Relevance of forward scatter and side scatter in aneuploidy detection by flow cytometry

The study of the nuclear DNA content by flow cytometry (FCM) has been classically accomplished by selecting the nuclear population on the biparametric forward scatter (FS)-DNA fluorescence or FS-DNA fluorescence peak histograms to determine ploidy and DNA index (DI). Different cellular factors such as nuclear morphological heterogeneity of the neoplastic cells, intratumoral variability, histological origin, displasia grade, necrosis, and size of the tumoral piece analyzed constitute important problems in ploidy studies and, consequently, residual or underrepresented clones with different ploidy levels can be masked by populations with a large cell number. In the present report, an alternative methodology is proposed for aneuploidy detection, since populations coinciding with DNA content may be different with respect to morphological criteria. The discrimination of aggregates and background noise by using peak or logarithmic fluorescence signal, and backgating in side scatter (SS)/FS histograms, permits the establishment of specific bounds through complete scatterplot mapping and to distinguish between scarce or minor populations in association with small or abnormal DNA peaks. Moreover, variations in the DNA modal channel value and the peak coefficient of variation value remained unmodified, also maintaining the quality of cytometric measurement data.