Direct immunofluorescence labeling provides an improved method for the glycophorin A somatic cell mutation assay

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.