Keio Journal of Medicine Vol. 17, No. 2, June 1008

THE ROLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA - SOME ASPECT FOR ETIOLOGY AND TREATMENT OF APLASTIC ANEMIA

MITSUTO HASEGAWA, YASUO MATSUKI, SHINJIRO OZAWA,

and YASUHIKO ANDO

Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

(Received for publication April 3, 1968)

It is widely accepted9,17,20,25,33,34,35 at the present time that the plasma erythropoietin (EP) level in aplastic anemia is markedly elevated and that the bone marrow of the patient with this type of anemia does not respond to EP. However, careful review of the literatures disclosed that highly elevated EP levels in aplastic anemia have all been estimated by single method measuring Feb9 incorporation in small animals although the assay method of measuring EP is still controversial, and the experimental proof for the unresponsiveness of the bone marrow to EP has also been lacking. From this point of view, in this study, an effort was made to reevaluate the plasma EP levels in aplastic anemia with use of three different methods simul taneously on the same subject, and clinical and experimental studies were also carried out to clarify whether or not the bone marrow in this anemia would be responsive to EP. The purpose of this paper is twofold to show that 1) the EP level in aplastic anemia varies depending on the assay methods used and that 2) the bone marrow of the patient with aplastic anemia may well be able to respond to EP either in vitro or in vivo.

I. EP LEVEL IN APLASTIC ANEMIA AND OTHER SUBJECTS

Material Plasma samples were collected from the following seven groups of subjects ,

109 110 MITSUTO HASEGAWA et al .

Plasma extracts were made from them according to the method of Borsook.2 The plasma and plasma extracts were tested by Fried's6 and modified Matoth's21 method and the untreated plasma sample by Krantz's19 method for assaying EP levels. The number of material in each group was showed in parenthesis. A) Idiopathic aplastic anemia (13) B) Anemic donors (10). C) Normal individuals (8). D) Anemia due to renal insufficiency (8). E) Untreated, male, rabbits weighing approximately 3 kilograms. Hema tocrit and reticulocyte counts of this group ranged 30 to 43% and 3 to 9%, respectively (12). F) Rabbits rendered anemic by daily exsanguation of 20 ml of whole blood per kilogram for two consecutive days. All rabbits of this group have hemato crits less than 20% and reticulocyte counts above 120%0 (12). G) Rabbits rendered polycythemic by transfusions of 20 ml of normal whole blood per kilogram per day for two consecutive days. Hematocrit and reticulocyte counts of this group were above 60% and less than 3% respectively (12).

Technique of assay The method of Fried, modified methods of Matoth and Krantz were used to assay EP levels. The assay technique of each method is briefly described below. a) Method of Fried: Three to five Wister strain starved, female, rats weighing approximately . 150 grams were used as assay animals for each sample. The samples and Fe59 were injected as described elsewhere6 and Fe59 incorpora tion into red blood cells in 18 hours was measured as indicator of erythropoietic activity. b) Modified method of Matoth : The method of Matoth was followed with the modification in which the bone marrow was placed on a thin cover glass instead of a culture tube bottom thus simplifying the making of sample smears. The doses of colchicine were increased to 100 micrograms per tube and no antibiotics were added to the culture medium. When the plasma extract was assayed, 1.0 ml of normal rabbit serum was added to the culture medium.

c) Method of Krantz : The in vitro synthesis of heme by the bone marrow cells was measured as an indicator of EP activity by a slight modification of the method of Krantz et al. The incubation mixture consisted of Eagle's basal medium with 10% calf serum, one ƒÊc of Fe69cls, human bone marrow cell sus pension (blood group 0, 1.2•~107 per tube) and patient's plasma. The rabbit ROLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA 111 bone marrow cells for assaying rabbit plasma were incubated for 3-45 hours at

37•Ž in a roller tube. At the end of the indicated incubation intervals, the heme was extracted from the cultured cells according to the method of Teale,36 and the Heme-Fe59 was measured in a well-type scintillation counter.

Results Significantly elevated EP levels in the anemic rabbits and anemic donors, and low EP levels in the polycythemic rabbits and the uremic patients were observed by any of applied assay methods (Tables 1, 2 and 3). On the other hand, results obtained from the plasma of patients with aplastic anemia varied according to the methods used, the method of Fried given an elevated EP and the modified Matoth's and Krantz's methods giving a lower EP.

Table 1 EP Levels of Rabbit Plasma

*1 Mitotic index , number of colchicine arrested cells per 1000 mitotable normoblasts. *2 Fe59 incorporation into 2 .4•~106 normoblasts during 24 hrs in vitro. *3 Fe59 incorporation in 18 hrs in vivo . *4 Numbers of rabbit .

Table 2 EP Levels of Anemic Subjects

* Number of cases 112 MITSUTO HASEGAWA et al.

Table 3

Comparison of EP Activity

•¨ nomal •ª elevated •« lowered

. RESPONSES OF THE BONE MARROW OF THE PATIENTS WITH APLAS TIC ANEMIA TO EP DEMONSTRATED BY IN VIVO AND IN VITRO STUDIES

A) In vitro studies The effect of EP on the bone marrow of aplastic anemia was observed in a culture medium. Material and Method : The bone marrow suspension was obtained from two cases of aplastic anemia (blood group 0) and was cultured with the plasma of anemic donors (rich in EP), with that of normal individuals and also with the plasma of the patient with aplastic anemia (poor in EP measured by modified methods of Matoth and Krantz). All procedures of assay technique were carried out according to the forementioned method of Krantz with the exception that heme synthesis was measured at three different time intervals, 3, 12 and 24 hours. Response of the bone marrow of the patient with aplastic anemia to each plasma was compared with that of normal individual. Results : The bone marrow cells of the patients with aplastic anemia showed significantly increased heme synthesis in response to erythropoietic stimulus, as did the bone marrow of normal individual (Fig. 1). Although the definite rate of Fe69 incorporation was generally lower when cultured with the bone marrow of aplastic anemia patients, particularly in the case with hypoplastic marrow, heme synthesis was noted to be significantly elevated by the EP rich donor's plasma and lowered by the plasma of aplastic anemia patients at all sampling

‡U ROLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA 113

Fig. 1 Response of bone marrow to EP in vitro.

intervals showing the same reactive pattern to EP as observed with the normal bone marrow (Fig. 1). B) In vivo (clinical) studies As the preliminary experimental studies suggested a favorable response of the bone marrow of aplastic anemia patients to EP, therapeutic trials of EP rich plasma to the patients with aplastic anemia were carried out . Material and Method: Seven cases of aplastic anemia admitted to Keio University Hospital were submitted for this therapeutic study with EP rich fresh plasma. All of these patients had a primary idiopathic type of aplastic anemia except for one (Case SM in table 4) who was considered to have a secondary aplastic anemia caused by anti-epileptic drug (diphenyl hydantoin) . The age, sex, hematological findings before plasma transfusion , the interval after onset of the disease and amount of plasma transfused are summarized in table 4. None of these patients had shown a favorable response to any drugs given before the plasma transfusion and the red blood cell count had been maintained at the constant level only by periodic transfusions of whole blood . Plasma was Table 4 Effect of Plasma Transfusion in Aplastic Anemia

Cases Treated with EP Rich Plasma

Cases Treated with Normal Plasma

* Ep level measured by modified method of Matoth. ROLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA 115

Table 5

Criteria to Evaluate the Effects of Plasma Transfusion

obtained from anemic donors whose EP levels was determined to be elevated. The EP rich plasma of these donors was then transfused to the patients within 2 hours of collection. Doses of two hundred ml of the plasma were transfused each time, a total of 2,800 to 6,600 ml of plasma per a patient during a period of 22 to 80 days. During plasma transfusion, all drugs which could effect the hematopoietic mechanism were avoided. Periodical examinations of complete blood counts, bone marrow, serum iron levels and hemorragic tendency were performed. In view of the data obtained, the effects of plasma transfusions were then evaluated according to the criteria showen in table 5. Four patients with aplastic anemia serving as controls received transfusions of plasma from normal, non-anemic, individuals by means of the above described procedure (table 4) . Results : Effects of EP rich plasma were shown in table 4. a) Effect of EP rich plasma on peripheral blood: Transfusions of EP rich plasma produced a striking increase in the peripheral RBC count in 3 116 MITSUTO HASEGAWA et al. patients, (case) maintained the constant RBC level of one patient without whole blood transfusions which had previously been essential, and reduced the number of whole blood transfusions needed in another patient (table 4). One representative case to indicate favorable effects of EP rich plasma transfusion is shown in fig. 2. No effects on peripheral RBC were observed in one of idiopathic anemia cases (GI) and the secondary case .

Fig. 2 A case of aplastic anemia. M.O. 33 yr. •Š

Favorable effects on the RBC count were not observed in all patients treated with the normal control plasma. The serum iron levels decreased in 5 patients that received EP rich plasma and in 2 patients that received normal plasma. No favorable changes in the peripheral WBC and thrombocyte counts were obtained either by EP rich plasma or by control plasma transfusions. b) Effect on the bone marrow: Studies of the serial aspirations of the bone marrow revealed favorable effect of plasma transfusions. ROLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA 117

c) Effect on hemorrhagic tendency : Improvement of hemorrhagic tendency was observed in only one out of 7 patients receiving transfusions of EP rich plasma, however, 3 out of the remainder showed no bleeding tendency through out the course of this treatment.

DISCUSSION

As no chemical techniques for the measurement of serum EP level are yet available to date, it is only estimated by means of bioassays 6,21,29,30 Numerous studies have recently been reported on EP and in all of them EP titer was obtained by a single method of bioassay, although the method itself remains controversial at present. In the present study, each sample was tested by means of three different methods of bioassay, and the results were compared. No difference was observed among the data obtained by three different methods in all subjects except when the serum tested was that of a patient with idiopathic aplastic anemia. The quite interesting results obtained from sera of patients with idiopathic aplastic anemia were apparently different, rather contradictory EP titers, which were elevated by method of Fried and lowered by Krantz and Matoth. In all of the previous studies,9,20,35 the EP was invariably reported as extremely elevated except in one case of chronic hypoplastic anemia reported by Pillielo 27 These investigators11,25,32,37 postulated that the cause of an elevated EP titer in aplastic anemia was due to the increased production or liberation of EP being stimulated by severe anemia and the decreased consumption of EP by the deteriorated bone marrow. These considerations appears justified on the ground of the fact that urine EP level of the patients with aplastic anemia was reported to be also elevated. However, as previously stated, all these results were obtained by a single assay method, mostly by method of Fried, while no report has been made as to the EP titer of serum from patients with aplastic anemia measured by the methods of Matoth or Krantz. On discussing the reason for contradictory EP titers obtained from sera of patients with aplastic anemia by the use of different methods, two major causes, could be considered, namely the difference of the indicators used and the dif ferent conditions under which the three bioassays were performed . As an indicator to measure EP titer, the incorporation rate of radio iron into the eryth roblast was used (stage of polychromatic to orthochromatic normoblast) in the method of Fried, while the mitotic rate of erythroblast applied in the Matoth's 118 MITSUTO HASEGAWA et al.

method (stage of stem cell to mitotable erythroblast). Although the point of action of EP is still debatable, most investigators agree7,17,33,35 that it is the earliest stage of erythropoiesis (stem cell to pro normoblast). From this point of view, the stage of erythropoiesis where the indicator for the measurement of EP was used in the method of Matoth is considered as being closer to the acting point of EP than that of Fried. Besides, the results obtained by the method of Fried might be interfered with the heme synthesis accelerating factor which was reported24 to be elevated in cases of aplastic anemia. From these considerations, it can be concluded that EP titer is measured more accurately by the method of Matoth rather than that of Fried. However the fact that serum EP titer obtained by the method of Krantz was shown as low as the one obtained by the Matoth's method implies that these contradictions were not sufficiently explained by simply difference of indicator, because the indicator applied in the Krantz's method is the same as that of Fried's method. The other major differences among the three methods may lie in the conditions of assay. Namely, the EP titer is assayed in vivo by the method of Fried, and in vitro by the methods of Krantz and Matoth. In the in vivo bioassay considered the variety of conditions which may easily interfere the results, such as condi tions of assay animals before and during an injection of samples, the various modes of reaction to the injected material and the possible changes of the specimen in the body of the assay animal before reaching the target organ. The in vitro assay on the other hand is considered free from the above stated problems, although other factors such as the compositions, pH, tempera ture of the culture medium, and the procedure for washing glass tube, may also interfere the results. However these factors were considered to be neglected in the present studies because the same results were obtained by three method in, all other cases. From these points of view, the following hypothesis may be postulated to explain two different EP titers obtained from sera of patients with idiopathic aplastic anemia by different methods of bioassay as we previously reported.14,23 In the plasma of patients with aplastic anemia, EP inhibitory factor(s) as well as EP may be elevated, and when the plasma is measured by an in vivo assay such as the method of Fried, destruction or degeneration of the EP inhibitor may occur in the assay animal resulting in a high EP titer. On the other hand, in the in vitro assay, both EP and the EP inhibitory factor act directly on bone marrow thus having a neutralizing effect which resulted in a low EP titer in the methods of Matoth and Krantz. To suport this hypothesis, several re ROLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA 119

ports12,16,18 propose the existence of anemia producing factor ana Nakao26 report ed on the inhibitory factor of leucopoiesis in the plasma of patients with aplastic anemia. However, there is no proof of the destruction of the EP inhibitory factor in the assay animals. Another possibility is that EP may exist mostly as a form of precursor in the sera of patients with idiopathic aplastic anemia. Thus, when measured in vivo assay, this precursor may be activated in the assay animal resulting in hioh EP titer by in vivo method. while lowered by in vitro method.

In any event. it is interesting from an etiological and therapeutic standpoint that the low level of EP, contrary to the previous reports, was obtained from the

plasma of individuals with idiopathic aplastic anemia by using in vitro assay techniques. Because this fact may lead to the possibility that the decrease in active EP may he one of the maior nathogenesis of idoniathic anlastic anemia which is refraeforv to all drugs. and transfusions of large amounts of EP may be a wea pon in combatting the disease. In order to nrove this hvnothesis and to clarify whether or not the bone marrow of the natient with anlastic anemia will respond to EP. further experimental studies, primarily in vitro, were carried out. These studies revealed that the bone marrow of anlastic anemia patients shows an increased incorporation rate of radio iron in resnonse to EP rich

plasma a.s observed with the bone marrow of normal individuals. On the grounds of these experimental rPenlts. FP rich nlasma was given to patients with anlastic anemia as a clinical. therapeutic trial. The favorable effect obtained in most of cases treated with a large amounts of VP rich plasma was considered not only to recomfirm the results of in vitro study in which the bone marrow of anlastic anemia patients responded to EP. but may also serve as a mite interesting fact for the pathogenesis and treatment of anlastic anemia. EP has been theoretically considered25 ineffective for this disease. despite of absence of clinical experiments except for that of Gurnev.10 because investi gators believed that EP is highly elevated in plasma of patients with anlastic anemia. Gurneylo renorted two cases of hypoplastic anemia of 1 month old babies treated with large amounts of EP rich plasma . Reticulocytosis occured in both cases about 5 weeks after the treatment and finally EP was considered as effec

tive in one of the babies treated. However, in his report , the EP titers prior to treatment were not stated. No other report of aplastic anemia patients treated with EP or EP rich plasma has been made, however, Hatta'5 reported favorable effects of ceruloplas min and apoceruloplasmin which were proven to have EP-like action in experi- 120 MITSUTO HASEGAWA et at.

ments performed on animals. Cobaltous hydrochloride, which Fountain and others5 reported to be effective in the treatment of aplastic anemia, was also proven to elevate EP activity either in clinical experiment by Robinson et al.,28 or in animal experiments by Goldwasser et al3 All of these reports are considered to show the cases of aplastic anemia successfully treate'd by indirect administration of EP. However, for the remarkable effect obtained with EP rich plasma in the

present study, the following questions may be raised. The first one is that if EP rich plasma obtained from anemic donors was effective, whole blood transfusion from the same people should also have been effective. The second question is whether the effect of EP rich plasma may be due merely to nutritional supply rather than EP, because nutritional deficiency is still believed by some workers to be one of the etiologic factors of aplastic anemia. As to the first question. many reportsh8'22 have been made on the inhtbitorv effects of whole blood transfusion on erythropoiesis of the bone marrow, and Smiths' has insisted upon the harm of whole blood transfusion for such cases as anlastie anemia whose major nathogenesis is hvpoplesia of the bone marrow. Medici22 and Elslev4 also reported that red blood cells have a direct inhibitory effect not through EP, on erythropoiesis of the bone marrow from their experi mental studies with animals. We13 have also reported supnressive action on erythropoiesis by transfusions. Accordingly EP rich plasma was considered to have an effect superior to that of whole blood transfusion which contains both EP and red blood cells. The nutrition supplying effect of this treatment is also a point to be dis cussed. however, this may be neglected by the fact that the administration of the same amount of plasma from normal persons (non-anemic) as controls. and high caloric diet, or large amounts of amino acid or glucose solution were inef fective in all cases. Besides, none of the patients treated here appeared to be in the state of nutritional deficiency. One of two cases which showed no favorable response in peripheral blood to EP rich plasma was a secondary aplastic anemia due to anti-epileptic drug and was included in this experiment as a control. Another case is the patient who had been suffering from aplastic anemia for 6 yearn and received 58,000 ml of whole blood transfusions by the time of this experiment, and showed the bone marrow fully occupied with adipose tissue. Besides, the patient died of gastric cancer 6 months after the first plasma trans fusion. In short, EP rich plasma was remarkably effective in improving hema tological findings including the peripheral blood, bone marrow and serum iron OLE OF ERYTHROPOIETIN IN APLASTIC ANEMIA 121 level in all patients treated in this study whose clinical history of idiopathic aplastic anemia did not exceed two years.

SUMMARY

1) The plasma EP levels of patients with idiopathic aplastic anemia and other cases were measured simultaneously by the methods of Fried, Krantz and Matoth. EP titer in aplastic anemia was found to be higher when serum (plasma) was tested by the method of Fried (in vivo assay) and lower when tested by the methods of Matoth and Krantz (in vitro assay), although results were equal by any of the three method in all other cases. 2) Causes of the different EP level in cases of aplastic anemia were dis cussed. 8) The bone marrow of patients with idiopathic aplastic anemia when tested in vitro revealed an increased incorporation rate in response to EP rich plasma as observed in the bone marrow of the normal individual. 4) Treatment with EP rich plasma showed a remarkable effect on almost all patients with idiopathic aplastic anemia which did not respond to the normal plasma given as the control. 5) Discussions whether or not EP if purified, will be a useful agent in the treatment of idiopathic aplastic anemia, were made.

ADDENDUM

Parts of this paper were reported in the 25th (1963) and 26th (1964) gen eral meeting of Japan Hematological Society, and in the symposium of 7th con gress (1966) of the Japanese Society of Clinical Hematology. Since submission of the foregoing article, a paper concerning with antiery thropoietin in the sera of aplastie anemia was reported39

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