Fig. 6: iCLOTS multiscale microfluidic cell accumulation applications characterize cell aggregation in a variety of experimental devices, including those that are commercially available.

Designed for use with fluorescence microscopy images such that multiple components of a cell suspension can be simultaneously monitored, users adjust threshold values for any image color channel(s) where immunofluorescence signal is present. a Line graphs representing occlusion and accumulation over time are automatically generated. Here, CD41+ platelets and CD45+ white blood cells from sickle cell disease patient whole blood accumulate on an ibidi chamber device coated with collagen at a faster rate than healthy control whole blood (n = 3 replicates). Data taken at ×20 magnification, scale bar represents 50 μm. b The application is designed to automatically generate a map of all signals present, e.g., the dimensions of a microfluidic device, shown here with a microvasculature-on-a-chip device designed to investigate the effect of crizanlizumab on sickle cell disease whole blood samples (n = 1 experiment). Data taken at ×20, scale bar represents 200 μm. Percent occlusion (c) and accumulation rate (d) changes over the course of an experiment, showing microvascular occlusion instability. e A microchannel-specific application is available for spatial analysis of one or many straight microchannel portions of a microfluidic device. Data taken at ×20, scale bar represents 50 μm. f Spatial quantification of microvascular occlusion is automatically performed by calculating an occlusion percentage for each pixel point along the length of each microchannel. ML algorithms enable further analysis in microchannels by treating each x-coordinate and corresponding occlusion measurement from each channel as a data point. At the initial time course timepoint, t = 7 min, CD45+ white blood cells in SCD whole blood (g) and CD45+ white blood cells in SCD whole blood treated with drug crizanlizumab (h) occlude endothelialized microchannels to variable degrees at each point along the 32 analyzed microchannels. i CD45+ white blood cells in SCD whole blood predominantly occlude distal ends of microchannels at early timepoints, while CD45+ WBCs in SCD whole blood treated with crizanlizumab occlude proximal entry points of microchannels at early timepoints. Source data are provided as a Source data file.