Improved accuracy and reproducibility of enumeration of platelet–monocyte complexes through use of doublet‐discriminator strategy

Abstract

Platelet-monocyte complex (PMC) formation is a marker of in vivo platelet activation and may be readily measured by flow cytometry. Due to the high frequency of free platelets relative to monocytes and PMCs, false-positive identification through coincidence remains a significant technical problem.To overcome this problem, we evaluated the use of a doublet-discriminator strategy (DDM) to allow faster sample acquisition whilst significantly reducing aberrant coincidence. Fourteen healthy volunteers and 20 patients with coronary artery disease (CAD) gave arterial and/or peripheral venous blood samples (NaCit). Whole blood was labelled in duplicate with anti-CD61 and anti-CD14 using a standard lyse/wash protocol. One of each paired sample was serially diluted before analysis; the second was analyzed at full concentration but using FL1-width to exclude co-incident platelet and monocyte events. Control experiments were performed with ex vivo thrombin activated samples. With the DDM use PMC frequencies in the peripheral blood of healthy individuals and in CAD patients fell significantly [6.27% ± 1.77 (mean ± sd) to 2.57% ± 0.99 (P = 0.02)] and from 16.04% (± 11.26) to 7.66% (± 5.18) (P < 0.01), respectively. DDM use significantly reduced the percentage of PMCs in the ex vivo thrombin activated samples (P < 0.05). Use of DDM effectively reduces the coincidence and enumerates true PMC in the samples of normal individuals and in patients with CAD and in ex vivo thrombin activated samples.