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HISTOLOGY OF THE BLOOD

NORMAL AND PATHOLOGICAL.

London: C. J. CLAY AND SONS, CAMBRIDGE UNIVERSITY PRESS WAREHOUSE, AVE MARIA LANE,

AND

H. K. LEWIS, 136, GOWER STREET, W.C.

Glasgow: 50, WELLINGTON STREET. Leipzig: F. A. BROCKHAUS. New York: THE MACMILLAN COMPANY. Bombay: E. SEYMOUR HALE.

Transcriber's note:

For Text: Words surrounded by a cedilla such as this signifies that the words are bolded in the text. Words surrounded by underscores like this signifies the words are in italics in the text. Words surrounded by equal signs (like this) means the letters in the words are spaced out (gesperrt). For numbers and equations, carats before bracketed numbers denote a superscript.

Minor typos have been corrected.

HISTOLOGY OF THE BLOOD

NORMAL AND PATHOLOGICAL

BY

P. EHRLICH AND A. LAZARUS.

EDITED AND TRANSLATED

BY

W. MYERS, M.A., M.B., B.Sc.

JOHN LUCAS WALKER STUDENT OF PATHOLOGY.

WITH A PREFACE

BY

G. SIMS WOODHEAD, M.D.

PROFESSOR OF PATHOLOGY IN THE UNIVERSITY OF CAMBRIDGE.

CAMBRIDGE: AT THE UNIVERSITY PRESS. 1900

[All Rights reserved.]

Cambridge: PRINTED BY J. AND C. F. CLAY, AT THE UNIVERSITY PRESS.



PREFACE.

In no department of Pathology has advance been so fitful and interrupted as in that dealing with blood changes in various forms of disease, though none now offers a field that promises such an abundant return for an equal expenditure of time and labour.

Observations of great importance were early made by Wharton Jones, Waller, and Hughes Bennett in this country, and by Virchow and Max Schultze in Germany. Not, however, until the decade ending in 1890 was it realised what a large amount of new work on the corpuscular elements of the blood had been done by Hayem, and by Ehrlich and his pupils. As successive papers were published, especially from German laboratories, it became evident that the systematic study of the blood by various new methods was resulting in the acquisition of a large number of facts bearing on the pathology of the blood; though it was still difficult to localise many of the normal haematogenetic processes. The production of the various cells under pathological conditions, where so many new factors are introduced, must necessarily be enshrouded in even greater obscurity and could only be accurately determined by patient investigation, a careful arrangement and study of facts, and cautious deduction from accumulated and classified observations.

The pathology of the blood, especially of the corpuscular elements, though one of the most interesting, is certainly one of the most confusing, of all departments of pathology, and to those who have not given almost undivided attention to this subject it is extremely difficult to obtain a comprehensive and accurate view of the blood in disease. It is for this reason that we welcome the present work in its English garb. Professor Ehrlich by his careful and extended observations on the blood has qualified himself to give a bird's-eye view of the subject, such as few if any are capable of offering; and his book now so well translated by Mr. Myers must remain one of the classical works on blood in disease and on blood diseases, and in introducing it to English readers Mr. Myers makes an important contribution to the accurate study of haemal pathology in this country.

Comparatively few amongst us are able to make a cytological examination of the blood, whilst fewer still are competent to interpret the results of such an examination. How many of our physicians are in a position to distinguish between a myelogenic leukocythaemia and a lymphatic leukaemia? How many of us could draw correct inferences from the fact that in typhoid fever there may not only be no increase in the number of certain of the white cells of the blood, but an actual leukopenia? How many appreciated the diagnostic value of the difference in the cellular elements in the blood in cases of scarlet fever and of measles, and how many have anything more than a general idea as to the significance of a hypoleucocytosis or a hyperleucocytosis in a case of acute pneumonia, or as to the relations of cells of different forms and the percentage quantity of haemoglobin found in the various types of anaemia?

One of the most important points indicated in the following pages is that the cellular elements of the blood must be studied as a whole and not as isolated factors, as "it has always been shown that the character of a leukaemic condition is only settled by a concurrence of a large number of single symptoms of which each one is indispensable for the diagnosis, and which taken together are absolutely conclusive." Conditions of experiment can of course be carefully determined, so far, at any rate, as the introduction of substances from outside is concerned, but we must always bear in mind that it is impossible, except in very special cases of disease, to separate the action of the bone-marrow from the action of the lymphatic glands; still, by careful observation and in special cases, especially when the various organs and parts may be examined after death, information may be gained even on this point. By means of experiment the production of leucocytosis by peptones, the action of micro-organisms on the bone-marrow, the influence of the products of decaying or degenerating epithelial or endothelioid cells, may all be studied in a more or less perfect form; but, withal, it is only by a study of the numerous conditions under which alterations in the cellular elements take place in the blood that any accurate information can be obtained.

Hence for further knowledge of the "structure" and certain functions of the blood we must to a great extent rely upon clinical observation.

Some of the simpler problems have already been flooded with light by those who following in Ehrlich's footsteps have studied the blood in disease. But many of even greater importance might be cited from the work before us. With the abundant information, the well argued deductions and the carefully drawn up statement here placed before us it may be claimed that we are now in a position to make diagnoses that not long ago were quite beyond our reach, whilst a thorough training of our younger medical men in the methods of blood examination must result in the accumulation of new facts of prime importance both to the pathologist and to the physician.

Both teacher and investigator cannot but feel that they have now at command not only accurate results obtained by careful observation, but the foundation on which the superstructure has been built up—exquisite but simple methods of research. Ehrlich's methods may be (and have already been) somewhat modified as occasion requires, but the principles of fixation and staining here set forth must for long remain the methods to be utilised in future work. His differential staining, in which he utilised the special affinities that certain cells and parts of cells have for basic, acid and neutral stains, was simply a foreshadowing of his work on the affinity that certain cells and tissues have for specific drugs and toxins; the study of these special elective affinities now forms a very wide field of investigation in which numerous workers are already engaged in determining the position and nature of these seats of election for special proteid and other poisons.

The researches of Metschnikoff, of Kanthack and Hardy, of Muir, of Buchanan, and others, are supplementary and complementary to those carried on in the German School, but we may safely say that this work must be looked upon as influencing the study of blood more than any that has yet been published. It is only after a careful study of this book that any idea of the enormous amount of work that has been contributed to haematology by Ehrlich and his pupils, and the relatively important part that such a work must play in guiding and encouraging those who are interested in this fascinating subject, can be formed.

The translation appears to have been very carefully made, and the opportunity has been seized to add notes on certain points that have a special bearing on Ehrlich's work, or that have been brought into prominence since the time that the original work was produced. This renders the English edition in certain respects superior even to the original.

G. SIMS WOODHEAD.



NOTE BY THE TRANSLATOR.

This translation of the first part of Die Anaemie, Nothnagel's Specielle Pathologie und Therapie, vol. VIII. was carried out under the personal guidance of Professor Ehrlich. Several alterations and additions have been made in the present edition. To my friend Dr Cobbett I owe a debt of gratitude for his kind help in the revision of the proof-sheets.

W. M.



CONTENTS.

PAGE

INTRODUCTION 1

DEFINITION. CLINICAL METHODS OF INVESTIGATION OF THE BLOOD 1

The quantity of the blood 2 Number of red corpuscles 4 Size of red corpuscles 12 Amount of haemoglobin in the blood 13 Specific gravity of the blood 17 Hygrometry 21 Total volume of the red corpuscles 21 Alkalinity of the blood 23 Coagulability of the blood 24 Separation of the serum 24 Resistance of the red corpuscles 25

THE MORPHOLOGY OF THE BLOOD 27

A. METHODS OF INVESTIGATION 29

[alpha]. Preparation of the dry specimen 32 [beta]. Fixation of the dry specimen 34 [gamma]. Staining of the dry specimen 36 Theory of staining 37 Combined staining 38 Triacid fluid 40 Other staining fluids 41 Recognition of glycogen in the blood 45 Microscopic determination of the distribution of the alkali of the blood 46

B. NORMAL AND PATHOLOGICAL HISTOLOGY OF THE BLOOD 48

The red blood corpuscles 48 Diminution of haemoglobin equivalent 49 Anaemic or polychromatophil degeneration 49 Poikilocytosis 52 Nucleated red blood corpuscles 54 Normoblasts and megaloblasts 56 The fate of the nuclei of the erythroblasts 57 The clinical differences in the erythroblasts 61

THE WHITE BLOOD CORPUSCLES 67

I. NORMAL HISTOLOGY AND CLASSIFICATION OF THE WHITE BLOOD CORPUSCLES 71

The lymphocytes 71 The large mononuclear leucocytes 73 The transitional forms 74 The polynuclear leucocytes 75 The eosinophil cells 76 The mast cells 76 Pathological forms of white blood corpuscles 77 The neutrophil myelocytes 77 The eosinophil myelocytes 78 The neutrophil pseudolymphocytes 78 Stimulation forms 79

II. ON THE PLACES OF ORIGIN OF THE WHITE BLOOD CORPUSCLES 81

[alpha]. The spleen 84 [beta]. The lymphatic glands 100 [gamma]. The bone-marrow 105

III. ON THE DEMONSTRATION OF THE CELL-GRANULES, AND THEIR SIGNIFICANCE 121

History of the investigation of the granules 121 Since Ehrlich. 123 Methods of demonstration 124 Vital staining of granules 124 The Bioblast theory (Altmann) 128 The granules as metabolic products of the cells (Ehrlich) 130 Secretory processes in granulated cells 134

IV. LEUCOCYTOSIS 138

Biological importance of leucocytosis 138 Morphology of leucocytosis 142 [alpha]. 1. Polynuclear neutrophil leucocytosis 143 Definition 143 Clinical occurrence 144 Origin 144 [alpha]. 2. Polynuclear eosinophil leucocytosis, including the mast cells 148 Definition 149 Clinical occurrence 150 Origin 154 [beta]. Leukaemia ("mixed leucocytosis") 167 Lymphatic leukaemia 170 Myelogenous leukaemia 171 Morphological character 187 Origin 187

V. LEUKOPENIA 188

The blood platelets. The haemoconiae 190

INDEX TO LITERATURE 195

INDEX 209

PLATES



INTRODUCTION.

DEFINITION OF ANAEMIA. CLINICAL METHODS OF INVESTIGATION OF THE BLOOD.

In practical medicine the term "anaemia" has not quite the restricted sense that scientific investigation gives it. The former regards certain striking symptoms as characteristic of the anaemic condition; pallor of the skin, a diminution of the normal redness of the mucous membranes of the eyes, lips, mouth, and pharynx. From the presence of these phenomena anaemia is diagnosed, and according to their greater or less intensity, conclusions are also drawn as to the degree of the poverty of the blood.

It is evident from the first that a definition based on such a frequent and elementary chain of symptoms will bring into line much that is unconnected, and will perhaps omit what it should logically include. Indeed a number of obscurities and contradictions is to be ascribed to this circumstance.

The first task therefore of a scientific treatment of the anaemic condition is carefully to define its extent. For this purpose the symptoms above mentioned are little suited, however great, in their proper place, their practical importance may be.

Etymologically the word "anaemia" signifies a want of the normal quantity of blood. This may be "general" and affect the whole organism; or "local" and limited to a particular region or a single organ. The local anaemias we can at once exclude from our consideration.

A priori, the amount of blood may be subnormal in two senses, quantitative and qualitative. We may have a diminution of the amount of blood—"Oligaemia." Deterioration of the quality of the blood may be quite independent of the amount of blood, and must primarily express itself in a diminution of the physiologically important constituents. Hence we distinguish the following chief types of alteration of the blood; (1) diminution of the amount of Haemoglobin (Oligochromaemia), and (2) diminution of the number of red blood corpuscles (Oligocythaemia).

We regard as anaemic all conditions of the blood where a diminution of the amount of haemoglobin can be recognised; in by far the greater number of cases, if not in all, Oligaemia and Oligocythaemia to a greater or less extent occur simultaneously.

The most important methods of clinical haematology bear directly or indirectly on the recognition of these conditions.

There is at present no method of ESTIMATION OF THE TOTAL QUANTITY OF THE BLOOD which can be used clinically. We rely to a certain extent on the observation of the already mentioned symptoms of redness or pallor of the skin and mucous membranes. To a large degree these depend upon the composition of the blood, and not upon the fulness of the peripheral vessels. If we take the latter as a measure of the total amount of blood, isolated vessels, visible to the naked eye, e.g. those of the sclerotic, may be observed. Most suitable is the ophthalmoscopic examination of the width of the vessels at the back of the eye. Raehlmann has shewn that in 60% of the cases of chronic anaemia, in which the skin and mucous membranes are very white, there is hyperaemia of the retina—which is evidence that in such cases the circulating blood is pale in colour, but certainly not less in quantity than normally. The condition of the pulse is an important indication of diminution of the quantity of the blood, though only when it is marked. It presents a peculiar smallness and feebleness in all cases of severe oligaemia.

The bleeding from fresh skin punctures gives a further criterion of the quantity of blood, within certain limits, but is modified by changes in the coagulability of the blood. Anyone who has made frequent blood examinations will have observed that in this respect extraordinary variations occur. In some cases scarcely a drop of blood can be obtained, while in others the blood flows freely. One will not err in assuming in the former case a diminution of the quantity of the blood.

The fulness of the peripheral vessels however is a sign of only relative value, for the amount of blood in the internal organs may be very different. The problem, how to estimate exactly, if possible mathematically, the quantity of blood in the body has always been recognised as important, and its solution would constitute a real advance. The methods which have so far been proposed for clinical purposes originate from Tarchanoff. He suggested that one may estimate the quantity of blood by comparing the numbers of the red blood corpuscles before and after copious sweating. Apart from various theoretical considerations this method is far too clumsy for practical purposes.

Quincke has endeavoured to calculate the amount of blood in cases of blood transfusion for therapeutic purposes. From the number of red blood corpuscles of the patient before and after blood transfusion, the amount of blood transfused and the number of corpuscles it contains, by a simple mathematical formula the quantity of the blood of the patient can be estimated. But this method is only practicable in special cases and is open to several theoretical errors. First, it depends upon the relative number of red blood corpuscles in the blood; inasmuch as the transfusion of normal blood into normal blood, for example, would produce no alteration in the count. This consideration is enough to shew that this proceeding can only be used in special cases. It has indeed been found that an increase of the red corpuscles per cubic millimetre occurs in persons with a very small number of red corpuscles, who have been injected with normal blood. But it is very hazardous to try to estimate therefrom the volume of the pre-existing blood, since the act of transfusion undoubtedly is immediately followed by compensatory currents and alterations in the distribution of the blood.

No property of the blood has been so exactly and frequently tested as the NUMBER OF RED CORPUSCLES PER CUBIC MILLIMETRE OF BLOOD. The convenience of the counting apparatus, and the apparently absolute measure of the result have ensured for the methods of enumeration an early clinical application.

At the present time the instruments of Thoma-Zeiss or others similarly constructed are generally used; and we may assume that the principle on which they depend and the methods of their use are known. A number of fluids are used to dilute the blood, which on the whole fulfil the requirements of preserving the form and colour of the red corpuscles, of preventing their fusing together, and of allowing them to settle rapidly. Of the better known solutions we will here mention Pacini's and Hayem's fluids.

Pacini's solution. Hydrarg. bichlor. 2.0 Natr. chlor. 4.0 Glycerin 26.0 Aquae destillat. 226.0

Hayem's solution. Hydrarg. bichlor. 0.5 Natr. sulph. 5.0 Natr. chlor. 1.0 Aquae destillat. 200.0

For counting the white blood corpuscles the same instrument is generally used, but the blood is diluted 10 times instead of 100 times. It is advantageous to use a diluting fluid which destroys the red blood corpuscles, but which brings out the nuclei of the white corpuscles, so that the latter are more easily recognised. For this purpose the solution recommended by Thoma is the best—namely a half per cent. solution of acetic acid, to which a trace of methyl violet has been added[1].

The results of these methods of enumeration are sufficiently exact, as they have, according to the frequently confirmed observations of R. Thoma and I. F. Lyon, only a small error. In a count of 200 cells it is five per cent., of 1250 two per cent., of 5000 one, and of 20,000 one-half per cent.

There are certain factors in the practical application of these methods, which in other directions influence the result unfavourably.

It has been found by Cohnstein and Zuntz and others that the blood in the large vessels has a constant composition, but that in the small vessels and capillaries the formed elements may vary considerably in number, though the blood is in other respects normal. Thus, for example, in a one-sided paralytic, the capillary blood is different on the two sides; and congestion, cold, and so forth raise the number of red blood corpuscles. Hence, for purposes of enumeration, the rule is to take blood only from those parts of the body which are free from accidental variation; to avoid all influences such as energetic rubbing or scrubbing, etc., which alter the circulation in the capillaries; to undertake the examination at such times when the number of red blood corpuscles is not influenced by the taking of food or medicine.

It is usual to take the blood from the tip of the finger, and only in exceptional cases, e.g. in oedema of the finger, are other places chosen, such as the lobule of the ear, or (in the case of children) the big toe. For the puncture pointed needles or specially constructed instruments, open or shielded lancets, are unnecessary: we recommend a fine steel pen, of which one nib has been broken off. It is easily disinfected by heating to redness, and produces not a puncture but what is more useful, a cut, from which blood freely flows without any great pressure.

The literature dealing with the numbers of the red corpuscles in health, is so large as to be quite unsurveyable. According to the new and complete compilation of Reinert and v. Limbeck, the following figures (calculated roundly for mm.^{3}) may be taken as physiological:

Men.

Maximum Minimum Average 7,000,000 4,000,000 5,000,000

Women.

Maximum Minimum Average 5,250,000 4,500,000 4,500,000

This difference between the sexes first makes its appearance at the time of puberty of the female. Up to the commencement of menstruation the number of corpuscles in the female is in fact slightly higher than in the male (Stierlin). Apart from this, the time of life seems to cause a difference in the number of red corpuscles only in so far that in the newly-born, polycythaemia (up to 8-1/2 millions during the first days of life) is observed (E. Schiff). After the first occasion on which food is taken a decrease can be observed, and gradually (though by stages) the normal figure is reached in from 10-14 days. On the other hand the oligocythaemia here and there observed in old age, according to Schmaltz, is not constant, and therefore cannot be regarded as a peculiarity of senility, but must be caused by subsidiary processes of various kinds which come into play at this stage of life.

The influence which the taking of food exercises on the number of the red blood corpuscles is to be ascribed to the taking in of water, and is so insignificant, that the variations, in part at least, fall within the errors of the methods of enumeration.

Other physiological factors: menstruation (that is, the single occurrence), pregnancy, lactation, do not alter the number of blood corpuscles to any appreciable extent. The numbers do not differ in arterial and venous blood.

All these physiological variations in the number of the blood corpuscles, are dependent, according to Cohnstein and Zuntz, on vasomotor influences. Stimuli, which narrow the peripheral vessels, locally diminish the number of red blood corpuscles; excitation of the vasodilators brings about the opposite effect. Hence it follows, that the normal variations of the number contained in a unit of space are merely the expressions of an altered distribution of the red elements within the circulation, and are quite independent of the reproduction and decay of the cells.

Climatic conditions apparently exercise a great influence over the number of corpuscles. This fact is important for physiology, pathology, and therapeutics, and has come to the front especially in the last few years, since Viault's researches in the heights of the Corderillas. As his researches, as well as those of Mercier, Egger, Wolff, Koeppe, v. Jaruntowski and Schroeder, Miescher, Kuendig and others, shew, the number of red blood corpuscles in a healthy man, with the normal average of 5,000,000 per mm.^{3}, begins to rise immediately after reaching a height considerably above the sea-level. With a rise proceeding by stages, a new average figure is reached in 10 to 14 days, considerably larger than the old one, and indeed the greater the difference in level between the former and the latter places, the greater is the difference in this figure. Healthy persons born and bred at these heights have an average of red corpuscles which is considerably above the mean; and which indeed as a rule is somewhat greater than in those who are acclimatised or only temporarily living at these elevations.

The following small table gives an idea of the degree to which the number of blood corpuscles may vary at higher altitudes from the average of five millions.

-+ -+ -+ - Author Locality Height above sea- Increase of level -+ -+ -+ - v. Jaruntowski Goerbersdorf 561 metres 800,000 Wolff and Koeppe Reiboldsgruen 700 " 1,000,000 Egger Arosa 1800 " 2,000,000 Viault Corderillas 4392 " 3,000,000 -+ -+ -+ -

Exactly the opposite process is to be observed when a person accustomed to a high altitude reaches a lower one. Under these conditions the correspondingly lower physiological average is produced. These interesting processes have given rise to various interpretations and hypotheses. On the one hand, the diminished oxygen tension in the upper air was regarded as the immediate cause of the increase of red blood corpuscles. Miescher, particularly, has described the want of oxygen as a specific stimulus to the production of erythrocytes. Apart from the physiological improbability of such a rapid and comprehensive fresh production, one must further dissent from this interpretation, since the histological appearance of the blood gives it no support. Koeppe, who has specially directed part of his researches to the morphological phenomena produced during acclimatisation to high altitudes, has shewn, that in the increase of the number of red corpuscles two mutually independent and distinct processes are to be distinguished. He observed that, although the number of red corpuscles was raised so soon as a few hours after arrival at Reiboldsgruen, numerous poikilocytes and microcytes make their appearance at the same time. The initial increase is therefore to be explained by budding and division of the red corpuscles already present in the circulating blood. Koeppe sees in this process, borrowing Ehrlich's conception of poikilocytosis, a physiological adaptation to the lower atmospheric pressure, and the resulting greater difficulty of oxygen absorption. The impediment to the function of the haemoglobin is to a certain extent compensated, since the stock of haemoglobin possesses a larger surface, and so is capable of increased respiration. So also the remarkable fact may be readily understood that the sudden rise of the number of corpuscles is not at first accompanied by a rise of the quantity of haemoglobin, or of the total volume of the red blood corpuscles. These values are first increased when the second process, an increased fresh production of normal red discs, takes place, which naturally requires for its developement a longer time. The poikilocytes and microcytes then vanish, according to the extent of the reproduction; and finally a blood is formed, which is characterised by an increased number of red corpuscles, and a corresponding rise in the quantity of haemoglobin, and in the percentage volume of the corpuscles.

Other authors infer a relative and not an absolute increase in the number of red corpuscles. E. Grawitz, for example, has expressed the opinion that the raised count of corpuscles may be explained chiefly by increased concentration of the blood, due to the greater loss of water from the body at these altitudes. The blood of laboratory animals which Grawitz allowed to live in correspondingly rarefied air underwent similar changes. Von Limbeck, as well as Schumburg and Zuntz, object to this explanation on the ground, that if loss of water caused such considerable elevations in the number, we should observe a corresponding diminution in the body weight, which is by no means the case.

Schumburg and Zuntz also regard the increase of red blood corpuscles in the higher mountains as relative only, but explain it by an altered distribution of the corpuscular elements within the vascular system. In their earlier work Cohnstein and Zuntz had already established that the number of corpuscles in the capillary blood varies with the width of the vessels and the rate of flow in them. If one reflects how multifarious are the merely physiological influences at the bottom of which these two factors lie, one will not interpret alterations in the number of the red corpuscles without bearing them in mind. In residence at high altitudes various factors bring about alterations in the width of the vessels and in the circulation. Amongst these are the intenser light (Fuelles), the lowering of temperature, increased muscular exertion, raised respiratory activity. Doubtless, therefore, without either production of microcytes or production de novo, the number of red corpuscles in capillary blood may undergo considerable variations.

The opposition, in which as mentioned above, the views of Grawitz, Zuntz, and Schumburg stand to those of the first mentioned authors, finds its solution in the fact that the causes of altered distribution of the blood, and of loss of water, play a large part in the sudden changes. The longer the sojourn however at these great elevations, the more insignificant they become (Viault).

We think therefore that from the material before us we may draw the conclusion, that after long residence in elevated districts the number of red blood corpuscles is absolutely raised. The therapeutic importance of this influence is obvious.

Besides high altitudes, the influence of the tropics on the composition of the blood and especially on the number of corpuscles has also been tested. Eykmann as well as Glogner found no deviation from the normal, although the almost constant pallor of the European in the tropics points in that direction. Here also, changes in the distribution occurring without qualitative changes of the blood seem chiefly concerned.

* * * * *

The same reliance cannot be placed on inferences based on the results of the Thoma-Zeiss and similar counting methods for anaemic as for normal blood, in which generally speaking all the red cells are of the same size and contain the same amount of haemoglobin. In the former the red corpuscles, as we shall shew later, differ considerably one from another. On the one hand forms poor in haemoglobin, on the other very small forms occur, which by the wet method of counting cannot even be seen.

Apart even from these extreme forms, 1,000 red blood corpuscles of anaemic blood are not physiologically equivalent to the same number of normal blood corpuscles. Hence the necessity of closely correlating the result of the count of red blood corpuscles with the haemoglobinometric and histological values. The first figure only, given apart from the latter, is often misleading, especially in pathological cases.

It is therefore occasionally desirable to supplement the data of the count by THE ESTIMATION OF THE SIZE OF THE RED BLOOD CORPUSCLES INDIVIDUALLY. This is effected by direct measurement with the ocular micrometer; and can be performed on wet (see below), as well as on dry preparations, though the latter in general are to be preferred on account of their far greater convenience.

Nevertheless the carrying out of this method requires particular care. One can easily see that in normal blood the red corpuscles appear smaller in the thicker than they do in the thinner layers of the dry preparation. We may explain this difference as follows. In the thick layers the red discs float in plasma before drying, whilst in the thinner parts they are fastened to the glass by a capillary layer. Desiccation occurs here nearly instantaneously, and starts from the periphery of the disc; so that an alteration in the shape or size is impossible. On the contrary the process of drying in the thicker portions proceeds more slowly, and is therefore accompanied by a shrinking of the discs.

Even in healthy persons small differences in the individual discs are shewn by this method. The physiological average of the diameter of the greater surface is, according to Laache, Hayem, Schumann and others, 8.5 mu for men and women (max. 9.0 mu. min. 6.5 mu.) In anaemic blood the differences between the individual elements become greater, so that to obtain the average value, the maxima, minima, and mean of a large number of cells, chosen at random, are ascertained. But with a high degree of inequality of the discs this microscopical measurement loses all scientific value.

However valuable the knowledge of the absolute number may be for a judgment on the course of the illness, it gives us no information about the AMOUNT OF HAEMOGLOBIN IN THE BLOOD, which is the decisive measure of the degree of the anaemia. A number of clinical methods are in use for this estimation; first direct, such as the colorimetric estimation of the amount of haemoglobin, secondly indirect, such as the determination of the specific gravity or of the volume of the red corpuscles, and perhaps also the estimation of the dry substance of the total blood.

Among the direct methods for haemoglobin estimation, which aim at the measurement of the depth of colour of the blood, we wish first to mention one, which though it lays no claim to great clinical accuracy has often done us good service as a rapid indicator at the bedside. A little blood is caught on a piece of linen or filter-paper, and allowed to distribute itself in a thin layer. In this manner one can recognise the difference between the colour of anaemic and of healthy blood more clearly than in the drop as it comes from the finger prick. After a few trials one can in this way draw conclusions as to the degree of the existing anaemia. Could this simple method which is so convenient, which can be carried out at the time of consultation, come more into vogue, it alone would contribute to the decline of the favourite stop-gap diagnosis, 'anaemia.' For neurasthenic patients also, who so often fancy themselves anaemic and in addition look so, a demonstratio ad oculos such as this is often sufficient to persuade them of the contrary.

Of the instruments for measuring the depth of colour of the blood, the double pipette of Hoppe-Seyler is quite the most delicate. A solution of carbonic oxide haemoglobin, accurately titrated, serves as the standard of comparison. The reliable preparation and conservation of the normal solution is however attended with such difficulties, that this method is not clinically available. In the last few years, Langemeister, a pupil of Kuehne's, has invented a method for colorimetric purposes, also applicable to haemoglobin estimations. The instrument depends on the principle, that from the thickness of the layer in which the solution to be tested has the same colour intensity as a normal solution, the amount of colour can be calculated. As a normal solution Langemeister uses a glycerine solution of methaemoglobin prepared from pig's blood. To our knowledge this method has not yet been applied clinically. Its introduction would be valuable, for in practice we must at present be content with methods that are less exact, in which coloured glass or a stable coloured solution serves as a measure for the depth of colour of the blood. There are a number of instruments of this kind, of which the "haemometer" of Fleischl, and amongst others, the "haemoglobinometer" of Gowers, distinguished by its low price, are specially used for clinical purposes. Both instruments give the percentage of the haemoglobin of normal blood which the blood examined contains, and are sufficiently exact in their results for practical purposes and for relative values; although errors up to 10% and over occur with unpractised observers. (Cp. K. H. Mayer.) Quite recently Biernacki has raised the objection to the colorimetric methods of the quantitative estimation of haemoglobin, that the depth of colour of the blood is dependent not only on the quantity of haemoglobin but also on the colour of the plasma, and the greater or less amount of proteid in the blood. These errors are quite inconsiderable for the above-mentioned instruments, since here the blood is so highly diluted with water that the possible original differences are thereby reduced to zero.

Among the methods for indirect haemoglobin estimation, that of calculation from the amount of iron in the blood appears to be quite exact, since haemoglobin possesses a constant quantity of iron of 0.42 per cent. This calculation may be allowed in all cases for normal blood, for here there is a really exact proportion between the amounts of haemoglobin and of iron. Recently A. Jolles has described an apparatus for quantitative estimation of the iron of the blood, called a "ferrometer;" which renders possible an accurate valuation of the iron in small amounts of blood. However for pathological cases this method of haemoglobin estimation from the iron present is not to be recommended. For if one tests the blood of an anaemic patient under the microscope for iron one finds the iron reaction in numerous red blood corpuscles. This means the presence of iron which is not a normal constituent of haemoglobin. Other iron may be contained in the morphological elements (including the white corpuscles) as a combination of proteid with iron, which is not directly recognisable. It is further known that in anaemias the amount of iron of all organs is greatly raised (Quincke), apparently often the result of a raised destruction of haemoglobin ("waste iron," "spodogenous iron"). In many cases too, it should be borne in mind that the administration of iron increases the amount of iron in the blood and organs.

From these considerations we see how unreliable in pathological cases is the calculation of the amount of haemoglobin from the amount of iron. We have been particularly led to these observations by the work of Biernacki, since the procedure of inferring the amount of haemoglobin from the amount of iron has led to really remarkable conclusions. For example, amongst other things, he found the iron in two cases of mild, and one of severe chlorosis quite normal. He concludes that chlorosis, and other anaemias, shew no diminution, but even a relative increase of haemoglobin: but that other proteids of the blood on the contrary are reduced. These difficult iron estimations stand out very sharply from the results of other authors and could only be accepted after the most careful confirmation. But the above analysis shews, that in any case the far-reaching conclusions which Biernacki has attached to his results are insecure. For these questions especially, complete estimations with the aid of the ferrometer of A. Jolles are to be desired.

Great importance has always been attached to the investigation of the SPECIFIC GRAVITY of the blood; since the density of the blood affords a measure of the number of corpuscles, and of their haemoglobin equivalent. It is easy to collect observations, as in the last few years two methods have come into use which require only a small quantity of material, and do not appear to be too complicated for practical clinical purposes. One of these has been worked out by R. Schmaltz, in which small amounts of blood are exactly weighed in capillary glass tubes (the capillary pyknometric method). The other is A. Hammerschlag's, in which, by a variation of a principle which was first invented by Fano, that mixture of chloroform and benzol is ascertained in which the blood to be examined floats, i.e. which possesses exactly the specific gravity of the blood[2].

According to the researches of these authors and numerous others who have used their own methods, the specific gravity of the total blood is physiologically 1058-1062, or on the average 1059 (1056 in women). The specific gravity of the serum amounts to 1029-1032—on the average 1030. From which it at once follows that the red corpuscles must be the chief cause of the great weight of the blood. If their number diminishes, or their number remaining constant, they lose in haemoglobin, or in volume, the specific gravity would be correspondingly lowered. We should therefore expect a low specific gravity in all anaemic conditions. Similarly with an increased number of corpuscles, and a high haemoglobin equivalent, an increase in the density of the total blood makes its appearance.

Hammerschlag has found in a large number of experiments that the relation between the specific gravity and the amount of haemoglobin is much closer than between the specific gravity and the number of corpuscles. The former in fact is so constant that it may be represented by a table.

Sp. gravity Quantity of Haemoglobin (Fleischl's method)

1033-1035 25-30% 1035-1038 30-35% 1038-1040 35-40% 1040-1045 40-45% 1045-1048 45-55% 1048-1050 55-65% 1050-1053 65-70% 1053-1055 70-75% 1055-1057 75-85% 1057-1060 85-95%

In a paper which has quite recently appeared Diabella has investigated these relations very thoroughly, and his results partly correct, and partly confirm those of Hammerschlag. Diabella found from his comparative estimations that differences of 10% haemoglobin (Fleischl) correspond in general to differences of 4.46 per thousand in the specific gravity (Hammerschlag's method). Nevertheless with the same amount of haemoglobin, differences up to 13.5 per thousand are to be observed; and these departures are greater the richer the blood in haemoglobin. Regular differences exist between men and women; the latter have, with the same amount of haemoglobin, a specific gravity lower by 2 to 2.5.

Should the parallelism between the number of red blood corpuscles and the amount of haemoglobin be considerably disturbed, the influence of the stroma of the red discs on the specific gravity of the blood will then be recognisable. Diabella calculates, that with the same amount of haemoglobin in two blood testings, the stroma may effect differences of 3-5 per thousand in the specific gravity.

Hence the estimation of the specific gravity is often sufficient for the determination of the relative amount of haemoglobin of a blood. It is only in cases of nephritis and in circulatory disturbances, and in leukaemia, that the relations between specific gravity and quantity of haemoglobin are too much masked by other influences.

The physiological variations which the specific gravity undergoes under the influence of the taking in and excretion of fluid do not exceed 0.003 (Schmaltz). From what has been said, it follows that all variations must correspond with similarly occurring variations in the factors that underlie the amount of haemoglobin and the number of corpuscles.

More recent authors, in particular Hammerschlag, v. Jaksch, v. Limbeck, Biernacki, Dunin, E. Grawitz, A. Loewy, have avoided an omission of many earlier investigators; for besides the estimation of the specific gravity of the total blood, they have carried out that of one at least of its constituents, either of the corpuscles or of the serum. The red blood corpuscles have consistently shewn themselves as almost exclusively concerned with variations in the specific gravity of the total blood; partly by variations in number, or changes in their distribution; partly by their chemical instability; loss of water and absorption of water, and variations in the amount of iron.

The plasma of the blood on the contrary—and there is no essential difference between plasma and serum (Hammerschlag)—is much more constant. Even in severe pathological conditions, in which the total blood has become much lighter, the serum preserves its physiological constitution, or undergoes but relatively slight variations in consistence. Considerable diminutions in the specific gravity of the serum are much less frequently observed in primary blood diseases, than in chronic kidney diseases, and disturbances of the circulation. E. Grawitz has lately recorded that in certain anaemias, especially posthaemorrhagic and those following inanition, the specific gravity of the serum undergoes perceptible diminutions[3].

There are still therefore many contradictions in these results, and it is evidently necessary in a scientific investigation always to give the specific gravity of the serum and of the corpuscles, in addition to that of the total blood.

A method closely related to the estimation of the specific gravity is the direct estimation of the dried substance of the total blood, "HYGRAEMOMETRY"; the clinical introduction of which we owe to Stintzing and Gumprecht. This method is really supplementary to those so far mentioned, and like them can be carried out with the small amounts of blood obtainable at the bedside without difficulty. Small quantities of blood are received in weighed glass vessels: which are then weighed, dried at 65 deg.-70 deg. C. for 24 hours and then weighed again. The figures so obtained for the dried substance have a certain independent importance; for they do not run quite parallel with those of the specific gravity, amount of haemoglobin or number of corpuscles. The normal values are, for men 21.26%, for women 19.8%.

A further procedure for obtaining indirect evidence of the amount of haemoglobin is the DETERMINATION OF THE VOLUME OF BLOOD CORPUSCLES IN 100 PARTS OF TOTAL BLOOD. For this estimation a method is desirable, which allows of the separation of the corpuscles from plasma in blood, that is as far as possible unaltered. The older methods do not fulfil this requirement; since they recommend either defibrination of the blood (quite impossible with the quantities of blood which are generally clinically available); or keeping it fluid by the addition of sodium oxalate or other substances which prevent coagulation. The separation of the two constituents can be effected by simply allowing the blood to settle, or with the centrifugal machine, specially constructed for the blood by Blix-Hedin and Gaertner ("Haematocrit").

For these methods various diluting fluids are used, such as physiological saline solution, 2.5% of potassium bichromate and many others. According to H. Koeppe they are not indifferent as far as the volume of the red blood corpuscles is concerned; and a solution which does not affect the cells must be previously ascertained for each specimen of blood. For this reason attention may be called to the proceeding of M. Herz, in which the clotting of the blood in the pipette is prevented by rendering the walls absolutely smooth by the application of cod-liver oil. Koeppe has slightly varied this method; he fills his handily constructed pipette, very carefully cleaned, with cedar wood oil, and sucks up the blood, as it comes from the fingerprick into the filled pipette. The blood displaces the oil, and as it only comes into contact with perfectly smooth surfaces, it remains fluid. By means of a centrifugal machine, of which he has constructed a very convenient variation, the oil as the lighter body is completely removed from the blood; and the plasma is also separated from the corpuscles. Three sharply defined layers are then visible, the layer of oil above, the plasma layer, and the layer of the red blood corpuscles. In as much as the apparatus is calibrated, the relation between the volumes of the plasma and corpuscles can be read off. No microscopical alterations in the corpuscles are to be observed.

Though this procedure seems very difficult of execution, it is nevertheless the only one, which has really advanced clinical pathology. The results of Koeppe—not as yet very numerous—give the total volume of the red corpuscles as 51.1-54.8%, an average of 52.6%.

M. and L. Bleibtreu have endeavoured indirectly to ascertain the relation of the volume of the corpuscles to that of the plasma. Mixtures of blood with physiological saline solution in various proportions are made, in each the amount of nitrogen in the fluid which is left after the corpuscles have settled is estimated. With the aid of quantities so obtained they calculate mathematically the volume of the serum and corpuscles respectively. Apart from the fact that a dilution with salt solution is also here involved, this method is too complicated and requires amounts of blood too large for clinical purposes. Th. Pfeiffer has tried to introduce it clinically in suitable cases, but has not so far succeeded in obtaining definite results. That, however, the relations between the relative volume of the red corpuscles and quantity of haemoglobin are by no means constant, is well shewn by conditions (for example the acute anaemias) in which an "acute swelling" of the individual red discs occurs (M. Herz), but without a corresponding increase in haemoglobin. The same conclusion results from recent observations of v. Limbeck, that in catarrhal jaundice a considerable increase of volume of the red blood corpuscles comes to pass under the influence of the salts of the bile acids.

As we have several times insisted, the quantity of haemoglobin affords the most important measure of the severity of an anaemic condition. Those methods which neither directly nor indirectly give an indication of the amount of haemoglobin are only so far of interest that they possibly afford an elucidation of the special pathogenesis of blood diseases in particular cases. To these belong the ESTIMATION OF THE ALKALINITY OF THE BLOOD, which in spite of extended observations has not yet obtained importance in the pathology of the blood.

A value to which perhaps attention will be more directed than it has up to the present time by clinicians is the RATE OF COAGULATION OF THE BLOOD, for which comparative results may be obtained by Wright's handy apparatus, the "Coagulometer." In certain conditions, particularly in acute exanthemata, and in the various forms of the haemorrhagic diathesis, the clotting time is distinctly increased, or indeed clotting may remain in abeyance. Occasionally a distinct acceleration in the clotting, compared with the normal, may be observed. Wright has further ascertained in his excellent researches, that the clotting time can be influenced by drugs: calcium chloride, carbonic acid raise, citric acid, alcohol and increased respiration diminish the clotting power of the blood.

Recently Hayem has repeatedly called attention to a condition, that is probably closely connected with the coagulability of the blood. Although coagulation has set in, the separation of the SERUM FROM THE CLOT occurs only very slightly or not at all. Hayem asserts, that he has found such blood in Purpura haemorrhagica, Anaemia perniciosa protopathica, malarial cachexia: and some infectious diseases.

For such observations large amounts of blood are needed, which are clinically not frequently available. Certain precautions must be observed, as has been ascertained in the preparation of diphtheria serum, so that the yield of serum may be the largest possible. Amongst these that the blood should be received in longish vessels, which must be especially carefully cleaned, and free from all traces of fat. If the blood-clot does not spontaneously retract it must be freed from the side of the glass with a flat instrument like a paper-knife without injuring it. If no clot occurs in the cold, a result may perhaps follow at blood temperature.

In spite however of all artifices and all care, it is here and there, under pathological conditions, impossible to obtain even a trace of serum from considerable amounts of blood. In a horse for example which was immunised against diphtheria, and had before yielded an unusually large quantity of serum, Ehrlich was able to obtain from 22 kg. of blood scarcely 100 cc. serum, when the animal was bled on account of a tetanus infection.

Perhaps a larger role is to be allotted in the diseases of the blood to these conditions. Hayem already turns the incomplete production of serum to account, for distinguishing protopathic pernicious anaemia from other severe anaemic conditions. A bad prognosis too may be made when for example in cachetic states this phenomenon is to be observed.

A few methods still remain to be mentioned which test THE RESISTANCE OF THE RED BLOOD CORPUSCLES to external injuries of various kinds.

Landois, Hamburger and v. Limbeck ascertain for instance the degree of concentration of a salt solution, in which the red corpuscles are preserved ("isotonic concentration," Hamburger) and those which cause an exit of the haemoglobin from the stroma. The erythrocytes are the more resistant, the weaker the concentration which leaves them still uninjured.

Laker tests the red blood corpuscles as regards their resistance to the electric discharge from a Leyden jar, and measures it by the number of discharges up to which the blood in question remains uninjured.

Clinical observation has not yet gained much by these methods. So much only is certain, that in certain diseases: anaemia, haemoglobinuria, and after many intoxications, the resistance, as measured by the methods above indicated, is considerably lowered.

FOOTNOTES:

[1] For the estimation of the numbers of white corpuscles, relatively to the red, and of the different kinds relatively to each other, see the section on the morphology.

[2] In Roy's method, mixtures of glycerine and water are used. By means of a curved pipette, the drop of blood is brought into the fluid, and its immediate motion observed. Lazarus Barlow has modified this method. He employs mixtures of gum and water, and instead of several tubes, one only; and into this the mixtures are introduced, those of higher specific gravity being naturally at the bottom. The alternate layers are coloured, and remain distinguishable for several hours.

[3] In conditions of shock experimentally produced, the specific gravity of the total blood is increased, that of the plasma, however, is diminished (Roy and Cobbett).



THE MORPHOLOGY OF THE BLOOD.



A. METHODS OF INVESTIGATION.

A glance at the history of the microscopy of the blood shews that it falls into two periods. In the first, which is especially distinguished by the work of Virchow and Max Schultze, a quantity of positive knowledge was quickly won, and the different forms of anaemia were recognised. But close upon this followed a standstill, which lasted for some decades, the cause of which lay in the circumstance that observers confined themselves to the examination of fresh blood. What in fact was to be seen with the aid of this simple method, these distinguished observers had quickly exhausted. That these methods were inadequate is best shewn by the history of leucocytosis, which after the precedent of Virchow was in general referred to an increased production on the part of the lymphatic glands; and further by the imperfect distinction between leucocytosis and incipient leukaemia, which was drawn almost exclusively from purely numerical estimations. It was only after Ehrlich had introduced the new methods of investigation by means of stained dry preparations, that the histology of the blood received the impulse for its second period.

We owe to them the exact distinction between the several kinds of white blood corpuscles, a rational definition of leukaemia, polynuclear leucocytosis, and the knowledge of the appearances of degeneration and regeneration of the red blood corpuscles, and of their degeneration in haemoglobinaemic conditions. The same process, then, has gone on in the microscopy of the blood that we see in other branches of normal and pathological histology: by advances in method, advances in knowledge full of importance result. It is therefore little comprehensible, that an author quite recently should recommend a reversion to the old methods, and emphatically announce that he has managed to make a diagnosis in all cases, with the examination of fresh blood. At the present time, after the most important points have been cleared up by new methods, in the large majority of cases, this is not an astonishing achievement. For any difficult case (for instance the early recognition of malignant lymphoma, certain rare forms of anaemia, etc.) as the experienced know, the dry stained preparation is indispensable. The object of examining the blood, is certainly not to make a rapid diagnosis, but to investigate exactly the individual details of the blood picture. To-day, we can only take the standpoint, that everything that is to be seen in fresh specimens—apart from the quite unimportant rouleaux formation, and the amoeboid movements—can be seen equally well, and indeed much better in a stained preparation; and that there are several important details which are only made visible in the latter, and never in wet preparations.

As regards the purely technical side of the question, the examination of stained dry specimens is far more convenient than that of fresh. For it leaves us quite independent of time and place, we can keep the dried blood with few precautions for months at a time, before proceeding to further microscopic treatment; and the examination of the preparation may last as long as required, and can be repeated at any time. On the contrary, the examination of the wet preparation is only possible at the bedside, and must be conducted within so short a time, on account of the changeability of the blood, clotting, destruction of white corpuscles and so forth, that a searching investigation cannot be undertaken. In addition the preparation and staining of dried blood specimens is amongst the simplest and most convenient of the methods of clinical histology. In the interest of its wider dissemination, it will be justifiable to describe it more in detail.

We must also mention here the use of the dry preparation in the estimation of the important relation between the number of the red and of the white corpuscles; and also of the relative numbers of different kinds of white blood corpuscles.

For this purpose, faultless specimens, specially regularly spread, are indispensable. Quadratic ocular diaphragms (Ehrlich-Zeiss) are requisite, which form a series, so that the sides of the squares are as 1:2:3 ... :10, the fields therefore as 1:4:9 ... :100. The eye-piece made by Leitz after Ehrlich's directions is more convenient, in which, by a handy device, definite square fractions of the field can be obtained. The enumeration is made as follows. The white blood corpuscles are first counted in any desired field with the diaphragm no. 10, that is with the area of 100. Without changing the field, the diaphragm 1, which only leaves free a hundredth part of this area, is now put in, and the red corpuscles are counted. The field is then changed at random, and the red corpuscles counted in a portion of the area which represents the hundredth of that of the white. About 100 such counts are to be made in a specimen. The average of the red corpuscles is then multiplied by 100 and so placed in proportion with the sum of the white. If the white corpuscles are very numerous, so that counting each one in a large field is inconvenient, smaller sections of the eye-piece 81, 64, 49, etc. may be taken.

The important estimation of the percentage relation of the various forms of leucocytes is effected by the simple "typing" of several hundred cells, a count which for the practised observer is completed in less than a quarter of an hour.

[alpha] Preparation of the dry specimen.

To obtain unexceptionable preparations cover-slips of particular quality are necessary. They should not be thicker than 0.08 to 0.10 mm., the glass must not be brittle or faulty, and must in this thickness easily allow of considerable bending, without breaking. Every unevenness of the slip renders it useless for our purposes. The glasses must previously be particularly carefully cleaned, and all fat removed. It is generally sufficient to allow the slips to remain in ether for about half-an-hour, not covering one another. Each one still wet with ether is then wiped with soft, not coarse, linen rag or with tissue-paper. The slips now are put into alcohol for a few minutes, are dried in the same manner as from the ether, and are kept ready for use in a dust-tight watch-glass. Bearing in mind, that these cover-slips are not cut out from a flat piece but from the surface of a sphere, it is evident that only with glasses thus prepared, can it be expected that a capillary space should be formed between two of them, in which the blood spreads easily. For with the smallest unevenness or brittleness of the glass it is an impossibility for the one to fit every bend of the other. And it is only then that the slips can be drawn away one from another, without using a force which breaks them.

To avoid fresh soiling of the cover-slips, and above all the contact of the blood with the moisture coming from the finger, the cover-glass is held with forceps[4] to receive the blood. We recommend for the under cover-glass a clamp forceps a, with broad, smooth blades; the ends may be covered with leather or blotting-paper for a distance of about 1/2 in. For the other cover-slip a very light spring forceps b, with smooth blades, sharp at the tips, is used, with which a cover-glass can be easily picked up from a flat surface. The lower slip is now fixed by one edge in the clamp forceps, and held ready in the left hand. The right hand applies the upper glass with the forceps b to the drop of blood as it exudes from the puncture, and takes it up, without touching the finger itself. The forceps b is then quickly brought to a and the slip with the little drop of blood allowed to fall lightly on the other. In glasses of the right quality the drop distributes itself spontaneously in a completely regular capillary layer. With two fingers of the right hand on the edge of the upper glass, it is now carefully pulled from the lower, which remains fixed in the clamp, without pressing or lifting. Frequently only one, the lower, shews a regular layer, but occasionally both are available for examination. During the desiccation in the air, generally complete in 10-30 seconds, the preparations must naturally be protected from any dampness (for example the breath of the patient).

The extent of surface which is covered depends on the size of the drop, the smaller the latter, the smaller the surface over which it has to be spread. Large drops are quite useless, for with them, the one cover-glass swims on the other, instead of adhering to it.

Although a written description of these manipulations makes the method seem rather intricate, yet but little practice is required to obtain an easy and sure mastery over it. We have felt compelled to describe the method minutely, since preparations so often come under our notice which, although made by scientific men, who pursue haematological investigations, are only to be described as technically completely inadequate.

The specimens so obtained, after they are completely dried in the air, should be kept between layers of filter-paper in well closed vessels till further treatment. In important cases, preparations of which it is desirable to keep for some considerable time, some of the specimens should be kept from atmospheric influences by covering them with a layer of paraffin. The paraffin must be removed by toluol before proceeding further. The preparations must naturally be kept in the dark.

[beta]. Fixation of the dry specimen.

All methods of staining available for the blood require the fixing of the proteids of the blood. A general formula cannot be given, since the intensity of the fixation must be regulated in accordance with the kind of stain that is chosen. Relatively slight degrees of hardening suffice for staining in simple watery solutions, for example, in the triacid fluid, and can be attained by a short, and not too intense action of several reagents. For other methods, in which solutions that are strongly acid or alkaline are employed, it is however necessary to fix the structure much more strongly. But here, too, an excess as well as an insufficiency must be guarded against. It is easy with the few staining fluids that are in use to ascertain the optimum for each.

The following means of fixation are employed.

1. Dry Heat.

A simple plate of copper on a stand is used, under one end of which burns a Bunsen flame. After some time a certain constancy in the temperature of the plate is reached, the part nearest to the flame is hottest, that farther away is cooler. By dropping water, toluol, xylol, etc. on to it, one can fairly easily ascertain that point of the plate which has reached the boiling temperature of the particular fluid.

Far more convenient is Victor Meyer's apparatus, used by chemists. This consists of a copper boiler, modified for our purpose, with a roof of thin copper-plate, perforated for the opening of the vapour tube. Small quantities of toluol are allowed to boil for a few minutes in the boiler, and the copper-plate soon reaches the temperature of 107 deg.-110 deg..

For the ordinary staining reagents (in watery fluids) it is enough to place the air-dried preparation at about 110 deg. C. for one half to two minutes. For differential staining mixtures, for instance the eosin-aurantia-nigrosin mixture, a time of two hours is necessary, or higher temperatures must be employed.

2. Chemical means.

a. To obtain a good triacid stain, the preparations may be hardened, according to Nikiforoff, in a mixture of absolute alcohol and ether of equal parts, for two hours. The beauty of specimens fixed by heat is however not quite fully reached by this method.

b. Absolute alcohol fixes dried specimens in five minutes sufficiently to stain them subsequently with Chenzinsky's fluid, or haematoxylin-eosin solution. It is an advantage in many cases, especially when rapid investigation is required, to boil the dried preparation in a test-tube in absolute alcohol for one minute.

c. Formalin in 1% alcoholic solution was first used by Benario for fixing blood preparations. The fixation is complete in one minute, and the granulations can be demonstrated. Benario recommends this method of fixing, especially for the haematoxylin-eosin staining.

These methods are described as the most suitable for blood-investigation in general. For special purposes, for instance, the demonstration of mitoses, blood platelets, etc., other hardening reagents may be used with advantage: Sublimate, osmic acid, Flemming's fluid, and so forth.

[gamma]. Staining of the dry specimen.

Staining methods may be classified according to the purpose to which they are adapted.

We use first those which are suitable for a simple general view. For this it is sufficient to use such solutions as stain haemoglobin and nuclei simultaneously. (Haematoxylin-eosin, haematoxylin-orange).

Occasionally a stain is desirable which only brings out, but in a characteristic manner, a special kind of cell, e.g. the eosinophils, mast cells, or bacteria. Single staining is attained on the principle of maximal decoloration. (Cp. E. Westphal.)

Finally, we have panoptic staining; that is, by methods which bring out, as characteristically as possible, the greatest number of elements. Although we must use high magnifications with these stains, we are compensated by a knowledge of the blood condition that cannot be reached in any other way. A double stain is generally insufficient, and at least three different dyes are used.

Successive staining was formerly used for this purpose. But everyone who has used this method knows how difficult it is to get constant results, however careful one may be in the concentration and time of action of the stain.

Simultaneous staining offers undoubted and important advantages. As there is much obscurity with regard to the principle on which it rests we may here shortly explain the theory of simultaneous staining.

We will begin with the simplest example: the use of picro-carmine, a mixture of neutral ammonium carmine and ammonium picrate. In a tissue rich in protoplasm, carmine alone stains diffusely, though the nuclei are clearly brought out. But if we add an equally concentrated solution of ammonium picrate, the staining gains extraordinarily in distinctness, in as much as now certain parts are pure yellow, others pure red. The best known example is the staining of muscle with picro-carmine, by which the muscle substance appears pure yellow, the nuclei pure red. If, however, instead of ammonium picrate we add another nitro dye which contains more nitro-groups than picric acid, for example the ammonium salt of hexa-nitro-diphenylamine, the carmine stain is completely abolished, all parts stain in the pure aurantia colour. The explanation of this phenomenon is obvious. Myosin has a greater affinity for ammonium picrate than for the carmine salt, and therefore in a mixture of the two combines with the yellow dye. Owing to this combination it is not now in a condition to chemically fix even carmine. Further, the nuclei have a great affinity for the carmine, and therefore stain pure red in this process. If, however, nitro dyes be added to the carmine solution, which have an affinity for all tissues, and also for the nuclei, the sphere of action of the carmine becomes continually smaller, and finally by the addition of the most powerful nitro body, the hexa-nitro compound, is completely abolished. Connective tissue and bone substance, however, behave differently with the picro-carmine mixture, in as much as here the diffuse stain depends exclusively on the concentration of the carmine, and is quite uninfluenced by the addition of a chemical antidote. This staining can only be limited by dilution, but not by the addition of opposed dyes. We must look upon the latter kind of tissue stain not as a chemical combination, but as a mechanical attraction of the stain on the part of the tissue. We may also say: chemical stains are to be recognised by the fact that they react to chemical antidotes; mechanical stains to physical influences; of course always assuming, that purely neutral solutions are employed, and that all additions, which alter the chemical relation of the tissues such as alkalis and acids, or which raise or limit the affinity of the dye for the tissues, are avoided. A further consequence of this view is, that all successive double staining may be serviceably replaced by simultaneous multiple staining, if the chemical nature of the staining process is settled. In contradistinction, in all double stains, which can only be effected by successive staining, mechanical factors are concerned.

In the staining of the dry blood specimen, purely chemical staining processes are concerned, and therefore the polychromatic combination stain is possible in all cases.

The following combinations are possible for the blood:

1. Combined staining with acid dyes. The best known example is the eosin-aurantia-nigrosin mixture, in which the haemoglobin takes on an orange, the nuclei a black, and the acidophil granulations a red hue.

2. Mixtures of basic dyes. It is possible straight away to make mixtures consisting of two basic dyes. As specially suitable we must mention fuchsin, methyl green, methyl violet, methylene blue. On the other hand, mixtures of three bases are fairly difficult to prepare, and the quantitative relations of the constituents must be exactly observed. For such mixtures, fuchsin, bismarck brown, chrome green, may be used.

3. Neutral mixtures. These have played an important part in general histology, from the time that they were first introduced by Ehrlich into the histology of the blood up to the present day; and deserve before all others a full consideration.

Neutral staining rests on the fact, that nearly all basic dyes (i.e. salts of the dye bases, for instance, rosanilin acetate) form combinations with acid dyes (i.e. salts of the dye acids, for instance, ammonium picrate) which are to be regarded as neutral dyes, such as rosanilin picrate. Their employment offers considerable difficulties as they are very imperfectly soluble in water. A practical application of them was first possible after Ehrlich had ascertained that certain series of the neutral dyes are easily soluble in excess of the acid dye, and so the preparation of solutions of the required strength, readily kept, was made possible. Among the basic dyes which are suitable for this purpose are those particularly which contain the ammonium group, especially methyl green, methylene blue, amethyst violet[5] (tetraethylsafraninchloride), and to a certain extent pyronin and rhodamin also. In contradistinction to these, the members of the triphenylmethan series, such as fuchsin, methyl violet, bismarck brown, phosphin, indazine, are in general less suited for the purpose, with the exception of methyl green already mentioned. The acid dyes specially suited for the production of soluble neutral stains are the easily soluble salts of the polysulpho-acids. The salts of the carbonyl acids and other acid phenol dyes are but little suitable: and least of all, the nitro dyes. Specially to be mentioned among the acid dye series are those which can be used for the preparation of the neutral mixtures: orange g., acid fuchsin, narcein (an easy soluble yellow dye, the sodium salt of sulphanilic acid—hydrazo-[beta]-naphtholsulphonic acid).

If a solution of methyl green be allowed to fall drop by drop into a solution of an acid dye, for instance orange g., a coarse precipitate first results, which dissolves completely on the further addition of the orange. No more orange should be added than is necessary for complete solution. This is the type of a simple neutral staining fluid. Chemically the above-mentioned example may be thus explained; in this mixture all three basic groups of the methyl green are united with the acid dye, so that we have produced a triacid compound of methyl green.

Simple neutral mixtures, which have one constituent in common, may be combined together straight away. This is very important for triple staining, which can only be attained by mixing together two simple neutral mixtures, each consisting of two components. A chemical decomposition need not be feared. We thus get mixtures containing three and more colours. Theoretically there are two possibilities for such combinations:

1. Staining mixtures of 1 acid and 2 basic dyes,

e.g. orange—amethyst—methyl green; narcein—pyronin—methyl green; narcein—pyronin—methylene blue.

2. Staining mixtures of 2 acids and 1 base, in particular the mixture to be described later in detail of

orange g.—acid fuchsin—methyl green. Further narcein—acid fuchsin—methyl green,

and the corresponding combinations with methylene blue, and amethyst violet may be mentioned.

The importance of these neutral staining solutions lies in the fact that they pick out definite substances, which would not be demonstrated by the individual components, and which we therefore call neutrophil.

Elements which have an affinity for basic dyes, such as nuclear substances, stain in these neutral mixtures purely in the colour of the basic dye; acidophil elements in that of one of the two acid dyes; whilst those portions of tissue which from their constitution have an equal affinity for acid and basic dyes, attract the neutral compound, as such, and therefore stain in the mixed colour.

* * * * *

The eosine-methylene blue mixtures are exceptional in so far, that it is possible with them, for a short time at least, to preserve active solutions, in which with an excess of basic methylene blue, enough eosin is dissolved for both to come into play. A drawback however of such mixtures is, that in them precipitates are very easily produced, which render the preparation quite useless. This danger is particularly great in freshly prepared solutions. In solutions, such as Chenzinsky's, which can be kept active for a longer time, it is less. Hence fresh solutions stain far more intensely and more variously than older ones, and are therefore used in special cases (see page 46). If the stain is successful the appearances are very instructive. Nuclei are blue, haemoglobin red, neutrophil granulation violet, acidophil pure red, mast cell granulation deep blue, forming one of the most beautiful microscopic pictures.

For practical purposes, besides the iodine and iodine-eosine solution described below (see page 46) the following are especially used:

1. Haematoxylin solution with eosin or orange g.

Eosin (cryst.) 0.5 Haematoxylin 2.0 Alcohol abs. Aqu. dest. Glycerine aa 100.0 Glacial acetic acid 10.0 Alum in excess

The fluid must stand for some weeks. The preparations, fixed in absolute alcohol, or by short heating, stain in from half-an-hour to two hours. The haemoglobin and eosinophil granules are red, the nuclei stain in the colour of haematoxylin. The solution must be very carefully washed off.

2. In the practical application of the triacid fluid, particular care must be taken, as M. Heidenhain first shewed, that the dyes are chemically pure[6]. Formerly granules, apparently basophil, were frequently observed in the white blood corpuscles, particularly in the region of the nucleus. They were not recognised, even by practised observers (e.g. Neusser) as artificial, but were regarded as preformed, and were described as perinuclear forms. Since the employment of pure dyes these appearances, whose meaning for a long time puzzled us, are but seldom seen.

Saturated watery solutions of the three dyes are first prepared, and cleared by standing for some considerable time. The following mixture is now made:

13-14 c.c. Orange-g. solution 6-7 c.c. Acid fuchsin solution 15 c.c. Aqu. dest. 15 c.c. Alcohol 12.5 c.c. Methyl green 10 c.c. Alcohol 10 c.c. Glycerine

These fluids are measured in the above-mentioned order, with the same measuring glass; and from the addition of methyl green onwards the fluid is thoroughly shaken. The solution can be used at once, and keeps indefinitely. The staining of the blood specimen in triacid requires only a little fixation, cp. page 35. The stain is completed in five minutes at most.

The nuclei are greenish, the red blood corpuscles orange, the acidophil granulation copper red, the neutrophil violet. The mast cells stand out by "negative staining" as peculiar bright, almost white cells, with nuclei of a pale green colour.

The triacid stain is very convenient. It is much to be recommended for good general preparations; it is indispensable in all cases where the study of the neutrophil granulations is concerned.

3. Basic double staining. Saturated, watery methyl-green solution is mixed with alcoholic fuchsin.

The stain, which only requires a small fixation, is completed in a few minutes, and colours the nuclei green, the red blood corpuscles red, the protoplasm of the leucocytes fuchsin colour. It is therefore specially suited for demonstration preparations of lymphatic leukaemia.

4. Eosin-methylene blue mixtures, for example Chenzinsky's fluid:

Concentrated watery methylene blue solution 40 c.c. 1/2% eosin solution in 70% alcohol 20 c.c. Aqua dest. 40 c.c.

This fluid is fairly stable, but must always be filtered before use. It only requires a fixation of the specimen for five minutes in absolute alcohol. The staining takes 6-24 hours (in air-tight watch-glasses) at blood temperature. The nuclei and the mast cell granulations stain deep blue, malaria plasmodia light sky blue, red corpuscles and eosinophil granules a fine red.

This solution is particularly suited for the study of the nuclei, the baso and eosinophil granulations, and it is used by preference for anaemic blood, and also for lymphatic leukaemia.

5. 10 c.c. of a 1 per cent. watery eosin solution, with 8 c.c. methylal, and 10 c.c. of a saturated watery solution of methylene blue are mixed, and used at once, see page 41. Time of staining 1, at most 2 minutes. The staining is characteristic only in preparations very carefully fixed by heat. The mast cell granulations are stained pure blue, the eosinophil red, the neutrophil in mixed colour.

6. Jenner's stain consists of a solution in methyl alcohol of the precipitate formed by adding eosine to methylene blue.

Grubler's water soluble eosine, yellow 1.25% } a.a. watery " medicinal, methylene blue 1% } solutions.

Precipitate allowed to stand 24 hours, and then dried at 55 deg.. It is then made up to 1/2% in methyl alcohol (Merck). The stain may be obtained from R. Kanthack, 18, Berners Street, London, ready for use. It is exceedingly sensitive to acids and alkalis. Fixation is effected by heat. Time of staining 1-4 minutes.

Before we pass to the histology of the blood, two important methods may be described, for which the dried blood preparation is employed directly, without previous fixation: 1. the recognition of glycogen in the blood; 2. the microscopic test of the distribution of the alkali of the blood.

1. Recognition of glycogen in blood.

This may be effected in two ways. The original procedure consisted in putting the preparation into a drop of thick cleared iodine-indiarubber solution under the microscope, as had been already recommended by Ehrlich for the recognition of glycogen.

The following method is still better. The preparation is placed in a closed vessel containing iodine crystals. Within a few minutes it takes on a dark brown colour, and is then mounted in a saturated laevulose solution, whose index of refraction is very high. To preserve these specimens they must be surrounded with some kind of cover-glass cement.

By the use of better methods the red blood corpuscles which have taken on the iodine stain stand out, without having undergone any morphological change. The white blood corpuscles are only slightly stained. All parts containing glycogen on the contrary, whether the glycogen be in the white blood corpuscles, or extracellular, are characterised by a beautiful mahogany brown colour. The second modification of this method is specially to be recommended on account of the strong clearing action of the laevulose syrup. In using the iodine-indiarubber solution a small quantity of glycogen in the cells may escape observation owing to the opaqueness of the indiarubber, and occasionally too by the separate staining of the same. The second more delicate method is for this reason recommended, in the investigation of cases of diabetes and other diseases[7].

2. The microscopic test of the distribution of alkali in the blood.

These methods rest on a procedure of Mylius for the estimation of the amount of alkali in glass. Iodine-eosine is a red compound easily soluble in water, which is not soluble in ether, chloroform, or toluol. But the free coloured acid, which is precipitated by acidifying solutions of the salt, is very sparingly soluble in water. It is, on the contrary, very easily soluble in organic solvents, so that by shaking, it completely passes over into an etherial solution, which becomes yellow. If this solution be allowed to fall on glass, on which deposits of alkali have been formed by decomposition, they stand out in a fine red colour as the result of the production of the deeply coloured salt.

In its application to the blood, of course the vessels used for staining as well as the cover-glasses must be cleaned from all adhering traces of alkali by means of acids. The dry specimen is thrown directly after its preparation into a glass vessel containing a chloroform or chloroform-toluol solution of free iodine-eosine. In a short time it becomes dark red. It is then quickly transferred to another vessel containing pure chloroform, which is once more changed, and the preparation still wet from the chloroform is then mounted in canada balsam. In such preparations the morphological elements have preserved their shape completely. The plasma shews a distinct red colour, whilst the red corpuscles have taken up no colour. The protoplasm of the white corpuscles is red, the nuclei appear as spaces, because unstained (negative nuclear staining). The disintegrated corpuscles and the fibrin which is produced, shew an intense red stain. These stains are peculiarly instructive, and shew many details which are not visible in other methods. The study of these preparations is really of the highest value, since they allow the products of manipulation of the dry preparation and every error of production to stand out in the most reliable manner, and so render possible a kind of automatic control. The scientific value of this method lies in the fact that it throws light on the distribution of the alkali in the individual elements of the blood. It appears that free alkali reacting on iodine-eosine is not present in the nuclei; these must therefore have a neutral or an acid reaction. On the contrary the protoplasm of the leucocytes is always alkaline, and the largest amount of alkali is held by the protoplasm of the lymphocytes. We call particular attention, in this connection, to the strong alkalinity of the blood platelets.

FOOTNOTES:

[4] Kloenne and Mueller, Berlin, supply these after Ehrlich's directions.

[5] Baden Anilin and Soda manufactory, Kalle and Co.

[6] At M. Heidenhain's instigation, the Anilin-dye Company of Berlin have prepared the three dyes in the crystalline form.

[7] It may also be used for the recognition of glycogen in secretions. For instance, gonorrhoeal pus always shews a considerable glycogen reaction of the pus cells. It is found, moreover, in cells which originate from tumours, whether these be present in exudations, or obtained by scraping.



B. NORMAL AND PATHOLOGICAL HISTOLOGY OF THE BLOOD.

In satisfactorily prepared dry specimens the red blood corpuscles keep their natural size and shape, and their biconcavity is plainly seen. They present a distinct round homogeneous form, of about 7.5 mu in diameter. They are most intensely coloured in a broad peripheral layer, and most faintly in the centre corresponding to their depression. With all stains mentioned above the stroma is quite uncoloured, and the haemoglobin exclusively attracts the stain, so that for a practised observer the depth of stain gives a certain indication of the haemoglobin equivalent of each cell, and a better one than the natural colour of the haemoglobin in the fresh specimen. Corpuscles poor in haemoglobin are easily recognised by their fainter staining, especially by the still greater brightness of the central zone. When somewhat more marked, they present appearances which from the isolated staining of the periphery Litten has happily named "pessary" forms. The faint staining of a red corpuscle cannot be explained, as E. Grawitz assumes, by a diminished affinity of the haemoglobin for the dye. Qualitative changes of this kind of the haemoglobin, expressing themselves in an altered relationship towards dyes, do not occur, even in anaemic blood. If in the latter, the blood discs stain less intensely, this is due exclusively to the smaller amount of haemoglobin.

A diminution in the haemoglobin content can in this way be shewn in all anaemic conditions, especially in posthaemorrhagic, secondary and chlorotic cases. On the contrary, as Laache first observed, in the pernicious anaemias, the haemoglobin equivalent of the individual discs is raised.

To appreciate correctly pathological conditions, it must always be borne in mind, that in normal blood the individual red blood corpuscles are by no means of the same value. Step by step some of the cells are used up and replaced by new. Every drop of blood contains, side by side, the most various stages of life of fully formed erythrocytes. For this reason influences which affect the blood—provided their intensity does not exceed a certain degree—cannot equally influence all red corpuscles. The least resistant elements, that is, the oldest, will succumb to the effect of influences, to which other and more vigorous cells adapt themselves.

To influences, of this moderate degree, belongs without doubt the anaemic constitution of the blood as such, the effect of which in this direction one can best investigate in cases of posthaemorrhagic anaemia.

In all anaemic conditions we observe characteristic changes in the blood discs.

A. Anaemic or polychromatophil degeneration.

This change in the red blood corpuscles, first described by Ehrlich, to which the second name was given later by Gabritschewski, is only recognisable in stained preparations. The red blood discs, which under normal circumstances stain in pure haemoglobin colour, now take on a mixed colour. For instance, the red corpuscles are pure red in preparations of normal blood, stained with haematoxylin-eosine mixture. But in preparations of blood of a chromic anaemia stained with the same solution, in which possibly all stages of the degeneration in question are present, one sees red discs with a faint inclination to violet; others which are bluish red; and at the end of the series, forms stained a fairly intense blue, in which scarcely a trace of red can be seen, and which by their peculiar notched periphery are evidently to be regarded as dying elements.

Ehrlich put forward the theory, that this remarkable behaviour towards dyes indicates a gradual death of the red blood corpuscles, that is of the old forms, leading to a coagulation necrosis of the discoplasm. The latter takes up, as is the case in coagulation necrosis, the proteids of the blood, and acquires thereby the power of combining with nuclear stains. At the same time the discoplasm loses its power of retaining the haemoglobin, and gives it up to the blood plasma in ever increasing quantity as the change proceeds. Hence the disc continues to lose the capacity for the specific haemoglobin stain.

Objection has been raised to these views from many quarters, especially from Gabritschewski, and afterwards from Askanazy, Dunin and others. The polychromatophil discs are not, they say, dying forms, but on the contrary represent young individuals. The circumstance, that in certain anaemias the early stages of the nucleated red corpuscles are variously polychromatic, was evidence for this opinion.

In view of the great theoretical importance which attaches to this subject, the grounds for regarding this change as degenerative, may be here shortly brought forward.

1. The appearance of the erythrocytes which shew polychromatophilia in the highest degree. By the notching of their margins they appear to eyes practised in the judgment of morphological conditions, in a stage almost of dissolution, and as well-pronounced degeneration forms.

2. The fact that by animal experiment, for instance, in inanition, their appearance in large numbers in the blood can be produced. That is, precisely in conditions, where there can be least question of a fresh production of red blood corpuscles.

3. The clinical experience, that in acute losses of blood in man, these staining anomalies, can be observed in numerous cells, within so short a time as the first 24 hours. Whilst in our observations, which are very numerous upon this point, embracing several hundred cases, and carried out with particular care, no nucleated red blood corpuscles in this space of time can be found in man[8].

4. The polychromatophil degeneration can frequently be observed in nucleated red blood corpuscles, particularly in the megaloblasts. This fact can be so easily established that it can hardly escape even an unpractised observer, and it was sufficiently familiar to Ehrlich, who first directed attention to these conditions. The fact that the normoblasts, which are typical of normal regeneration, are as a rule free from polychromatophil degeneration, gave the key for the interpretation of this appearance. And similarly for the nucleated red blood corpuscles of lower animals. Askanazy asserts that the nucleated red blood corpuscles of the bone-marrow, which he was able to investigate in a case of empyema, shew, immediately after the resection of the ribs, complete polychromatophilia. This perhaps depends on the peculiarities of the case, or on the uncertainty of the staining method: eosine-methylene blue stain, which is for this purpose very unreliable, since slight overstaining towards blue readily occurs. (We expressly advise the use of the triacid solution or of the haematoxylin-eosine mixture for the study of the anaemic degenerations.)

After what has been adduced, we hold in agreement with the recent work of Pappenheim, and Maragliano, that the appearance of polychromatophilia is a sign of degeneration. To explain the presence of erythroblasts which have undergone these changes we must suppose that in severe injuries to the life of the blood these elements are not produced in the usual fashion, but from the very beginning are morbidly altered. Analogies from general pathology suggest themselves in sufficient number.

B. A second change that we find in the red blood corpuscles of the anaemias, is poikilocytosis.

By this name a change of the blood is denoted, where along with normal red blood corpuscles, larger, smaller and minute red elements are found in greater or less number. The excessively large cells are found in pernicious anaemia, as Laache first observed, and as has since been generally confirmed. On the contrary in all other severe or moderate anaemic conditions, the red corpuscles shew a diminution in volume, and in their amount of haemoglobin. This contradiction, which Laache first mentioned, but was unable to explain, has found a satisfactory solution in Ehrlich's researches on the nucleated precursors of the myelocytes and normocytes (see below).

The blood picture of the anaemias is made still more complicated in that the diminutive cells do not preserve their normal shape, but assume the well-known irregular forms: pear-, balloon-, saucer-, canoe-shapes. Nevertheless in good dry preparations the smallest forms usually still shew the central depression. The so-called "microcytes" constitute an exception to this statement. These are small round forms, to which was allotted in the early days of the microscopic investigation of the blood, a special significance for the severe anaemias. They are however nothing but contraction forms of the poikilocytes, as the crenated are of the normal corpuscles. Consequently microcytes are but seldom found in dried specimens, whilst in wet preparations they are easily seen after some time. It is further of importance to know, that in fresh blood the poikilocytes exhibit certain movements, which have already given rise to mistakes in many ways. Thus at one time the poikilocytes were considered to be the cause of malaria. More recently the somewhat larger sizes were regarded by Klebs, Perles as amoebae and similar organisms. In agreement with Hayem, who from the very first described these forms as pseudo-parasites, a warning must be given against attributing a parasitic character to them.

The origin of poikilocytosis, previously the subject of much discussion, is now generally explained in Ehrlich's way. For the mere fact, that by careful heating, poikilocytosis can be experimentally produced in any blood, forces one to the assumption that the poikilocytes are products of a fragmentation of the red blood corpuscles ("schistocytes," Ehrlich). And correspondingly the smallest fragments shew the biconcave form in the dry specimen; for they too contain the specific protoplasm of the disc "which possesses the inherent tendency to assume the typical biconcave form in a state of equilibrium."

Qualitative changes in the protoplasm of the poikilocytes are not to be observed, even by staining; and one may therefore ascribe to them complete functional power, and regard their production as a purposeful reaction to the diminished number of corpuscles. For by the division of a larger blood corpuscle into a series of homologous smaller ones, the respiratory surface is considerably increased.

C. A third morphological variation which anaemic blood may shew in the more severe degrees of the disease, is the appearance of nucleated red blood corpuscles.

Though we do not wish to enter here upon the latest questions concerning the origin of the blood elements, we must shortly indicate the present state of our knowledge of the nucleated red corpuscles.

Since the fundamental work of Neumann and Bizzozero, the nucleated forms have been generally recognised as the young stages of the normal red blood corpuscles. Hayem's theory, on the contrary, obstinately asserts the origin of the erythrocytes from blood-platelets, and has, excepting by the originator and his pupils, been generally allowed to drop.

Ehrlich had in the year 1880 pointed out the clinical importance of the nucleated red blood corpuscles, in as much as he demonstrated that in the so-called secondary anaemias, and in leukaemia, nucleated corpuscles of the normal size, "normoblasts"; in pernicious anaemia excessively large elements, "megaloblasts," "gigantoblasts" are present. At the same time Ehrlich mentioned that the megaloblasts also play a prominent part in embryonic blood formation.

In 1883 Hayem likewise proposed a similar division of the nucleated red blood corpuscles into two,

(1) the "globules nuclees geantes" which he found exclusively in the embryonic state, (2) the "globules nuclees de taille moyennes" which he found invariably present in the later stages of embryonic life, and in adults. Further, W. H. Howell (1890) found in cats' embryoes two kinds of erythrocytes, (1) very large, equivalent to the blood cells of reptiles and amphibia ("ancestor corpuscles"), and (2) of the usual size of the blood corpuscles of mammalia. And similarly more recent authors, H. F. Mueller, C. S. Engel, Pappenheim and others, have adhered to the division of haematoblasts into normo- and megaloblasts. And it is on the whole recognised, that, physiologically, normoblasts are always present in the bone-marrow of adults, as the precursors of the non-nucleated erythrocytes; that the megaloblasts, however, are never found there under normal circumstances, but only in embryonic stages, and in the first years of extra-uterine life.

S. Askanazy on the contrary has expressed the view, that the normoblasts may arise from the megaloblasts, and thereby denies the principal distinction between them. Schaumann also holds that the separation of the two kinds rests on doubtful foundation, since occasionally it is questionable whether particular cells are the normoblasts or the megaloblasts.

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