Serial Thrombin Time (STT) Method which does not need any particular skill in the procedure is very practical for the evaluation of circulating plasmin activity. But there have been only a few informations of basic studies on STT, and very few invenstigators have used this method in daily practice.
We evaluated Reid's method adding some modification to it, developed new one, and then performed in vitro and in vivo investigations by the latter.
STT determinations were carried out upon patients with various diseases and they were compared with the results by other methods for fibrinolytic activity, such as Euglobulin Lysis Time (Eug. L.T.) and Hemagglutination Inhibition Titer (HIT) which is used for estmating fibrinogen breakdown products.
Results: (1) The prolongation of STT was regarded as the result of the minimal increase in plasmin activity which is not enough for giving a change in the plasma fibrinogen concentration and for lysis of the standard bovine fibrin plate. (2) Prolonged STT was observed in chronic hepatitis, cirrhosis of the liver, malignant neoplasms, and in myocardial infarction. (3) STT demonstrated the minimal increase in fibrinolytic activity which could not be detected by Eug. L.T.. (4) About 88% of increased fibrinolytic activity detected by STT-30' showed abnormal rises in HIT. (5) Dynamically determined STT-30' in a patient administered urokinase went in parallel with Eug. L.T.. (6) STT in a patient with disseminated cancer of the stomach went in parallel with the bleeding tendency.

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A principal limitation in the use of currently available cancer chemotherapy is bone marrow toxicity. Bone marrow transplants have been tried as a means of correcting marrow aplasia in animals and men. Immunologic incompatibility, however, has proved to be a limiting factor in the use of this technic. Furthermore, if transplantation is successfully performed, hemologous disease is likely to ensue. To avoid these difficulties, the author has introduced the repopulation of suppressed marrow with autologous marrow stored, i, e, the patient's own marrow. For this, frozen and stored in glycerin seems to be ideal. However, this procedure is too complex for routine use. For this purpose, therefore, the author has chosen the marrow stored at 4°C which is a simpler procedure.
In the matter of preliminary observations, the viability of human marrow after storage at 4°C was assessed in vitro, in this paper. For this purpose, two methods to assess the viability of marrow were used. One is the modified INK-method to assess the viability of the aspirated marrow after storage at 4°C, for determine the activity of total tissue dehydrogenase in vitro. The other is viable cell counting by Nigrosin staining with marrow-cell suspension.
Results are as follows: After storage at 4°C, sudden decrease of the viability of the marrow was observed, showing about 50% survival of normal, after 24 hours. The observations demonstrate that human marrow can be preserved satisfactory at 4°C for 24 hours.

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It has been previously reported that some fragile cells were found in reticulocytes which appeared after massive hemorrhage.
The present studies were made on the phenomenon and mechanism of the early disappearance of abnormal reticulocytes.
From the change in the peripheral erythrocyte indicies after massive hemorrhage, the abnormal reticulocytes or “stress reticulocytes”, which were suggested by us to be formed by the early denucleation of intermediate erythroblasts, were thought to belong to large size cells with a low hemoglobin concentration.
As these macroerythrocytes were confirmed to be earsily sequestered in sinuses of the RES organs, the influences on the cells and the changes in media were studied under sequestration.
During the in vitro 37°C incubation of reticulocyte-rich blood, decreases were observed both in pH of media and in reticulocyte count.
Under similar in vitro stasis, increases of osmotic fragility and autohemolysis were found in the newly formed erythrocytes due to bleeding.
These immature cells seem to be much more influenced by some metabolic inhibitor, but they are resistant to mechanical fragility tests.
The above data, as summarized, suggest that stress reticulocytes are easily sequestered in sinuses and are more selectively conditioned by factors during sequestration resulting in their early destruction.

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Three members of a family, a father, his daughter and son, suffered from a congenital hemolytic anemia, which was characterized by the presence of inclusion bodies in erythrocytes after splenectomy and dark brown pigmenturia.
Their hemoglobins were thermolabile, and electrophoretically, a minor component of their Hb-A₁ moved slightly faster to the anode than that of normal control at pH 6.2. The SH-radicals of patient's Hb were not blocked. The G-6-P dehydrogenase activity of the erythrocytes was elevated and catalase activity was in the normal range.
The brown pigment in the urine, isolated with Al₂O₃ column chromatography, was proved to have similar chemical, chromatographic and spectroscopic properties to those of mesobilfuscin, described by Siedel.
It was unsettled whether the urinary pigment was anabolic or catabolic product of heme. To persue the question, the porphyrin synthesis from ¹⁴C-δ-ALA and bile pigments formation from hemolysate by the coupled oxidation with L-ascorbic acid were performed in vitro. The porphyrin synthesis was more active in patient's hemolysate than in the control, and the formed bile pigments were not different quantitatively, chromatographycally, or spectroscopically from those of normal hemolysate. ‘Labile-iron’ was not elevated in the patient's hemoglobin.
By the Ouchterlony's methods, no haptoglobin was detected in the serum of the mother, who was not clinically affected.
The daughter was administered ATP, folic acid and pyridoxine. By this treatment, her anemia and pigmenturia were markedly improved.
On the basis of these findings pathogenesis of the disease was discussed.

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A 65-year-old male was admitted to our clinic on December 5, 1966 with the chief complaint fever ranged from 38° to 39°C for two months. On admission the patient was found emaciated without petechia. The liver was palpable 2 fingerbreadths beneath the right costal margin. Examination of the blood disclosed red-cell count of 4,480,000/cmm with hemoglobin content of 11.9 gm/dl, and white-cell count of 17,600/cmm with 84 per cent neutrophil and 1 per cent myelocyte. The thrombocyte count was 200,000/cmm on January 6, 1967, 1,200,000 on February 17, 1967; subsequently it ranged from 442,000 to 1,342,000. He was given antibiotics. On the 9th hospital day the body temperature dropped to normal level but the fever sometimes was kept at the level of 38°C until March 1967 and fluctuated thereafter between normal and feverous. He suddenly died on August 21, 1967. Autopsy revealed that in lung there were small abscesses and bacterial colonies in the right lower, left upper and lower lobes. Spleen had 410 gms of weight and looked brownish red. Its cut surface was grossly muddy and microscopically infectious. Sternal bone marrow revealed hyperplasia of blood-forming elements, especially granulopoiesis. No maturation arrest of megakaryocytic series was seen.
In addition to them, there were meningitis, pyelonephritis and tuberculosis. (lungs, hilar lymph nodes, retroperitoneal lymph nodes and liver)

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A 38 year-old male with congenital metemoglobinemia was reported.
His hemolysate of the patient showed a distinct absorption band in 630 mμ by spectrophotometry and the content of methemoglobin was 23% by Evelyn Malloy's method.
In enzymatic study, the DPNH-diaphorase activity in red cells was 18 units and younger sister of the patient showed also slightly low activity of this enzyme.
Studying the causes of congenital methemoglobinemia, it was considered that it was not coused by the deficiency of the DPNH-diaphorase, but by the anomaly of this enzyme as the previous report by Nakao et al.

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A four-year-old boy was addmitted on Dec. 10, 1963, complaining of urinary retention, constipation, and paraplegia of the lower extremities.
He had hemorrhagic tendencies and revealed deficiency of antihemophilic globulin.
He was treated with fresh blood transfusions, and completely recovered on March. 19, 1964.

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An instance of familial leukemia, occurring in a father and his daughter, was presented. Father, aged 46 years, was hospitalized in August 1963 with a 2-month history of anemia. Hematological diagnosis was monocytic leukemia. He was placed on corticosteroid and 6MP, but died on September 23, 1963. His daughter, aged 12 years, was hospitalized in February 1964 with a 1.5-month history of sore leg and anemia. Diagnosis of acute myelogenous leukemia was made. She was placed on antileukemic drugs, and clinical improvements were attained, but she died on April 15, 1964.
The environmental and genetic factors in the etiology of leukemia were discussed.

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It has been repeatedly confirmed that testosterone has erythropoiesis-stimulating effects in various experimental animals. The mechanism of action of the hormone is controversial and several modes of action have been proposed. In this study, mice were used in an attempt to elucidate the mechanism of action of testosterone propionate.
1. Two daily injections of 2 mg testosterone propionate caused a significant increase in the red cell ⁵⁹Fe incorporation in normal adult male mice.
2. In plasma of male mice which had been injected with 2 mg testosterone propionate for two days, an elevation of erythropoietic activity was detected when assayed in transfusion polycythemic mice.
3. Bilateral nephrectomy caused a prompt, marked decrease in the splenic ⁵⁹Fe uptake and plasma ⁵⁹Fe disappearance rate in mice which had been treated with testosterone propionate as above and had shown elevations in both of the erythropoietic parameters. On the other hand, neither the splenic uptake or plasma disappearance rate of ⁵⁹Fe showed any significant decrease in mice subjected to bilateral ureter ligation following injections of testosterone propionate.
4. A rapid fall was observed in plasma erythropoietic activity following bilateral nephrectomy in mice which had previous injections with testosterone propionate, while little change was found in mice subjected to bilateral ureter ligation.
5. Light mitochondrial fraction of the kidney of testosterone-treated mice showed the erythropoietic activity only comparable in degree to that of normal mice on incubation with normal mouse plasma, possibily because the renal erythropoietic factor was released swiftly into the circulating blood when the production was accelerated.
6. In rabbit bone marrow culture, testosterone suppressed ⁵⁹Fe incorporation into heme at higher concentrations.
The results support the concept that testosterone stimulates erythropoiesis by increasing the plasma erythropoietic activity via kidney. Suppressive effects of testosterone in the bone marrow culture were unexpected and further studies are in progress to confirm the restlt of this preliminary observation.

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Serum erythropoietin levels of various anemic patients, polycythemic and anemic rabbits were determined by Fried's, Matoth's and Krantz's method. No discrepancy of the results in accordance with these three methods was obtained in all cases except for aplastic anemia, in which the erythropoietin activities were found to be elevated by Fried's method, while lowered by both Matoth's and Krantz's method.
The responses of the bone marrow cells of patients with aplastic anemia to the sera from (a) normal volunteers, (b) patients with chronic bleeding and (c) patients with aplastic anemia were studied by Krantz's method. The heme Fe⁵⁹ synthesis was increased when cultured with serum (b), but decrease with serum (c). These responses were almost epual to those seen in normal bone marrow cell cultures.
Parenteral administration of erythropoietin-rich plasma obtained from patients with chronic bleeding resulted in apparent increase of erythroid series in most cases with aplastic anemia, while administration of normal plasma showed no favorable effects.
The role of erythropoietin in aplastic anemia was discussed.

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The kidney cells and bone marrow cells were cultured together in the same culture medium containing Fe⁵⁹, and heme Fe⁵⁹ synthesis was observed in vitro. When they were cultured in anoxic state, heme Fe⁵⁹ synthesis was increased. Two kinds of gas mixture were prepared to induce anoxic state. The mixture consisted of 97% nitrogen and 3% oxygen was more effective to accelerate the heme Fe⁵⁹ synthesis in the parabiotic culture, however, the gas mixture consisted of 99% nitrogen and 1% oxygen failed to stimulate the heme Fe⁵⁹ synthesis because of the extreme scantiness of oxygen. The erythropoietic activity of the kidney cell culture medium determined by Fried's method was found to be elevated when the culture was performed in anoxic state.

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Since the experimental results of Alpen et al, it is widely accepted that erythropoietin promotes differentiation of haematopoietic stem cells.
On this basis, Gurney et al designed the stem cell assay system by measuring the ability of polycythaemic mice to respond to definite dose of erythropoietin.
On the other hand, Till et al claimed to measure the stem cells contents by examining the colony forming ability of injected bone marrow cells in spleens of supralethally irradiated mice.
Previously, we have shown the results of the comparative studies of these two techniques to estimate the stem cell content of haematopoietic tissues after single 150R irradiation. The difference between the results obtained from these two methods suggested that Erythropoietin Responsive Cells (called as ERC) are not the same cell population as Colony Forming Cells (called as CFC).
To clarify this question, the progressive growth and development of CFC and ERC was studied in the spleens of transfusion induced polycythaemic C3H/He strain mice which have been heavily irradiated and transplanted with bone marrow cells.
Firstly, the contents of CFC in the spleen were estimated by the following procedures; Bone marrow cells were injected into irradiated polycythaemic mice and at various times after transplantation the spleens were removed. This spleen cells suspension was assayed of their colony forming ability in the second heavily irradiated recipient mice by the method of measuring the radioactive iron uptake of recipient's spleen according to our original method. The growth pattern of CFC showed that following an initial lag phase of 4 days after transplantation, exponential proliferation with a doubling time of 55 hours.
Secondly, the contents of ERC in the spleens were estimated by the following procedures; At varying intervals after transplantation, 6.0 units of erythropoietin was injected into polycythaemic mice which have been irradiated and transplanted with bone marrow cells.
48 hours after erythropoietin administration, radioactive iron was injected and 5 hours thereafter the splenic uptake of radioactive iron was measured and compared to the results of control group which was transplanted but not challenged by erythropoietin.
Following the application of erythropoietin on 2 days after transplantation, hardly any reversal of the suppressed haematopoietic state was observed. Since 3 days after transplantation, ERC was detectable definitely and thereafter increased but in different way of CFC growth curve.
These experimental results clarified the ERC are not the same population but some progeny of CFC.
This research was supported by research grant from International Atomic Energy Agency. (Research Contract No. 352/Rl/RB)

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It has been well recognized that eryrhropoietin exist in the serum and urine of anemic patients when the hemoglobin level decreased below some degree except in case of renal anemia. On the other hand, we have reported that erythrocyte stimulating factor (s) also exist in the cord plasma of normal full-term neonate. It is the purpose of this paper to describe the relationship of erythropoietic stimlating factor of these material, immunologically.
The antigen for the preparation of the anti-etythropoietin-serum was obtained from urine of a patient with congenital hypoplastic anemia and also sera of phenylhydrazine-treated rabbits.
Crude erythropoietin extracted from above materials were administered for preparing the anti-erythrpoietin-serum into rabbits. After exanguinating the rabbits, sera was separated and stored for study.
In summary, following results were observed:
1. No relationship between the anti-E potency and RBC series findings in the immunized rabbits were noted.
2. No autoimmune phenomena of the erythropoietin were proved in the anemic children even who had high ESF level in the plasma and urine.
3. The potency of anti-E sera were different in each rabbit even immunized by same antigen with same amount.
4. Erythropoietic anti-sera could neutralized not only hemoantigen but also heteroantigen.
5. The neutralizing ability of anti-erythropoietic sera given into the normal rat are not remarkable as far as our study is concern.
6. No parallel results were obtained by two different method, i.e., Diffusion-in-Gel and Bioassay method.

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It has been shown by several investigators that erythropoietin induces the differentiation of stem cells into erythroblasts. By incubating the spleen of the polycythemic mouse, we also demonstrated that the stem cells could be differentiated into erythroblasts by erythropoietin in vitro. In this study, bone marrow cells obtained from normal and aplastic anemia patients were incubated in vitro with erythropoietin to observe whether the stem cells in human bone marrows can respond to erythropoietin in physiological and pathological states.
Marrow cells aspirated by the sternal puncture were washed 2 times with NCTC 109 solution and suspended in an incubation media consisting: 60% NCTC 109 solution, 40% of human AB plasma 50 U/ml of penicilln, and purtially parified urinary erythropoietin (0.1∼0.8 CoU/ml). Eight tenth ml of the above marrow cell suspension with a cellularity 1000∼4000 nucleated cells per cu. mm. was applied tissue culture dish (Falcon plastics) and incubated under 5% CO₂ and 95% air, at 37°C for 72 hours. Six hours prior to the termination of the incubation, 3 microcuries of radioiron (Fe⁵⁹Cl₃) was added to each dish. At the termination of incubation, cells were washed 3 times with buffered saline. The heme was extracted from the sediment by the method of Teale. The extracted heme was dried on a stainless steel planchets and its radioactivity was counted in a gas flow counter. Bone marrow cell incubated without the addition of erythropoietin were served as the controls.
Bone marrow cells obtained from 6 normal volunteers responded to erythropoietin in vitro with an increase in heme synthesis. In two cases (one of them was in clinical remission) of aplastic anemia, their bone marrow cells responded to erythropoietin. In other 3 cases, however, the bone marrow cells were irresponsive to erythropoietin.
There was no difference in the degree of stimulation by erythropoietin between normal and erythropoietin responsive aplastic anemia bone marrow cells. Considering the mechanism of stem cell differentiation by erythropoietin, we supporse some abmormalities in the stem cells in some cases of the aplastic anemia. From the data of our present study, however, it is impossible to conclude whether these abnormalities in the stem cell pool are quantitative or qualitative. Further studies accumulating more cases of aplastic anemia will give us some clues on elucidating the nature of the abnormalities.

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In order to study the biochemical events taking place in acordance with the erythroid cell differentiation, transfusion-induced polycythemic mice were injected with 6 CoU of partially purified urinary erythropoietin. After the indicated intervals, the polycythemic mice were sacrificed and enzyme activities related to heme synthesis were measured in these mice. Incorporation of ¹⁴C-glycine into globin fraction of the spleens was also measured in these polycythemic mice.
ALA-synthetase activity, which had not been detectable in the spleen of the polycythemic mouse, started to appear on the 8th hour of erythropoietin injection. The activity reached to the maximum on the 52nd hour of the injection. ALA-dehydrase activity was demonstrable in the spleen of the polycythemic mouse probably because of the contamination of peripheral red blood cells. The enzyme activity started to increase on the 12th hour of the erythropoietin injection, and reached to the maximum on the 52nd hour. Heme-synthetase activity was not detectable in the spleen of the polycythemic mouse. It started to apear in the spleen on the 28th hour of the erythropoietin injection. The activity was maximum on the 52nd hour of the injection. Incorporation of ¹⁴C-glycine into globin was also not detectable in the spleen of the polycythemic mouse. The incorporattion was first observed on the 16th hour of the erythropoietin injection and reached the maximum on the 52nd hours.
It was noted that appearance of the globin synthesis started earlier than that of the heme-synthesis after erythropoietin injection. From the above described sequential appearance of the enzymes of heme synthesis, it was speculated that the erythropoietin works on the hematopoietic stem cells to induce the synthesis of ALA synthetase. Whether this hormone is necessary for the further development of the erythroid cells or the substrate induction of the enzyme activities are responsible for the accomplishment of heme synthesis in the differentiated cells, are sill conjectural.

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