Flow Cytometric Analysis of Micronuclei in the CD2 ± Subpopulation of Human Lymphocytes Enriched by Magnetic Separation

Abstract

An improved flow cytometric method for the scoring of micronuclei in human lymphocytes irradiated in vitro is presented. Because, especially in cultivated human lymphocytes, unspecific DNA-containing debris from dying cells can influence the measured frequency of micronuclei, a preselection of CD2 + population was performed before preparation of the suspension of micronuclei and nuclei. Magnetic separation using anti-CD2 antibody-conjugated magnetic beads were used for this purpose. The results obtained by this improved flow cytometric technique were compared with results obtained by microscopic scoring using the CB technique. No correlation was found when the individual values in unirradiated controls were compared, due mainly to the presence of DNA-containing particles from fragmented cell nuclei and other unspecific debris. The averaged data from nine dose–effect curves simultaneously analysed by both techniques showed a linear–quadratic dose dependence with α and β's that were similar for flow cytometry and for microscopic scoring. Only the constant term was higher for the flow cytometric results. A correlation between both techniques applied to individual data at doses > 0·2 Gy could also be demonstrated. It is concluded that a dose estimation of man exposed to low doses of ionizing radiation can at present not be improved by the flow cytometric technique.