Pattanapanyasat K, Kyle D E, Tongtawe P, Yongvanitchit K, Fucharoen S
Thalassemia Center, Faculty of Graduate Studies, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Cytometry. 1994 Dec 15;18(4):199-208. doi: 10.1002/cyto.990180403.
Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three-color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71-fluorescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, glyco A-FITC negative cells, or CD45-PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non-lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two-color antibodies. Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six-tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of non-lymphoid cells.
由于未成熟红细胞比例较高，地中海贫血患者外周血的流式细胞术（FCM）免疫表型分析存在技术难题。前向散射（FSC）和侧向散射（SSC）与人白细胞抗原/单核细胞抗原（CD45/CD14）相关荧光的组合无法识别地中海贫血患者的淋巴细胞群体；因此，有必要排除未成熟红细胞以分析这些患者的淋巴细胞亚群。基于转铁蛋白受体（CD71）或血型糖蛋白A（glyco A）存在于所有未成熟红细胞上，但在CD45阳性白细胞上不表达，开发了一种同步三色FCM方法。在本研究中，通过使用FSC/CD71-异硫氰酸荧光素（FITC）、FSC/glyco A-FITC或FSC/CD45-多甲藻叶绿素蛋白（PerCP）图谱排除不需要的细胞群体来识别淋巴细胞群体。识别出CD71-FITC阴性细胞、glyco A-FITC阴性细胞或CD45-PerCP阳性细胞，然后根据FSC/SSC进行分析，以排除FSC/SSC门中任何剩余的非淋巴细胞。然后使用其他不相关的双色抗体分析FSC/SSC门中的细胞。在三种门控策略中，CD45-PerCP和glyco A-FITC方法优于CD71-FITC门控方法。两种方法均显著提高了分析门中淋巴细胞的纯度。两种方法都简单、直接，需要一套六管试剂管，并为含有大量非淋巴细胞的制剂中的淋巴细胞亚群免疫表型分析提供了可靠的方法。
