Proof without prejudice revisited: Immunofluorescence histogram analysis using cumulative frequency subtraction plus ratio analysis of means

Background: Apart from the work of Lampariello and colleagues (Cytometry 15:294-301, 1994; Cytometry 32: 241-254, 1998), very little analytical work has been carried out for analysis of immunofluorescence distributions containing an overlapping mixture of labeled and unlabeled cells. The methods developed tend to rely on fitting theoretical distributions to the relevant populations. However, the method described here attempts to produce an analytical solution. Methods: A new method for immunofluorescence histogram analysis is presented. It uses cumulative frequency distribution subtraction of the test sample from the control to predict the mean of a labeled cell component embedded within a histogram containing unlabeled cells. Ratio analysis of means (RAM) was then carried out to calculate the labeled fraction. The results were submitted to Kolmogorov-Smirnov analysis and Student's t-test for validation at a given level of probability. Results: The method was developed with a data set ex hibiting a small "positive" shoulder, which was predicted to contain a labeled fraction comprising 8.0% of the total at the 99% confidence limit. It was then tested with data analyzed and published previously where the Johnson Su family of distributions was used in curve fitting. Conclusions: There was good agreement between the known and predicted proportions of labeled cells. However, the method is dependent on the symmetry of the distributions. Some minor systematic errors were encountered due, in part, to skewed experimental distributions. Cytometry 43:55-68, 2001. (C) 2001 Wiley-Liss,Inc.