Groeneveld K, te Marvelde J G, van den Beemd M W, Hooijkaas H, van Dongen J J
Department of Immunology, Erasmus University Rotterdam, The Netherlands.
Leukemia. 1996 Aug;10(8):1383-9.
Intracellular antigens are of major importance for immunophenotyping of normal leukocytes as well as leukemias and malignant lymphomas. Immunofluorescence microscopic evaluation of cytocentrifuge preparations has remained the preferred technique for detection of intracellular antigens for a long time. Recently, flow cytometric detection of intracellular antigens has been improved by the development of new permeabilization/fixation solutions. We compared four commercially available solutions: FACS Brand Lysing Solution (FACS Brand; Becton Dickinson, San Jose, CA, USA), Fix & Perm cell permeabilization kit (Fix & Perm; An der Grub, Vienna, Austria), OptiLyse B lysing solution (OptiLyse B; Immunotech, Marseille, France), and ORTHO PermeaFix(PermeaFix; Ortho Diagnostic Systems, Raritan, NJ, USA). These solutions were evaluated for the complexity and duration of the intracellular staining procedure, the effects on light scatter patterns, and the staining results for the intracellular antigens terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD3 (CyCD3), myeloperoxidase (MPO), and cytoplasmic immunoglobulin light chains (CylgL). The four methods could easily be introduced in our laboratory and had only minor effect on the light scatter patterns of the tested cell samples. Each of the four tested antigens was detectable with at least one of the four methods. Only the Fix & Perm cell permeabilization kit could be used for reliable detection of all four intracellular antigens. In a large series of 450 BM and PB samples containing various percentages of TdT+ cells, the results of flow cytometric TdT staining with FACS Brand Lysing Solution were highly comparable to the results obtained by immunofluorescence microscopy (P = <0.00001). Our comparative study shows that flow cytometric detection of the intracellular antigens TdT, CyCD3, MPO, and CylgL can now reliably be performed on a routine basis.
细胞内抗原对于正常白细胞以及白血病和恶性淋巴瘤的免疫表型分析至关重要。长期以来，细胞离心涂片的免疫荧光显微镜评估一直是检测细胞内抗原的首选技术。最近，新型通透化/固定溶液的开发改进了细胞内抗原的流式细胞术检测。我们比较了四种市售溶液：FACS品牌溶血溶液（FACS品牌；美国加利福尼亚州圣何塞市贝克顿·迪金森公司）、Fix & Perm细胞通透化试剂盒（Fix & Perm；奥地利维也纳安德尔格鲁布公司）、OptiLyse B溶血溶液（OptiLyse B；法国马赛免疫技术公司）和ORTHO PermeaFix（PermeaFix；美国新泽西州拉里坦市奥多诊断系统公司）。对这些溶液进行了评估，包括细胞内染色程序的复杂性和持续时间、对光散射模式的影响以及细胞内抗原末端脱氧核苷酸转移酶（TdT）、细胞质CD3（CyCD3）、髓过氧化物酶（MPO）和细胞质免疫球蛋白轻链（CylgL）的染色结果。这四种方法可以很容易地引入我们的实验室，并且对测试细胞样本的光散射模式只有轻微影响。四种测试抗原中的每一种都可以用这四种方法中的至少一种检测到。只有Fix & Perm细胞通透化试剂盒可用于可靠检测所有四种细胞内抗原。在一系列包含不同百分比TdT+细胞的450份骨髓和外周血样本中，用FACS品牌溶血溶液进行流式细胞术TdT染色的结果与免疫荧光显微镜获得的结果高度可比（P = <0.00001）。我们的比较研究表明，现在可以在常规基础上可靠地进行细胞内抗原TdT、CyCD3、MPO和CylgL的流式细胞术检测。
