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.

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 larvae 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 larvae 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 deg. 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 anaemia (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, Polychaetes, 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 Mueller, these results of Hirschfeld's must be described as inaccurate. After many vain endeavours, Dr Mueller 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 laevulose 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 haemorrhagic 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 haematological 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, Loewy 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. Loewit 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," Loewit) 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 Loewit has given to leukopenia. A 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 Loewit'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 Loewit 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, Tuerk 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 (Loewy 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.

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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 lymphaemia, including that of leukaemia. 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; "myelaemia."

[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 Tuerk 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' exochen]—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 aetiology, 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 Tuerk. Of Tuerk'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). Stienon, 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 haemoglobinuria (sulphonal poisoning?) as well as after protracted chloroform narcosis.

3. The leucocytosis which accompanies acute and chronic anaemic conditions, especially posthaemorrhagic.

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 Tuerk. 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).

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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 anaemic 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 versa.

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 leukaemia a considerable time elapsed before an eosinophilia was found in other diseases, an eosinophilia however that differs in its essential traits from the leukaemic type. To Friedrich Mueller 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. Mueller 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 leukaemia 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 leukaemia, 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 leukaemia, 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 Mueller 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 faeces. In a third case of Ankylostomiasis Zappert found no increase of eosinophil cells in the blood, nor the crystals in the faeces. Almost simultaneously, Siege made similar observations.

For a detailed working out of this important branch we are greatly indebted to Leichtenstern. Under his direction Buecklers 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]. Buecklers 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 Taenia 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 faeces. Since eosinophil cells and Charcot's crystals have elsewhere been observed to be interconnected phenomena (for example in bronchial asthma, in nasal polypi, in myelaemic 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 leukaemia.

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. Tuerk 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 leukaemia 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 Mueller 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 a 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 Mueller and Rieder. Were the hypothesis true, transitional stages ought to be found with ease in every sample of normal blood. Rieder and Mueller 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, Mueller 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 depot, 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. Mueller 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 anaemic conditions," we believe we may content ourselves with the above objections to the Mueller-Rieder theory, although it has even quite recently found supporters (e.g. B. Lenhartz). H. F. Mueller 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 Duehring's disease. The bullae 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 bullae, 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 bullae 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 depot 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 Mueller-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 leukaemia, 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 leukaemia 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 leukaemia, 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. Michaelis 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 Michaelis' 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 Duehring's disease, that the eosinophil cells, which are also present in the cutaneous tissue in large numbers, apart from the contents of the bullae, 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. Mueller, 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 anaemic 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 faeces; 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 anaemia, 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 anaemia. Many observations, the absence, for example, of eosinophilia in Bothriocephalus anaemia (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 anaemic condition is due.

Leukaemia.

("Mixed leucocytosis.")

In spite of the enormous extent of the haematological observations of the last decennia, of which a very considerable portion deals with the problem of leukaemia, 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 leukaemia.

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

Neumann first shewed that the lymphoid proliferation in lymphatic anaemia 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 leukaemia, or the condition of the blood. In spite of the splenic tumour we have to deal then with a pure lymphatic leukaemia. In customary clinical language, a case of this kind would be described as lieno-lymphatic leukaemia. The unreliability and incorrectness of this terminology is best illustrated by another form of leukaemic metastasis. In lymphatic leukaemia the liver may swell by lymphomatous growth, to a large tumour, and we ought then to speak of a "hepato-lymphatic" form of leukaemia. 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 leukaemia is totally unwarranted from haematological investigations. The possibility of a specific blood change, depending solely upon disease of the spleen, appears a 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 leukaemia 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" leukaemia must also be described as illogical and misleading.

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

1. leukaemic processes with proliferation of lymphoid tissue:

"lymphatic leukaemia";

2. leukaemic processes with proliferation of myeloid tissue:

"myelogenic leukaemia."

The accompanying clinical phenomena may be indicated by simple unequivocal amplifications, for instance, "lymphatic leukaemia with enlargement of the spleen or of the liver"; "myelogenic leukaemia 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 leukaemia have quite a different aetiology. The recent discovery of Loewit should be decisive on this point, for he demonstrated in myelogenic leukaemia the presence of forms like plasmodia within the white blood corpuscles, but was unable to find them in lymphatic leukaemia.

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

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

The second form of lymphatic leukaemia 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 leukaemia represents a single disease, or should be etiologically subdivided. Haematologically, all lymphatic leukaemias 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 leukaemia, 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 leukaemia, 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 leukaemia presents a picture that is different in every particular. In former years the distinction between myelogenic leukaemia 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 leukaemia 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 leukaemia brings elements into the blood that are abnormal. The cells here introduced are so characteristic as to render the diagnosis of leukaemia 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 leukaemia 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 leukaemia 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 leukaemic 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 haemorrhage.