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

Abstract

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 PAR 1‐ 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 PAR 1‐ 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 PAR 1‐ 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 PFT s 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 PFT s in situations with low sample platelet counts, a careful optimization of experimental parameters is still required in order to eliminate platelet count‐related effects.

Harmonizing light transmission aggregometry in the Netherlands by implementation of the SSC-ISTH guideline

“…Furthermore, promising results are accomplished with platelet aggregation studies under flow conditions [28]. Advantages of these new methods are the use of whole blood and low sample preparation, but they also require trained technicians and specialized equipment [29][30][31][32][33][34]. Likewise, standardization of the traditional LTA method is still essential here.…”

Platelet Phenotyping and Function Testing in Thrombocytopenia

“…Flow cytometry-based single cell analysis provides platelet assays, which are largely independent of platelet count and thereby feasible in blood from patients even with severe thrombocytopenia [ 95 , 96 ]. However, a recent study indicated that a platelet count of ≤10 × 10 9 /mL might influence in vitro platelet activation assays due to the platelet count-related decreased release of ADP, which serves as important amplifier of platelet activation [ 97 ]. Figure 2 gives an overview of important platelet phenotype and activation markers, which are determined by flow cytometry for the diagnostic purposes of platelet function defects potentially associated with thrombocytopenia.…”

Advances in Platelet Function Testing—Light Transmission Aggregometry and Beyond

“…In recent years, several flow cytometry approaches have been successfully used to assess platelet function in patients with severe chronic immune thrombocytopenia, showing that impaired platelet function is associated with bleeding, independent of platelet count [ 76 , 77 , 78 ]. However, it should also be noted that simply decreasing the platelet count induces an impaired platelet function phenotype as measured by flow cytometry, due to the loss of paracrine amplification of platelet responses by ADP release [ 79 ]. This indicates that flow cytometric assessment of platelet function is affected by platelet count, admittedly to a lesser extent than other traditional platelet function assays, and highlights the importance of deriving platelet-count-adjusted reference ranges.…”