solution of the problem of the significance of the cell granules. The work undertaken with this view has proved of great importance to hfematology, and it is now clear that a number of important questions wliich have still to be answered are intimately associated with the study of granules. It would therefore appear to be advisable in this place to deal with the history, methods, and results obtained up to the present from the investigations concerning cell granules in a comprehensive manner. The credit of having first pointed out the great importance of granules, and of having obtained practical results by systematic long-continued investigations of this subject, undoubtedly belongs to Ehrlich. It seems necessary to emphasise this fact, as Altmann has repeatedly maintained that it is not the case, in spite of the 134 ' ANEMIA fact that his attention has been called to the real state of affairs. After Ehrlich replied to Altmann's priority claim in a special article in 1891,^ Altmann stated in the second edition of his Elementarorganisme7i, which was published in 1894, that he was the first to recognise the specific importance of the granules, and that although these bodies had been observed by a few authors, they had only been regarded as " specialities and isolated appearances." It is therefore necessary to quote a few pregnant passages from Ehrlich's work. It is quite clear that Ehrlich did not regard the granules as "isolated appearances" in one of his earliest publications on this subject, which appeared in 1878, that is, ten years before Altmann's contributions. It must be admitted that an author who devotes ten years' work almost exclusively to a single subject could not but regard the subject of his investigations as of considerable biological importance. In respect to this matter, Ehrlich wrote : " The word ' granu- lated' has been employed with predilection since the beginning of histology to indicate a constitution of cellular structures The choice of this expression is not a very happy one, since very many circumstances may lend the appearance of granulation to protoplasm. Modern methods of examination have shown that many elements which were described by earlier authors as granu- lated owe this impression to the presence of a reticulated superimposed protoplasmic network. These cells are just as little granulated as are cells which show granulated albumin precipita- tion resulting from cadaveric coagulation or from the influence of certain chemical reagents such as alcohol. The term should therefore be reserved for the elements, which include substances in granular form during life, that can be distinguished by chemical means from the normal albuminous substances of the cells. Only a few of these granulations, like fat and pigment, are easily recognisable ; the majority cannot be characterised, or can only be indistinctly characterised with the help of the methods now in ^ See Ehrlich, Farhenanalytische UntersucJningen XII,, zur GescMdtte der Granula, p. 134. THE WHITE IJLOOI) (OIMM JSCIJIS i;jV general use. It used to be cou8i(l(!i(;i-;iin form. The majority of the mammals whose blood has Ijeen examined possesses granulated polynuclear cells. Hirschfeld lias dealt with this subject in an exhaustive work, in which a large nundjer of very remarkable details are contained. He found that in the majority of the animals examined the polyrmclear cells were provided with neutrophile granules, and only in one animal, the white mouse, did he fail to find this or an analogous form of granulation. The statements made by Hirschfeld cannot be accepted, in view of some investigations undertaken by Dr. Franz Miiller in Ehrlich's laboratory. After many fruitless attempts Dr. Miiller discovered a method by means of which he was able to demon- strate numerous but extremely fine granules in the polynuclear cells of the mouse. This shows that it is not permissible to assume the absence of granules, even if the ordinary staining methods do not suffice to reveal any. Just as there is no universal method of demonstrating bacteria, so there is none for rendering granules visible. All the granules which consist of soluble substances must necessarily disappear when the ordinary triacid method is employed, thus simulating a homogeneous cell protoplasm. The foregoing, however, is not intended to indicate that the occurrence of non-granulated polynuclear cells is denied in certain animal species. Hirschfeld states that such cells exist side by side wdth granulated cells — for example, in the dog, and from this find he deduces far-reaching conclusions with regard to the significance of the granules. It must, however, be pointed out that Kurloff's work tends to show that there is no reason for assuming that the non-granulated polynuclear cells are 144 ANEMIA identical with the granulated cells. Kurloff was able, at all events as far as the blood of the guinea-pig is concerned, to prove that, these two different elements may be sharply dis- tinguished from one another and that they each have a separate genesis. The fact that in general only those cells of the blood which are meant for migration and chemotaxis, and which are capable of carrying out these functions, contain granules must be regarded as highly important. This applies to all species of animals. It is a very suggestive assumption, which can scarcely be disproved, that the migration of the granulated cells has a certain degree of nutritive character, and for this purpose just those cells which enclose abundant quantities of reserve material would be peculiarly adapted. On the other hand, the lymphocytes do not contain the kind of granulation met with in the myeloid cells, nor are they involved in the chemotactic process. A further indication that the granulation actually is coimected with a specific activity of the cells is found in the fact that one cell is the carrier of only one specific kind of granule. Ehrlich was able, on the basis of investigations undertaken specially to clear up this point, to show that the contrary opinion, which recognised the simultaneous occurrence of neutrophile and eosino- phile granulation or of eosinophiie and mast cell granulation in one and the same cell, was not in correspondence with fact. This contention has been fully confirmed during the past ten years in an almost innumerable series of control experiments. The author, who has carried out an extraordinarily large number of examinations has never met with the combination in question even when the most severe pathological conditions have existed, either in the blood or in the blood-forming organs. Ehrlich has never observed the alleged transformation of the pseudo- eosinophile cell of the rabbit into the true eosinophile cell.^ ^The cause of this kind of misiinclerstanding is to be sought in the develop- mental stages of the granules, when the tinctorial characters show variations, as has been described at some length on a preceding page. How little tinctorial variations alone suffice to determine the chemical identity of granules becomes quite clear when the granules of other organs are taken into consideration. No one would THE WHITE IJLOOD CORPUSCLES J 45 With regard to the transformation in tho rabhit, it may be stated that the best method of proviiiL()()I) COllJMJSCLKS 157 phenomenon, the form of wliicli i.s largely detoi'minorl ])y Llio power of reacting on the part oi the bone marrow. The definition of the word chemotaxia might, it is true, he extended to include a distant action of chemical .suhstiMiccH in gcncial on the blood and blood-producing organs, instead of simply applying to the attraction and local movement of the leucocytes. On the application of a suitable dose of the chemical substance a stimulation of the cells present in the bone marrow would take place, which would be evidenced Ijy a proliferation of the marrow cells and as a rule by an increased output into the blood of these cells. When other doses are applied a hyper- sensibility of the medullary elements would be produced, under the influence of which the immature mononuclear forms would leave the marrow, and in this way all pronounced increase of cells in the central organs would cease. Eegarded in the more extended light, the chemotaxis as defined formerly, i.e. the locomotion of the leucocytes, would then only form a part of the whole process. Leucocytes may be divided from this point of view into : (1) Simple leucocytes, usually endowed with distant action (formerly spoken of as active), and (2) leucocytes without the capability of distant action (formerly termed passive). The lymphocytes would belong to the latter group. Having regard to the fact, mentioned above, that the same substance may produce leucocytosis or not according to the dose, it can scarcely be supposed that a diminution in the number of leucocytes — leucopenia — is a process which has no connection with leucocytosis. Both conditions can very well be pro- duced by the same cause, and must therefore be regarded as differ- ing only in degree, in correspondence with the dose of toxin. The close relationship may further be shown to exist in the fact that a diminution or even total disappearance of one kind of leucocyte frequently occurs even when the total number is greatly increased. In other words, there may be a leucocytosis, say, of the neutrophile cells simultaneously with a leucopenia of the eosinophiles ; this occurs quite frequently. Leucocytosis 158 ANEMIA and leucopenia are thus the morphological expression of bio- logical processes in the function of the leucocyte-forming organs. Marked diminution in number of the white blood corpuscles is very characteristic of certain diseases, especially enteric fever. The neutrophile elements are most frequently affected, especially when the disease has reached its acme and in the final stages. The conclusion to be derived from the foregoing is that the toxin of typhoid fever specially damages the function of bone marrow. This assumption has been confirmed by animal experiment (JSTaegeli, Studor), and has received support from the fact that a neutrophile leucocytosis does not occur in severe cases, even when certain factors are present which tend to produce leucocytosis, such as pneumonia, abscesses, turpentine injections, etc. Considerable degrees of leucopenia are met with in severe cases of enteric fever, at the onset of morbilli, frequently in •cirrhosis of the liver, as well as in severe forms of anaemia of various origin, and especially as a regular find in Biermer's pernicious anaemia, under which condition the cause of the ■disease does not play any part. The characteristic changes in these cases include those cells which are derived from the bone marrow, — that is, neutrophile cells, transition forms, and eosinophile cells, which are much diminished in absolute numbers, often as low as one-fourth to one-sixth of the normal number, while the lymphocytes appear in abnormally high percentages, although their absolute values correspond approximately to the normal. A steady, slow increase in the number of the leucocytes is seen regularly during remissions in this disease. The explanation for this peculiar character of the blood in pernicious antemia is obvious. The leucopoiesis, like the ery- thropoiesis, is markedly inhibited and insufficient. Excluding leucopenia, which would result from a destruction of a part of the white blood corpuscles (Lowit) on the ground that it is non-proven, the following causes of the phenomenon •of leucocytosis may be accepted : — rilE WHITE J5LOOD COIUMJSC LKS 159 1. Abnorinal disirihution. — This in nivn. .'iiid tran.sitoiy. The leucocytes colloot in the capillaries of tin; inteiDul organs after intravenous injcsctioiis. 2. Ahnurvifdljj hhihII fiupj)ly of leucocytes. — {<() Duo to toxic, functional inhibition of the foiniation of leucocytes, as in infectious diseases, ])()is(»iiing, and anaemia. {])) Duo to anatomical destruction of the If-ucopoietic organs, as in extensive tuberculosis or .car- cinosis of tlie lymphatic system, in which case the lymphocyte values are ]»ermanently and markedly lowered. It lias never been proved that negative chemotaxis affects the cells of the blood. .The morphological character of leucocytosis is Ijy no means uniform, and it is therefore necessary to divide the increase of the leucocytes into various groups, according to the kind of cell which participates in the increase. Ehrlich formerly recognised an active leucocytosis in which the cells obeyed a chemotactic law and migrated spontaneously into the blood, and a passive form in which the cells were washed mechanically into the circulation. In accordance with his view, that the lymphocytes are not endowed with any active movement, Ehrlich included all forms of lymphocytosis, including lymphatic leukeemia, among the passive leucocytoses. The extension, of the conception of chemotaxis rendered it impossible to adhere to this division, especially since the chief importance is invested in the influence exercised on the formation of cells in the organs, or in other words in organ function. "Whenever any kind of cell is present in the blood in increased numbers a more intense activity of the organ in which these cells are formed undoubtedly takes place. Leucocytosis may be divided according to the class of cell which is increased. An increase of more than one kind of leucocyte may not infrequently be met with in one disease. 160 ANEMIA A. — Polymorpho-nuclear NeutropMle Leucocytosis By far the most common form of leucocytosis is that in which the polynuclear neutrophils leucocytes are increased in numbers. A larffe number of the most different conditions and influences O lead to its occurrence. Virchow, the discoverer of leucocytosis, was of opinion that leucocytosis depended on an increased stimulation of the lym- phatic glands. He taught that the stimulation of the lymphatic glands consists in " the taking on of an increased production of cells and in the enlargement of the follicles, in which, after a time, many more cells are contained than before." The swelling of the lymphatic glands was supposed to induce an increase in the number of lymph corpuscles, and from this followed an increase in the number of white blood corpuscles in the blood. Ehrlich's researches necessitated the relinquishing of this view, since they showed that the migration of the polynuclear neutrophile cells was to a large extent responsible for the leucocytosis. Exact cell counts were first carried out by Einhorn under Ehrlich's direction, and later on the results obtained were generally confirmed. In correspondence to the increase which was limited to the neutrophile corpuscles, the percentage of the lymphocytes was always found to be diminished, at times to such an extent that these cells only represented 2 per cent, or less of the total number of white cells. It must, however, be remembered that the percentage of the lymph cells may be markedly diminished without their absolute number being altered. But it has frequently been found that, associated with the polynuclear leucocytosis, a decrease in the absolute number of lymphocytes takes place. The transition forms often show considerable increase in neutrophile leucocytosis, and single neutrophile myelocytes may be found * among these cells. This increase may even reach a moderately high percentage. Apart from the appearance of myelocytes and of numerous immature leucocytes, the fact that a few nucleated red blood corpuscles may be found in the peripheral THE WHITE BLOOD CORPUSCLES lOi blood in the absence of anaemia, speaks strongly in favoui- f)f the view that the bon(3 marrow is working at very iiigh pressure. In the ordinary forms of polynuclear neutrophile leucocytosis the eosinophile cells are usually absolutely diminished in number, as Ehrlich pointed out in his first publication on this subject. Tlie diminution is frequently a considerable one, and at times these cells may be absent altogether. In a few pathological conditions there may, however, be an increase of eosinophile cells in association with a polynuclear neutrophile leucocytosis. This will be dealt with under a separate heading. Polynuclear neutrophile leucocytosis — leucocytosis par excellence — may be divided into several groups according to its clinical occurrence. The following forms are recognised : — (i) PHYSIOLOGICAL LEUCOCYTOSIS The leucocytosis of digestion must be included in this group. This is said to occur after the ingestion of albuminous food. Japha, however, is of opinion that this is merely a physiological diurnal variation. While the older authors found that the lymphocytes were increased, more recently an increase of the neutrophiles has been said to occur. According to the more recent investigations, the leucocytosis of pregnancy only affects primiparce regularly, and even in them is but slight. It affects the neutrophiles chiefly. The leucocytosis of new-born infants is only marked in the first four days of life and is of a neutrophile character. Increased numbers of leucocytes can also be found in the peripheral blood after bodily over-exertion and thermic stimuli. It is, however, possible that vasomotor changes may be responsible for this, at all events in part. (ii) ATHOLOGICAL LEUCOCYTOSIS 1. The increase in the number of the polynuclear cells which occurs in infectious processes has been called inflammatory, in accordance with the principle: a potior i fit dcnominatio. They II 162 ANEMIA are nevertheless inflammatory toxic processes, since the toxins of the infective microbes determine the character of the leucocytosis. This has been proved beyond all doubt by innumerable experi- mental researches. It is particularly important to note that the majority of febrile conditions, c.^r. pneumonia, erysipelas, diphtheria, septic conditions of various origin, acute articular rheumatism, etc., are accompanied by a definite more or less marked leucocytosis. Uncomplicated enteric fever and morbilli alone occupy an exceptional position in this connection. The absolute number of white blood corpuscles in these diseases is decreased often at the cost of the polynuclear neutrophile cells. The reader is referred to the various text-books on hematology, and to the publications of Tiirk, Stienon, Schindler, Eeckzeh, Zappert, and others for the details with regard to this behaviour, and also for the course and termination of leucocytosis associated with the infectious diseases. The chronic infective processes, and above all tuberculosis, produce extremely slight changes in the blood, so that no constant variations from the normal can be ascertained. As a rule the acute infectious diseases begin with a consider- able neutrophile leucocytosis. This may, as is the case in measles, fall during the incubation stage, or, as is the case in typhoid fever, may last for an extremely short time ; as a rule it lasts for a considerable time. During this stage the lymphocytes are diminished and the eosinophiles are either greatly reduced in number or disappear from the blood altogether. As the infection passes off the lymphocyte curve rises again, as does that of the eosinophiles, and during convalescence the curves may reach a level higher than normal, which is spoken of as post-infective lymphocytosis and eosinophilia. This is in accordance with a general biological law which states that a diminished activity of a tissue is followed by an activity after recovery which exceeds the normal. 2. Toxic leucocytosis is met with especially in poisoning with the so-called blood poisons. The majority of blood poisons, such as potassium chlorate, the derivatives of phenyl-hydrazin, THE WHITE BLOOD CORPUSCLES 1 03 pyrodin, phenacetin, etc., in general appear to x^roduce a consider- able increase of leucocytes in human beings, as well as to destroy the red blood cells. This has been confirmed experimentally. It must further be mentioned that marked leucocytosis may be produced by the injection of tissue extracts containing nuclein and of nuclein alone. 3. The leucocytosis of ansemic conditions and ha-moirhages is especially well known as post-h[emorrhagic leucocytosis. It indicates a strong bone marrow reaction, which affects inter alia the white blood corpuscles. 4. The leucocytosis of malignant tumours is not constant, but may be very marked. The cause must be sought in various factors, e.g. in the absorption of toxic substances in the decom- position of fouling discharges. A particularly great increase is met with in metastases occurring in the bone marrow. In these cases nucleated red blood corpuscles and numerous myelocytes may pass into the blood, so that the blood presents an appearance similar to that seen in leukaemia. The so-called cachectic or agonal leucocytosis does not depend on cachexia or agony as such, as used to be held. It is frequently absent in both conditions. When it is present it is the result of the condition producing the cachexia or agony. It is quite clear that the conditions of the cells of the blood in the various diseases may be of considerable clinical importance. It is only possible to touch on a few of the more salient points in this place, and to refer the reader to the text-books on morpho- logical liEematology for further details. (a) The great importance in the differential diagnosis which attaches to the leucopenic blood condition in enteric fever as contrasted with nearly all other infectious diseases. The early diagnosis of measles during the incubation period. The recognition of trichinosis and the extraordinarily easy differential diagnosis between trichinosis and typhoid fever, which used to be difficult to make. 164 ANEMIA The importance of leucocytosis for the recognition of suppura- tive processes, and of the tendency of these processes to become extended. (h) The prognostic importance of changes of this kind in the blood, e.g. the absence of leucocytosis in severe diseases, in which a marked increase of the neutrophile cells would otherwise have- been expected from the nature of the process, would indicate an insufficiency of the bone marrow, and would therefore point to a very severe affection (examples : pneumonia, perityphlitis,, peritonitis, etc.). Ehrlich teaches that the origin of polymorpho-nuclear neutro- phile leucocytosis lies in the bone marrow. It is not necessary now to support this view with as many arguments as it was ten years ago. This does not mean that there are no persons left who believe that the origin of leucocytosis should be sought for in the inflammatory and suppurative foci, in the intestinal wall, in the mucous membrane of the uterus, and so on, but views of this kind may be treated to-day as curiosities. The origin of the neutro- phile elements in the bone marrow is firmly established, because- in no other organ are the precursors of the neutrophiles of the blood — the myelocytes — to be found. They are present in these organs in tissue formations in very large numbers. In this situa- tion all forms of transposition of the nucleus, and all forms intermediate to those of the cells found in the blood, are present. Mitosis is found in this situation, and even if there is no doubt that in certain pathological conditions myeloid formations appear in other organs, the functional significance of these extraneous formations is only very subordinate, save perhaps in leukaemia. B. — Polynuclear Eosinophile Leucocytosis. After Ehrlich had demonstrated the constant increase of the- eosinophile cells in leukaemia, a long time passed before eosino- philia was found in any other form of disease, the characters of which differed essentially from leuksemia. The first advance in this direction was made by Friedrich Miiller, on whose advice^ THE WHITE lU.OOI) CORPUSCLES 1G5 ■CJollasch examined tlio l)lf)()(I of asthmatics and found therein a •distinct increase in the nuinhor of eosinophile colls. Following this, H. F. Miiller and Kiedcr discovered that eosinophilia exists with great frequency in children and in connection with chronic splenic tumours. Next followed the well-known work of Edm. Neusser, in which he proved that a very marked increase of the oxyphilic elements occurs in pemphigus and almost simultaneously analogous observations by Canon in chronic skin diseases. It is only necessary to mention the comprehensive survey of this subject published by Zappert and K. Meyer from among the •enormous number of other communications. By the term eosinophilia is meant an increase of the cells of the blood affecting the eosinophile polyuuclear cells alone. It is quite impossible to confuse this form of leucocytosis with leukaemia, because a whole series of other characteristic signs is necessary for the recognition of the latter. These signs will be dealt with in the following chapter. It is not permissible to regard the presence of mononuclear eosinophile cells in the blood as absolute proof of a leukaemia, as some authors have done, since these cells are found in some cases of ordinary leucocytosis. The increase in the number of the eosinophile cells is in every case not only a relative, but also an absolute one. The percentage of these cells under normal conditions is from 2 to 4 per cent., but rises in eosinophilia to 10, 20, 30 per cent, and higher. Polynuclear eosinophile leucocytosis is found in manifold pathological conditions, as well as in healthy children, and for the sake of clearness these conditions may be divided into the following groups. 1. Bronchial Asthma. — In this disease an increase, which is frequently very considerable, in the number of eosinophile cells in the blood was first discovered by Gollasch, and this was confirmed later by a large number of other observers. The percentage may rise to 10 and 20 per cent, or higher. In hay asthma and hay fever absolutely similar conditions are met with. (For the special clinical course of eosinophilia in asthma, see below.) 166 ANEMIA 2. Pemphigus. — Neusser was the first to find an extra- ordinarily marked almost specific eosinophilia in some cases of pemphigus. This interesting observation has been confirmed by a number of workers, among whom Zappert should be mentioned. The latter found in one case as many as 4800 oxyphile cells in a c.mm, of blood. 3. Acute and Chronic Skin Diseases.— Canon was the first to notice that in a large number of skin diseases, especially in prurigo and psoriasis, the eosinophile cells may be increased up to 17 per cent. Canon pointed out one remarkable fact, that it is not so much the kind of disease or its local intensity as the extent of the process which determines the degree of the increase of the eosinophile elements. In one case of acute very extensive urticaria A. Lazarus found the eosinophiles representing 60 per cent, of all the leucocytes ; within a few days this enormous number of eosinophile cells diminished to the normal level. 4. Helminthiasis. — The first observations with regard to the occurrence of eosinophilia in helminthiasis emanated from H. F. Mliller and Kieder, who demonstrated fairly high values (8*2 and 9*7 per cent.) in two men suffering from ankylostomum duodenale. Shortly afterwards Zappert reported that he had found a considerable increase of these cells in the blood of two further cases of the same disease ; the value reached 17 per cent. He also found Charcot's crystals in the faeces. In a third case of ankylostomum, however, Zappert failed to find the eosinophiles of the blood increased, or any crystals in the fseces. Seige also found similar conditions. Leichtenstern, whose work on parasitology is well known, has published an extensive essay on this important subject. Under his direction Biicklers discovered the interesting fact that ankylostomum does not take an exceptional position among the diseases produced by worms with regard to the production of eosinophilia. He found that all the forms of worms observed in the Cologne Hospital, from the thread-worm which is generally regarded as harmless to the pernicious strongylus, produced an increase of the eosinophile cells in the blood, which at times THE WHITK KLOOD COJlPliSCLKS 107 reached a great height. BiicklerH reported that he found 16 per cent, of eosinophiles in oxyuris, 19 per cent, in Ascaris lura- hricoides, and Leichtenstern announced in a later communication that he had come aci'os.s a ca.se of ankylostomiasis with 72 per cent, of eosinophiles and one case of Tmnia mediocanelkUa witli 34 per cent. It is very remarkable that Leichtenstern was able to find large numbers of eosinophile cells especially in those cases in which the fcieces contained quantities of Charcot's crystals. Since eosinophile cells and Charcot's crystals have on other occasions been frequently found associated with one another (e.r/. in bronchial asthma, nasal polypi, in myelremic blood and bone marrow) it is quite reasonable to accept Leichtenstern's thesis, that eosinophile cells may be present in the intestinal mucosa in ankylostomiasis. This has, however, not yet been demonstrated. The almost constant increase in numbers of eosinophile cells in trichinosis has proved to be of great diagnostic value. This- fact was first discovered by T. E. Brown, working under the- direction of Thayer. He found the eosinophile value as high as 68 per cent, and the absolute value as high as 20,400. Since Brown's communication, analogous observations have been made by Schleip among others in a large epidemic, and by Opie and Straubli in experimental trichinosis. It has on many occasions been possible to clear up clinically obscure or doubtful cases by means of haematological examination. 5. The Post- infective Form of Eosinophilia (after the termination of various infectious diseases). — As has been men- tioned in the chapter dealing with polyuuclear neutrophile leucocytosis, a relative diminution of the number of eosinophiles or even a total disappearance of these cells may be seen at the height of the fever in the infectious diseases, with the one ex- ception of scarlatina. In the post-febrile stages, however, the highest values which can still be considered normal are often met with, or there may even be a distinct eosinophilic leucocytosis. When this occurs it is usually quite moderate in degree. Not infrequently, however, very considerable increases may 168 ANEMIA be noted, as in the case of typhoid fever, when the numbers may be as high as from 1200 to 1500; this can be seen more often if the blood is examined for a considerable time after recovery (two or three months). The eosinophilia appearing after, injections of tuberculin should also be included in this group. It must, however, be mentioned that, according to Fauconnet's researches, this condition cannot be regarded as proved. 6. Malignant Disease. — A number of authors have observed a moderate degree of eosinophilia in the cachexia of malignant disease ; the values do not exceed 7 to 10 per cent, in these cases. Eeinbach only found the eosinophile cells increased four times in forty cases of this kind. The values found were .7"8 per cent, in sarcoma of the forearm, 8'4 per cent, in sarcoma of the leg, and 11'6 per cent, in an abdominal malignant tumour. He also recorded a case of lymphosarcoma of the neck with secondary growths in the lymphatic glands, in which an enormous increase of the white blood corpuscles and especially of the eosinophile cells was present. The absolute number of the latter at one time was 60,000, which is equivalent to three hundred times the normal value ; such an increase has never been seen in any condition save perhaps leukaemia. A moderate degree of increase is not uncommon in the early stages, especially of carcinoma. The more the cachexia advances the greater is the tendency for the values to sink below the normal level. In the immediate neighbourhood of cancerous nodules veritable nests and collections of eosinophile cells are often met with. 7. Compensatory Eosinophilia (after elimination of the spleen). — This form of eosinophilia has been dealt with in detail in the chapter on the function of the spleen. It has been pointed out that the increase of eosinophile cells which has been found by Rieder, Weiss, and others in chronic splenic tumours may be attributed to the elimination of the spleen. The data with regard to this point need supplementing. THE WHITE BLOOD CORPUSCLES 1G9 8. Medicamentous Eosinophilia. — The only observation of thi.s kind published hitherto was made by von Noorden. He found eosinophilia up to per eent. in the blood of two chlorotie girls after they had taken camphor. The phenomenon could not be induced in other patients. An increase of the eo8inoi>hile8 can be noted in connection with other preparations; in these cases there is always a preliminary decrease. As is the case in the post-infective form of eosinophilia, this condition is un- doubtedly due to a toxic (toxic-infective) influence on the pro- duction of the eosinophile cells in the bone marrow, and not to a chemotactic effect. The function and cell production of the marrow is first diminished, and after recovery takes place the function is likewise restored and may even be increased above the normal level. It is possible that a direct casting out of eosinophile cells occurs without any destruction of the same in the early stages, or in other words that there is a negative chemotaxis in this case; but this question requires further careful investigation. 9. Nervous Eosinophilia. — The origin of this phenomenon is still obscure, but there is no doubt that it takes place. Quite considerable increases in number of eosinophile cells are seen not infrequently in neurasthenia. The author has found as high a value as 10 per cent, in nervous diarrhoea associated with colic. The analogy with the conditions obtaining in bronchial asthma is so obvious that it is unnecessary to dwell upon it. 10. Scarlatinal Eosinophilia. — Scarlatina is the only bacterial infection which yields an increase of the eosinophiles at the height of the fever. This usually takes place on the second day of the fever. As a rule, only moderately high numbers are found, such as 500 to 1000, but at times they may be as high as 2000 or 3000 during the acute stages. It is quite possible that this form of eosinophilia is related in some way to the exanthem ; in scarlatina without a rash no increase is observed. 1 1 . Leukaemic Eosinophilia. — The increase in the numbers of the eosinophile cells in myeloid leukaemia is practically a 170 ANAEMIA regular occurxence, and the absolute numbers at all events are often . very high. This subject will be considered in detail subsequently. Various theories have been enunciated with regard to the origin of the polynuclear eosinophile leucocytes. Ehrlich found it necessary to establish his view of the bone marrow genesis of these cells in the first edition of this work by the application of much ingenuity. It is no longer necessary to defend this view. The theories which assume that the eosinophile cells are derived from the neutrophile cells within the blood vessels are not supported by any histological evidence. Such an occurrence can never have been observed, and this view need therefore not be taken seriously. The bone marrow genesis is quite clear, and may be demon- strated histologically by means of modern section staining. As is the case with the neutrophile cells, the bone marrow is the only site where eosinophile myelocytes occur under normal con- ditions and in which all the forms intermediate between the myelocytes and the polymorpho-nuclear cells are met with. Several authors believe that the eosinophile cells can be formed locally. This is, however, not the case under ordinary conditions. The infiltrations around the trichinee, the enormous peribronchial collections in asthma, and the collections in the intestinal walls have been proved to be chemotactic, since only polymorpho- nuclear cells and no myelocytes are present. Mononuclear eosinophiles are, it is true, seen in sputum, but these cells are involution forms, for they are not so large as myelocytes. The nucleus is very small, and it is not possible to demonstrate a chromatin network in them. Nevertheless, under quite exceptional conditions, an extra- medullary genesis of the eosinophile cells may take place, but not from any chance connective-tissue cell. This can only occur in connection with a vessel, and the formation takes place either from adventitial cells or in consonance with Schridde's views, from " cells of the vascular wall." Such formations, however, are never exclusively eosinophile. They always reveal myeloid com- THE WHLTK JJLOOI) C OKPIJSCLKS J71 plexes, in whicli iioutropliilo chUh find even nucleated red cells are also formed. These extraniedullary myeloid I'nci am v(;iy widely dissemin- ated and fre(|uent in the embryo. In some animals they are also met with in the adult. They are found in man in infective processes, in intoxications, and in anremias, more especially in leukaemia, and may be very extensive. That the collections in bronchial asthma are really chemotactic is proved by the way in which the proportional numbers of the eosinophile cells vary to a large extent. According to Heineke and Deutschmann, the number of eosinophile cells in the blood in bronchial asthma sinks very materially after the attack. This would imply that a temporary functional eosinophilia is present which, as is the case in the analogous conditions of the neutrophiles, can only be the result of an increased activity of the bone marrow. The question which cells produce chemotactically active substances out of their disintegration products is a very important one, but is one which cannot be decided at present with certainty. The ordinary pus cells and the lymphocytes do not appear to produce any such substances on disintegration ; on the other hand, there are many reasons for believing that the dissociation products of epithelial cells and of epithelioid cells act chemotactically. In this way the frequent occurrence of eosinophilia in the various forms of skin diseases might be explained. The same explanation would hold good for the appearance of local collections of eosino- phile cells in all atrophic conditions of the gastric, intestinal, and bronchial mucosa, and also for the increase of these cells in the neighbourhood of carcinomata. A further argument in favour of this view is the fact that the eosinophile cells are more numerous in bronchitis and asthma when the secretion contains but few pus cells. In the last place, the observation made in scarlatina, that when no rash is present no eosinophilia takes place, also speaks in favour of this view. On the other hand, there is no doubt that foreign substances circulate in the body which are able to exercise a positive chemo- tactic influence on the eosinophile cells. Goldmann found in 172 ANiEMlA sections of the pancreas which contained Proteus sanguineus, that the eosinophile cells were markedly increased in number in the neighbourhood of the encapsuled parasites, while he sought for these cells in vain in other areas. Opie came across similar con- ditions in connection with encapsuled trichinae, but Straubli emphasised the presence of interstitial foci of eosinophile cells under these conditions. Proscher reported a very striking condition, by producing an eosinophilic pleurisy experimentally by means of extracts of the tape-worm. In this connection the observations made in the various forms of helminthiasis (see p. 166) are of considerable importance. It was formerly thought that the action of worms was a purely local one. The view that their action is due to toxic substances which they produce is now gaining considerable support. Linstow has pointed out that the general typhoid condition, and also the fatty degeneration of the liver and kidneys, i.e. of organs in which the Trichince are not found, necessitate the assumption of a toxin in trichinosis. The symptoms produced by the Bothriocephalus latus are now regarded as being due to a specially produced poison. Even the ordinary tape- worms effect a damage to the organism not infre- quently, and this damage is to be ascribed to the production of a poison (Peiper). These considerations justify the deduction that tape-worms not only take up substances from their hosts, but also give off other substances which may be taken up by the intestine of the host and which may exercise a distant action. One sign of this distant action, as Leichtenstern has pointed out, is the appearance of eosinophilia of the blood. The author believes that the above- mentioned facts absolutely exclude the possibility that the sub- stances which attract the eosinophile cells are identical with the substances which produce ansemia. Several observations, such as that of the absence of eosinophilia in BothriocepJialus anosmia (Schauman and Naegeli) point to the probability of the existence of two separate functions. At all events, the substance which THE WHITE 15IX)()I) ClOliPUSCLES 173 produces the eosinopliilia is iiiucli more widely distributed than the substance wliich is responsible for the anjemia. In order to consider how polynuclear eosinopliilia is pro- duced, it may be advisalde to have regard, in the first place, to an experiment wliich was performed by E. Neusser. Neusser found that the contents of the bulla' in a case of pemphigus con- sisted ahnost exclusively of eosinophile cells. The blood of this patient showed a considerable increase of eosinophiles. Neusser thereupon produced a non-specific inflammatory blister by means of a vesicant, and found that the cellular contents of this blister were exckisively polynuclear neutrophile pus cells such as are met with in all simple inflammations. Leredde and Perrin met with conditions which were absolutely analogous to those of Neusser's case, without the assistance of any experimentally produced lesions, in what is known as Diihring's disease. The vesicles which occur in this dermatosis contained only polynuclear eosinophile cells as long as the fluid remained clear. In a later stage, as is usually the case, bacteria invaded the vesicles, and when this occurred they were found to contain cells with neutrophile granules only. ISTeusser's experiment and Leredde and Perrin's observations can only be explained, in accordance with the modern views of the nature of suppuration, by assuming that the eosinophile and the neutrophile cells possess chemotactic susceptibility. This view has already been supported in this work. According to this conception, the eosinophile cells would only migrate towards those sites which contain substances which specifically stimulate these cells. All the experiments and clinical observations on eosino- pliilia which have hitherto been recorded may be explained lege arfis on this assumption. ISTeusser's experiment may be analysed as follows. A substance is present in the bullfe of the pemphigus ease which attracts the eosinophile cells chemotactically. The eosinophile cells which are normally present in the blood migrate from the circulation and produce an eosinophilic suppuration. When the disease is not very severe the process may be regarded as being limited to a great extent to the localised phenomenon. 174 ANJEMIA A totally different picture is developed, however, when the disease* involves considerable areas of the body. Under these conditions a large quantity of the specific active agent is taken up into the blood stream by diffusion and absorption, and from this situation it exerts a strong chemotactic action on the physiological depots of the eosinophile cells, i.e. on the bone marrow. This leads to a more or less marked increase of the eosinophile cells in the blood. The bone marrow, in consequence of the increased migration, is stimulated to produce more cells, in accordance with general biological laws, and thiis retains the power, even when the disease lasts for a long time, of keeping up a continuous eosino- philia. Other clinical experiences may also be explained satisfactorily in this manner. Gollasch found that the sputum of asthmatics only contains eosinophile cells in addition to Charcot's crystals. It must therefore be supposed that the interior of the bronchial tree contains a substance which attracts the eosinophile cells. The close relationship which has been shown by numerous clinical observations to exist between the severity of the disease and number of eosinophile cells in the blood may also be regarded as pointing to this view, von Noorden was able to show that the eosinophile cells in the blood are more numerous about the time of an attack than when a considerable time since the last attack has elapsed. They were present in especially large numbers when the attacks followed one another rapidly during the course of several days. That the increase in number of the eosinophile cells in this case depends directly on the attack and is not merely a sign of a persistent anomaly of constitution is proved by a case which von Noorden reports. During the attack he found 25 per cent, eosinophiles, while a few days later he only found one single cell in twelve cover- slip specimens, which means that there was actually a diminution of this group of blood cell. Canon had the same experience in skin diseases. He was able to show that the degree of the eosinophilia depended on the local extent of the affection rather than on the intensity. THE WIHTK 15L()()1) COIMHJSCLES 17r, This means thaC the eosinophilia depondH on that factor whir^h determines the quantity of the .specific agent in the blood. All tliose laws which have heen described as governing neutrophile leucocytosis ar.; ai.plicable in the case of eosinophile leucocytosis also. One of the most important facts in this connection is that no eosinophilia occurs when the bone marrow function is paralysed, even if ('hcniotactically active substances are present. Striiubli produced very severe experimental trichinosis which ran its course to a fatal termination with a complete absence of acidophile cells; while Liermberger's ankylostomum patients only showed low percentages of eosino- phile cells during a very severe illness. On the application of arsenic the number increased from 3-2 per cent, to 33-7 per cent., in spite of the fact that the worms were not driven out. Leichtenstern noticed on a previous occasion that a croupous pneumonia in an ankylostomum patient could depress the number of eosinophile cells from 72 per cent, to 6 or 7 per cent., and that the former values were closely approached at a later date again. The presence of chemotactically active substances is there- fore not to be regarded as the final determining cause. A capability of reacting on the part of the bone marrow in addition is necessary. The quantity of toxin may be even too large, as in experimental trichinosis, so that the eosinophilia may be partly or completely prevented. Opie was able to show that an overdose of the agent does not produce a stimulation of the marrow, but actually kills the cells. These considerations prove that the eosinophile curve records the function of an organ, and it therefore follows that it is quite impossible for the changes in the blood and tissues to depend on a local histogenic formation. C— Mast Cell Leucocytosis The increase of this form of white blood corpuscle is undoubtedly rare, and with the exception of the case of 176 ANiEMIA leukaemia this increase is small. It must, however, be borne in mind that normal blood contains a very small number of these cells,- so that even a triple or quadruple increase would scarcely be of importance in consideration of the number of the other cells. Mast cell leucocytosis has been seen in the following con- ditions apart from leukaemia. In skin diseases, in suffusion of milk of the human breast (Unger), in urticaria pigmentosa (Sabrazes). Levaditi has produced mast cell leucocytosis in animals. The real mast cells of the blood with their characteristic polymorphous form of nucleus must not be confused with the mononuclear mast cells of the tissues, which are often found in large numbers in chronic inflammations, in indurations of the lung, in skin diseases, and in inflamed lymphatic glands. These latter cells are absolutely different structures, and possess practically no relationship to the mast cells of the blood. The only character which they possess in common with the blood cells is the presence of basophile metachromic granulation. The tissue cells have small round nuclei, which take on the stain of nuclear dyes well. The mast myelocytes of the bone marrow, which represent the precursors of the blood mast cells, are quite different from these tissue mast cells, and it needs only slight histological knowledge to see that an intimate connection between these two kinds of mast cells cannot exist. VL— LEUKEMIA, OR LEUCOCYTH.ffiMIA The interest which the investigator and the clinician has taken in leukaemia has not diminished during the past ten years. This is shown by the innumerable publications on the subject, and by the many theories which have been evolved on the pathogenesis of this remarkable disease. Much that has been suggested in this connection has enjoyed but a short life and has soon been forgotten. It has been shown THE WIIITK HI.OOI) COIMMJSCLKS 177 tli;i,t tli(! study of Liu; lihjod mid its cells is inc;i));i,Mc oF llnow iiig light on the genesis of tlie disease, hut that advance can only be expected from a most careful examination of the organs, in combination no doul)t with a iiiiiiut(; aiudysis of cells in the microscopical sections of thesci organs. TJie icsulls of tiiis kind of investigation became so jdentifid when this was recognised that our knowledge of leuktemia thereby lias been very materially extended. It may be worth while first to follow the march of research since the discovery of lenkicmia. In this way those problems which engage the attention of the investigator at present will present themselves automatically. At first a lymphatic, a splenic, a spleno-medullary, and a pure medullary or myelogenous form of leukcemia or leucocythtemia were distinguished on the basis of their clinical appearances. This classification depended on purely external and gross signs which could not be recognised in hematology. Such a classification takes the extent of the changes in the organs into consideration in the first place, but not the nature of these changes. It ignores the most important factor of all,- the kind of cell proliferation. Neumann was the first to show that in lymphatic leuktemia the lymphatic proliferation is not limited to the lymphatic glands, but may affect the spleen and bone marrow. These proliferation processes may cause an enormous enlargement of the spleen, without any change taking place in the specific character of the leukemic process or of the appearances of the blood. In spite of the splenic tumour, the case is one of lymphatic leukaemia. In ordinary clinical terminology such a case is spoken of as " lymphatic splenic leucocy thtemia." The unreliability and incorrectness of such a term can best be demon- strated in another form of leuksemic proliferation. The liver may become enlarged to the size of a large tumour in lymphatic leukaemia by the production of lymphomata, and logically speaking this should be termed a " lymphatic hepatic leukcemia." This term would not be so prone to lead to error as the term 12 178 ANEMIA " lymphatic splenic leuksemia " ; for no one would imagine that in the- former the liver cells pass over into the circulating blood, while' the latter term suggests that the specific splenic cells take a part in the changes in the blood. The recognition of a pure splenic form of leuksemia is to be regarded as unjustifiable from the point of view of hsematological and histological investigations. After what has been said with regard to the physiological participation on the part of the spleen in the formation of blood, the probability of a blood change which is specifically due to an affection of the spleen is almost excluded. This view has received full confirmation from the results of pathological investigations. There is not a single case in the whole of the literature of the subject in which a pure splenic leuksemia could be accepted. The conditions obtaining with regard to myeloid leukaemia are similar to those mentioned above, in so far as the occurrence of myeloid tissue in the spleen and lymphatic glands is con- cerned. Since the proliferation of this tissue and not the accompanying swelling of the spleen or lymphatic glands is the specific factor in the process, it follows that the term " spleno-medullary " or " medullary lymphatic " leukaemia is also illogical and likely to lead to error. Ehrlich therefore recognised from the hsematological stand- point two forms of leuksemia. 1. Leuksemic processes with proliferation of lymphatic tissue — Lymphatic Leuksemia. 2. Leuksemic processes with proliferation of myeloid tissue — Myeloid Leuksemia. If it be found advisable, there would be no objection to indicate the accompanying clinical signs by the addition of words which would not give rise to misunderstanding ; for example, "lymphatic leuksemia with swelling of the spleen or of the liver," or " myeloid leuksemia with enlargement of the lymphatic glands," and so on. This classification, which was made on the basis of THE WTTITR lU.OOD CORPUSCLES 170 cytological coiiBidonitioiiH, Iiiis in-ovail Lo Ix; absolutely satis- factory in the light ol' Hubsequent histological researches. The study of the organs showod f(uit(', dcntiitdy that there are only two forms of leukicinic, ])rolir(M;i,tioii. In tlie one form the lymphatic tissue and on the, otbci' tin; niodulbuy tissue is aCleeted in o()i'res])ondene(; with LIk; bict tli;it there ;tr(; only two forms of tissue which form leucocytes nornjally. liefore these aspects of this disease are dealt with it may be advisable briefly to survey its clinical manifestations, since, in view of the radical differences of the clinical appearances of the two forms of leukasmia, both should be recognised. Lymphatic leuka3mia may be divided into two clinically distinct forms. First, acute lymphatic leuksemia may be recognised by its rapid course, by the small degree of swelling of the spleen, by the tendency of petechial haemorrhages to appear, and by the general hasmorrliagic diathesis. This form of disease has given the majority of clinicians the impression of an acute infective process on account of its fulminating course. The second form of lymphatic leuktemia is distinguished from the preceding form by its chronic, frequently protracted course. The spleen usually participates in the disease, in taking on a very considerable swelling. Hsematologically, all forms of lymphatic leukaemia are characterised by a marked predominance of lymph cells. Either the small or the large lymphocytes may be present, or a variable mixture of both forms. It must be emphatically pointed out that the pre- ponderance of large lymph cells is by no means characteristic of the acute form of lymphatic leukaemia, since the same blood changes are found in very slowly advancing chronic cases. In a case of this kind, which was being treated in Gerhardt's clinic, all the observers who examined the blood (G-rawitz, von jSToorden, Ehrlich) were able to find the large cells during the whole course of illness. The histological examination of the organs has yielded an indisputable explanation of the origin of this form of leukaemia. The foci of lymphatic production are not only very extensive, 180 ANiEMIA but, as is proved by the occurrence of mitosis, are in a state of considerable activity. The disease therefore depends on an enormous increase in the output of cells, and the cells thus formed are passed over to the blood obviously in the same way as under normal conditions. Chemotactic laws do not come into play at all, neither are the cells passively washed out of the organs. Hyperfunction of the tissue is the characterising factor of the process. Myeloid leukaemia presents a picture which is totally different from every point of view. In former years great difficulty was experienced in differen- tiating between myeloid leukaemia and simple leucocytosis ; the two phenomena were even regarded as different degrees of the same pathological process, and it was thought that when the proportion of the white to the red blood corpuscles exceeded a definite limit (1 : 50) leucocytosis ended and leukaemia began. The fundamental differences of the two conditions were only recognised when the cells were analysed with the assistance of staining methods. Leucocytosis is now recognised as a condition in which the normal polynuclear neutrophile leucocytes are merely increased in number, while in myeloid leukaemia elements are introduced into the blood stream in large numbers which are not normally present in it. The cell forms which are thus introduced into the blood are so characteristic that the diagnosis of leukaemia is possible even in those very rare cases in which the total number of white blood corpuscles is not materially increased or is actually diminished. It is, however, necessary in cases of this kind to exercise a critical judgment and to rely on experience, since marked dis- turbances of leucopoiesis may be present in severe anaemias without the condition necessarily being leukaemic. It is possible with the assistance of modern hsematological technique, in accordance with Ehrlich's principles, to recognise leukaemia with certainty in practically every case from the appearance of the blood. Difficulties are only temporarily met THE WHITE HLOOI) CORPUSCLES 181 with in those cases in which either the disease is still in a very early stage — this stage, however, is very rarely seen — or when the leuk.'cinic characters are temporarily ohsciired by the occurrence of complications siK^h as a,n infective disease. The opposition to the recognition of the changes in the blood in this disease has now been finally removed. Although this found its way into the text-book on ha^matology of ten years ago (v. Limbeck), the arguments used then seem quite incompre- hensible now. The microscopical appearances of myeloid leukii-mia are characterised by an increase in the number of white blood corpuscles, which is almost always considerable, and by the variegated and changeable character of the cells. The latter is due to the complication of several anomalies, which consist in : — 1. That besides the polynuclear cells their precursors, the mononuclear granulated leucocytes, the myelocytes circulate in the blood ; 2. That in the increase of the white blood corpuscles all three types of granules are met with, i.e. the neutrophile, the eosinophile, and the mast- cell granules; 3. That atypical forms of cells, e.g. dwarf forms of various kinds of white blood corpuscles and also mitotic figures are seen ; and 4. That the blood always contains nucleated red blood corpuscles, often in great numbers. 1. It is advisable to begin by dealing with the Mononuclear neutrophile cells, Ehrlich's Myelocytes. These cells are present in such large numbers in the blood of medullary leukaemia that they give the whole picture, at all events in the later stages, a predominating mononuclear character. Under normal conditions the myelocytes only occur in the bone marrow, as has been stated repeatedly, and never in the cir- culating blood. The signal diagnostic importance of the presence of these cells is not detracted from by the fact that they occur temporarily in feome other conditions. Even if they are found as one of the signs of a general leucocytosis in the critical period of a pneumonia, it is unlikely that the condition could 182 ANEMIA be confused with the blood changes of leuksemia. This is safe- guarded : (1) By the much smaller increase of the white cells generally ; "(2) by the diminution of the eosinophile and mast cells ; (3) by the predominating polynuclear character of the leuco- cytosis, which is not obscured by the presence of a small number of myelocytes ; and (4) by an incomparably smaller absolute number of myelocytes. If Turk's most extreme case be taken, namely, one of croupous pneumonia in which the percentage of the myelocytes reached as high as 11 '9 per cent, of the total number of leucocytes, it will be seen that the absolute number of these cells per cubic millimetre was 1000 at most. This number is one which cannot compare with the number of myelocytes in leuksemic blood, which may reach in an average and certainly not exceptional case, 50,000 to 100,000 per cubic millimetre and higher. Some difficulty may be experienced in connection with the so-called atypical leuksemias. The conditions obtaining with regard to this form are so complicated that it appears inadvisable to discuss them in detail in this work. The author therefore prefers to reserve this for a further communication on the subject. He also refers the reader to the chapter dealing with this subject in his text-book (page 364). 2. The Mononuclear Eosinophile Cells. — Mosler described large coarsely granulated cells, medullary cells, as characteristic of the myelogenous form of leukaemia even before the intro- duction of modern staining technique. These cells must be regarded as being to a great extent identical with mononuclear eosinophile cells, to which Mliller and Eieder called attention as a special form of cell, and which they appropriately described as the eosinophile analogies of the myelocytes. These cells are large, rather bulky elements with oval nuclei which stain feebly. In spite of the fact that these cells are undoubtedly a valuable sign of a leukasmic affection, their importance in the diagnosis of this condition is not so great as that of the mononuclear neutropliile cells, on account of the numerical superiority of the latter. It is not permissible to diagnose a TIIK WIMTK VAAH)\) ( OIMMJSCLKS \H'4 leukaemia alone from Llic pru.senco of " eoHiuoijIiile myelocyteH," becaiLse they do occur, alhoit in small TiuiribfirH, in other afCections. 3. The Absolute Increase of the Eosinophile Cells. — Ehrlich has always iauglit, since lie first published his opinions on leukicmia, that the absolute numljer of the polyniiclear eosinophiles is always much increased in myeloid leuka-mia. This statement of Khrlich's did not remain uncontradicted, von Limbeck thus speaks of the " alleged " increase of the eosinophile cells in his text-book. It was chiefly the well-known work of Miiller and Kieder which stimulated this opposition and which awakened doubt with regard to tlie diagnostic significance of the eosinophile cells. These authors, however, founded their opposition on false premises. Ehrlich did not speak of an increase in the percentage of eosinophile cells, but only of an increase in their absolute numbers. Even if a normal percentage of eosinophiles is found in a case of leukaemia, this must indicate a great increase in the absolute numbers, and Miiller and Kieder would have been able to have confirmed Ehrlich's statements if they had only calculated the absolute numbers in their cases. Out of the seven cases given in the work bearing on this subject, only three are given in sufficient detail to enable the absolute number of the eosinophile cells to be calculated. From these data the values are : — Case 29. — 3-5 per cent, eosinophiles = 14,000 per c.niin. Case 30.— 3-9 per cent. „ = 8000 Case 31.— 3-4 per cent. „ =11,000 Zappert calculated that 250 per cubic millimetre was the highest number of eosinophiles which could still be considered normal. As compared with this number, the average of the three cases cited, which works out at 11,000, is nearly fifty times as great. In this way the results of Midler and Eieder's own counts fully confirm Ehrlich's statement. Since this time a very large number of further observations 184 ANEMIA have confirmed the correctness of the view that a marked increase of eosinophile cells takes place. In exceptional con- ditions, however, this increase may be absent, as when the disease is complicated by septic or infective processes, and also in the atypical and acute cases. At the time when Ehrlich set up the doctrine of the diagnostic importance of eosinophilia in leuksemia, simple eosinophile leucocytosis (see p. 164) had not yet been recognised. This was only found at a later date in comiection with asthma, etc. But even this further discovery did not overthrow the correctness of the doctrine. Confusion between those con- ditions which accompany eosinophilia and leukaemia is quite excluded, since there is not the slightest resemblance between the clinical aspects of these conditions. But apart from this, the appearance of the blood offers plentiful differentiating characteristics. (1) The total increase in the number of white cells rarely reaches a degree which could remind the hsemat- ologist of leuksemic blood ; (2) eosinophile leucocytosis is exclus- ively polynuclear; (3) mast cells and neutrophile myelocytes are almost completely absent. A further argument in favour of the diagnostic value of the absolute increase in the number of eosinophile cells is found in those cases which present a blood picture which is extremely like that of leuksemia, but in which the diagnosis of leukaemia can be excluded by the absence of eosinophile cells. An instance of this is found in a case of carcinomatosis of the bone marrow described by Epstein. The blood in this case presented an appearance of anaemia such as is nearly always found in leukaemia, and revealed further an increase of the white cells similar to that seen dn leukaemia, with numerous neutrophile myelocytes and nucleated red blood corpuscles. Every one who, like Mliller and Eieder, holds that the number of eosinophile cells need not be taken into account in making the diagnosis, would have diagnosed this as a case of myeloid leukaemia. This was, in accordance with Ehrlich's teaching and with the actual state of affairs, excluded by the absence of eosinophile cells. THE WIiriK liLOOI) COKIMJSCLKS 185 In view oi' all these consideratioiiH il In lulvisjihlr;, in accord- ance with Ehrlich's teaching, to regard an ab.solute increfiwe in the numbers of eosinophile cells as a very important symptom in the diagnosis of leukseniia, wliich actually Ixjlongs to the nature of the disease. Great caution should be exercised in making the diagnosis in the absence of this symptom, and it should always be borne in mind that this indicates a very unusual condition, for which some exjilanation will liavc to be found. 4. The Absolute Increase in the Num,ber of Mast Cells. — Mast cells are nearly always increased in number in myohjid leuktemia. It is possible to count these elements in leuktemic blood when the films are stained by triacid or by eosin-methylene- blue. When stained by the former they appear like polynuclear non-granulated cells, since their granules do not take on any stain from the triacid mixture, and these cells have therefore been described by Uthemann in his dissertation and classified as non-granulated cells. It was only at a later date that Ehrlich recognised them as mast cells. The mast cells are more easily recognised after staining with Giemsa, or still better with Jenner's stain, since the granules are not dissolved in the methyl alcohol as they are in watery solutions. The increase of mast cells is an absolute and very considerable one in nearly every case of myeloid leukaemia. They are usually half as or quite as numerous as the eosinophiles, and at times they may even be present in still greater numbers than the latter. It follows from this that the mast cells increase at a relatively higher rate than do the eosinophiles, as their normal percentage of the total number of the leucocytes is only about 0-28 per cent. The diagnostic value of the increase of these cells in myeloid leukffimia is perhaps even more valuable than that affecting the eosinophile cells, especially because at present no other condition is known in which a marked increase of mast cells is met with. It must, however, be borne in mind in this connection, that in certain exceptional conditions, such as acute and atypical cases, the increase is usually not present or these cells may be altogether absent from the blood. 186 ANEMIA 5. Atypical Forms of White Blood Corpuscles. — These are : («) Dwarf forms of polynuclear neutrophile or eosinophile elements. • They were first described in connection with leukaemia by Spilling. As a rule they are merely small specimens of normal polynuclear cells, (b) Dwarf forms of mononuclear neutrophile and eosinophile leucocytes. The significance of the dwarf forms of the leucocytes in leuksemia is not yet sufficiently explained, and it is difficult to decide whether they enter the blood as small structures or whether they become smaller in the blood by fission and by constriction. It is, however, more probable that their production was faulty from the beginning, in correspondence with the overproduction of cells, (c) Cells show- ing mitosis. It was formerly believed that the detection of mitosis in leuktemic blood was of considerable importance, since it was held that this phenomenon signified that the increase of the white blood corpuscles took place in the circulating blood as a result of the process of fission. This view was defended more especially by Lowit. A number of authors (H. F. Mtiller, Wertheim, Eieder) have demonstrated the occurrence of mitosis, more especially of the myelocytes in leuksemia in the circulating blood. The mitosis, however, is not of any diagnostic importance. In the first place, it can only be demonstrated by the application of special methods ; and secondly, it is present only in very few cells. Miiller stated that he had to examine many thousands of white blood corpuscles to find one single instance. Only in one case did he meet with somewhat more numerous specimens, but even then the proportion was one nucleus undergoing mitosis to several hundred leucocytes. This find, which must be regarded as practically a negative one, teaches that mitosis only plays a negligible part in the increase of cells in the blood. It is of no value in the diagnosis of leukaemia. 6. Myeloblasts.^ — -The blood of every case of myeloid leu- ksemia contains a certain number of non-granulated cells of the myeloid system, — myeloblasts (Naegeli). These structures were formerly confused with Ehrlich's so-called large mononuclears THE WHITE IJEOOI) COHTUSCLES 187 (they can be readily distinguiHlied i'rom tliese by the iincleuH) or with the lai'<4'e lymphocytes, or else they were all included in one elass. A more exact analysis, howcjver, reveals tliat tljey are totally different cells. These cells stiike the experienced morphological investigator at once as a s])(!c,i;il kind of cell, and the marked essential correspondence with tlio myelocytes is clearly noted. The points which indicate the analogy to the myelocytes are the colour of the staining, the size of the nuclei and its proportion to the protoplasm of the cell. The nucleus usually includes several nucleoli (from two to four), which are well seen when stained by Giemsa. The protoplasm is basophile. At times early granulation of a neutrophilic nature may be seen in these cells, and when stained by Giemsa and triacid stains a large number of every conceivable intermediate form between myeloblasts and myelocytes may be met with. It is quite clear and obvious for many reasons that these cells are not lymphocytes. In the first place, the development of a neutrophile granulation proves that they cannot be cells of lymphatic tissue, since this tissue is not capable under any circumstances of producing neutrophile granules. In the next place, it would be necessary to ascertain where the lymphocytes could come from, since histological research shows that the lymphatic tissue is eliminated and substituted by myeloid tissue. The final proof against the lymphatic nature of these cells is obtained by staining with Schridde-Altmann's dye mixture. These cells stained in this way do not show any fuchsinophile granulation, which is always present in lymphocytes. There are besides biological reasons for deciding that the non-granulated cells must belong to myeloid and not to lymphatic tissue. These cells increase very extensively immediately before death and in acute exacerbations of the disease, as Ehrlich first noticed and as will be described later. It would be most extraordinary if under such conditions a lymphatic cell production should become prominent. Much more probable would be the production of the least mature and most indiiierent form of myeloid cell. This suggestion has actually been made by Turk. However, histolo- ■188 ANEMIA logical tests must decide primarily in such cases, and these tests have decided that lymphatic tissue does not proliferate in myeloid leukaemia, but is crushed out of existence, and that the myeloid character of the proliferation is actually proved by the presence of large numbers of myeloblasts. A further argument in favour of the view sketched above, and one which in the opinion of the author is very convincing, is that all acute forms of myeloid leukaemia (see p. 191) show high and steadily increasing myeloblast values from the beginning. The sending forth of such an immature medullary cell must therefore be regarded as a sign of exhaustion, and of an absolutely pre- cipitated cell formation of the myeloid tissue. Ehrlich mentioned these forms of changes in leuksemic blood in the first edition of this work, and pointed out that such occurr- ences at times might give rise to serious difficulties in the diagnosis. He wrote on page 126 of the first edition of this work : — , Zappert reports the case of a patient who presented the typical appearances of a myeloid leukaemia in February 1892. Inter alia, the proportion of the white to the red blood cells was found to be as 1 : 4'92, and 1400 eosinophile cells per c.mm. (3 '4 per cent.) were found. The patient was admitted in a very pitiable condition into hospital toward the end of September of the same year and died soon afterwards. During this period of observation the counts showed a ratio of whites to reds of 1 : 1"5 ; a percentage of 0"43 eosinophiles, the majority of the mononuclear cells were free from all traces of neutrophile granulation, and represented about 70 per cent, of the white cells. Zappert emphatically points out that these cells were not in the least like lymphocytes. Zappert found at the post-mortem examination that the bone marrow was infiltrated with a large number of non- granulated mononuclear cells, while the eosinophile cells were considerably less numerous than they usually are in the bone marrow in leukaemia. Dr. Blachstein, under Ehrlich's direction, examined a second case of this kind. The patient had likewise been under careful clinical observation on account of a myeloid THE WHITE HLOOD COIMMISCLES 189 leukfiomia for a long tiiiio. During- his la«t slay in luispital the examination could only be carried out one day before he died. The (loath was due to a septic complication. It was found that the blood showed all the marked chjuaclfiiistics of leukii-mic blood. There were 62 per cent, polynuclear ceils and 17'5 per cent, mononuclear non-granulated myelocytes of about the size of ordinary myelocytes, 0*75 per cent, eosinophile cells, and moderate quantities of nucleated red blood corpuscles. The preponderance of polynuclear and the small number of eosinophile cells was accounted for by the presence of the septic infection ; on the other hand, the absence of granules in the mononuclear cells was very curious. Both these cases can only be adequately explained by pre- suming that in certain terminal stages the organism loses the power of forming neutrophile substance. Analogous conditions occur in uon-leuksemic affections ; for example, in a case of post- hsemorrhagic anaemia described by Ehrlich. In such cases it is of great importance to keep in mind these rare cases, which are usually not taken into consideration at all, since this want of knowledge could easily give rise to gross errors with regard to the nature and origin of the mononuclear cells, and might lead to the assumption of a splenic form of leukaemia. It will be seen how both these investigators adhered to the myeloid character of the blood formation. Since this publication a large number of further observations have been reported in this connection (Naegeli, Hirschfeld, Billings and Capps, Warburg, von Jaksch, Mager and Sternberg), and minute histology, biology, and detailed morphology have proved concurrently that these cells are not lymphocytes, but really myeloblasts. There still remains one thing to be proved, whether, as Ehrlich and Helly have assumed, the myelocytes have lost theii- granulations, or whether these myelobasts are to be regarded as a new form of cell, a precursor of the myelocytes. The latter view is the more favoured one at the present date, for these cells cannot be distinguished from the myeloblasts which are normally present in the bone marrow, and the study of the cells themselves 190 ANiEMIA shows that they are young immature cells, because their protoplasm still has a marked basophile reaction, and because granules very frequently appear immature in young forms. This has been observed in cells with commencing neutrophile, and especially well marked in cells with eosinophile, granulations. 7. Nucleated Red Blood Corpuscles. — These cells are constantly found in the blood of leukaemia. Their number in the various cases is very variable ; at times they are very sparse, and at other times every microscopical field contains numbers of them. The normoblastic type is the most frequent, but this form of cell is not infrequently found in conjunction with megaloblasts and intermediate forms. Mitosis has been described in the nuclei of the red discs by various authors, but this only possesses a small theoretical or clinical significance. The occurrence of erythroblasts in the blood of leuksemia might be a specific phenomenon of the disease, or only a sign of the anaemia accompanying the leukeemia. The author is inclined to adopt the former view, since such a ])rofuse occurrence of nucleated red cells has never been observed in other forms of anaemia of a similar degree. These are the individual characters of leukaemic blood, on which the diagnosis of the disease is based. It must, however, still be pointed out that even if each individual factor which has been described may be detected in every case of medullary leukaemia, the manner in which they appear, and their numeric ratio to one another and to the total cells of the blood, vary considerably. Apart from the degree of the increase in number of the leucocytes, one case rarely resembles another as far as the other anomalies are concerned. In one case the blood picture possesses a large mononuclear neutrophilic character; in a second case the preponderance of the eosinophile cells is most striking, and in a third case the nucleated red blood corpuscles predominate. Again, the blood may be overwhelmed by mast cells. This shows that there is such a limitless number of possible combinations that each case must possess its own individual type. It is true that the stages of the disease differ markedly from one THE WHITE P>L()()D COIirUSCEES Hi l another. For example, tlio nuiubcr of inyolocytes in smaller at first, and later on in(n'oas(}S steadily. It is of especial importance to study Mm! clianges wliicli liie blood in medullary leucocythaimia und(;in-o(!S duiiii^- tiir- course of an intercurrent disease, and also under tiie inlluence of successful treatment by arsenic or Koentil)liogra])liy). GoLLASCH. — " Zur Kenntnis dcs asthnialisclicii SpuLuiu.s," Forlxcliritie d. Medizin, 1889, vol. vii. Grawitz and (Juunki'-krg. — Die Zellen des mensrhlichen Bluten im idtra- violetten Lichte, Leipzig, 1906. Grawitz. — Klinische Patholoijie des Blutes, 3 Edit., Leipzig, 1906. Grunberg. — " Beitriige zur vergleichenden Morphologic der Leukocytcn," Virchow's Archiv, 1901, vol. clxiii. Grunwald. — "Studien ilber Zellen im Auswurf iind in entziindliclicn Au.s- scheidnngen des Mensclien," Virchow's Archiv, vol. clviii. GiJTiG. — "Ueber die Beziehungen der Hypeideukocytose zuni Knucken- mark," Berl. Min. JVochenschr., 1905. GuLLAND. — " On the Granular Leucocytes," Journ. of Fhy.noL, 1896, vol. xix. Hahn, M. — " Ueber die Beziehungen der Leukocyten zur bakteriziden Wirkung des Blutes," Arch.f. Hygiene, 1895, vol. xxv. Hayem. — Du sawj et de ses alterations pathologiques, Paris, 1889. Heineke. — " Ueber die Einwirkung der Rontgenstrahlen auf innere Organe," Mitteil. aus den Grenzgebieten der Medizin und Chirurgie, 1904, vol. xiv. Experimentelle Untersuchungen iiber die Einwirkung der Rontgen- strahlen auf das Knochenmark," u.s.w. Deutsche Zeitschr. f. Chirurgie, 1905, vol. Ixxviii. Heineke and Deutschmann. — "Das Verhalten der weissen Blutzellen wahrend des Asthmaanfalles," Manchen. med. JVochenschr., 1907, No. 17. Helly. — " Zur Morphologic der Exsudatzellen und zur Spezifitat der weissen Blutkorperchen, Ziegler's Beitrage, 1905, yo\. xxxvii. "Experimentelle Untersuchungen iiber weisse Blutkorperchen und Exsudatzellen,'' Wien. Min. JVochenschr., 1904. " Die liaematopoetischen Organe," Wieu, 1906, A. Holder, Nothnagelsche Sammlung. " Weitere Versuche iiber Exsudatzellen und deren Beeinflussung durcli Bakterien," Zentralbl. f. Balcteriologie, 1905, vol. xxxix. Hesse. — " Zur Kenntnis der Granula der Zellen des Knochenmarks, bezieh- ungsweise der Leukocyten," Virchoio's Archiv, 1902, vol. clxvii. HiRSCHFELD. — "Beitriige zur vergleichenden Morphologie der Leukocyten," J^irchoids Archiv, 1897, vol. cxlix. HoRwiTZ. — " Ueber die Histologic des embryonalen Knochenmarkes," Wien med. Wochenschr., 1904. Jakob. — "Ueber Leukocytose," XV. Kongress innere f. Medizin, 1897, Zeitschr. f. Min. Medizin, vols. xxx. and xxxii. Jaksch, R. v. — "Ueber die Zusanimensetzung des Blutes gesunder und kranker Menscheu," Zentralbl. f. Min. Medizin, 1893, vol. xxiii. 196 ANEMIA Jaksch, v. — " Ueber die prognostisclie Bedeutung der bei crouposer Pneumonie auftretenden Leukocytose," Zentrcdhl. f. hlin. Medizin, 1892, No. 5. Jaksch, v., and Kretz. — "Fall von Leukaemie," Wien. med. JVochenschr., 1908. Japha. — "Die Leukocyten beim gesunden und ktanken Saiigling," Jahrb.f. Kinderh., 1900, vol. lii. and 1901, liii. Jolly.- — " Sur les mouvements des myelocytes," Compt. rend. Soc. Biol., 1901, vol. liii. Jones, Wharton. — Philosophical Transactions, 1846, vol. i. Fo. 82 (quoted by Scbultze). Jordan. — " Die Exstir|)ation der Milz, ihre Indikation und Resultate," Mitteil. aus den Grenzgebieten XL, 1903. Keuthe. — " Ueber die f unktionelle Bedeutung der Leukocyten im zirkulierenden Blute bei verschiedener Ernalirung," Deutsche med. Wochenschr., 1907. KiKODSE. — " Die patliologische Anatomie des Blutes bei der crouposen Pneumonie," Inaugural Dissertation, 1890 (Russian) Abstract, Zentralhl. f. allgem. Pathologic u. patholog. Anatomie, 1891, No. 3. KoTHE. — " Ueber die Leukocytose bei der Appendicitis," Deutsche Zeitschr. f. Chirurgie, 1907, vol. Ixxxviii. Laache. — Die Anaemie, Christiania, 1883. Labadie-Lagrave. — Traits des maladies du sang, Paris, 1893. Limbeck, v. — Orundriss einer klinischen Pathologic des Blutes, 2nd Edition, Jena, 1896. Lengemann — " Ueber die Eutstehung der Leukocytose imd von Zellver- scbleppungen aus dem Knochenmark," Deutsche med. Wochenschr., 1899. Lengemann. — " Knochenmarksveranderungen als Grundlage von Leuko- cytose," U.S.W., Ziegler's Beitrdge, 1901, vol. xxix. LiERMBERGER. — " Beitrag zur Behandlung der Ankylostomiasisanaemie und der Tropenanaemie," Berl. Min. TFochenschr., 1905. LiTTEN. — " Die Krankheiten der Milz und die haemorrhagisclien Diathesen," NothnageVs spez. Pathol, u. Therapie, 1899, vol. viii. LoBENHOFPER. — "Ueber extravaskulare Erythropoese in der Leber unter pathologischen und normalen Verhaltnissen," Ziegler's Beitrdge, 1908, vol. xliii. LoEWiT. — " Bezieliungen der einzelnen Leukocytenformen untereinander, Leukocytose, Leukaemie, Pseudoleukaemie," Lubarscli u. Ostertag, Ergeh- nisse, vol. vii. Leredde and Perrin. — " Anatomie pathologique de la Dermatose de Dlihring," Annal. de Dermat. et Syphiligraph., Ill"'® Ser. 6, pp. 281 and 452. Leyden, E. — "Ueber eosinophile Zellen aus dem Sputum von Bronchial- asthma," Deutsche med. Wochenschr., 1891, No. 38. LiCHTHEiM. — " Leukaemie mit komplizierender tuberkuloser Infektioii," Vereinf. wissenschaftl. Heilkunde zu Konigsherg, February 22, 1897. LoEWY, A. — " Ueber Veranderungen des Blutes durcli thermisclie Einfliisse," Berl. Min. Wochenschr., 1896, No, 4. THE WHITE BrX)()I) (OllPUSCLES 197 LoKWY, A., and V. F. JtuMtTicii. " Uchcr dcu lOinllii.s.s von Fioljcr luid Lciuko- cytose auf den Verlanf von Inf'cktionHkrankhciten," DeuUche med. JVocIunnchr., 1895, No. 15. " Ziir I'.ioloj^nc, rlcr L(;idared 10 per cent, solution of sodium lucta-phosphate to dilute the blood. Another method is the relative counting of the blood platelets in stained dry films. This method has been employed by the majority of observers in recent times. Ehrlich found that in specimens treated by the iodine eosin method (see p. 53) the blood platelets are conspicuous by their intense red colour, which corre- sponds to the high alkali content of these elements, and in this way they can be easily counted. Biirker recommended a sort of accumulation method for the purpose of obtaining these cells for examination. He allowed a drop of blood to fall on a smooth surface of paraffin, and then placed the paraffin in the moist chamber. The heavier erythro- cytes and leucocytes soon sedimented, while nearly all the blood platelets were found on the surface of the drop after about twenty to thirty minutes. They were easily picked up on to a cover glass, and then examined directly. Levaditi, Eosin and Bibergeil, Puchberger, and later on a large number of other observers employed another method. A drop of a weak alcoholic solution of a dye {e.g. brilliant cresyl-blue) was allowed to dry on a cover-slip, and the blood was received directly on this cover-slip. More recently Eomanowsky-Giemsa's staining has been employed with good results for the study of blood platelets. No reliable results have as yet been obtained in the counting of these elements, either with regard to their relative or their absolute numbers. The normal values have been fixed at 200,000 to 300,000 by Ebner, 635,000 by Brodie and Eussell, 730,000 to 962,000 by Kemp. From this it will be seen how little value can be attached to counts in cases of disease at present, and conse- quently how misleading any deduction must be when based on such counts. 204 AN.^M1A Deetjen has described a special method of studying the platelets. " 5 grms. of agar-agar are dissolved by boiling for a half-hour in 500 grms. of distilled water, and the solution while still hot is filtered through a folded filter paper, through which it passes readily without using a hot filter funnel. 0-6 grm. of NaCl, 6 to 8 c.c. of a 10 per cent, solution of ]SraP03, and 5 c.c. of a 10 per cent, solution of KgHPO^ are added to each 100 c.c. of the filtrate. For the examination of the blood a little of the agar solution is poured on to a slide and allowed to set. After the agar is quite cold a strip about 2 mm. broad is cut out of it, and the drop of blood gained from the finger is applied to this strip. This is then covered with a cover-slip." The amoeboid movements of the platelets may be seen by means of this method, and they can thus be more closely studied than in any other way. With regard to the origin and significance of the blood plate- lets, the majority of authors (of whom Hayem, Bizzozero, Laker, and Arnold should be especially mentioned) have come to the conclusion that they are preformed in living blood. The author believes that this view is correct. The opposite view, which is held especially by Lowit, that these structures are formed in the blood after it has left the blood vessels, is denied by the author, on the ground of his own extensive observations. Some authors, including Deetjen, Argutinsky, and others, have, within recent times, suggested that the blood platelets should be regarded as independent complete cells. This opinion is based on the generally accepted structure of the majority of the platelets, which at times even in unstained specimens show a strongly refractile internal substance and a less strongly refractile external substance ; this division is confirmed by the tinctorial behaviour of the elements. The internal substance stains with strong basic dyes and when nuclear dyes are applied in strong solution (Pappenheim). Morawitz deduced from the part which the platelets play in the coagulation of blood (see below), and from their thrombogen THE BLOOD PLATELETS: H^^MOCONLV 205 content whioh ])laceH tluiin in a (lif'ferfMit cato^ory to all other elements of the hlood, tli.'tt tiiey niuHt ha independent cells. These facts, howc-vfir, nvc, insiidieient to show that the platelets should be regarded as real cells. The chief objection to this view, wliioh has been held by other and especially the older authors, is an observation ol' Schwalbe's. lie found these platelets especially numerous in tied vessels between the two ligatures. Whether they are intravital fragments of plasmatic substances or whether they are cast off from the cells cannot at present be decided witli certainty, even if there is reason to believe that the latter view is probably correct. The glycogen content of the platelets (see p. 52) certainly suggests that they are derivatives of the blood cells. Some authors believe that the platelets are derived from the leucocytes (F. Mliller, Arnold, Grrawitz). The most striking argument against this supposition is the fact, which was demon- strated by Helber, that in the blood of mammalian embryos, platelets can be found before the leucocytes have been formed. If the platelets were derived from leucocytes it would be necessary to assume that they are formed in a different manner during embryonal life than during post-embryonal life, or that a second method of production comes into force after birth. The suggestion that the blood platelets are derived from the red blood corpuscles receives more support at present than any other suggestion. There are, however, three different conceptions with regard to this mode of origin. According to Arnold, Schwalbe, and their pupils, both pig- mented and pigment-free plates originate by means of erythror- rhexis and erythroschisis. G-reat caution is necessary with regard to the acceptation of this supposition, since it is scarcely possible to differentiate between schistocytes and platelets, and since the complicated structure of the platelets would be difficult to account for if this were true. The last-mentioned objection would also apply to the suggestion made by Weidenreich, that the blood platelets are detached par- ticles from the surface of the erythrocytes. 206 ANiEMIA The so-called nucleoid theoiy appears to be well founded, and is supported by the majority of htematologists. According to this theory, the blood platelets owe their origin to the remains of nuclear substance, which result from the endo-corpuscular karyolysis of the erythrocytes. This theory has recently received considerable support from the observations made by its founder, Pappenheim, with the assistance of the dark field microscope. Blood platelets are, according to this theory, nothing else than discharged nucleoids. One or two very striking facts speak greatly in favour of it. These include the staining of the chro- matin and also direct morphological analysis. Not infrequently the specimens give the appearance as if the blood platelets com- pletely formed were issuing from the bodies of the red blood corpuscles (Koppe, Engel, Maximow, Hirschfeld). The pictures seen in these specimens are often so suggestive that Naegeli's objection can scarcely lessen the likelihood of the correctness of the doctrine. Naegeli was of opinion that the specimens seen by him were merely instances of blood platelets superimposed on the middle or edges of erythrocytes. A view which is held by a number of observers (Schwalbe, Grrawitz), that the platelets may be derived from various sources, is the least likely of all. If it were accepted it would be necessary to give up regarding the platelets as uniform elements of the blood. J. H. Wright has quite recently observed tlie production of the blood platelets in bone marrow and in the spleen ; he has seen them being formed by detachment of the plasma of megakaryocytes. The knowledge possessed at present with regard to the physiological function of the blood platelets is also extremely defective. The original view which was put forward by Hayem, that the platelets are the precursors of the red blood discs and should therefore be called heematoblasts, is regarded by the majority of hsematologists as untenable. On the other hand, the intimate connection between the blood platelets and coagulation which was first noticed by Bizzozero has been recognised in THE IH.OOI) lM>y\TRLETS: HTRMOCOXIA 207 iioai'ly all the recent works on tlic Kuljject (of. JJiwit and Schvvalbe's reviews). It is still nnccrtain wliethcr tin; substance of the platelets yields tlic iniit(;iial for 1,1k; I'oiination of fibrin as liizzozoro suL!,'ii;osts, oi' wlietli(;i' th(!y onl)' jdiiy the ])ait of inter- mediators, in accordance with the observations of Eljertii and Schimnielbusch on the formation of thrombi. It would occupy too much space to enter into a discussion of the cbemical aspect of this (juestion in this ])la('.e, and for this reason only a few clinical observations will be mentioned, which point to the relations between the coagulability of the blood and the platelet content. A considerable increase of blood platelets is found especially in chlorosis (Muir), and also in post-ha^morrhagic ana-mia (Hayem). In both these conditions there is a marked raising of the coagulability of the blood. An important observation of Denys should, however, be mentioned, who found that in two cases of purpura the only morphological change of the blood was a very considerable decrease of blood platelets. It is well known that the coagulability of the blood is markedly diminished in purpura, or may be abolished altogether. Ehrlich also was able to examine a similar case, in which the blood platelets were entirely absent. A number of authors (Cesaris-Demel, Hayem, Levaditi, Eowley) have described an increase in volume of the platelets in various ana:'mias, while Pappenheim described the same in poly- globuhemia. The size may be as large as that of a normal erythrocyte. Le Sourd and Pagnier have suggested another method of gaining information with regard to blood platelets. By injecting the blood platelets of rabbits into guinea-pigs they obtained a serum from the guinea-pig which was capable of exercising a specific action on the rabbit's platelets. This serum destroyed the platelets in vitro, and in the body of the living rabbit it caused them to disappear altogether wdthout in any way damaging the erythrocytes or leucocytes. The deduction which these authors drew from these experiments, that the platelets could not 208 ANEMIA be derivatives of either red or white blood corpuscles, does not appear to be justified ; it could be assumed that the platelets, being the products of disintegration, would possess a smaller or different kind of resistance to that of the mother cells. G-ruber and Futaki extracted a substance from blood platelets which acted bactericidally against tetanus. Tschistowitsch is inclined on the strength of his blood platelet counts to apply this observation generally, and to regard these elements as being possessed of the function of carrying the protective substances of the blood. Ottolenghi regards the blood platelets as the originators of alexin, because he ascribes to them the capability of reactivating donkey's or rabbit's serum, which had been robbed of a bacteri- cidal action towards tetanus bacilli by heat. H. F. Miiller has described a fourth element of blood, and has given it the name of hsemoconia. These are found in blood plasma in the form of very minute, colourless, highly refractile corpuscles, like granules or cocci. They are possessed of active molecular movement, which can be watched for a very long time without any special precautionary measures. They do not turn black with osmic acid (Miiller), and therefore do not contain fat. They do not appear to have any connection with the formation of fibrin, since they are always found outside the fibrin network. Miiller found them in every specimen of normal blood, but noted that their number varied very considerably. They were very markedly increased in number in one case of Addison's disease, and diminished in starvation and in cachexia. BIBLIOGEAPHY. Argutinsky, quoted by Naegeli, Anat. Anzeiger, 1901, vol. xix. No. 21. Arnold. — "Zur Morphologie und Biologie der roten Blutkorperchen," Firchow's Archiv, 1896, vol. cxlv. " Ueber die Herkunft der Blutplatt- cLen," Zentralhlf. allyein. Pathol, und pathol. Anat., 1897, vol. viii. THE BLOOD PLATELETS: Ilyl^MOCOX L\ 20'.) Bl/ZOZKRO. " IJelicn' ciiieii iicmn l<'()iiiilM--l;ui'lliil '\''H ]'A\iU'H iiikI dcHseii Rolle Ix'i (Icr Tlimniliosc unil ili-i- l;liil;.n-riiiiiiMi^'," Virrhow'ti Archiv, 1882, vol. xc. Brodik and Kusskll. — " 'I'Ik; luimiicnili'in of JJlood I'laleletK," Journ. of Fhy.Hiolufjy, 1897, Noh. 4 iuul T). BiJiiKEK. — M/inchen. wed. lVochev!!r}i.r., 1004, No. 27. Cbsakis-Demel.— "]jC!ol)aclitiing(!ii iiln-r das Blul," />« SperwuiUali, 1905^ vol. lix. ((luoted in FoL Juion., 1900). Deetgen. — " IJntersuclnin;^ iibci' ilic I'-lul plat Iclu.-ii," I'irchovh Arrhiv, 1901, . vol. clxlv. Denys. — "Un noiiveau cas de Purpura avec diminution conaiderable des plaquette.?," Revue La Cellule^ vol. v. part 1. Enqei., C. S. — Leitfaden zur klinisrhen Untersurhamj den Jllutes, 3rd Edition, Berlin, 1908. Grawitz. — Klinische Pathologic des Bluies, 3rd Edition, Leipzig, 190G. Gruber and Futaki. — " Ueber die Resistenz gegen Milzlirand und iiber die Herkunft der milzbrandfeindliclien Stoffe," Milnchen. med. JFochenschr., 1907, No. 6. Hayem.— -Dtt sang, Paris, 1889. Helber. — "Ueber die Entstehung der Blutplattcben," u.s.w., Deutsch. Arch. f. Min. Medizin, 1905, vol. Ixxxii. Hirschpeld.— " Demonstration mikroskopischer Blutphiparate und Blut- plattcben," Ver.f. innere Medizin, February 4, 1901. Kemp, Catham, and Harris.—" The Blood Plates ; their Enumeration in. Physiology and Pathology," Journ. of the Amer. Med. Assoc, April 7^ 1906 (quoted in Fol. haem.). Laker. — "Die Blutscheiben sind konstante Formeleniente des normal zirkulierenden Saugetierblutes," Virchoio's Archiv, 1889, vol. cxvi. Levaditi, qrioted by Schwalbe. Maximow.— " Ueber die Struktur und Entkernung der roten Blutkorperchen der Siiugetiere und die Herkunft der Blutplattcben," Archiv f. Anat. u. Enticickhmgsgesch., 1899. MuiR, E.— " Contribution to the Physiology and Pathology of the Blood,"^ Journ. of Anat. and Physiol, 1891, vol. xxv. p. 475. Muller, H. F.— " Ueber einen bisher nicht beachteten Fornibestandteil des Blutes," Zentralbl.f allgem. Pathol, und pathol. Anat., 1896, p. 929. Naegeli. — Blutkrankheiten und Blutdiagnostik, Leipzig, 1907. Ottolenghi.— " Die Blutplattcben als Alexinerreger," Miinchen. m^d. TVochenschr., 1907, No. 17. Pappenheim.— "Dunkelfeldbeleuchtung," Fol. haem., 1908, vol. vi. p. 190. " Demonstration von Blutplattcben," Milnchen. med. JVochenschr., 1901,. No. 24, p. 989. Rosin and Bibergeil.— "Ueber vitale Blutfiirbung," u.s.w., Zeitschr.f. khn. Medizin, 1902, A'ol. liv. (Bibliography). Rowley.— " Note on the Morphologie of Blood Plates," Journ. of Amer. Med. Assoc, March 10, 1906 (i'oZ. haem.). S&-Bhi.--Klinische Untersuchungsmethoden, 4th Edition, Leipzig, 1905. 14 210 ANEMIA ScHiMMELBUSCH. — " Die Blutplattclieu und die Blutgerinnung," Virchow's Archiv, 1885, vol. ci. ScHWALBE.— t" Thrombose, Gerinnung, Blutplattclien," Liibarscli-Ostertag's Ergebnisse, 1907. SouRD, Le, acd Pagniez. — "Recherches experimentales sur le role des hematoblastes dans la coagulation," Compt. rend. Soc. de Biol., 1907, vol. Ixii. p. 934. TscHiSTOWiTSCH. — " Ueber die Blutplattchen bei akuten Infektionskrank- heiten," Fol. haem., 1907, No. 3. Wright, J. H. — " Die Entstehung der Blutplattcben," Virchow's Archiv, 1906, vol, clxxxvi. DESCRIPTION OF PLATES DESCRIPTION OF PLATE I (Magnification, 700 1)iamkter,s) 1-6. — Myeloblasts (Gieinsa staining). Cells 1-3 are from a case of chronic myeloid leuktemia. Cells 4-6 from a case of acute myeloid leukiumia. The nucleus corresponds to the nucleus of a myelocyte, .showing a delicate chromatin structure and three or four distinct blue nucleoli. The protoplasm pos.sesses a basophile reticulum, reach- in i,^ right up to the nucleus. There are no granules. 7-9.— Development of Myeloblasts to Neutrophile Myelo- cytes (Giemsa). Cell 7 (chronic myeloid leuka?uiia). The reticulum is .still markedly basophilic ; there are but few granules, and the nucleoli are still visible. Cell 8 (chronic myeloid leukaunia). Similar to Xo. 7. The nucleoli are still distinct, but the granulations are more plentiful. Cell 9 (another case of chronic myeloid leuktemia). Xo nucleoli are visible ; the granulation is very sparse. 10-17. — Neutrophile Myelocytes (Giemsa). Cells 10-14 and 17 are from cases of myeloid leukfemia. Cells 15-16 are from croujaous pneumonia. The granules are very numerous. The basophilic character of the protoplasm has diminished. Cell 17, slightly crushed (demonstration of isolated granules). 18. — Metamyelocytes (Leuksemia) (Giemsa). Transition of myelocyte nucleus to polymorphous form. 19-25. — Polym.orpho-nuclear Neutrophile Leucocy-tes (Giemsa). Normal blood and leucocytosis. Cell 25 crushed (isolated granules). 26-30. — Eosinophile Myelocytes (Giemsa). From two cases of chronic mveloid leukaemia. 211 212 DESCRIPTION OF PLATES Cell 26. — Nearly all tlie granules are blue (basopliile pre- liminary stage). At the edge some red granules are seen. Cells 27-28 and 30 (crushed). The red granules are pre- dominating ; only a few blue granules are present. Cell 29. — All the granules are red (oxyphil e, ripe), but the protoplasm is distinctly reticulated blue. 31-35. — Eosinophile Leucocytes (Giemsa). Cell 31. — Metamyelocyte from a case of myeloid leukaemia. The nucleus is just taking on the polymorphous structure. Cell 25 (crushed). Showing isolated granules. DESCRIPTION OF PLATE II (Magnification, 700 Diameters) 36-43. — Mast Myelocytes (Giemsa). From a case of chronic myeloid leuksemia with very numerous mast cells. Cell 36. — The blue precursors of the mature granules are differentiated in the protoplasm. Cell 37. — Numerous blue granules. Cells 38-40. — A mixture of immature blue and mature mauve- coloured granules. Cells 41-42. — Mauve-coloured granules, — markedly resistant to water. Cell 43. — Mature mauve-coloured granulation ; no longer resistant to water. 44. — Mast Cell Myelocyte (Giemsa). Prom case of myeloid leuksemia. 45-48. — Mast Leucocytes (Giemsa). The granules are readily soluble in water. Cells 45-46. — Normal blood. Cells 47-48. — Chronic myeloid leuksemia. 49-51. — Mast Leucoc3rtes (May Griinwald staining). From a case of myeloid leuksemia. 52-57. — Large Mononuclear Leucocytes and Transition Forms (Giemsa). ISTormal blood and leucocytosis. The granulation is very fine and plentiful, and the protoplasm slate coloured. The series shows a gradually increasing transformation of the nucleus. The much more intense nuclear staining and the much finer granu- lation than in the myelocytes should be noted. 58-62. — Stimulation Forms = Pathological Myeloblasts (Giemsa). Vacuoles in the deep blue protoplasm. Cells 58-59. — From a case of pernicious anaemia. Cells 60-62. — From a case of encephalitis (child aged six years). DESCJlirTION OF IM.AIKS 2J3 63-87. — Lymphocytes (fJioniH.'i). Cki:i,s ()3-(;0. Normal Ijlood, C/.i-C)!') witlioiif, ;izur(; granules, GH-nii wiLli ;i/,iin; gnumles. (.\;]\ G'J HOiiicwhiit crushed. Cklls 70-72. — SoiiH'wliat, l;irf,'or l^iiipliooyteH (four years' old cl.ild). CiOLLS 73-87. — ^Lynipliocylcs irDiii a case of lyuijdiaLic louk;x;niia. Ckt.lh 73-75. — Nucleus almost free. Cklls 76-81. — Large forms. 78 cruslied. 70-81 with azure granules. Cklls 82-87. — Transformation of nucleus into Rieder's form. DESCRIPTION OF PLATE III (Magnification, 700 Diameters) 88-97. — Erythroblasts (Giemsa). From a case of carcinoma of the bone marrow. Cells 88-90. — Megaloblasts, showing polychromatic proto- plasm. Cells 91-93. — Intermediate forms between megaloblasts and normoblasts ; two are almost orthochromatic. Cells 94-95. — Polychromatic and orthochromatic normoblasts. Cells 96-97.- — Dissociation of nucleus in polychromatic and basophilic granulated cells. 98-1 05.— Erythrocytes (Giemsa). Megalocytes and normocytes in all stages from marked poly- chroniasia up to orthochromasia. Case of carcinoma of the bone marrow. 106-118.— Mitosis of Erythroblasts (Giemsa). All the cells possess characteristic basopliile granulation of the protoplasm. Cells 106-115. — All stages of mitosis, from a case of infantile pseudo-leuksemic ansemia. Cells 116-118.— A typical pathological mitosis, from a case of acute myeloid leuktemia. Cell 118.— Triple mitosis and triple division of the nucleus. 119-122.— Remains of Nuclei and Nuclear Debris (Giemsa). From a case of infantile pseudo-leukaemic ansemia. Cells 119-121. — Solution of the nuclear remains. 123-142.— Ring Bodies (Giemsa). At times with red or blue, or red and blue granulations. The rings are in part free in. the plasma. Cells 123-134. — From a case of pernicioiis anremia. 214 DESCRIPTION OF PLATES Cells 135-142. — From a case of acute myeloid leukaemia. By an unfortunate mistake, ^vhicll could not be corrected after it was discovered, tlie cells of the series 88-93, as well as cells 102 and 103, were reproduced on a smaller scale than in the drawings. This reduction is considerable and likely to mislead. The diameter of the cells should be about one-third larger than they appear. DESCRIPTION OF PLATE IV (Magnification, 700 Diameters) 143-156. — Red and Blue Punctation in Erythrocytes (Giemsa). Cells 143-153. — From cases of pernicious ansemia. Cells 154-156. — From cases of acute myeloid leukaemia. A mixture of the blue and red punctation is at times seen, while at other times only the red appears. Two cells show a. diffuse red coloration of the protoplasm. The size of the red granules varies at times. 157. — Red Punctation in Erythrocytes (Giemsa). From a case of pernicious anaemia. The cells show very well-marked red granules, singly or in numbers. In the middle there is a polychromatic megalocyte with numerous red granules. 158-169. — Blue Basophile Punctation (Giemsa). Lead poisoning. All the cells depicted are orthochromatic. Cells 163-169. — Severe chlorosis in the stage of recovery. There are numerous iDolychromatic cells with blue basophile punctation. One red grain is also seen. DESCRIPTION OF PLATE V (Magnification, 700 Diameters) 170. — Blood Platelets (Giemsa). From the blood in chlorosis. a-g. Cells stained by Triacid Solution. (a) Neutropliile myelocytes. (b) Polynuclear neutropliile leucocytes. (c) Eosinophile cells. {d) Mast cells. (e) Normoblasts. (/) Megaloblasts. (g) Erythrocyte.s. The illustrations 1-170 have been drawn in colours by the academie painter, Mr. L. Schrotter (Zurich-Heidelberg), from preparations supplied by Dr. Naegeli, and under his supervision. AncemicL. PLaU I \SS' ■if 10 /;>,-/ J^ -S" ^? ^^anf^' 11. 12. 'J*V\.l;j./ 13. 1-f. IS 16- 17. 18. 19. "-mi^^ 20. 21 '?v-.'. 22. 23. ^%1^ ^#. 25. m 26. 27 28. 29. 30. *iv:.;^i^s,r 31. 32 33. 34. . ^ 35. ■ A" Rebman Limiled. London AncBirUa.. PLcLte JI AncemtcL. PtaXe m. AncemicL. PLale JV H8. / ■!■ i-r/ 1 * '•% 1 /■/■'/ ,:• • • •'' ■ • ■'-? ]53. Ibd:. 0^-"%, 158 259. 160. 261. '#|s*i?' *f^" %#- fep 16:-- 164\ 165. i^-f?. ,■■ i6'**> w' '?'V'oi!& '^^5^'* ^. § r. m y- Rebman Limited, London INDEX Acid dyes, 41. „ fuchsin, 42, 43. Acidopliile granules, 41, 46. Adventitial cells, 149, 150. Alcohol fixation, 38. Alkali in blood, 25, 26, 53. Altmann - Schridde's granules, 91, 102, 136. „ ,, stain, 91. Anaemia, definition of, 1. „ pernicious, 27, 63, 66, 67, 69, 76, 79, 100, 175. " Ancestor " corpuscles, 69. Anisocytosis, 67. Ankylostonium ansemia, 78. Amethyst violet, 42, 43. Asthma, 165, 171. Aurautia, 37, 40. Autolysis, 96. Azure, 45. Azurophile granules, 102, 105. Basic dyes, 41. „ staining (double), 47. Basophile granules, 47, 49. „ mast cells, 125. Biermer's anaemia, 27, 63, 66, 67, 69, 76, 79, 100, 175. Bioblast theory, 141. Bismarck brown, 41. Blood platelets, 38, 52, 202-207. Bone marrow, 109, 124. Bothriocephalus ana3mia, 78, 172. Bremer's diabetes reaction, 54. Bronchial asthma, 165, 171. Carcinoma, 75. Carmine, 40. Chemutaxis, 121, 129, 145, 154, 157, 159, 171. Chenzinski's solution, 38, 44, 48. Chromic green, 41. Chronic nephritis, 75. Climate, influence of, on blood cells, 8, 9, 12. Coagulability of blood, 3, 22, 26, 27, •206, 207. CO hseraoglobin, 4, 5. Colour index, 17. "Combined" staining, 39, 41. Cover glasses, 33-35. Cresyl violet R, 98. Dahlia glycerin, 202. Dark field illumination, 31. " Dedifierentiation," 151. Definition of anaemia, 1. Diabetes reaction (Bremer), 54. „ „ (Williamson), 5.i. Differential staining, 37. Discoplasm, 56, 68. Double basic staining, 47. Dry films, 32-35. „ substance in blood, 11. Dualistic doctrine, 146. Dye mixtures, neutral, 42. Dyes, acid, 41. „ basic, 41. Eatin, 98. Electric discharge, effect on blood, 28. Eosin, 44, 46, 48, 49, 203. Eosin-aurantia-nigrosin mixture, 37. Eosin and heematoxylin, 46. Eosinophile cells, 97, 113, 125, 164, 165, 173, 183, 184. 216 INDEX Eosinophile granules, 49, 97, 145. ,, mononuclear cells, 182, 183. „ ■ myelocytes, 100. Eosinopliilia, compensatory, 168. „ leuksemic, 169. „ nervous, 169. „ medicamentous, 169. „ scarlatinal, 169. „ post-infective, 167. Erythroblasts, 71-73. Erythrocytes, 3, 6, 7, 17, 18,38, 48, 56. ,, nucleated, 60, 68, 69, 70, 115, 190-192. „ origin of, 71, 72. ,, punctated, 61-64, 66. Erythropoiesis, 124. Estimation of hsemoglobin content, 13-15. Ej^e-pieces (microscope), 32. Films, dry, 32-35. Fixing by heat, 37. Fixation of films, 36. Formal fixation, 38. Fuchsin, 41-43. Fuchsinophile granules, 91, 102, 136. Giemsa's stain, 38, 50, 93, 94. Gigantoblasts, 69. " Globules nuclees geantes," 69. " Globules nuclees de taille moyenne," 69. Glycogen in blood, 52. Granules, 62, 110, 133-135. Granules, acidophile, 41, 46. „ Altmann's, 91, 102, 136. „ azurophile, 102. „ basojihile 47, 49. „ eosinophile, 49, 97, 145. „ fuchsinophile, 91, 102, 136. ,, metachromic, 107. „ neutrophile, 43, 94, 96, 99, 141-143, 188. „ perinuclear, 96. Guinea-pig's blood. 111. Hsemacytometer, 5, 10. Hfematoxylin, 38, 39, 46. Haemoconia, 208. Hfemoglobin, 2, 4, 5, 11, 13, 20, 24, 41, 57, 58. Hsemoglobinsemia, 76. Heemoglobinometer, 14, 15. Hgemometer, 14, 15. Hsemorrhage, 22, 63. Haldane's and Lorrain - Smith's method of determining the quantity of blood, 4, 5. Hayem's fluid, 6. Helminthiasis, 166, 172. Hygrometry, 22. Indacine, 42. Iodide of eosin, 53, 203. Iodine test for glycogen, 52. lodophile substance, 96. Iron in blood, 16. Jenner's stain, 50, 98. Karyolysis, 72, 74. Karyorrhexis, 72, 74. Kottmann's method of determining the quantity of blood, 4. Lead poisoning, 63. Leucocytes, 30, 87, 151-155, 157, 158. ,, large mononuclear, 94, . ,, polynuclear, 95, 126. ,, transition form, 94, 95. Leucocytosis, 30. ,, mast cell, 175. „ pathological, 161-164. „ physiological, 161. ,, polymorpho-nucl ear neu- trophile, 30, 89, 160, 164. ,, polynuclear eosinophile, 164. Leucopenia, 154, 158. Leukfemia, 30, 69, 73-75, 89, 106, 163, 176-193. Lymphsemia, 132. Lymphatic glands, 109, 116, 117. Lymphatic leukgemia, 177-180. „ system, 146. INDEX 217 Lymphatic tissue, 101, 103. Lympliocytcs, 87, 88, 90. „ (pathological), 105. Lymphocytosis, 114, 117, 120, 121, 130, 131. Lymphocytoiuatosis, 119. Malignant disease, 75. Mast cells, 47, 98, 175, 185. „ basophile, 125. Mast myelocytes, 101. Mav-Griinwald's stain, 49. Megaloblasts, 70, 71, 75-77, 79. Megalocytes, 67. Metrachroniasia, 98. Metachromic granulation, 107. Methylene-blue, 41, 43, 44, 48, 137. ,, blue-azure, 45, 50. „ blue-eosin, 44, 48, 50. Methyl-green, 41-43, 91. Methyl- violet, 41. Microblasts, 70. Microcytes, 38, 67. Mitosis, 11,63, 65, 180, 186. Mononuclear, large leucocytes, 94. „ cells with neutrophile granules, 99. ,, eosinophil e cells, 182, 183. „ cells (non-granulated), 125. Morawitz's method of determining the quantity of blood, 4. Mlillern's staining. 49. Myeloblasts, 101, 103. „ (pathological), 104. Myelocytes, 99, 137, 138. „ eosinophile, 100. Myeloid leukeemia, 178, 180, 181. Myeloid tissue, 103, 146, 147. Narcein, 42, 43. NegatiA'e nuclear staining, 54. Neutral mixtures of dyes, 42. Neutral red, 137-139. Neutrophile cells, 108. Neutrophile granules, 43, 94, 96, 99, 141-143, 188. NikiforolfH fi.xution, 38. Normoblasts, 69, 71, 74, 76, 77. Normocytes, 67. Nucleated erythrocytes, 60-69, 70, 115, 190-192. Nuclei of lyiiipljocyti;H, 91. Oikoid, 56. Oligochromscmia, 2. Oligocythtcniia, 2. Orange G, 42, 43. Origin of leucocyte.", 109. Oxygen, 12. Ozonophores, 141. Pacini's fluid, 6. Panoptic staining, 39. Pappenheim's mixture, 48. Pathological leucocytes, 161-164. Pathological lymphocytes, 105. Pathological myeloblasts, 104. Perinuclear granules, 96. Pemphigus, 166, 173. Pernicious anaemia, 27, 63, 66, 67, 69, 79, 100, 175. Phagocytosis, 153. Physiological leucocytes, 161. Phosphorus poisoning, 75. Picrate of ammonium, 40, 42. Picro- carmine, 40. Plasma cells, 106. Plasmodia, 40. Platelets (blood), 38, 52, 202- 207. Pletch's method of determining the quantity of blood, 4. Poikilocytosis, 11, 67, 77. Polychroniasia, 66. Polychromatic staining, 41. Polychromatophilia, 58-60. Polycytheemia, 5, 8. Polymorpho-nuclear neutrophile leu- cocytes, 95, 126. Polymorpho-nuclear leucocytosis, 30, 8*9, 160, 164. Polynuclear eosinophile leucocytes, 164. Pseudo-eosinophile cells, 112, 126. 218 INDEX Punctated erythrocytes, 61-64, 66. Pyronin, 41, 48, 91. Quadratic ocular diaphragms, 32. Quantity of blood, 3, 4. Quincke's method of determining the quantity of blood, 3. Keaction of blood, 24, 25. Eed blood corpuscles, 3, 6, 17, 18, 38, 48, 56. ,, nucleated, 60, " 68, 69, 70, 115, 190- 192. origin of, 71, 72. ,, punctated, 61- " 64, 66. site of produc- tion, 124, 125. size of, 57. Ehodamin, 42. Kieder's cells, 88, 91, 105. Eomanowsky's dye, 44, 93. Eosanilin picrate, 42. Schistocytes, 68. Separation of serum from clot, 27. Simultaneous staining, 39, 41. Size of blood corpuscles, 18. Size of red blood corpuscles, 57. Skin diseases, 161. Slides, 35. Specific gravity of blood, 11, 19-21, 29. Spleen, 108, 111. „ removal of, 113. Spodogenous tumours, 115. "Stimulation" form of corpuscles, 104. Tarchanoff' s method of determination of the quantity of blood, 3. Thrombogen, 204. Tonicity of blood, 27. Transition forms (leucocytes), 94, 95. Triacid staining, 46, 103. Urticaria, 176. Victor Mayer's apparatus, 37. Vital staining, 51. Volume of erythrocytes, 22, 23. Volume of the blood, 3, 4. White blood corpuscles, 30, 87, 151- 155, 157, 158. Williamson's test, 55. Ziemann's solution, 49. Zooid, 56. Printed hy Morrison & Gibb Limited, Edinburgh •" 1 Date Due ^ii • -., <'' \ ' 9 ■ I 3 U ^ ■"-! ?!fiij!5:?SLi2;HrSiiS_"?'«!5#'"sp5=;8?tf=''^ ■■t-'^S^M^/M^'^^^^T^'^rir^j?^^ *S.i