Baatout S, Chatelain B, Staquet P, Symann M, Chatelain C
Laboratory of Experimental Hematology and Oncology, Oncology Unit, UCL, Brussels, Belgium.
Haematologia (Budap). 1998;29(3):213-28.
A flow cytofluorometric measurement of megakaryocyte ploidy has been adapted from Tomer's method. Briefly, bone marrow is aspirated through a medium containing theophyllin, prostaglandin-E1 and other antiaggregant agents. Megakaryocytes-enriched buffy coats are recovered. Megakaryocytes are then stained for GP IIIa coupled with FITC and for DNA (propidium iodide). A Becton Dickinson FACScan flow cytometer is used for measuring the ploidy distribution of GP IIIa positive cells. The method we developed presents several advantages. Firstly, the time required is greatly decreased in comparison with other studies. Secondly, the washing steps are limited in number allowing a diminution of the cell loss. Thirdly, the method ensures a better megakaryocyte preservation. Finally, selection of megakaryocytes by ploidy and expression of GP IIIa can be made easily because of the simultaneity of the two stainings as well as by the use of a precise gating on the flow cytometer. Based on these results, we conclude that the present method provides a better means for the isolation and analysis of human normal megakaryocytes. This technique has been applied to the analysis of megakaryocyte populations from patients with abnormal platelet counts. In chronic myeloid leukemia patients, analysis of ploidy distribution shows a shift toward the low ploidy while in patients with immune thrombocytopenic purpura, polycythemia vera and essential thrombocythemia the ploidy distributions are shifted toward the high ploidy.
巨核细胞倍性的流式细胞荧光测定法是根据托默的方法改编而来。简要来说，通过含有茶碱、前列腺素 - E1和其他抗聚集剂的培养基抽吸骨髓。回收富含巨核细胞的血沉棕黄层。然后将巨核细胞用与异硫氰酸荧光素（FITC）偶联的血小板糖蛋白IIIa（GP IIIa）以及DNA（碘化丙啶）进行染色。使用贝克曼库尔特公司的FACScan流式细胞仪测量GP IIIa阳性细胞的倍性分布。我们开发的方法具有几个优点。首先，与其他研究相比，所需时间大幅减少。其次，洗涤步骤数量有限，减少了细胞损失。第三，该方法能更好地保存巨核细胞。最后，由于两种染色同时进行以及在流式细胞仪上使用精确的门控，可轻松根据倍性和GP IIIa的表达来选择巨核细胞。基于这些结果，我们得出结论，目前的方法为分离和分析人类正常巨核细胞提供了更好的手段。该技术已应用于分析血小板计数异常患者的巨核细胞群体。在慢性髓性白血病患者中，倍性分布分析显示向低倍性偏移，而在免疫性血小板减少性紫癜、真性红细胞增多症和原发性血小板增多症患者中，倍性分布向高倍性偏移。
