The local formation of blood pigments

616-003.829. I

THE LOCAL FORMATION OF BLOOD PIGMENTS."

ROBERT Mum and JANET S. F. NWEN. F r m the Pathology Department of the University, Glaagow,

and the G h 3 o w Weatem Injrmary.

THE observations recorded in this paper were initially part of an enquiry into the disposal of red corpuscles and of haemoglobin respectively when introduced into the tissues. The intracellular formation of haematoidin, previously observed in tissue cultures by Rich (1924) and also by one of us (Niven, 1935) was found to be a prominent feature and is the chief subject dealt with in the following pages. The actual crystallisation of haematoidin within cells can be readily followed by the methods to be described but a large number of observations at different stages have been found necessary in order to .arrive 'at a definite view with regard to the sequence of events and their significance. Although we have not found evidence that in the conditions of these experiments hsmatoidin is formed outside cells, we wish to make it clear that the possibility of this is by no means excluded. The general question of the splitting of haemoglobin by extracellular enzymes, however, is outside the scope of the present paper.

The division of pigments derived from haemoglobin into the two classes of iron-containing and iron-free is, of course, Universally recognised. And although there is no generally accepted view as to their relation it has been commonly stated that they are met with in different conditions and formed by different processes. In particular, the intracellular formation of haemosiderin has been emphasised whilst, on the other hand, the formation of haematoidin has usually been assigned to an extracellular site. This mode of viewing the matter has been largely due to the work of German observers toward the end of last century. The presence of haematoidin within cells, which has been often described, has as a rule been interpreted as resulting fiom phagocytosis of crystals formed extracellularly. The question of site of formation is of

* Towards the expenses of this research a grant was received from the Rankin Medical Research Fund of the University of Glasgow, for which we have pleaawe in expressing our indebtedness.

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184 R. MUIR AND J . S. F . NIVEN

course to be distinguished from that of distribution. The general position, however, must be regarded as theoretically unsatisfactory since whether hzemoglobin is split up intracellularly or extracellularly, iron-containing and iron-free moieties have to be considered in both cases. As the literature on the subject has been fully reviewed by Rich (1925), we need refer only to a few of the more important points.

Virchow (1847), in his paper containing the first observation on hiematoidin crystals, described pathological blood pigments as occurring in three forms, viz. diffuse, granular and crystalline, and stated that all forms are met with both inside and outside cells. In the plate attached to the paper a cell is shown containing some hematoidin crystals, but the actual formation of haematoidin within cells is not stated. He described the formation of pigments from diffused hzmoglobin and also from masses of red cells. Langhans (1869-70) studied the question experimentally in fowls, guinea- pigs and rabbits, homologous blood being used, and described the phagocytosis of injected erythrocytes. He found that hrematoidin crystals were formed a t an early stage in the first mentioned and occurred both as rhomboids and needles. He considered that the formation of the former a t least occurred extracellularly. Hzematoidin crystals were found in a guinea-pig twelve days after injection of blood but were never observed in rabbits. Cohnheim (1877) in his Lectures on geneTal pathology expresses the view in agreement with the prevalent belief that hiematoidin is formed from free hemoglobin without the aid of cellular elements. Quincke (1884) also studied the question experimentally by injecting dog’s blood into dogs. He found that when red corpuscles are taken up by phagocytes granular pigment (hmmosiderin) is formed. On the other hand, when the haemoglobin diffuses out of corpuscles bile pigment (hematoidin) is produced and crystallises, while the iron of the hzmoglobin is dissolved in some way in the tissue fluids and .is carried away. The formation of crystals is extracellular and it occurs especially in yellow spots or patches. Neumann (1888) came to quite definite conclusions with regard to the origin of blood pigments and his work confirmed the general opinion on the subject. From observations on human material his two main conclusions were -(a) living cells form hsemosiderin either from erythrocytes or from hemoglobin, and (a) the formation of hiematoidin represents a chemical process of destruction independent of the living activity of the tissues. He observed hzmatoidin crystals within cells but wm quite definite that they were not formed there. Skrzeczka (1887-88) supported Neumann’s views and stated that haematoidin is formed only in comparatively large extravasatiom of blood apart from the influence of living tissues. More recently, Hueck (1912), in a long paper on pathological pigments, came to a like conclusion.

The summary of these papers represents the basis of the current belief up to recent times with regard to the formation of blood pigments. An entirely new light was thrown on the subject by the observations of Rich (1924) on tissue culture. By this method he showed that erythrocytes added to cultures were taken up by mesodermal cells, that they underwent disintegration within them and that there appeared intracellular crystalline haematoidin both in rhomboidal form and as needles. The crystals gave the

LOCAL FORMATION OF BLOOD PIGMENTS 185

typical Gmelin reaction. Granular hemosiderin also appeared as a residue in the cells. No crystals were formed outside cells before the time of their crystallisation within cells.

Methoda.

The technique of the experiments was of a simple nature. The blood injected subcutaneously was ordinarily homologous blood and was used in the fresh condition so that it might coagulate in the tissues and form a definite hzematoma. In this way the blood is kept fairly well restricted to a single area. The blood was allowed to remain for varying lengths of time and then the tissues were examined. Hzemoglobin when used for injection was always rabbit hzemoglobin prepared by the method described by Muir and Young (1932) and the solution had always been passed through a Seitz filter to free it of stromata. The concentration of the hzemoglobin solution corresponded to about 60 per cent. in the Haldane hzmoglobinometer standard.

The tissues were examined by means of spread preparations and also by sections. We consider the former essential; in fact, we believe that it would be hardly possible to trace the changes without them. A “ spread ” is made by snipping off a small piece of the delicate connective tissue to be examined and stretching it as fully as possible on a cover-glass by means of blunt glass needles. The preparation is then examined either (a) in the fresh condition, or ( b ) after fixation. For the former the preparation is simply mounted in normal saline on a slide; the saline may be suitably tinted with neutral red. One can obtain much information by this simple method-the types of cells, the presence of mitoses, etc.-and after a little experience one can usually distinguish iron-containing from iron-free pigment. If the preparation is to be fixed the spread on the cover-glass immediately after being made is simply dropped on to the surface of the fixing fluid; after fixation it is treated like a section. For permanent preparations in which crystals are present the most suitable method was found to be mounting in “Euparal” immediately after dehydration in alcohol. In specimens prepared in this way crystals are still well preserved after several months. Other methods tried were less satisfactory. Pieces of tissue a t the site of injection were also fixed in the usual way and sections cut in paraffi.

Various fixing fluids were tried but it was found that a saturated solution of corrosive sublimate in normal saline gave the best results for all-round purposes ; the iron reactions are specially clear after its use. The ordinary staining methods were employed, and one need only note that in the case of sections Mann’s methyl blue eosin method was most suitable for showing phagocytosis of red corpuscles and their condition.

The occurrence of crystals of course is readily observed.

Experiments with injections of erythrocytes.

Mouse blood (0.5-1.0 c.c.) was used for the injections. A few points with regard to the naked-eye appearances of the local hsmat,oma at different stages may be mentioned. At first the hsmatoma simply consists of a collection of clotted blood of dark red colour and of rather loose consistence. Within a few days its margin comes to have a somewhat brownish tint, and this is very distinct after six days. There is also some faint brownish

A. Mice.

186 R. MUIR AND J . S. F. NIVEN

colouration of the subcutaneous tissue in the neighbourhood. Further changes consist in the gradual diminution of the collection of blood and the deepening and extension of the brown colour. At this stage we may state that the difFuse brown colouration is essentially due to phagocytes containing hsmosiderin, these cells wandering widely in the tissues around. We have found no evidence of diffusion of haemoglobin with subsequent extracellular formation of brown pigment from it. In our series of experiments some red colour was still present on the eleventh day but it had quite gone by the fifteenth day. Another fact of importance is that about the tenth day small yellow areas may begin to appear in the brown tissue at the margin of the clot. These are paler and of a more distinctly yellowish tint than their surroundings, from which they are often sharply defined. One or more of these patches may be present. As described below they are formed by collections of cells containing pigment which represents a pre- haematoidin stage. For a time after the disappearance of the haematoma its site is marked by a deeper brown colour which may be accompanied by orange-yellow patches.

The microscopic changes are to be associated with the naked-eye appearances and may appropriately be grouped under the following headings :-(a) phagocytosis of red corpuscles, ( 6 ) formation of hsmosiderin, and (c) formation of hsmatoidin.

(a) Phagocytosis of red cmpuscles. The destruction of red corpuscles occurs essentially by phagocytic action. Evidence of this is clearly found within 24 hours after the blood is injected, when cells containing many corpuscles can be seen. After 48 hours the amount of phagocytosis is very remarkable. The phagocytes are mainly local histiocytes which become enlarged and multiply and also assume various forms. In some preparations numerous mitoses have been seen. On staining with neutral red, their segregation granules are often large and very prominent, especially when phagocytosis is occurring. The process of phagocytosis continues active till all the red colour of the injected blood has disappeared. It is noteworthy that the erythrocytes up to the time of their being actually ingested by the phagocytes are well preserved, and there is practically no evidence of diffusion of haemoglobin such as is seen in the case of the rat. In fact the appearances on the 7th or 8th day where phagocytosis is going on are similar to those at quite an early stage, many of the cells containing numerous erythrocytes in a well preserved condition

( b ) Formation of hmosiderin. Phagocytosis is soon followed by the formation of hzmosiderin within the cells and at an early period some of the ingested erythrocytes also give a piussian blue reaction. Evidence of this may be found even after 24 hours.

(fig. 1).


Some of the phagocytes containing red corpuscles may show a shght diffuse bluish tinting of the cytoplasm and even some minute reacting granules may be present. After 36 hours a few cells giving the prussian blue reaction may be seen in sections to be within the blood clot and these are probably monocytes of the injected blood which have ingested erythrocytes; but most of the pigment-holding cells are found in the tissue around the clot and their origin from histiocytes can be readily traced. As time goes on they increase in size and come to contain pigment in large amount (fig. 7, d and e). They wander widely into the tissues around and are found not only in the subcutaneous tissue but also deeply between the bundles of striped muscle fibres. They are specially numerous around blood vessels but their accumulation in large numbers around the small nerves is an even more prominent feature. We cannot say definitely that there is no extracellular destruction of erythrocytes with diffusion of hsmoglobin but we have found no evidence of it. If it occurs a t all it can only be of very slight degree, and we consider that the brownish colouration extending diffusely around the haematoma is essentially due to the wandering of phagocytes containing haemosiderin.

As time goes on the amount of hsmosiderin becomes very large and when treated with hydrochloric acid and ferrocyanide the pigment is found to be in the form of flne and coarse granules, some of the latter being large and of irregular form. There is also in varying degree a diffuse blue staining of the cytoplasm. The haemosiderin is practically restricted to cells, within which it may be amociated with red corpuscles both intact and in various stages of disintegration. At later stages the pigment-holding celh in places are of relatively large size and stuffed with pigment. We have noted no formation of multinucleated cells of " foreign body '' type.

The essential reaction to the presence of blood in the tissue is thus a phagocytosis of red corpuscles by histiocytes, this being followed by the formation of haemosiderin within them and the wandering of the pigment-holding cells. Such are the changes observed within the f i s t week.

(c) Formation of hcematoidin. The next occurrence is the formation of haematoidin crystals. We have found this to be quite distinct in certain instances eight days after the injection of the blood. The characteristic crystals of haematoidin are f i s t formed within cells ; of this there is no doubt. They may occur in small numbers associated with haemosiderin or, on the other hand, even a t an early stage the cytoplasm of some cells appears to be crowded with them. In the latter case the process of crystallisation would appear to be a comparatively rapid one.

Two points are to be considered, viz. (1) the mode of formation

188 R. M U I R AND J . S. F. NI?'EN

of hsmatoidin, and (2) the relation of the hsmatoidin to hsmosiderin. As regards the formation of hcenmtoidin, this may be said to occur in two ways-(a) in a massive fashion in certain areas or ( 6 ) in a disseminated fashion. The former is seen in the discrete yellow spots referred to above, the latter in the diffusely stained subcutaneous tissue around. If one examines microscopically a yellow area before crystals have appeared, one finds a characteristic appearance, namely collections of fairly large cells of a bright lemon-yellow colour, the tint differing from that of the brownish cells seen a t an earlier date and still present. It may be noted, too, as an important fact that the colour is restricted to the cells ; there is no colouration around them. On examination with a high power the colouration is seen to be diffuse or with very fine granu- larity, the appearance being as if the cytoplasm were stained. This characteristic appearance we have found to precede the formation of haematoidin-we may call it the pre-haematoidin stage. An important point is that the cells of a typical yellow spot often give no iron reaction ; on the other hand some cells may give a faint green colouration or contain some granular hsmosiderin. After some experience one can recognise the latter even in unstained specimens. It is more granular and of a browner tint than the yellow pigment, which does not give the iron reaction.

Examination of a yellow patch about the eighth day or later may show the.next occurrence, namely the separation of hsmatoidin in the crystalline form; this would appear often to be a comparatively rapid process, many cells becoming packed with small rhomboidal crystals in a comparatively short time. The number of crystals may be very remarkable, the cytoplasm of some cells appearing to be largely occupied by them. No trace of crystallisation of haematoidin outside the cells has been observed ; it is essentially an intracellular process. I n many areas the phagocytes are stuffed with crystals, while no crystals can be seen in the tissue spaces. At a later period, however, crystals may become free (fig. 4).

The earliest formation of haematoidin crystals which we have observed has been chiefly of this massive kind in certain areas in the neighbourhood of the haematoma, but crystal formation soon appears in pigment-holding cells widely scattered and gradually becomes more marked. In this slower mode of formation one or two crystals first appear in phagocytes containing abundant hsmosiderin. The crystals then increase in number while the haemosiderin diminishes (fig. 7, f -h) .

With regard to the relation of hcematoidin crystub to hmmosiderin the general statement may be made that they are present in inverse proportion. As has been stated, up till the eighth day the pigment within the phagocytes is haemosiderin only, and in relation

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LOCAL FORMATION OF BLOOD PIQMENTS

PLATE XXIII

WG. I.--TWo phagocytes from sub- FIG. 2.-Macrophege st,uffed with small cutaneous tissue of mouse containing hiematoidin crystals ; rat, 31 days numerous erythrocytes and some after injection of erythrocytes. Very hemosiderin granules, 6 days after numerous similar cells were present. injection. x 750. Unstained preparation. x 800.

FIQ. 3.-Two macrophages of mouse FIG. 4.-Later intracellular formation showing early intracellular formation of hematoidin ; mouse, 29 days. of hematoidin, 8 days after injection Some of tho crystals are now extra- of erythrocytes. x 800. cellular. x 600.

FIG. 5. - Intracellular formation of FIG. 6.-Fat globule containing hlenia- hematoidin after injections of b m o - toidin crystals (see text) ; mouse, globin; mouse, 16 days. X600. 20 days after injection of rabbits'

All figures are from " spread " preparations, which, except those in figs. 1 and 6, have been treated with hydrochloric acid and ferrocyanide of potassium and stained with carmalum.

erythrocytes. x 750.

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J. 8. F. R. FIG. 7 . 4 4 . Macrophages of mouse after subcutaneous injections of haemoglobin ;

a, 2 days, showing diffuse iron reaction ; b, 12 days, showing early formation of crystals of hamatoidin; c, 16 days, later stage with diminution of hemosiderin.

d-h. d and e, types of cells containing abundant hemosiderin several days after injection ; f-h, types of hematoidin formation in isolated cells, 10, 15 and 29 days respectively after injection. x 850.

Macrophages after injections of erythrocytes;

J. 5. F. N.

FIG. 8,Spread preparation of subcutaneoua tissue of mouae 12 days after injection of mouse erythrocytes. The brown area is a large collection of iron-free phagocytes containing crystalline hrematoidin and represents its formation on a massive scale. This area is surrounded by collections of hamosiderin- containing phagocytes free from haematoidin. At the junction of tho two areas phagocytes containing both kinds of pigment are seen. x 50.


to the interpretation of the changes which follow an important point is that at no period is there any evidence of extracellular formation of pigment. In parts where extensive formation of haematoidin is going to occur the first changes are the disappearance of haemosiderin or rather of the iron-reacting substance, and the formation of the diffuse yellow pigment. Thereafter the yellow pigment crystallises out and thus we find collections of phagocytes packed with haematoidin crystals, whilst hsmosiderin is scanty or absent. Such collections or depots of haematoidin-holding cells are thereafter a prominent feature, and in their neighbourhood one finds other areas in which haemosiderin alone is present in large amount (fig. 8). Both the diffuse yellow pigment and the hamatoidin crystals give a definite Gmelin reaction. If one treats a yellow patch where both are present with weak caustic potash solution to dissolve the crystals and then exposes it to the fumes of nitric acid one gets the typical play of colours-green, blue, violet, red-the colour thereafter disappearing. The reaction is given by the cells containing t,he diffuse pigment as well as by those containing crystals. It is thus clear that the former is bilirubin which afterwards separates out as haematoidin crystals.

In the case of haematoidin formation in the cells scattered in the tissues, a similar law holds-the more haematoidin the less haemosiderin-and we may add that when crystals are well formed the haemosiderin often gives an imperfect prussian blue reaction or a sort of washed-out appearance. In fact, we may say that as the crystals increase in number we get all stages of iron reaction down t,o complete disappearance (fig. 7, f-h). The changes seen in such cells suggest a more gradual change in the pigment than is seen in the areas where crystal formation is extensive. They correspond in a way with what has been observed in tissue cultures and might bc interpreted t-a indicating the splitting of haemoglobin into an iron-containing and an iron-free moiety. It is to be noted, however, that in our experiments the formation of haemosiderin always preceded the separation of haematoidin.

The changes which we have described do not occur in a uniform way. The yellow patches in which abundant formation of hematoidin takes place are found especially close to the original hsmatoma and usually at some distance from blood vessels. A not uncommon site is at the margin of adipose tissue and often the patches are actually in its substance. Phagocytes containing haemosiderin only are most abundant round blood vessels and nerves and in these situations haematoidin formation is unusual. Even after several weeks the cells in these situations may be quite free from haematoidin. The formation of haematoidin by the slower process in isolated cells is seen especially in intermediate regions but it is not possible to say on what conditions it depends.

190 R. MUIR A N D J . S. F . NIVEN

The latest period at which we have examined the tissue mas 36 days after the injection of blood. In this case haemosiderin and hamatoidin were still present in large quantity. In fact, the amount of haematoidin crystals waa the greatest met with in any of the experiments and some of the crystals were of large size. There mas evidence that some of the haemosiderin was undergoing absorption, many of the cells when stained for iron giving a diffuse and slight reaction. We have, however, not traced the ultimate fate of the two chief varieties of pigment. As has been said, at a latcr period haematoidin crystals are found outside cells, being either liberated by the cells or set free by their disintegration (fig. 4). In one experiment the process could be readily traced. In this case the mouse had been injected with rabbit’s corpuscles and the tissues were examined on the 20th day. Many of the phagocytes contained fat droplets in large numbers and in some of them haematoidin crystals were present also. Further, some of the crystals were actually within the fat globules. The fat then apparently became free and large extracellular globules containing crystals could be seen (fig. 6). Such globules then ran together and thus there resulted large collections of crystals in a matrix of fat, as it were. Rich, in his summary, says that hsmatoidin has a tendency to crystallise out in fat droplets. The appearances found by us seem, however, to be capable of explanation on the view that the crystals owing to physical properties are wetted by the fat. Thus when a crystal comes in contact with a fat globule the crystal or crystals pass into the globule just as two globules of fat become confluent to form a larger one. Fat droplets containing crystals may afterwards be set free and become confluent. Some of the extracellular globules were quite large and contained numerous crystals.

The changes following injections of rats’ blood into rats need not be given in any detail as they correspond in all essentials with those observed in mice. The amount of blood injected was larger than in the case of mice, viz. 2-3.5 c.c., and examinations were made at various periods from 36 hours up to 43 days. The naked-eye changes are similar to those already described-gradual disappearance of the blood with diffuse brown colouration around. At the end of 7 days little blood remained and after 15 days the red colour had quite gone. Phagocytosia of red corpuscles and formation of haemosiderin were observed after 36 hours and continued until the blood had disappeared. Unlike what occurs in mice, however, there is evidence of a certain amount of diffusion of hzemoglobin, most of the blood as seen in sections having a sort of fused appearance after a few days. Nevertheless active phagocytosis of red corpuscles is seen up till their disappearance. Though a certain amount of diffusion of haemoglobin may

B. Rats.

LOCAL FORMATION OF BLOOD PIQilfENTS 191

occur it is important to note that no extracellular formation of pigment was found ; this is in accordance with what occurred after injection of haemoglobin solution (vide infra), “ Yellow patches ” were found after about 8 days and, as in the case of the mice, the cells contained m u s e pigment of lemon-yellow colour, many giving no iron reaction. In these cells the pigment soon began to separate out in the form of minute rhomboidal crystals, usually of smaller size than those observed in mice (fig. 2). At a later period crystals of larger size were met with. Sometimes the haematoidin separated out in the form of granules or coccoid bodies with characteristic mahogany colour, and this might be seen over a considerable area. The process was noted in cells which had a m u s e yellow colour and gave a negative iron reaction. In some of the cells there were also imperfectly formed crystals. We have been unable to determine on what factor this phenomenon depends ; it seemed to occur in quite an irregular fashion. At the latest period of examination (43 days) hzmatoidin and haemosiderin were still present in large amount. Nany of the phagocytes containing the hsmosiderin mere of unusually large size and some of this pigment was in the form of small spheres up to 6 p in diameter, as well as in ordinary granule form. These spheres or globules gave a greenish tint when treated with ferrocyanide and hydrochloric acid, instead of the usual blue. We have not traced the fate of the hsmosiderin at a later stage.

The results of experiments on rats are thus essentially similar to those obtained in mice. They show clearly that the formation of bilirubin and haematoidin occurs within cells and follows the hsmosiderin stage. Also, the haematoidin crystals may form (a) rapidly in cells containing diffuse yellow pigment without iron reaction, or ( 6 ) more slowly in cells containing hsmosiderin ; a few crystals appear and then their number increases as the hsmosiderin diminishes. The interpretation of these changes is discussed below.

A number of these experiments were carried out in the usual way, fresh rabbits’ blood being injected subcutaneously and examined a t various dates up to the 35th day ; but they were not continued in detail as formation of haematoidin was not found. There was phagocytosis of corpuscles and this went on until the hzmatoma had disappeared, but the phagocytes usually contained only a few erythrocytes and one did not find the cells stuffed with them as in the experiments in mice and rats. Abundant formation of hsmosiderin within phagocytes occurred as usual and the pigment increased in amount as the blood was disposed of. At the end of 12 days red corpuscles well preserved were found in the hsmatoma but there were also at places signs of lysis of the corpuscles. The disposal of the blood thus appeared to be chiefly due to a somewhat slow process of phagocytosis. At the later

C. Rabbits.

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stages some of the intracellular pigment had an imperfectly crystalline character and somewhat reddish tint but one could not convince oneself that this was hsmatoidin. X o rhomboidal crystals of hsmatoidin were found. As already stated, Langhans also obtained negative results in the rabbit.

The difference between the mouse and rat on the one hand and the rabbit on the other, as regards the formation of hzematoidin in coniparable conditions, was noteworthy, and it appeared that this might depend on the red corpuscles or on the phagocytes. To test this point we injected a series of mice with rabbits’ corpuscles and examined them on the 5th, gth, 20th and 24th days ; the earlier stages were not investigated. On the 5th day there was a moderate degree of phagocytosis of red corpuscles and extensive formation of hsmosiderin. Some cells showed a diffuse lemon colour with negative iron reaction. On the 9th day the changes were more marked and there were a number of distinct yellow patches as described above. Microscopic examination showed haeniatoidin cryst,als within celh and also some diffuse yellow staining. On the 20th day there was extensive formation of haematoidin crystals and many of the cells containing them did not give an iron reaction ; the crystals were still more abundant on the 24th day- It was thus clear that haematoidin formation occurred from rabbit’s corpuscles as readily as from mouse’s corpuscles. This is in accordance with the results obtained by injecting rabbit haemoglobin into the mouse (p. 193). The difference between the rabbit and the mouse (or rat) thus depends on difference in the action of the phagocytes.

Experiments with injections of hamoglobin.

In these experiments a solution of rabbit’s haemoglobin in normal saline was injected into mice. The solution was prepared as above described and it is important to note that it was freed of stromata by filtration. The concentration was about 60 per cent. according to the Haldane hremoglobinometer and the amount injected at one time was 1 C.C. When the experiments were continued over several days the injections were repeated daily, aa much of the haemoglobin becomes rapidly absorbed. This of course complicates the picture as haemoglobin or its derivatives are present at different stages of change. One can, however, estimate the earliest appearance of any change in the haemoglobin from the time elapsing from the first injection. The injections of haemoglobin had a peculiar action in leading to edema of the tissues, a change not observed after a single injection of fresh blood. Another noteworthy feature was a considerable emigration of polymorphonuclear leucocytes ; these cells, however, were never found to contain pigment.

The naked-eye changes cannot be properly interpreted in view

LOCAL FORMATION OF BLOOD P I Q M E N T S 193

of the repeated injections of fresh haemoglobin but it may be stated that some brownish colour is to be noted at the periphery of the site at the end of 24 hours and this gradually becomes more distinct. After about a week the central part of the site of injection haa a deep brown colour and this gradually fades off to a pale yellow at the periphery. There is a t the same time little evidence of red colour such as one might expect from the haemoglobin injected on the day before the animal waa killed. We wish to make it clear that in this series of experiments we have not dealt with the question as to whether the haemoglobin undergoes change outside cells. We can say, however, that all the derived pigment when first formed waa found to be within cells and also that there waa no evidence at any time of extracellular formation of pigment in either granular or crystalline form. The results are to be interpreted along with what wae found when fresh corpuscles were injected.

When the site of injection was examined microscopically after 24 hours there were found numerous spherical macrophages (free histiocytes or monocytes) and, on being treated with ferrocyanide of potassium and hydrochloric acid, the cytoplaam of these was found to give a slight iron reaction ; the blue tint was quite diffuse and very faint though distinct (fig. 7, a). At the end of two days the blue staining was more pronounced and, in addition, there waa slight blue stippling owing to formation of some haemosiderin in granular form. The macrophages were of larger size and varied more in form. On subsequent days the hamosiderin increased in amount and the granules became coarser, the appearances coming to resemble those seen after injections of corpuscles.

Small haematoidin crystals were observed first on the eighth day, when they were seen within cells containing a considerable amount of hzmosiderin. Later they became more numerous and of larger size, whilst haemosiderin diminished in quantity (fig. 7, b and c). We did not carry the experiments beyond a period of 27 days and have not traced the subsequent fate of the pigments. The results of these experiments correspond closely with what we found in the case of injection of corpuscles; they appear to show that the haemoglobin is taken up as such by macrophages, that it then rapidly undergoes change and gives a M u s e iron reaction, that granular haemosiderin then appears and that later haematoidin crystals are formed. The changes thus clearly support the view that at a time after hamosiderin is formed it still contains the bilirubin moiety and that the latter becomes split of f and finally crystallises aa haematoidin when it is in sufficient concentration. Along with these latter changes there is a distinct diminution of iron-reacting pigment.

In a recent paper Kedrowski (1933), studying the cellular

This is a matter for further enquiry.

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reaction following the intraperitoneal injection of hsmoglobin in mice, describes the occurrence of a diffuse iron reaction in macrophages as early as 6 hours after injection. (We ourselves have not examined the tissues aRer hemoglobin injection at an earlier period than 24 hours.) Kedrowski, however, makes no mention of the occurrence of haematoidin a t a later stage and, so far as we can find, intracellular formation of crystalline liaematoidin from hemoglobin in solution has not been demonstrated experimerhally.

Discussion.

We have given an account of t)Iie actual fads which have been established by numerous observations and have now to consider their interpretation. We shall deal with the results in the mouse, but those in the rat correspond with the exception that there is some diffusion of hemoglobin from the erythrocytes. At a certain stage (about the seventh day) all the pigment within cells is haemosiderin ; further, there is no extracellular pigment. A little later, various cells especially in the yellow patches cont,ain a rather diffuse yellow pigment which does not give the iron reaction. This is found by the Gmelin test to be bilirubin. So far as we can see there is only one explanation of this, namely, that the hsmosiderin )’ when first formed still contains bilirubin constituents and that a t a certain time and under certain conditions there occurs a change whereby the iron becomes discharged from the cells and bilirubin is set free, thereafter erystallising as haematoidin. The three stages in this rapid or massive formation are accordingly (a) hemosiderin formation, ( b ) formation of diffuse yellow pigment, and (c) formation of hsmatoidin crystals. Thus in the last stage numerous crystals occur in cells with negative iron reaction. But in other parts one meets with cells in which one or two crystals are present along with much haemosiderin and it is seen that, in such cells, as the crystals increase in number the haemosiderin diminishes till it completely disappears (fig. 7, f-h). Here the same process is apparently concerned-a splitting-off of bilirubin from haemosiderin with discharge of the iron. Such is our interpretation of the changes found and, seeing that there is no evidence of extracellular formation of pigment, it seems to us to be the only one possible.

It is, of course, well recognised that hamosiderin does not represent a defhite compound, but ia merely a derivative of hsmoglobin which gives the iron reaction ; the iron may be in vrtrious conditions of combination and present in varying proportion. Most writers, however, still consider, as has been stated, that haemosiderin and haematoidin are separate derivatives of haemoglobin, formed under different circumstances. The conclusion drawn that in the conditions of our experiments the haematoidin


is split off from the iron-reacting pigment is quite in conformity with other facts. For example, the earliest formation of an iron- reacting substance from haemoglobin may be seen in the cells of the convoluted tubules of the kidney in haemoglobinuria. Within 24 hours after the occurrence of the latter these cells give a diffuse iron reaction and this is apparently due to some slight change in the haemoglobin. There is no justification for supposing that at that period bilirubin has been split off from the iron-reacting substance. Brown (1911), moreover, has shown that haemoglobin may give an iron reaction after oxidking with hydrogen peroxide, and probably the changes in the kidney cell are of somewhat similar nature. Two or three days after haemoglobin is absorbed by kidney cells the substance giving the iron reaction passes from a diffuse form into a granular state, the granules giving a pure prussian blue reaction. Again there is no evidence that the bilirubin molecule has been split off when this stage is reached. Our experiments with injections of haemoglobin into the subcutaneous tissue give quite similar results. A diffuse prussian blue reaction is given by the histiocytes within 24 hours and somewhat later the hsmosiderin appears in granular form. But further, these experiments give conclusive evidence that the bilirubin is still present, seeing that hEmatoidin crystals form some days later within cells containing haemosiderin. Whilst we consider that the conclusions which we have drawn are justifiable, they of course apply only to the conditions of the experiments and we make no statement with regard to the constitution of hsmosiderin in general. In other conditions the iron is no doubt part of a simpler molecule. Cook (1929), for example, has investigated the nature of the haemosiderin occurring normally in the horse’s spleen and has found that it consists of organic granules impregnated with some form of ferric oxide. The iron compound can be recovered from the granules by treatment with acid, leaving the substrate practically inert. In haemochromatosis also the proportion of iron in the hsmosiderin is very high and apparently it is in relatively simple form.

We wish to make it clear that the observations which we have recorded apply only to the conditions of the experiments. In particular, we do not wish to pronounce with regard to extracellular formation of haematoidin crystals, which is so often indicated by what is seen in human material. We may mention, however, that we have found in a case of cerebral haemorrhage similar appearances to those described in the experiments. The hzmorrhage was one of three weeks’ duration and in film preparations all stagea of pigment formation were found. There were cells containing diffuse yellow pigment, in some of which hsmatoidin crystals were present ; others contained crystals alone, others

196 R. MUIR AND J . S. F. NIVEN

granular haemosiderin alone and again others both varieties of formed pigment. In sections there was seen a zone of phagocytes containing numerous hamatoidin crystals next to the clot, many of the cells giving no iron reaction, while outside this zone there was another in which numerous cells containing abundant granular hemosiderin were present ; between the two zones the pigment in the cells was intermediate in character. We are, however, not yet in a position to speak further with regard to the changes in human lesions.

CONCLUSIONS. (1) In mice and rats the formation of granular and crystalline

pigment from erythrocytes injected subcutaneously is intracellular. (2) Phagocytosis of erythrocytes is followed by rapid forniation

of hsemosiderin, evident from 24 hours onward after the injection of blood. The haemosiderin is found in both diffuse and granular forms. These changes go on till all the erythrocytes are disposed of.

(3) Formation of haematoidin is first seen at about the seventh day and may occur in two ways :-(a) in cells containing haemosiderin a few crystals first appear and then their number increases ; (b ) in cells which have a bright yellow protoplasm and give no iron reaction haematoidin often forms rapidly in a massive fashion by crystallisation from the diffuse yellow pigment. The yellow pigment and the crystals give the Gmelin reaction for bile pigment. The interpretation made is that the diffuse yellow pigment is formed from “ hsemosiderin,” the iron being apparently diecharged in some form.

(4) The changes following injections of haemoglobin are of a corresponding nature. Iron reaction of a diffuse character may be detected in cells within 24 hours and granular hzemosiderin appears later. The formation of haematoidin crystals may be observed on the seventh day and takes place in cells containing hzmosiderin ; at a later period it may be relatively abundant. No formation of granular or crystalline pigments has been observed outside cells.

( 5 ) In rabbits the formation of hsmatoidin has not been observed after injections of erythrocytes or haemoglobin.

The photomicrographs have becn made by Mr John Kirkpatrick, technical assistant in the Pathology Department of Glasgow University, and we have pleasure in thanking him for the skilful way in which he has executed them, also for his valuable assistance in preparing hmnoglobin solutions and in other ways.

REFERENCES.

BROWN, W. H. . . . . J . Exper. Med., 1911, xiii. 477. CORNHEIM, J. . . . . . Vorlesnngen uber allgemeine Pathologie,

Berlin, 1877, i. 329.


COOK, S. F.. . . . . HUECK, W. . . . . .

KEDROWSKI,B, . . . .

LANGHANS, T. . . . . . MUIR, R., AND YOUNG, J. s. . NEUMm, E. . . . . . NIVEN, J. S. F.. . . . . QUINCEE, H. . . . . . RICH, A. R. , . , , .

SKRZECZKB, 0. . . . . . . . . . . 9 7 , I

VIRCROW, R. . . . . .

J . Bwl. Chem., 1929, lxxxii. 595. Ziegler’s Beitr. z. path. Anat. u. z. allg. Path.,

Ztschr. f. Zellforsch. u. mikr. Anat., 1933,

Virchow’s Arch., 1869-70, xlix. 66. this Journal, 1932, xxxv. 113. Virchow’s Arch., 1888, cxi. 25. this Journal, 1935, xli. 177 Virchow’s Arch., 1884, xcv. 125. Bull. Johna Hoplcina Hosp., 1924, xxxv. 415. Phys. Reviews, 1925, v . 182. Ziegler’s Beitr. z. path. Anat. u. z. allg. Path..

virchozu’s Arch., 1847, i. 379.

1912, liv. 68.

xvii. 587.

1887-88, ii. 275.

JOUIU?. OF PATH.-VOL. XLI. N 2

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The local formation of blood pigments