Optimized flow cytometric assay for the measurement of platelet microparticles in plasma: pre-analytic and analytic considerations

Abstract

Platelet microparticles (PMP) are submicroscopic membrane vesicles released by platelets during activation. Flow cytometry is the most widely used method for quantifying PMP, but the optimization of the technical method has not yet been fully evaluated. This study was designed to assess the pre-analytical variables including blood sampling conditions, and to evaluate the analytical variations including effect of the platelet-specific antibodies and quantitative beads, precision, linearity and accuracy in comparison with beta-thromboglobulin, which is one of the platelet activation markers. Numbers of PMP collected into citrate-theophylline-adenosine-dipyridamole (CTAD) tubes were increased with time, but to a lesser extent than when collected into sodium citrate tubes. The precision of the PMP assay was relatively high. Excellent linear correlation was observed for dilution linearity. Regarding the platelet-specific antibodies used, anti-CD41a-labeled samples resulted in higher PMP levels than those labeled with anti-CD61 and anti-CD42a. There was no significant difference of PMP counts according to the quantitative beads. The PMP assay is well correlated with beta-thromboglobulin levels. Our findings suggest that blood samples for the PMP assay should be collected in a CTAD tube and delayed measurement is not allowed to avoid artefactual platelet activation. The PMP assay can be used successfully as a useful marker of the detection of in vivo platelet activation, provided that pre-analytical and technical points are optimally taken into consideration.

Measurement of microvesicle levels in human blood using flow cytometry

“…Prior studies have reported that delays in processing the sample can lead to increases in platelet and annexin V1 microvesicles (Table 3) (12)(13)(14)(15)(16). A 1-h delay resulted in about a 50 2 100% increase in platelet and annexin V1 microvesicle counts with longer delays associated with larger increases in microvesicle counts.…”

“…Most studies evaluating the effect of anticoagulant type on microvesicles in blood have focused on platelet and/or annexin V1 microvesicles. Citrate, citrate-theophylline-adenosine-dipyridamole (CTAD), and acid-citrate-dextrose (ACD) anticoagulants have all been reported to be preferred anticoagulants compared to heparin or EDTA (9)(10)(11)(12)(13)(14). Use of EDTA or heparin resulted in higher platelet and annexin V1 microvesicle counts in most studies, likely due to platelet activation and release (10).…”

Application of Flow Cytometry in the Studies of Microparticles

“…The delay between sampling and further processing should be as short as possible (less than 1 hour), as storage results in increased number of PMP [Simak & Gelderman, 2006, Kim et al, 2002.…”

Value of Platelet Activation Markers as Prothrombotic Risk Indicators

“…To prepare PMV-containing PPP, various centrifugation protocols are used, e.g. 15 min at 700 × g and up to 5 min at 5,000 × g. For isolating PMV from plasma high speed centrifugation, such as 1 h at 13,000 × g, is necessary [86,117,118]. A further problem concerns the enumeration of PMV.…”