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Objective: To investigate the role of the burden of abnormal hematopoietic clone in the development of myelodysplastic syndromes (MDS)., Methods: The ratio of the bone marrow cells with abnormal chromosomes to the total counted bone marrow cells was regarded as the index of MDS clone burden. The disease severity related parameters including white blood cell count, hemoglobin, platelet count, lactate dehydrogenase level, bone marrow blast, myeloid differentiation index, micromegakaryocyte, transfusion, interleukin-2, tumor necrosis factor (TNF), CD4+ and CD8+ T cells of MDS patients were assayed, and the correlations between those parameters and MDS clone burden were also analyzed., Results: The clone burden of MDS patients was 67.4% +/- 36.2%. MDS clone burden positively correlated with bone marrow blasts (r = 0.483, P < 0.05), negatively with hemoglobin level (r = -0.445, P < 0.05). The number of blasts, hemoglobin, and erythrocytes in high clone burden (> 50%) and low clone burden ( < or = 50%) groups were 7.78% +/- 5.51% and 3.45% +/- 3.34%, 56.06 +/- 14.28 g/L and 76.40 +/- 24.44 g/L, (1.82 +/- 0.48) x 10(12)/L and (2.32 +/- 0.66) x 10(12)/L, respectively (all P < 0.05). CD4+ T lymphocytes of MDS patients and normal controls were (0.274 +/- 0.719) x 10(9)/L and (0.455 +/- 0.206) x 10(9)/L, respectively (P < 0.05). CD8+ T lymphocytes of MDS patients and normal controls were (0.240 +/- 0.150) x 10(9)/L and (0.305 +/- 0.145) x 10(9)/L, respectively. The serum level of interleukin-2 of MDS patients (6.29 +/- 3.58 ng/mL) was significantly higher than normal control (3.11 +/- 1.40 ng/mL, P < 0.05). The serum level of TNF of MDS patients and normal control group were 2.42 +/- 1.79 ng/mL and 1.68 +/- 0.69 ng/mL, respectively. The ratio of CD4 to CD8 was higher in high clone burden MDS patients (1.90 +/- 0.52) than that in low clone burden patients (0.97 +/- 0.44, P < 0.05)., Conclusion: The quantitive clonal karyotype abnormalities and deficient T cell immunity are important parameters for evaluating MDS severity and predicting its progression.

This study was aimed to evaluate expression levels of CD166, Fas and apoptosis-related proteins in bone marrow neutrophils of PNH patients and normal controls, and to analyze their correlation in order to explore whether exist apoptosis abnormality in BM neutrophils of PNH patients. The expression levels of CD16b, Fas and Bax, Bcl-2 in BM neutrophils of PNH patients and normal controls were assayed by flow cytometry; the difference of expression levels between patients and controls, and expression correlation between CD16b and apoptosis-related proteins were compared. The results showed that (1) the expression levels of CD16b on BM neutrophils of patients and controls were (20.36 +/- 9.05)% and (71.34 +/- 26.8)% respectively (P = 0.01); (2) the expression levels of CD95 on BM neutrophils of patients and controls were (62.83 +/- 32.11)% and (48.00 +/- 38.52)% respectively, there were no significant difference between CD95 expressions in BM neutrophils of PNH patients and controls and no significant correlation between expression of CD95 and CD16b on BM neutrophils of PNH patients (P > 0.05); (3) the expression levels of Bcl-2 in BM neutrophil cytoplasma of patients and controls were (8.64 +/- 5.40)% and (16.82 +/- 15.39)% respectively, there were no significant difference between Bcl-2 expression of patients and controls, and no significant correlation between the expression of Bcl-2 and CD16b in BM neutrophil cytoplasma of PNH patients (P > 0.05); (4) the expression levels of Bax in BM neutrophil cytoplasma of patients and control were (30.47 +/- 22.15)% and (48.47 +/- 15.99)% respectively, there were no significant difference between the Bax expressions of patients and controls, and no significant correlation between the Bax and CD16b expressions in BM neutrophil cytoplasma of PNH patients. In conclusion, BM neutrophils of PNH patients expressed apoptosis-related CD95, Bcl-2 and Bax without significant difference from the normal controls, and without significant correlation with the CD16b expression. It is suggested that the cell growth and decrease of PNH patients possibly are independent of abnormal apoptosis.

Objective: To investigate the abnormal hematopoietic clone burden of the patients with myelodysplastic syndromes (MDS) and its clinical implication., Methods: The ratio of the metaphase with abnormal karyotypes to the total was regarded as the index of MDS clonal burden. Thirteen parameters were assayed and the correlations between these parameters and MDS clone burden were analysed., Results: The clonal burden of MDS patients was (67.4 +/- 36.2)%. It correlated positively with bone marrow blasts (r = 0.483, P < 0.05), negatively with hemoglobin level (r = -0.445, P < 0.05). The number of blasts, hemoglobin and erythrocytes in high clonal burden (>50%) and low clonal burden (< or = 50%) groups were significantly different (P < 0.05). CD4+ T lymphocytes of MDS patients and normal controls were (274.18 +/-71.85) x 10(6)/L and (454.82 +/- 205.88) x 10(6)/L (P < 0.05) respectively. CD8+ T lymphocytes between MDS patients and normal controls had no difference. The serum level of IL-2 of MDS patients and normal control groups were (6.29 +/- 3.58) g/L and (3.11 +/- 1.40) microg/L (P < 0.05) respectively; but no difference in the serum level of TNF between MDS and control groups. The ratio of CD4+ to CD8+ in high clonal burden patients was 1.90 + 0.52, and in low clonal burden patients was 0.97 +/- 0.44 (P < 0.05)., Conclusion: The clonal burden and deficient T cell immunity are the indicators for predicting MDS patients clinical progression.

Objective: To study the response of hematopoietic cells (HSC) to granulocyte colony stimulating factor (G-CSF) in paroxysmal nocturnal hemoglobinuria (PNH) patients., Methods: (1) Bone marrow mononuclear cells (BMMNC) from 17 PNH patients and 12 normal subjects were inoculated into semisolid culture media containing or not G-CSF (50 ng/ml). The cluster/colony forming unit-granulocyte/monocyte (CFU/cFU-GM) were counted and compared. (2) BMMNC of 20 PNH patients and 12 normal controls were triply stained for CD34, CD59 and G-CSF receptor CD114/stem cell factor receptor (C-KIT) CD117 and assessed by FCM. The CD34(+) cells were identified as CD34(+)/CD59(+) and CD34(+)/CD59(-). Percentage of CD114 and CD117 expression in each cell population was calculated., Results: (1) PNH cFU-GM without G-CSF were (112.41 +/- 22.74)/10(5) BMMNC, while with G-CSF: (133.82 +/- 25.85)/10(5) BMMNC and normal cFU-GM were (190.33 +/- 36.05)/10(5) BMMNC, (309.42 +/- 92.94)/10(5) BMMNC, respectively. Whether with or without G-CSF, PNH BMMNC formed less cFU-GM than control did, both of the two kinds of BMMNC responded to G-CSF well (P < 0.05), but the increment of PNH cFU-GM yields was less than that of the normal control (P < 0.05). CFU-GM yields of PNH BMMNC without G-CSF were (24.29 +/- 9.05)/10(5) BMMNC, with G-CSF were (27.53 +/- 10.65)/10(5) BMMNC, while normal control were (77.42 +/- 36.01)/10(5) BMMNC and (98.00 +/- 43.14)/10(5) BMMNC, respectively. Whether with or without G-CSF, PNH BMMNC showed less CFU-GM yields than that of control (P < 0.05). (2) The percentage of CD114 positive cells in PNH CD34(+)CD59(+) BMMNC was (73.34 +/- 29.40)% and that in PNH CD34(+)CD59(-) BMMNC and in control CD34(+)CD59(+) BMMNC were (32.70 +/- 6.89)% and (58.52 +/- 29.99)%, respectively. The percentage of CD114 expression in PNH CD34(+) CD59(-) BMMNC was less than that in the other two groups (P < 0.05). The percentages of CD117 positivities on the PNH CD34(+)CD59(+) BMMNC were (76.90 +/- 22.08)%, PNH CD34(+) CD59(-) (36.03 +/- 7.69)% and control CD34(+) CD59(+) (80.28 +/- 13.36)%, respectively (P < 0.01)., Conclusion: In vitro, BMMNC of normal control grow better, and respond better to G-CSF than PNH BMMNC do. PNH CD34(+)CD59(-) BMMNC express less G-CSF receptor and C-KIT than PNH CD34(+)CD59(+) and normal CD34(+)CD59(+) BMMNC do, which may be the reason that abnormal PNH clone grow worse than the normal clones do.

Objective: To investigate the expression of apoptosis receptor FAS (CD95) and apoptosis related protein Bcl-2 and Bax in CD34 positive bone marrow cells of the patients with polycythemia vera (PV)., Methods: The expressions of apoptosis receptor FAS (CD95) and apoptosis related protein Bcl-2 and Bax in bone marrow CD34(+) cells from 21 PV patients, 8 essential thrombocythemia (ET) and 11 normal persons were assessed by bicolor flow cytometry (FCM), and the expressions of Bcl-2 and Bax mRNA were assessed by RT-PCR, and their correlation was analysed., Results: There was no difference between the expressions of CD95 in CD34(+) bone marrow cells of PV patients (42.65 +/- 15.56)%, and that of ET patients (45.31 +/- 17.62)% and of normal person (37.55 +/- 15.19)% (P > 0.05). There was no difference between the expression of Bax in CD34(+) bone marrow cells of PV patients (35.83 +/- 9.33)% and of normal persons (41.65 +/- 9.04)% (P > 0.05). The expression of Bcl-2 in CD34(+) bone marrow cells of PV patients (79.35 +/- 14.43)% was significantly higher than that of normal controls (55.84 +/- 13.43)% (P < 0.01). The ratio of Bax/Bcl-2 of PV patients (0.47 +/- 0.14) was significantly lower than that in normal controls (0.76 +/- 0.24) (P < 0.01). The expression of Bcl-2 mRNA in PV patients' bone marrow hematopoietic cells was higher than that of normal controls (P < 0.01). There was no difference between the expression of Bax mRNA in bone marrow hematopoietic cells of PV patients and that of normal controls. Bcl-2 expression was negatively correlated with Annexin V expression in CD34(+) bone marrow cells of PV patients., Conclusion: Over-expression of Bcl-2, one of anti-apoptosis genes, in CD34(+) bone marrow cells might be involved in the lower apoptosis of PV patients.

Objective: To study the apoptosis and proliferation of CD(34) positive (CD(34)(+)) bone marrow cells (BMC) in patients with polycythemia vera (PV)., Methods: The expression of Annexin V and Ki67 of the CD(34)(+) BMC in 20 PV patients and control cases [10 essential thrombocythemia (ET), 12 normal persons] were assessed by bicolor flow cytometry (FCM), and the correlation between apoptosis and clinical situation was analysed in PV patients., Results: The Annexin V expressions of CD(34)(+) BMC were (15.96 +/- 1.45)% in PV patients and (15.53 +/- 1.76)% in ET patients which were lower than that in normal subjects [(23.61 +/- 3.89)%, (P < 0.05)]. The Ki67 expression of CD(34)(+) BMC was (48.79 +/- 11.68)% in PV patients and (49.60 +/- 9.98)% in ET patients, which were significantly higher than that in normal controls (33.87 +/- 6.82)%. The ratio of apoptosis/proliferation in PV patients was 0.33 +/- 0.10 and in ET patients 0.32 +/- 0.02 which were significantly lower than that in normal controls 0.72 +/- 0.11 (P < 0.01). The apoptosis of CD(34)(+) BMC was negatively correlated with the hemoglobin (Hb) levels (r = -0.481, P = 0.037), white blood cells (WBC) (r = -0.538, P = 0.026) and the numbers of endogenous erythroid colony (EEC) (r = -0.632, P = 0.50), and the ratio of apoptosis/proliferation was negatively correlated with the Hb (r = -0.537, P = 0.018) and WBC (r = -0.667, P = 0.003) in PV patients., Conclusion: There were lower apoptosis and higher proliferation in CD(34)(+) BMC of PV patients. Lower apoptosis was correlated with the severity of the disease.