Patent ID: 8831889

Claim:
A method of quantifying and validating differences between measured values of fluorescence intensities obtained using a flow cytometer or other fluorescence-based instrument, the method comprising: a) running multiple sets of standardized particles through the instrument to generate at least one calibration histogram for each set of particles, the histograms containing fluorescence intensity values; b) calibrating, with a computer coupled with the instrument, a distance function configured to measure distances between the calibration histograms generated by the sets of standardized particles, comprising: i) constructing a quadratic form (QF) metric using the distance function, where the QF metric comprises QF ⁡ ( h , f ) = ( h - f ) T ⁢ A j i ⁡ ( h - f ) = ∑ i = ⁢ a n ⁢ ∑ j - 1 n ⁢ a ij ⁡ ( h i - f i ) ⁢ ( h j - f j ) , where h and f are histograms, and A comprises a bin-to-bin dissimilarity matrix that is an n×n positive semi-definite matrix, n being the number of bins in histograms h and f; and ii) populating entries of the dissimilarity matrix with elements using parameters that control at least groups of the elements such that the metric maximizes the following conditions at substantially the whole range of fluorescence intensity values for the sets of standardized particles: (A) the distance function of two histograms representing two sets of different standardized particles substantially equals the difference of their fluorescence intensity values or the logarithms of their fluorescence intensity values; and (B) the distance function of two histograms representing fluorescence of two sets of identical particles is substantially zero; wherein each set of particles is characterized by a fluorescence intensity corresponding to a known number of fluorochromes of interest, wherein the dissimilarity matrix further includes distances between bins i,j of histograms h,j which list counts of detected fluorescence intensities corresponding to each of the cytometry histogram bins; A j i = exp ⁡ ( γ ⁢ ⅆ ij ⅆ max ) δ , ⁢ where d i , j =  i - j  , d max = max i , j ⁢ { d i , j } ; iii) computing and iv) iteratively determining γ and δ of A j i to control a global shape of the dissimilarity matrix such that maximizes the conditions of b)ii)A) and b)ii)B); c) applying, by the computer, the constructed metric to a set of flow cytometry histograms generated by the instrument for a plurality of biological samples, to determine distances between the histograms; d) constructing and applying a statistical test, by the computer, using the constructed metric to determine a statistical significance of the distances between the histograms; and e) determining, by the computer, whether the histogram results from the biological samples are reliable based on the statistical significance.