CHAPTER XII: Final discussion

CHAPTER XII.

Final discussion.

In chapter 1, page 25, the problems which remain unsolved after a survey of the literature are stated in the form of 5 questions. Some of these questions may now be answered by means of the results, of the investigations reported in chapters VIII-XI.

1) When does macrocytosis appear in liver diseases? How fast does the phenomenon appear and disappear ?

The present investigation gives the same results as previously reported by a number of authors: A constant increase in the mean diameter, without a simultaneous increase in the cell volu~~le, is seen in all cases of hepatitis. In hepatitis there is therefore a macroplaniao of the blood cells, as previously pointed out by ARCHI, SCIIULTEN and DEDICHEN. This fact explains the discre- pancy between the authors who have determined the mean diameter of the red cells, and the authors who have determined the mean corpuscular volume. In patients with obstructive jaundice, the size of the cells is normal.

The macrocytosis persists during the whole course of the disease, provided the patient does not receive any special treatment (see later).

IVhen the p atient recovers, the macrocytosis disappears, and 1ID becomes normal. The change in MD average in my material 0.014 my per day, which agrees well with observations by MOGEN- SEN and C. GRAM.

1 70

2) Is the macrocytosis due to changes in the peripheral blood, or is the cause a changed erythropoesis?

In chapter I X i t is definitily shown that none of the changes in the peripheral blood with which earlier authors have tried to explain the phenomenon, can be the cause. In chapter X i t is shown that the macrocytosis is due to a changed erythropoiesis: Mac- rocytes, pathological large cells, with an average increase in diameter: 0.85 my, are produced, and the blood cell population in the peripheral blood becomes heterogeneous. The altered mean din- meter of the blood sample is only an expression of the number of macrocytes present in the peripheral blood. Since the blood cell population becomes heterogeneous, the distribution curves of the red cell diameters will be broader than normal, with increased standard deviation. These findings explain the clinical observations made by HAMMARSTEN, that an increased standard deviation of the frequency 'curve indicates that the liver disease is still active. When the patient recovers, the macrocytes are substituted by normocytes. The production of normocytes occurs in concordance with the biological growth formula, and a t approximately the same rate as that with which erythrocytes are produced in pernicious anaemia during treatment. I t seems reasonable to assume that the production of macrocytes in the beginning of the disease occurs a t the same rate.

3) Has the macrocytosis in liver disease the same cause as the macrocytosis in pernicious anaemia?

In chapter X I i t is shown that macrocytosis in liver diseases is not identical with macrocytosis observed in untreated pernicious anaemia. In untreated pernicious anaemia we find megalocytes and microcytes, two cell forms which are not observed in liver diseases. But we also find macrocytes, and these macrocytes cannot be distinguished from those seen in liver diseases by any of the criteria which are utilized in this work. When patients with pernicious anaemia receive adequate treatment the cells which are typical for this disease disappear. But the macrocytosis remains in spite of the treatment. According to calcula- tions based on MOGENSEN'S investigations this macrocytosis re-

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mains €or months and years after tlie specific treatment has com- menced, and in spite of full remission of the haemoglobin and the number of red blood cells.

This, together with the arguments which are given in the introduction (page 20), makes i t unlikely tha t the macrocytosis in liver diseases is caused by any lack in the antipernicious principle, as usually assumed (see page 22).

4) If the macrocytosis in liver disease and in pernicious anaemia is not due to the same cause, what is then

the cause of this phenomenon?

This question cannot be answered by the previous investigations. But these investigations may possibly indicate how a solution may be attempted.

The normal erythropoiesis.

The normal erythropoiesis demands that two processes must follow a normal course: Firstly, a sufficient number of erythrocytes must be produced in the bone marrow, to replace those blood cells which perish in the peripheral circulation. Secondly, tlie haemoglobin synthesis must be normal, so that these newly formed blood cells become saturated with haemoglobin.

CAPPS, already in 1903, showed that we here have two distinct processes, which do not necessarily follow a parallel course. And this has later been confirmed by a number of authors: By SJO- WALL in bleeding experiments, by ~ Y H I P P L E , ROBSCHEIT- ROBBINS and others in feeding experiments, and by HAWKINS, HAHN &: others in experiments with radio-active iron. SCHIODT, and later GJERDSP), have shown that the synthesis of liaemoglobin also occurs in concordance with the equation for mono-molecular auto-catalytic reactions, but with another value €or K than the one which applies to the production of the cells. And finally, THORELL has recently, by analysis of the single cells, shown that the haemoglobin synthesis is an independent process.

The formation of the erythrocytes takes place in the bone marrow from a relatively small number of parent cells. The forma- Lion depends upon two distinct processes, growth and maturation

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of the cells. The growth, i. e. the formation of a sufficient number of cells, is connected with the youngest precussors of the blood cells. Already the number of the different cells in the bone marrow seems to indicate this: 2-G yo of young immature proerythroblasts gives rise to 35-40 yo basophile normoblasts and to 50-60 yo orthochromatic normolhsts (\\'HITBY & BRITTON). Some liaemato- logists (ROHR, KIENLE, HABELMAN) are opposed to this view, but THORELL, in the above mentioned work, has shown that the new- formation of the cells (the growth proper) takes place at the earliest stages of development, and that i t is normally finished, or nearly finished, when the maturation starts.

The maturation of the cells, or the development from the younger forms to the mature erythrocyte, also takes place in the bone marrow. There is some disagreement as to whether this development from the immature to the mature cell is a continuous process (\\'HITBY & BRITTON, HADES (8), SCHILLING, NAEGELI), or if the process is discontinuous. This is maintained by KIENLE (mitosis studies), FREERIGEN (comparative anatomical studies) and by FREERKSEN, ALDER and ROHR, based on clinical observations and morphological studies.

If thc haemoglobin synthesis is reduced, while the cell produc-

If the grozulh of the cells is restricted, we get hypoplastic, re-

If the maturation process is disturbed, we ohlain changes ol the

tion remains normal, hypochromic anaemia develops.

spectively aplastic anaemias.

kind which are discussed in the present work.

Disturbed mnlurafion of the blood cells.

There is, as menlioned, no agreement whether the maturation is a continuous or a discontinuous process: \\.%ether one cell, as for instance a pro-erythroblast, may divide into two proerythroblasts (homoplastic division) or into two macroblasts (heteroplaslic division).

\Vithout taking any side in this controversial question, one must, however, be allow+ed to maintain that the development of the cells passes through one or more (critical stagesn. In order

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to bring the development of the cells through these slages certain substances or principles, only partly known, are necessary.

The proerythroblast represents one such ((critical stager. The development of the cells will be completely or partly arrested here if there is a lack in the ((anti-pernicious principle)). The growlh will continue as before (THORELL) and the hone marrow presents the picture typical for untreated pernicious anaemia - a marrow increased in volume and invading the fat ty marrow. The greatly increased number of cellular elements mainly consist of the younger forms of the erythropoietic cells, the proerythroblasts. Simultane- ously, an absolute and relative reduction in the number of normoblasts occurs. The proerythrohlasts mature into megaloblasts, cells with an abnormal pycnotic nucleus, partly or wholly filled with haemoglobin. These meglohlasts pro1)ably give rise to the megalocyles in the peripheral blood. A salient feature is t h a t these cells are not reduced in size during maturation, in contrast to erythrocytes formed in normal marrow’. As an expression for a materially reduced maturation and production of erythrocytes. anaemia develops.

When these patients receive tlie canti-pernicious principle)) the maturation continues. Already the day after the treatment has started, the marrow is seemingly normoblaslic and apparently normal blood cells are streaming into the blood. The whole picture is what we might expect if a blockade had heen lifted.

But the erythropoiesis is slill not normal. The ccnormoblasts) of the bone marrow are still larger than normal (NORDERSON (4) and this macroblastic marrow remains long after the reticulocyte crisis is finished. (NORDENSON (4), SCHULTEN (3). A t the same time macrocyles circulate in the peripheral blood, and this macrocytosis may persist for months and years after the treatment has been started, and long after the blood counts have returned to normal values (KIRK (2), MOGENSEN (l) , ScHuLTm (1) (see also page 167). I t is reasonable to see these t w o conditions as related, and to assume tha t the macrocytes in the peripheral lilood originale from the macroblasts in the bone niarrow.

The similarity between the changes seen in the bone marrow in pernicious anaemia and in liver diseases makes i t probable tha t the macrocytosis as wdl, is due to a deficient maturation, but at a later stage in the developrncnt than the mcgaloblastic stage.

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Even in macrocytosis we find an increase in the number of the younger erythropoietic cells, and an increase in the volume of the red bone marrow. (ROSSIER, BLEICHROEDER). The increased number of erythropoietic cells is mainly due to macroblasts (ISAACS, ALDER), as if the growth and the development progressed normally until this stage. These macroblasts seem to be more easily developed in to macrocytes than the megaloblasts are developed into megalocytes, since we do not obtain the same grave anaemias as observed in pernicious anaemia. But a moderate degree of anaemia is observed here as well (see page 120). In liver diseases the number of macroblasts in the bone marrow is greater than normal. IYhen the blockade is lifted the maturation progresses, and normoblasts with subsequent formation of normocytes are formed. The production of normocytes then occurs a t the same rate as the production of erythrocytes in pernicious anaemia during specific treatment. This also confirms that the two pheno- mena in principle are of the same nature. Both in pernicious anaemia and in liver diseases, we are faced by a deficient maturation.

The cause of deficient maturation.

CASTLE & PIIisoT have shown that the rnegaloblastic bone marrow in pernicious anaemia is due to a lack in ((the antipernicious principle)). \Ye now know that this principle is not one single substance, but consists of a series of components where each component has its specific action (DEDICHEN, BJQRNsON, BARFRED). For some time after the discovery of folic acid one believed that one had found the specific substance, necessary lo mature the proerythroblast into the macroblast, and that a deficiency in this substance leads to the development of megaloblasts. More recent researches (HEINLE, DINGLE & WEISBERGER, VILTER, VILTER & SPIES, HADEN (9) and others), seem to indicate that a lack of folic acid is no1 always the cause of deficient maturation. I t is more probable that folk acid exists in the food in conjugated form, and that we need another principle to release the folk acid so that it may be utilized in the organism.

Folk acid belongs to the vitamin-B group and i t is probable that other members of this group also take a part in the erythropoiesis.

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Thus GOODHART, VAUCHAN (1) and WILLS mention that they have seen improvement in macrocytic anaemias after administration of yeast and manitt and i t has also been shown in animal experiments that a series of the Vitamins B are of importance (see survey by WINTROBE, 7, page 97).

CAYER, RAFFIN & PERLZWEIG have shown tha t the amount of niacin, riboflavin and thiamin is sub-normal in patients with pernicious anaemia and AHLSTROM has found sub-normal values for vitamin-B in patients with liver cirrhosis. VILLA and BEICL- BOCK & SPIESS-BERTSCHINCER finally report tha t liver diseases and the accompanying anaemias improve by the administration of large doses of niacin.

These observations may possibly indicate that the cause of the deficient maturation is due to a lack in one of the members of the vitamin-B group.

Does a lack of uitamin-B play a role?

To answer this question the present material of chronic hepatitis may be used. These patients have, as shown in table 46, a macrocytosis which persists through weeks and months. If the reason is tha t these patients lack a certain principle, then one might expect that the macrocytosis would disappear when this principle was supplied. The maturation should become normal, and normocytes ought to appear in the peripheral blood a t a rate which may be predicted by the equation for biologic growth (see page 152-156).

If this hypothetical principle was again withdrawn, one might expect that the macrocytosis in the peripheral blood would re- appear, and again at a rate which may be predicted by the formula.

The author has carried out some experiments to test this hypothesis :

Fig. 38 shows the number of normocytes in millions per mm3 in 4 patients with chronic hepatitis who did not receive any treatment. The curves show that the number of normocytes remains fairly uniform through several months.

Fig. 39 shows similar curves from 4 patients with spontaneous improvement in their chronic hepatitis. The number of normo-

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cytes increases to some extent, and the agreement with the biological equation of growth is in all cases fairly good.

Fig. 40 shows the curves of 3 patients who received liver extract as used in pernicious anaemia without an increase in the number of the normocytes.

Fig. 41 shows the curves from 2 patients who received folic acid without any effect.

Fig. 42 shows the curves from 3 patients who received liver extract with an addition of the vitamins-B (Hepto-B), without effect.

Fig. 43 shows that vitamin-K has no effect. Fig. 44 shows thal aneurin has no effect and fig. 45 shows

that neither does lactoflavin lead to any increase in the numl~er of normocytes.

Fig. 38-50: The number of normocytes (in mill. pr. mm3) in cases of chronic hepatitis. The stippled curves represent the curve of biologic growth and indicate the rise and fall one must expect in the number of normocytes if the treatment

is of any significance. The numbers refer to case-numbers in table 46.

I ID---- 20 10 4 0 SO--- 60 10 e m

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-------- lo 20 so 4 0 so b0 10 oa Dart

Fig. 42, 43.

12

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20 5 0 4 0 50 (0 I0 10 D o y i

Wiacln.O.2Oq dally Im. I - 7 I '

' ,'

- - -

,I+/, -

10 2 0 SO 4 0 50 60 70

Fig. 46, 47.

Fig. 46 and 47 shows similar curves from patients nos. 29 and 30. These two patienls received niacin, 0.20 daily, i . m. The number of normocytes in the blood of these patients increased during the treatment and reached nearly normal values. The increase occurred in close concordance with the biological equation of growth. When the nieacin treatment is discontinued the number of normocytes recede to the original values at the expected rate.

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Fig. 48 is from patient no. 34. The dose of niacin was in this case only half of the former, 0.10 daily. Even here we find an increase in the number of normocytes, but the increase appears somewhat later than in the two first cases. But when the niacin treatment is discontinued, the normocytes recedes to the original values in concordance with the theoretical curve.

Fig. 49 shows a similar curve from patient no. 43 who received niacin in three pcriods. The dose was the first two times, 0.10 daily and the increase and the decrease of the normocytes appeared as expected. The third time the dose was only 0.05 gr. daily, and this time the effect was absent.

Finally, fig. 50 shows the curves from 3 patients who also received niacin, but where the effect was absent. In cases nos. 38 and 40 there is an increase in the number of normocytes, but the increase is not in concordance with the theoretical curve and in case no. 40 at least, the rise starts before i h e treatment is begun. This is probably a case of spontaneous remission. In case no. 32 there was definitely no effect.

These experiments are too few, and give too conflicling resulls to be regarded as decisive. Niacin does perhaps play some role in the ripening of the macroblast into a normoblast. The reason for the failure in the last three cases may be that the organism needs a sufficiently large dose, and that the niacin is without any effect in insufficient quantities. Perhaps the demonstrated curves are only due to spontaneous remissions and therefore of no conse- quence.

The question can not be decided without further investigation. One might examine more patients with chronic hepatitis. I have had no opportunity to do this, owing to lack of material. I t would be better to investigate the condition of the blood cells in experimentally-caused jaundice. Such an experimentally produced jaundice might also definitely answer the question whether the macrocytosis appears and disappears at the same rate. Even if this is probable, i t has not been proved in the present work (see page 156). 0. HANSSEN and P. HANSSEN have shown that one may cause a jaundice resembling acute benign hepatitis by administering lactofenin. I have carried out several such experiments, but have been unable t o produce jaundice, perhaps because the lactofenin used was of a poor quality.

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Finally one might answer the question by animal experiments, as mentioned by HIGGINS & STASNEY and HEINLE, CASTLE & ROSE. This comes however outside the frame of the present investigation.

5) Which diagnostic and prognostic conclusions may be drawn from the presence of macrocytosis?

This, the fifth and last question put forward in the introduction, page 25, may now be answered: The demonstration of macrocytosis in a case of jaundice signifies a disturbed erythropoiesis connected with hepatocellular damage. In jaundice due to occlusion, without simultaneous hepatocellular damage, macrocytosis does not occur. This is borne out by the present investigation which confirms the earlier findings of SCHALM, HAMMARSTEN & CO-WORKERS, and others. HAMMARSTEN and CO-WORKERS have for a number of years studied the relationship between macrocytosis and other signs of liver disease, and have found a close relationship. Recently, LINDGREN (2), in an extensive survey, has shown that the degree of macrocytosis in liver disease is closely related to tests based on the hepatic excretory and secre- tory actions, as well as on the functions of the liver in the inter- mediary metabolism. He finds, on the other hand, no significant relation between the red blood cell mean diameter and tests based on the detoxicating ability of the liver, and tests em- pirically found to be of value in liver diagnostics. The present investigation confirms this, and the presence of macrocytosis may therefore be used both in the diagnosis and the prognosis of liver diseases. If the mean diameter of the red blood cells is definitely increased, whereas the mean corpuscular volume remains normal, i t is overwhelmingly probable that hepatocellular damage exisl.

But the method has a serious drawback: The range of the normal values for MD is so great that an observed value may be well within normal limits even with manifest macrocytosis. A deter- mination of the mean diameter alone, either by micrometry or by halometry, is therefore only of conditional value. To ascertain if macrocytosis is present, the complete analysis of the frequency curve is necessary. But this involves so much work, that the method can hardly be of any practical value as a method of routine.

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If one, by halometry or by micrometry, finds that tlie mean diameter of the red cells exceeds the upper normal value, one may conclude that macrocytosis is present. But the reverse conclusion, that a normal value for MD indicates a normal erythropoiesis, is not permissible. If an observed, too high value for MD gradually becomes normal during the course of the disease, then one may conclude that the erythropoiesis is becoming normal and that the palient will recover. If the MD remains too high, one may conclude that the hepatocellular damage persists.

The methods used for mehsuring the blood cells must be tried out by each individual investigator to ascertain the technical and personal factors described in chapter IV.

RCsum6 of Chapter XII.

In this chapler the questions stated in the introduction, pg. 25, are discussed.

The macrocytosis observed in liver diseases is a ccmacroplaniao, increased diameter of the blood cells without simultaneous increase of the corpuscular volume. The cause of the phenomenon is an altered erythropoiesis which occurs simultaneously with damage of the liver cells. The cause of the macrocytosis in liver diseases is not tlie same as the cause of macrocytosis in untreated pernicious anaemia. But both conditions seem to be due to an arrest of the maturation of the blood cells, occurring at different levels in the maturation process. While the disturbance seen in untreated pernicious anaemia seems to be related in one way or another to f,olic acid, this has no effect on the condition seen in liver disease.

Some experiments (figs. 38-50) seem to indicate that the cause of the disturbance in liver diseases in some way may be related to niacin. But the experimental results are too few, and too controversial to settle the question.

Finally, the practical use of the phenomenon in the diagnosis of liver diseases is discussed.

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CHAPTER XII: Final discussion