Fig. 4: Removal of autofluorescence through compensation with an additional autofluorescence channel.

a, b Examples, for the unstained control of the HS1 dataset, of compensation of spillover from the autofluorescence channel (y-axes) to two secondary channels (x-axes). Uncompensated data is displayed in blue and compensated data in black. Resulting compensation errors (slope coefficients of the regressions on compensated data) are shown at the bottom left or right of each panel. Vertical green dashed lines are shown as a reference for perfectly compensated data. c–f Compensation of two channels (one case per row) severely affected by autofluorescence in the HS1 dataset (left, (c, e), without autofluorescence channel; right, (d, f), with autofluorescence channel), with primary channels in y-axes and the secondary channels in x-axes. Same color and line code, and number with compensation error, as in a, b. g, h Comparison of probability density functions of spillover skewness in the HS1 dataset, without (g) or with (h) autofluorescence channel. Errors are displayed in log-scale of absolute values, separated in positive (solid lines) and negative (dashed lines) values to document any bias between positive and negative errors. Autofluorescence, causing spurious positive spillover, corresponds to anomalously large positive skewness in the affected channels (left). Raw datasets are available at FlowRepository with IDs FR-FCM-Z2SS (MM1) [https://flowrepository.org/id/FR-FCM-Z2SS] and FR-FCM-Z2ST (HS1) [https://flowrepository.org/id/FR-FCM-Z2ST].