Colony formation in in vitro culture by hemopoietic stem and progenitor cells consists of three major components; (1) a cell(s), (2) hemopoietic factors, and (3) culture medium and supporting matrix. Single stem or progenitor cell may be separated or picked up by using micromanipulation technique and several types of hemopoietic factors have been highly purified by biochemical and immunological methods. In addition, hemopoietic factors produced by molecular cloning technique would be available in near future. Thus, chemically defined culture medium is now urgently required instead of the conventional serum-containing culture medium because of the disadvantage of the latter culture medium that serum contains numerous undefined substances and factors which make it difficult to investigate the interaction between a cell(s) and hemopoietic factors. In this paper, serum-free culture for hemopoietic stem and progenitor cells as well as leukemic cell lines is reviewed. The word “serum-free culture” is used at present to mean a culture system supplemented with serum components, such as bovine serum albumin, human transferrin, etc., instead of “whole serum”. There is no report that colony formation by hemopoietic stem or progenitor cells in completely serum-free or serum component-free medium is possible. Establishment of such culture systems would greatly contribute to further studies on the proliferation and differentiation of hemopoietic stem and progenitor cells.

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A panel of monoclonal antibodies reactive with myeloid cells at different stages of developement was produced and was used to characterize the leukemic cells of 62 patients. Monoclonal antibodies included TG8/TG28 (anti-granulocytes), TM2 (anti-platelets, granulocytes, monocytes and cALL), TM15 (anti-monocytes, granulocytes and null cells), TM18 (anti-monocytes), TM1.30 (anti-monocytes and extra), TM2.7 (anti-cell lines?), TP80 (anti-platelet glycoprotein IIb/IIIa complex), TP82 (anti-cALL associated antigen, p 24), NC1 (anti-HLA-DR framework), and NC6 (anti-HLA-ABC framework).
TG8/TG28 were reactive with not only mature granulocytes but also cells from some AML, APL and AMML. TM2 reacted with leukemic cells from patients with AML, APL, AMML, EL, CML in chronic phase and myeloid blastic crisis and also common ALL. TM15 reactive cells were observed in 6/11 cases of AML, 2/3 cases of APL, 3/3 cases of AMoL or AMML but none of T or B cell malignancies. However, TM18 had narrower specificity against AML (4/11), APL (2/3), AMoL or AMML (2/3) and no positive cells were present in lymphoid malignancies. Common ALL-associated antigen (p24) recognized by TP82, was shown to be expressed on platelets, cALL and on some other acute lymphoblastic or myelo (mono) blastic leukemias. NC6 confirmed the expression of major histocompatibility complex class I antigen on lymphoid, myeloid and erythroid leukemias. TM2.7 reacted with only a part of cell lines but not with any leukemic cells. No definitive reactivity pattern of TM1.30 was observed against leukemic cells or cell lines.
Cells from all patients with CML in chronic phase were reactive with TG8/TG28 and TM15 with more than 50% in reactivity but cells bearing these antigens were decreased in myeloid blastic crisis. In contrast, blast cells gave abundant expression of HLA-DR antigen. Three out of eight cases with myeloid crisis had TP80 positive cells in their blood were observed. This result suggested that megakaryocytic lineage cells might take more part in myeloid crisis than has been expected. It was shown that HLA-DR antigen, that was recognized by NC1, was more abundantly expressed on blast cells in crisis than on cells in chronic phase.

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Twelve patients with treatment refractory leukemia were administered high dose cytosine arabinoside (Ara-C). As a rule, patients were treated with Ara-C at a dose of 3 g/m², twice daily for 6 days. The clinical effect in five patients with acute non-lymphocytic leukemia (ANLL), 2 with acute lymphoblastic leukemia (ALL) and 1 with blast crisis of chronic myelogenouse leukemia (CML-BC) were evaluable.
In patients with ANLL, 3 patients achieved complete remission (CR) and other 2 partial remission (PR). Duration of the complete remission was 2.5, 4 and 4 months, respectively. No remission was observed in patients with ALL and CML-BC.
Nausea, vomitting, diarrhea, conjunctivitis, fever and alopecia appeared frequently and symptomes of central nervous system, liver dysfunction and skin rash were found occasionally. In general, the toxicity of this treatment was well tolerated.

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Hemophiliacs are considered to be one of the high risk groups in AIDS and anti-hemophiliac factor (Factor VIII) is thought to contain a promoting factor for transmitting the causative virus (HTLV-III) of AIDS. Immunologically, the OKT4/OKT8 ratio of peripheral lymphocytes from hemophilia patients was significantly lower than controls although clinical symptons of immune deficiency have not been observed in hemophiliacs in Japan.
A previous study was carried out to determine whether the OKT4/OKT8 ratio in hemophiliacs was influenced by repeated intravenous injection of anti-hemophiliac factor used for therapy. A decrease of the ratio was found to be enhanced by prolongation of therapy Factor VIII.
In the study, lymphocyte markers in patients with hemophilia were tested using OKT4, Leu3a and KOLT-1 monoclonal antiobodies. The lymphocytes reacted with Leu3a and KOLT-1 but not with the OKT4 antibody.
Furthermore, the expression of OKT4 antigen on T lymphocytes of patients with hemophilia was restored by serum-free culture and enhanced by cultivation with ConA at 37°C for 96 hours while T lymphocytes from healthy donors showed no changes with these treatment.
From the above results, the experssion of OKT4 antigen on T lymphocytes in patients with hemophilia seems to be altered by repeated injection of anti-hemophiliac factor and the OKT4/OKT8 ratio in these patients is reversible.

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Increased proportions of peripheral blood lymphocytes from hemophilia patients treated with anti-hemophilic factor express activation-linked cell surface antigens. The alteration of lymphocytes surface markers in hemophilia is considered to be correlated with the duration of clotting factor therapy.
In this study, cultured lymphocytes from patients with hemophilia were analysed for changes in activation-linked cell surface antigens. The results revealed a decrease in the proportion of the cells expressing certain activation-linked cell surface antigens when compared to normal controls following cultivation in serum free condition. In contrast, the expression of these antigens was enhanced during lymphocyte cultivation with ConA than in controls. The results suggest that hemophilia may have a reservoir for immune defense function based on the above cellular mechanism.

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The significance of clinical stage and primary site of tumor of T- and B-lymphomas for prognosis was analyzed in the 208 patients with non-Hodgkin's lymphoma who had been treated in National Cancer Center Hospital from the beginning of 1975 to the end of 1981. Patients with B-lymphoma of Waldeyer's ring or stomach in stage I had the best prognosis and about 80% or more patients could be cured by local radiation therapy (Waldeyer's B-lymphoma) or surgical resection (stomach B-lymphoma). On the contrary, those in stage II had poor prognosis, suggesting that local therapy was not satisfactory. In the case of other extranodal B-lymphoma and nodal B-lymphoma, prognosis was poor and not related to the clinical stage, although 50% survival time was much longer in the earlier stages than in the more advanced stages. In the cases of T-lymphoma, primary sites of lymphoma had also much influence to the prognosis. Some of T-lymphomas of the skin and nasal cavity had good prognosis. In nodal T-lymphoma and T-lymphoma of thymus, prognosis was poor and not related to the clinical stage.
These results suggest that T-lymphoma as well as B-lymphoma are comprised of heterogenous diseases and tumor cells of a certain type of lymphoma disseminate not only through lymph-born but also through blood-born route.

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A 16-year old male patient with acute T-lymphoblastic leukemia had bone marrow transplantation (BMT) from HLA phenotype identical mother during the second remission. He was first diagnosed on April 1982, achived remission on May and relapsed 5 months later with concurrent CNS leukemia. Serological typing for HLA revealed that the patient and his mother were phenotypically identical, and MLC showed weakly positive response. After he achieved his second remission and his CNS leukemia was improved by cranial and spinal irradiation, CY plus TBI was given, then 2×10⁸/kg of bone marrow cells were transfused on June 7, 1983. Methotrexate was given until day 60 to prevent GVHD. Chronic GVHD first appeared on day 110, subsided without treatment but worsened on day 250 with eosinophilia, pulmonary shadow and dermatomyositis-like skin lesions, which were treated with cyclospolin A, followed by prednisolone and azathioprine. Other complications such as interstitial pneumonia, hepatitis and listeria monocytogenes meningitis were also encountered. Throughout the course after BMT, hypogammaglobulinemia, poor lymphocyte blastogenic rsponse to mitogens and inversion of OKT 4/8 ratio were observed. He is now alive with Karnofsky's score 95 without any signs of leukemia relapse and neurological abnormalities 21 months after BMT.

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A 79-year-old woman, complaining of malaise and fever, was admitted to our hospital on January 11th, 1984, when pea-sized lymphadenopathies in axillary and inguinal region, ascites and hepatomegaly were noticed.
Hematological examination on admission showed a WBC of 36,200/mm⁸ with 89% blasts, and a nucleated cell count in the bone marrow of 74,000/mm⁸ with 84.6% blasts. The blasts were negative on peroxidase staining. ATLA (adult T-cell leukemia associated antigen) antibody was negative. TdT (terminal deoxynucleotidyl transferase) activity showed 1.9 U/10⁸ cells. The concentration of serum immunoglobulin was normal. Chromosome examination of bone marrow cells showed an abnormal karyotype of 49, XX, +9, +18, -19, +mar₁, +mar₂. The examination of blast surface markers showed 8% E-rosette, 1% s-Ig, 84.1% B1, 73.2% OKIa1, 35.8% OKT10 and 14.1% J5. After VP (vincristine, prednisolone) therapy, the patient initially showed a decrease in the number of blasts. A month later, however, the blast number increased and the patient died.
The clinical and pathological diagnosis in this case was ALL (L₂ type by FAB classification).
On autopsy, there was no cytoplasmic-IgM in the blasts.
ALL with surface markers of B cell (B1⁺) in an elderly patient is rarely found.

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The life-threatning intracranial hemorrhage in a patient of hemophilia B with inhibitor (high responder) was successfully treated with high dose of FEIBA infusion.
However, disseminated intravascular coagulation (DIC) caused by FEIBA was appeared during the treatment. The level of antigen of antithrombin III (ATIII) remained within normal limits during the therapy with FEIBA. On the other hand, the level of the activity of ATIII was decreased during the treatment. In addition, the pattern of crossed immunoelectrophoresis of ATIII of the patient's plasma (on the 4th hospital day, immunological: 90%, biological: 52%) was different from control. The qualitative change of ATIII might be related to DIC. When high dose of FEIBA is required, it is necessary to check coagulation system carefully.
The treatment of life-threatning severe bleeding in a patient of hemophilia B with inhibitor has not been established and has remained difficult problem. However, according to the experience of this case, careful treatment with high dose of FEIBA is thought to be very helpful therapy for such patients.

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Although familial Pelger-Hüet anomaly has been known as usually asymptomatic, a case of systemic lupus erythematosus associating with this anomaly appears in the literature.
This paper reports on a 32-year-old female with famillal Pelger-Hüet anomaly who had clinical symptoms of Sjögren's syndrome and was positive for antibodies to SS-A. Immunological studies of the patient's peripheral lymphocytes disclosed decreased number of OKT8 cells and diminished blastoid transformation induced both by PHA and ConA.
Judging from the fact that other authors also observed immunological abnormalities accompanying Pelger-Hüet anomaly, it is possible that the association of Sjögren's syndrome with this nuclear anomaly was not a mere coincidence but immunological defects found in the latter was responsible for the manifestation of the former.

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A one-year old girl was admitted to the Okayama Red Cross Hospital on August 22, 1984 with fever and multiple furuncles. She was pale; peripheral blood examination revealed pancytopenia, and bone marrow aspiration showed a very hypoplastic marrow with only 4.5 percent of hematopoietic cells. Immediately anabolic steroid was administered but it failed to improve her hematological condition. She had a HLA identical brother and was transferred to the Department of Pediatrics of Nagoya University Hospital for bone marrow transplantation. After gut sterilization and an intravenous catheter were prepared, she received 500 mg of cyclophosphamide for successive 4 days followed by 750 rads of subtotal lymphoid irradiation, and 5×10⁹ bone marrow cells were infused from her brother. Bone marrow aspiration on day 13 showed an increase in hematopoietic cells, and engraftment was confirmed by examinations of red blood cell type and sex chromosome. Hepatic transaminase increased from day 19, but was normalized by cessation of methotrexate and administration of betamethasone. Decreased immunoglobulin level after transplantation has recovered, and inverted OKT 4/8 ratio has also been normalized. After one year from transplantation, she is in a good hematological condition and is enjoying her life without any complication.

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A 64-year-old man was admitted to our hospital with pancytopenia and hypoplastic bone marrow on December 23, 1983. The patient had been treated for gastric ulcer with cimetidine 800 mg/day from September, 1982 to December, 1982, when the drug was reduced to 200 mg/day. In July 1983, facial pallor and edema developed. A month later, he was found to have pancytopenia and cimetidine was discontinued. His previous history revealed alcoholic hepatitis treated scince 1975. Following admission, a solitary hepatoma, measuring 3.4 cm in diameter, was found and was successfully treated by endoarterial embolization. The hematologic condition was diagnosed as hypoplastic anemia and was treated with androgens with some improvement. Cimetidine was considered the most likely cause of his hypoplastic anemia because no other drugs given were known to be myelosuppressive. And he had IgM-M proteinemia. Recently, tumorenhancing effect and IgM response stimulating effect of cimetidine have been reported. It is unknown, however, whether or not cimetidine played any role in the progression of hepatoma and IgM-M proteinemia in our case.

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A 34-year-old female with Budd-Chiari syndrome was admitted for the abnormal findings on hemostatic examinations to our clinic in 1983. PTT and aPTT were remarkablly prolonged and PT was slightly prolonegd, but whole blood clotting time and each coagulation factor were within normal range. The plasma from this patient showed prolonged kaolin clotting time (KCT) which failed to normalize with normal plasma, while the platelet rich plasma from her normalized normal KCT. Those findings were consistent with the presence of lupus anticoagulant. Further examination clarified that the anticoagulant activity from patient's plasma was found in the second peak on Sephadex G-200 chromatography, and reacted with phosphatidylserine but not with other phospholipids on immunodiffusion. we also demonstrated the accelerated genaration of procoagulant activity in a modified thromboplastin generation time test and prolongation of Kaolin-induced fibrinolytic activity indicating lowered intrinsic fibrinolytic activity. Those results might be associated with the thrombotic events in this patient.
Furthermore, immunological studies indicated increased IgG, positive anti-DNA antibody and anti-nuclear antibody, and positive direct Coombs' test.

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