Jv. Watson, Proof without prejudice revisited: Immunofluorescence histogram analysis using cumulative frequency subtraction plus ratio analysis of means, CYTOMETRY, 43(1), 2001, pp. 55-68
Background: Apart from the work of Lampariello and colleagues (Cytometry 15
:294-301, 1994; Cytometry 32: 241-254, 1998), very little analytical work h
as been carried out for analysis of immunofluorescence distributions contai
ning an overlapping mixture of labeled and unlabeled cells. The methods dev
eloped tend to rely on fitting theoretical distributions to the relevant po
pulations. However, the method described here attempts to produce an analyt
ical solution.
Methods: A new method for immunofluorescence histogram analysis is presente
d. 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 validatio
n at a given level of probability.
Results: The method was developed with a data set ex hibiting a small "posi
tive" shoulder, which was predicted to contain a labeled fraction comprisin
g 8.0% of the total at the 99% confidence limit. It was then tested with da
ta analyzed and published previously where the Johnson Su family of distrib
utions was used in curve fitting.
Conclusions: There was good agreement between the known and predicted propo
rtions of labeled cells. However, the method is dependent on the symmetry o
f the distributions. Some minor systematic errors were encountered due, in
part, to skewed experimental distributions. Cytometry 43:55-68, 2001. (C) 2
001 Wiley-Liss,Inc.