Multiplexed imaging and automated signal quantification in formalin-fixed paraffin-embedded tissues by ChipCytometry.

Deciphering the spatial composition of cells in tissues is essential for detailed understanding of biological processes in health and disease. Recent technological advances enabled the assessment of the enormous complexity of tissue-derived parameters by highly multiplexed tissue imaging (HMTI), but elaborate machinery and data analyses are required. This severely limits broad applicability of HMTI. Here we demonstrate for the first time the application of ChipCytometry technology, which has unique features for widespread use, on formalin-fixed paraffin-embedded samples, the most commonly used storage technique of clinically relevant patient specimens worldwide. The excellent staining quality permits workflows for automated quantification of signal intensities, which we further optimized to compensate signal spillover from neighboring cells. In combination with the high number of validated markers, the reported platform can be used from unbiased analyses of tissue composition to detection of phenotypically complex rare cells, and can be easily implemented in both routine research and clinical pathology.