Data-Driven Compensation for Flow Cytometry of Solid Tissues
(a) shows events of a negative control (NC). (b) shows the events of the matching TEST. Both graphs are uncompensated 2-color plots, from sentinel lymph node (SLN) tissue. (c) shows the TEST after compensation, using a trace line fitted through the common counts of the NC and SSC (dashed line). (d) shows the TEST values, after compensation with a compensation trace fitted through the CC of the NC and the TEST (the full line). Each dot plot consists of 100.000 counts. The grey dots represent the individual counts in each dot plot. The black dots represent the common counts. The median values of each of the 5 distinct clusters (ploidy level 2C, 4C, 6C, 8C, and 10C) are marked with an “X.” For optimal visual representation, the pairs of compensation trace lines are white in the upper graphs and black in the lower graphs. Note that the 2 different compensation trace lines seem to be parallel in (a) and (b), which is an optic illusion because the ordinate is logarithmic. In the linear domain these 2 compensation trace lines diverge. The dot plots represent logical (, , ) , FITC fluorescence (ordinate) versus linear PI fluorescence (abscissa). The negative control was incubated with a negative mouse Ig and labeled with FITC, and the test case was incubated with a monoclonal cytokeratin antibody (clone MNF116). In all cases the DNA content was labeled with PI. There was only cross-over from PI in the FITC detector, which can be seen in the slope of the dotted compensation trace lines, and no cross-over from FITC in the PI detector, because no slope, in vertical direction, can be seen in the FITC positive counts in the 2C, 4C, 6C, 8C, and 10C clusters in (b). Ideally, the CC would form a straight line, slightly deformed by the logarithmic scaling of the ordinate. In practice the ideal line is scattered due to the influence of auto fluorescence, photon count statistics and noise. Additional peaks of positive counts are visible in the 2C, 4C, 6C, 8C, and 10C clusters in the TEST, which are not included in the CC.
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