Debris compensation of DNA histograms and its effect on S-phase analysis

Debris compensation is an important variable affecting S-phase fraction (SPF) analysis in flow cytometric DNA histograms. The SPF was estimated in fresh frozen breast carcinomas using the following four debris subtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moel (EXP-SC); the random cutting of nuclei into multiple pieces of varying sizes (MC); and a combination of both nuclear cutting models (SC-MC). Comparison of SPF estimates indicated that the various debris subtraction models yielded differences in SPF, with SPF values obtained using the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms based on nuclear slicing and fragmentation. However, SPF estimates could be affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G0/1 peak, and the relative amount of debris. Using the ratio of the height of the G0/1 peak to the height of the debris between the chicken red blood cells (CRBC) and G0/1 peaks as an objective measurement of nuclear debris, debris compensation was necessary in diploid DNA histograms where this ratio was as low as 1.5%. Taken in the context of SPF prognostic cutoff levels, variation in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF categories, thereby making the comparison of SPF values derived from different studies difficult.