Khuhapinant A, Bunyaratvej A, Sahaphong S, Pattanapanyasat K, Fucharoen S
Department of Pathobiology, Faculty of Science, Mahidol University, Thailand.
Southeast Asian J Trop Med Public Health. 1997;28 Suppl 3:82-92.
Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.
地中海贫血是一种遗传性血液系统疾病，通常可根据受影响的珠蛋白失衡（α-或β-珠蛋白）分为两种主要类型，即α-地中海贫血和β-地中海贫血。与地中海贫血红细胞相关的细胞异常范围很广，如低色素性、小红细胞症、细胞变形性降低和膜氧化损伤。红细胞异常导致过早破坏，伴有骨髓红系增生和无效造血。如先前在骨髓红系前体细胞和网织红细胞（红系分化的倒数第二阶段）中所示，除成熟阶段外，在地中海贫血红细胞的红系分化途径中也发现了异常。然而，由于骨髓穿刺困难且有风险以及所获得细胞的异质性，我们对更原始的红系阶段的了解存在滞后。我们采用了一种新型的β-地中海贫血/HbE红系前体细胞两相液体培养（TPLC）方法，而不是传统的半固体培养。这种液体培养可以获得更高的细胞产量，细胞分化阶段相当同步，并且很容易应用于其他细胞分析技术。从未行脾切除术和已行脾切除术的β-地中海贫血/HbE患者获得的外周血单个核细胞（PBMC）首先在补充有5637条件培养基的培养基中培养6天（第一阶段），然后转移到补充有重组人促红细胞生成素的培养基中，以使红系前体细胞进行终末分化（第二阶段）。在第一阶段或第二阶段期间，定期对培养的细胞进行取样以确定细胞数量，将细胞离心到载玻片上，并用瑞氏染色法染色，以对其分化阶段进行形态学评估，并通过用荧光素化抗转铁蛋白受体（抗CD71）和藻红蛋白偶联抗血型糖蛋白A染色进行流式细胞术分析。通过流式细胞术评估后，将剩余的染色细胞离心到载玻片上，并用荧光显微镜和激光扫描共聚焦显微镜拍照。将呈现形态学评估、流式细胞术分析和显微镜图像的结果。
