DNA DENATURATION SENSITIVITY MAY INVALIDATE BROMODEOXYURIDINE-DNA FLOW CYTOMETRIC ANALYSIS OF POTENTIAL DOUBLING TIMES IN COLORECTAL TUMORS

Background Flow cytometric analysis of 5-bromo-2'-deoxyuridine (BrdU)- substituted DNA has been used to calculate tumour cell proliferation r ate. In this methodology, DNA denaturation, commonly by hydrochloric a cid, is essential to expose incorporated BrdU for quantification with monoclonal antibodies. This study was designed to establish the validi ty of this technique by examining the change in flow cytometric DNA pr ofiles introduced by DNA denaturation procedures. Methods Four experim ents were performed using suspensions of nuclei derived from human col orectal tumours exposed in viva to 150 mg/m(2) BrdU 8-11 h before samp ling. Results After denaturation with hydrochloric acid 2 mol/l a sign ificant decrease was observed in the DNA aneuploid G1 population (P < 0.001) with a concurrent increase in the DNA aneuploid S phase fractio n (P < 0.05). These changes were independent of the washing-centrifuga tion step and were maximal at different hydrochloric acid concentratio ns for different tumours. Conclusion Hydrochloric acid denaturation in troduces a tumour-specific non-linear variation in the analysis of Brd U.