Background: Although flow cytometry is often brought forward as a preferable method in the setting of thrombocytopenia, the relative effects of low sample counts on results from flow cytometry-based platelet function testing (FC-PFT) in comparison with light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) has not been reported.
Objectives: To compare the effects of different sample platelet counts (10, 50, 100, and 200 × 109 L-1) on platelet activation measured with FC-PFT, LTA, and MEA using the same anticoagulant and agonist concentrations as for the commercial MEA test.
Methods: Platelets were stimulated with two commonly used platelet agonists (ADP [6.5 μmol L-1] and PAR1-AP [TRAP, 32 μmol L-1]). The specified sample platelet counts were obtained by combining platelet-rich and platelet poor hirudinized plasma in different proportions with or without red blood cells.
Results: For FC, P-selectin exposure and PAC-1 binding was reduced at 10 × 109 L-1 after stimulation with PAR1-AP (by approximately 20% and 50%, respectively), but remained relatively unchanged when ADP was used as agonist (n = 9). The platelet count-dependent effects observed with PAR1-AP were eliminated when samples were pre-incubated with apyrase, implying that reduced purinergic signaling was the main underlying factor (n = 5). Both aggregometry-based PFTs showed a 50% reduction at 50 × 109 L-1 and more than 80% reduction at 10 × 109 L-1, irrespective of agonist used (n = 7).
Conclusions: Although FC-PFT is generally preferable to aggregometry-based PFTs in situations with low sample platelet counts, a careful optimization of experimental parameters is still required in order to eliminate platelet count-related effects.
Keywords: platelet activation; platelet aggregation; platelet count; platelet function tests; thrombocytopenia.