 The lymphatic glands As it is impossible experimentally to prevent the lymphatic glands as a whole from contributing to the formation of the blood, we are dependent almost entirely on clinical and anatomical researches for an elucidation of their function. Since Virchow's definition of the lymphocyte, it has been admitted that the lymphocytes of the blood, both the small and the larger kinds, are identical with those of the lymphatic glands and the rest of the lymphatic system. This is proved by the complete agreement in general morphological character, in staining properties of the protoplasma and nucleus, and from the absence of granulation. Abundant clinical experience testifies that the lymphocytes of the blood really do arise from the lymphatic system. Aerolich had previously observed that when extensive portions of the lymphatic glandular system are put out of action by new growths and similar causes, the number of the lymphocytes may be considerably diminished. These observations have since that time been confirmed by various authors. For example, Reimbach describes several cases of malignant tumor, particularly sarcoma, in which the percentage of lymphocytes, which normally amounts to about 25%, was very considerably lowered. In one case of lymphosarcoma of the neck, they only made up 0.6% of the total number. These conditions are quite easily and naturally explained by the exclusion of the lymphatic glands. It is difficult for the advocates of the view that the lymphocytes are the early stages of all white blood corpuscles to reconcile it with these facts. According to their scheme, the low number of lymphocytes is to be explained in such cases by their unusually rapid transformation to the polynuclear elements, the old forms, or to appropriate the expression of Uskov by a too rapid aging of the lymphocytes. Further evidence for the origin of the lymphocytes of the blood from the lymphatic glands is to be obtained from those cases in which we find an increase of the lymphocytes in the blood. These lymphocytoses occur, in comparison with other leukocytoses, relatively seldom. Under certain conditions in which a hyperplasia of the lymphatic glandular apparatus makes its appearance, we often see at first an increase of the lymphocytes in the blood. Ehrlich and Karouski, in some unpublished work, have investigated together a large number of typical cases of lymphoma malignum, and were able constantly to observe a lymphocytosis, which in some cases was of high degree and bore almost a leukemic character. Relying on these facts, Ehrlich and Wasserman made the diagnosis in vivo of malignant lymphoma in a rare skin disease, chiefly from the absolute increase of the lymphocytes alone, although no swelling of the glands was palpable. The post-mortem showed that the chief condition was a swelling of the retroperitoneal lymph glands to lumps as large as a fist. The lymphocytosis following extirpation of the spleen also belongs to this category, since a vicarious enlargement of the lymph glands is always to be observed in these cases. On investigating the conditions under which in healthy individuals an increased number of lymphocytes enter the bloodstream, we have in the first place to notice the digestive canal, whose wall contains a thick layer of lymphatic tissue. According to the results of Ryder, the proportion of the lymphocytes to polynucleus is practically normal in the leukocytosis of digestion, indeed the lymphocytes are rather in excess. The eosinophils, on the other hand, show a marked relative reduction in this condition. The leukocytosis of digestion consequently differs essentially from the other kinds in which the neutrophil elements are chiefly increased. This simultaneous increase of lymphocytes and polynucleus is doubtless brought about by a superposition of a raised income of lymphocytes and an ordinary leukocytosis caused by the assimilated products of metabolism. The influence of the digestive tract is still more evident in certain diseases, more particularly in intestinal diseases of infants. A considerable increase of the lymphocytes in the bloodstream is here to be observed. Thus, Weiss found an important increase of the white blood corpuscles in simple Qatar of the stomach and intestines, which presented the main features of a lymphocytosis. Hooping cough, according to the recent observations of Myrnier, also belongs to the small number of diseases which are accompanied by a pronounced lymphemia. In the convulsive period of this disease, both the polynucleus cells and the lymphocytes are increased, the latter in preponderating amount. The former cells are increased to twice, the lymph cells to four times their normal amount. Doubtless in these cases also, the lymphocytosis is due to the stimulation and swelling of the tracheobronchial glands. An increase of the lymphocytes from chemical stimuli is exceedingly rare, though, as is well known, a large number of substances, bacterial products, proteins, nucleans, organic extracts, and so forth, can call forth a polynuclear leukocytosis. In quite isolated cases, an increase of the lymphocytes in the blood, in consequence of the injection of tuberculin into tuberculous individuals, has been seen. From the rarity of these cases, it can scarcely be doubted that here a tuberculous disease of the glands also plays a part, so that the increased immigration of lymphocytes is brought about, not by a chemical property of the tuberculin, but by the extensive specific reaction of the diseased glands. Only one single substance has so far been mentioned in the literature, as capable in itself of producing a lymphocytosis. Feldstein asserts that he has produced by injection of pylocarpene a lymphemia which undergoes a progressive increase with a rise in number of the injections. The origin also of lymphocytosis is therefore sharply marked off from that of the ordinary leukocytosis, which consists in an increase of the neutrophil elements. Whilst the latter is admittedly the expression of chemiotactic action, and arises by action at a distance of soluble substances on the bone marrow, lymphocytosis is due to a local stimulation of certain glandular areas, thus in the leukocytosis of digestion, of intestinal diseases of children. We refer it to the excitation of the lymphatic apparatus of the intestine. In tuberculin lymphemia we recognize mainly a reaction of the diseased lymph glands. Hence we conclude that a lymphocytosis appears when a raised lymph circulation occurs in a more or less extended area of lymphatic glands, and when in consequence of the increased flow more elements are mechanically washed out of the lymph glands. The pilocarpine lymphocytosis does not contradict this view, for pilocarpine causes extraordinary though transient variations in the distribution of water, whereby the inflow into the blood of fluid containing lymph cells is increased. We therefore regard lymphocytosis as the result of a mechanical process, whilst leukocytosis is the expression of an active chemiotactic reaction of the polynuclear elements. This view finds its best support in the fact that the polynuclear leukocytes possess lively amoeboid movement, which is completely wanting in the lymphocytes. Corresponding to the absence of contractility in the lymphocytes, it is also observed that in inflammatory processes, in contradistinction to the polynuclear neutro and oxyfils, the lymphocytes are not able to pass through the vessel wall. A very interesting experiment on this point was described by Newman years ago. Newman produced sub-puration in a patient with lymphatic leukemia, in whom the blood contained only a very small number of polynuclear cells. Investigation of the pass showed that it consisted exclusively of polynuclear cells, and that not a single lymphocyte had come into the exudation, although this kind of cell was present so abundantly in the blood. Histological examination of all fresh inflammatory processes, in which mainly polynuclear elements are found, leads to accordant results. It is well known that small cell infiltration occurs in the later stage of inflammation, apparently consisting of lymph cells. Nevertheless, this does not in the least prove that these lymphocytes have emigrated here from the blood vessels. This is not the place to enter into the very extensive controversy on this point. We are content to refer to the most recent, very thorough paper of Ribbert. Ribbert regards these foci of small cell infiltration as the analogues of the lymphatic nodules, and explains their origin by an increase in size of the foci of lymphatic tissue, normally present though in a condition but little developed. It consequently follows from clinical and morphological researches, as well as from the observations on inflammatory processes, that the lymphocytes are in no way connected with the polynuclear leukocytes. We shall reach the same result in another way in the following section. Gamma, the bone marrow. The spleen and lymphatic glands were at first regarded as the sole places of formation of the blood corpuscles. The almost simultaneous researches of Newman and Bitzer-Zero first attracted general attention to the importance of the bone marrow. These authors showed that the early stages of the red blood corpuscles are produced there, a discovery which was quickly and generally recognized, and which soon became pathologically useful through the observations of Kuhnheim and others. In this connection the observation was of great value, that after severe loss of blood, the fatty marrow of the larger hollow bones again changes to red marrow, as it is evidence of the increased demands on the regenerative function of the bone marrow. We are unaware of a second place of formation of the red blood corpuscles in man. In other mammalia however, as we have above mentioned, the spleen may also take a small share in the production of erythrocytes. The type which the normal blood formation follows in adults, and the deviations therefrom shown in pernicious anemia have been described in the chapter on the red blood corpuscles. Ehrlich's view that the blood formation in pernicious anemia belongs to a different type, which is analogous to the embryonic, was also described there. In this section we have therefore to deal chiefly with the white blood corpuscles and their connection with the bone marrow. In man, as in a large number of animals, for example the monkey, guinea pig, rabbit, pigeon, and so forth, the bone marrow exhibits the peculiarity that the cells it produces bear a specific granulation in sharp contrast to the lymphatic glandular system, which contains elements free from granules in the whole animal series. The granulated cells of the bone marrow fall into two groups. The first group of the cells with special granules is very important, since it constitutes a characteristic for certain species of animals. According to the class of animal, they show different tinctorial and morphological properties. Man and monkey, for example, have neutrophil granulation, guinea pig and rabbit, the pseudo-eosinophil granulation described by Kerloff. In birds we find two specific granulations present side by side, which both are oxyphil and of which one is embedded in the protoplasm in crystalline form, the other in the form of granules. The kinds of special granulations so far investigated have the common property that they stain in acid and neutral dyes respectively. They show a much smaller affinity for the basic dyes. The fact that they greatly exceed the other elements of the bone marrow in all classes of animals is evidence of the importance of these granules. The second group of bone marrow cells contains granules which we find in the whole vertebrate series from the frog to man, and which therefore are not characteristic for any one species of animal. They are one, the eosinophil cells, two, the basophil mast cells. The bone marrow forms which are free from granules consist mostly of mononuclear cells of different type. They are not nearly so numerous or so important as the granulated kind, more especially as the first and predominant group. Amongst the granule free forms the giant cells deserve special mention, for they are an almost constant constituent of the bone marrow of the mammalian class. According to the recent researches of Pugliesi the giant cells are considerably increased after extirpation of the spleen in the hedgehog, an organ of quite extraordinary size in this animal, and doubtless therefore possessing important hematopoietic functions. Pugliesi asserts that in the hedgehog after splenectomy the nucleated giant cells pass into leukocytes by amyototic nuclear division. Unfortunately in his preliminary communication there are no notes of the granules of the bone marrow cells. On examining a stained dry preparation of the bone marrow of the guinea pig, rabbit, man, etc. it is seen that the characteristic finely granular cells are present in all stages of development, from the mononuclear through the transitional to the polynuclear, note polymorphously nucleated, end note, forms which we meet with in the circulating blood. A glance at a preparation of this kind shows that the bone marrow is clearly the factory where typical polynuclear cells are continuously formed from the granule containing mononuclear. Here also the same process of ripening can be seen in the polynuclear eosinophil leukocytes. Erlich has been able by special differential staining to bring forward proof that the constitution of the granulation changes during the metamorphosis of the mononuclear to the polynuclear cells. In the young granules there is prominent a basophil portion that becomes less and less marked as the cell grows older. The pseudo eosinophil granules of the mononuclear cells, of the guinea pig for example, stain bluish red in eosin methylene blue after long fixing in superheated steam. In the transitional stages this admixture is gradually lost and finally completely vanishes in the granules of the polynuclear leukocytes which stain pure red. Analogous observations may be made in the eosinophil cells of man and animals and in the neutrophils of man, hence it is even possible to decide whether an isolated granule belonged to an old or to a young cell. It is still impossible to judge with certainty the rate at which the ripening of the mononuclear to the polynuclear cells proceeds or further to decide if the ripening of the granules always runs parallel in point of time with that of the whole cell. On the grounds of our observations we would suppose that in general the two processes run their course side by side, but that in special cases the morphological ripening of the cell may proceed more rapidly than that of the granules. It is particularly easy to observe this point in eosinophil cells. Ehrlich had already mentioned in his first paper, 1878, that side by side with the typical eosinophil granules isolated granules are often found which show a deviation in tinctorial properties. For instance they stain more of a black colour in eosin-orantionigrosin. In eosin-methylene blue bluish-red to pure blue. Ehrlich had already described these as young elements in his first paper. The same differences are found more sharply marked in leukemia even in the circulating blood in the neutrophil as well as in the eosinophil group. Ehrlich has repeatedly found in leukemic blood polynuclear eosinophil cells whose granules must almost exclusively be regarded as young forms. Footnote, many authors, e.g. Arnold, explain this double staining of the eosinophils by the presence of the eosinophil and mast cell granulation side by side. That, this is certainly not the case, is shown by the fact that the basophil granulation of the eosinophil cells does not in metachromatic staining show the metachromacia characteristic for the mast cells. End footnote. Ehrlich regarded these as typical examples of a relative acceleration of the morphological ripening of the cells as compared with the development of the granules. In normal blood we find only the ripe forms of the specific granulated cells of the bone marrow. The mononuclear and transitional forms of the neutrophil group do not, under normal circumstances, pass over into the blood stream. Ehrlich regarded the mononuclear neutrophil granulated cells as characteristic for the bone marrow, since they are found exclusively in the bone marrow, never in the spleen or lymph glands, and for this reason named them myelocytes. Footnote. A. Frankel has recently reported histological investigations in which he could demonstrate in one case true myelocytes in inflamed lymph glands. He says, for some time past I have had systematic examinations carried out by my assistant Dr. Jaffa on the granulations of the leukocytes 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 coverslip 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 is immigrated there. Every past preparation shows that the polynuclear neutrophil leukocytes can change in the tissues to mononuclear, and the isolated observations of Jaffa should be explained in this manner. When myelocytes, no matter of what size, appear in considerable numbers in the blood of an adult, a leukemia of myelogenic nature is nearly always present. Note, for the very rare exceptions to this rule, which it may be added, can never be confused with leukemia, see later pages. End note. Exactly similar conditions hold good for the eosinophil cells in as much as the singly nucleated cells, which one may call eosinophil myelocytes, occur almost exclusively in leukemic blood. These forms which were first recognized by H. F. Mueller are however of less importance, for in myelogenic leukemia the chief part of the foreign admixture of the blood is made up of aelix myelocytes. Very important conclusions on the interesting question of leukocytosis can be drawn from these observations, bearing in mind that polynuclear neutrophil cells are developed and stored up only in the bone marrow, that in ordinary leukocytosis only the polynuclear forms are increased in the bloodstream, it is evident that leukocytosis is purely a function of the bone marrow, as Eelich has always insisted with all distinctness. It is only on this assumption that the frequently sudden appearance of leukocytosis, as has so often been observed in morbid and experimental conditions, can be satisfactorily explained. In these cases the space of time amounting often only to minutes is far too short for a new formation of leukocytes to be conceivable. There must be places in which these cells are already completely formed and able then to emigrate on any suitable stimulus. This place is single and is the bone marrow alone. Here all mononuclear forms gradually ripen to the polynuclear contractile cells, which obey each chemiotactic stimulus by emigration and which thus bring about sudden leukocytosis. The bone marrow thus fulfills, amongst others, the extremely important function of a protective organ, by which definite injurious influences which affect the organism may be quickly and energetically combated. Just as in a fire station ample means of assistance is continuously in readiness immediately to answer an alarm from any quarter. We wish to insist once more that the large mononuclear leukocytes and the transitional forms of the normal blood are not concerned in the increase in ordinary leukocytosis. In leukocytosis of high degree their relative number may indeed be lowered in consequence of the exclusive increase of the polynuclear cells. It appears then that these elements do not react to chemiotactic stimuli and that possibly they reach the blood by entirely different ways than the polynucleus do. We believe that these non-granulated mononuclear cells of man are to be regarded as analogous to those of the guinea pig, described by Kerloff. The mononuclear cells of man, however, are finally transformed into the neutrophil granulated cells, whilst the cells of Kerloff remain free from granules in the course of their metamorphosis. In acute leukocytosis in the guinea pig only the pseudo-eosinophil polynuclear cells are increased, which wander as such out of the bone marrow, but not the polynucleated non-granulated forms, which but slowly grow to maturity in the blood. Thus the peculiarities of guinea pig's blood, in which two kinds of polynuclear cells are recognizable, throw light upon the corresponding conditions in human blood. The distinction in the latter is more difficult. Since it is not evident in this case that the fully formed polynuclear neutrophil leukocytes have a two-fold origin, for the majority wander fully formed from the bone marrow into the blood, and only a considerably smaller number grow to maturity within the bloodstream from the mononuclear and transitional forms. No definite statement can as yet be made as to the places of formation of the non-granulated large mononuclear leukocytes. Kerloff has demonstrated that in the guinea pig these cells are present both in the bone marrow and in the spleen, but that after extirpation of the spleen the absolute number does not change. The bone marrow then in the guinea pig can also preserve the balance of the large mononuclear non-granular cells in the blood. The numbers we found in our blood investigations in man, after splenectomy, were also normal. We may then doubtless assume that the large mononuclear granulis cells of human blood also arise for the most part from the bone marrow. In this tissue they are to be picked out in the medley of the different kinds of cells only with the utmost difficulty, owing to their small number and their but little characteristic properties. Consequently, an exact investigation of their origin could probably only be successful if it were possible experimentally to produce a disease in which these forms in particular underwent important increase. This advance is not quite hopeless, since in man at least an absolute increase of the large mononuclear cells is observed in the post-thebrile stage of measles. On the grounds merely of microscopic investigations we conclude that the bone marrow is by far the most important of the blood-forming organs for its function is the exclusive production of red blood discs as well as of the chief group of the white corpuscles the polynuclear neutrophil. The physiological experimental investigation of the functions of the bone marrow offers insurmountable difficulties. An exclusion of the whole bone marrow or of larger portions only is an impossible operation, nor can we ascribe any value to the researches which endeavor to obtain a result by comparative enumeration of the arterial and venous blood of a bone marrow area. J. P. Retzky working under Uskov's direction has recently made counts of this kind in the dog from the nutrient artery of the tibia and the corresponding vein. He found that the number of white corpuscles of the vein is slightly greater, that on the other hand the absolute number of young corpuscles, note Uskov, end note, i.e. of the lymphocytes, has been considerably diminished, while the number of ripe corpuscles which for the most part correspond to our polynuclear is considerably increased. He gives the following table. Total number of cells in arterial blood 15,000, in venous blood 16,400. Young corpuscles in arterial blood 1,950, that's 13%, in venous blood 656, 4%. Ripe corpuscles in arterial blood 840, 5.6%. In venous blood 2,788, 17%. Old corpuscles in arterial blood 12,210, 81%. In venous blood 12,956, 79%. The argument based on figures such as these assumes that the function of the bone marrow is continuous, an assumption which Uskov indeed seems to make. But if the bone marrow is constantly absorbing the lymphocytes to such an extent, it is quite incomprehensible how the normal condition of the blood can be preserved, bearing in mind the extent of the bone marrow and the rate of the circulation. All evidence indeed tends to show that on the contrary the bone marrow performs its functions discontinuously, in as much as elements continually grow to maturity in the bone marrow, as we have already explained, but they only emigrate at certain times as a result of chemical stimuli. It is obvious a priori from this consideration how inconclusive must be the result of experiments such as these of Roitsky. Footnote. Moreover, the investigations of Roitsky are quite without foundation in as much as the tibiae 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 Professor Shirtz, no red marrow, but fatty marrow only, which, as is well known, is incapable of the smallest hematopoietic function. End footnote. Far more important for the elucidation of the function of the bone marrow are clinical observations on cases in which considerable portions of the bone marrow are replaced by tissue of another kind. We may best divide the observations on this point into two groups. One, malignant tumours of the bone marrow. Two, the so-called acute leukemia. There are, unfortunately, very few available observations as yet upon the first group. Still rarer are the cases in which, as is necessary, the whole bone marrow has been subjected to an exhaustive examination, which alone affords adequate evidence of the extent of the defect. Amongst the changes of the bone marrow arising from tumours, one may distinguish two groups, according to the nature of the condition of the blood. The first type is exemplified by a case of North Nagel published in his work on Lymphadenia Osium. Here, during life, the blood showed in the main the features of a simple severe anemia, but in addition isolated norma blasts, small marrow cells, and moderate leukocytosis. The autopsy, at which the whole skeletal system was subjected systematically to an exact examination, showed a complete atrophy of the bone marrow, and replacement of the same by the tumour masses. In this case, then, the condition of the blood in vivo is satisfactorily explained by the absence of function of bone marrow. North Nagel conjectured that the formation of the scanty-nucleated red blood corpuscles occurred vicariously in the spleen, that of the leukocytes in the lymph glands. In the second series, to which the cases of Israel and Laden, as well as the recently published one of Jay Epstein from Neusers wards, belong, the blood shows, besides the usual anemic changes, other anomalies which are peculiar, partly to pernicious anemia, partly to myelogenic leukemia. In Epstein's case of metastatic carcinoma of the bone marrow, there was found a considerable anemia with numerous nucleated red blood corpuscles, both of the normal and megaloblastic types. Their nuclei presented the strangest shapes, due not merely to typical nuclear division, but also to nuclear degeneration. The white blood corpuscles were much increased, their proportion to the red was 1 in 25 to 1 in 40. The increase concerned in the main, the large mononuclear forms, which bore for the most part, neutrophil granulation, and were, therefore, to be called myelocytes. In all the specimens, only two eosinophil cells were found. Footnote. 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 leukemia. End footnote. The explanation of a blood picture of this kind, apart from the purely anemic 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 leukocytes 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 to 1% of the white blood corpuscles. The autopsy showed isolated yellowish white foci of growth in the bone marrow, reaching the size of a sixpony piece. Bearing in mind that, in none of the other 39 cases where myelocytes demonstrated, one does not hesitate to explain their presence in the blood in this single case by the metastasis 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 anemia. In the cases of anemia 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 anemia, 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 bloodstream 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 leukemia, according to the well-known results of Newman, 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. Foot's note, in contrast to this lymphatic metamorphosis of the bone marrow, in myelogenous leukemia, a myeloid transformation of the other blood-forming organs, especially of the lymph glands, is found. A transformation sufficiently characterized as myeloid by the presence of myelocytes, eosinophils, and nucleated red blood corpuscles. End foot's note. The most convincing results are obtained from cases of acute lymphatic leukemia, the pretty frequent occurrence of which was first noticed by Epstein, and which has lately been very thoroughly studied by A. Frankel. For the purpose in question, acute leukemia 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 earlicks. The polynucleolucocytes 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. Doc has also arrived at similar results, as we see from a preliminary report, and he similarly explains the absence of neutrophil cells in lymphatic leukemia by the replacement of the myeloid by lymphatic tissue. Thus, lymphatic leukemia 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 Frankel, after accurately examining and analyzing eight cases of acute lymphatic leukemia, 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. Frankel, 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 leukemia, but of an absolute hindrance 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 aging 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 Frankel's monograph. Frankel distinguishes the acute from the chronic forms of leukemia by the fact that in the former the newly formed elements emigrate from their places of formation into the bloodstream with extraordinary rapidity, hence there is not time for further local metamorphosis. In chronic leukemia the emigration takes place very probably much more slowly. This distinction is contradicted by the facts, for there are chronic forms of lymphatic leukemia whose microscopic picture is identical with that of acute leukemia, and hence the starting point of all Frankel's deductions is rendered insecure. Chapter 3 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 hematology 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 Ehrlich refuted Altmann's claim to priority, nevertheless Altmann, in the second edition of his elementary organisms, 1894, stated that before him no one had recognized the specific importance of the granules, though some authors had viewed them as, quote, rare and isolated phenomena, end quote. We may quote a passage published by Ehrlich in 1878, that is, ten years before Altmann's papers, quote, 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 shown 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 protein precipitate to 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 protein are embedded in a granular form. We can readily distinguish but few of these substances, such as fat and pigment. Most of them we cannot present characterize but imperfectly or not at all. End quote. Quote. Earlier observations, special on the mast cells, let me to expect that these granulations, though they had long been inaccessible to chemical analysis, could be distinguished by their behavior with certain stains. I found in fact granules of this kind characterized 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 subgroups, 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 coverslips in as thin a layer as possible, dried at room temperature, and after a convenient length of time stained. I had chosen this apparently Koch's 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, for example water or alcohol, or as oxidizing 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 today as the most valuable, and the only practical means of grouping the leukocytes. 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 Altman'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 Altman's hardening method lies in the circumstance that the cell proteins 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 artifact. Since A. Fisher'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 Altman's forms have been raised from various quotas. 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. Footnote? Altman'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. And footnote? 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. Schütze, who placed a larvae of frogs in dilute methylene-blue solution, and after a short period found the granules of the stomach, the red blood corpuscules, 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 granules. Teichmann directs a detailed analysis to this point, and regards most of the granules described by Schütze as artificial products. Neutral red is highly suitable for the study of vital granules staining, a dye recommended by Ehrlich, and employed successfully since that time are Prashmiki, Pražek, Esmaja, Solger, Friedmann, Pappenheim, and others. This dye was prepared by O. N. Witt from Nitrozodymethylamine and Methetoluelandiamine, that is the hydrochloric acid sold of a base which is soilable in pure water, yielding a fuchsine-red color, but which in weak alkaline solution, the alkalinity of mineral water suffices, is a yellow-orange hue. Now neutral red is characterized 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 a simplest conceivable, as subcutaneous or intravenous injection, or even feeding, in the higher animals, stains the granules, with frox 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 affected by allowing small pieces to float in physiological salt solution, to which a trace of neutral red is added under plentiful excess 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, for example flies eggs, or the malpigean 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 in 50,000 to 1 in 100,000 is recommended, so that the protoplasm and nucleus remain quite uncolored. Artificial products with this method cannot entirely be excluded, and, for example, implant cells containing tenon are to be explained by the production and precipitation of the salt of tenic acid. However, it is not difficult for the experienced to recognize artificial products as such in individual cases. The kind of granulation, the typical distribution, a comparison with neighboring cells, the combination of various methods, the comparison of the same object and their 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 fixin color, 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. Frogs 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 three-fold 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 reserved 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 showed 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 color 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 colored brown with glycogen. Quote, 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 show by staining that the membrane was chemically different from the granules, since with eosin, oranthia, indulin, glycerin, the membrane stained black, but the granules orange-red. End quote. To these observations Ehrlich added the following conclusion. Quote, 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. Quote has shown, first for the liver cells, and this is now recognized 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 helium-ground substance in preponderating amount, and a more scanty, finely granular, fibrillary substance embedded in it. Kupfer calls the first paraplasm, the letter protoplasm. On warming the preparation to about 22 degrees Celsius manifest though feeble movements appear to 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 center of the particular specific cell function. In any case, it is desirable to give a special name such as microzomes, handstein, to these forms, which in the liver cells are recognizable as distinct round or oval granules, coloring yellow with iodine and easily and deeply staining in other ways. End quote. It was necessary to quote in full from this older paper to show that Ehrlich regarded the granules as the special carriers of the cell function so long ago as 1883, a view that Altman advocated many years later under the name Theory of Bioblasts. Altman's ever-repeated assertion that no one before him had a lot of so high an importance to the granules is consequently in disagreement with the facts we have above made sufficiently clear. The importance Altman ultimately gave to the granules, which he also calls by the name Ozono Force, is shown by his own words in elementary organisms first edition, page 39. Quote. Our conception of the Ozono Force 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 summarizing the properties of the Ozono Force, as oxidant carriers, they can perform reduction and oxidation and can thus affect the decompositions and synthesis 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 Altman. Studies in particular on the oxygen requirements of the organism showed that the Ozono Force 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 recognized by ordinary methods. Finally, a pathological observation made untenable the view that the granules are the bearers of the cell function. In the case of pernicious anemia, sie farben analytische 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 secretary 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 Altman 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-granula. End quote. 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 color 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, for example in birds, the analogues of the granules of mammalian blood are characterized by a decided crystalline form, and a strong oxophyllia. 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 colorless blood cells has been demonstrated in the most various classes of animals, and even in the blood of many invertebrates, particularly, as Gnoll has shown, in the lemily brunquates, polyquates, petates, tunicates, and cephalopods. Concerning vertebrates, especially the higher classes, accurate and ample researches are to hand. In birds, we recognize two oxophyll 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 granulation's analogous to them, completely wanting. According to the investigations carried out some years back in Ehrlich's laboratory by Dr. Franz Müller, these results of Hirschfeld's must be described as inaccurate. After many vain endeavors, Dr. Müller was able to find a method by which numerous, though very minute granules could be found in the polynuclear cells of the mouse. The case shows 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 protoplasma 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 Kurlov's work, we must insist on the contrary, that there is no evidence that the non-granulated polynuclears are identical with the granulated cells. Kurlov has shown, at least for guinea pigs 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 the 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 reserved 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, eerlich regards as unfounded, from extensive researches specially directed to this point. Nor has eerlich seen a pseudo-eosinophil cell of the rabbit change to a true eosinophil. Footnote. The cause of these misunderstandings is the tinctorially different stages of development of the granules as we are 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 gypsin 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 glendular cells however with various simultaneous functions several kinds of granules may be contained. End footnote. That such a transition does not occur is most distinctly shown by the fact that the various granulations behave entirely differently towards solvents. With the aid of acids for example the pseudo-eosinophil 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 especially striking expression is afforded by the study of the various forms of leukocytosis as will be shown in detail in the following chapter neutrophil and eosinophil leukocytes 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 in as much 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 corpuscules seem to be a 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 guanine, 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 Parker the eosinophil granulations appear to contain iron. The keystone of the hypothesis of the secretary nature of the granules is a direct observation of a secretary 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 neighborhood to be followed. Kantak and Hardy have succeeded in demonstrating the secretary 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 Kantak 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. One, appearance of granules within the cells. Two, discharge of these granules externally. The mast cells too seem suited for this purpose since their specific substance is strongly characterized by its peculiar metachromatic staining and is further especially readily recognizable since by its great affinity for basic dyes it remains plainly stained even in preparations that are almost quite decolorized. In fact appearances of the mast cells are not infrequently found which must be referred to excretory process 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 of a colorless 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 showing the 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 Una whose notice Ehrlich's remark had no doubt escaped described in an analogous condition as follows. Quote, In some nodules the mast cells appeared in part twice as large as usual especially with the new mast cell stain polychrome-metalline blue glycerine ether mixture. This was caused by the staining of a large round halo in the center of which lay the peculiar long-known mast cell consisting of blue nucleus and an areola of deep red granules. Higher magnification showed that the halo was not granular but very finely reticular although it exhibited exactly the same red color as the granules. It was consequently a spongioplasm peculiar to these mast cells. End quote. The appearance of the mast cells described by Una may also be artificially produced by allowing a preparation that is stained with the oxygen-containing analog of cyanine, oxamine, to remain for some time in levilose, syrup, or watery glycerine. Evidently part of the diet mast cell substance is dissolved and retained in the immediate neighborhood but as Una 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 they 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. Footnote. From a paper of Kayecha we learn that Raamon Ikairal recognized the halos of the mast cells and interpreted them in a manner we have above. Kayecha also describes these halos and the method of demonstrating them in detail, thionine 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. End footnote. A condition that Prus has brought forward in the so-called perpura of the horse is also to be interpreted as a secretory process of the mast cells. He describes young mast cells from the hemorrhagic 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, quote, 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, end quote. Evidence that the substance of the granules is given off externally may sometimes be seen in the polynuclear neutrophil or their analogs, thus in rabbit's blood in which he had experimentally produced leukocytosis. Hankin found a distinct progressive decrease of the pseudo-ineosinophil granules on allowing the samples of blood to remain sometime in the thermostat. Further, in separating foci and man, especially when separation has lasted long, are the pus has remained for some time in the place in question, Janowski. A rare infection, almost a complete disappearance of the polynuclear neutrophil granules occurs, and is to be explained by 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 leukocytes. End of section 8