Use of leukocytes infiltrating bladder tumors as an internal reference for determining the DNA index by flow...

The use of leukocytes infiltrating bladder tumors as an internal standard for flow cytometric measurements of DNA and SG2M indices was investigated in comparison with the conventional use of chicken erythrocytes (chicken red blood cells; CRBC). The leukocytes were identified by indirect fluorescence, using a mouse anti-CD45 monoclonal antibody and a fluorescein isothiocyanate-conjugated goat anti-mouse Ig F(ab')2 fragment. DNA was stained with propidium iodide. The percentage of CD45-positive cells averaged 37% in 23 unimodal bladder tumors and 30% in 27 bimodal ones. The diploid peak of CD45-positive cells was always easily identified and permitted a more accurate calculation of the DNA index and the percentage of cells in SG2M phases than did the use of CRBC. Seven tumors that were classified as unimodal using CRBC as the standard did, in fact, contain a minor clone (with a DNA index of 1.80) that could be detected only when CD45-positive leukocytes were used as the internal standard.