Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry.
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.
Johns Hopkins University, Baltimore, MD, USA.
Oliver D
OD
Atar
Christian
C
Eisert
Ilya
I
Pokov
Victor L
VL
Serebruany
D001792:Blood Platelets / Q000737:chemistry / Q000166:cytology; D001793:Blood Preservation; D005434:Flow Cytometry / Q000379:methods*; D005557:Formaldehyde; D006801:Humans; D019007:P-Selectin / Q000032:analysis; D015539:Platelet Activation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D010980:Platelet Membrane Glycoproteins / Q000032:analysis*; D011108:Polymers; D044463:Receptor, PAR-1 / Q000032:analysis; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods* / Q000592:standards
D019007:P-Selectin; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D010980:Platelet Membrane Glycoproteins; D011108:Polymers; D044463:Receptor, PAR-1; D005557:Formaldehyde; C003043:paraform