Figure 4: Illustration of the identification of the common counts and the calculation of the cross-over value. Each 2-color experiment consists of 2 acquisitions; a negative control (NC) and a TEST. Matrices are shown for the NC (a) and TEST (b) based on an experiment with a total of 4 counts. Cross-over exists from fluorochrome B into detector DA and from fluorochrome A into detector DB. The matching rows of the NC and TEST matrices (grey) are defined as the matrix of the Common Counts (c). The CCs matrix contains counts with zero primary fluorescence for fluorochromes (D0A) and (D0B). Separation is required into counts with zero primary fluorescence for fluorochrome A and counts with zero primary fluorescence for B. Cross-over values can be calculated directly from the common count. In this example, = 42/800 = 0.05 and = 250/8500 = 0.03.