Analysis of cellular DNA distributions

Abstract

An analytical formula for calculating peak channel ratios in fluorescent cytophotometric determinations of DNA content per cell was derived to assess the effects of inaccuracies in the model-dependent derivation of S-phase cell populations and of systematic instrumental errors. The DNA distribution histograms usually have two peaks, corresponding to the 2C DNA content of G1 cells and to the 4C DNA content of G2 and M cells. In the presence of S-phase cells, the ratio of peak channels G2/G1 becomes less than 2. The calculation uses the model-dependent number of S-phase cells per channel and instrumental resolution to obtain G2/G1. The peak channel ratio calculated in this way decreases with increasing coefficient of variation and increasing proportion of S-phase cells. The calculated G2/G1 peak channel ratios were compared with 17 experimental values ranging from 1.68 to 2.08. Significant differences were found for two experiments, and the calculated G2/G1 ratios were systematically low by ≈4% for the other experiments. When this systematic difference in predicted peak channel ratios is taken into account, the formula predicts the observed ratios with an accuracy of 1% showing the dominant effect of S-phase cells in modifying the observed spectrum. The possible experimental effects leading to the observed systematic discrepancy are discussed A programmable pocket calculator program to perform these calculations is also described in detail.