Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry

Abstract

Sample fixation for storage and/or transportation represents an unsolved challenge for multicenter clinical trials assessing serial changes in platelet activity, or monitoring various antiplatelet regimens. Whole blood flow cytometry represents a major advance in defining platelet function, although special training and expensive equipment is required. We sought to determine how fixation with 2% paraformaldehyde (PFA), and storage of blood samples over 1 week affects the flow cytometry readings for both intact and thrombin-activating four major surface platelet receptors. Whole blood platelet expression of PECAM-1, P-selectin, PAR-1 inactive receptor (SPAN-12), and cleaved (WEDE-15) epitope was assessed immediately after blood draw, after staining with 2% PFA, and at day 1, 3, 5, and 7. The study was performed in 6 volunteers with multiple risk factors for vascular disease, not receiving any antiplatelet agents. Staining with PFA resulted in a slight decrease of fluorescence intensity, especially for PECAM-1, while antigen expression at day 1, 3 and 5 remains consistent, and highly reproducible. At day 7 there was a small but inconsistent trend towards diminished fluorescence intensity. The platelet data were consistent while validated with the isotype-matched irrelevant antibody. These data suggest that there is a 5 day window to perform final flow cytometry readings of whole blood PFA-fixed inactivated platelet samples. In contrast, thrombin activation cause gradual loss of flow cytometry signal, and cannot be recommended for long-term storage. This is critical logistic information for conducting multicenter platelet substudies within the framework of major clinical trials.

A comprehensive assessment of immunophenotyping performed in cryopreserved peripheral whole blood

“…It has also been useful from an epidemiological standpoint for the comprehensive assessment of health and predicting disease risk (6,7). Considering this needs to be accomplished within a timeframe that is not necessarily feasible for all study designs (24 h or less depending on the nature of the study (8)), researchers have investigated strategies to extend the time to analysis; for example, immediate staining followed by fixation and storage (9), storage for extended periods at 48C, followed by staining and immediate analysis (10), immediate lysis and fixation, followed by storage and subsequent staining and analysis (11), and lastly, the isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) (12). A major barrier is the logistics required to transport biospecimens from the site of collection to the analysis centre where flow cytometry can be conducted.…”

“…A major barrier is the logistics required to transport biospecimens from the site of collection to the analysis centre where flow cytometry can be conducted. Considering this needs to be accomplished within a timeframe that is not necessarily feasible for all study designs (24 h or less depending on the nature of the study (8)), researchers have investigated strategies to extend the time to analysis; for example, immediate staining followed by fixation and storage (9), storage for extended periods at 48C, followed by staining and immediate analysis (10), immediate lysis and fixation, followed by storage and subsequent staining and analysis (11), and lastly, the isolation and cryopreservation of peripheral blood mononuclear cells (PBMCs) (12). Although these strategies allow for substantial improvements to inter-assay precision due to the potential reduction of analytical batches, they can be technically demanding to implement and can limit the quality and informativeness of data obtained (e.g., isolating and freezing PBMCs for future immunophenotyping eliminates the possibility of analyzing granulocytes).…”

On the Use of the Platelet Activity State Assay for the In Vitro Quantification of Platelet Activation in Blood Recirculating Devices for Extracorporeal Circulation

“…However, flow cytometry implies repeated sample manipulation, required for fixation, antibody labeling, and washing, which may affect the accuracy of the analysis. Moreover, fixative agents (formaldehyde or paraformaldehyde) have been reported to potentially induce a decrease of the sample fluorescence intensity (29,30), as well as a time-dependent modification of the PA level (31). Furthermore, flow cytometry requires expensive reagents (antibodies) and equipment (FACS) and highly specialized dedicated personnel.…”

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)

“…However, paraformaldehyde itself affects flow cytometry readings. Staining with PFA has been shown to result in a slight decrease in fluorescent intensity [ 44 ] and a decrease in forward scatter and side scatter [ 45 ]. Our analysis of changes in fluorescence intensity for 68 leukocyte surface receptors for up to seven days generally agrees with a previous analysis showing that staining generally remained consistent and highly reproducible at days 1, 3 and 5 post-staining [ 44 ].…”