Bidimensional PCA of the effect of additional surface markers on the separation of normal and malignant cell populations. Bone marrow samples obtained at diagnosis and after induction therapy (day 78) were obtained from 2 pediatric ALL patients (A-B). Samples were stained with the usual backbone of antibody combinations including CD19, CD34, CD45, CD10, and CD20 in all tubes and supplemented with either CD18 and CD157, CD31 and CD97, CD63, CD84, CD100, CD102, or CD305. After data acquisition, .fcs files were merged using the Infinicyt software.27 ALL cells at diagnosis (red) and normal regenerating BCPs at day 78 in MRD-negative samples (blue) were identified based on the backbone markers and plotted in a bidimensional PCA. The first principal component (PC) is shown on the x-axis, and the second PC on the y-axis, using the Automatic Population Separator (APS) graphical representation of the Infinicyt software.27 Data represent the mean and first and second standard deviation of the 2 populations. Backbone: APS plots using backbone markers only. Both populations show some overlap, which impacts the resolution of the analysis. The effect of the addition of 1 additional marker to this backbone as indicated in the figure legends is visualized by PCA. The separation between the ALL and normal BCPs improves when some of these markers are added. The relative contribution of each marker varies in the 2 samples.
