Purification of human hemopoietic stem cells for transplantation in Thailand.

Stem cell transplantation (SCT) has become the therapy of choice for many hematologic and immunologic disorders. At present, only 25% of patients have suitable HLA-identical donors. In an attempt to increase the donor pool for SCT in Thailand and Southeast Asia, we developed a program whereby parents and mismatched siblings can be used as donors. In this preliminary study, after granulocyte-colony-stimulating factor (G-CSF) was given to adult donors, peripheral blood stem cells (PBSC) were collected and CD34+ cells purified using a CliniMACS immunomagnetic device (Miltenyi Biotec, Germany). In seven experiments, purified CD34+ cells could be obtained from G-CSF-stimulated PBSC in large numbers (1.71 +/- 0.19 x 10(8)), with high purity (93 +/- 2.4%) and excellent recovery (64.28% - 85.62%). Immune reactive T and NK cells were adequately depleted to less than 0.2%. The purification procedure can be completed within 3 hours. In conclusion, a clinical stem cell purification program using this novel device is now established in Thailand and for the first time in Southeast Asia. This should allow further development of advanced SCT therapy including haploidentical and mismatched CD34+ SCT for patients' lacking HLA-identical donors in this region.

Study of hematopoietic progenitor cells in thalassemia after bone marrow transplantation.

The hematopoietic committed progenitor cells (BFU-E and CFU-GM) in blood and bone marrow were studied in thalassemic patients before and after bone marrow transplantation. Eighteen transplants were performed in 17 patients with thalassemia. Five were homozygous beta-thalassemia and 12 were beta-thalassemia/hemoglobin E disease. The age ranged from 1.2-14 years (median = 3.4 years). The conditioning regimen comprised busulfan 3.5-4 mg/kg/day for 4 days and cyclophosphamide 50 mg/kg/day for 4 days. Cyclosporin in combination with methotrexate were administered post transplant for GVHD prophylaxis. Before transplantation, BFU-E and CFU-GM in the blood of the patients were significantly higher compared with normal (p < 0.05) but were normal in the bone marrow. Only the CFU-GM in the bone marrow of the successful cases after transplantation recovered to the normal level at the first month through the 12th month whereas the BFU-E were low. Both CFU-GM and BFU-E in the blood were lower than normal after follow up to the 12th month. Inspite of the low number of progenitor cells, there was hematological recovery in the blood of the patients. It may be due to the capacity of the hematopoiesis react to stress which could be amplified the differentiation compartment or the shortened-transit time through the stem cell compartment.

Cytochemical stainings in acute promyelocytic leukemia: chloroacetate esterase reaction as a prognostic index.

Cytochemical studies including peroxidase, sudan black B and esterases were used for staining peripheral blood and bone marrow smears from 42 patients with acute promyelocytic leukemia. The most sensitive methods were sudan black B (mean 98%, range 81-100%) and peroxidase (mean 92% range 70-100%). Naphthol AS-D chloroacetate esterase activity was less sensitive and was positive in only 49.4 per cent (range 2-100%). All of the population of leukemic cells contained less than 3 per cent of alpha-naphthyl acetate esterase staining. For stability tests of the storage specimens compared to fresh stains, there was no difference in naphthol AS-D chloroacetate esterase (mean 45% vs 49% P greater than 0.7) and sudan black B (mean 74% vs 98% P greater than 0.3), but the enzyme activity was significantly decreased in peroxidase staining (mean 42% vs 92% P greater than 0.05). When the patients were divided into 2 groups according to the degree of AS-D chloroacetate esterase activity, those with lower activity had a higher number of white blood cells, promyelocytes and shorter survival compared to those with higher activity. Therefore, naphthol AS-D chloroacetate esterase may be useful as a prognostic index.

Massive chemotherapy and autologous bone marrow transplantation in Burkitt's lymphoma: the first two patients successfully treated in Thailand.

Massive chemotherapy and autologous bone marrow transplantation (ABMT) have been successfully used for the treatment of Burkitt's lymphoma. We report our first success with such treatment in two children with Burkitt's lymphoma in Thailand. Both patients had massive abdominal tumors with ascites and minimal bone marrow metastasis at the first presentation. They received induction chemotherapy and intensive treatment including central nervous system prophylaxis with cranial irradiation and intrathecal methotrexate until being in complete remission before starting massive chemotherapy, comprising the combination of BCNU, cytosine arabinoside, cyclophosphamide and 6-thioguanine followed by ABMT. Both patients recovered completely following intensive supportive treatments post ABMT and are still good health without evidence of the disease for 18 and 8 months after transplantation respectively. The role of massive chemotherapy and ABMT for the treatment of non-Hodgkin's lymphoma especially Burkitt's lymphoma was discussed.

Abnormalities of hematopoietic progenitor cells in patients with aplastic anemia after hematologic recovery.

Hematopoietic progenitor cells were studied in 11 patients with aplastic anemia who had hematologic recovery after androgen therapy. The mean numbers of colonies derived from erythroid and granulocyte-macrophage progenitor cells (BFU-E and CFU-GM) were markedly decreased compared to normal controls. Cell-mediated suppression of colony growth as detected by coculture studies was observed in 5 patients; 4 patients for CFU-GM and one for both CFU-GM and BFU-E. It is thus concluded that the pool of hematopoietic stem cells in patients after hematologic recovery is still not fully reconstituted. In addition, this impaired reconstitution appears due in some cases to cell mediated suppression of progenitor colony growth.

Studies on the pathogenesis of aplastic anemia in Thailand: evidence of immune-mediated mechanism.

The pathogenesis of aplastic anemia in Thailand was studied by using in vitro progenitor cells culture. In 37 patients who had active disease, the numbers of colonies derived from erythroid and granulocyte-macrophage progenitor cells (BFU-E and CFU-GM) were markedly decreased both in the blood and bone marrow as compared to normal controls. Co-culture of patients' cells with normal blood cells was performed in order to verify an immunologically mediated mechanism. In 8 of 26 patients, there were very low numbers of colonies both BFU-E and CFU-GM in the blood and bone marrow with significant suppression of colony formation in co-culture. Suppressor cells may have caused the aplasia in these patients. The rest had low colony formation and no suppression in co-culture. These patients may have absent or defective stem cells. None had normal colony formation. Therefore, aplastic anemia in Thailand may result mostly from defects involving the stem cells. Only some patients had cell mediated suppression of hematopoiesis as detected by co-culture.

Methylprednisolone treatment in aplastic anaemia.

Twenty patients with aplastic anaemia were treated with a short term bolus of methylprednisolone. Seven patients were refractory to anabolic steroids and were observed in very long follow-up periods of 14-104 months. Thirteen patients had never been treated. The latter group also received anabolic steroids. Five out of 20 patients responded to the treatment while the remaining patients did not or died. Responders among patients refractory to anabolic steroids had shorter duration of disease compared to non-responders. No recurrence of aplasia was observed in all responders.