We purified platelets from whole blood (obtained from healthy volunteers) that had undergone anticoagulation with adenine citrate dextrose solution A. None of the volunteers had been taking antiplatelet drugs or had been vaccinated in the previous 10 days. We prepared platelets using methods that have been described previously.2 (link),3 (link) In a subgroup of experiments, platelets were preincubated in buffer with ChAdOx1 nCov-19 (1:2000 dilution) and washed before use. Washed platelets (75 microliters) were incubated with either buffer, a low-molecular-weight heparin (reviparin [Abbott]), or PF4 (Chromatec) in either the presence or absence of the FcγIIa receptor–blocking antibody IV.3. In some experiments, unfractionated heparin (100 IU per milliliter) was added to inhibit PF4-dependent reactions, or ChAdOx1 nCov-19 (1:50 dilution) was added per well. Serum was coincubated with PF4 and platelets in the presence of immune globulin (Privigen IVIG [CSL Behring]) at a concentration of 10 mg per milliliter. After establishing assay conditions using serum from the initial four patients, we investigated another 24 serum samples that tested positive on immunoassay to validate our findings. We refer to this modified platelet-activation test as the PF4-enhanced platelet-activation test.
To measure direct antibody binding, we used two immunoassays, a PF4–heparin enzyme-linked immunosorbent assay (ELISA) and a PF4 ELISA, with antibody binding measured by a secondary antihuman IgG, as described previously.4 (link) In addition, antibodies from two serum samples were affinity purified by immobilized PF4–heparin and immobilized PF4, and the purified antibodies were tested in the assays. (Details about this method are provided in theSupplementary Appendix , available with the full text of this article at NEJM.org.)
We defined reactivity on ELISA according to the optical-density units as strong (≥2.00), intermediate (1.00 to 1.99), or weak (0.50 to 0.99). On the PF4-enhanced platelet-activation test, reactivity was graded according to the time that had elapsed until platelet aggregation,5 (link) with shorter reaction times indicating stronger platelet activation (strong activation, 1 to 5 minutes; intermediate activation, >5 to 15 minutes; and weak activation, >15 to 30 minutes).
To measure direct antibody binding, we used two immunoassays, a PF4–heparin enzyme-linked immunosorbent assay (ELISA) and a PF4 ELISA, with antibody binding measured by a secondary antihuman IgG, as described previously.4 (link) In addition, antibodies from two serum samples were affinity purified by immobilized PF4–heparin and immobilized PF4, and the purified antibodies were tested in the assays. (Details about this method are provided in the
We defined reactivity on ELISA according to the optical-density units as strong (≥2.00), intermediate (1.00 to 1.99), or weak (0.50 to 0.99). On the PF4-enhanced platelet-activation test, reactivity was graded according to the time that had elapsed until platelet aggregation,5 (link) with shorter reaction times indicating stronger platelet activation (strong activation, 1 to 5 minutes; intermediate activation, >5 to 15 minutes; and weak activation, >15 to 30 minutes).