Platelet function testing at low platelet counts: When can you trust your analysis?

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 × 10(9) 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 × 10(9) 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 × 10(9) L(−1) and more than 80% reduction at 10 × 10(9) 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.