Technical Report No. 9-88
Detection of somatic mutations at the glycophorin-A locus in erythrocytes of atomic bomb survivors using a single beam flow sorter
Kyoizumi S, Nakamura N, Hakoda M, Awa AA, Bean MA, Jensen RH, Akiyama MEditor’s note: A publication based on this report was published in Cancer Res 49:581-8, 1989.
Summary
A modified method was developed for measuring the frequency of variant erythrocytes at the glycophorin A (GPA) locus using a single beam cell sorter (SBS). Fluorescein- or phycoerythrin-labeled monoclonal antibodies specific for the M or N GPA alleles were used for the SBS assay. Filter cross-talk of fluorescence emitted from these two dyes was corrected using the SBS electronic compensator. To prevent contamination of nucleated cells in the sorting windows, the nucleated cells in the fixed red blood cell 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 variants of the M or N GPA allele were measured by the SBS. For the three types of variant, hemizygotes for M and N allele (N phi and M phi ) and homozygotes for M allele (MM), the variant frequency measured by the SBS showed a good correlation with that previously determined by the dual beam cell sorter. Variant frequencies of the N phi, M phi, and MM cell types in A-bomb survivors, as determined by SBS measurements, were found to increase with radiation dose (DS86, kerma), as well as with the frequency of chromosome aberrations in lymphocytes.
A modified method was developed for measuring the frequency of variant erythrocytes at the glycophorin A (GPA) locus using a single beam cell sorter (SBS). Fluorescein- or phycoerythrin-labeled monoclonal antibodies specific for the M or N GPA alleles were used for the SBS assay. Filter cross-talk of fluorescence emitted from these two dyes was corrected using the SBS electronic compensator. To prevent contamination of nucleated cells in the sorting windows, the nucleated cells in the fixed red blood cell 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 variants of the M or N GPA allele were measured by the SBS. For the three types of variant, hemizygotes for M and N allele (N phi and M phi ) and homozygotes for M allele (MM), the variant frequency measured by the SBS showed a good correlation with that previously determined by the dual beam cell sorter. Variant frequencies of the N phi, M phi, and MM cell types in A-bomb survivors, as determined by SBS measurements, were found to increase with radiation dose (DS86, kerma), as well as with the frequency of chromosome aberrations in lymphocytes.