TY - JOUR
T1 - A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia
AU - Lee, Christine S.M.
AU - Liang, Hai Po Helena
AU - Connor, David E.
AU - Dey, Agnibesh
AU - Tohidi-Esfahani, Ibrahim
AU - Campbell, Heather
AU - Whittaker, Shane
AU - Capraro, David
AU - Favaloro, Emmanuel J.
AU - Donikian, Dea
AU - Kondo, Mayuko
AU - Hicks, Sarah M.
AU - Choi, Philip Y.I.
AU - Gardiner, Elizabeth E.
AU - Clarke, Lisa Joanne
AU - Tran, Huyen
AU - Passam, Freda H.
AU - Brighton, Timothy Andrew
AU - Chen, Vivien M.
N1 - Funding Information:
This study is partly funded by NSW Health Pathology. V.M.C. is funded by a NSW Ministry of Health Senior Clinician Scientist Cardiovascular Capacity Building Grant.
Publisher Copyright:
© 2022 by The American Society of Hematology.
PY - 2022/6/14
Y1 - 2022/6/14
N2 - Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti–platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcgRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein–coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine–based activation motif–linked receptors (FcgRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry–based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n 5 23), but not healthy donors (n 5 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n 5 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay–negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcgRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.
AB - Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti–platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcgRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein–coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine–based activation motif–linked receptors (FcgRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry–based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n 5 23), but not healthy donors (n 5 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n 5 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay–negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcgRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.
UR - http://www.scopus.com/inward/record.url?scp=85132801981&partnerID=8YFLogxK
U2 - 10.1182/bloodadvances.2021006698
DO - 10.1182/bloodadvances.2021006698
M3 - Article
C2 - 35359002
AN - SCOPUS:85132801981
SN - 2473-9529
VL - 6
SP - 3494
EP - 3506
JO - Blood Advances
JF - Blood Advances
IS - 11
ER -
