Abstract
The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Despite growing interest in studying extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) as potential biomarkers, how blood processing and freeze/thaw impacts the profiles of these analytes in plasma was not thoroughly understood. We utilized flow cytometric analysis to examine the effect of differential centrifugation and a freeze/thaw cycle on EV profiles. Utilizing flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering and fluorescence, we revealed how differential centrifugation and post-freeze/thaw processing removes and retains EV subpopulations. Additionally, cf-mRNA levels measured by RT-qPCR profiles from a panel of housekeeping, platelet, and tissue-specific genes were preferentially affected by differential centrifugation and post-freeze/thaw processing. Critically, freezing plasma containing residual platelets yielded irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze/thaw. Our findings suggest the importance of minimizing confounding variation attributed to plasma processing and platelet contamination.
Highlights
The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors
The median light scatter statistic of each bead size gated population was inputted into flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering[32,33,34]
The collection half-angle of our system, which is important to quantify the amount of light reaching a detector in absolute units, was determined to be 45.3° using FCMpass software
Summary
The discovery and utility of clinically relevant circulating biomarkers depend on standardized methods that minimize preanalytical errors. Despite growing interest in studying extracellular vesicles (EVs) and cell-free messenger RNA (cf-mRNA) as potential biomarkers, how blood processing and freeze/ thaw impacts the profiles of these analytes in plasma was not thoroughly understood. Utilizing flow cytometry postacquisition analysis software (FCMpass) to calibrate light scattering and fluorescence, we revealed how differential centrifugation and post-freeze/ thaw processing removes and retains EV subpopulations. Cf-mRNA levels measured by RT-qPCR profiles from a panel of housekeeping, platelet, and tissue-specific genes were preferentially affected by differential centrifugation and post-freeze/thaw processing. Freezing plasma containing residual platelets yielded irreversible ex vivo generation of EV subpopulations and cf-mRNA transcripts, which were not removable by additional processing after freeze/thaw. Despite the growing body of literature describing the impact of blood processing on EVs, standardization through light scatter calibration was not widely adopted in these studies to analyze EV subpopulations using flow cytometry. Given an effective refractive index of EVs, established scatter-diameter curves yielded reproducible EV measurement between instruments[30,31,32,33,34]
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