Detection of somatic mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors using a single beam flow sorter.

TLDR

A single‑beam cell sorter assay using fluorescently labeled anti‑glycophorin A antibodies and propidium iodide staining was developed to quantify variant erythrocytes at the glycophorin A locus in blood from atomic bomb survivors. SBS‑measured variant frequencies of hemizygous and homozygous glycophorin A cells correlated with dual‑beam results and increased with radiation dose and lymphocyte chromosome aberration frequency.

Abstract

A modified method was developed for measuring the frequency of variant erythrocytes at the glycophorin A locus using a single beam cell sorter (SBS). Fluorescein- or phycoerythrin-labeled monoclonal antibodies specific for the M or N glycophorin A alleles were used for the SBS assay. To prevent contamination of nucleated cells in the sorting windows, the nucleated cells in the fixed erythrocyte sample were stained with propidium iodide before flow sorting. Blood samples were obtained from atomic bomb survivors who were heterozygous for the MN blood type, and the frequencies of the hemizygous and homozygous variant of the M or N glycophorin A allele were measured by the SBS. For the three types of variants, hemizygotes for M and N allele (Nø and Mø) and homozygotes for M allele (MM), the variant frequency measured by the SBS correlated well with that previously determined by a dual beam cell sorter. Variant frequencies of the Nø, Mø, and MM cell types in atomic bomb survivors determined by SBS measurements were found to increase with radiation dose (DS86, kerma) as well as with the frequency of chromosome aberrations in lymphocytes.