Evaluation of the PDQ™ centrifuge for preparing platelet rich, platelet poor and platelet free plasma samples for light transmission aggregometry and microparticle measurement

Light transmission aggregometry (LTA) is considered the gold standard for platelet function testing [1]. This test requires the preparation of platelet rich plasma (PRP) and platelet poor plasma (PPP) from each blood sample [2]. Preparation of PRP and PPP by standard protocols is time consuming and poorly standardized (in terms of choice of g values and time of centrifugation) normally taking around 30– 45 minutes [3–5]. There is now also much interest in the measurement of microparticles within platelet free plasma preparations (PFP) [6]. Again the preparation of PFP is also poorly standardized but is important for eliminating the generation of cell derived microparticles from any residual contaminating platelets or other cells during sample storage [6]. Recently, a new centrifuge (PDQ, Bio Data Corporation, Alpha Laboratories Hampshire, UK) has been introduced which is intended for the rapid preparation of PRP, PPP, and PFP samples using fixed g values and centrifugation times. The main advantage of the PDQ centrifuge is its ability to prepare these samples rapidly (less than 5 minutes) in a standardized manner which could be of significant benefit to clinical and research laboratories. The aim of this study was to therefore compare and contrast the properties of PRP, PPP and PFP samples prepared by the PDQ with our normal laboratory sample preparations. Whole blood samples (n1⁄4 10) from apparently healthy individuals (six males and four females) who were not taking any anti-platelet drugs were collected into four Vacutainer tubes (Becton Dickinson, Oxford, UK) containing 0.105 M sodium citrate (1:9 v/v) using a 21 gauge needle, an initial discard tube and minimal tourniquet. Two samples were centrifuged at 170 g for 10 minutes to prepare PRP. For the preparation of PPP, samples were further centrifuged at 2000 g for 20 minutes. PFP samples were prepared by double centrifugation at 2000 g for 20 minutes followed by a rapid short centrifugation at 13,000 g for 2 minutes [7]. All supernatants (top two thirds) were carefully removed and transferred to capped tubes. Two samples were also simultaneously centrifuged in the PDQ to rapidly prepare PRP, PPP, and PFP samples. The instrument is preprogrammed to spin at 8500 rpm (4400 g) and prepares PRP in 30 seconds (2220 g-min), PPP in 120 seconds (8880 g-min), and PFP in 180 seconds (13,320 g-min). Platelet counts including mean platelet volume (MPV), platelet distribution width (PDW), and platelet large cell ratio (P-LCR) were measured in all PRP, PPP and PFP samples using an impedance hematology analyzer (KX-21, Sysmex, Milton Keynes, UK). Platelet aggregation within unadjusted PRP samples was also measured in response to ADP (0.5, 1, 2.5, and 5 mM final concentrations) within a four channel platelet aggregometer (Aggram aggregometer, Helena Biosciences, Gateshead, UK). 100% transmission values were calibrated using PPP samples prepared by the method being tested. As the impedance counter has a lower limit of 1 10/L a modified flow cytometric reference method was also used to determine residual platelet counts in PPP and PFP samples (FacsCalibur, Becton Dickinson, Oxford, UK) [8]. Briefly, 5 mL of samples were incubated with 5 mL of a FITC-conjugated anti-CD61 antibody (Becton Dickinson, Oxford, UK) within 40 mL of