DEBRIS COMPENSATION OF DNA HISTOGRAMS AND ITS EFFECT ON S-PHASE ANALYSIS

Debris compensation is an important variable affecting S-phase fractio n (SPF) analysis in now cytometric DNA histograms. The SPF was estimat ed in fresh frozen breast carcinomas using the following four debris s ubtraction algorithms: modeling debris as an exponential curve (EXP); the incorporation of nuclei cut a single time into the exponential moe l (EXP-SC); the random cutting of nuclei into multiple pieces of varyi ng sizes (MC); and a combination of both nuclear cutting models (SC-MG ). Comparison of SPF estimates indicated that the various debris subtr action models yielded differences in SPF, with SPF values obtained usi ng the exponential model having considerable variation compared to SPF estimates from histograms where debris was modeled by algorithms base d on nuclear slicing and fragmentation. However, SPF estimates could b e affected by initial placement of the nuclear debris boundaries, the coefficient of variation of the G(0/1) peak, and the relative amount o f debris. Using the ratio of the height of the G(0/1) peak to the heig ht of the debris between the chicken red blood cells (CRBC) and G(0/1) peaks as an objective measurement of nuclear debris, debris compensat ion was necessary in diploid DNA histograms where this ratio was as lo w as 1.5%. Taken in the context of SPF prognostic cutoff levels, varia tion in debris models and boundaries can change the classification of cases with borderline SPF levels into the poor prognostic high SPF cat egories, thereby making the comparison of SPF values derived from diff erent studies difficult. (C) 1995 Wiley-Liss, Inc.