Assays for quality control of platelets for transfusion

Quality control of platelets relies on the determination of the extent by which platelets are still able to react with known agonists, and here knowledge of the biochemistry of platelet activation may guide to decide which tests are useful. In vivo, platelets will adhere to collagen exposed upon vessel wall damage, and especially under high shear conditions through von Willebrand factor (VWF) that forms a bridge between collagen and platelet glycoprotein (GP) Ib. Binding of GPVI to collagen next results in the activation of a tyrosine kinase cascade, finally resulting in phosphorylation and activation of phospholipase C (PLC) γ2, which leads to an increase in cytosolic Ca²⁺ levels. High Ca²⁺ levels trigger the production of thromboxane A2 (TXA2) and release of platelet granules containing among others, adenosine diphosphate (ADP). TXA2 and ADP now will activate their receptors on platelets which are, among others, linked to a G-protein that activates PLCβ2 with more Ca²⁺ increase. This ultimately provokes activation of the integrin α_IIbβ₃ (or GPIIbIIIa), which now can bind the symmetrical fibrinogen, thus allowing cross-linking of platelets or platelet aggregation. From the above it is clear that testing whether platelets are fully reactive ideally should be looking at as many of the above parameters as possible, while at the same time being simple and fast. Systems in which blood is flown over collagen surfaces mimic best the physiological situation; however, these tests are not fit for stored platelet testing as haematocrit is a critical factor in these experiments. Thromboelastography at best provides a test for G-protein dependent activation (through thrombin) and integrin α_IIbβ₃ involvement. Platelet agglutination induced by ristocetin (surrogate for the adhesion phase) next to aggregation (involvement of integrin α_IIbβ₃) induced by collagen (Tyr kinase pathway) and ADP (G-protein mediated pathway) can give a comprehensive view on the platelet quality. However, flow cytometry is also an excellent technique to detect (i) bound VWF to platelets in the presence of ristocetin, (ii) collagen- or ADP-induced activation of integrin α_IIbβ₃ (by determining the binding of fibrinogen or of the activation-dependent PAC-1 antibody) and (iii) secretion (by detecting surface expression of P-selectin).