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OBJECTIVE To investigate the effect of amifostine(Amf)on the differentiation of human megakaryocyte cell line-Dami. METHODS Dami cells were treated with Amf 0.01-5.0 mmol · L-1 for 12 d. Dami cells were counted every day for the growth curve:only cells with a diameter>20μm. The platelet demarcation membrane system was observed by transmission electron microscopy. The expression of CD33,CD34,CD41a and DNA ploidy was detected by flow cytometry. RESULTS Amf 0.1-1.0 mmol · L-1 promoted the differentiation of Dami cells ,but inhibited their proliferation at a concentration>1.0 mmol · L-1. When these cells were treated with Amf 1.0 mmol · L-1 for 12 d,the platelet demarcation membrane system was observed,the percentage of cells with a diameter >20 μm was increased by 24.6%(P1.0 mmol·L-1).
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Objective To setup a measurement of human bone marrow micromegakaryocyte which based on CD41a and PI double‐labeled flow cytometric analysis ,and study the significance in the diagnosis of MDS .Methods In 42 cases of MDS patients , their bone marrow megakaryocytes were obtained by Percoll density gradient separation medium .The megakaryocyte glycoproteinⅡb/Ⅲa(CD41a)were marked with fluorescein isothiocyanate through its corresponding monoclonal antibody ,and their DNA were marked with PI .Then the megakaryocyte ploidy was analyzed by flow cytometry(FCM ) .Results The method for micromegakaryo‐cyte identification and analysis was established .In 42 patients with MDS ,the detection rate of micromegakaryocyte was 90 .5 per‐cent by FCM analysis ,but only 54 .8 percent by Wright‐Giemsa staining test and 64 .3 percent by immunohistochemistry ,the differ‐ence among them was statistically significant(χ2 = 13 .640 ,P= 0 .001) .The 42 patients with MDS were divided into two groups (low‐risk group and high‐risk group) .The detection rates of micromegakaryocyte were 81 .8 percent in low‐risk group and 100 per‐cent in high‐risk group separately by FCM analysis ,the difference was statistically significant(χ2 =4 .019 ,P=0 .045) .Conclusion The detection rate of micromegakaryocyte by FCM with CD41a and PI double marker is higher than that by cytochemical staining . The detection rate of micromegakaryocyte in the high‐risk group is higher than that of the low‐risk group ,which shows that the de‐tection of micromegakaryocyte is of great significance for MDS prognosis assessment .
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Aims: The aim of this study was to investigate of the roles of CD5+ and CD19+ on lymphocytes, CD5+ on B lymphocytes, CD41a+ on platelets and CD55+ and CD59+ on erythrocytes in platelet destruction; and evaluate them according to the patient response status to steroid therapy and platelet counts in chronic immune thrombocytopenic purpura (ITP). Study Design: This study included 20 chronic ITP patients and 20 healthy controls. We investigated the roles of CD5+ and CD19+ expression on lymphocytes, CD5+ expression on B lymphocytes, CD41a+ expression on platelets, and CD55+ and CD59+ expression on erythrocytes, as well as the platelet counts in healthy and chronic ITP patients. Additionally, these markers were evaluated according to the patient response status to steroid therapy and platelet counts. Place and Duration of Study: This study took place at the Department of Internal Medicine and Haematology, Meram Medical Faculty at Selçuk University in Turkey, between November, 2008 and July, 2009. Methodology: A total of 40 patients (26 women, 14 men, age range: 19-79 years) were studied. The study group included 20 chronic ITP patients (12 women and 8 men, age range: 19-78 years) and the control group included 20 healthy volunteers (14 women and 6 men, age range: 22-79 years). The platelet counts and expressions of CD5+ and CD19+ on lymphocytes, CD5+ on B lymphocytes, CD41a+ on platelets, and CD55+ and CD59+ on erythrocytes were analysed in the patients and control subjects. The chronic ITP patients were evaluated according to their requirements of treatment. Five patients whose platelet counts were above 50,000 mm–3 were observed without treatment. The other 15 patients whose platelet counts were under 50.000 mm–3 and had bleeding, or whose platelet counts were under 20,000 mm–3, were given methylprednisolone treatments (1 mg/kg/day orally). Three of the 15 patients discontinued treatment for various reasons. The twelve patients who continued the methylprednisolone treatment were divided into two subgroups according to their responder status of steroid treatment. The patients whose platelet counts slowly increased above 30,000 mm–3 within three months included the steroid treatment responder subgroups. The chronic ITP patients were also divided into two subgroups according to the severity of their thrombocytopenia. The limit of the platelet count was 30,000 mm–3 for severe thrombocytopenia. These parameters were analysed according to the response status of the steroid treatment and platelet counts. The platelet counts, and the expressions of these markers, were compared between the subgroups. Results: The level of CD5+ on B lymphocyte expression (2.19 ± 1.65) in peripheral blood lymphocytes was significantly higher in the immune thrombocytopenic purpura patients than in the controls (P = .05). The CD55+ + CD59+ expression on erythrocytes (98.03 ± 1.77) was significantly higher in the ITP patients than in the controls (P = .05). There was no significant relationship between the expression of CD5+, CD19+ or CD5+ on B lymphocytes, CD41a+ expression on platelets or CD55+ and CD59+ expression on erythrocytes, according to the response status to steroid therapy in the patient group (P > 0.05). Additionally, the patients were evaluated according to platelet counts, and there was a significantly positive correlation between the level of CD41a+ expression on the platelets and the platelet count (P = .05). Conclusion: The level of CD5+ on B lymphocytes was significantly higher in the ITP patients than in the controls. A relationship between CD55+ plus CD59+ expression on erythrocytes and immune destruction of platelets was not observed in the chronic ITP patients.
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BACKGROUND: The binding of some monoclonal antibodies platelet glycoprotein (GP) IIb/IIIa, which is frequently used for flow cytometric immnophenotyping, is known to be inhibited by EDTA. To select the ideal antibodies to be included in the 'Acute Leukemia Panel' for immunophenotyping of acute leukemia, we compared the inhibitory effect of EDTA on the binding of 5 different clones of monoclonal antibodies to platelet GP IIb/IIIa. We also discovered a simple method to neutralize this inhibitory effect. METHODS: Flow cytometric measurement of the number of platelet GP IIb/IIIa binding sites with different anticoagulants was performed using a panel of 5 clones of monoclonal antibodies against CD41 (clone PM6/248), CD41a (clone 96.2C1 & clone HIP8), CD41b (clone HIP2) and CD61 (clone VI-PL2), and the results are expressed as the mean equivalent soluble fluorochrome (MESF) values. RESULTS: The MESF value of the EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody showed a significantly lower value than the MESF of platelets anticoagulated with heparin or citrate (P<0.001). The inhibitory effect of EDTA on the binding of anti-CD41a, clone 96.2C1 antibody to the platelets was neutralized by addition of heparin and CaCl2. The mean MESF value of EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody was significantly increased by the addition of heparin and CaCl2 (P=0.0001). CONCLUSION: The false-negative results of the binding of anti-CD41a, clone 96.2C1 antibody to the platelets seem to be due to the calcium chelating property of EDTA, and the addition of CaCl2 and heparin could be used as an easy compensatory measure for the inhibitory effect of EDTA on other antibodies as well.