Quality control of highly multiplexed proteomic immunostaining with quantum dots: Correcting for crosstalk

The process of developing molecular assays for disease diagnosis and prognosis requires cross-disciplinary research which monitors quality and reproducibility at all levels. This paper discusses challenges in the quality control of highly multiplexed Quantum Dot (QD) staining and provides a method for improving accuracy of QD quantification in two phases. Phase one is the estimation of unintended crosstalk between multiplexed QD-antibody reporters, and phase two is digital correction of this crosstalk. Results show that crosstalk varies among tissues and reagents, and in some cases it can be on the same order of magnitude as the original intended signal. In cases where target protein expression is assumed to be independent, crosstalk can be empirically estimated from imaging data and corrected for. This work is expected to improve the overall reproducibility and quantification of multiplexed QD staining.