Comprehensive phenotyping of human platelets by single‐cell cytometry

Abstract

Platelets are small anucleate blood cells that contribute to hemostasis, immunity, and inflammation. Circulating platelets are heterogeneous in size, age, receptor expression, and reactivity. They inherit many features from megakaryocytes and are further modified on exposure to bioactive substances in the bloodstream. Among these substances, prothrombotic agonists, vasodilators, and bloodborne pathogens modulate platelet phenotypes via distinct signaling cascades. The ability of platelets to respond to (patho)physiologic signals is incompletely understood but likely depends on their repertoire of surface receptors, which may partition them into discrete subsets with specialized functions and divergent abilities. The single-cell resolution of flow and mass cytometry is ideal for immunophenotyping and allows the identification of platelet subsets in remarkable detail. In this report, we describe the surface markers and gating strategies needed for the comprehensive characterization of platelets.

Platelet Phenotyping by Full Spectrum Flow Cytometry

“…Antibody‐mediated activation is unpredictable but can sometimes be tempered by titration. Indeed, CD36 (clone CB38) and GPVI (clone HY101) induce activation at high concentrations, but careful titration can find dilutions that allow good staining without inducing/potentiating activation (Spurgeon and Frelinger, 2022b). Mechanisms of activation can differ; anti‐CD36 may cause stimulation of ligand‐binding sites (Alessio et al., 1993), while anti‐GPVI may cause dimerization and/or clustering of GPVI/FcγRIIa (van Dievoet, Brusa, Octave, Horman, and Stephenne, 2022).…”

“…For most applications, we recommend that staining be performed on fresh (unfixed) samples within 30 min of blood collection. Prefixation is discouraged, as it obscures epitopes and leads to staining artifacts for some markers (Spurgeon and Frelinger, 2022b). Ideally, stained and fixed samples are acquired within a few hours, as longer‐term stability is unknown and likely depends on the antibodies/reagents in use.…”

“…Our 16-color panel is thoroughly optimized and validated (Spurgeon and Frelinger, 2022b), and all reagents are known to provide quality results (Table 1). The panel was developed using a three-laser (16V-14B-8R) Cytek Northern Lights TM but should be compatible with similar instruments.…”

“…As with all computational output, users are advised to verify novel discoveries by manual interrogation with appropriate biological controls (e.g., basal versus stimulated platelets). Figure 3 shows a simple application of FAUST, and the use of Astrolabe is shown elsewhere (see Spurgeon and Frelinger, 2022a). High‐parameter phenotyping is too recent for there to be consensus on the best option(s) for platelet analysis, but new trends are always emerging, and routine uptake of automatic gating will widen the scope and awareness of platelet heterogeneity (Cheung et al., 2021).…”

“…Anticoagulant choice is an important consideration, with sodium citrate preferred because it inhibits clotting but leaves enough free calcium to support the structure and function of key markers (Spurgeon, Michelson, and Frelinger, 2021c). Heparin and EDTA may suffice for certain markers but are unlikely to be useful for large (and diverse) panels, as they can induce activation and conceal important subsets by inhibiting calcium‐dependent events such as (1) annexin V binding, (2) complement deposition, (3) glycoprotein cleavage, and (4) PAC‐1 binding (Spurgeon and Frelinger, 2022b). For most applications, we recommend that staining be performed on fresh (unfixed) samples within 30 min of blood collection.…”