Chapter 9

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.