In order to detect the presence of activated platelets in vivo, platelet factor 4 (PF 4) in the platelet-free plasma was measured by radioimmunoassay in healthy subjects and diabetics with various vascular complications such as retinopathy (Scott III-V), nephropathy, ischemic heart disease and ischemic cerebral attack.
Plasma concentration of PF4 in 16 healthy subjects was 8±2 ng/ml (M±SD, range 5∼11 ng/ml). No close correlation was observed between plasma PF 4 and platelet PF 4 levels.
Thirteen of 43 patients with diabetes and vascular complications had higher PF 4 levels than 20 ng/ml. Plasma PF 4 level was examined repeatedly in 7 patients without anti-platelet therapy. Among three patients with elevated plasma PF 4 two showed continuously high PF 4 levels. PF 4 value of remaining 4 patients stayed in normal range.
Correlation coefficient between plasma PF 4 and beta-thromboglobulin (βTG) level measured by radioimmunoassay in normal subjects and diabetics were 0.76 and 0.78, respectively. In 9 out of 84 determinations high plasma βTG level was observed without elevation of PF 4 value. The opposite phenomenon was obtained in 8 determinations.
Although the raised plasma PF 4 levels are observed also in careless preparation of platelet free plasma, the measurement of PF 4 is useful for the study of platelet pathophysiology.

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Kit for serum ferritin assay (SPAC ferritin kit) which was offered by Daiichi Radioisotope Labs. was studied on the basic points and serum ferritin was measured in various hematological diseases. Concerning to the basic points, a reproducibility test, a recovery test and a dilution test were examined. From these results, a good correlation was obtained in each test, however, when the values showed more than 150∼200 ng/ml, a proper dilution and a reexamination should be recommended because of an increasing error.
Concerning to the clinical examinations, sera form the normal subjects and the patients with hematological diseases, for example, iron deficiency anemia, aplastic anemia, hemolytic anemia, polycythemia vera, leukemia, multiple myeloma, myelofibrosis and so on were examied. In normal subjects, a significantly higher value in men was shown than in woman. Iron deficiency anemia showed a markedly low value and serum ferritin elevated after an effective treatment. In other hematological dieases, serum ferritin showed various values. Based on these results, it was suggested that they may reflect an iron deficiency state due to a bleeding, a disturbed iron utilization due to an impaired erythropoiesis, an iron overload state due to blood transfusions, a production by leukemia cells, a release from liver cells and so on.

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The kinetics and distribution of methotrexate in intraventricular fluid were studied in a patient with central nervous system leukemia after drug administration by intravenous infusion, indwelling intraventricular subcutaneous reservoir (Ommaya).
Methotrexate administered by Ommaya reservoir, at a dose of 15 mg/M², rapidly distributed in the subarachnoid space. The peak ventricular concentration of methotrexate was 2.54±0.9×10^-4M, and remained in 10^-6M level for 72 hours with a half-life of 10.5 hours. During an intravenous 6 hour-infusion at a dose of 750∼1,500 mg/M² and 2,250∼3,000 mg/M², the ventricular cerebrospinal-fluid concentration of methotrexate rose to 7.55±1.33×10^-7M and 2.35±0.89×10^-6M, respectively.
The patient was achieved CNS-remission following 6 doses of intraventricular methotrexate (15 mg/M²/dose) and hydrocortisone (50 mg/M²/dose), and maintained with intraventricular chemotherapy every 6 weeks. Over eighteen months after Ommaya reservoir insertion the patient has been still in CNS-remission.
Our data suggested that intraventricular chemotherapy via Ommaya reservoir has improved significantly for CNS-leukemia with monitoring methotrexate concentration in the cerebrospinal fluid.

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Chemotaxis of peripheral monocytes was measured by agarose plate method in children and adults. Mononuclear cells (MNC) were collected from heparinized peripheral blood by Ficoll-Hypaque centrifugation and they were suspended in Hanks balanced salt solution at a concentration of 1×10⁸/ml. The center well on agarose plate received 10 μl of the MNC suspension, and the outer and inner wells received chemotactic factor (zymosan-activated human serum) and non-chemotactic control medium, respectively. The agarose plate was incubated at 37°C in a humidified atmosphere containing 5% CO₂ in air for 20 hours. Chemotaxis assay was done by measuring the linear distance the cells had moved from the margin of the well toward the chemotactic factor (chemotaxis) or the control medium (random migration).
As the appropriate incubation time was 20 hours and MNC concentration was 1×10⁸/ml, the following studies were done under such conditions. Monocyte chemotaxis was not demonstrated when culture filtrate of E. coli was used as a chemotactic factor. There was no significant difference of monocyte chemotaxis and random migration between children and adults: chemotaxis values (mean±S. D.) were 8.5±0.7 (1∼5 years of age), 8.6±1.5 (6∼11 years), 8.9±1.0 (12∼19 years), and 9.9±1.3 (adults).
The method is simple and reproducible and can provide further information as to monocyte functions.

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The functions of platelets collected by the Haemonetics blood processor Model 30 in 20 healthy volunteers were investigated. Aggregability and release ability of these platelets were compared with platelets from the same donors before plateletpheresis. ADP-, epinephrine- or collagen-induced platelet aggregation was markedly decreased in platelets isolated by Haemonetics Model 30. It was, however, less reduced than that of platelets obtained from Japan Red Cross Blood Center. ATP release induced by collagen was also decreased when examined with Chrono-log lumiaggregometer. During plateletpheresis, significant amount of β-thromboglobulin and possibly ATP were released from platelets indicating that the activation of platelets occured. Haemonetics-isolated platelets were transfused into 5 year-old boy, in whom platelet count was only 1.0×10⁴/μl due to aplastic anemia. Following transfusion platelet count was increased to 15×10⁴/μl resulting in shortening of Ivy bleeding time from over 15 min. to 10 min. Aggregation of platelets obtained from the patient after transfusion was also considerably improved indicating that defects of Haemonetics-isolated platelet functions appeared reversible. Our study showed that Haemonetics-isolated platelets were hemostatically effective although they were defective in aggregation and release activity in vitro.

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Concentrations of antithrombin III (AT-III) in plasma in 20 healthy subjects and in 43 patients with disseminated intravascular coagulation (DIC) were determined simultaneously using synthetic chromogenic substrate (Tos-Gly-Pro-Arg-pNA), single radial immunodiffusion method and latex agglutination method, which is based on observation of agglutination of latex particles coated with anti-AT-III-serum.
Levels of AT-III in plasma markedly decreased in cases of DIC.
A statistically significant positive correlation between values of AT-III, measured using three different methods in the same plasma samples from normal subjects and patients with DIC, was observed. Correlation coefficient between values obtained using amidolytic method and single radial immunodiffusion method was +0.94 (p<0.001).
Activity to antigenicity ratio was slightly lower in cases of DIC than in healthy subjects, although the difference was not statistically significant. Abnormalities in pattern of two-dimensional electrophoresis of plasma AT-III were observed in some cases of DIC.
From these results, it was concluded that antithrombin activity and antigenicity of AT-III in plasma were generally correleted well in healthy subjects and cases of DIC.

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Effect of Melphalan and prognostic factors were evaluated in the patients with multiple myeloma.
Effect of Melphalan was examined in 33 patients who were treated with intermittent courses of Melphalan-Prednine combination (MP therapy). Good response to MP therapy was obtained in 24 cases out of 33 cases, while 9 cases did not respond to MP therapy. The most remarkable effect was decrease of plasma cells in the bone marrow aspirates.
The prognostic significance of laboratory data on admission and clinical features has been analysed in all 34 cases. Low hemoglobin concentration, increased ratio of plasma cells in the bone marrow, hypercalcemia, high serum uric acid level, hypoalbuminemia, excretion of Bence Jones protein in urine and light chain of λ type were all associated with shortened lifespan. The statistical analysis confirmed that low hemoglobin level (less than 10 g/100 ml), increased plasma cell count (more than 20%) and hypoalbuminemia (less than 2.5 g/100 ml) were strongly correlated with a poor prognosis (p<0.05). As complications of multiple myeloma, infection and uremia were frequent and these two were main causes of death. Postmortem examination in 15 cases revealed myeloma kidney in 8 cases and pulmonary infection in 8 cases. Infections and uremia were considered to affect the survival of the patients with this disorder.

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A case of agranulocytosis is reported in which discovery of the offending drug was attempted by using the in vitro cloning technique.
A 45-year-old housewife presented with a 10-day history of fever and sore throat. She had received flurazepam and carbamazepin over the preceding period of three weeks. On examination she had a pyrexia of 38.6°C and necrotic ulcers in the buccal mucosa, palate and pharynx. The initial blood count revealed a WBC of 1.5×10⁹/l with total absence of neutrophils, and bone marrow aspiration showed an entire lack of mature granulocytes. Flurazepam and carbamazepin were withdrawn, and a potent antibiotic chemotherapy was immediately started. Six days after the last dose of the drugs the neutrophils began to appear in the peripheral blood. Fever and sore throat subsided, and her blood findings returned to normal after a transient neutrophilia.
Influence of flurazepam and carbamazepin on the growth of granulocyte-macrophage progenitor cells (CFU-C) was investigated in the semisolid ager culture system. At a concentration comparable to that attained in vivo, flurazepam significantly suppressed CFU-C colony formation from the patient's marrow taken 10 months after the agranulocytic episode but not from a normal marrow with or without addition of the patient's serum to the culture system. Cluster formation either from the patient's or normal marrow was not inhibited by flurazepam. The result appears to indicate that the patient's bone marrow is susceptible to toxic action of this drug which affects progenitors younger than cluster forming cells. Carbamazepin was found not to be inhibitory on CFU-C in the patient's bone marrow.

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Peripheral blood polymorphonuclear neutrophils (PMNs) from 356 patients of various diseases including three cases with chronic granulomatous disease (CGD) and their mothers, 130 cases with bacterial infections, 70 cases with non-bacterial diseases and 150 cases with hematological disorders, were examined by the newly devised histochemical nitroblue tetrazolium (NBT) reduction test. The principles of the method was previously reported in the first part⁷ of this clinical research. Spontaneous NBT test and endotoxin-stimulated NBT test were simultaneously performed.
Most of the cases with systemic bacterial infections such as pyothorax, panperitonitis, septicemia and acute lobar pneumonia showed moderate (above 200) to extreme (above 400) elevations of spontaneous score (normal range 92∼121, mean 101.5 in 50 healthy controls). Rapid decline and normalization of the elevated scores were excellent indicators of good response to chemotherapy.
Three cases with CGD during bacterial infections, two cases with typhoid fever, 33 cases with tuberculosis and most of the cases with localized bacterial infection such as tonsilitis showed no elevation of spontaneous score, i. e., false negative results. Among non-bacterial diseases false positive results were obtained in some of the cases with systemic lupus erythematosus, advanced carcinoma, aplastic anemia, polycythemia vera, hepatitis A and transfusion reaction.
Spontaneous test can be utilized as a diagnostic aid for systemic bacterial infections in conjunction with other laboratory tests.
By stimulated test a complete absence of score elevation (normal range 260∼470, mean 357 in 50 healthy controls) was detected in all of three cases with CGD and a intermediately low score (166) in a mother of a male CGD case. Among hematological disorders abnormally low scores (122∼240) were found in 9 (24.3%) of 37 cases with AML, 4 (28.5%) of 14 cases with transformed CML and 9 (45%) of 20 cases with hemopoietic dysplasias (preleukemic states). Other than hematological disorders one case with SLE, two cases with rheumatoid arthritis and one case with ITP following rubella showed abnormally low stimulated scores.
The stimulated test can be used as a reliable screening tool to find out cellular defects in oxygen dependent anti-bacterial metabolism.

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A 27-year old male patient with acute lymphocytic leukemia who had shown resistance to repeated chemotherapies, was successfully grafted with bone marrow from his HLA-matched sister. Prior to bone marrow transplantation (BMT), he received the combined chemotherapy (DCMP) and was conditioned with cyclophosphamide 60 mg/kg/day on two successive days, followed by 1000 rads total body irradiation (TBI). Marrow cells (1.5×10¹⁰) were infused intravenously 24 hours after TBI. Successful engraftment has been demonstrated karyotypically (sex marker) by replacement of bone marrow and peripheral blood cells with those of donor. He received intermittent amethopterin after BMT to prevent graft-versus-host disease (GVHD). Three weeks after BMT, however, developed moderate GVHD of grade I-II in the skin and liver, which was fortunately well controled with predonisolone. In addition interstitial pneumonia developed 140 days after BMT and again was well controled with high dose of prednisolone. Now 700 days after BMT, he has been well in complete remission.
This case is the longest survivor among those who were treated with allogeneic BMT for hematological malignancy in Japan.

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A 43-year-old male was admitted to Jichi Medical School Hospital because of pancytosis. Three years before this admission, his peripheral blood values were WBC 8,300/mm³, Hb 17.0g/100 ml. Eight weeks before the admission, WBC increased to 17,000/mm³. RBC 505×10⁴/mm³, Hb 17.0 g/100ml and platelet 66×10⁴/mm³.
On admission, physical examination revealed a presence of hepatosplenomegaly. RBC 539×10⁴/mm³, Hb 15.7 g/100ml, platelet 84.8×10⁴/mm³, WBC 20,200/mm³ with shift to the left, three per cent of eosinophils and eight per cent of basophils. Bone marrow aspiration revealed a marked increase in myeloid series of various stages and megakaryocytes. Chromosome analysis showed 46 XY with Ph¹ chromosome. Leukocyte alkaline phosphatase score was 160 (normal 180∼340). Total red cell volume was 56.7 ml/kg. From these findings he was diagnosed as having chronic myelogeneous leukemia with erythrocytosis. The number of CFU-E in the marrow was increased to 596 per 1×10⁵ bone marrow cells (control 237±47). Secondary erythrocytosis was not possible because causative diseases such as pulmonary diseases, cardiac diseases, renal diseases, hemangioblastoma or hepatoma were not found. Moreover, serum erythropoietin was not detectable by polycythemic mouse assay. This increase in the number of erythroid progenitor cells in the marrow is considered to be related to the pathogenesis of erythrocytosis observed in this case.

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A case of childhood acute leukemia associated with Down's syndrome is reported. The patient was three year-old girl at the time of diagnosis of acute leukemia, and had typical clinical features of Down's syndrome with 21 trisomy.
She was transferred to the National Cancer Center Hospital because of hepatosplenomegaly, severe anemia and marked leukocytosis.
At first, the patient responded well to a combination chemotherapy with daunorubicin, cytosine arabinoside, vincristine and predonisolone. Complete remission was obtained for four months. She relapsed, however, and died of septicemia of pseudomonas aeruginosa fifteen months after the diagnosis.
Children with Down's syndrome have a higher incidence of acute leukemia than normal children. The cell type of the acute leukemia, i. e. myeloblastic or lymphoblastic, is still controversial. Leukemic cells in this case had both myeloid and lymphoid expression of the surface markers, such as E-receptor, complement and Fc receptors, T-cell and Ia-like antigens as well as myeloid antigen. TdT activity was not recognized. The presence of both myeloid and lymphoid expression suggested that the neoplastic change occurred at the level of myeloid-lymphoid stem cell. These findings may be concerned with chromosomal abnormality of Down's syndrome.

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A case of lymphoblastic lymphoma with high terminal transferase (TdT) activity in the tumor cells was reported, which showed frequent infiltrations in various organs and sites.
The patient, a 41-year-old man was admitted because of cervical and axillary lymph node swelling, and showed a large mediastinal mass and a massive pleural effusion in the X-ray films of the chest. Microscopical examination of the lymph node revealed a diffuse infiltration of uniform blastic cells with scanty cytoplasm and round or indented nuclei. Frequent mitotic figures and scattered starry-sky patterns were present. Localized positive granule for acid phosphatase stain was detected in the cytoplasm in the touch imprint of the specimen.
Histological diagnosis was malignant lymphoma, poorly differentiated lymphocytic, diffuse (PDL-D), and VEPA therapy was started. After 2 weeks complete remission was achieved, however CNS involvement developed immediately, and then frequent infiltrations appeared in CNS, neck muscle, CNS, bone marrow, skin and ascites. Eleven months after admission he died of leukoencephalopathy which seemed to be induced by cranial radiation and intrathecal chemotherapy with MTX and Ara-c etc.
Clinical pictures, cytological findings and high levels of TdT, which were detected in the tumor cells of lymph node, pleural effusion, bone marrow and ascites, indicated that the case was lymphoblastic lymphoma (convoluted cell type) that has been included in PDL-D. Although no surface markers were demostrated in the lypmh node cells, E-rosette formation was detected in the tumor cells of ascites.
The relationship between tumor cell origin and clinical pictures and therapy was discussed.

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A 37-year-old female had been admitted to our hospital 4 times since January 1976, because of nausea, vomiting, diarrhea, numbness of her hands and feet and generalized skin pigmentation. From these findings and high levels of arsenic in her hair and nail, she was diagnosed as being involved in arsenic poisoning.
On her first and second admission, leukopenia and normocytic normochromic anemia with marked reticulocytosis were noted. This reticulocytosis suggested the existence of hemolysis. On her third admission, it was noteworthy that she had typical laboratory manifestations of ineffective erythropoiesis due to megaloblastic process, including mild pancytopenia, low reticulocyte index, increased marrow cellularity with erythroid hyperplasia and morphological evidence of megaloblastic maturation in the marrow. The serum levels of vitamin B₁₂ and folate were within normal ranges, and the administration of vitamin B₁₂ was not effective. On her last admission, severe pancytopenia and hypocellular marrow were noted. Since all these hematological abnormalities were regressed with the regression of other symptoms and signs, it was suggested that these hematological abnormalities including megaloblastic anemia were also caused by the arsenic poisoning.

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A fifty-year-old male was admitted with a chief complaint of cervical lymphadenopathy in January 24, 1979. He noticed fever up to 38°C and swelling of the tonsils and cervical lymph nodes in February 1978 and visited a university hospital, where a biopsy of a tonsil was done. Pathological diagnosis was non-specific tonsillitis and oral administration of antibiotics resulted a diminution of the lymphadenopathy but fever continued. ⁶⁰Co irradiation 42 times was applied to the both mandibular and cervical regions with beneficial results. In September 1978 lymph node swelling reappeared on axillar, inguinal and cervical regions and the pathological diagnosis of the biopsied inguinal lymph node was immunoblastic lymphadenopathy. ⁶⁰Co irradiation was applied five times to the thoracic, abdominal and both inguinal regions.
On admission marked lymph node enlargement, up to 4 cm. in diameter, was observed on cervical, axillar and inguinal regions. There was no hepato-and splenomegaly. Pathological diagnosis of the left axillar lymph node biopsy material was immunoblastic sarcoma. Study of the lymphocyte subpopulation of the lymph node biopsy material revealed a decrease in E rosette and an increase in IgGλ positive cells on the cell surface. After BACOP therapy fever and lymph node swelling subsided but a complicated interstitial pneumonitis led to the death.

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A case of T cell CLL complicated with Mikulicz syndrome was reported.
A 40-year-old man, who had been diagnosed T cell CLL two years before, was admitted to the Kyushu University Hospital because of the swelling of bilateral lacrimal and submandibular glands, which was thought Milculicz syndrome.
Biopsy of the lacrimal gland showed infiltration of leukemic cells and the swelling of lacrimal and submandibular glands was soon reduced with VEMP therapy.
Maintenance chemotherapy with cyclophosphamide and predonisolone has been continued and he is still under remission for about ten years.
Underlying diseases of Mikulicz syndrome in Japan were mainly discussed.

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