PB0791 - An approach for whole blood platelet flow cytometry and standardization across instruments

Background: Instrument variability is a major challenge in the standardization of flow cytometry-based assays. For platelet assays, monitoring instrument performance is crucial for ensuring the optimal resolution of platelet subpopulations and distinguishing between background noise (Br) and fluorescence signals emitted from dimly expressed surface antigens, such as basal p-selectin (CD62P) and PAC1 expression. Since platelets are heterogenous in size and surface receptor expression, benchmarks for robust and reproducible characterization of various platelet populations is vital. There is no commercially available marker for determining platelet viability and easily quantifying and monitoring variations in platelet populations for longitudinal and multicenter studies.

Aims: The aim of the study was to design and standardize a high-sensitivity whole blood platelet flow cytometry panel.

Methods: We designed a multicolor panel to identify various platelet populations. This included a single platelet lineage marker GPIIb/IIa (CD61), a dense granule marker (CD63), and a marker for alpha granule exocytosis (CD62P). We tested conjugated monoclonal antibodies from two different vendors and selected the optimal antibody clone and fluorochrome conjugates based on titrated signal-to-noise ratios and fluorochrome spill-over spread using fluorescence minus one experiments. To measure the instrument performance, Levey-Jennings plots were generated for Qr and Br values for each fluorescence channel across a timeframe from September 13th 2022 to December 21st 2023.

Results: The panel accommodates three agonists (ADP, PAR-4, and TRAP-6) in addition to natural saline, and requires a low volume of citrated blood.

Conclusion(s): By characterizing standards for size, levels of fluorescence, and the variation of controls themselves, we were able to identify rare populations which may otherwise be attributed to outliers or background noise. Additionally, molecules of equivalent fluorochrome (MEFs) and the appropriate standard to perform this calculation were reported for each platelet marker, for the purpose of data reproducibility and cross-platform calibration.