key: cord-0953368-xl7hvhgo
authors: Vayne, Caroline; Rollin, Jérôme; Gruel, Yves; Pouplard, Claire; Galinat, Hubert; Huet, Olivier; Mémier, Vincent; Geeraerts, Thomas; Marlu, Raphael; Pernod, Gilles; Mourey, Guillaume; Fournel, Alexandra; Cordonnier, Charlotte; Susen, Sophie
title: PF4 Immunoassays in Vaccine-Induced Thrombotic Thrombocytopenia
date: 2021-05-19
journal: N Engl J Med
DOI: 10.1056/nejmc2106383
sha: f70e2a640d9a9de46ee9e46a9c7eaa6033a8cb21
doc_id: 953368
cord_uid: xl7hvhgo

nan

sorbent assays and obtained variable results (Fig. 1B) . Significant levels of IgG antibodies to PF4 were detected in seven patients only by the assay that used PF4-poly(vinyl sulfonate) (PVS) complex as the antigenic target. In addition, optical density values were variable and lower than those previously reported with a similar test. 2 The diagnosis of VITT was confirmed by PF4-serotonin release assay 3 in all seven patients with IgG antibodies to PF4-PVS (Fig. 1C) , whereas a standard serotonin release assay was negative in two patients. Platelet activation was suppressed by IV.3, a monoclonal antibody that binds FcγRIIA receptors, but also by IdeS (IgGdegrading enzyme derived from Streptococcus pyogenes) (Fig. 1C) , a protease that also inactivates heparin-induced thrombocytopenia IgG antibodies. 4 Intravenous immune globulins may be inappropriate for severe cerebral vein thrombosis with intracranial hypertension. IdeS (imlifidase) may be an effective treatment and needs to be evaluated.

Our results provide further support to show that rapid immunoassays should be avoided in the detection of PF4-specific antibodies in patients with suspected VITT. Therefore, the use of a sensitive, quantitative, immunologic test is strongly recommended, because according to the recently proposed algorithm, 1,5 nonheparin anticoagulants should be preferred when clinically significant levels of anti-PF4 antibodies are detected. Panel A shows that rapid immunoassays do not detect antibodies to platelet factor 4 (PF4) associated with vaccine-induced immune thrombotic thrombocytopenia (VITT). All the plasma samples from the patients with findings suggestive of VITT were tested with two rapid immunoassays (STic Expert HIT, Stago, and HemosIL AcuStar HIT-IgG, Werfen), and negative results were obtained in every case. Two other rapid assays had also been performed in some patients (each represented by a specific symbol) in the referring centers (ID-PaGIA H/PF4, DiaMed, and HemosIL HIT-Ab, Werfen), and they also showed negative or doubtful (in patient *, who had an initial positive result but then tested negative) results.

Panel B shows that the sensitivity of enzyme-linked immunosorbent assays (ELISAs) to detect PF4-specific IgG antibodies depends on the antigen target. Levels of PF4-specific IgG antibodies were evaluated in plasma samples from the nine patients with suspected VITT (each represented by a specific symbol) with the use of three different ELISAs with varying antigen targets ( Panel C shows that a serotonin release assay (SRA) should be performed with the use of PF4 to detect platelet-activating antibodies to VITT. SRA was performed by incubating 75 μl of washed platelets obtained from healthy persons with 20 μl of plasma obtained from each patient, either in the absence (standard SRA) or the presence (PF4-SRA) of 10 μg per milliliter of PF4. All tests were performed with or without unfractionated heparin at 0.1 IU per milliliter (UFH 0.1), 0.5 IU per milliliter (UFH 0.5), and 10 IU per milliliter (UFH 10). Clinically significant and strong platelet activation, with maximum release ranging from 36 to 99%, was measured in seven of the nine patients only when PF4 was present in the reaction mixture. Moreover, platelet activation was not inhibited by therapeutic concentrations of UFH 0.1 or UFH 0.5. In contrast, platelet activation was completely abolished by 10 μg per milliliter of IV.3, a monoclonal antibody specific for FcγRIIA, or 6 U of IdeS, an IgG-degrading enzyme of Streptococcus pyogenes, preincubated for 15 minutes at 37°C in the plasma sample of each patient tested (five patients) before SRA. 

Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination

Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination

Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparininduced thrombocytopenia antibodies

Cleavage of anti-PF4/heparin IgG by a bacterial protease and potential benefit in heparin-induced thrombocytopenia

Diagnosis and management of vaccine-related thrombosis following Astra-Zeneca COVID-19 vaccination: guidance statement from the GTH

Correspondence Copyright © 2021 Massachusetts Medical Society

Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.This letter was published on May 19, 2021, at NEJM.org.