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 _a 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 leukaemia.
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 anaemia; 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 anaemic changes, other anomalies which
are peculiar partly to pernicious anaemia, partly to myelogenic
leukaemia. In Epstein's case of metastatic carcinoma of the bone-marrow,
there was found a considerable anaemia, 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
anaemic 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 anaemia. In the cases of anaemia 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 anaemia 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 leukaemia 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)
leukaemia, the pretty frequent occurrence of which was first noticed by
Epstein, and which has lately been very thoroughly studied by A.
Fraenkel. For the purpose in question, acute leukaemia 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 leukaemia by the replacement of the myeloid by lymphatic
tissue.
Thus lymphatic leukaemia 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 Fraenkel,
after accurately examining and analysing eight cases of acute lymphatic
leukaemia, 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;
Fraenkel 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 leukaemia, 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 Fraenkel's
monograph. Fraenkel distinguishes the acute from the chronic forms of
leukaemia 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 leukaemia the emigration takes place very
probably much more slowly." This distinction is contradicted by the
facts; for =there are chronic forms of lymphatic leukaemia whose
microscopic picture is identical with that of acute leukaemia=. And hence
the starting-point of all Fraenkel'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
leukaemia. 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. Fraenkel 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. Schuetz--no red marrow, but
fatty marrow only, which as is well known is incapable of the smallest
haematopoietic 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 leukaemia.
[19] In contrast to this lymphatic metamorphosis of the bone-marrow, in
myelogenous leukaemia 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 haematology, 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 larvae 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.