Histology of the Blood - Normal and Pathological

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Title: Histology of the Blood - Normal and Pathological
Author: Ehrlich, Paul, 1854-1915, Lazarus, Adolf, 1867-1925
Language: English
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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

P. EHRLICH AND A. LAZARUS.

EDITED AND TRANSLATED

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

JOHN LUCAS WALKER STUDENT OF PATHOLOGY.

WITH A PREFACE

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 hæmatogenetic 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 hæmal 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 leukocythæmia and a lymphatic leukæmia?
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 hæmoglobin found in the various types of anæmia?

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 leukæmic 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 hæmatology 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 Anæmie, 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 hæmoglobin 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 hæmoglobin equivalent 49
Anæmic 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]. Leukæmia ("mixed leucocytosis") 167
Lymphatic leukæmia 170
Myelogenous leukæmia 171
Morphological character 187
Origin 187

V. LEUKOPENIA 188

The blood platelets. The hæmoconiæ 190

INDEX TO LITERATURE 195

INDEX 209

PLATES

INTRODUCTION.

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

In practical medicine the term "anæmia" has not quite the restricted
sense that scientific investigation gives it. The former regards certain
striking symptoms as characteristic of the anæmic 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
anæmia 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 anæmic
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 "=anæmia=" 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 anæmias we can at once exclude from our consideration.

_À priori_, the amount of blood may be subnormal in two senses,
quantitative and qualitative. We may have a diminution of the amount of
blood--"=Oligæmia=." 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 Hæmoglobin (=Oligochromæmia=), and
(2) diminution of the number of red blood corpuscles (=Oligocythæmia=).

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

The most important methods of clinical hæmatology 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. Ræhlmann
has shewn that in 60% of the cases of chronic anæmia, in which the skin
and mucous membranes are very white, there is hyperæmia 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 oligæmia.

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
Aquæ destillat. 226.0

Hayem's solution. Hydrarg. bichlor. 0.5
Natr. sulph. 5.0
Natr. chlor. 1.0
Aquæ 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, polycythæmia (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
oligocythæmia 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, Kündig 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.

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 Reiboldsgrün, 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 hæmoglobin is to a certain extent compensated, since the
stock of hæmoglobin 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 hæmoglobin, 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 hæmoglobin, 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 (Fülles), 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 anæmic as for normal
blood, in which generally speaking all the red cells are of the same
size and contain the same amount of hæmoglobin. In the former the red
corpuscles, as we shall shew later, differ considerably one from
another. On the one hand forms poor in hæmoglobin, 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
anæmic 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 hæmoglobinometric
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
µ for men and women (max. 9.0 µ. min. 6.5 µ.) In anæmic 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 HÆMOGLOBIN IN THE BLOOD, which is the decisive measure of
the degree of the anæmia. A number of clinical methods are in use for
this estimation; first direct, such as the colorimetric estimation of
the amount of hæmoglobin, 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 hæmoglobin 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 anæmic 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 anæmia. 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, 'anæmia.' For neurasthenic patients also, who so often fancy
themselves anæmic 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 hæmoglobin, 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 Kühne's, has invented a method for colorimetric purposes, also
applicable to hæmoglobin 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 methæmoglobin 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
"hæmometer" of Fleischl, and amongst others, the "hæmoglobinometer" of
Gowers, distinguished by its low price, are specially used for clinical
purposes. Both instruments give the percentage of the hæmoglobin 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 hæmoglobin,
that the depth of colour of the blood is dependent not only on the
quantity of hæmoglobin 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 hæmoglobin estimation, that of
calculation from the amount of iron in the blood appears to be quite
exact, since hæmoglobin 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
hæmoglobin 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
hæmoglobin estimation from the iron present is not to be recommended.
For if one tests the blood of an anæmic 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
hæmoglobin. 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 anæmias
the amount of iron of all organs is greatly raised (Quincke), apparently
often the result of a raised destruction of hæmoglobin ("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 hæmoglobin 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 hæmoglobin
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 anæmias, shew no diminution, but even a relative increase of
hæmoglobin: 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 hæmoglobin 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 hæmoglobin, or in volume,
the specific gravity would be correspondingly lowered. We should
therefore expect a low specific gravity in all anæmic conditions.
Similarly with an increased number of corpuscles, and a high hæmoglobin
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 hæmoglobin 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 Hæmoglobin
(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% hæmoglobin (Fleischl)
correspond in general to differences of 4.46 per thousand in the
specific gravity (Hammerschlag's method). Nevertheless with the same
amount of hæmoglobin, differences up to 13.5 per thousand are to be
observed; and these departures are greater the richer the blood in
hæmoglobin. Regular differences exist between men and women; the latter
have, with the same amount of hæmoglobin, a specific gravity lower by 2
to 2.5.

Should the parallelism between the number of red blood corpuscles and
the amount of hæmoglobin 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
hæmoglobin 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 hæmoglobin of a blood. It is
only in cases of nephritis and in circulatory disturbances, and in
leukæmia, that the relations between specific gravity and quantity of
hæmoglobin 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 hæmoglobin 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 anæmias, especially
posthæmorrhagic 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,
"HYGRÆMOMETRY"; 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°-70° 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 hæmoglobin 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
hæmoglobin 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 Gärtner ("Hæmatocrit").

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 hæmoglobin are by no means constant, is well shewn by conditions (for
example the acute anæmias) in which an "acute swelling" of the
individual red discs occurs (M. Herz), but without a corresponding
increase in hæmoglobin. 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 hæmoglobin affords
the most important measure of the severity of an anæmic condition. Those
methods which neither directly nor indirectly give an indication of the
amount of hæmoglobin 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 hæmorrhagic
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 hæmorrhagica, Anæmia 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 _rôle_ 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 anæmia from
other severe anæmic 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 hæmoglobin 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: anæmia, hæmoglobinuria, 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 anæmia 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 leukæmia, 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 leukæmia, polynuclear
leucocytosis, and the knowledge of the appearances of degeneration and
regeneration of the red blood corpuscles, and of their degeneration in
hæmoglobinæmic 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 anæmia, 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 hæmatological
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°-110°.

For the ordinary staining reagents (in watery fluids) it is enough to
place the air-dried preparation at about 110° 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 hæmatoxylin-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 hæmatoxylin-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 hæmoglobin and
nuclei simultaneously. (Hæmatoxylin-eosin, hæmatoxylin-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 hæmoglobin 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, narcëin (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;
narcëin--pyronin--methyl green;
narcëin--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 narcëin--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,
hæmoglobin 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. =Hæmatoxylin solution with eosin or orange g.=

Eosin (cryst.) 0.5
Hæmatoxylin 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 hæmoglobin and eosinophil granules are red, the nuclei stain in the
colour of hæmatoxylin. 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 leukæmia.

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
anæmic blood, and also for lymphatic leukæmia.

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°. 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 lævulose
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 lævulose 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] Klönne and Müller, 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 µ 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
hæmoglobin exclusively attracts the stain, so that for a practised
observer the depth of stain gives a certain indication of the hæmoglobin
equivalent of each cell, and a better one than the natural colour of the
hæmoglobin in the fresh specimen. Corpuscles poor in hæmoglobin 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 hæmoglobin for the dye. Qualitative changes of this kind
of the hæmoglobin, expressing themselves in an altered relationship
towards dyes, do not occur, even in anæmic blood. If in the latter, the
blood discs stain less intensely, this is due exclusively to the smaller
amount of hæmoglobin.

A diminution in the hæmoglobin content can in this way be shewn in all
anæmic conditions, especially in posthæmorrhagic, secondary and
chlorotic cases. On the contrary, as Laache first observed, in the
pernicious anæmias, the hæmoglobin 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 anæmic
constitution of the blood as such, the effect of which in this direction
one can best investigate in cases of posthæmorrhagic anæmia.

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

A. =Anæmic 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 hæmoglobin colour, now take on a
mixed colour. For instance, the red corpuscles are pure red in
preparations of normal blood, stained with hæmatoxylin-eosine mixture.
But in preparations of blood of a chromic anæmia 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
hæmoglobin, 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 hæmoglobin 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
anæmias 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 hæmatoxylin-eosine
mixture for the study of the anæmic 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
anæmias, 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 anæmia, as Laache first observed, and as has since been
generally confirmed. On the contrary in all other severe or moderate
anæmic conditions, the red corpuscles shew a diminution in volume, and
in their amount of hæmoglobin. 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 anæmias 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 anæmias. 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 amoebæ 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 anæmic 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 anæmias, and in leukæmia, nucleated corpuscles
of the normal size, "normoblasts"; in pernicious anæmia 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 nuclées géantes" which he found exclusively in the
embryonic state, (2) the "globules nuclées 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. Müller, C. S. Engel, Pappenheim and others, have adhered to the
division of hæmatoblasts 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.

We distinguish three kinds of nucleated red blood-corpuscles on the
grounds of the following characters;

1. =The normoblasts.= These are red corpuscles of the size of the usual
non-nucleated disc, whose protoplasm as a rule shews a pure hæmoglobin
colour, and which possess a nucleus. Occasionally there may be 2-4
nuclei. The sharply defined nucleus lies generally in the centre,
comprises the greater part of the cell, and is above all distinguished
by its intense colour with nuclear stains, which exceeds that of the
nuclei of the leucocytes, and indeed of all known nuclei. This property
is so characteristic that the free nuclei, which occur occasionally in
anæmias, and particularly often in leukæmia, may be recognised as nuclei
of normoblasts, although surrounded by traces only of hæmoglobin, or by
none at all.

2. =The megaloblasts.= These are 2-4 times as large as normal red blood
corpuscles. Their protoplasm, which constitutes by far the chief portion
of the body of the cell, very often shews anæmic degeneration to a
greater or less degree. The nucleus is larger than that of the
normoblasts, but does not form so considerable a fraction of the cell as
in the latter. It is often not sharply defined, and is of a rounded
shape. It is distinguished from the nucleus of the normoblast by its
much weaker affinity for nuclear stains, which may often be so small
that little practised observers perceive no nucleus.

Occasionally very large cells are present of the kind just described,
which are therefore called =gigantoblasts=, but which are not
distinguishable in other respects from the megaloblasts.

It cannot be denied that it is often difficult to decide whether a
particular cell is to be regarded as a specially small megaloblast or a
large normoblast. In such cases one would naturally search the
preparation for perfect forms of hæmatoblasts, and for the presence of
free nuclei or of megalocytes, in order to obtain an indirect conclusion
concerning the cells in question.

3. =The microblasts.= These are occasionally present, _e.g._ in traumatic
anæmias, but they are very seldom found, and have not so far attracted
particular attention.

* * * * *

The question of the meaning of the =normoblasts= and =megaloblasts= has led
to lively and significant discussions, partly in favour of, partly
against the distinction between these two cell forms. After surveying
the literature, we are forced to separate the megaloblasts from the
normoblasts, in the first place because of their subsequent histories,
and the peculiarities of their nuclei, and secondly because of clinical
observation.

[alpha]. =The fate of the nuclei.= For some time past two views, almost
diametrically opposed, have been in existence with regard to the nature
of the change of the nucleated to the non-nucleated erythrocytes. The
chief exponent of the one, Rindfleisch, taught that the nucleus of the
erythroblasts leaves the cell, which thereby becomes a complete
erythrocyte, whilst the nucleus itself, by the aid of the small remnant
of protoplasm which surrounds it, takes up new material from the
surrounding plasma, manufactures hæmoglobin and so becomes a fresh
erythroblast. According to the second theory the erythroblasts change
to non-nucleated discs by the destruction and solution of the nucleus
within the cell body. ("Karyorrhexis," "Karyolysis.") The authors who
support this view and also describe it as the only kind of erythrocyte
formation are chiefly Kölliker and E. Neumann.

Rindfleisch arrived at his theory by direct observation of the process
described, as it occurred in physiological saline solution with the
blood of foetal guinea-pigs and teased preparations of bone-marrow.

E. Neumann regards Rindfleisch's doctrine as untenable, since the
process which he observed is chiefly the result of a severe injury of
the blood from the sodium chloride solution and the teasing. If a method
of preparation be chosen which protects the blood as far as possible,
and avoids every chemical and physical alteration, the exit of the
nucleus as described by Rindfleisch does not occur.

The view of Kölliker and Neumann that the nuclei gradually decay in the
interior of the cell is not supported by the observation of a process,
but by the fact that in suitable material, for instance, foetal
bone-marrow, liver blood, and leukæmic blood, the transition from
erythroblast to erythrocyte is shewn by all phases of nuclear
metamorphosis. v. Recklinghausen professes to have directly observed the
dissolution of the nucleus within the cell in rabbit's blood, kept
living in a moist chamber. Pappenheim's opinion however, that in this
case processes are concerned such as Maragliano and Castellino have
described as artificial necrobiosis, seems in this connection worthy of
consideration.

Just as with regard to the formation of erythrocytes the views differ
one from another, so also with regard to the "free" nuclei which come
under observation in numerous preparations. Kölliker has taught that
these nuclei are not quite free, but are always surrounded by a minute
border of protoplasm. On the other hand Rindfleisch regards these nuclei
as having migrated from, or having been cast off by the erythroblasts;
and Neumann explains them as the early forms of erythroblasts. Ehrlich
was the first to endeavour to effect a compromise between the directly
opposed views of Rindfleisch and Neumann. He taught that both kinds take
part in the production of the red discs. From blood preparations which
contain numerous normoblasts, for instance in "blood crises" (see p.
62), an unbroken series of pictures can easily be put together shewing
how the nucleus of the erythroblast leaves the cell, and at last
produces the appearance of the so-called free nucleus. It must be
expressly mentioned that these pictures are only to be found in
specimens in whose preparation pressure of any kind upon the blood has
been avoided. Further, however rich a blood may be in normoblasts, the
metamorphosis of the nucleus as described by Neumann, is practically
never to be observed. It is quite otherwise with the megaloblasts.
Amongst them, few examples are to be found in which traces at least of
the destruction and solution of the nucleus are not shewn, and in a
blood preparation of pernicious anæmia, which is not too poor in
megaloblasts, one can construct step by step the unbroken series from
megaloblasts with a complete nucleus through all stages of Karyorrhexis
and Karyolysis to the megalocytes, as the process is described by
Neumann[9].

From Ehrlich's observations it follows, that the normoblasts become
normocytes by extrusion or emigration of the nucleus, the megaloblasts
become megalocytes by degeneration of the nucleus within the cell.

M. B. Schmidt without making use of the principal distinction made by
Ehrlich, also concludes from his researches on sections of the
bone-marrow of animals in extra-uterine life, that both kinds of
erythrocyte formation occur.

Quite recently Pappenheim, partly in conjunction with O. Israel, has
carried out very thorough researches on these particular points. As the
subject for observation he chose the blood of embryonic mice. He was
able in the first place, like Rindfleisch, to produce the exit of the
nuclei from the cells by the addition of "physiological" salt solution
to fresh blood, and is of the opinion that the exit of the nucleus from
the erythroblasts only takes place artificially.

In embryonic blood the metamorphosis to erythrocytes occurs exclusively
by nuclear destruction and solution within the cell, be it in the case
of megalo- or gigantoblasts or of cells of the size of the normal red
blood corpuscle.

The free nuclei that are observed, whose appearance Pappenheim explains
by a preceding solution of the protoplasm (plasmolysis), he regards, in
opposition to Rindfleisch and Neumann, not as the beginnings of a
developmental series, but as the surviving remnants of the degenerated
dying blood cells. Clinical observation, certainly, does not support
this conception of Pappenheim's; in as much as in suitable cases with
numerous free nuclei (leukæmia, blood crises) transitional forms, which
according to Pappenheim must necessarily be present, are not to be
found. Moreover, in alluding to a case of leukæmia of this kind, this
author himself admits that the appearance of free nuclei can be
explained in this instance by the exit of the nucleus.

Although Pappenheim, as above mentioned, recognises no difference
between megaloblasts and normoblasts in embryonic blood as far as the
fate of the nucleus is concerned, he nevertheless decidedly supports
Ehrlich's separation of the erythroblasts into these two groups, as two
hæmatogenetically distinct species of cells. He does not regard as
distinguishing characteristics, the size and hæmoglobin content of the
cells--although as we have described above, these are in general
different in normo- and megaloblasts--for these two properties undergo
such great variations as to increase considerably under certain
circumstances the difficulty of diagnosis of individual cells. The chief
characteristic is, as Ehrlich has always particularly insisted, the
=constitution of the nucleus=. The nuclei of cells which are with
certainty to be reckoned among the normoblasts are marked by the absence
of structure, their sharply defined contour, their intense affinity for
nuclear stains. That is by properties which histology sums up under the
name =Pyknosis= (Pfitzner) and recognises as signs of old age. The nuclei
of the megaloblasts are round, shew a good deal of structure, and stain
far less deeply.

[beta]. The clinical differences. Normoblasts are found almost
invariably in all severe anæmias that are the result of trauma,
inanition or organic disease of some kind. They are however mostly
rather scanty, so that a preparation must be searched for some time
before an example is found. But occasionally, most often in acute, but
also in chronic anæmias, and even in =cachectic= conditions, every field
shews one or more normoblasts.

V. Noorden was the first to describe a case in which in the course of a
hæmorrhagic anæmia normoblasts temporarily appeared in such overwhelming
numbers in the circulating blood, that the microscopic picture, which at
the same time comprised a marked hyperleucocytosis, was almost similar
to that of a myelogenous leukæmia. And as in addition to this occurrence
the number of blood corpuscles was nearly doubled, v. Noorden gave it
the distinctive name "blood crisis."

The following procedure is to be recommended for the
investigation of the blood crisis:

1. Estimation of the absolute number of red blood corpuscles.

2. Estimation of the proportion of white to red corpuscles.

3. Estimation of the proportion of nucleated red to white
corpuscles by means of the quadratic ocular diaphragm (see page
31) in the dry preparation.

For instance if we find in a case of anæmia, 3-1/2 millions of
red blood corpuscles, the proportion of white to red corpuscles
= 1/100 and that of the nucleated red to the white = 1/10, then
in 1 cubic millimeter there are 3500 nucleated red corpuscles,
that is for 1000 ordinary there is 1 nucleated corpuscle.

=Megaloblasts= on the contrary are never found in traumatic anæmias. And
in chronic anæmias of the severest degree, the result for example of old
syphilis, carcinoma of the stomach and so forth, one looks for them
almost always in vain, although they are sometimes to be found in
leukæmia. On the contrary, the conditions, apparently much milder, in
which from the clinical history, ætiology and general objective symptoms
pernicious anæmia is suggested, are almost without exception
characterised by the appearance of megaloblasts in the blood.
Nevertheless in very late stages of the disease they are always scanty,
and a very tedious search through one or more specimens is often
required to demonstrate their presence. Hence follows the rule, that the
investigation of a case of severe anæmia should never be considered
closed, before three or four preparations at least have been minutely
searched for megaloblasts under an oil immersion objective.

This clinical difference between the two kinds of hæmatoblasts admits of
but one natural conclusion, which primarily leaves untouched the
question, so much discussed at the present time, whether the megalo- or
normoblasts can change one to the other. In all cases of anæmia, in
which the fresh formation occurs according to the normal type, only in
greater quantity and more energetically, we find normoblasts. Almost all
anæmias resulting from known causes: acute hæmorrhages, chronic
hæmorrhages, poverty of blood from inanition, cachexias, blood poisons,
hæmaglobinæmia and so forth,--in short all conditions rightly called,
secondary, symptomatic anæmias,--may shew this increase of normal blood
production. In the conditions, which Biermer, on the grounds of their
clinical peculiarities, has distinguished as "essential, pernicious
anæmia" megaloblasts on the contrary occur, and represent an embryonic
type of development. The extent to which this type participates in the
blood formation in pernicious anæmia is most simply demonstrated by the
fact that megaloblasts are present in all cases of pernicious anæmia, as
Laache first shewed, and in some cases form the preponderating portion
of the blood discs. Whilst, therefore, in the ordinary kinds of anæmia
we find that the red corpuscles tend to produce small forms, in
pernicious anæmia, on the other hand, and exclusively in this form, we
find a tendency in the opposite direction. This constant difference
cannot be a chance result, but must depend on some constant law: in
pernicious anæmia excessively large blood corpuscles are produced.
Ehrlich's demonstration of megaloblasts has sufficed for this logical
advance. =All researches, which try to obscure or totally deny the
distinction between megaloblasts and normoblasts are wrecked by the
simple clinical fact that in pernicious anæmia the blood is
megaloblastic.=

The appearance of megaloblasts and megalocytes is therefore evidence
that the regeneration of the blood in the bone-marrow is not proceeding
in the normal manner, but in a way which approximates to the embryonic
type. The extreme cases are naturally seldom, such as that of
Rindfleisch, in which the whole bone-marrow was found full of
megaloblasts. It is sufficiently conclusive for the pernicious nature of
the case, "if only considerable portions but not the whole marrow, have
lapsed into megaloblastic degeneration." We can now say that the
megaloblastic metamorphosis is not a purposeful process, and for the
following reasons: 1. Since the fresh formation of red blood corpuscles
by means of the megaloblastic method is clearly much slower. This is
especially borne out by the fact that the megaloblasts are present in
the blood always in small numbers only, whilst the normoblasts, as above
mentioned, are often found in much larger quantities. In agreement with
this, "blood crises" are not to be observed in the megaloblastic
anæmias. 2. Since the megalocytes which are formed from the megaloblasts
possess in proportion to their volume a relatively smaller respiratory
surface, and so constitute a type disadvantageous for anæmic
conditions[10]. This is still more evident when we remember that the
production of poikilocytes is on the contrary a serviceable process.

The megaloblastic degeneration of the bone-marrow is no doubt due to
chemical influences, which alter the type of regeneration in a
disadvantageous manner. We do not for the most part yet know the
exciting causes of the toxic process; consequently we are unable to put
a stop to it, and its termination is lethal. The Bothriocephalus
anæmias, which in general as is well-known offer a good prognosis, by no
means contradict this view. They hold their privileged position amongst
the anæmias of the megaloblastic type, only for the reason that their
cause is known to us, and can be removed. As in many infectious
diseases, individuals react quite differently to the presence of the
Bothriocephalus. Some remain well; others show the signs of simple
anæmia, ultimately with normoblasts; whilst a third group presents the
typical picture of pernicious anæmia. For many years, so long as its
ætiology was unknown, Bothriocephalus anæmia was not separated on
clinical grounds from pernicious anæmia. Severe Bothriocephalus anæmia
may be described as a pernicious anæmia, with a known and removable
cause. Good evidence for this point of view is afforded by a case of
Askanazy, who describes a severe pernicious anæmia, with typical
megaloblasts, in which after the complete expulsion of the
Bothriocephalus, the megaloblastic character of the blood formation
quickly vanished, was replaced by the normoblastic, and the patient
rapidly recovered. This observation is so unequivocal, that it is a
matter of surprise that Askanazy chooses to deduce from it, the ready
transition from megaloblasts to normoblasts; whereas it is clear and
definite evidence that =megaloblasts are only produced under the
influence of a specific intoxication=. And in this way the presence of
megaloblasts in the pernicious anæmias is to be explained. The
megaloblastic degeneration of the bone-marrow depends on the presence of
certain injurious influences, of which unfortunately we are as yet
ignorant. Were it possible to remove them, it is quite certain _à
priori_ that the bone-marrow--if the disease were not too
advanced--would resume its normal normoblastic type of regeneration.
Clinical observation supports this contention in many cases. In
megaloblastic anæmias apparent cures are by no means rare, but sooner or
later a relapse occurs, and finally leads with certainty to a lethal
issue. These cases, familiar to every observer, prove with certainty
that the megaloblastic degeneration as such may pass away, and that in
isolated cases the conventional treatment by arsenic suffices to bring
about this result. A definite cure however under these conditions is not
yet attained, since we do not know the ætiological agent, still less can
we remove it. =For this reason, the prognosis of megaloblastic anæmia,
apart from the group of Bothriocephalus anæmia, is exceedingly bad.=

FOOTNOTES:

[8] Dunin, on the contrary, designates the appearance of nucleated red
blood corpuscles within the first 24 hours after the loss of blood as
normal and regular. This view does not correspond with the facts. A
single case on one occasion may exhibit a rarity of this kind.

[9] Probably the dot-like and granular enclosures in the red corpuscles,
which stain with methylene blue, and which Askanzy and A. Lazarus have
observed in numerous cases of pernicious anæmia are also products of a
similar nuclear destruction.

[10] It does not seem superfluous in this place expressly to emphasise,
that what has been said on the diagnostic importance of the megaloblasts
only holds for the blood of adults. For the conditions of the blood in
children, which vary in many respects from that of adults see "Die
Anæmie," Ehrlich and Lazarus, Pt. II. (Anæmia pseudoleukæmica infantum).

THE WHITE BLOOD CORPUSCLES.

The physiological importance of the _white blood corpuscles_ is so many
sided that they form the most interesting chapter of the subject. That
the white corpuscles play a significant part in the physiology and
pathology of man has been recognised but slowly, obviously because there
was at first some hesitancy in ascribing important functions to elements
that are present in the blood in such relatively small numbers. A place
in pathology was first assured to them by Virchow's discovery of
leukæmia. The interest in the question was increased by Cohnheim's
discovery that inflammation and suppuration are due to an emigration of
the white blood corpuscles, and these conditions were particularly
suitable for throwing light on normal processes. The fact that in
diffuse inflammations, large quantities of pus are often produced in a
short time, without the blood being thereby made poorer in
leucocytes,--that the opposite indeed occurs,--necessitated the
supposition that the source of the leucocytes must be extraordinarily
productive. Hence in contradistinction to the red blood corpuscles,
their small number is fully compensated by their exceptional power of
regeneration.

Nevertheless, a considerable time elapsed before the powerful impulse
that started from Cohnheim, bore fruit for clinical histology. As we
have mentioned this was due to the circumstance that an exact
differentiation of the various forms of leucocytes was very difficult
with the methods in use up to that time. Although such distinguished
observers as Wharton Jones and Max Schultze had been able to distinguish
different types of leucocytes, Cohnheim's work remained clinically
fruitless since the criteria they assigned were far too subtle for
investigation at the bedside. Virchow indeed, the discoverer of
leucocytosis, interpreted it as an increase of the lymphocytes; whereas
it is chiefly produced by the polynuclear cells. Only after the
distinction was facilitated by the dry preparation and the use of
stains, did interest in the white corpuscles increase, and continue
progressively to the present day. This is borne out by the exceptionally
exhaustive hæmatological literature, and particularly by that of
leucocytosis.

In spite of these advances, a retrograde movement in the doctrine of the
leucocytes has gained ground surprisingly, especially in the last few
years. Ever since Virchow's description of the lymphocytes, observers
have tried to separate the various forms of leucocytes one from another,
and if possible to assign different places of origin to these different
kinds. There now suddenly appears an endeavour to bring all the white
blood corpuscles into one class, and to regard the different forms as
different stages merely of the same kind of cells. The following
sections will show that this tendency is unwarranted and unpractical.

I. NORMAL AND PATHOLOGICAL HISTOLOGY OF THE WHITE BLOOD CORPUSCLES.

The classification of the white corpuscles of normal human blood, drawn
up by Ehrlich, has been accepted by most authors, and we therefore give
a short summary of it, as founded on the dry specimen.

1. =The Lymphocytes.= These are small cells, as a rule approximating in
size to the red blood corpuscles. Their body is occupied by a large
round homogeneously stained nucleus centrally situated, whilst the
protoplasm surrounds the nucleus as a concentric border. Between nucleus
and protoplasm there is often found a narrow areola, which doubtless
results from artificial retraction. Nucleus and protoplasm are basophil,
nevertheless in many methods of staining the protoplasm possesses a much
stronger affinity for the basic stain than does the nucleus. The nucleus
in these cases stands out as a bright spot from the deeply stained mass
of protoplasm, which is reticulated in a peculiar manner.

Within the nucleus are often to be found one or two nucleoli with a
relatively thick and deeply stained membrane. With methylene blue and
similar dyes the protoplasm stains unequally, which is not to be
considered as the expression of a granulation, as Ehrlich first
assumed, but rather of a reticular structure. The contour of the
lymphocytes is not quite smooth as a rule, at least in the larger forms,
but is somewhat frayed, jagged, and uneven (Fig. 1). Small portions of
the peripheral substance may repeatedly bud off, especially in the large
forms, and circulate in the blood as free elements. In stained
specimens, especially from lymphatic leukæmia, these forms, which
completely resemble the protoplasm of the lymphocytes in their staining,
may from their nature and origin be readily recognised.

As far as the further metamorphosis of the nucleus is concerned, a sharp
notching of the border of the nucleus may occasionally be found, the
further fate of which is shewn in the accompanying figure (Fig. 3). It
is evident that in this case the resulting nuclear forms are quite
different from those which are characteristic of the polynuclear
elements.

The protoplasm possesses no special affinity for acid and neutral dyes,
and hence in triacid and hæmatoxylin preparations the small lymphocytes
are seen chiefly as lightly stained nuclei, apparently free. In the
larger cells the protoplasm can be seen even in these preparations to be
slightly stained. By the aid of the iodine-eosine method the reaction of
the protoplasm of the lymphocytes is shewn to be strongly alkaline. They
do not contain glycogen.

These properties taken as a whole constitute a picture completely
characteristic of the lymphocytes; and these elements can thereby be
diagnosed and separated from other forms, even when their size varies.
Generally speaking, these cells, as above mentioned, are distinguished
in the blood of the healthy adult by their small size, approximating to
that of the red blood corpuscles. In the blood of children on the
contrary larger forms are found even in health; and in lymphatic
leukæmia particularly large forms occur, which are mistaken in various
ways by unpractised observers. Thus Troje's "marrow cells" still figure
in the literature, but have absolutely nothing to do with the marrow.
They are large lymphocytes, as was established by A. Fränkel years
afterwards.

[Illustration: Fig. 1.

Fraying out of the protoplasmic border in large lymphocytes. Free plasma
elements formed by budding. ("Plasmolysis.")

(From a photograph of a preparation from chronic lymphatic leukæmia.)

_To face page 72_]

[Illustration: Fig. 2. (From Rieder's Atlas.)

Metamorphosis of the nucleus of the lymphocytes. (Combined picture from
a preparation from acute leukæmia.)

_To follow Fig. 1_]

In the normal blood of adults the number of the lymphocytes amounts to
about 22-25% of the colourless elements.

Increase of the lymphocytes alone occurs, but in comparison with that of
the other forms, much more seldom, and will be conveniently called by
the special names of "lymphocytosis" or "lymphæmia."

2. Sharply to be distinguished from the lymphocytes is the second group:
the "large mononuclear leucocytes." These are large cells about twice to
three times the size of the erythrocytes. They possess a large oval
nucleus, as a rule eccentrically situated and staining feebly, and a
relatively abundant protoplasm. The latter is free from granulations,
feebly basophil, and in contrast to the protoplasm of the lymphocytes
stains less deeply than the nucleus. This group is present in normal
blood in but small numbers (about 1%). They are separated from the
lymphocytes because they are totally different in appearance, and
because forms transitional between the two are not observed. It cannot
yet be decided from which blood-producing organs these forms arise,
whether from spleen or bone-marrow, although there are many reasons for
regarding the latter as their place of origin.

These large mononuclear leucocytes change in the blood to the following
kind:

3. "=The transitional forms.=" These resemble the preceding, but are
distinguished therefrom by deep notchings of the nucleus, which often
give it an hour-glass shape, further by a somewhat greater affinity of
the nucleus for stains, and by the presence of scanty neutrophil
granulations in the protoplasm. The groups 2 and 3 comprise together
about 2-4% of the white blood corpuscles[11].

4. The (so-called) "=polynuclear leucocytes=." These arise in small part,
as will be described later in detail, from the above-mentioned No. 3,
within the blood stream. By far the larger part is produced fully formed
in the bone-marrow, and emigrate to the blood. These cells are rather
smaller than Nos. 3 and 2 and are distinguished by the following
peculiarities: firstly by a peculiar polymorphous form of nucleus which
gives the relatively long, irregularly bulged and indented nuclear rod
the appearance of an S, Y, E or Z. The complete decomposition of this
nuclear rod into three to four small round single nuclei may occur
during life, as a natural process. Ehrlich first discovered it in a case
of hæmorrhagic small-pox; it is frequently found in fresh exudations.
Formerly when various reagents, for instance acetic acid, were
customarily used, the decomposition of the nucleus into several parts
was more frequently observed, and Ehrlich for this reason chose the not
wholly appropriate name "polynuclear" for this form of cell. As this
name has now been universally adopted, and misunderstandings cannot be
expected, it is undoubtedly better to keep to it. The expression "Cells
with polymorphous nuclei" would be more accurate.

[Illustration: Fig. 3.

Nucleoli in larger lymphocytes.

(From a photograph of a preparation from chronic lymphatic leukæmia.)

_To face page 74_]

The nucleus stains very deeply with all dyes; the protoplasm possesses a
strong attraction for most acid stains, and is unmistakeably
characterised by the presence of a dense neutrophil granulation. The
reaction of the protoplasm is alkaline, to a less degree however than in
the lymphocytes. No free glycogen is contained in the polynuclear cells
as a rule; nevertheless in certain diseases cells are always found which
give a marked iodine reaction. In this manner the appearance of cells
containing glycogen in diabetes was first proved. (Ehrlich,
Gabritschewsky, Livierato.) The iodine reaction in the white blood
corpuscles is also seen in severe contusions and fractures, in
pneumonias, in rapidly progressing phlegmata from streptococcus and
staphylococcus, after protracted narcosis (Goldberger and Weiss).

Ehrlich explains the appearance of glycogen as follows. The glycogen is
not present in the cell as such, but in the form of a compound, which
does not stain with iodine. This compound readily splits off glycogen,
which then gives the iodine reaction[12].

We cannot regard the perinuclear green granules, described by Neusser in
the polynuclear cells, as pre-existing. (See p. 42.)

The number of polynuclear leucocytes in the blood of the healthy adult
amounts to about 70-72%, of the total white corpuscles. (Einhorn.)[13]

5. =The eosinophil cells.= These are characterised by a coarse, round
granulation, staining deeply with acid dyes, and similar in other
respects to the polynuclear neutrophils. With faint staining, a thin
peripheral layer of the eosinophil granule is seen more deeply stained
than the interior. The nucleus as a rule is not so deeply stained as in
the polynuclear neutrophil, but otherwise in its general shape is
completely similar. Both forms have in common a considerable
contractility, which renders possible their emigration from the vessels,
and their appearance in exudations and in pus. The size of the
eosinophils frequently exceeds that of the neutrophils. Their number is
normally about 2-4% of the white cells.

6. The mast cells. These are present, though very sparingly, in every
normal blood; 0.5% is their maximum number in health.

Their intensely basophil granulation, of very irregular size and unequal
distribution, must specially be mentioned. The granulation possesses the
further peculiarity, in that with the majority of basic dyes it stains,
not in the pure colour of the dye, but metachromatically--most deeply
with thionin. As Dr Morgenroth found, the deviation from the colour of
the dye is still more marked with Kresyl-violet-R (Mülheim manufactory),
when the granules stain almost a pure brown.

The staining power of the nuclei is very small, and it is therefore hard
to make out the shape of the nucleus without the use of difficult
methods. In triacid preparations the granulation is unstained, and the
mast cells appear as clear, polynuclear cells, free from granules.

* * * * *

So much for the colourless cells in the blood of the normal adult.

In pathological cases, not only do the forms so far mentioned occur in
altered numbers, but abnormal cells also make their appearance. To these
belong:

1. =Mononuclear cells with neutrophil granulation.= ("=Myelocytes=,"
Ehrlich.) Generally they are bulky, with a relatively large, faintly
staining nucleus, often fairly centrally placed, and equally surrounded
by protoplasm on all sides. A fundamental distinction from the large
mononuclear cells lies in the fact that the protoplasm exhibits a more
or less numerous neutrophil granulation. Besides the larger myelocytes,
much smaller forms, approximating to the size of the erythrocytes are
also found. All transitions between these two stages are likewise met
with. In contradistinction to the polynuclear neutrophil elements, these
mononuclear forms shew no amoeboid movement on the warm stage. They
form a constant characteristic of myelogenic leukæmia, and in these
cases generally occur in large numbers.

Reinbach has found them in a case of lymphosarcoma with metastases in
the bone-marrow. A. Lazarus observed their transitory occurrence in
moderate number in a severe posthæmorrhagic anæmia. M. Beck observed
them in the blood of a patient with severe mercury poisoning. They are
also frequently found in children's diseases, especially in _anæmia
pseudoleukæmica infantum_. K. Elze established their presence in a boy
of 15 months, suffering from a slowly progressing tuberculosis of the
lymphatic glands.

The appearance of myelocytes in infectious diseases is particularly
interesting. Rieder had previously demonstrated that myelocytes may be
present in acute inflammatory leucocytoses; and recently a thorough
work by C. S. Engel has appeared upon the occurrence of myelocytes in
diphtheria. Engel discovered the interesting fact, that myelocytes are
often to be found in children suffering from diphtheria, and further
made the important observation that a high percentage of myelocytes
(3.6-16.4% of the white elements) only occurs in severe cases, and
points to an unfavourable prognosis. Myelocytes are also present in mild
cases, though not constantly and in much smaller number. Türk has
recently undertaken a very exact and thorough analysis of their
occurrence in infectious diseases, in the course of which he accurately
tabulated the white corpuscles in a large number of cases. The results
he obtained in pneumonia are especially characteristic, for he found at
the commencement of the disease that myelocytes are not seen at all or
only very scantily: and it is only at the time of the crisis, or
directly afterwards, that they become specially numerous. In isolated
cases the increase at this time was very considerable; and in one case
amounted almost to 12% of all neutrophil cells.

2. Mononuclear eosinophil cells ("=eosinophil myelocytes="). H. F. Müller
was the first to point out their importance. They constitute the
eosinophil analogue of the previous group, and are much larger than the
polynuclear eosinophils; medium and small sized examples are often found
in leukæmia. Eosinophil myelocytes are almost constantly present in
myelogenous leukæmia and in anæmia pseudolymphatica infantum. Apart from
these two diseases they are very rarely found; Mendel saw them for
example in a case of myxoedema, Türk quite exceptionally in some
infectious diseases.

3. =Small neutrophil pseudolymphocytes.= They are about as large as the
small lymphocytes, possess a rounded deeply stained nucleus, and a small
shell of protoplasm studded with a neutrophil granulation. The
relatively deep stain of the nucleus and the small share of the
protoplasm in the total cell body prevent confusion with the small forms
of myelocytes, which never reach such small dimensions. The neutrophil
pseudolymphocytes are exceedingly infrequent, and represent products of
division of the polynuclear cells; they were first described by Ehrlich
in a case of hemorrhagic small-pox. The process of division goes on in
the blood in such a manner that the nuclear rod first divides into two
to four single nuclei, and then the whole cell splits up into as many
fragments. These cells occur also in fresh pleuritic exudations. After a
time the nucleus of these cells becomes free, and the little masses of
protoplasm thus cut off are taken up mostly by the spleen substance. The
free nucleus likewise shares in the destruction. It is of the greatest
importance that these cells, which up to the present have not elsewhere
been described, should receive more attention. They must be of
significance, in particular for the question of transitory
hyperleucocytosis, which is by some referred to a destruction, by others
to an altered localisation of the white blood corpuscles.

4. "=Stimulation forms=" were first described by Türk, and are mononuclear
non-granulated cells. They possess a protoplasm staining with various
degrees of intensity, but in any case giving with triacid solution an
extraordinarily deep dark-brown, and further a round simple nucleus
often eccentrically situated, stained a moderately deep bluish-green,
with however a distinct chromatin network. The smallest forms stand
between the lymphocytes and the large mononuclear leucocytes, but
approach the first named as a whole in their size and general
appearance. According to Türk's investigations, these cells often occur
simultaneously with, and under the same conditions as the myelocytes.
Their importance cannot at present be accurately gauged. Possibly they
form an early stage of development of the nucleated red blood
corpuscles, as the deeply staining and homogeneous protoplasm seems to
indicate.

With the description of these abnormal forms of white corpuscles all
occurring forms are by no means exhausted. We are here excepting
completely the variations in size which particularly affect the
polynuclear and eosinophil cells, and which lead to dwarf and giant
forms of them. For however considerable the difference in size, these
cells always possess characteristics sufficient for an exact diagnosis.
But besides these, isolated cells of an especially large kind are found
particularly in leukæmic blood, and concerning their importance and
relationship we are up to the present in the dark.

FOOTNOTES:

[11] In enumerating the blood corpuscles, 2 and 3 may be counted
separately or in one group.

[12] The assumption of Czerny, that the cells which react to iodine
emigrate from suppurating foci, is without foundation. A simple
investigation of freshly inflamed tissue is sufficient to show that the
cells which have wandered from the blood stream soon contain glycogen.

[13] Kanthack described this group as "finely granular oxyphil" cells.
Their granules stain red in eosine and in eosine-methylene blue
solutions, but the colour is different from that of the true eosinophil
cells, and much less intense. In the latter mixture they stain really
with the methylene blue salt of eosine. Their true nature is shown by
their behaviour with the triacid solution.

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

For the comprehension of the histology of the blood as a whole, it is of
great importance to obtain an exact knowledge how and to what extent the
three organs, which are undoubtedly very closely connected with the
blood, lymphatic glands, bone-marrow, and spleen, contribute to its
formation. The most direct way of deciding the question experimentally
by excision of the organs in question, is unfortunately only available
for the spleen. The part played by the lymphatic glands and bone-marrow,
whose exclusion _in toto_ is not possible, must mainly be determined by
anatomical and clinical considerations. But only by a careful
combination of experiments on animals, of anatomical investigations, and
especially, of clinical observations on a large scale, can light be
thrown on these very difficult questions. It cannot be emphasised
sufficiently how important it is that everyone engaging in hæmatological
work should first of all collect a large series of general observations;
otherwise errors are bound to occur. For instance, the endeavour is
often made to compensate the lack of personal experience by careful
literary studies; but in this way the histology of the blood falls into
a vicious circle, of which the new phase of blood histology affords
many examples. And it is characteristic of this kind of work that from
the investigation of a single rare case, most far-reaching conclusions
on the general pathology of the blood are at once drawn; _e.g._ Troje's
paper, in which having failed to recognise the lymphocytic character of
a case of leukæmia, and believing therefore that he had to do with a
myelogenous leukæmia, the author denied and completely reversed all that
had been previously established about this disease. It is equally hard
to avoid errors if one confines oneself exclusively to animal
experiments, without supplementing these by clinical experience, as is
shewn by the numerous papers of Uskoff. Not the anatomist, not the
physiologist, but only the clinician is in the position to discuss these
problems.

In the introduction to this chapter we have already alluded to the
striking retrograde movement in hæmatology at the present time, brought
about by the view that the white corpuscles as a whole are derived from
the lymphocytes. If we disregard the embryological investigations on
this point (Saxer), anatomists, physiologists, and clinicians alike have
taken up a similar point of view. Among anatomical papers we may refer
to those of Gulland, according to whom all varieties of leucocytes are
but different stages of development of one and the same element. He
distinguishes hyaline, acidophil and basophil cells, and derives all
from the lymphocytes. Arnold advocates similar views, though in a
negative form. He says that a distinction between so-called lymphocytes
and the leucocytes with polymorphous nuclei, on the grounds of the form
of the cell and nature of the nucleus, is not possible at the present
time. Neither is a classification based on the granules admissible,
since the same granules occur in different cells, and different
granules in the same cell. The work of Gulland and Arnold takes into
consideration the differential staining of the granules in various ways.
In spite of their facts we disagree with their conclusions; and we shall
therefore have to analyse them in the special description of the
granulated cells and granules.

Recently (since 1889) Uskoff has in particular published experimental
work in this province of hæmatology. This has led him to see in the
white blood corpuscles the developmental series of one kind of cell, and
to distinguish in it, three stages: (1) "young cells," which correspond
to our lymphocytes; (2) "ripe cells" (globules mûrs), large cells with
fairly large and irregularly shaped nucleus, which are therefore our
large mononuclear and transitional forms; (3) "old cells" (globules
vieux), which represent our polynuclear cells. The eosinophil cells are
completely excluded from this classification. Amongst clinicians A.
Fränkel has recently gone in the same direction, and on the grounds of
his experience in acute leukæmias has supported the view of Uskoff, that
the lymphocytes are to be regarded as young cells, and early stages of
the other leucocytes. But few authors (for instance C. S. Engel,
Ribbert) have raised a protest to this mixing of all cell forms of the
blood, and have held to the old classification of Ehrlich. But as it is
emphatically taught in numerous medical works that all these cells are
closely related, the grounds for sharply separating the lymphocytes from
the bone-marrow group may here be shortly summarised, and stress laid on
the great importance which this apparently purely theoretical question
has for clinical observation. We shall come to most important
conclusions upon this point when we consider more closely the share
which the various regions of the hæmatopoietic system take in the
formation of the blood, and especially of the colourless elements.

[alpha]. The Spleen.

The question whether the ~spleen~ produces white blood corpuscles has
played a large part from the earliest times of hæmatology.

Endeavours were first made to investigate the participation of the
spleen in the formation of the white blood corpuscles by counting the
white corpuscles in the afferent and efferent vessels of the spleen. It
was thought that the blood-forming power of the spleen was proved by the
larger number of corpuscles in the vein as compared with the artery. The
results of these enumerations however are very varying; the
investigators who found a relative increase in the vein are opposed by
other investigators equally reliable; and with the experience of the
present day one would not lay any value on these experiments.

We must emphasise the fact, established by later researches, that after
extirpation of the spleen, an enlargement of various lymphatic glands
occurs. The alterations of the thyroid, which have been observed by many
authors, cannot be described as constant.

Further, the blood investigations which Mosler, Robin, Winogradow,
Zersas and others have carried on in animals and man after removal of
the spleen must here be mentioned. These have already proved that a
leucocytosis occurs after some considerable time. Prof. Kurloff carried
out detailed investigations in 1888 in Ehrlich's laboratory, and
carefully studied the condition of the blood after extirpation of the
spleen. As the work of Prof. Kurloff has so far only appeared in
Russian, his important results may be here recorded more fully. For his
researches, Kurloff employed the guinea-pig, as this animal by its
peculiar blood is specially suited for this purpose.

In order to give a systematic account of the results of these
important investigations, we must first shortly sketch the
normal histology of the blood of the guinea-pig according to
Kurloff.

In the blood of the healthy guinea-pig the following elements
are found.

I. Cells bearing granules.

1. =Polynuclear, with pseudoeosinophil granulation.= This
granulation, which Ehrlich had previously found in the rabbit,
is easily distinguishable from the true eosinophil, since it is
much finer, and stains quite differently in
eosine-aurantia-nigrosin mixtures. One principal distinction
between these two forms of cells lies in the fact that,
according to Kurloff, this granulation is very easily dissolved
by acid, but remains unchanged in alkaline solutions; doubtless
an indication that the granulation consists of a basic body
soluble with difficulty, which with acids forms soluble salts.
The true eosinophil granulation remains, on the other hand,
quite unchanged under these conditions.

=These pseudoeosinophil, polynuclear cells, correspond
functionally to the neutrophil polynuclear of man=; their number
amounts to 40-50% of the total white cells. The red bone-marrow
is to be regarded as the place of origin of this kind of cell.
It contains very many pseudoeosinophil cells, and indeed all
stages are to be found in it, from the mononuclear cells
bearing granules to the fully formed polynuclear.

2. The typical =eosinophil leucocytes=, which fully correspond to
those found in man, and amount to about 10% of the number of
the white.

3. The "=nigrosinophil cells=," as they are called by Kurloff. In
their general appearance, in the size of the cell and the
granulation, they completely correspond to the eosinophil cell.
The only distinction between them consists in a chemical
difference in the granulation. These cells stain in the colour
of nigrosin in the aurantia-eosin-nigrosin mixture, whilst the
eosinophil cells become red. The two granulations always show
different shades in the triacid preparation as well; for the
nigrosinophil cells stain a blacker hue.

II. Cells free from granules.

([alpha]) Cells with vacuoles.

This is a quite peculiar group, characteristic for the blood of
the guinea-pig. It shews transitions in the blood, from large
mononuclear to transitional and polynuclear forms, but is
marked by the lack of any kind of granulation. Instead of the
latter, we find in these cells a roundish, nucleus-like form in
the protoplasm, which also takes the nuclear stains, and
possibly is to be considered an accessory nucleus. We have
received the impression that we have here to deal with a
vacuole filled with substance secreted by the cell. In a large
series of preparations, it is possible to obtain some
elucidation of the development and fate of these appearances.
They first appear as point-like granules in the protoplasm,
bearing no relation to the cell nucleus; they gradually
increase, and acquire a considerable circumference. When they
have attained about the size of the cell nucleus, they, or
rather their contents, appear to break through the protoplasmic
membrane and to leave the cell.

The number of the vacuole containing cells is 15-20% of the
colourless blood corpuscles.

([beta]) Typical lymphocytes.

Their appearance completely corresponds with that of human
lymphocytes as described above. They make up 30-35% of the
total number of leucocytes.

Now Kurloff in the course of extremely careful and laborious
researches, estimated the total number of leucocytes, and then
from the percentage numbers, the total quantity of
pseudoeosinophil, neutrophil, eosinophil, vacuole containing
cells, and lymphocytes, and could thus demonstrate that in
uncomplicated cases of removal of the spleen, where
inflammatory processes, accompanied by an increase of the
polynuclear neutrophil corpuscles, were avoided, a =gradual
increase of the lymphocytes= alone in course of time results.
This may be a two- or threefold increase, whereas the numbers
of all other elements remain unchanged.

Kurloff obtained his figures as follows: first he estimated the relative
proportion of the different kinds of white blood corpuscles one to
another in a large number of cells (500 to 1000). A count of this kind
however gives no evidence as to whether one or other kind of cell is
absolutely increased or diminished. A fall in the percentage of the
lymph cells may be brought about by two quite different factors: (1) by
a diminished production of lymphocytes, (2) by an increased influx of
polynuclear forms, which naturally lowers the relative count of the
lymphocytes. It was therefore necessary to obtain a method which would
show alterations in the absolute number of the individual forms of
leucocytes. Kurloff used for this purpose the "comparative field"; that
is, he counted by the aid of a moveable stage the different forms which
lay on a definite area (22 sq. mm.) of the dried blood preparation. This
procedure gave very exact results, as only faultlessly prepared, and
regularly spread preparations were used. The following figures (from
Exp. II.) illustrate the method and its results:

April 12 52% pseudo-eos. 10% lymphocytes counted.
Sept. 2 (one month
after the operation) 22% " 53% " "

By the aid of the comparative surface, these figures were supplemented
by the following averages. On each surface used for comparison were
found:

April 12 38 white = 19.8 pseudo-eos. 10.6 lymphocytes.
Sept. 2 81 " 18.0 " 46.9 "

From this example it follows without doubt, that the =total number of the
white blood corpuscles had about doubled itself=, but that in this
increase the lymphocytes exclusively were concerned, and the
pseudo-eosinophil cells had not undergone the smallest increase.

The results which Kurloff obtained by means of this method in animals
whose spleens had been removed, may be illustrated by one of his
original researches and its accompanying chart and table.

Exp. I. Young female, weight 234 gr. Number of red corpuscles
in a cubic millimeter of blood 5,780,000. Number of white
10,700. On April 19, 1888, the spleen was removed, the wound
healed by first intention. The results of the further
investigation of the blood are found in the following table.

From the chart and table, the number on the surface of
comparison of the white blood corpuscles is seen to have more
than doubled itself in the first seven months, and that this
increase was solely dependent on the flooding of the blood by
lymphocytes. The nucleated or bone-marrow elements and the
large mononuclear cells remained continuously at the same level
during the whole period. The changes in the percentage
proportions ran somewhat differently. The percentages rose from
35 to 66% for the lymphocytes only, whilst for the other forms
they distinctly fell: for the nucleated from 44% to 22% and for
the large mononuclear from 18% to 9%. It was only in the course
of the second year that a very considerable relative and
absolute increase of the eosinophil cells appeared: the values
rose gradually from about 1.0% to 28.9% or from 0.5 to 13.9 on
each comparison area. The last examination of the blood in this
animal was made on April 30, 1890, that is, two years after the
removal of the spleen. The animal was quite healthy, bore four
healthy young guinea-pigs by a father whose spleen had been
removed. The young have a completely normal spleen, and their
blood likewise shows no abnormalities.

[Illustration: CHART TO EXPT. No. I. (cp. Table, page 89. The figures in
the chart refer to comparative surfaces.)

Thick line--total number of leucocytes
Broken line--lymphocytes
Thin line--number of nucleated, pseudo-eosinophil cells
Double line--large mononuclear cells
Dotted line--eosinophil cells]

TABLE I.

Key to columns:
A - Leucocytes
B - Pseudo-eosinophil cells
C - Lymphocytes
D - Large mononuclear cells
E - Eosinophil cells
F - Nigrosinopil cells
G - On comparative surfaces

---------------------------------------------------------------------------
|| A || B || C || D || E || F |
------------------------------------------------------------------
Date ||Total| G|| % | G || % | G || % | G || % | G || % | G |
---------------------------------------------------------------------------
1888 || | || | || | || | || | || | |
April 19|| 500|--||44.7| -- ||35.4| -- ||18.4| -- || 1.1| -- || 0.5| -- |
23|| 990|24||40.4| 9.7||35.6| 8.5||21.6| 5.2|| 1.9| 0.4 || 0.4|0.09|
May 1|| 858|28||47.0|13.6||32.6| 9.1||18.0| 5.0|| 0.9| 0.2 || 0.3|0.08|
8|| 934|28||45.2|12.6||40.3|11.3||14.3| 4.0|| 0.6| 0.2 || 0.4|0.1 |
16|| 1122|30||38.4|11.5||47.7|14.3||10.3| 3.1|| 3.3| 0.9 || 0.2|0.06|
24|| 1722|35||40.1|14.0||35.0|12.2||23.6| 8.3|| 1.0| 0.3 || 0.1|0.03|
30|| 900|30||36.6|10.9||44.4|13.3||18.4| 5.5|| 0.1| 0.03|| 0.3|0.09|
June 5|| 825|33||28.4| 9.4||49.3|16.2||20.0| 6.6|| 1.7| 0.6 || 0.4|0.1 |
12|| 1314|33||28.0| 9.3||49.0|16.2||20.0| 6.6|| 2.2| 0.7 || 0.8|0.3 |
19|| 917|37||32.4|11.9||52.3|19.3||14.5| 5.4|| 0.6| 0.3 || 0.2|0.07|
28|| 802|42||30.5|12.8||56.4|23.7||11.7| 4.9|| 0.7| 0.3 || 0.4|0.2 |
July 2|| 1062|56||16.5| 9.2||57.1|31.9||25.6|10.3|| 1.2| 0.7 || 1.2|0.7 |
9|| 1245|51||17.6| 8.9||59.1|30.1||21.8|11.1|| 0.8| 0.4 || 0.8|0.4 |
16|| 974|69||17.5|12.0||66.4|45.8||15.7|10.8|| 0.2| 0.1 || 0.2|0.1 |
23|| 1156|58||21.7|12.6||67.2|38.9|| 9.5| 5.5|| 1.5| 0.9 || 0.2|0.1 |
30|| 802|54||20.2|10.7||65.4|34.6||12.8| 6.8|| 1.4| 0.7 || -- | -- |
Aug. 6|| 910|52||21.7|11.3||67.3|34.9|| 9.7| 4.9|| 1.0| 0.5 || 0.3|0.2 |
Sept. 6|| 815|51||23.0|11.7||65.3|33.5|| 9.8| 4.9|| 0.9| 0.5 || 0.4|0.2 |
Oct. 5|| 625|62||26.4|16.3||64.4|39.9|| 8.5| 5.2|| 0.6| 0.4 || -- | -- |
Nov. 4|| 800|58||22.5|13.0||66.4|38.5|| 9.6| 7.3|| 0.9| 0.5 || 0.5|0.2 |
|| | || | || | || | || | || | |
1889 || | || | || | || | || | || | |
April 10|| 700|--||29.8| -- ||53.3| -- ||14.8| -- || 1.2| -- || 0.6| -- |
June 6|| 900|71||28.2|20.0||50.1|35.6||12.9| 9.1|| 8.2| 5.8 || 0.6|0.4 |
Aug. 1|| 670|62||30.6|18.9||44.2|27.4||15.2| 9.4|| 9.6| 5.9 || 0.4|0.2 |
Dec. 4|| 731|63||36.0|22.0||38.3|24.1||11.3| 7.1||13.3| 8.7 || 0.6|0.4 |
|| | || | || | || | || | || | |
1890 || | || | || | || | || | || | |
Feb. 2|| 622|51||32.3|16.5||30.1|15.3||11.1| 5.6||26.0|13.2 || 0.5|0.2 |
April 30|| 500|48||36.5|17.5||24.5|11.7|| 9.4| 4.5||28.9|13.9 || 0.6|0.3 |
---------------------------------------------------------------------------

The results of further investigations, which we here shortly repeat in
tabular form, shew that in this experiment No. I. we are not dealing
with an abnormal phenomenon of an exceptional animal.

------------------------------------------------------
| Number of white blood corpuscles
No. of |---------------------------------------------
Expt. | Before the | At the end of | At the end of
| splenectomy| the first year| the second year
------------------------------------------------------
1 | 10,700 | 14,200 | 18,000
2 | 12,000 | 27,600 | 32,000
4 | 15,000 | 19,200 | 19,000
------------------------------------------------------
Average | 12,600 | 20,333 | 23,300

By estimating the percentage proportion of the single kinds of white
corpuscles, Kurloff obtained the following result:

Key:
A - Number of the Experiment
B - Polynuclear granular cells
C - Lymphocytes
D - Mononuclear
E - Eosinophil

----------------------------------------------------------------------------
|| Before the operation || At the end of the || At the end of the
|| || first year || second year
A || B | C | D | E || B | C | D | E || B | C | D | E
----------------------------------------------------------------------------
1 || 4782| 3788| 1969| 117|| 4232| 1568| 2101| 170|| 6570| 4410| 1692| 5202
2 || 6276| 3360| 2244| 72|| 5464|16615| 2980|2539|| 5824|20861| 2688| 2240
4 || 6715| 5250| 2595| 450|| 6568|10041| 3686| 96|| 7108| 3009| 2138| 7543

From these researches we draw the following conclusions.

1. The spleen is not an indispensable, vitally important organ for the
guinea-pig, since that animal bears splenectomy without loss of health,
developes normally, and gains well in weight.

2. The hypertrophy and hyperplasia of the lymph glands, particularly of
the mesenteric glands, which develop after the operation correspond to a
=lymphocytosis=, which makes its appearance in the course of the first
year after the operation so constantly that it may be looked upon as a
=characteristic sign of the absence of the spleen=. This increase may
amount to double and more. We must therefore assume that the deficiency
of splenic function may be met by the lymphatic glandular system. This
period of lymphæmia may doubtless in some animals persist for years in
exceptional cases; in the majority, however, the lymphæmia diminishes in
the course of the first year, and indeed subnormal quantities of
lymphocytes may then be produced.

3. The cells of the bone-marrow, on the contrary, and the polynuclear
pseudoeosinophil cells do not show the least variation in the course of
the first year. Bearing in mind that under normal conditions these cells
are met with exclusively in the bone-marrow, and that inflammation in
animals after removal of the spleen is accompanied by an acute
pseudoeosinophil leucocytosis, exactly as in normal animals, one must
admit that the production and function of this kind of cell are quite
independent of the spleen. Hence there can be no doubt about their
myelogenic nature.

4. It is especially important that the mononuclear and the leucocytes
associated with them, undergo no increase. As these cells under normal
circumstances occur both in the spleen and in the bone-marrow, we must
assume that normally also the bone-marrow is responsible for the
majority of this kind in the blood, and that the deficiency in the
splenic contribution can be easily covered by a slightly raised activity
of the bone-marrow. Were the share of the spleen important, from
general biological considerations, an over-production of the kind of
cell in question must occur in the vicarious organs.

5. The increase of the eosinophil cells, which constantly makes its
appearance in the second year after the operation, is highly
interesting, and leads to a really enormous rise in their absolute and
relative numbers. Their percentage number once rose to 34.6%, and their
absolute quantity amounted at the end of the second year on the average
to 30-50-fold their original number (see table).

=Hence it follows from Kurloff's researches that the spleen of the
guinea-pig plays quite an unimportant part in the formation of the white
blood corpuscles, and that after splenectomy in the first year
compensation occurs only in the lymph-glands, followed in the second
year by a great increase of the eosinophil cells. It is to be
particularly insisted once again that the spleen has nothing at all to
do with the formation of the pseudoeosinophil polynuclear cells, which
are the analogues of the polynuclear neutrophils of man.=

* * * * *

How do observations on man stand in the light of Kurloff's observations,
which might be regarded as depending on peculiarities of the particular
kind of animal?

Completely analogous material is afforded by cases, in which in healthy
people a splenectomy has been necessary in consequence of trauma.
Unfortunately the material available for this purpose is extremely rare;
and it would be of the utmost value if the alterations of the blood in
such a case were systematically studied for a period of years. We have
ourselves begun our observations in two patients directly after the
operation, but were unable to continue them, as death occurred within
the first week after the extirpation. Up to the present only seven cases
of rupture of the spleen with subsequent splenectomy have been
published, as is stated in the collection of cases of v. Beck. In two
only, of these seven cases, one of Riequer's (Breslau) the other of v.
Beck's (Karlsruhe) was a cure effected. Through the courtesy of the
above-mentioned gentlemen, we were able to investigate specimens from
these two patients.

In the case of v. Beck the operation was performed on June 15,
1897. We received a dry blood preparation about 6 months after
operation. Investigation showed a considerable lymphæmia: the
bulk of the lymphocytes belonged to the larger kinds: the
eosinophil cells were certainly not increased. For other
reasons an exact numerical analysis could not be undertaken. We
hope to be able to follow the further course of this case.

In the second case the operation was performed on May 17, 1892,
by Dr Riequer of Breslau, for trauma, and later described. We
made counts in oldish and fresh preparations. It is worthy of
notice that this case is not uncomplicated, as an amputation of
the thigh was performed shortly after the splenectomy on
account of gangrene.

We found the following figures.

------------------------------------------------------------------------
Preparations from | Polynuclear | Lymphocytes | Eosinophil | Large
| | | | mononuclear
------------------------------------------------------------------------
June 12, 1892 | 81.9% | 15.9% | 1.3% | --
October 11, 1892 | 80.0% | 13.7% | 4.0% | 1.7%
September, 1897 | 56.8% | 33.1% | 3.5% | 1.5%
------------------------------------------------------------------------

It is much to be regretted that dry preparations only at the beginning
and at the end of the five year period of observation were at our
disposal. It appears from the paper of Riequer as if in this case the
lymphocytosis had established itself one month after the operation, and
had lasted for a very long time, just as Kurloff has found in some
animal experiments. Just as little as a polynuclear increase is
abnormal, is an increase of the lymphocytes remarkable; and in this case
the lymphocytic increase was recognisable after the end of the fifth
year. The eosinophil cells oscillate at this period about the upper
normal limit. From all that we know, it is probable that their number in
the meantime had undergone an intercurrent increase.

The cases are more frequent in which a splenectomy has been undertaken
on account of disease of the spleen. Amongst these, the clearest results
are _à priori_ to be expected from splenic cysts, since the part of the
spleen not affected by the cyst formation often shews quite a normal
structure, and therefore is physiologically active. On the other hand,
the excision of chronic splenic tumours may be--for the blood
condition--of no importance inasmuch as the function of the spleen may
have previously long been eliminated by pathological changes.

Amongst these cases, we must in the first place mention the well-known
and carefully investigated case of B. Credé. In a man 44 years of age
the spleen was extirpated on account of a large splenic cyst. Within two
months of the operation there developed a thoroughly leukæmic condition
of the blood, exclusively brought about by the increase of the
lymphocytes, as is seen from the results of Credé and the table
contained in his paper. It is further remarkable that four weeks after
the operation a painful doughy swelling of the whole thyroid appeared,
which remained, with variations, for nearly four months. With the
general recovery of the patient this shrank to a small remnant. We
notice further that this very interesting swelling of the thyroid,
which doubtless stands in the closest connection with the splenectomy,
is nevertheless no constant accompaniment of this operation, as for
instance in the case of v. Beck, where it was not present.

The most recent work on extirpation of the spleen for tumours is from
Hartmann and Vasquez. As the result of their researches the authors
arrive at the following conclusions:

1. A slight post-operative increase of the red blood corpuscles and a
true acute hyperleucocytosis occur and pass rapidly away.

2. The hæmoglobin equivalent of the corpuscles sinks at first but
recovers its original value by degrees.

3. 4-8 weeks afterwards a lymphocytosis of varying duration is
established.

4. Later, after many months, a moderate eosinophilia occurs.

We have ourselves been able to investigate three conclusive cases.

The first was a patient, which we were ourselves enabled to
investigate by the courtesy of Dr A. Neumann. The patient's
spleen was removed by E. Hahn on account of an echinococcus on
Feb. 5, 1895. One may well assume that before the operation the
spleen no longer discharged its normal function. On Sept. 2,
1897, we found the following numerical proportions:

Polynuclear neutrophil 76.5%,
Lymphocytes 18.4%,
Eosinophil 3.4%,
Large mononuclear 1.1%,
Mast cells 0.4%.

A condition therefore which was quite normal. In this
connection it must be mentioned that an incipient phthisis
pulmonum existed at the time, to which we must attribute an
increase of the polynuclear elements, and without which the
percentage figures of the lymphocytes and eosinophils would
perhaps have been greater.

For the knowledge of the two other cases we are indebted to the
kindness of Professor Jounescu of Bucharest. The one case was
of a man of about 40 years of age, in whom splenectomy was
undertaken on Sept. 27, 1897, for an enlarged spleen. Healing
by first intention. The white blood corpuscles were permanently
increased. The proportion of white to red was 1:120 to 1:130,
the average number of red was 3,000,000. Our own examination of
preparations obtained some two months after the operation,
shewed a distinct lymphæmia, and also a preponderance of the
larger lymph cells. The eosinophil and mast cells were plainly
increased. We are unable to give more exact numerical data, as
the preparations sent to us were not spread with sufficient
regularity.

From the second case, which was also operated upon for
enlargement of the spleen, we unfortunately only obtained much
damaged preparations. Nevertheless so much could with certainty
be established--that there was no considerable increase of the
lymphocytes. The eosinophils on the contrary were increased
distinctly, the mast cells to a lesser extent. It is probable
that the increase of both of the latter kinds of cell was not a
consequence of the extirpation of the spleen alone, but rather
the expression of the reactive changes, which had already begun
before the operation, from the exclusion of the splenic
function.

Cases of splenectomy of this kind are transitional to the chronic
diseases of the spleen. The latter present great difficulties, for one
never knows how far in the most chronic diseases the other organs are
damaged or influenced by the general illness.

An increase of the lymphocytes, so long as an affection of the lymphatic
glands may be excluded, should be referred to functional exclusion of
the spleen.

On the other hand, an increase of eosinophil cells associated with a
chronic tumour of the spleen, is analogous to Kurloff's secondary
reaction of the bone-marrow. Such cases are frequently found in the
literature. For instance Müller and Rieder bring forward three cases of
splenic tumour caused by congenital syphilis, cirrhosis of the liver,
neoplasm in the cranial cavity, and in which the numbers of the
eosinophils amounted to 12.3%, 7.0%, 6.5% respectively. In three cases
of acute splenic tumour in typhoid fever the figure 0.31% with a maximum
of 0.82%, was found. These authors have already raised the question
"whether the increase of the eosinophil cells is connected with the
splenic tumour or the bone-marrow? Perhaps the functional activity of
the latter is vicariously raised to meet the more or less complete
exclusion of the spleen from the formation of the blood; since Ehrlich
has distinctly asserted that the probable place of formation of the
eosinophil cells is the bone-marrow."

From what has been brought forward no doubt can now remain that the
question has been decided quite in Ehrlich's favour.

But what then are the physiological functions of the spleen, since that
organ is unnecessary for the persistence of life? Doubtless its chief
duty is the taking up of the greater part of the decaying fragments of
red and white blood corpuscles in the blood-stream, so that this
valuable material is not quite lost for the organism. Thus Ponfick has
found that after destruction of the red corpuscles the spleen takes up a
portion of their "shadows," and for this reason calls the splenic tumour
a spodogenous splenic tumour ([Greek: spodos], ruins). Ehrlich has made
a corresponding observation for the products of dissolution of the white
blood corpuscles, and has proved that the splenic tumour which occurs in
many infectious diseases and in phosphorus poisoning is to a large
extent caused by the parenchyma of the spleen taking up the remains of
the neutrophil protoplasm.

The question of the relation of the spleen to the =fresh formation of red
blood corpuscles= is a problem of comparative anatomy. Observations on
this point made on one kind of animal can certainly not claim validity
for other kinds. In lower vertebrates, as in fishes, frogs, tortoises,
and also in birds, the blood-forming activity of the spleen is
pronounced and of great importance. In mammalia on the other hand, in
some cases this function cannot be demonstrated, and in others only to a
very small degree. In the spleen of normal mice nucleated red blood
corpuscles are seen in relatively large numbers; in the rabbit they are
less numerous and often only to be found with difficulty. In the dog
they only make their appearance after anæmia from loss of blood,
normally they are absent. =In the human spleen nucleated red blood
corpuscles are not to be found normally or in cases of severe anæmia,
but exclusively in leukæmic diseases.= U. Gabbi in his recently published
work on the hæmolytic function of the spleen, also emphasises the
difference between the various animal species. In guinea-pigs he found
that the spleen acts largely as a scavenger of the red blood corpuscles;
in rabbits very slightly. Consequently after removal of the spleen in
guinea-pigs the number of red blood corpuscles rose 377,000 in the cubic
millimetre, and the amount of hæmoglobin 8.2%. After splenectomy in
rabbits the increase in these values is absent.

Shortly summarising our analysis of the facts before us, we must say
that =the importance of the spleen for the production of the white blood
corpuscles can in no respect be considerable=, and that if these cells
really are produced by it, they must be free from granulations. The
spleen therefore stands functionally in closer connection with the
lymphatic gland system than with the bone-marrow. =The spleen has not the
least connection with ordinary leucocytosis[14].=

([beta]) The Lymphatic Glands.

As it is impossible experimentally to prevent the lymphatic glands as a
whole from contributing to the formation of the blood, we are dependent
almost entirely on clinical and anatomical researches for an elucidation
of their function.

Since Virchow's definition of the lymphocyte it has been admitted that
the lymphocytes of the blood, both the small and larger kinds, are
identical with those of the lymphatic glands and the rest of the
lymphatic system. This is proved by the complete agreement in general
morphological character, in staining properties of the protoplasm and
nucleus, and from the absence of granulation.

Abundant clinical experience testifies that the lymphocytes of the blood
really do arise from the lymphatic system. Ehrlich had previously
observed that when extensive portions of the lymphatic glandular system
are put out of action by new growths and similar causes, the number of
the lymphocytes may be considerably diminished. These observations have
since that time been confirmed by various authors. For example, Reinbach
describes several cases of malignant tumour, particularly sarcoma, in
which the percentage of lymphocytes, which normally amounts to about
25%, was very considerably lowered; in one case of lymphosarcoma of the
neck they only made up 0.6% of the total number. These conditions are
quite easily and naturally explained by the exclusion of the lymphatic
glands. It is difficult for the advocates of the view that the
lymphocytes are the early stages of all white blood corpuscles to
reconcile it with these facts. According to their scheme the low number
of lymphocytes is to be explained in such cases by their unusually rapid
transformation to the polynuclear elements--the old forms; or to
appropriate the expression of Uskoff, by a too rapid ageing of the
lymphocytes.

Further evidence for the origin of the lymphocytes of the blood from the
lymphatic glands is to be obtained from those cases in which we find an
increase of the lymphocytes in the blood. These "lymphocytoses" occur,
in comparison with other leucocytoses, relatively seldom. Under certain
conditions in which a hyperplasia of the lymphatic glandular apparatus
makes its appearance, we often see at first an increase of the
lymphocytes in the blood. Ehrlich and Karewski in some unpublished work
have investigated together a large number of typical cases of lymphoma
malignum, and were able constantly to observe a lymphocytosis, which in
some cases was of high degree and bore almost a leukæmic character.

Relying on these facts Ehrlich and Wassermann (_Dermatolog.
Zeitschr._ Vol. I., 1894) made the diagnosis _in vivo_ of
malignant lymphoma in a rare skin disease, chiefly from the
absolute increase of the lymphocytes alone, although no
swelling of the glands was palpable. The post-mortem shewed
that the chief condition was a swelling of the retro-peritoneal
lymph glands to lumps as large as a fist.

The lymphocytosis following extirpation of the spleen also belongs to
this category, since a vicarious enlargement of the lymph glands is
always to be observed in these cases.

On investigating the conditions under which in healthy individuals an
increased number of lymphocytes enter the blood-stream, we have in the
first place to notice the digestive canal, whose wall contains a thick
layer of lymphatic tissue. According to the results of Rieder the
proportion of the lymphocytes to polynuclears is practically normal in
the leucocytosis of digestion, indeed the lymphocytes are rather in
excess. The eosinophils on the other hand shew a marked relative
reduction in this condition. The leucocytosis of digestion consequently
differs essentially from the other kinds, in which the neutrophil
elements are chiefly increased. The simultaneous increase of lymphocytes
and polynuclears is doubtless brought about by a super-position of a
raised income of lymphocytes, and an ordinary leucocytosis caused by the
assimilated products of metabolism.

The influence of the digestive tract is still more evident in certain
diseases, more particularly in intestinal diseases of infants. A
considerable increase of the lymphocytes in the blood-stream is here to
be observed. Thus Weiss found an important increase of the white blood
corpuscles in simple catarrh of the stomach and intestines, which
presented the main features of a lymphocytosis.

Whooping-cough, according to the recent observations of Meunier, also
belongs to the small number of diseases which are accompanied by a
pronounced lymphæmia. In the convulsive period of this disease both the
polynuclear cells and the lymphocytes are increased, the latter in
preponderating amount. The former cells are increased to twice, the
lymph cells to four times their normal amount. Doubtless in these cases
also the lymphocytosis is due to the stimulation and swelling of the
tracheobronchial glands.

An increase of the lymphocytes from chemical stimuli is exceedingly
rare, though, as is well known, a large number of substances (bacterial
products, proteins, nucleins, organic extracts, and so forth) can call
forth a polynuclear leucocytosis. In quite isolated cases, an increase
of the lymphocytes in the blood in consequence of the injection of
tuberculin into tuberculous individuals has been seen. (E. Grawitz.)
From the rarity of these cases it can scarcely be doubted that here a
tuberculous disease of the glands also plays a part, so that the
increased immigration of lymphocytes is brought about not by a chemical
property of the tuberculin but by the extensive specific reaction of the
diseased glands.

Only one single substance has so far been mentioned in the literature as
capable in itself of producing a lymphocytosis. Waldstein asserts that
he has produced by injection of pilocarpine, a lymphæmia which undergoes
a progressive increase with a rise in number of the injections.

The origin also of lymphocytosis is therefore sharply marked off from
that of the ordinary leucocytosis, which consists in an increase of the
neutrophil elements. Whilst the latter is admittedly the expression of
chemiotactic action, and arises by action at a distance of soluble
substances on the bone-marrow, lymphocytosis is due to a local
stimulation of certain glandular areas. Thus in the leucocytosis of
digestion, of intestinal diseases of children, we refer it to the
excitation of the lymphatic apparatus of the intestine, in tuberculin
lymphæmia we recognise mainly a reaction of the diseased lymph glands.
Hence we conclude that a lymphocytosis appears when a raised lymph
circulation occurs in a more or less extended area of lymphatic glands,
and when, in consequence of the increased flow, more elements are
mechanically washed out of the lymph glands. The pilocarpine
lymphocytosis does not contradict this view, for pilocarpine causes
extraordinary though transient variations in the distribution of water,
whereby the inflow into the blood of fluid containing lymph cells is
increased. We therefore regard =lymphocytosis= as the result of a
=mechanical= process; whilst =leucocytosis= is the expression of =an active
chemiotactic reaction= of the polynuclear elements.

This view finds its best support in the fact that the polynuclear
leucocytes possess lively amoeboid movement, which is completely
wanting in the lymphocytes.

Corresponding to the absence of contractility in the lymphocytes it is
also observed that in =inflammatory= processes in contradistinction to the
polynuclear neutro-and oxyphils, the lymphocytes are not able to pass
through the vessel wall. A very interesting experiment on this point was
described by Neumann years ago. Neumann produced suppuration in a
patient with lymphatic leukæmia, in whom the blood contained only a
very small number of polynuclear cells. Investigation of the pus shewed
that it consisted exclusively of polynuclear cells, and that not a
single lymphocyte had come into the exudation, although this kind of
cell was present so abundantly in the blood.

Histological examination of all fresh inflammatory processes, in which
mainly polynuclear elements are found, leads to accordant results. It is
well known that small-celled infiltration occurs in the later stage of
inflammation, apparently consisting of lymph cells; nevertheless this
does not in the least prove that these lymphocytes have emigrated here
from the blood vessels. This is not the place to enter into the very
extensive controversy on this point. We are content to refer to the most
recent very thorough paper of Ribbert. Ribbert regards these foci of
small-celled infiltration as the analogues of the lymphatic nodules, and
explains their origin by an increase in size of the foci of lymphatic
tissue, normally present, though in a condition but little developed.

It consequently follows from clinical and morphological researches, as
well as from the observations on inflammatory processes, =that the
lymphocytes are in no way connected with the polynuclear leucocytes=. We
shall reach the same result in another way in the following section.

([gamma]) The Bone-marrow.

The spleen and lymphatic glands were at first regarded as the sole
places of formation of the blood corpuscles. The almost simultaneous
researches of Neumann and Bizzozero first attracted general attention to
the importance of the bone-marrow. These authors showed that the early
stages of the red blood corpuscles are produced there; a discovery which
was quickly and generally recognised, and which soon became
pathologically useful through the observations of Cohnheim and others.
In this connection the observation was of great value, that after severe
loss of blood the fatty marrow of the larger hollow bones again changes
to red marrow, as it is evidence of the increased demands on the
regenerative function of the bone-marrow.

We are unaware of a second place of formation of the red blood
corpuscles in man. In other mammalia however, as we have above mentioned
(see page 99), the spleen may also take a small share in the production
of erythrocytes. The type which the normal blood formation follows in
adults, and the deviations therefrom shewn in pernicious anæmia, have
been described in the chapter on the red blood corpuscles. Ehrlich's
view that the blood formation in pernicious anæmia belongs to a
different type, which is analogous to the embyronic, was also described
there.

In this section we have therefore to deal chiefly with the white blood
corpuscles and their connection with the bone-marrow. In man as in a
large number of animals (for example the monkey, guinea-pig, rabbit,
pigeon and so forth) =the bone-marrow exhibits the peculiarity that the
cells it produces bear a specific granulation, in sharp contrast to the
lymphatic glandular system, which contains elements free from granules,
in the whole animal series=.

The granulated cells of the bone-marrow fall into two groups.

The first group of the cells with "=special granules=" is very important
since it constitutes a characteristic for certain species of animals.
According to the class of animal they shew different tinctorial and
morphological properties. Man and monkey for example have neutrophil
granulation; guinea-pig and rabbit the pseudo-eosinophil granulation
described by Kurloff; in birds we find two specific granulations present
side by side, which both are oxyphil, and of which one is imbedded in
the protoplasm in crystalline form, the other in the form of granules.

The kinds of special granulations so far investigated have the common
property, that they stain in acid and neutral dyes respectively; they
shew a much smaller affinity for the basic dyes. The fact that they
greatly exceed the other elements of the bone-marrow in all classes of
animals, is evidence of the importance of these granules.

The second group of bone-marrow cells contains granules which we find in
the whole vertebrate series from the frog to man, and which therefore
are not characteristic for any one species of animal. They are, (1) the
eosinophil cells, (2) the basophil mast cells.

The bone-marrow forms which are free from =granules= consist mostly of
mononuclear cells of different type. They are not nearly so numerous, or
so important as the granulated kind, more especially as the first and
predominant group.

Amongst the granule-free forms the =giant cells= deserve special mention,
for they are an almost constant constituent of the bone-marrow of the
mammalian class. According to the recent researches of Pugliese the
giant cells are considerably increased after extirpation of the spleen
in the hedgehog; an organ of quite extraordinary size in this animal and
doubtless therefore possessing important hæmatopoietic functions.

Pugliese asserts that in the hedgehog after splenectomy the nucleated
giant cells pass into leucocytes by amitotic nuclear division.
Unfortunately in his preliminary communication there are no notes of the
granules of the bone-marrow cells.

On examining a stained dry preparation of the bone-marrow of the
guinea-pig, rabbit, man, etc. it is seen that the characteristic finely
granular cells are present in all stages of development, from the
mononuclear through the transitional to the polynuclear (polymorphously
nucleated) forms, which we meet with in the circulating blood. A glance
at a preparation of this kind shews that the bone-marrow is clearly the
factory where typical polynuclear cells are continuously formed from the
granule-containing mononuclears.

Here also the same process of ripening can be seen in the polynuclear
eosinophil leucocytes.

Ehrlich has been able by special differential staining to bring forward
proof that the constitution of the granulation changes during the
metamorphosis of the mononuclear to the polynuclear cells. In the young
granules there is prominent a basophil portion that becomes less and
less marked as the cell grows older. The pseudo-eosinophil granules of
the mononuclear cells, of the guinea-pig for example, stain bluish-red
in eosine-methylene blue after long fixing in superheated steam: in the
transitional stages this admixture is gradually lost, and finally
completely vanishes in the granules of the polynuclear leucocytes which
stain pure red. Analogous observations may be made in the eosinophil
cells of man and animals, and in the neutrophils of man. Hence it is
even possible to decide whether an isolated granule belonged to an old
or to a young cell.

It is still impossible to judge with certainty the rate at which the
ripening of the mononuclear to the polynuclear cells proceeds, or
further to decide if the ripening of the granules always runs parallel
in point of time with that of the whole cell. On the grounds of our
observations we would suppose that in general the two processes run
their course side by side, but that in special cases the morphological
ripening of the cell may proceed more rapidly than that of the granules.
It is particularly easy to observe this point in eosinophil cells.
Ehrlich had already mentioned in his first paper (1878) that side by
side with the typical eosinophil granules isolated granules are often
found which shew a deviation in tinctorial properties: for instance,
they stain more of a black colour in eosine-aurantia-nigrosin; in
eosine-methylene-blue, bluish-red to pure blue. Ehrlich had already
described these as young elements in his first paper. The same
differences are found more sharply marked in leukæmia even in the
circulating blood, in the neutrophil as well as in the eosinophil group.
Ehrlich has repeatedly found in leukæmic blood polynuclear eosinophil
cells, whose granules must almost exclusively be regarded as young
forms[15].

Ehrlich regarded these as typical examples of a relative acceleration of
the morphological ripening of the cells, as compared with the
development of the granules.

=In normal blood we find only the ripe forms of the specific granulated
cells of the bone-marrow. The mononuclear and transitional forms of the
neutrophil group, do not under normal circumstances pass over into the
blood-stream.=

Ehrlich regarded the mononuclear neutrophil granulated cells as
characteristic for the bone-marrow, since they are found exclusively in
the bone-marrow, never in the spleen or lymph glands, and for this
reason named them "=myelocytes=," [Greek: kat' exochên][16]. When
myelocytes, no matter of what size, appear in considerable numbers in
the blood of an adult, a leukæmia of myelogenic nature is nearly always
present. (For the very rare exceptions to this rule, which it may be
added can never be confused with leukæmia, see pages 77, 78.)

Exactly similar conditions hold good for the eosinophil cells, in as
much as the singly nucleated forms, which one may call eosinophil
myelocytes, occur, almost exclusively, in leukæmic blood. These forms,
which were first recognised by H. F. Müller, are however of less
importance, for in myelogenic leukæmia the chief part of the foreign
admixture of the blood is made up of Ehrlich's myelocytes.

Very important conclusions on the interesting question of leucocytosis
can be drawn from these observations. Bearing in mind that polynuclear
neutrophil cells are developed and stored up only in the bone-marrow,
that in ordinary leucocytosis only the polynuclear forms are increased
in the blood-stream, it is evident that =leucocytosis is purely a
function of the bone-marrow=, as Ehrlich has always insisted with all
distinctness. It is only on this assumption that the frequently sudden
appearance of leucocytosis, as has so often been observed in morbid and
experimental conditions, can be satisfactorily explained. In these cases
the space of time, amounting often only to minutes, is far too short for
a new formation of leucocytes to be conceivable; there must be places in
which these cells are already completely formed, and able thence to
emigrate on any suitable stimulus. This place is single, and is the
bone-marrow alone. Here all mononuclear forms gradually ripen to the
polynuclear contractile cells, which obey each chemiotactic stimulus by
emigration, and which thus bring about sudden leucocytosis.

The bone-marrow thus fulfils, amongst others, the extremely important
function of a protective organ, by which definite injurious influences
which affect the organism may be quickly and energetically combated.
Just as in a fire-station ample means of assistance is continuously in
readiness immediately to answer an alarm from any quarter.

We wish to insist once more, that the =large mononuclear leucocytes= and
the transitional forms of the normal blood are not concerned in the
increase in ordinary leucocytosis; in leucocytosis of high degree their
relative number may indeed be lowered, in consequence of the exclusive
increase of the polynuclear cells. It appears then that these elements
do not react to chemiotactic stimuli, and that possibly they reach the
blood by entirely different ways than the polynuclears do.

We believe that these non-granulated mononuclear cells of man
are to be regarded as analogous to those of the guinea-pig
described by Kurloff (see page 86). The mononuclear cells of
man however are finally transformed into the neutrophil
granulated cells, whilst the cells of Kurloff remain free from
granules in the course of their metamorphosis. In acute
leucocytosis in the guinea-pig only the pseudo-eosinophil
polynuclear cells are increased, which wander as such out of
the bone-marrow, but not the polynucleated non-granulated
forms, which but slowly grow to maturity in the blood. Thus the
peculiarities of guinea-pig's blood, in which two kinds of
polynuclear cells are recognisable, throw light upon the
corresponding conditions in human blood. The distinction in the
latter is more difficult, since it is not evident in this case
that the fully formed polynuclear neutrophil leucocytes have a
twofold origin: for the majority wander fully formed from the
bone-marrow into the blood, and only a considerably smaller
number grow to maturity within the blood-stream from the
mononuclear and transitional forms.

No definite statement can as yet be made as to the places of formation
of the non-granulated large mononuclear leucocytes.

Kurloff has demonstrated, that in the guinea-pig these cells are present
both in the bone-marrow and in the spleen, but that after extirpation of
the spleen the absolute number does not change. The bone-marrow then in
the guinea-pig can also preserve the balance of the large mononuclear,
non-granular cells in the blood.

The numbers we found in our blood investigations in man after
splenectomy were also normal. We may then doubtless assume that the
large mononuclear granuleless cells of human blood also arise for the
most part from the bone-marrow. In this tissue they are to be picked out
in the medley of the different kinds of cells only with the utmost
difficulty, owing to their small number and their but little
characteristic properties. Consequently an exact investigation of their
origin could probably only be successful if it were possible
experimentally to produce a disease in which these forms in particular
underwent important increase. This advance is not quite hopeless, since
in man at least an absolute increase of the large mononuclear cells is
observed in the post-febrile stage of measles.

On the grounds merely of microscopical investigations we conclude that
the bone-marrow is by far the most important of the blood-forming
organs, for its function is the exclusive production of red blood discs
as well as of the chief group of the white corpuscles, the polynuclear
neutrophil.

=The physiological, experimental investigation= of the functions of the
bone-marrow offers insurmountable difficulties. An exclusion of the
whole bone-marrow or of larger portions only is an impossible operation.
Nor can we ascribe any value to the researches which endeavour to obtain
a result by comparative enumerations of the arterial and venous blood of
a bone-marrow area. J. P. Roietzky working under Uskoff's direction has
recently made counts of this kind in the dog, from the nutrient artery
of the tibia and the corresponding vein. He found that the number of
white corpuscles of the vein is slightly greater, that on the other hand
the absolute number of "young corpuscles" (Uskoff), _i.e._ of the
lymphocytes, has been considerably diminished, whilst the number of
"ripe" corpuscles, which for the most part correspond to our
polynuclear, is considerably increased. He gives the following table:

-------------------------------------------------------------
Total number | Young | Ripe | Old
| corpuscles | corpuscles | corpuscles
-------------------------------------------------------------
Arterial blood 15000| 1950 (13%) | 840 (5.6%) | 12210 (81%)
Venous " 16400| 656 (4.0%)| 2788 (17.0%)| 12956 (79.0%)

The argument based on figures such as these assumes that the function of
the bone-marrow is =continuous=; an assumption which Uskoff indeed seems
to make.

But if the bone-marrow is constantly absorbing the lymphocytes to such
an extent, it is quite incomprehensible how the normal condition of the
blood can be preserved, bearing in mind the extent of the bone-marrow
and the rate of the circulation. All evidence indeed tends to shew that
on the contrary the bone-marrow performs its functions discontinuously,
inasmuch as elements continually grow to maturity in the bone-marrow, as
we have above explained, but they only emigrate at certain times as the
result of chemical stimuli. It is obvious _à priori_ from this
consideration how inconclusive must be the results of experiments such
as these of Roietzky[17].

Far more important for the elucidation of the function of the
bone-marrow are =clinical observations= on cases in which considerable
portions of the bone-marrow are replaced by tissue of another kind. We
may best divide the observations on this point into two groups: 1.
malignant tumours of the bone-marrow, 2. the so-called acute leukæmia.

There are unfortunately very few available observations as yet upon the
first group. Still rarer are the cases in which as is necessary the
whole bone-marrow has been subjected to an exhaustive examination, which
alone affords adequate evidence of the extent of the defect.

Amongst the changes of the bone-marrow arising from tumours one may
distinguish two groups, according to the nature of the condition of the
blood. The first type is exemplified by a case of Nothnagel published in
his work on lymphadenia ossium. Here during life the blood shewed, in
the main, the features of a simple severe anæmia; but in addition
isolated normoblasts, small marrow cells, and moderate leucocytosis. The
autopsy, at which the whole skeletal system was subjected systematically
to an exact examination, shewed a complete atrophy of the bone-marrow,
and replacement of the same by the tumour masses. In this case then the
condition of the blood _in vivo_ is satisfactorily explained by the
absence of function of bone-marrow. Nothnagel conjectured that the
formation of the scanty nucleated red blood corpuscles occurred
vicariously in the spleen, that of the leucocytes in the lymph glands.

In the second series to which the cases of Israel and Leyden, as well as
the recently published one of J. Epstein from Neusser's wards, belong,
the blood shews, besides the usual anæmic changes, other anomalies which
are peculiar partly to pernicious anæmia, partly to myelogenic
leukæmia. In Epstein's case of metastatic carcinoma of the bone-marrow,
there was found a considerable anæmia, with numerous nucleated red blood
corpuscles both of the normo- and megaloblastic type; their nuclei
presented the strangest shapes, due not merely to typical nuclear
division, but also to nuclear degeneration. The white blood corpuscles
were much increased, their proportion to the red was 1/25 to 1/40; the
increase concerned in the main the large mononuclear forms, which bore
for the most part neutrophil granulation, and were therefore to be
called myelocytes. In all the specimens, only two eosinophil cells were
found[18].

The explanation of a blood picture of this kind, apart from the purely
anæmic changes, is by no means easy, as Epstein rightly observes. The
appearance of myelocytes is most readily explained by a direct
stimulation of the remaining bone-marrow by the surrounding masses of
tumour. In this, the mechanical factor is less concerned than the
chemical metabolic products of the tumour masses; which at first act on
the adjacent tissue in specially strong concentration, and also in a
negatively chemiotactic manner on the wandering cells. This view
receives support from the careful work of Reinbach on the behaviour of
the leucocytes in malignant tumours. Out of 40 cases examined, in only
one, of lymphosarcoma complicated with tuberculosis, were myelocytes
found in the blood, amounting to about 0.5-1.0% of the white blood
corpuscles. The autopsy shewed isolated yellowish white foci of growth
in the bone-marrow, reaching the size of a sixpenny piece. Bearing in
mind that in none of the other 39 cases were myelocytes demonstrated,
one does not hesitate to explain their presence in the blood in this
single case by the metastases in the bone-marrow. The small extent of
the latter is likewise the cause of the small percentage of the
myelocytes.

In explaining the presence of the megaloblasts in the blood of Epstein's
patient we must keep before us what we have said elsewhere on this kind
of cell. They are not present in the normal bone-marrow; they arise on
the contrary, according to our view, when a specific morbid agent acts
upon the bone-marrow, as we must assume is the case in the pernicious
forms of anæmia. In the cases of anæmia from tumours, in which we find
megaloblasts in large numbers in the blood, we must likewise assume that
chemical stimuli proceed from the tumours, leading to the formation of
megaloblasts in the bone-marrow.

The presence of megaloblasts in the bone-marrow does not itself cause
their appearance in the blood, for in pernicious anæmia the bone-marrow
may be filled with megaloblasts, and yet only very scanty examples are
to be found in the blood. Whether the emigration of the megaloblasts
from the bone-marrow into the blood-stream is in general to be referred
to chemical stimuli, as it is in the particular case of Epstein's, or to
mechanical causes, cannot at present be decided.

The bone-marrow may be replaced by typical lymphatic tissue, as well as
by the substance of malignant tumours. The former occurs constantly in
lymphatic leukæmia according to the well-known results of Neumann, which
have since been generally confirmed. In these cases extensive tracts of
bone-marrow are replaced not by masses of malignant growth but by an
indifferent tissue, so to speak, a tissue which is unable to exercise
the above-described stimulating influence upon the remaining
bone-marrow. It is owing to this circumstance that we are able to
observe in the cases of lymphatic degeneration of the bone-marrow the
phenomena due to its exclusion, in their most uncomplicated form[19].

The most convincing results are obtained from cases of acute (lymphatic)
leukæmia, the pretty frequent occurrence of which was first noticed by
Epstein, and which has lately been very thoroughly studied by A.
Fränkel. For the purpose in question, acute leukæmia is specially
suited, since the abnormal growth of the lymphatic tissue takes place
very rapidly, and for this reason brings about a quick and uncomplicated
exclusion of the bone-marrow tissue; as it were, experimentally. Under
its influence the neutrophil elements of the bone-marrow vanish rapidly,
and in many cases so completely that it needs some trouble to find a
single myelocyte, as for example in a case of Ehrlich's. The polynuclear
leucocytes are produced in the bone-marrow, consequently where the
bone-marrow is destroyed, as in this case, it is clear that their
numbers must be absolutely very much diminished in the blood.

Dock has also arrived at similar results, as we see from a preliminary
report; and he similarly explains the absence of neutrophil cells in
lymphatic leukæmia by the replacement of the myeloid by lymphatic
tissue.

Thus lymphatic leukæmia affords a striking proof that the lymphocytes
are cells of a peculiar kind, and which are quite independent of the
polynuclear cells. It is therefore exceedingly surprising that Fränkel,
after accurately examining and analysing eight cases of acute lymphatic
leukæmia, believes he has found in them imperative reasons for the
assumption that the lymphocytes are transformed to polynuclear cells.
This can only be explained by the confusion which Uskoff's doctrine of
"young cells" has brought about.

We define lymphocytosis as an increase of the lymphocytes of the blood;
Fränkel like Uskoff regards it as the emigration of the young forms of
the white blood corpuscles into the blood. He concludes logically from
the diminution of the polynuclear cells in this form of disease "that
the conditions of the transformation of the young forms have undergone a
disturbance." But if one assumes that the lymphocytes are young forms,
and the polynuclears their older stages, it is much nearer to the facts
to speak, not of a disturbance in lymphatic leukæmia, but of an absolute
hinderance to the ripening process. It is easy to conceive any
particular stimulus or injury bringing about an acceleration of the
normal process, that is, a premature old age, but it is equally
difficult to represent clearly to oneself conditions which retard or
completely prevent the normal ageing of the elements. The discovery of
such conditions would be really epoch-making, both for general biology,
and for therapeutics. The only escape from this dilemma would be the
assumption of a very premature death of the lymphocytes, for which
however not the smallest evidence is to be found, even in Fränkel's
monograph. Fränkel distinguishes the acute from the chronic forms of
leukæmia by the fact, "that in the former the newly formed elements
emigrate from their places of formation into the blood-stream with
extraordinary rapidity. Hence there is not time for further local
metamorphosis. In chronic leukæmia the emigration takes place very
probably much more slowly." This distinction is contradicted by the
facts; for =there are chronic forms of lymphatic leukæmia whose
microscopic picture is identical with that of acute leukæmia=. And hence
the starting-point of all Fränkel's deductions is rendered insecure.

FOOTNOTES:

[14] C. S. Engel has recently proposed to call acute leucocytosis
"=lienal leucocytosis=," in analogy with the clinical idea of a lienal
leukæmia. This terminology should only be used if the polynuclear cells
did in fact arise from the spleen, an assumption which Engel himself
does not once appear to make, since he expressly warns against drawing
any conclusions from this name as to their origin. Since, however, the
acute leucocytoses, as we shall shew in the next section, are
exclusively to be referred to the bone-marrow, the term lienal
leucocytosis seems to us quite mistaken, for it must logically lead to a
conception of the origin of the leucocytes, exactly opposed to their
actual relationships.

[15] Many authors, _e.g._ Arnold, explain this double staining of the
eosinophil cells by the presence of eosinophil and mast cell granulation
side by side. That this is certainly not the case is shewn by the fact
that the "basophil" granulation of the eosinophil cells does not in
metachromatic staining shew the metachromasia characteristic for the
mast cells.

[16] A. Fränkel has recently reported histological investigations in
which he could demonstrate in one case true myelocytes in inflamed lymph
glands. He says (xv. _Congress f. innere Medecin_): "For some time past
I have had systematic examinations carried out by my assistant, Dr
Japha, on the granulations of the leucocytes contained in these glands
in a large number of infectious diseases, which are accompanied by acute
swelling of the lymphatic glands, such as scarlet fever, diphtheria,
typhoid. They were performed in the following way: dry cover-slip
preparations were made from the juice of the glands removed shortly
after death, and were stained in the usual way by Ehrlich's triacid
mixture. Amongst a large number of cases thus examined, it was possible
in only one case of scarlet fever--but in this beyond all doubt--to
demonstrate the presence of mononuclear cells with neutrophil
granulation." The extreme rarity of this condition supports our opinion
that the formation of neutrophil mononuclear elements cannot be regarded
as a normal function of the lymphatic glands. Polynuclear neutrophil
cells are nearly always naturally present in inflamed lymph glands, as a
product of the inflammation which has immigrated there. Every pus
preparation shews that the polynuclear neutrophil leucocytes can change
in the tissues to mononuclear, and the isolated observations of Japha
should be explained in this manner.

[17] Moreover the investigations of Roietzky are quite without
foundation, inasmuch as the tibia of the dog, upon which this author
performed his experiments, contains in all races of dogs--according to
the information very kindly given us by Prof. Schütz--no red marrow, but
fatty marrow only, which as is well known is incapable of the smallest
hæmatopoietic function.

[18] We draw particular attention to the small number of eosinophil
cells, since according to Ehrlich's postulates this absence of
eosinophil cells is incompatible with the diagnosis of a leukæmia.

[19] In contrast to this lymphatic metamorphosis of the bone-marrow, in
myelogenous leukæmia a myeloid transformation of the other blood-forming
organs, especially of the lymph glands is found; a transformation
sufficiently characterised as myeloid by the presence of myelocytes,
eosinophils, and nucleated red blood corpuscles.

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

During the last ten years a large amount of valuable work has been done
on the cell-granules from histological, biological and clinical sides.
This has particularly assisted hæmatology, where a number of problems
remain whose solution is only possible by the aid of a knowledge of the
granules. We must therefore consider the history, methods, and results
of this work.

Ehrlich was undoubtedly the first to insist on the importance of the
cell-granules, and to obtain practical results in this direction. We are
obliged to mention this, since Altmann has, in spite of express
corrections, repeatedly asserted the contrary. In 1891[20] Ehrlich
refuted Altmann's claim to priority, nevertheless, Altmann in the 2nd
edition of his _Elementary Organisms_ (1894) stated that before him no
one had recognised the specific importance of the granules, though some
authors had viewed them as "rare and isolated phenomena."

We may quote a passage published by Ehrlich in 1878[21], that is, ten
years before Altmann's papers. "Since the beginning of histology the
word 'granular' has been used to describe the character of cellular
forms. This term is not a very happy one, since many circumstances
produce a granular appearance of the protoplasm. Modern work has shewn
that many cells, formerly described as granular, owe this appearance to
a reticular protoplasmic framework. And we have no more right to call
cells granular in which proteid precipitates occur, either spontaneously
as in coagulation, or from reagents (alcohol). The name should be kept
exclusively for cells in which during life substances, chemically
distinct from normal proteid, are embedded in a granular form. We can
readily distinguish but few of these substances, such as fat and
pigment; most of them we can at present characterise but imperfectly, or
not at all."

"Earlier observations, especially on the mast cells, led me to expect
that these granulations, though they had long been inaccessible to
chemical analysis, could be distinguished by their behaviour with
certain stains. I found, in fact, granules of this kind, characterised
by their affinity for certain dyes, and which could thereby be easily
followed through the animal series and in various organs. I further
found that certain granules only occurred in particular cells, for which
they were characteristic, as pigment is for pigment cells, and glycogen
for cartilage cells (Neumann) and so forth. We can diagnose the
variously shaped mast cells only by the staining of their granules in
dahlia solution, that is by a microchemical test. And in the same way we
can separate tinctorially other granulated cells, morphologically
indistinguishable, into definite sub-groups. And for this reason, I
propose to call these granulations =specific=."

"The investigations were performed after Koch's method in the following
manner. The fluid (blood) or the parenchyma of the organs (bone-marrow,
spleen, etc.) was spread on cover-slips in as thin a layer as possible,
dried at room temperature, and after a convenient length of time
stained. I had chosen this apparently coarse method for the special
reason that for the histological recognition of new, possibly definite
chemical combinations, corresponding to the granulations, all substances
must be avoided that might act as solvents, _e.g._ water or alcohol, or
as oxydising agents, such as osmic acid. In this instance only such
procedures may be employed as will leave the simple drying of each
single chemical substance as much as possible unchanged."

A more detailed study of the process of staining, and of the relation
between chemical constitution and staining power, enabled a further
advance to be made. And the first result in this previously unworked
direction, was the sharp distinction between acid, basic, and neutral
dyes, and between the corresponding, oxy-, baso-, and neutrophil
granulations. The =triacid solution= was only found after trial of many
hundred combinations; and up to the present day this stain in its
original form or in slight modifications has played a prominent part in
various provinces of histology.

The classification of the cell-granules of the blood according to their
various chemical affinities which was drawn up by this method is
accepted to-day as the most valuable, and the only practical means of
grouping the leucocytes. From the first Ehrlich has insisted, =that
different kinds of cells possess different granules=, distinguished not
only by their tinctorial properties, but also by their various reactions
to solvents.

It is in this connection indeed, that Altmann's method, consisting of a
complicated hardening process, and the use of a single, always similar
stain, constitutes a retrograde step, in as much as it tends to obscure
the principle of the specificity of each kind of granulation.

A further disadvantage of Altmann's hardening method lies in the
circumstance, that the cell proteids are precipitated by it in a
spherical form, and stain in the subsequent treatment. Hence it is
extremely difficult to distinguish what is preformed, and what is
artefact. Since A. Fischer's publication, where the formation of
granule-like precipitates under the influence of various reagents is
experimentally demonstrated, grave doubts as to the reality of Altmann's
forms have been raised from various quarters. Ehrlich's dry process, on
the contrary, is entirely free from error. Granules cannot be
artificially produced, by desiccation, and the stained appearances
correspond precisely to what is seen in fresh living blood. The greatest
value of the dry method is that the =chemical nature= of the single
granules remains unchanged, so that attempts at differentiation are made
on a nearly unaltered object[22].

Another means of studying the nature of the granules depends on the
principle of =vital staining=. The "vital methylene blue staining"
(Ehrlich) that has since become so important, especially in neurology,
led to the first attempts at staining the granules in living animals.
One of the first publications on this subject is that of O. Schultze,
who placed the larvæ of frogs in dilute methylene blue solution, and
after a short period found the granules of the stomach, the red blood
corpuscles and other cells stained blue. This method, however, cannot
pass as entirely free from error, as Ehrlich frequently found that when
the experiment lasts some time the methylene blue often forms granular
precipitates that may be confused with the granules. Teichmann directs a
detailed analysis to this point, and regards most of the granules
described by Schultze as artificial products.

=Neutral red= is highly suitable for the study of vital granule-staining,
a dye recommended by Ehrlich, and employed successfully since that time
by Przesmycki, Prowazek, S. Mayer, Solger, Friedmann, Pappenheim and
others. This dye was prepared by O. N. Witt from nitrosodimethylamin and
metatoluylendiamin, and is the hydrochloric acid salt of a base which is
soluble in pure water, yielding a fuchsin red colour, but which in weak
alkaline solution--the alkalinity of mineral water suffices--is a
yellow-orange hue.

Now neutral red is characterised by a really maximal affinity for the
majority of the granules. Ehrlich was able by the aid of this dye to
demonstrate granules, even in some vegetable cells. Moreover the method
of using it is the simplest conceivable, as subcutaneous or intravenous
injection, or even feeding, in the higher animals stains the granules;
with frog's larvæ and invertebrates, to allow them to swim in a dilute
solution of the dye is often sufficient. The staining also succeeds in
"surviving" organs, and is best effected by allowing small pieces to
float in physiological salt solution, to which a trace of neutral red is
added, under plentiful access of air. When the object is macroscopically
red it is ready for examination.

The finest results are naturally given by organs that are easily teased
out, _e.g._ flies' eggs, or the Malpighian canals of insects. The
staining solution is to be chosen so that the act of staining does not
last too long, but on the other hand too high a concentration must not
be used. About 1/50000 to 1/100000 is recommended, so that the
protoplasm and nucleus remain quite uncoloured. Artificial products with
this method cannot entirely be excluded, and, _e.g._ in plant-cells
containing tannin, are to be explained by the production and
precipitation of the salt of tannic acid. However it is not difficult
for the experienced to recognise artificial products as such in
individual cases. The kind of granulation, the typical distribution, a
comparison with neighbouring cells, the combination of various methods,
the comparison of the same object under vital and "=survival=" staining,
facilitate judgment and obviate mistakes of this kind.

The majority of the granules of vertebrates are stained orange-red by
neutral red, corresponding with the weakly alkaline reaction of these
forms. Granules staining in pure fuchsin colour and which hence possess
a weak acid reaction are much more rarely found.

Combination staining may be recommended as a valuable aid to the neutral
red method. Ehrlich has used a double stain with neutral red and
methylene blue. Frog's larvæ were allowed to remain in a solution of
neutral red, to which a trace of methylene blue had been added. He then
found red granulations almost exclusively, only the granules of the
smooth musculature of the stomach were stained intensely blue. With the
aid of a threefold combination Ehrlich obtained a still further
differentiation of the living cell-granules. There is no doubt whatever
that a thorough study of this neutral red method would lead to
important conclusions as to the nature and function of the granules, and
lead us to the most real problems of cell life. With our present
information even we can get definite conceptions founded upon facts, as
to the =biological importance of the cell-granules=.

* * * * *

In his first publication Ehrlich described the =granules as products of
the metabolism of the cells=, deposited within the protoplasm in a solid
form, in part to serve as reserve material, in part to be cast off from
the cell. On the ground of observations on the liver cells, described in
detail in a paper of Frerichs (1883, page 43), Ehrlich gave up this
position, though only temporarily. Ehrlich shewed that the liver cells
of a rabbit's liver, rich in glycogen, appear in dry preparations as
bulky polygonous elements, of a uniform homogeneous brown colour,
surrounded by a thin, well-defined yellow membrane. In cells that were
not too rich in glycogen, small roundish bodies, clearly of a
protoplasmic nature, of a pure yellow, can be seen embedded in the
homogeneous cells that are coloured brown with glycogen. "The hyaline
cellular ground substance, carrying the glycogen, could not under any
circumstances be stained, but the cell-granules above mentioned stained
easily with all kinds of dyes. It was further possible to shew by
staining that the membrane was chemically different from the granules,
since with eosin-aurantia-indulin-glycerine, the membrane stained black,
but the granules orange-red."

To these observations Ehrlich added the following conclusion, "that the
cells of the liver after food really possess a thin =protoplasmic=
membrane, and a homogeneous glycogen-bearing substance, in which the
nucleus and =round granules= (? functionally active) of protoplasm are
embedded.

"On comparing these results with those of more recent investigation of
the cells, it is easy to determine the location of the glycogen very
accurately. Kupffer has shewn, first for the liver cells--and this is
now recognised as generally valid--that their contents do not represent
a microscopically single substance. In the 'survival' preparation he
found, in addition to the nucleus, two clearly distinct substances: a
hyaline ground substance in preponderating amount, and a more scanty,
finely granular, fibrillary substance embedded in it. Kupffer calls the
first paraplasm, the latter protoplasm. On warming the preparation to
about 22° C. manifest though feeble movements appeared in the network.
It can hardly be doubted, that of these two substances the granular
reticulated one--the protoplasm--is the more important; and it should
not be erroneous to suppose that the granulations of the network form
the =centre of the particular (specific) cell function=. In any case, it
is desirable to give a special name, such as microsomes (Hanstein) to
these forms, which in the liver cells are recognisable as distinct,
round or oval granules, colouring yellow with iodine, and easily and
deeply staining in other ways."

It was necessary to quote in full from this older paper, to shew that
Ehrlich regarded the granules as the special carriers of the cell
function so long ago as 1883, a view that Altmann advocated many years
later, under the name "theory of bioblasts." Altmann's ever repeated
assertion that no one before him had allotted so high an importance to
the granules is consequently in disagreement with the facts we have
above made sufficiently clear.

The importance Altmann ultimately gave to the granules, which he also
calls by the name "=Ozonophores=" is shewn by his own words (_Elementary
Organisms_, 1st edit., p. 39):

"Our conception of the ozonophores may therefore replace that of the
living protoplasm, at least so far as vegetative function is concerned;
and may serve us as an explanation of complicated organic processes.
Once again, shortly summarising the properties of the ozonophores; as
oxygen carriers they can perform reduction and oxydation, and can thus
effect the decompositions and syntheses of the body, without losing
their own individuality."

In the meantime Ehrlich had made various observations which could not be
completely brought into line with his own earlier hypothesis or the
far-reaching conclusions of Altmann. Studies in particular on the oxygen
requirements of the organism, shewed that the "ozonophores" could
certainly not be an important part of the cell. In addition it was found
that normally cells occur in which no granules can be recognised by
ordinary methods. Finally a pathological observation made untenable the
view that the granules are the bearers of the cell function. In a case
of pernicious anæmia (cp. _Farbenanalytische untersuchungen_) Ehrlich
found the polynuclear cells of the blood and bone-marrow and their early
forms free from all neutrophil granulation. On the grounds of this
observation Ehrlich returned to his original assumption that the
granules are secretory products of the cells, and defined his standpoint
at that time as follows:

"Did the neutrophil granulations really represent the bodies which
supply these cells with oxygen, as Altmann supposes, a condition such as
we have here brought forward would be impossible, since with the
disappearance of the granules death of the cells must follow. But from
the point of view of the secretion theory the condition described is
easily explainable. Just as under certain conditions fat-cells may
completely lose their contents without dying, so the bone-marrow cell,
if the blood fails to yield to it the necessary substances, may
occasionally be unable to produce more neutrophil granules. And thus it
becomes non-granular."

The view, that the granules are special metabolic products of the
specific cellular activity, is strongly supported by the great chemical
differences between them. Ehrlich made these peculiarities clear for the
blood-cells, and found that their granulations differ from one another,
not only in their colour reactions, but also in their =shape= and
=solubility=; so that they must be sharply distinguished.

Whilst for instance the majority of the granules are more or less
rounded forms, in some classes of animals, _e.g._ in birds, the
analogues of the granules of mammalian blood are characterised by a
decided crystalline form, and a strong oxyphilia. The substance of the
mast cell granulations is also crystalline in some species of animals.

The size of the individual granules is constant in any animal species
for every kind of granule--excepting only the mast cells. The eosinophil
granulation reaches its greatest size in the horse, where really
gigantic examples are found.

The presence of granulated colourless blood-cells has been demonstrated
in the most various classes of animals, and even in the blood of many
invertebrates, particularly, as Knoll has shewn, in the
Lamellibranchiates, Polychætes, Pedates, Tunicates and Cephalopods.
Concerning vertebrates, especially the higher classes, accurate and
ample researches are to hand. In birds we recognise two oxyphil
granulations, of which one is embedded in the cells in the crystalline,
the other in the usual granular form. Amongst the vertebrates most
investigated classes possess granulated polynuclear cells. To this
circumstance Hirschfeld has recently devoted a thorough paper containing
many details worthy of note. In the majority of the animals observed, he
found too that the polynuclear cells contained neutrophil granules; in
only one animal, the white mouse, did he find them, or granulations
analogous to them, completely wanting.

According to the investigations carried out some years back in Ehrlich's
laboratory by Dr Franz Müller, these results of Hirschfeld's must be
described as inaccurate. After many vain endeavours, Dr Müller was able
to find a method by which numerous though very minute granules could be
found in the polynuclear cells of the mouse. The case shews that it is
not permissible to assume the absence of granules, when the ordinary
staining methods are not at once successful. There is no universal
method for the staining of granules, any more than for the staining of
various kinds of bacteria. Indeed all granules, that are easily soluble,
vanish when the triacid method is used, and so a homogeneous cell
protoplasm is simulated.

But naturally, the occurrence of non-granulated polynuclear cells in
certain classes of animals is not to be denied from these
considerations. Hirschfeld asserts that such cells occur side by side
with granulated cells, for instance in the dog; and draws from them
far-reaching conclusions as to the meaning of the granules. From
Kurloff's work (see p. 85) we must insist, on the contrary, that there
is no evidence that the non-granulated polynuclears are identical with
the granulated cells. Kurloff has shewn, at least for guinea-pig's
blood, that these two heterogeneous elements are to be sharply separated
one from the other, and that they have an entirely different origin.

Specially important for a theory of the nature of the granules is the
circumstance, that generally speaking in all species of animals =they are
present in those cells of the blood only which are adapted to and
capable of emigration=. That a certain nutritive function is to be
ascribed to the emigration of the granulated cells is a very obvious
supposition, scarcely to be denied; and naturally cells with a plentiful
store of reserve material are eminently suited for this purpose. The
lymphocytes on the contrary, incapable of emigration, are almost totally
devoid of specific granulations.

A further indication that =the granulations really are connected with a
specific cell activity lies in the fact, that one cell bears but one
specific granulation=. The contrary assertions that neutrophil and
eosinophil, or eosinophil and mast cell granulations occur in the same
cell Ehrlich regards as unfounded, from extensive researches specially
directed to this point. Nor has Ehrlich seen a pseudoeosinophil cell of
the rabbit change to a true eosinophil[23]. That such a transition does
not occur is most distinctly shewn by the fact that the various
granulations behave entirely differently towards solvents. With the aid
of acids, for example, the pseudoeosinophil granules can be completely
extracted from the cells, whilst the eosinophil granules remain whole
under this process, and can now be stained by themselves.

The clearest proof that the neutrophil, eosinophil, and mast cells are
entirely separated from one another by the fundamental diversity of
their protoplasm, of which the granulation is but a specially striking
expression, is afforded by the study of the various forms of
leucocytosis. As will be shewn in detail in the following chapter,
neutrophil and eosinophil leucocytes behave quite differently in their
susceptibility to chemiotactic stimulation. Substances strongly
positively or negatively chemiotactic for one cell group are as a rule
indifferent for the other; frequently indeed there is an exactly opposed
relationship, inasmuch as substances which attract the one kind repel
the other. Still greater is the difference between the mast cells and
the other two cell groups; for so far as present investigations go, they
are quite uninfluenced by substances chemiotactic for the neutrophil or
eosinophil cells.

As specific cellular secretions, various kinds of granules must also be
sharply marked off from each other by their chemical properties. The
granules of the blood corpuscles seem to be of very simple chemical
constitution. We have special grounds for the assumption that the
crystalline granulations are for the most part composed of a single
chemical compound, not necessarily highly complex even, but which seems
to be a relatively simple body such as guanin, fat, melanin, etc.
Doubtless other granulations have a more complicated constitution, and
very often are a mixture of various chemical substances. The most
complicated granules of the blood are the eosinophil, which are, as has
elsewhere already been mentioned, of a more complex histological
structure. For a peripheral layer is plainly distinguishable from the
central part of the granule. It should be mentioned that according to
Barker the eosinophil granulations appear to contain iron.

The key-stone of the hypothesis of the secretory nature of the granules
is the direct observation of a secretory process in the cells bearing
the granules. Naturally these researches offer extraordinary
difficulties since only the coincidence of a number of lucky
circumstances would allow the passage of dissolved granule substance
into the neighbourhood to be followed. Kanthack and Hardy have succeeded
in demonstrating the secretory nature of the eosinophil granules of the
frog. When, for example, anthrax bacilli are introduced into the dorsal
lymph sac of the frog they exert a positive chemiotaxis on the
eosinophil cells. The latter come in contact with the bacilli, and
remain for some time attached to them. During this period Kanthack and
Hardy observed a discharge of granules from these cells, which now
possess a protoplasm relatively homogeneous. Afterwards these cells move
away from the bacilli, and are succeeded by the polynuclear neutrophil
cells, as will be mentioned later. These authors were further able to
observe gradual accumulation of granules in eosinophil cells in lymph
kept under microscopic observation as a hanging drop, and thus
demonstrated that they undergo the two stages characteristic of
secretion, (1) appearance of granules within the cells, (2) discharge of
these granules externally.

The mast cells too seem suited for this purpose since their specific
substance is strongly characterised by its peculiar metachromatic
staining, and is further especially readily recognisable, since by its
great affinity for basic dyes it remains plainly stained, even in
preparations that are almost quite decolorised. In fact appearances of
the mast cells are not infrequently found, which must be referred to
excretory processes of this kind.

In the first place it is occasionally seen that the mast cell
granulation is dissolved within the cell, and diffuses in solution into
the nucleus. In place of the well-known picture of the mast cell (see
page 76) of a colourless nucleus, surrounded by a deeply stained
metachromatic granulation, a nucleus is present intensely and
homogeneously stained in the tint of the mast cell granulation,
surrounded by a protoplasm shewing but traces of granules.

Still more convincing is the presence of a peculiar halo of the mast
cells, described by various authors. Ehrlich first shortly mentioned
this halo in his book on the oxygen requirements of the organism. A few
years ago, Unna, whose notice Ehrlich's remark had no doubt escaped,
described an analogous condition as follows: "in some nodules the mast
cells appeared in part twice as large as usual, especially with the new
mast cell stain (polychrome methylene blue, glycerine ether mixture).
This was caused by the staining of a large round halo, in the centre of
which lay the peculiar long-known mast cell, consisting of blue nucleus,
and an areola of deep red granules. Higher magnification shewed that the
halo was not granular, but very finely reticular; although it exhibited
exactly the same red colour as the granules. It was consequently a
spongioplasm peculiar to these mast cells."

The appearance of the mast cells described by Unna may also be
artificially produced, by allowing a preparation that is stained with
the oxygen containing analogue of thionin, oxamine, to remain for some
time in lævulose syrup or watery glycerine. Evidently part of the dyed
mast cell substance is dissolved and retained in the immediate
neighbourhood. But as Unna possesses great experience of the mast cells
and is a complete master of the methods of their demonstration, one must
suppose that the halos described by him were preformed, and did not
arise during the preparation of the specimen.

It must hence be concluded that an analogous process may go on during
life, that these halos are the expression of a vital secretion of the
substance of the mast cells externally[24].

A condition that Prus has brought forward in the so-called purpura of
the horse, is also to be interpreted as a secretory process of the mast
cells. He describes young mast cells from the hæmorrhagic foci of the
wall of the gut, on the margins of which bodies of various sizes
appeared, and which differ essentially from the mast cells themselves by
their staining. Nevertheless from their whole configuration and position
it is evident that these bodies have arisen in the mast cells
themselves; and Prus comes to the conclusion "=that the degenerating
young mast cells secrete a fluid or semi-fluid substance, which as a
rule sets on the surface of the cells, but also, more rarely, in their
interior=."

Evidence that the substance of the granules is given off externally may
sometimes be seen in the polynuclear neutrophil or their analogues. Thus
in rabbit's blood in which he had experimentally produced leucocytosis,
Hankin found a distinct progressive decrease of the pseudoeosinophil
granules on allowing the samples of blood to remain some time in the
thermostat. Further in suppurating foci in man, especially when
suppuration has lasted long, or the pus has remained for some time in
the place in question (Janowski) a rarefaction almost to complete
disappearance of the polynuclear neutrophil granules occurs, and is to
be explained by a giving up of the granulations to the exterior.

These facts and considerations, on the whole, lead then to the
conclusion, =that in general the granules of the wandering cells are
destined for excretion. This elimination of the granules is probably one
of the most important functions of the polynuclear leucocytes.=

FOOTNOTES:

[20] _Farbenanalytische Untersuchungen_ XII. _zur Geschichte der
Granula_, p. 134.

[21] _loc. cit._ pp. 5, 6.

[22] Altmann's freezing process would be similar to the advance always
insisted on by Ehrlich. It offers such great technical difficulties,
however, that it has up to now been little used.

[23] The cause of these misunderstandings is the tinctorially different
stages of development of the granules, as we have fully explained above.
How little adequate tinctorial differences by themselves are to settle
the chemical identity of a granulation, is at once evident on
consideration of the granules of other organs. No one surely would
assert, that a liver, muscle, or brain cell could occasionally secrete
trypsin, simply because the granules of the pancreas stain similarly and
analogously to those of the cells mentioned. We would here expressly
insist that we only assume a distinct character for each kind of
granulation, in the strict sense of the term for the cells of the blood,
since they possess a relatively simple function. In very complex
glandular cells, however, with various simultaneous functions, several
kinds of granules may be contained.

[24] From a paper of Calleja we learn that Ramon y Cajal recognised the
halos of the mast cells, and interpreted them in the manner we have
above. Calleja also describes these halos and the method of
demonstrating them in detail (thionin staining, and mounting the
sections in glycerine). We must mention, however, that we do not
consider this method suitable for the recognition of preformed halos,
for the reasons above mentioned.

IV. LEUCOCYTOSIS.

The problem of leucocytosis is one of the most keenly debated questions
of modern medicine. An exhaustive account of the various works devoted
to it, of the methods and results, could fill by itself a whole volume,
and would widely exceed the limits of an account of the histology of the
blood. We can only deal fully therefore with the purely hæmatological
side of the subject.

Virchow designated by the name "=Leucocytosis=," a transient increase in
the number of the leucocytes in the blood; and taught that it occurred
in many physiological and pathological conditions. In the period that
followed particular attention was paid to the leucocytosis in infectious
diseases, and to the investigators of the last 15 years in this province
we owe very important conclusions as to the ~biological meaning~ of this
symptom. Above all Metschnikoff has done pioneer service in this
direction by his theory of phagocytes, and though his theory has been
shaken in many essential points, yet it has exercised a stimulating and
fruitful influence on the whole field of investigation.

To sketch Metschnikoff's doctrine in a few strokes is only possible by a
paraphrase of the very pregnant words "Phagocytes, digestive cells."
These words express the view, that the leucocytes defend the organism
against bacteria by imprisoning them by the aid of their pseudopodia,
taking them up into their substance, and so depriving them of the power
of external influence. The issue of an infectious disease would chiefly
depend on whether the number of leucocytes in the blood is sufficient
for this purpose.

This engaging theory of Metschnikoff has undergone important limitations
as the result of further investigation. Denys, Buchner, Martin Hahn,
Goldscheider and Jacob, Löwy and Richter, and many others have
demonstrated, that the most important weapon of the leucocytes is not
the mechanical one of their pseudopodia, but their chemical products
("Alexine," Bucher). By the aid of bactericidal or antitoxic substances
which they secrete, they neutralise the toxines produced by the
bacteria, and thus render the foe harmless by destroying his weapon of
offence, even if they do not exterminate him.

An explanation of the almost constant increase of the leucocytes of the
blood in bacterial diseases is given by the chemiotactic as well as by
the phagocytic theory of leucocytosis. The principle of =chemiotaxis=
discovered by Pfeffer asserts that bacteria, or rather their metabolic
products, are able to attract by chemical stimulus the cells stored up
in the blood-forming organs ("positive chemiotaxis"). In the cases in
which a diminution of the leucocytes in the blood is found, it is the
result of a repulsion of the leucocytes by the bodies mentioned,
=negative chemiotaxis=.

As the experimental investigation of leucocytosis was carried further,
it was found that leucocytosis, quite similar to that occurring in
infectious diseases, could also be brought about by the injection of
various chemical substances (bacterio-proteins, albumoses, organic
extracts and so forth); and it became evident that the explanation of
the process by chemiotaxis must be supplemented in many respects. Löwit
for instance found that when substances of this kind are injected, two
different stages can be distinguished in the behaviour of the
leucocytes. First came a stage in which they were diminished
("leukopenia," Löwit) and in such a way that only the polynuclear cells
were concerned in the diminution, whilst the number of the lymphocytes
was unchanged. After this came the phase of increase of the white blood
corpuscles; and here too exclusively of the polynuclear cells; the
=polynuclear leucocytosis=. This behaviour seemed to indicate that during
the first period a destruction of white blood corpuscles brought about
by the foreign substances took place, and that it was only the dissolved
products of the latter which caused the emigration of fresh leucocytes
by chemiotaxis. But new objections were raised against this view.
Goldscheider and Jacob, in particular, shewed by exact experiments that
the transient leukopenia of the blood was not true but merely apparent;
and was caused by an altered distribution of the white blood corpuscles
within the vascular system. For whilst in the peripheral vessels from
which the blood for investigation was usually obtained, there was in
fact a diminution of the leucocytes, "=hypoleucocytosis=," in the
capillaries of the internal organs, especially of the lungs, a marked
increase of the leucocytes, "=hyperleucocytosis=," was found.

There are other objections to the great importance that Löwit has given
to leukopenia. _À priori_ it is quite incomprehensible that the various
substances, which in the fundamental test-tube experiment are able to
exercise a distinct chemiotactic influence on the leucocytes, should
under other circumstances need the intervention of the products of
decomposition of the white blood corpuscles. Moreover clinical
experience speaks in general against Löwit's theory. For in infectious
diseases hyperleucocytosis is very common; and a transient leukopenia is
equally rare.

This contradiction to the experimental results obtained by Löwit is
easily explained when one reflects how different from the natural
processes of disease are the circumstances of experiment. In this case
the animal is by intravenous injection flooded at once with the morbid
substance, and a violent acute reaction of the vascular and blood
systems is the natural consequence. In natural infection, insidious and
increasing amounts of poison come quite gradually into play, and for
this reason, perhaps, hypoleucocytosis in the normal course of
infectious diseases is much rarer than in the brusque conditions of
experiment.

Upon the clinical importance of leucocytosis, particularly for the
infectious diseases and their various stages, an enormous mass of
observations has accumulated. Selecting pneumonia as the best studied
example, in the typical course of this disease the constant occurrence
of leucocytosis is undisputed; the increase usually continues up to the
crisis, and then gives place to a diminution of the leucocytes until a
subnormal number is reached.

Of special importance are the observations on an absence of leucocytosis
in particularly severe or lethally ending cases (Kikodse, Sadler, v.
Jaksch, Tschistowitch, Türk and others).

In many other diseases as well, the observation has been made that
hyperleucocytosis as a rule is only absent in specially severe, or in
some way atypical cases. Several observers (Löwy and Richter, M. Hahn,
Jacob), have been able to demonstrate experimentally for various
infections, that artificial hyperleucocytosis influences the course of
an artificial infection most favourably. The question, in what way does
this process contribute to the protection of the body, is at the present
time under discussion, and introduces the most difficult problems of
biology.

* * * * *

The ~morphological character of leucocytosis~ is certainly not simple, and
we must sharply separate various groups, according to the kind of
leucocyte increased.

The most important consideration is, whether cells capable of
spontaneous movement, and of active emigration into the blood, are
increased ("~active leucocytosis~"); or whether the number of those cells
is raised, to which an independent mobility cannot be ascribed, which
therefore are only passively washed into the blood-stream by mechanical
forces ("~passive leucocytosis~").

The passive form of =leucocytosis= corresponds to the different kinds of
lymphæmia, including that of leukæmia. In the section on the lymphatic
glands, we have established this view in detail, and we have
particularly insisted that a suppuration, consisting of lymph cells,
does not occur.

In sharp contrast to this form there are for every specific kind of
active leucocytosis, analogous products of inflammation (pus,
exudations), composed of the same kind of cell.

We divide =active leucocytosis= into the following groups:

([alpha]) =polynuclear leucocytoses=:

1. =polynuclear neutrophil leucocytosis=,
2. =polynuclear eosinophil leucocytosis=;

([beta]) =mixed leucocytoses= in which the granulated mononuclear
elements take part; "=myelæmia=."

[alpha] 1. ~Polynuclear neutrophil leucocytosis~, is the most frequent
of all forms of active leucocytoses.

Virchow, the discoverer of leucocytosis, advocated the view, that it
resulted from an increased stimulation of the lymph glands. The
stimulation of the lymph glands consists in "that they are engaged in an
increased formation of cells, that their follicles enlarge, and after a
time contain many more cells than before." The swelling of the lymphatic
glands has as a consequence an increase of the lymph corpuscles in the
lymph, and through this an increase again of the colourless blood
corpuscles.

This standpoint had to be abandoned, when Ehrlich shewed that it is
chiefly the emigration of the polynuclear neutrophil cells, which brings
about leucocytosis. Exact figures on this point were first given by
Einhorn, who worked under Ehrlich, and were later generally confirmed.
Corresponding with the increase of neutrophil blood corpuscles alone,
there is always a relative decrease of lymphocytes, often to 2% and even
lower. It must here be borne in mind, that the percentage of the lymph
cells may be much diminished, without change in their absolute number.
It has however been conclusively demonstrated that occasionally in
polynuclear leucocytosis, the absolute number of the lymphocytes may
decrease. Einhorn had already described a case of this kind, and
recently Türk has for the first time established the fact by an
abundance of numerical estimations[25].

The eosinophil cells are as a rule diminished in ordinary polynuclear
leucocytosis, as Ehrlich had already mentioned in his first
communication. The diminution is often considerable, often indeed
absolute.

A few diseases shew, besides the neutrophil leucocytosis, an increase of
the eosinophils as well, as we shall describe in detail in the next
section.

Polynuclear neutrophil leucocytosis--leucocytosis [Greek: kat'
exochên]--may be divided into several groups =according to their clinical
occurrence=. We distinguish:

A. =physiological leucocytosis=,

which appears in health as an expression of changes in the physiological
state. To this group belongs the leucocytosis of digestion, the
leucocytosis from bodily exertion (Schumburg and Zuntz) or from cold
baths, and further the leucocytosis of pregnancy.

B. =pathological leucocytosis=.

1. The increase of polynuclear cells occurring in infectious processes,
often called inflammatory, after the principle "_a potiori fit
denominatio_." The majority of febrile infectious diseases, pneumonia,
erysipelas, diphtheria, septic conditions of the most varied ætiology,
parotitis, acute articular rheumatism, etc. are accompanied by a
leucocytosis of greater or less extent. In this connection uncomplicated
typhoid fever and measles occupy a peculiar position. In them the
absolute number of white blood corpuscles is diminished, and chiefly at
the expense of the polynuclear neutrophil cells.

For the details we have quoted, and for the course and variations of
leucocytosis in infectious diseases we refer to the thorough monograph
of Türk. Of Türk's observations we will mention only that in the final
stage of the process of leucocytosis, which occurs at the time of the
crisis in diseases which run their course critically, mononuclear
neutrophil cells and stimulation forms as well often make their
appearance in the blood. In still later stages, in which the blood has
once more a nearly normal composition, a moderate increase of the
eosinophils--gradually waxing and again waning--is very frequently found
(Zappert and others). Stiénon, who has likewise devoted special
researches to the occurrence of leucocytosis in infectious diseases,
shews this point very well in his curves.

2. =Toxic leucocytosis= occurring in intoxications with the so-called
blood poisons. This important group has not yet received adequate
treatment in the literature. In general the majority of blood poisons,
potassium chlorate, the derivatives of phenyl hydrazin, pyrodin,
phenacetin call forth even in man a considerable increase of the
leucocytes besides the destruction of the red blood corpuscles. This has
been observed experimentally by Rieder.

We observed marked increase of the white blood corpuscles after
poisoning from arsenurietted hydrogen, from potassium chlorate, further
in a fatally ending case of hæmoglobinuria (sulphonal poisoning?) as
well as after protracted chloroform narcosis.

3. The =leucocytosis= which accompanies acute and chronic anæmic
conditions, especially posthæmorrhagic.

4. =Cachectic leucocytosis= in malignant tumours, phthisis, etc.[26]

To enter here more precisely into the special clinical importance of
blood investigation in different forms of disease would lead us too far,
and we refer for this subject to the excellent and thorough monograph on
leucocytosis by Rieder and to the papers of Zappert and Türk. In this
place we will only touch on the most weighty points.

[alpha]. The importance for differential diagnosis of the leukopenic
blood condition in typhoid fever as compared with other infectious
diseases, and in measles as against scarlet fever.

[beta]. The prognostic importance of the enumeration of the white
blood corpuscles. Thus for example the absence of leucocytosis
influences the prognosis of pneumonia unfavourably (Kikodse and others);
and the appearance of numerous myelocytes in diphtheria is ominous, as
demonstrated by C. S. Engel (see page 78).

* * * * *

Finally, we may dismiss in a few words the origin of ~polynuclear
neutrophil leucocytosis~, and refer to what has been said in another
place on the function of the bone-marrow.

In agreement with Kurloff's researches, Ehrlich formulated ("On severe
anæmic conditions" 1892) his views on this subject as follows: "The
bone-marrow is a breeding place in which polynuclear cells are produced
in large numbers from mononuclear pre-existing forms. These polynuclear
cells possess above all other elements the power of emigration. So soon
as chemiotactic substances circulate in the blood, which attract the
white elements, this power comes into play. This readily explains the
rapid and sudden appearance of large numbers of leucocytes, which so
many substances bring about, and particularly the bacterio-proteins,
recognised by Buchner as leucocytic stimuli. I regard leucocytosis
therefore, in agreement with Kurloff, as a function of the bone-marrow."

Of great theoretical interest is the contrast between eosinophil and
neutrophil cells. At the height of ordinary leucocytosis, the number of
eosinophil cells is diminished often to disappearance; whilst during its
decline they occur in abnormally high numbers. Hence it follows that the
eosinophil and neutrophil cells must react towards stimulating
substances completely differently, and in a certain sense oppositely[27].

It seems, generally speaking, that the bacterial =metabolic products
formed in human diseases which are positively chemiotactic for the
polynuclear neutrophil cells are negatively chemiotactic= for the
eosinophils, and _vice versâ_.

The explanation of the individual clinical forms of leucocytosis is
self-evident from the above description. The occurrence of physiological
and inflammatory leucocytosis is exclusively to be explained by
chemiotaxis. In the other forms, however, other factors also come into
play, in particular the increased activity of the bone-marrow, or the
extensive transformation of fatty to red marrow, causing a large fresh
formation of leucocytes.

[alpha] 2. ~Polynuclear eosinophil leucocytosis. Mast cells.~

Our knowledge of eosinophil leucocytosis is still of comparatively
recent date. After Ehrlich demonstrated the constant increase of the
eosinophil cells in leukæmia a considerable time elapsed before an
eosinophilia was found in other diseases, an eosinophilia however that
differs in its essential traits from the leukæmic type. To Friedrich
Müller we owe the first researches in this direction, at whose
suggestion Gollasch investigated the blood of persons suffering from
asthma; in which he was able to demonstrate a considerable increase of
the eosinophil cells. This was followed by the researches of H. F.
Müller and Rieder, who discovered the frequency of eosinophilia in
children, and its presence in chronic splenic tumours; further by the
well-known work of Ed. Neusser, who observed a quite astounding
increase of the oxyphil elements in pemphigus, and by the almost
simultaneous analogous observations of Canon in chronic skin diseases.
From amongst the flood of further papers upon this condition we will
only mention the comprehensive account of the subject by Zappert.

By =eosinophilia= we understand an =increase only of the polynuclear
eosinophil cells in the blood=. Confusion of this form of leucocytosis
with leukæmia is quite impossible, because a good number of
characteristic signs are necessary for the diagnosis of the latter, as
we shall have to explain in the next section. The presence of
mononuclear eosinophil cells in the blood should not be regarded, as is
the case in many quarters, as an absolute proof of leukæmia, for they
are also found in isolated cases of ordinary leucocytosis.

The increase of eosinophil cells is not always relative, but may be
absolute. The relative number, normally 2 to 4% of all leucocytes, rises
in eosinophilia to 10, 20, 30% and over; in a case described by Grawitz
90% indeed was found. The thorough researches of Zappert, carried out on
moist preparations by a suitable method, are particularly instructive
with regard to their absolute number. As the lowest normal value he
gives 50-100 eosinophil cells per mm.^{3}, as mean value 100 to 200, as
a high normal value 200-250. The highest absolute number he has ever
found was 29,000 per mm.^{3} in leukæmia, the highest number in simple
eosinophil leucocytosis 4800 (in a case of pemphigus). Reinbach indeed
once found about 60,000 eosinophil cells per mm.^{3} in a case of
lymphosarcoma of the neck with metastases in the bone-marrow.

Polynuclear eosinophil leucocytosis, apart from the form observed
in healthy children, occurs in varied conditions, and for
comprehensiveness we divide them into several groups. We distinguish
eosinophilia:

1. =In bronchial asthma.= Increase of the eosinophil cells of the blood,
often considerable, amounting to 10 and 20% and more has been regularly
found, first by Gollasch, later by many other observers. (For the
special clinical course of the eosinophilia in asthma see below.)

2. =In pemphigus.= Neusser first recorded that an extraordinarily great,
indeed a specific eosinophilia was found in many cases of pemphigus.
This interesting observation has been confirmed on many sides, in
particular by Zappert, who once observed 4800 oxyphil per mm.^{3}

3. In acute and chronic =skin-diseases=. Canon was the first to notice
that in a fairly large number of skin-diseases, especially in prurigo
and psoriasis, the eosinophil cells are increased up to 17%. The
observation of Canon is worthy of attention, that the increase of the
eosinophils is connected with the degree of extension of the disease,
rather than with its nature or local intensity. In a case of acute
widely distributed urticaria, A. Lazarus found the eosinophils increased
to 60% of the leucocytes, a number which after the course of a few days
again sank to normal.

4. =In helminthiasis.= The first observations on the occurrence of
eosinophilia in helminthiasis we owe to Müller and Rieder, who obtained
fairly high values (8.2 and 9.7%) in two men suffering from Ankylostomum
duodenale. Shortly afterwards Zappert stated that he had found a
considerable increase of the eosinophil cells in the blood, reaching 17%
in two cases of the same disease; at the same time he demonstrated
Charcot's crystals in the fæces. In a third case of Ankylostomiasis
Zappert found no increase of eosinophil cells in the blood, nor the
crystals in the fæces. Almost simultaneously, Siege made similar
observations.

For a detailed working out of this important branch we are greatly
indebted to Leichtenstern. Under his direction Bücklers established the
interesting fact that Ankylostomiasis in its relation to eosinophilia
does not occupy a special place in diseases caused by worms. All kinds
of Helminthides, from the harmless Oxyuris to the pernicious
Ankylostoma, may bring about an increase of the eosinophil cells in the
blood, often to an enormous extent[28]. Bücklers reports an observation
of 16% eosinophils in Oxyurides, of 19% in Ascarides; and Prof.
Leichtenstern, as we learn from a private communication, has quite
recently found 72% eosinophil cells in a case of Ankylostomiasis, and
34% in a case of Tænia mediocanellata.

It is well worthy of note that Leichtenstern was able to observe
numerous eosinophil cells in the blood in those cases where Charcot's
crystals were abundantly contained in the fæces. Since eosinophil cells
and Charcot's crystals have elsewhere been observed to be interconnected
phenomena (for example in bronchial asthma, in nasal polypi, in myelæmic
blood and bone-marrow) one must fall in with Leichtenstern's supposition
that eosinophil cells ought also to be found in the intestinal mucus in
cases of Ankylostomiasis. Positive observations on this point as yet are
wanting.

T. R. Brown, who worked under direction of Thayer, has lately
communicated the interesting observation that in =trichinosis= there is
constantly an extraordinary relative increase in the oxyphil leucocytes
in the blood, up to 68%. The absolute figures were also much raised, and
attained values (20,400 for example) which are by no means frequent even
in leukæmia.

Brown regards this astonishing phenomenon as pathognomic for
trichinosis, so much so, that in a case that was clinically obscure, he
made, from the marked eosinophilia, the diagnosis of trichinosis which
was later fully confirmed.

5. =Post-febrile form of eosinophilia= (after the termination of various
infectious diseases). In the section on polynuclear neutrophil
leucocytosis we have already mentioned that at the height of most of the
acute infectious diseases, with the single exception of scarlet fever,
the eosinophils undergo a relative decrease and may even entirely
disappear. In the post-febrile period, however, abnormally high values
for the eosinophil cells are often found, or even a well-marked
eosinophil leucocytosis, which generally attains but moderate degree.
Türk for example in pneumonia found a post-critical eosinophilia of
5.67% (430 absolute), after acute articular rheumatism 9.37% (970
absolute); Zappert in malaria, one day after the last attack 20.34%
(1486 per mm.^{3}).

The eosinophilia observed as the result of tuberculin injections, we
include, in agreement with Zappert, in the group of post-febrile
leucocytosis. For it appears only after considerable rises of
temperature. During the real reaction period the number of eosinophil
cells sinks, and only goes up again after the termination of the fever.
The rise may be very considerable. In one case of Zappert's the number
of the oxyphils increased to 26.9%; in another of his cases the highest
absolute figure formed after tuberculin injections was 3220 per mm.^{3}
In a case of Grawitz' the eosinophilia was quite extraordinary. The most
marked changes in the blood occurred some three weeks after cessation of
the tuberculin injections, of which eight altogether (from 5 mg. to 38
mg.) were given. Investigation shewed 4,000,000 red blood corpuscles per
mm.^{3}, 45,000 white. Amongst the latter there were ten eosinophils to
one non-eosinophil. The total number of eosinophil cells amounted to
some 41,000 per mm.^{3}, whilst the other cells as a whole made up some
4000. Inasmuch as the latter contained polynuclears, lymphocytes and
other forms, it follows that in this case the polynuclear neutrophils
must have been very much decreased, not only relatively but also
absolutely; so that this case represents precisely the contrary
condition to ordinary leucocytosis and the infectious form in
particular.

6. =In malignant tumours.= In the cachexia from tumours an increase of the
eosinophil cells has been observed by various authors. It is however of
moderate degree and does not exceed 7-10%. Out of 40 decided cases
Reinbach found the eosinophils increased only in four, in a case of
sarcoma of the forearm he found 7.8%; of the thigh 8.4%; malignant
tumour of the abdomen 11.6%. Besides these he describes a case of
lymphosarcoma of the neck with metastases in the bone-marrow, in which
an unexampled increase of the white blood corpuscles, and especially of
the eosinophil cells was found. The absolute number of the latter
amounted on one day to some 60,000! This is an increase of 300 fold the
normal, which apart from leukæmia has doubtless never before been
found.

7. =Compensatory eosinophilia= (after exclusion of the spleen). We have
entered in detail into this form in the chapter on splenic function; and
have there already mentioned that the increase of the eosinophils found
in chronic splenic tumours by Rieder, Weiss and others, must also be
referred to the exclusion of the splenic function.

8. =Medicinal eosinophilia.= Under this group occurs only a single
observation of v. Noorden's, who observed the appearance of an
eosinophilia up to 9% in two chlorotic girls after internal
administration of camphor. In other patients this occurrence did not
repeat itself. But probably researches specially directed to this
province of pharmacology would bring to our knowledge many interesting
facts.

On the origin of ~polynuclear eosinophil leucocytosis~ authors have put
forward various theories, which we will here critically discuss in
succession.

An experiment frequently quoted as explanatory is that of Müller and
Rieder's; these authors do not derive the eosinophil cells of the blood
from the bone-marrow, but assume, as very probable, that the finely
granular cells grow into eosinophils within the blood-stream. This
developmental process seems very improbable for many reasons. Since the
polynuclear cells circulating in the blood are all under the same
conditions of nutrition, it is _à priori_ inconceivable why only a
relatively small portion of them should undergo the transformation in
question. And it is quite inexplicable why in infectious leucocytosis,
where the number of the polynuclears is increased so enormously, their
ripening to the eosinophils should remain completely interrupted.

But the fact, that a transition from neutrophil to oxyphil cells has
never really been observed in the blood, is decisive evidence against
the hypothesis of Müller and Rieder. Were the hypothesis true,
transitional stages ought to be found with ease in every sample of
normal blood. Rieder and Müller themselves are unable to bring forward
any positive result of this kind, else they would hardly have been
contented to fall back on the authority of Max Schultze, who professed
to shew the transitional forms between the finely and coarsely granular
leucocytes in the circulating blood. The authority of Max Schultze in
morphological questions stands high, and very rightly; but one ought not
to rely upon it for support in problems that are really histo-chemical,
and which should be solved by their appropriate methods.

As a logical consequence of their view, and in decided opposition to
Ehrlich, Müller and Rieder assume that the eosinophil cells of the
bone-marrow "are far rather the expression of a storage than of a fresh
formation there. =The bone-marrow therefore should be regarded in
reference to the coarsely granular cells of the blood more as a storage
depôt, where these cells serve other purposes, which for the present
cannot be more closely defined.="

The chief reason for this assumption, these authors see in the fact,
that the majority of the eosinophils in the bone-marrow are mononuclear,
whilst those of normal blood possess a polymorphous nucleus. Müller and
Rieder should themselves have raised the obvious objection that the same
holds good for the nucleus of the neutrophils. They would then have seen
the fault in their theory; for according to it the most important blood
preparing organ constitutes as it were, not the cradle of the blood
cells, but their grave. The simplest and readiest explanation, based
too upon histological observation, is surely this: that the mononuclear
eosinophil cells grow into polynuclear in the bone-marrow, but that the
latter only reach the blood by means of their power of emigration. As
this view has been accepted by the great majority of authors since
Ehrlich's paper "On severe anæmic conditions," we believe we may content
ourselves with the above objections to the Müller-Rieder theory,
although it has even quite recently found supporters (_e.g._ B.
Lenhartz). H. F. Müller moreover in his paper on bronchial asthma (1893)
takes a position different from his earlier, and approaching that of
Ehrlich.

In considering the production of polynuclear eosinophilia we may best
start from an experiment of E. Neusser's. Neusser found in a pemphigus
patient, whose blood shewed a considerable increase of the eosinophils,
that the contents of the pemphigus bulla consisted almost entirely of
eosinophil cells. Neusser now produced a non-specific inflammatory bulla
in the skin by a vesicant, and found that the cellular elements in it
were exclusively the polynuclear neutrophil concerned in all ordinary
inflammations.

Exactly analogous conditions, occurring spontaneously, have been
demonstrated by Leredde and Perrin in the so-called Dühring's disease.
The bullæ which appear in this dermatosis contain, so long as their
contents are clear, chiefly polynuclear eosinophil cells. In a later
stage, as is usually the case, bacteria effect an entrance into the
bullæ, which now become filled with neutrophils.

According to modern views on suppuration, the experiment of Neusser and
the observation of Leredde and Perrin can only be explained by the
hypothesis, that the eosinophil and neutrophil cells, as we have
already several times mentioned, are of different chemiotactic
irritability. Hence the eosinophil cells only emigrate to those parts
where a specific stimulating substance is present. From this point of
view experiments and clinical observations known up to the present on
eosinophilia may be readily explained. Neusser's experiment for instance
may be explained in the following way. In the pemphigus bullæ a
substance is present that chemiotactically attracts the eosinophils.
Hence the cells normally contained in the blood emigrate into them, and
produce the picture of an eosinophilous suppuration. Should the disease
assume from the first a localised distribution only, the essential
feature of the process is excluded. =A totally different appearance,
however, is produced= when the disease has attacked large areas. =Under
these circumstances large amounts of the specific active agent reach the
blood-stream by absorption and diffusion. Here it exercises a strong
chemiotactic influence on the physiological storage depôt of the
eosinophils, the bone-marrow; leading to an increase of the eosinophils
of the blood to a greater or less degree. The bone-marrow, according to
general biological laws, is by the increased emigration now further
stimulated to a fresh production, and during a protracted illness can
hence keep up the eosinophilia.=

In this way other clinical observations may be explained. Gollasch has
found that the sputum of asthmatic patients contains, in addition to
Charcot-Leyden's crystals, eosinophil cells only. One must therefore
assume that within the bronchial tree there exists material which
attracts the eosinophils. This supposition is also supported by the
close connection that obtains, according to many observations, between
the severity of the disease and the eosinophilia. Thus v. Noorden
records that the eosinophil cells are more numerous about the time of an
attack. They accumulated in especially large numbers after attacks had
rapidly occurred several days in succession. =That the increase of the
eosinophil cells in this instance is directly connected with the
attacks, and is not the expression of a permanent constitutional
anomaly=, is shewn by a case in which v. Noorden found 25% eosinophils
during the attack, and a few days later could only observe one example
in twelve cover-slip preparations: a diminution therefore of this group
of cells.

The observations of Canon in skin-diseases are quite similar, for he
shewed that the extension of the disease determines the degree of
eosinophilia more than its intensity. And it is the former factor which
directly determines the quantities of the specific agent that pass into
the blood.

To the Müller-Rieder hypothesis, and the chemiotactic theory of
eosinophil leucocytosis a third has lately been added, which may be
shortly called =the hypothesis of the local origin= of the eosinophil
cells. A. Schmidt has, with special reference to asthma, raised the
question "whether in the extensive production of eosinophil cells in
asthma, local production in the air passages is not more probable than
origin from the blood. One may well regard the increase of the
eosinophil cells in the blood of an asthmatic as secondary." This view,
which has also been advocated by other authors, rests more particularly
on the following facts and considerations:

1. That in various diseases of the nose, especially in mucous polypi
and hyperplasia of the mucous membrane (Leyden, Benno Lewy and others),
a great accumulation of eosinophil cells is found in these tissues,
whilst they are apparently not increased in the blood. This objection is
easily laid aside from the chemiotactic point of view. For if in the
places in question substances are present which act chemiotactically on
the eosinophil leucocytes, in the course of time marked accumulation
must occur, without an increase of their number in the blood. One might
as well conclude from Neumann's experiment in lymphatic leukæmia, for
example, where the artificial suppuration consisted only of polynuclear
neutrophil cells, that the polynuclear cells were formed in the tissue,
since in the blood they were present in very small percentage. For in
this case too the same incongruity between the blood and the particular
tissue exists.

2. Adolph Schmidt has urged the converse argument. He shewed that in the
sputum of patients with myelogenic leukæmia no more eosinophil cells
were present than are commonly to be found in the bronchial secretion,
although the blood was unusually rich in eosinophil cells. In our
opinion however this observation does not support the hypothesis of
local origin, but on the contrary is clear evidence that not the larger
or smaller number of eosinophil cells in the blood decides their
emigration, but the presence of specifically active chemical stimuli.
For we know from our observations on leucocytosis in infectious diseases
that the bacterial stimulating substances act on the eosinophil cells
rather in a negative than in a positive sense. And if ordinary sputum is
not rich in eosinophils in spite of a marked eosinophilia of the blood,
this only corresponds to our experience in general. Indeed, this
phenomenon is quite similar to Neusser's pemphigus experiment, where
the specific foci of disease shewed an eosinophilia, whilst abscesses
produced artificially, on the contrary, only neutrophil cells. Finally
we may employ, to support our view, another analogous experiment of
Schmidt himself. He found numerous eosinophil cells in the sputum of an
asthmatic patient, but only neutrophil cells in an artificially produced
suppuration of the skin.

Thus we see that the chief reasons brought forward by the supporters of
the theory of local origin are not proof against the most obvious
objections that can be raised from the chemiotactic standpoint.
Moreover, neither histological nor experimental proof has been given for
this theory in spite of numerous investigations in this direction. All
the same, it should not be out of place to explain the possibilities
that are given for a local origin of the eosinophil cells. First, the
eosinophil cells might be the result of a progressive metamorphosis of
the normal tissue cells. That such a process is possible, is proved by
the local origin of the mast cells. These may arise, as Ehrlich and his
school have always assumed, by transformation of pre-existing connective
tissue cells[29]; but that the same holds good for the eosinophil cells
as well, has nowise as yet been proved. Secondly, it is conceivable,
that isolated eosinophil cells, pre-existing in the tissues, should
rapidly multiply, and so produce the local accumulation only. Numerous
mitoses could be considered an adequate proof of this process. But so
far no figures of nuclear division have been observed; indeed A.
Schmidt, who has directed special experiments thereto from the
standpoint of his theory, has found them entirely absent.

As a =third= possibility for the local origin of the eosinophil cells,
their direct descent from neutrophil cells is conceivable, and is by
many regarded as a kind of ripening. This assumption nevertheless must
be described as unsound, since the necessary condition of its
foundation, namely the observation of corresponding transitional stages,
has not so far been fulfilled.

By the inductive method then we conclude that a local origin of the
eosinophil cells can hardly come under discussion. And this conclusion
is strengthened by comparison with the behaviour of the mast cells,
which are related to the eosinophils in many points, and only differ
from them essentially in the nature of their granulation. The mast cells
too, like the eosinophils, form a normal constituent of the bone-marrow,
and occur regularly besides in normal blood, though in very small
number--according to Canon they amount to 0.28% of the leucocytes. We
know that the mast cells are produced in large quantities locally,
wherever an over-nutrition of the connective tissue occurs, for instance
in chronic diseases of the skin, elephantiasis, brown induration of the
lungs. In the case of the mast cells, then, we see the conditions
actually realised, which the supporters of the theory of the local
origin of the eosinophil cells only assume. We should therefore expect
that an increase of mast cells in the blood or in certain inflammatory
exudations would be by no means seldom. With this point in mind Ehrlich
has subjected the sputum in emphysema and brown induration of the lungs
to exact examination for 20 years. Nevertheless he has obtained =entirely
negative= results. The special blood investigations of Canon have
likewise proved to be practically negative. In 22 healthy persons Canon
entirely failed to find the mast cells on nine occasions, in the others
he found on the average 0.47%; the highest percentage number obtained
was 0.89%. Only in a few cases of skin disease was a slight increase
indicated. The average amounted to 0.58%, a number, therefore, which is
often to be found in healthy individuals. A leucocytosis of mast cells,
comparable with the eosinophil or neutrophil forms of leucocytosis, has
not been demonstrated in the cases of Canon or other observers. On the
other hand, the mast cells undergo a considerable increase in myelogenic
leukæmia, in many cases equalling or even exceeding that of the
eosinophils. We shall not err in deriving the mast cells of the blood
solely from the bone-marrow, on the grounds of this fact; or in
conjecturing that their origin is not from the connective tissue, even
when they are there excessively increased[30].

We think we have shewn in the preceding paragraphs that the evidence, so
far brought forward for a local origin of the eosinophil cells, does not
withstand the objections that have been raised. The task now lies before
us, to produce positive proof that the accumulations of eosinophil cells
in the organs and secretions must be explained by emigration from the
blood.

This proof offers great difficulties in as much as we normally find
eosinophil cells in many places. Here then we cannot trace a process
step by step, but we have to deal with final conditions. Could we
observe the genesis of eosinophil cells in organs usually free from
them, it would be easier to clear up this question. Up to the present
but a single observation on this point is available. Michælis
established the interesting fact, that on interrupting lactation in
suckling guinea-pigs, in the course of a few days numerous eosinophil
cells collect in the mammary glands, but not in the lumen of the
canaliculi. The eosinophil cells are further polynuclear, exactly
corresponding to those of the blood, and therefore to be regarded as
immigrants. We may explain this condition according to modern views as
follows. Under certain conditions the mammary gland is capable of an
internal secretion, by means of which substances are produced that are
specifically chemiotactic for the eosinophil cells. When the external
secretion of milk is disturbed, the internal secretion is abnormally
increased. The fact too that in Michælis' researches no eosinophil cells
passed into the true secretion of the gland may be thus explained[31].

Exactly similar observations have been made on pathological material,
first recorded in the brilliant and fundamental work of Goldmann. In a
case of malignant lymphoma Goldmann found a considerable accumulation of
eosinophil cells within the tumour, and demonstrated anatomically, that
it was brought about by an emigration of the cells from the vascular
system. Hence Goldmann concluded that the eosinophil cells pass over
into the tissue in question, at the call of certain chemiotactic
products. Goldmann, and later Kauter, shewed that these eosinophil cells
were not merely due to an ordinary inflammation; for in a large number
of other diseases of the lymph glands--particularly the tuberculous,
they were entirely absent. Similarly Leredde and Perrin have shewn in
their investigations of Dühring's disease, that the eosinophil cells,
which are also present in the cutaneous tissue in large numbers, apart
from the contents of the bullæ, are due to an emigration from the
blood-stream.

Thus it is evident from a number of various facts, that the eosinophil
cells found in the tissues are not formed there, but have immigrated
from the blood-stream. It naturally often happens that this appearance
is not preserved equally distinctly in all cases. For, as has been seen
in the ordinary polynuclear leucocytes, the immigrated polynuclear
eosinophils may similarly change to mononuclear cells; they may perhaps
settle down, and approximate to the character of fixed connective tissue
cells. Such appearances may readily give rise to the view that in this
case the reverse nuclear metamorphosis has occurred; that is a
progressive development from mononuclear eosinophil to polynuclear
cells.

In agreement with Goldmann, Jadassohn and H. F. Müller, we believe that
the only admissible explanation for the facts mentioned above is that
the eosinophil cells obey specific chemiotactic stimuli. By this
hypothesis we can easily understand eosinophil leucocytosis, the
presence of eosinophil cells in exudations and secretions, and the local
accumulation of this kind of cell.

As to the nature of these chemiotactically active substances, we can so
far only surmise. From amongst the clinical phenomena capable of
throwing light on this subject we mention once more the fact, that the
metabolic products of bacteria repel the eosinophil cells.

The opposed behaviour of eosinophil and neutrophil cells is
very well illustrated by a case of Leichtenstern:

"In a very anæmic almost moribund patient with Ankylostomias
there were found 72% eosinophil cells in the blood in 1897. The
patient contracted a croupous pneumonia, and in the high
febrile period of the disease the number of eosinophils sank to
6-7%, and rose again after the termination of the pneumonia to
54%. After removal of the worm the number at once fell to 11%.
In the year 1898 the patient harboured but a very few
Ankylostomata; Charcot's crystals were no longer present in the
fæces; the number of the eosinophils amounted to 8%."

The question, what cells produce on their destruction actively
chemiotactic substances, is of very great importance; but cannot be
answered with the material at present available. The breaking up of
ordinary pus cells or lymphocytes does not appear to give rise to any
such substances; but there is much evidence that the decomposition
products of epithelial and epithelioid cells act chemiotactically. Thus
we can explain the frequent occurrence of eosinophilia in all kinds of
skin-diseases. Again, in all atrophic conditions of the gastric,
intestinal and bronchial mucous membrane there occurs a local
accumulation of eosinophil cells; further, this kind of cell is
increased in the neighbourhood of carcinoma. Additional support for this
view is seen in the fact that in bronchitis and asthma the less the
suppurative element of the secretion is developed, the more numerous are
the eosinophil cells. An observation of Jadassohn is worthy of mention
in this connection. He observed abundant eosinophil cells in foci of
lupus after injection of tuberculin. In these foci then, by the
destruction of the epithelioid cells brought about by the tuberculin,
substances must have been produced which act chemiotactically on the
eosinophil cells.

The specific substances are absorbed and reach the blood, and impart to
it also the chemiotactic power. =The direct cause then of most forms of
eosinophilia seems actually to lie in a destruction of tissue, and in
the products thus produced.=

On the other hand, it cannot be doubted that substances foreign to the
organism, circulating in the body, may act chemiotactically on the
eosinophil cells[32]. The observations quoted above, of the well-marked
eosinophilia in the different forms of Helminthiasis, may here be
specially mentioned. The action of the Helminthides was formerly
regarded as purely local, but the indications that they act also by the
production of poisonous substances continue to increase. Thus Linstow
has pointed out that the general typhoid state, and the fatty
degeneration of liver and kidneys, that is of organs which the Trichina
does not reach, necessitate the assumption of a poisonous substance. And
in several varieties of Ankylostoma as well, there is distinct evidence
of the production of a poison. We gather from Husemann's article on
"animal poisons" (Eulenberg's _Realenencyclopoedie_ 1867) that just as
Ankylostomum in man produces the well-known severe anæmia, so
Ankylostomum trigonocephalum in the dog, and Ankylostomum perniciosum
in the tiger, causes analogous general effects.

Bothriocephalus latus too is now generally accredited with the
production of a definite toxic substance; and the common tapeworm even,
by no means infrequently brings about injuries to the body which are to
be referred to the action of a poison.

So much follows from these observations, that the tapeworms can not only
absorb but also can give out substances that are absorbed from the
intestine of the host, and are able to bring about distant effects. One
expression of these distant actions is, as Leichtenstern insists, the
eosinophilia of the blood. We do not think we should assume on the
evidence before us, that the substance which attracts the eosinophil
cells is identical with the cause of the anæmia. Many observations, the
absence, for example, of eosinophilia in Bothriocephalus anæmia
(Schauman), render probable the existence of two different functions. In
any case the substance causing the eosinophilia is more widely
distributed than that to which the anæmic condition is due.

Leukæmia.

("Mixed leucocytosis.")

In spite of the enormous extent of the hæmatological observations of the
last decennia, of which a very considerable portion deals with the
problem of leukæmia, the literature shews many obscurities and
misconceptions, even on important fundamental ideas. This is especially
the case with the weighty question of the distinction between various
forms of leukæmia.

From the purely clinical standpoint it is usual to describe a lienal, a
lienomedullary, and a pure medullary (myelogenic) form of leukæmia. But
the distinguishing characteristics in this classification are crude and
purely external, and they find no place in hæmatology.

Neumann first shewed that the lymphoid proliferation in lymphatic anæmia
is not confined to the lymph glands, but may extend to the spleen and
bone-marrow. These proliferative processes may give rise to a
considerable enlargement, for example, of the spleen, without any change
in the specific character of the leukæmia, or the condition of the
blood. In spite of the splenic tumour we have to deal then with a pure
lymphatic leukæmia. In customary clinical language, a case of this kind
would be described as lieno-lymphatic leukæmia. The unreliability and
incorrectness of this terminology is best illustrated by another form of
leukæmic metastasis. In lymphatic leukæmia the liver may swell by
lymphomatous growth, to a large tumour, and we ought then to speak of a
"hepato-lymphatic" form of leukæmia. This term is by no means so
misleading as lieno-lymphatic; for no one would conclude from the former
that any liver-cells passed into the blood, whilst the latter implies
the idea, that specific splenic cells take part in the blood changes.

Further, the assumption of a pure =lienal= variety of leukæmia is totally
unwarranted from hæmatological investigations. The possibility of a
specific blood change, depending solely upon disease of the spleen,
appears _à priori_ almost excluded, after what has been said on the
physiological participation of the spleen in the formation of the blood.

Pathological data completely confirm this view. Ehrlich at least, in an
enormous number of cases, has never once succeeded in confirming the
existence of a purely splenic form from the blood examination[33].

The conditions in myelogenic leukæmia are quite similar, for foci of
myeloid tissue may appear in the spleen or lymph glands according to the
kind of metastasis. As it is the proliferation of the myeloid tissue and
not the accompanying swelling of spleen or lymph glands that is specific
in the process, the nomenclature "lienomedullary or medullary-lymphatic"
leukæmia must also be described as illogical and misleading.

We distinguish then, from the histological standpoint, but two forms of
leukæmia:

1. =leukæmic processes with proliferation of lymphoid tissue=:

"~lymphatic leukæmia~";

2. =leukæmic processes with proliferation of myeloid tissue=:

"~myelogenic leukæmia~."

The accompanying clinical phenomena may be indicated by simple
unequivocal amplifications, for instance, "lymphatic leukæmia with
enlargement of the spleen or of the liver"; "myelogenic leukæmia with
enlargement of the lymph glands," &c.

From our present knowledge, which, it may be remarked, is still far from
full, we may assume that lymphatic and myelogenous leukæmia have quite
a different ætiology. The recent discovery of Löwit should be decisive
on this point, for he demonstrated in myelogenic leukæmia the presence
of forms like plasmodia within the white blood corpuscles, but was
unable to find them in lymphatic leukæmia.

The necessity of separating lymphatic from myelogenic leukæmia is
further shewn by the fundamental clinical differences between them.

~Lymphatic leukæmia~ falls clinically into two readily distinguishable
forms. In the first place acute lymphatic leukæmia, characterised by its
rapid course, the small splenic tumour, the tendency to petechiæ and to
the general hæmorrhagic diathesis. By its startling course this disease
has given all observers the impression of an acute infectious process.

The second form of lymphatic leukæmia is marked off from the preceding
by its chronic, and often very protracted course. The spleen shews its
participation in the disease, as a rule by very considerable
enlargement. We have at present no investigations adequate to decide
whether chronic lymphatic leukæmia represents a single disease, or
should be etiologically subdivided. Hæmatologically, all lymphatic
leukæmias are characterised by a great preponderance of lymph cells, in
particular of the larger varieties. It should here be expressly
mentioned, that richness of the blood in large lymph cells, is by no
means characteristic of the acute form of leukæmia, for chronic, very
slowly progressing cases shew the same condition. Thus in a case of this
kind under observation in Gerhardt's wards, all observers (Grawitz, v.
Noorden, Ehrlich) found the large cells during its whole course. In
agreement with our remarks elsewhere (see p. 104), we assume with
regard to the origin of lymphatic leukæmia, =that the increase of the
lymph cells is brought about by a passive inflow into the blood; and not
by an active emigration from chemical stimuli=.

~Myelogenic leukæmia~ presents a picture that is different in every
particular. In former years the distinction between myelogenic leukæmia
and simple leucocytosis offered great difficulties. These conditions
were regarded as different stages of one and the same pathological
process, and when the proportion of white to red corpuscles exceeded a
certain limit (1:50) it was said that leucocytosis ceased, and leukæmia
began. By the aid of the analytic colour methods the fundamental
difference between the two conditions was first disclosed. Leucocytosis
is now recognised to be chiefly an increase of the normal polynuclear
neutrophil leucocytes; whereas myelogenic leukæmia brings elements into
the blood that are abnormal. The cells here introduced are so
characteristic as to render the diagnosis of leukæmia possible, even in
the very rare cases where the total number of the white blood corpuscles
is not to any extent increased. The best example of which we are aware
is a case observed by v. Noorden, in which the proportion of white to
red was only 1:200.

Although the blood picture of myelogenic leukæmia has been so clearly
drawn by Ehrlich, misconceptions and obscurities still occur in the
literature. And they are due to great errors in observation. It has for
instance happened that unskilled observers have regarded and worked up
cases of lymphatic leukæmia as myelogenic. The apparent deviations
discovered in this manner are copied, as specially remarkable, from one
book to another. Through insufficient mastery of the staining method,
the characteristic and diagnostically decisive elements (neutrophil
myelocytes for example) are frequently mistaken. A further source
productive of misconceptions lies in the circumstance that the typical
leukæmic condition of the blood may essentially change under the
influences of intercurrent diseases. Thus the intrusion of a
leucocytosis, brought about by secondary infection, is able to
obliterate more or less the specific character of the blood. Such
conditions must naturally be considered apart, and should not be used to
overthrow the general characteristics of the picture. No one surely
would deny the diagnostic value of glycosuria for diabetes, because in
conditions of inanition, for instance, the sugar of a diabetic may
completely vanish, although the disease continues. And one does not deny
the diagnostic value of the splenic tumour in typhoid fever, because the
enlargement of the spleen may occasionally subside, under the influence
of an intestinal hæmorrhage.

From these considerations it is obviously necessary to derive the
description of leukæmic blood from pure uncomplicated cases; and to
construct it with the aid of standard methods. In this manner a type is
obtained so characteristic, as to render diagnosis absolutely certain
from the blood alone.

It is needful here to emphasise this hundred-fold repeated experience
with special distinctness, for some recent authors do not even yet allow
the full diagnostic importance of the blood examination. v. Limbeck says
in the latest edition of his clinical _Pathology of the Blood_, "That
one should not regard the blood changes as an invariably reliable
diagnostic resource in myelogenic leukæmia; and that the diagnosis of
leukæmia should not rest on the presence or significance of one or more
cells. Not only the general features of the case, but the blood
condition as well should be considered." To these remarks the objection
must be made that up to the present no serious hæmatologist will have
had to diagnose a leukæmic disease principally "from the presence of one
or more cells." In the work of Ehrlich and his pupils at least, it has
always been shewn that the character of a leukæmic 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. With these premises it is indisputable =that
the microscopic examination of the blood alone on dry preparations,
without the assistance of any other clinical method, can decide whether
a patient suffers from leukæmia, and whether it belongs to the lymphatic
or myelogenic variety=.

The microscopic picture of =myelogenic leukæmia=, disregarding the almost
constant increase of the white blood corpuscles, has a varied, highly
inconstant character. This arises from the co-operation of several
anomalies, namely:

A. =that in addition to the polynuclear cells, their early stages, the
mononuclear granulated corpuscles likewise circulate in the blood=;

B. =that all three types of granulated cells, the neutrophil, eosinophil,
and mast cells participate in the increase of the white blood
corpuscles=;

C. =that atypical cell-forms appear=, _e.g._ =dwarf forms of all the kinds
of white corpuscles; and further mitotic nuclear figures=;

D. =that the blood always contains nucleated red blood corpuscles, often
in great numbers=.

1. We begin with the discussion of the =mononuclear neutrophil cells=,
Ehrlich's "=myelocytes=." They are present so abundantly in the blood of
medullary leukæmia as to impart to the whole picture a predominantly
mononuclear character. As we have frequently mentioned, myelocytes occur
normally only in the bone-marrow, not in the circulating blood. Their
eminent importance for the diagnosis of myelogenic leukæmia, where they
have been regularly found by the best observers, is in no way diminished
by their transitory appearance in a few other conditions (see pages 77,
78). Though they have been occasionally found, according to Türk's
investigations, in the critical period of pneumonia as parts of a
general leucocytosis, the danger of confusion with leukæmic blood
changes is non-existent. This is guarded against by (1) the much smaller
increase of the white cells; (2) the diminution of the eosinophil and
mast cells; (3) the fact, that the myelocytes of leukæmic blood are
nearly always considerably larger; (4) the preponderating polynuclear
character of the leucocytosis, which is not effaced by the small
percentage amount of myelocytes (at most 12%): (5) the incomparably
smaller absolute number of myelocytes. In the most pronounced case of
Türk's, for example, in which the percentage number of myelocytes
amounted to 11.9, calculation of their absolute number gives at most
1000 myelocytes per mm.^{3} This is a figure which bears no comparison
with that obtaining in leukæmia, where 50,000-100,000 myelocytes per
mm.^{3} and over occur in cases that are in no way extreme.

2. =The mononuclear eosinophil cells.= Before the introduction of the
staining method, Mosler had described large, coarsely granulated cells,
"marrow cells," as characteristic for myelogenic leukæmia. These are to
be regarded as for the most part identical with the mononuclear
eosinophil cells, noticed by Müller and Rieder as peculiar, and aptly
described by them as the eosinophil analogues of the preceding group.
They appear as large elements with oval, feebly staining nucleus.
Undeniably a valuable sign of leukæmia, they are not nearly so important
as the mononuclear neutrophil cells, as follows from the numerical
superiority of the latter. To regard the presence of "eosinophil
myelocytes" as absolute proof of the existence of a leukæmia is
inadmissible, since they are occasionally present in small numbers in
other diseases.

3. =The absolute increase of the eosinophil cells.= In his first paper on
leukæmia, Ehrlich stated that the absolute number of polynuclear
eosinophils is always much increased in myelogenic leukæmia. This
assertion of Ehrlich has been received under some protest; v. Limbeck in
his text-book even speaks of an "alleged" increase of the eosinophil
cells. The well-known work of Müller and Rieder has more particularly
given rise to this opposition, and thrown doubt on the diagnostic
importance of the eosinophil cells. These authors however base their
contradiction on false premises.

For Ehrlich did not speak of a rise of the percentage of the eosinophil
cells, but only of an increase in their absolute number. If in a case of
leukæmia only the normal percentage number of eosinophils is found, it
indicates, all the same, a great absolute increase; and Müller and
Rieder would themselves have fully confirmed Ehrlich's statement, had
they only calculated the absolute figures in a few of their cases.
Selecting from the seven cases in this paper, those where it is possible
from the given data to obtain the absolute number of the eosinophil
cells, we get the following results:

Case 29 3.5% eos. 14,000 per mm.^{3}
" 30 3.9% " 8,000 "
" 31 3.4% " 11,000 "

The figure given by Zappert as a high normal value is 250. In these
cases there is an average number of 11,000, that is 50 times as great.
The observations then of Müller and Rieder themselves suffice fully to
confirm Ehrlich's statement.

The absolute number of eosinophil cells depends naturally to a certain
extent on the relative proportion of white to red corpuscles, and the
greater the relative number of leucocytes, the greater should be the
number of eosinophils. Zappert, for instance, found the following
figures in his cases:

Proportion of white Absolute number
to red corpuscles. of eosinophils.

1:24 3,000-4,560
1:18 3,300
1:15 7,000
1:13 8,700
1:11 6,000
1:7.6 8,300
1:7.0 7,600
1:7.0 29,000
1:5.0 14,000
1:3.8 34,000.

Apart from the approximate parallelism between the two rows of figures,
this abstract shews that the minimal value--3,000 eosinophils with a
proportion of white to red of 1:24--still amounts to 15 times the
normal. The maximal figure found by Zappert of 30,000 is moreover by no
means to be considered extreme. Cases of leukæmia are not infrequent in
which we find 100,000 eosinophils per mm.^{3} and over.

From these figures it must be admitted that the absolute increase of the
eosinophil cells in medullary leukæmia is not "alleged" (v. Limbeck) but
on the contrary is very real and considerable.

That the absolute and relative number of eosinophil cells may markedly
sink in certain complications of leukæmia, constitutes no exception to
the law that the eosinophil cells are increased in myelogenic leukæmia.
In this connexion the self-evident principle must be observed, that only
analogous conditions are comparable. The standard of comparison for a
leukæmic patient suffering from severe sepsis is not the blood of a
healthy person with normal numerical proportions, but that of a patient
similarly attacked by a severe sepsis. Now we know that in sepsis the
number of eosinophil cells is enormously diminished, so that Zappert, in
five cases of this nature, was unable to recognise any eosinophils in
the blood. In contrast to this stands a case of myelogenic leukæmia
described by Rieder and Müller, complicated by a severe and lethally
ending suppurative process. In consequence of the acute neutrophil
leucocytosis brought about by the septic infection, the number of
eosinophils sank rapidly from 3.5% to 0.43% (4 hours before death). The
absolute number of eosinophil cells however in this terminal stage still
amounted to 1400-1500 per mm.^{3}, and was therefore, in comparison with
an uncomplicated sepsis, very much raised. Writers should not have
disputed the importance of the eosinophil cells for the diagnosis of
leukæmia from cases like these; on the contrary they should have seen in
them a decisive confirmation of the constancy of the absolute increase
of the eosinophils in leukæmic blood.

At the time when Ehrlich formulated his proposition on the diagnostic
importance of the eosinophil cells in leukæmia, the simple eosinophil
leucocytosis (see p. 148), first discovered later by the investigation
of asthma etc., was unknown. For no confusion can arise between
leukæmia, and conditions accompanied by eosinophilia, as they can be
distinguished on clinical grounds alone. The blood moreover provides
ample means for a differential diagnosis: (1) the total increase of the
white cells in this case seldom reaches degrees that remind one of
leukæmia; (2) the eosinophil cells are exclusively polynuclear; (3) mast
cells and neutrophil myelocytes are almost entirely absent.

In favour of the diagnostic value of the absolute increase of the
eosinophil cells are those cases too, where with a blood condition
closely recalling leukæmia, the absence of eosinophil cells excludes the
diagnosis of that disease. In a case of carcinoma of the bone-marrow,
described by Epstein, with an anæmic constitution of the blood (nearly
always present it may be mentioned in leukæmia), there was found a
marked increase of the white blood corpuscles, numerous neutrophil
myelocytes and nucleated red corpuscles. Anyone holding, as Müller and
Rieder do, that the number of eosinophil cells need not be considered in
the diagnosis, must in this case have diagnosed myelogenic leukæmia.
This however was according to Ehrlich's system impossible owing to the
complete absence of eosinophil cells.

From all these observations it follows that an absolute increase of
eosinophil cells is indispensable for the diagnosis of leukæmia.

4. =The absolute increase of the mast cells.= The mast cells are always
increased in myelogenic leukæmia. They may be counted in leukæmic blood
with the aid of the triacid or eosine-methylene blue stain. As shewn by
the former they appear as polynuclear cells free from granules, since
their granulation takes on no dye of the triacid mixture.

In all cases of myelogenic leukæmia the increase of mast cells is
absolute and considerable. Generally they are equally or half as
numerous as the eosinophils, occasionally they may exceed the latter in
number. Hence it follows that the mast cells undergo an increase in
number relatively greater than the eosinophil cells, for they normally
amount only to some 0.28%. They are perhaps of greater diagnostic value
than the eosinophils, because up to the present time we know of no other
condition (in contradistinction to eosinophil leucocytosis) in which a
marked increase of the mast cells occurs.

5. =Atypical forms of the white corpuscles.= Amongst these are to be
mentioned: (_a_) dwarf forms of the polynuclear neutrophils and of the
eosinophil elements respectively. As a rule they resemble normal
polynuclear cells on a small scale. (_b_) Dwarf forms of the mononuclear
neutrophil and eosinophil leucocytes, which correspond to the
pseudo-lymphocytes described elsewhere (see p. 78). The importance of
these dwarf forms for leukæmia is as yet insufficiently explained; and
it is difficult to decide whether they are already small forms on
reaching the blood-stream, or whether they are there produced by
division of a large cell. (_c_) =Cells with mitoses=. Formerly particular
weight was laid on the observation of mitoses, for they were regarded as
evidence that the increase of white blood corpuscles was brought about
in the circulating blood itself, an assumption specially supported by
Löwit.

A large number of authors (H. F. Müller, Wertheim, Rieder) have
demonstrated mitoses, particularly of the myelocytes, in the circulating
blood in leukæmia. No =diagnostic= importance of any kind can however be
ascribed to them. They are found in all cases only in very small
numbers. Thus Müller says that he generally must look through many
thousands of white cells before meeting one mitosis. Only in one case
did he find the figures of nuclear division somewhat more abundant,
where there was one mitosis only to several hundreds of leucocytes.

These really negative observations shew that the mitoses play a
completely negligeable part in the increase of the cells in the blood
itself. For the diagnosis of leukæmia they are valueless.

6. =Nucleated red corpuscles= form a constant constituent of leukæmic
blood. In different cases their number is very varying; in one case they
occur extremely sparingly, in another every field contains very many.
The normoblastic type is found most frequently, but side by side with
it, megaloblasts and forms transitional between the two are occasionally
found. Mitoses within the red blood discs have been described by
different authors, but possess no theoretic or clinical importance. The
appearance of erythroblasts in leukæmia may be either a specific
phenomenon, or merely the expression of an anæmia accompanying the
leukæmia. We are inclined to the first supposition, since the occurrence
in such numbers of nucleated red cells is hardly ever observed in other
anæmias of the same severity.

So much for the characteristics of leukæmic blood, upon which the
diagnosis of the disease is made. We must add that although in any case
of medullary leukæmia each particular factor described is to be
recognised, yet the manner of its appearance, its numerical relation to
the others and to the total blood varies extremely. Apart from the
degree of increase of the leucocytes, no one case is the same as another
with regard to the other anomalies. In one case the blood bears a
large-celled, mononuclear neutrophil character; in another the increase
of the eosinophil cells predominates; in a third the nucleated red blood
corpuscles preponderate; in a fourth we see a flooding of the blood with
mast cells. And hence results a multiplicity of combinations, and each
single case has its own individual features[34].

* * * * *

It is of special importance to study the changes due to certain
intercurrent diseases in the blood picture of medullary leukæmia. This
point has recently been the object of detailed investigation, in
particular by A. Fraenkel, Lichtheim and others[35]. According to these
authors, under the influence of febrile diseases the total number of
leucocytes may be enormously decreased. The blood moreover is altered,
so that the myelæmic characteristics become less marked, and the
polynuclear neutrophil elements largely preponderate. The latter may
attain the percentage numbers of common leucocytosis up to 90% and
over.

We will here mention a few rare cases, demanding special attention,
shewing the alterations leukæmic blood may undergo, and occasionally
presenting almost insuperable difficulties in diagnosis. We find but a
single case of this kind mentioned in the literature. Zappert reported a
patient, who in February, 1892, had shewn the typical signs of
myelogenic leukæmia. Amongst others the relation of white to red cells
was found to be 1:4.92, and 1400 eosinophil cells per mm.^{3} (3.4%)
were counted. At the end of September of the same year the patient was
brought in a miserable condition to the hospital, where she soon died
with gradually failing strength. During this period of observation the
proportion of white to red was 1:1.5; the percentage of eosinophils,
0.43; the mononuclears, most of which had no neutrophil granulation,
amounted to 70% of the leucocytes. Zappert expressly mentions that these
mononuclear cells were in no way similar to the lymphocytes in general
appearance. At the autopsy Zappert found the bone-marrow studded with
non-granulated mononuclear cells, and the eosinophil cells were much
more scanty than is usually the case in leukæmic bone-marrow.
Blachstein, under Ehrlich's direction, investigated a second case of
this kind. This patient had also been the subject of exact clinical
investigations for some time on account of a myelogenic leukæmia. During
the time he was last in hospital the blood could only be examined a day
before the fatal termination, the direct consequence of a septic
complication. With a markedly leukæmic constitution of the blood there
were found 62% polynuclear cells, 17.5% mononuclear about the size of
the ordinary myelocyte, 0.75% eosinophil cells, nucleated red blood
corpuscles in moderate amount. The preponderance of polynuclear and the
small number of eosinophil cells is readily explainable from the septic
infection; on the other hand the absence of granules in the mononuclear
cells is most surprising.

These two observations can only be interpreted by assuming a loss, in
certain terminal stages, on the part of the organism, of its power of
forming neutrophil substances. Similar conditions occur in non-leukæmic
conditions; for example in a striking case of posthæmorrhagic anæmia
described by Ehrlich. It is of great importance to direct attention to
these cases, which up to the present have been practically
disregarded--for ignorance of their occurrence may easily give rise to
gross errors concerning the nature and origin of the mononuclear cells,
and to the manufacture of a lienal form of leukæmia.

* * * * *

Finally we have to discuss the important question, how ~the origin of
myelæmic blood~ is to be explained. According to our conceptions two
possibilities come under consideration. Either we have to deal with a
passive inflow of bone-marrow elements, or with an active emigration
from the bone-marrow into the circulation. This important and difficult
question is certainly not fully ripe for discussion. The most weighty
objection to be raised against an active emigration of the bone-marrow
cells is derived from the behaviour of the white blood corpuscles on the
warm microscopic stage. These investigations have been performed by a
number of authors of whom may be mentioned Biesiadecki, Neumann, Hayem,
Löwit, Mayet, Gilbert, and particularly H. F. Müller on the ground of
his summary of this subject. Concerning the behaviour of the forms of
cell here involved, all authors are agreed that under no conditions do
the lymphocytes shew the smallest spontaneous movement; whilst the
polynuclear neutrophil cells always exhibit vigorous contractility. With
regard to the forms most characteristic of leukæmic blood the statements
are partially contradictory. Some authors deny all spontaneous movement
of these cells; but most of them report observations from which it
follows that a certain power of spontaneous movement is not to be
gainsaid. It will be admitted that in questions of this kind, negative
results are weakened by positive data. Thus Jolly recently described
similar observations as follows: "C'étaient des changements de forme sur
place, lents et peu considérables, formations de bosselures à grands
rayons, passage d'une forme arrondie à une forme ovulaire ou bilobée
etc. Ces mouvements étaient visibles dans les observations I et IV et
appartenaient surtout à des globules de grande taille." It is naturally
impossible to decide if these minute movements suffice for a spontaneous
locomotion. But one cannot exclude off-hand the supposition that they
do. It is indeed supported by a further observation of Jolly on the
mononuclear eosinophil cells of the marrow. Hitherto it was taken for
established that these cells are completely devoid of spontaneous
movement. Jolly however was recently able to examine a specimen from a
case of typical leukæmia, =in which nearly all the eosinophil cells
shewed active movement=. He says: "Ces globules granuleux actifs
présentaient des mouvements de progression et des changements de forme
caractéristiques et rapides; cependant je n'ai pas vu ces globules
présenter de pseudopodes effilés; de plus, leurs contours restaient
presque toujours assez nettement arrêtés. Ces particularités
correspondent exactement à la description, qu'a donnée depuis longtemps
Max Schultze des mouvements des cellules granuleuses du sang normal."
Examination of dry specimens from the same case shewed, as Jolly
expressly mentioned, that the blood contained, as leukæmic blood always
does, polynuclear and mononuclear eosinophil cells. In contrast then
with all earlier observations, Jolly has demonstrated an active
spontaneous movement of the mononuclear eosinophil cells. The amoeboid
movement of the mononuclear cells is so seldom seen, not because they
lack this function, but obviously from defects in the methods of
investigation, which as is manifest are rather rough and wholly unsuited
for delicate biological processes. There are many instances in the
literature of the failures of this method, even in the case of cells
with undisputed mobility. Thus Rieder failed to observe any
contractility in the majority of polynuclear leucocytes in a case of
malignant lymphoma, whereas according to all other observations they
possess this property without exception.

We think then we must draw the conclusion that the feeble mobility of
the mononuclear cells, both eosinophilous and polynuclear, is only
apparent, and is owing to the gross method of investigation. In reality
they doubtless have mobility sufficient for emigration.

A further, but much less weighty objection to the view that myelogenic
leukæmia is an active leucocytosis is, that pus artificially produced in
leukæmic patients has nearly always the histological constitution of
normal pus. But from our previous detailed remarks we should only expect
a myelæmic constitution of the pus, if the specific morbid agent of
leukæmia were present in a concentrated form at the place of
inflammation. Just as we saw in pemphigus, Neusser's eosinophilous
suppuration occurred only in the specific pemphigus bullæ, but not in
the foci of suppuration that were artificially produced. We know that
the myelocytes are in no way positively influenced by the chemiotactic
stimuli of ordinary infectious agents. On the contrary, it clearly
follows from the above-mentioned observations on the transformation of
leukæmic blood under the influence of infectious diseases, that the
common bacterial poisons act in a negatively chemiotactic sense, both on
the eosinophil and on the neutrophil mononuclear cells. Under these
circumstances we should indeed expect that artificially produced
suppuration in leukæmic patients would have, not a myelæmic, but a
polynuclear neutrophil character.

It will be the task of further investigations to examine accurately
inflammatory products, _e.g._ pleuritic exudations, in leukæmic
patients, with the object of elucidating the question, whether under
special conditions of disease all the leucocytes characteristic for
leukæmia may not be able to wander from the blood. Thus in a case of
pleurisy in a leukæmic patient, Ehrlich received the impression from the
preparations that a "myeloid" emigration had in fact occurred, carrying
all the elements in the blood into the exudation. This observation does
not prove the point, for numerical estimation of the proportion of white
to red blood corpuscles in the exudation was not made. And these
estimations are necessary in order to prove indisputably the active
emigration of the white blood corpuscles into the exudation, and to
exclude their purely mechanical passage, _per rhexin_, from the
blood-stream.

The hypothesis of the active origin of myelæmia is considerably
supported by a further train of argument. In leukæmia, besides the
myelocytes, the polynuclear leucocytes are also enormously increased,
and their active emigration is beyond doubt. And the view, that the
mononuclear cells are washed into the blood, excludes that of a single
mode of origin of the leukæmic blood condition; and commits us to a
highly artificial explanation of its production.

The morphological changes of leukæmic blood under the influence of
infectious diseases can only be explained from the standpoint of the
emigration theory. For if the white blood corpuscles were mechanically
carried out of the bone-marrow as a whole, it is incomprehensible that a
bacterial infection should alter this process to a polynuclear
leucocytosis. This change of character is easily explained on the other
hand, as we have above shewn more in detail, by the assumption that
ordinary bacterial poisons act positively chemiotactically only on the
polynuclear neutrophil cells, but negatively on the other forms.

=We explain the origin of leukæmic blood by the emigration into the blood
under the influence of the specific leukæmic agent, not only of the
formed polynuclear elements, but also of their mononuclear, eosinophil
and neutrophil early stages; and to classify myelogenic leukæmia with
the active leucocytoses.=

FOOTNOTES:

[25] Naturally an ordinary leucocytosis may be combined with a
lymphæmia. We have already mentioned elsewhere (see page 102) that in
the leucocytosis of digestion or of diseases of the intestine in
children, such a coincidence occurs.

[26] The so-called agony leucocytosis we do not regard as a true
leucocytosis, but only as the expression of a stoppage of the
circulation caused by that condition. This produces an accumulation of
the white corpuscles on the vessel walls, especially in the peripheral
parts of the body which are as a rule used for clinical investigation. A
leucocytosis is thus simulated.

[27] It is also of interest to notice the behaviour of the eosinophil
cells in the passive form of leucocytosis, lymphæmia. _À priori_ both
conditions could be combined. As C. S. Engel has established in the
congenital syphilis of children a simultaneous marked increase of
lymphocytes and eosinophil cells is found. The lymphocytosis in these
cases is probably due to the anatomical changes of the lymph glands, and
the eosinophilia to specific chemiotactic attraction.

[28] In his monograph on Bothriocephalus anæmia Schauman, with reference
to the behaviour of the eosinophil cells, states that he has found them
in but few cases of this disease.

[29] This view has lately received striking confirmation from the
interesting experiment of Bäumer, who produced on himself by means of
continued stimulation with _Urticaria ureus_ a considerable increase in
four days of the mast cells in the irritated portions of the skin.

[30] That a well-marked basophil leucocytosis has not so far been
observed may be thus explained. The substances which attract the mast
cells are very rarely produced in the body; much more seldom than the
corresponding substances attractive for the eosinophils. In morbid
conditions, where substances attracting the mast cells were present, it
might be possible to find a suppuration of mast cells, or a mast cell
leucocytosis as well. In this connection an observation of Albert
Neisser is of the greatest interest. He met with (private communication)
one, out of numberless cases of gonorrhoea, in which the purulent
secretion consisted entirely of mast cells.

[31] Unger has recently published completely analogous observations on
the human breast for the mast cells. Under the influence of stagnation
of the milk he saw an invasion of the gland tissue by typical mast
cells.

[32] A very interesting observation of Goldmann's deserves mention here.
Goldmann found in preparations of the pancreas of proteus sanguineus,
containing parasites, that the eosinophil cells in the neighbourhood of
the encapsuled parasites were much increased, whereas they were sought
for in vain, in more distant parts.

[33] A case observed some time back by Ehrlich may here be mentioned as
a characteristic example. A woman received a blow in the region of the
spleen by a fall from the roof, which gradually led to a marked splenic
enlargement. As no other symptoms appeared, the surgeon in charge
proposed splenectomy, on the assumption of a pure splenic leukæmia.
Examination of the blood, however, shewed a condition fully
corresponding with myelogenic leukæmia, and thus prevented surgical
interference.

[34] Ehrlich was once able to recognise, by balancing the different
forms of cells, the blood preparations after the loss of their labels
from some ten cases of leukæmia.

[35] Literature given by A. Fraenkel.

V. DIMINUTION OF THE WHITE BLOOD CORPUSCLES (LEUKOPENIA).

Diminution of the white blood corpuscles plays--comparatively with their
increase--a very unimportant _rôle_ in clinical observations. It occurs
in but few groups of diseases, and but seldom attains a marked degree.
Koblanck has described a most marked fall in the number of the
colourless cells, in the following remarkable blood condition. In a
strong man, 25 years of age, whose internal organs were found to be
healthy, short epileptiform attacks occurred, in one of which death took
place. The autopsy gave no indication of the cause of death. Two
examinations of the blood were made in the course of the three days he
was under observation. In one of these, out of =ten cover-glass
preparations, not a single white blood corpuscle was found=, and in the
second only one example.

We have mentioned this case here, because it is remarkable as an extreme
leukopenia never before observed. An explanation however is impossible
owing to the obscurity of the general clinical condition.

For the rest the conditions, under which a considerable diminution of
the leucocytes occurs, are very well-known. We distinguish two chief
groups:

1. Leukopenia from =destruction of a portion of the white blood
corpuscles= (Löwit);

2. Leukopenia from =deficient inflow of white corpuscles=:

[alpha]. in infectious diseases from =negative chemiotaxis=;

[beta]. in anæmia etc. from =defective action of the bone-marrow=.

We have entered more fully into the leukopenia experimentally produced
by Löwit, in the chapter on leucocytosis. We there explained, that
according to present views, we have to deal, not with an actual
destruction of the white elements, but merely with an altered
distribution within the blood-stream.

Amongst the infectious diseases where an hypoleucocytosis occurs,
typhoid fever must first be mentioned. The diminution is chiefly at the
expense of the polynuclear cells. Uncomplicated measles too, generally
runs its course with a marked leukopenia, specially distinct during the
breaking out and at the height of the exanthem. These cases of
infectious leukopenia are to be explained, not by a destruction of white
corpuscles, but rather by a diminished inflow, brought about by the
circulation of substances negatively chemiotactic for the polynuclear
elements.

Leukopenia has still another meaning in certain cases of severe anæmia,
where it indicates a highly unfavourable prognosis. Ehrlich has
described (_Charité Annalen_ 1888) a case of posthæmorrhagic anæmia
ending fatally, where an extreme diminution of the leucocytes occurred.
Exact figures shewed that the greater proportion (80%) of white blood
corpuscles consisted of lymphocytes, whilst the polynuclears amounted to
14% (instead of 70-72% normally). Eosinophil cells and nucleated red
blood corpuscles were entirely absent. Ehrlich explained these phenomena
by a deficient activity of the bone-marrow, which found expression in
the insufficient formation of red and white blood corpuscles. As the
anatomical basis of this deficient activity, he conjectured that in this
case the fatty marrow of the big long bones could not have been changed
to blood forming red marrow, as is the rule in severe anæmias. In two
cases the autopsy fully confirmed this diagnosis made during life.

The blood platelets.--The hæmoconiæ.

The ~blood-platelets~ were first described by Hayem, later by Bizzozero,
as a third formed element of normal blood. They are roundish or oval
discs free from hæmoglobin. They are extremely unstable under
mechanical, thermal, and chemical influences. Their size amounts to some
3 µ. Specially characteristic is their tendency, the result of their
extraordinary stickiness, to run together into largish clumps, "grape
clusters." This circumstance greatly facilitates the distinction of the
blood platelets from the other formed elements, but renders their
enumeration most difficult. The apparatus usually used for counting the
blood corpuscles is, for this reason, deceptive; for the platelets
rapidly cling to its walls and remain there. All early authors (_e.g._
Bizzozero) endeavoured to obviate this error by some particular diluting
fluid; but a number of these elements still remained fastened to the
walls of the capillary tube of the mixing apparatus.

Recently Brodie and Russell have recommended a new mixture in which the
platelets remain quite isolated, and are stained at the same time. They
allow the drop of blood as it comes from the puncture to enter a drop of
the fluid, and then estimate the relative proportion of red blood
corpuscles to platelets[36]. The prescription for their solution is as
follows:

Dahlia-glycerin,
2% solution of common salt ... equal parts.

Another method, used by the majority of more recent authors, is the
relative enumeration of blood platelets in the stain dry specimen.
Ehrlich found that the blood platelets were picked out by their deep red
colour, corresponding to the amount of alkali they contain, in
preparations treated by the iodine eosine method (see p. 46). Rabl's new
method is much more complicated and in no way more serviceable,
depending on a stain with iron hæmatoxylin recommended by E. Haidenhain
for demonstration of the centrosomes. A process of Rosin's, not yet
published, is more convenient. It consists in fixing the dry preparation
for 20 minutes in osmic acid vapour, and staining in a concentrated
watery solution of methylene blue.

* * * * *

With regard to the significance of the blood platelets, most authors, of
whom we should before all mention Hayem, Bizzozero, Laker, assume
justifiably that they are preformed in the living blood. The view
opposed to this, advocated more particularly by Löwit, that these forms
first arise in the blood after it has left the vessels, we may describe
on the grounds of our own extensive observations as inaccurate.

The blood platelets, on the grounds of their small size and complete
lack of nuclear substance, are generally regarded as not analogous to
real cells. Whether they represent intravital precipitation of
substances of the plasma, or whether they are budded off from the cells,
cannot at the present be decided with certainty, though many facts seem
to support the latter assumption. That they contain glycogen (see p.
45), marks them as descendants of the blood cells. Moreover, appearances
are often met with in dry preparations that arouse the suspicion that
the platelets arise from the red blood corpuscles (Koeppe). Arnold has
further observed processes of budding in the red blood corpuscles not
only extravascularly but also intravascularly in the mesentery of young
guinea-pigs, and has seen the elements that were cut off change into
forms free from hæmoglobin.

Our knowledge too of the physiological function of the blood platelets
still needs much amplification. The original view of Hayem, who regards
the blood platelets as early stages of the red blood discs, and for this
reason calls them "hæmatoblasts," is, according to the judgment of most
hæmatologists, untenable.

Nearly all more recent papers, on the other hand (cp. Löwit's
compilation), recognise the =close connection of the blood platelets with
coagulation=, first observed by Bizzozero. Whether the substance of the
platelets directly yields the material for fibrin formation, as
Bizzozero holds, or whether according to the observations on thrombus
production of Eberth and Schimmelbusch they play but a subordinate part,
is not yet decided. To enter here into the chemical side of this
complicated problem, would lead us much too far, and we will only refer
to a few clinical observations which illustrate the relations between
the clotting power of the blood and the number of platelets it contains.

Marked =increase of the blood platelets= occurs in chlorosis (Muir) and in
posthæmorrhagic anæmia (Hayem). In both conditions there is a decided
=increase in the clotting power of the blood=. In contrast, is the
important observation of Denys, who found in two cases of purpura, where
as is well-known the =clotting power of the blood is always much lowered=
or may even be entirely destroyed, only one morphological blood change,
a very marked =diminution of the blood platelets=. Ehrlich likewise had
occasion to examine a similar case, in which the blood platelets were
entirely absent.

* * * * *

H. F. Müller has described a fourth formed constituent of the blood, and
given it the name of "=hæmoconiæ=" or "=blood atoms=," "blood dust." It is
found in the plasma of the blood as very small granule- or coccæ-like
colourless corpuscles, highly refractile, with a very active molecular
movement, which keep their shape under observation for a very long time
without any special precautions. According to Müller these bodies are
not blackened by osmic acid, and probably contain no fat; they seem to
have no connection with fibrin formation, as they always lie outside
the fibrin network. Müller found them in every normal blood, in varying
numbers however; much increased in a case of Morbus Addisonii;
diminished in hunger and cachexias.

More detailed observations are necessary to determine the chemical
nature of these forms. Experiments in this direction by extraction with
ether, or by the use of fat staining substances, alkanna, Soudan dye,
and comparative investigations on lipæmic blood should be successful.

FOOTNOTES:

[36] The physiological figures found by Brodie and Russell with the aid
of this method exceed considerably those of earlier authors. They found
a proportion of platelets to erythrocytes of 1:85 or an absolute number
of about 635,000 per mm.{^3}

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---- Über Bothriocephalus-Anæmie, und die prognostische Bedeutung der
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~Barker.~ On the presence of iron in the granules of the eosinophil
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---- Über Hydræmie. _Zeitschr. f. klin. Med._ 1892, vol. XXI.

---- Über Blutbefunde bei Chlorose. _Wiener Med. Presse_, 1894, no. 27.

~Hankin, E. H.~ Über den Ursprung und das Vorkommen von Alexinen im
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---- ~und Richter, P. F.~ Über den Einfluss von Fieber und Leucocytose auf
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~v. Noorden.~ Untersuchungen über schwere Anæmie. _Charité Ann._ 1889,
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1880.

---- Über den Fehler der Blutkörperchenbildung bei der perniciösen
Anæmie. _Virchow's Archiv_, 1890, vol. CXXI.

~v. Roietzky.~ Contributions à l'étude de la fonction hæmatopoïétique de
moëlle osseuse. _Arch. des sciences biol. Pétersbourg_, 1877. T. V.

~Sadler.~ Klinische Untersuchungen über die Zahl der corpusculären
Elemente und den Hæmoglobingehalt des Blutes. (Quoted by ~Türk~.)
_Fortschr. d. Med._ 1892.

~Schauman.~ Zur Kenntniss der sogenannten Bothriocephalus-Anæmie.
_Berlin_, 1894.

---- und Rosenquist. Zur Frage über die Einwirkung des Höhenklimas auf
die Blutbeschaffenheit (Prelim. Comm.). _Congr. f. inn. Med._ 1896, no.
22.

~Schiff.~ Über das quantitative Verhalten der Blutkörperchen und des
Hæmoglobins bei neugeborenen Kindern und Säuglingen unter normalen und
pathologischen Verhältnissen. _Zeitschr. f. Heilkunde_, 1890, vol. II.

~Schimmelbusch.~ Die Blutplättchen und die Blutgerinnung. _Virchow's
Archiv_, 1885, vol. CI.

~Schmaltz.~ Die Untersuchung des specifischen Gewichtes des menschlichen
Blutes. _Deutsch. Arch. f. klin. Med._ 1891, vol. XLVII.

---- Die Pathologie des Blutes und der Blutkrankheiten. _Leipzig_, 1896.

~Schmidt, A.~ Demonstration mikroscopischer Präparate zur Pathologie des
Asthma. _Congr. f. inn. Med._

~Schultze, Max.~ Ein heizbarer Objecttisch und seine Verwendung bei
Untersuchung des Blutes. _Arch. f. mikr. Anat._ 1865, vol. I.

~Schultze, O.~ Die vitale Methylenblaureaction der Zellgranula. _Anatom._
_Anzeiger_, 1887.

~Schumburg and Zuntz, N.~ Zur Kenntniss der Einwirkungen des Hochgebirges
auf den menschlichen Organismus. _Pflüger's Archiv_, 1896, vol. LXIII.

~Seige.~ Über einen Fall von Ankylostomiasis. _Inaug. Dissert._ _Berlin_,
1892.

~Spilling.~ See ~Ehrlich~, Farbenanalytische Untersuchungen.

~Stiénon.~ Recherches sur la leucocytose dans la Pneumonie aigue.
_Bruxelles_, 1895.

---- De la leucocytose dans les maladies infectueuses. _Bruxelles_,
1896.

~Stierlin.~ Blutkörperchenzählung und Hæmoglobinbestimmung bei Kindern.
_Deutsches Arch. f. klin. Med._ 1889, vol. XLV.

~Stintzing und Gumprecht.~ Wassergehalt und Trockensubstanz des Blutes
beim gesunden und kranken Menschen. _Deutsches Arch. f. klin. Med._
1894, vol. XLIII.

~Tarchanoff, J. R.~ Die Bestimmung der Blutmenge am lebenden Menschen.
_Pflüger's Archiv_, vols. XXIII, XXIV.

~Teichmann.~ Mikroscopische Beiträge zur Lehre von der Fettresorption.
_Inaug. Dissert._ _Breslau_, 1891.

~Thoma und Lyon.~ Über die Methode der Blutzählung. _Virchow's Archiv_,
vol. LXXXIV.

~Troje.~ Über Leukæmie und Pseudoleukæmie. _Berl. klin. Woch._ 1892, no.
12.

~Tschistowitsch.~ Sur la quantité des leucocytes du sang dans les
pneumonies fibrineuses à issue mortelle. Review: _Centralbl. f. d. Med.
Wissensch._ 1894, no. 39.

~Türk.~ Klinische Untersuchungen über das Verhalten des Blutes bei acuten
Infectionskrankheiten. _Wien u. Leipzig_, 1898.

~Unger.~ Das Colostrum. _Virchow's Archiv_, vol. CLI. 1898.

~Unna.~ Über mucinartige Bestandtheile der Neurofibrome und des
Centralnervensystems. _Monatshefte f. prakt. Dermatologie_, 1894, vol.
XVIII.

~Uskoff~, and the papers of his pupils. See _Archiv des sciences
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_Compt. rend. d. l'Acad. des Sciences_, III.

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---- Cellular-Pathologie. 4th Ed. _Berlin_, 1871.

~Waldstein.~ Beobachtungen an Leucocyten u. s. w. _Berl. klin. Woch._
1895, no. 17.

~Weiss.~ Hæmatologische Untersuchung. _Wien_, 1896.

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quantitativen Zusammensetzung des Menschenblutes unter pathologischen
Verhältnissen. _Zeitschr. f. klin. Med._ 1894, vol. XXV.

---- ---- Bestimmung des Volumens und des Stickstoffgehaltes des
einzelnen roten Blutkörperchens in Pferde- und Schweineblut. _Pflüger's
Archiv_, vol. LII.

~Westphal.~ Über Mastzellen. _Inaug. Dissert._ _Berlin_, 1880. (Cp.
~Ehrlich~, Farbenanalytische Untersuchungen.)

~Winternitz.~ Weitere Untersuchungen über Veränderungen des Blutes unter
thermischen Einwirkungen. _Wiener klin. Woch._ 1893, no. 47.

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med. Woch._ 1893, no. 11.

~Wright.~ Remarks on methods of increasing and diminishing the
coagulability of the blood. _Brit. Med. Journal_, 1894.

~Zangemeister.~ Ein Apparat für colorimetrische Messungen. _Zeitschr. f.
Biologie_, 1896, vol. XXII.

~Zappert, J.~ Über das Vorkommen der eosinophilen Zellen im anæmischen
Blut. _Zeitschr. f. klin. Med._ vol. XXIII. (Literature.)

---- Neuerliche Beobachtungen über das Vorkommen des Ankylostomum
duodenale bei den Bergleuten. _Wiener klin. Woch._ 1892, no. 24.

~Zenoni, C.~ Über das Auftreten kernhaltiger roter Blutkörperchen im
circulierenden Blut. _Virchow's Archiv_, 1895, vol. CXX.

~Zesas, G.~ Beitrag zur Kenntniss der Blutveränderung bei entmilzten
Menschen und Tieren. _Langenbech's Arch._ 1883, vol. XXVIII.

FOOTNOTES:

[37] Owing to the enormous extent of hæmatological literature, we have
been able to refer only to more recent publications. We have, however,
indicated several papers, in which bibliographies of particular parts of
the subject are to be found.

INDEX.

Active leucocytosis, 142

Acute lymphatic leukæmia, 170

---- ---- ---- marrow in, 118

Acute swelling of red corpuscles, 23

Administration of iron, 16

Agony leucocytosis, 146

Alexines, 139

Alkali distribution in blood, 47

---- test for in blood, 46

Anæmia, 1

---- bothriocephalus, 65

---- definition, 2

---- hæmoglobin in, 49

---- iron in blood and organs in, 16

---- isotonic point of corpuscles in, 25

---- leukopenia in, 189

---- pernicious _see_ Pernicious anæmia

---- platelets in, 193

---- posthæmorrhagic, 49, 51

---- ---- erythroblasts in, 51

---- ---- platelets in, 193

---- pseudoleukæmica infantum, 77

---- pulse in, 3

---- retinal vessels in, 3

---- specific gravity of blood in, 18

---- volume of corpuscles in, 23

Anæmic degeneration of corpuscles, 49-52

---- leucocytosis, 146

Arsenurietted hydrogen, leucocytosis from, 145

Asthma

---- eosinophilia in, 150, 158

---- sputum in, 157

---- suppuration in, 160

Atypical leucocytes, 77-80

---- in leukæmia, 179

Basic double staining, 38

Baths, leucocytosis from, 144

Bioblasts, theory of, 128

Birds, eosinophil cells of, 107, 130

Blood, quantity of, 2

---- ---- in anæmias, 3

---- specific gravity of, 17

Blood corpuscles, enumeration of, 5, 6

---- ---- enumeration of in anæmia, 12

Blood corpuscles, estimation of, 12

---- ---- red _see_ Red blood corpuscles

---- ---- volume of, 13

---- ---- white _see_ Leucocytes

---- count, factors in, 11

---- crises, 62

---- dust, 193

---- platelets, 190

---- ---- alkali in, 47

---- ---- in chlorosis, 193

---- ---- origin of, 192

---- poisons, leucocytosis from, 145

Bone-marrow, 105, 112

---- ---- carcinoma of, 178

---- ---- cells of, in various animals, 106

---- ---- changes in anæmia, 117

---- ---- changes in lymphatic leukæmia, 118

---- ---- changes in pernicious anæmia, 117, 129

---- ---- connection with leucocytosis, 155

---- ---- connection with leukopenia, 189

---- ---- development of leucocytes in, 108-110

---- ---- giant cells in, 107

---- ---- of dog, 114

---- ---- protective organ, 111

---- ---- tumours of, 115

Cachectic leucocytosis, 146

Camphor, eosinophilia from administration of, 154

Catarrhal jaundice, volume of corpuscles in, 23

Charcot's crystals, 151

Chemiotaxis, 139

Chemiotaxis, positive, 139

---- negative, 140

Chenzinsky's fluid, 43

Chloroform narcosis, leucocytosis from, 145

Chlorosis, platelets in, 193

Coagulation, rate of, 23

---- connection with platelets, 192, 193

---- in purpura, 193

Coagulometer, 23

Compensatory eosinophilia, 154

Congenital syphilis, leucocytosis in, 147

Corpuscles, at high altitudes, 8

---- enumeration of, 5

---- influences on, 7

---- red _see_ Red corpuscles

---- volume of, 21

---- white _see_ Leucocytes

Crisis, blood, 62

---- in infective fevers, 145

Diabetes, glycogen in blood in, 75

Differential count of corpuscles, 31

---- staining, theory of, 37

Digestion, leucocytosis of, 102

Diphtheria, myelocytes in, 78, 146

Dog, bone-marrow in, 114

Dry preparations, 32

---- substance of blood, 20

Dühring's disease, eosinophilia in, 156

Electricity, resistance to, of corpuscles, 25

Enumeration of corpuscles, 5, 31

---- ---- ---- in anæmia, 12

Eosine-methylene blue methods, 43

Eosinophil cells, 78, 185

Eosinophil cells after splenectomy, 89, 94

---- ---- development of, 109

---- ---- in birds, 130

---- ---- in leukæmia, 176, 177

---- ---- in malignant lymphoma, 163

---- ---- in mammary glands, 163

---- ---- origin of, 160, 161

---- ---- secretion in, 134

---- leucocytosis, 148

---- ---- causes of, 165

---- ---- distinction from leukæmia, 178

---- ---- occurrence, 150-154, 158

---- ---- origin of, 154

---- myelocytes, 78

---- ---- in leukæmia, 174

Erythroblasts, in leukæmia, 180

---- in spleen, 99

---- nuclei of, 57, 61

---- origin of, 55

---- varieties of, 55, 56

Exercise, leucocytosis from, 144

Ferrometer, 16

Fevers, complication of leukæmia, 177, 181

---- leucocytosis in, 144, 146

---- spleen in, 98

Fixation of films, 34

Formalin as fixative, 35

Giant cells in bone-marrow, 107

Gigantoblasts, 56

Glycogen in gonorrhoeal pus, 46

---- in platelets, 192

Glycogen in polynuclear leucocytes, 75

---- stain for, 45

Gonorrhoeal pus, glycogen in, 46

---- ---- mast cells in, 160

Granulation, absence of, 118, 129, 182

---- chemical nature of, 134

---- ripening of, 108

Granules, 121

---- connection with emigration, 132

---- distribution of, 130

---- function of, 127

---- history of, 121-130

---- intravital staining of, 124

---- intravital staining of by neutral red, 125

---- reaction of, 126

---- secretory nature of, 134

---- specificity of, 133

---- survival staining of, 125

Guinea-pig, leucocytes in, 85, 112

Hæmatocrit, 21

Hæmatocytometer, 4

Hæmatoxylin-eosine mixture, 42

Hæmoconiæ, 193

Hæmoglobin, amount of, 13

---- connection with specific gravity, 18

---- equivalent in anæmias, 49

---- estimation of, 14-17

Hæmoglobinometer, 15

Hæmorrhages, acute, 51

Hæmorrhagic small-pox, pseudo-lymphocytes in, 79

Hayem's solution, 5

Heat as fixative agent, 34

Hedgehog, splenectomy in, 107

Helminthiasis, eosinophilia in, 150

High altitudes, erythrocytes at, 8-12

---- ---- poikilocytosis at, 9

Hodgkin's disease (malignant lymphoma), 101, 102

---- ---- eosinophil cells in, 165

Hyaline cells _see_ Large mononuclear leucocytes

Hygræmometry, 20

Infectious diseases, leucocytosis in, 146, 147

---- ---- myelocytes in, 77

---- ---- splenic enlargement in, 99

Intestinal diseases, leucocytosis in, 102

Intravital staining, 124

Iron, administration of, 16

---- connection of, with hæmoglobin, 16-18

---- in blood, 16

---- in eosinophil cells, 134

Isotonic point of corpuscles, 25

Jenner's stain, 44

Kresyl-violet-R, metachromatism with, 76

Kurloff's researches, 85

Lactation, eosinophil cells in, 163

---- mast cells in, 163

Large mononuclear leucocytes, 73, 112, 185
---- mononuclear leucocytes in measles, 113

Leucocytes, 71

Leucocytes, atypical forms, 77, 179

---- enumeration of, 5

---- in birds, 131

---- in guinea-pig, 85-87, 112

---- places of origin, 81

Leucocytosis, 138

---- active, 142

---- agony, 146

---- cachectic, 146

---- chemically produced, 189

---- classification of, 142

---- diagnostic importance of, 146

---- eosinophil _see_ Eosinophil leucocytosis

---- function of bone-marrow, 110

---- in anæmia, 146;
congenital syphilis, 147;
measles, 113, 145, 189;
parotitis, 144;
pneumonia, 144, 174;
rheumatism, 144;
tumours of bone-marrow, 116;
typhoid, 146, 189

---- mixed, 143, 167

---- of digestion, 102

---- origin of, 146

---- passive, 142

---- physiological, 144

---- polynuclear neutrophil, 143

---- theories of, 138

Leukæmia, acute lymphatic, 118, 170;
chronic, 170;
suppuration in, 105

---- myelogenic, 169

---- ---- atypical cases, 182

Leukæmia, myelogenic, atypical leucocytes in, 179

---- ---- characteristics of, 174-181

---- ---- complicated with other diseases, 174, 181

---- ---- contrasted with eosinophilia, 178

---- ---- contrasted with pneumonia, 174

---- ---- diagnosis from blood, 171

---- ---- eosinophilia in, 176

---- ---- mast cells in, 179

---- ---- mitoses in, 180

---- ---- mononuclear eosinophils in, 175

---- ---- myelocytes in, 174

---- ---- origin of blood condition, 183, 187

---- ---- pleuritic exudation in, 186

---- ---- polynuclear myelocytes in, 175

Leukopenia, 188-190

---- experimental, 140

---- in various diseases, 189

Liver, granules in, 127

Lymphæmia _see_ Lymphocytosis

Lymphatic glands, 100

---- ---- myelocytes in, 110

Lymphocytes, 71

---- in disease of lymphatic glands, 101;
lymphatic tumours, 101;
polynuclear leucocytosis, 143

Lymphocytosis, 101, 103, 144

---- after splenectomy, 90, 94

---- causes of, 101-103

---- in leukæmia, 170

---- origin of, 104

Lymphoma malignum (Hodgkin's disease), 101, 102

Malignant tumours, eosinophilia from, 153

Mast cells, 76, 160

---- ---- halo round, 135

---- ---- in gonorrhoea, 162;
leukæmia, 179;
purpura (horse), 136

---- ---- in skin, 160

---- ---- origin of, 162

---- ---- secretion in, 135

Measles, leukopenia in, 145, 189

Medicinal eosinophilia, 154

Megaloblasts, 56-62

---- in bothriocephalus anæmia, 65;
leukæmia, 180;
pernicious anæmia, 62, 66;
tumours of bone-marrow, 117

Megalocytes, 64

Metachromatism in mast cells, 76

Methylal method, 44

Microblasts, 57

Mitoses in leukæmic blood, 180

Mononuclear leucocytes, 73, 112, 116

---- ---- origin of, 113

---- ---- power of motion, 185

Mouse, polynuclear cells in, 131

---- spleen in, 99

Myelæmia, origin of, 183, 187

Myelocytes, 77, 110

---- importance in blood, 110;
in diphtheria, 78

---- in anæmia pseudoleukæmica infantum, 77;
bone-marrow tumours, 117;
leukæmia, 174;
lymphatic glands, 110;
lymphatic leukæmia, 118;
pneumonia, 78

Myelocytes, eosinophil, 78, 175

Myelogenic leukæmia _see_ Leukæmia, myelogenic

Nasal polypi, eosinophil cells in, 159

Neutral red, 125

Neutrophil granulation, absence of, 118, 129

Neutrophil leucocytes _see_ Polynuclear neutrophil leucocytes

Neutrophil pseudolymphocytes, 79

Normoblasts, 56, 57-62

---- in anæmias, 62;
leukæmia, 180

Nose, diseases of, eosinophil cells in, 159

Nucleated red corpuscles _see_ Erythroblasts

Old granulations, 109

Oligæmia, 2

Oligochromæmia, 2

Oligocythæmia, 2

Ozonophores, 129

Pacini's solution, 5

Paralysis, unilateral, corpuscles in, 6

Parotitis, leucocytosis in, 144

Passive leucocytosis, 142

Pemphigus bullæ, 156

---- ---- eosinophilia in, 150

Pernicious anæmia, bone-marrow in, 129

Pernicious anæmia, hæmoglobin equivalent in, 49

---- ---- megaloblasts in, 63, 65

---- ---- prognosis, 66

Phagocytosis, 139

Phosphorus poisoning, spleen in, 98

Pilocarpine, lymphocytosis from, 103

Platelets _see_ Blood platelets

Pleuritic exudations, in leukæmia, 186

---- ---- pseudo-lymphocytes in, 79

Pneumonia, contrast with leukæmia, 174

---- glycogen reaction in, 75

---- leucocytosis in, 141, 146, 152

---- myelocytes in, 174

Poikilocytosis, 52

---- at high altitudes, 9

---- explanation of, 54

---- from heating, 54

Polychromatophil degeneration, 49

Polynuclear neutrophil leucocytes, 74, 112, 185

---- ---- ---- diminution of in lymphatic leukæmia, 118;
in myelogenic leukæmia, 182

---- ---- ---- increase of _see_ Leucocytosis

---- ---- ---- movement in, 184

---- ---- ---- secretion in, 137

Post-febrile eosinophilia, 152

Posthæmorrhagic anæmia, blood in, 51

---- ---- ---- platelets in, 193

Potassium chlorate, leucocytosis from, 155

Pregnancy, leucocytosis in, 144

Pseudo-eosinophil cells, 85, 133

---- ---- ---- secretion in, 137

Pseudo-lymphocytes, 79

Purpura, of horse, mast cells in, 136

---- platelets in, 193

Pyknosis, 61

Pyrodin, leucocytosis from, 145

Quadratic ocular diaphragm, 31

Quantity of blood, estimation of, 2

Red corpuscles, 48

---- ---- acute swelling of, 21

---- ---- at high altitudes, 8-12

---- ---- connection with spleen, 99

---- ---- in anæmia, 49-57

---- ---- isotonic point of, 25

---- ---- nucleated _see_ Erythroblasts

---- ---- number of, in health, 7

---- ---- number of differences in age, 7;
in sex, 7;
food, 7;
from vasomotor influences, 11

---- ---- polychromatophil degeneration in, 49

---- ---- size of, 12

---- ---- volume of, 21

Retinal vessels in anæmia, 3

Rheumatic fever, leucocytosis in, 152

Scarlet fever, leucocytosis in, 146

---- ---- lymphatic glands in, 110

Separation of serum, 24

Serum, specific gravity of, 20

Sex, influence on red corpuscles, 7

---- ---- specific gravity of blood, 17

Shock, effect of, on specific gravity of blood, 18

Size of red corpuscles, 12

Skin diseases, eosinophilia in, 150

Small-celled infiltration, 105

Specific gravity of blood, 17

---- ---- ---- ---- connection with hæmoglobin, 18

---- ---- ---- ---- in anæmias, 18

---- ---- ---- ---- influences acting on, 18

---- ---- ---- ---- of serum, 20

Spleen, 84

---- as blood forming organ, 84, 99

---- enlargement of, in fevers and phosphorus poisoning, 98

---- excision of _see_ Splenectomy

---- functions of, 98

---- in guinea-pig, 91, 93, 99

---- tumours of, 97, 98

Splenectomy, effect of on blood in hedgehog, 107;
in guinea-pig, 88;
in man, 93, 95, 96, 113

Staining, theory of, 36

---- vital, 124

Stimulation forms of leucocytes, 79

Stroma of corpuscles, influence on specific gravity of blood, 19

Suppuration in lymphatic leukæmia, 104;
in myelogenic, 177;
in pemphigus, 157

Survival staining, 125

Thyroid, changes after splenectomy, 84

Time of life, influence of, on corpuscles, 7

Toxic leucocytosis, 145

Transitional forms of leucocytes, 74, 112

Triacid stain, 42, 131

Trichinosis, eosinophilia in, 152

Tropics, influence of, on red corpuscles, 12

Tuberculin injections, eosinophilia from, 152

---- ---- lymphocytosis in, 103

Tumours of bone-marrow, 115

Typhoid fever, leukopenia in, 146, 189

Urticaria, eosinophilia in, 150

Vital staining, 124

Volume of corpuscles, 21

White blood corpuscles _see_ Leucocytes

Whooping cough, lymphocytosis in, 103

Worms _see_ Helminthiasis

Wright's coagulometer, 24

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

*** End of this LibraryBlog Digital Book "Histology of the Blood - Normal and Pathological" ***

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