Koníková E, Kusenda J, Babusíková O, Glasová M
Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia.
Neoplasma. 1995;42(5):227-34.
Double immunofluorescence studies using both surface and cytoplasmic antigens were performed on cells of some human hematopoietic lines. We tested several permeabilization protocols in order to optimize, improve and simplify flow cytometric assay to detect the combinations of two markers present in one cell which could be regarded as leukemia-related markers. It was found, that buffered formaldehyde-acetone (BFA) fixation renders the cell membrane permeable without destroying surface antigens so that intracellular and cell surface markers could be measured simultaneously by flow cytometry. Cell lines used for the experiments reported here included MOLT4 T cell line, mature B cell lines DAUDI and U-266, and early B cell line REH-6. Results from our studies demonstrated, that in the absence of CD3 antigen on the surface membrane of viable MOLT4 blast cells, double labeling of fixed, permeabilized cells revealed 97% mCD7+, cCD3+ double positive cells. Two color staining with anti-CD19 and anti-CD22 monoclonal antibodies (MoAbs) in DAUDI cells showed, that larger part of cCD22+ cells expressed mCD19 antigen. CD22 antigen was absent on DAUDI cell membrane. Of great interest was the finding, that the marker detected by anti-CD19 MoAb which was absent on the membrane of U-266 cells was detected in their cytoplasm. Double staining of these cells revealed, that the number of mCD22+, cCD19+ double positive cells was 80%. Cytoplasmic CD22 antigen along with surface membrane CD19 was used to define early B cell line REH-6 as well. Our results demonstrate majority of double positive cells among tested population (mCD19+, cCD22+). To our knowledge the presence of cytoplasmic IgM detectable by flow cytometry in REH-6 cells, which could be so regarded as a precise and adequate counterpart to pre-B acute leukemia cell phenotype in children, is an original finding. Immunological typing plays an important part in the multiple marker analysis of hematopoietic malignancies. Through these surface and cytoplasmic marker combinations minor neoplastic cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for monitoring minimal residual disease in acute leukemia patients.
使用表面抗原和细胞质抗原进行的双重免疫荧光研究在一些人类造血细胞系的细胞上进行。我们测试了几种通透化方案，以优化、改进和简化流式细胞术检测，以检测存在于一个细胞中的两种标志物的组合，这些标志物可被视为白血病相关标志物。结果发现，缓冲甲醛 - 丙酮（BFA）固定可使细胞膜通透而不破坏表面抗原，从而可以通过流式细胞术同时测量细胞内和细胞表面标志物。用于此处报道实验的细胞系包括MOLT4 T细胞系、成熟B细胞系DAUDI和U - 266以及早期B细胞系REH - 6。我们的研究结果表明，在活的MOLT4原始细胞表面膜上不存在CD3抗原的情况下，对固定、通透化细胞进行双重标记显示97%的细胞mCD7 +、cCD3 +双阳性。在DAUDI细胞中用抗CD19和抗CD22单克隆抗体（MoAbs）进行双色染色显示，大部分cCD22 +细胞表达mCD19抗原。DAUDI细胞膜上不存在CD22抗原。有趣的是，发现在U - 266细胞膜上不存在的抗CD19 MoAb检测到的标志物在其细胞质中被检测到。这些细胞的双重染色显示，mCD22 +、cCD19 +双阳性细胞的数量为80%。细胞质CD22抗原与表面膜CD19一起也用于定义早期B细胞系REH - 6。我们的结果表明，在测试群体中大多数细胞为双阳性（mCD19 +、cCD22 +）。据我们所知，通过流式细胞术在REH - 6细胞中可检测到细胞质IgM，这可被视为儿童前B急性白血病细胞表型的精确且合适的对应物，这是一项原始发现。免疫分型在造血恶性肿瘤的多标志物分析中起着重要作用。通过这些表面和细胞质标志物组合可以检测到少量肿瘤细胞群体。人类造血细胞系可作为监测急性白血病患者微小残留病的可靠模型系统。
