Platelet immunophenotyping in health and inherited bleeding disorders, a review and practical hints

Abstract

Inherited platelet function disorders are rare hemorrhagic diseases. The gold standard for their exploration is optical aggregometry; however, investigations by flow cytometry (FCM) are being increasingly used. In this review, the physiology of platelets is first recalled, setting the stage for the compartments of platelets that can be apprehended by specific and appropriate labeling. As this requires some preanalytical precautions and specific analytical settings, a second part focuses on these characteristic aspects, based on literature and on the authors' experience in the field, for qualitative or quantitative explorations. Membrane labeling with antibodies to CD42a or CD41, respectively, useful to assess the genetic-related defects of Glanzmann thrombocytopenia and Bernard Soulier syndrome are then described. Platelet degranulation disorders are detailed in the next section, as they can be explored, upon platelet activation, by measuring the expression of surface P-Selectin (CD62P) or CD63. Mepacrin uptake and release after activation is another test allowing to explore the function of dense granules. Finally, the flip-flop anomaly related to Scott syndrome is depicted. Tables summarizing possible FCM assays, and characteristic histograms are provided as reference for flow laboratories interested in developing platelet exploration.

Effect of DDAVP on Platelet Activation and Platelet-Derived Microparticle Generation

“…Expression at T0 was consistent with our usual standards. 21 P-selectin expression was significantly decreased at T1 compared with T0, with a mild yet significant mean decline in MFI (3,543 vs. 3,320; p = 0.029), whereas there was a 28% decrease of labeled platelets which failed to reach statistical significance ( p = 0.061; Fig. 4 ).…”

“…Indeed, it has been reported 22 that there was no impact of DDAVP on the expression of P-selectin nor PAC-1 binding on circulating platelets, while we observed a significant decrease in P-selectin expression and a significant decrease in PAC-1-positive platelets. Nevertheless, Colucci at al 21 found a decrease in both P-selectin and expression and PAC-1 binding after platelet activation by ADP or thrombin. A possible explanation for this discrepancy is again the time course of DDAVP's effects.…”

“…A second tube was prepared in parallel for each sample where anti-CD62P was replaced by a murine-irrelevant IgGk (BD Biosciences) as isotypic control. After 15 minutes of incubation in the dark, 1 mL of PBS was added and suspensions directly processed for flow cytometry according to Fouassier et al 21 At least 10,000 platelets were acquired at low speed. A platelets' gate was drawn based on light scatter properties and FITC-CD41 fluorescence to assess platelet numbers.…”

Issue Highlights – November 2020

“…Such function may be categorized initially by platelet adhesion followed by their activation and finally collective aggregation. How platelets implement these, and other functions are the subject of the review by Fouassier, et al, 2020. In this fascinating review, the general physiology of the platelet is described, as well as antigens detectable by cytometry to track these cells, such as annexin, CD41, CD42a-d, CD61, CD62P and CD63.…”

Issue Highlights—May 2022

“…The functional pro‐coagulant activity of platelets can be easily appreciated both in vivo and in vitro, with a number of reliable and established laboratory procedures (Fouassier et al, 2020). Flow cytometry has the unique capability to appreciate the heterogeneity of platelet subpopulations in response to functional stimuli and agonists, while other assays measure just an average functional activity.…”