Jensen R H, Bigbee W L
Department of Laboratory Medicine, University of California, San Francisco, USA.
Cytometry. 1996 Apr 1;23(4):337-43. doi: 10.1002/(SICI)1097-0320(19960401)23:4<337::AID-CYTO10>3.0.CO;2-U.
The glycophorin A (GPA) somatic cell mutation assay is being applied widely as an in vivo biomarker in molecular epidemiologic studies of human populations. The assay uses two-color immunolabeling and flow cytometry of peripheral blood samples to enumerate allele-loss variant erythrocytes that appear as a result of mutations at the GPA locus in bone marrow erythroid cells. We have developed an improved version of the assay in which both anti-GPA monoclonal antibodies are directly conjugated with distinguishable fluorophores, fluorescein and phycoerythrin. Parallel analyses of 77 blood samples using the existing BR6 assay and our new DB6 assay demonstrate that the DB6 assay produces cleaner bivariate flow histograms with generally lower variant cell frequencies and lower coefficients of variation on replicate analyses of individual blood samples. With the BR6 assay, an artifact was shown to exist that results in enumeration of high frequencies of variant erythrocytes in a small fraction of samples that have been subjected to poor shipping and/or storage conditions. Using DB6, these same samples display acceptable histograms and low frequency of variant cells. High speed cell sorting followed by immuno analysis indicates that the BR6 artifact results from inhibited binding of the very high molecular weight antibody plus secondary reagent, which is used for the BR6 assay. We therefore recommend that DB6 be adopted as standard protocol for the GPA assay, and to assist other researchers in standardization and comparison, we are making available a set of calibrated, fixed blood samples.
血型糖蛋白A（GPA）体细胞突变检测作为一种体内生物标志物，正广泛应用于人群分子流行病学研究。该检测采用双色免疫标记和外周血样本流式细胞术，以计数因骨髓红细胞系GPA基因座突变而出现的等位基因缺失变异红细胞。我们开发了一种改进版检测方法，其中两种抗GPA单克隆抗体均直接与可区分的荧光团（荧光素和藻红蛋白）偶联。使用现有的BR6检测方法和我们新的DB6检测方法对77份血样进行平行分析，结果表明DB6检测方法能产生更清晰的双变量流式直方图，个体血样重复分析时变异细胞频率通常更低，变异系数也更低。使用BR6检测方法时，发现存在一种假象，即在一小部分运输和/或储存条件不佳的样本中会导致变异红细胞的高频率计数。使用DB6检测方法时，这些相同的样本显示出可接受的直方图和低频率的变异细胞。高速细胞分选后进行免疫分析表明，BR6假象是由用于BR6检测的非常高分子量抗体加二级试剂的结合受抑制导致的。因此，我们建议将DB6作为GPA检测的标准方案，并且为帮助其他研究人员进行标准化和比较，我们正在提供一组经过校准的固定血样。
