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

J Thromb Thrombolysis. 2010 Jul;30(1):79-83. doi: 10.1007/s11239-009-0402-7.

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.

MeSH terms

  • Blood Platelets / chemistry
  • Blood Platelets / cytology
  • Blood Preservation
  • Flow Cytometry / methods*
  • Formaldehyde
  • Humans
  • P-Selectin / analysis
  • Platelet Activation
  • Platelet Endothelial Cell Adhesion Molecule-1 / analysis
  • Platelet Membrane Glycoproteins / analysis*
  • Polymers
  • Receptor, PAR-1 / analysis
  • Time Factors
  • Tissue Fixation / methods*
  • Tissue Fixation / standards

Substances

  • P-Selectin
  • Platelet Endothelial Cell Adhesion Molecule-1
  • Platelet Membrane Glycoproteins
  • Polymers
  • Receptor, PAR-1
  • Formaldehyde
  • paraform