Abstract
It is widely believed that assays of platelet activation are susceptible to preanalytical variables related to blood draw technique. We assessed platelet activation by whole blood flow cytometry and investigated the effects of: (1) drawing blood into vacuum tubes or manually aspirated syringes, and (2) discarding the first drawn blood sample (discard tube). Platelet P-selectin expression and platelet-monocyte complexes were measured by flow cytometry under both basal conditions and following stimulation with 0.1, 1, or 10 µM ADP. Bland-Altman plots demonstrated agreement between results for vacuum tube and syringe-aspirated samples with an a priori-defined clinically relevant agreement limit of 5%. Agreement of results was also observed between discard tube and second draw samples for both vacuum-driven and manually aspirated blood. We conclude that a vacuum tube or a manually-aspirated syringe can be used when assessing platelet activation by flow cytometry and that there is no need for a discard tube.
Highlights
Unresponsiveness or hyper-reactivity of platelets can lead to disease, and assays of platelet function are commonly used diagnostically and experimentally
Since platelets are prone to become artificially activated ex vivo, rigorous attention is given to pre-analytical variables [1], the technique used for blood collection [2]
When assessing agreement between methods using Bland-Altman plots, good agreement was demonstrated by the 95% limits of agreement (LOA) and the a priori 5% agreement limit for platelet-monocyte complexes and platelet P-selectin expression for all comparisons (Fig 1)
Summary
Unresponsiveness or hyper-reactivity of platelets can lead to disease, and assays of platelet function are commonly used diagnostically and experimentally. Since platelets are prone to become artificially activated ex vivo, rigorous attention is given to pre-analytical variables [1], the technique used for blood collection [2]. Guidelines for the diagnostic evaluation of platelet disorders recommend either vacuum- or syringe-driven aspiration of blood into primary collection tubes [3]. Many authorities believe that vacuum systems subject blood to higher levels of shear stress, resulting in artificial platelet activation [4]. The Clinical Laboratory Standards Institute (CLSI) recommends discarding the first drawn tube when blood is taken through a butterfly needle [5]. The rationale is that dead space in the tubing leads to underfilling of the primary collection tube leading to an incorrect anticoagulant/blood ratio [6,7]
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