Fig. 5: iCLOTS cell adhesion applications provide indexed single-cell measurements of biological functionality.

After adjustment of relevant parameters, iCLOTS calculates numerical area and circularity values for individual cells within brightfield microscopy images, including a dark/dense platelets adhered on fibrinogen-coated surfaces (n = 231 platelets) and collagen-coated surfaces (n = 47 platelets) and b biconcave RBCs from patients with sickle cell trait (AS) genotypes (n = 134 RBCs) and sickle cell disease (SS) genotypes (n = 110 RBCs). Platelets adhered to fibrinogen surfaces spread less than those adhered to collagen (*p < 0.0001 via two-sided Mann–Whitney test). Data taken at ×30 magnification, scale bars represent 10 μm (a and b). To analyze fluorescence microscopy imaging data the user indicates pixel value thresholds for membrane and optional secondary stains and additional texture and staining intensity metrics are calculated per-cell. c Users may count regions of a secondary stain, here the number of nuclei lobes in neutrophils (n = 207 neutrophils). Data taken at ×20 magnification, scale bar represents 10 μm. d A cell protrusion characterization application calculates the number of filopodia-like protrusions present in an individual cell using additional application-specific parameters designed to apply objective requirement criteria. Data taken at ×20 magnification, scale bars represent 10 μm. e Transient adhesion time of individual cells to a biochemically-coated surface is calculated from videomicroscopy data, shown here with neutrophils (n = 1 experiment, n = 185 neutrophils) in a fibronectin-coated channel. Users adjust additional parameters designed to ensure veracity of returned data points. Data taken at ×20 magnification, scale bar represents 50 μm. f Analysis of fluorescence microscopy data of platelets reveals differences in the density of adhered platelets, the spreading area of individual platelets, and phosphatidylserine exposure in individual platelets from healthy controls and a Hermansky-Pudlak Syndrome patient. Data taken at ×40 magnification, scale bars represent 200 μm. g K-means ML analysis separates combined healthy (n = 1112) and HPS (n = 2674) platelets into two groups representing low- and high-PS exposure (n = 1 experiment). h The proportion of cells in cluster 2, the high-PS cluster, is greater in HPS samples than in healthy controls (**p < 0.0001, Chi-squared test). Source data are provided as a Source data file.