Citrate-Theophylline-Adenine-Dipyridamol Buffer Is Preferable to Citrate Buffer as an Anticoagulant for Flow Cytometric Measurement of Platelet Activation

摘要

A simple, rapid method is needed for collection of platelets for flow cytometric measurement of platelet activation in investigations relating to coronary heart disease, stroke, and peripheral arterial disease (1)(2)(3)(4)(5)(6). Specimen collection and sample preparation must minimize activation of platelets (7)(8)(9).The most frequently used anticoagulant for platelet analysis is sodium citrate, but it is deficient because of the difficulty in controlling osmolarity in functional assays (10). Other anticoagulants (EDTA and recombinant thrombin inhibitors such hirudin or low-molecular weight heparin) offer no alternative because of possible interactions with other substances used for analysis (10). In addition, platelets stimulated with ADP in citrate blood usually aggregate if stirred. To prevent clotting and cell-to-cell-adhesion, blood must be diluted and stirring reduced (7) when unfixed platelets are used. However, the advantage of unfixed samples is the opportunity to check the influence of substances added in vitro (11).The use of citrate-theophylline-adenine-dipyridamol (CTAD) buffer rather than citrate decreased platelet activation as indicated by lowering the plasma concentrations of platelet factor 4 (12). To investigate the influence of this buffer on platelet activation, we measured flow cytometrically detectable activation of platelets isolated from citrate-buffered blood and from commercially available CTAD-buffered blood.Blood was taken from 10 healthy volunteers who had not ingested drugs that affect platelet function for at least 14 days. Venipuncture was performed using a butterfly needle (21G; Sarstedt). Blood was aspirated directly into four Monovette tubes (Sarstedt) containing citrate or CTAD buffer.Platelets were isolated by a modification of the method described by Faraday et al. (13). The Monovette tubes were centrifuged at 180 g for 18 min. Platelet-rich plasma (PRP) was obtained and carefully mixed with H-d-Phe-Pro-Arg-chloromethylketone …