key: cord-0858560-rh5xshkr
authors: Handtke, Stefan; Wolff, Martina; Zaninetti, Carlo; Wesche, Jan; Schönborn, Linda; Aurich, Konstanze; Ulm, Lena; Hübner, Nils-Olaf; Becker, Karsten; Thiele, Thomas; Greinacher, Andreas
title: A Flow cytometric assay to detect platelet activating antibodies in VITT after ChAdOx1 nCov-19 vaccination
date: 2021-05-08
journal: Blood
DOI: 10.1182/blood.2021012064
sha: bc57a1688aaa2c47259bd3fa84357c355666efe1
doc_id: 858560
cord_uid: rh5xshkr

Vaccination is crucial in combatting the SARS-CoV-2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4. We present a widely applicable whole blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories.

Coronavirus disease 2019 (COVID-19) is caused by the single stranded RNA virus "acute respiratory syndrome coronavirus 2" (SARS-CoV-2). Currently, four different vaccines to prevent symptomatic COVID-19 have been approved by the European Medical Agency 1 .

Recently, several cases of thrombosis combined with moderate to severe thrombocytopenia were observed in patients vaccinated with the ChAdOx1 nCov-19 (AstraZeneca) vaccine [2] [3] [4] [5] and in patients vaccinated with the Johnson and Johnson vaccine 6, 7 . We have identified IgG antibodies directed against platelet factor 4 (PF4) activating platelets via FcγRIIa to be the likely cause of vaccine induced immune mediated thrombotic thrombocytopenia (VITT) 5 .

VITT shows striking similarities with autoimmune heparin-induced thrombocytopenia (HIT) 8, 9 . In contrast to HIT, platelet activation in VITT occurs in the presence of PF4 rather than low heparin concentrations. The antibodies associated with recent ChAdOx1 nCov-19 vaccination can be detected by some PF4/heparin enzyme-immunoassays (EIA) 10 , which are widely available to diagnose HIT. However, these assays may not be specific for VITT related antibodies. Not all antibodies binding to PF4/heparin complexes by EIA are functionally active and others may be typical HIT antibodies. We modified the functional heparin-induced platelet activation test, 11,12 a washed platelet assay, to detect vaccine-related antibodies and to differentiate them from HIT antibodies. We have named the modified assay the PF4induced platelet activation test (PIPA, details see supplementary material). As washed platelet assays are restricted to specialized laboratories, we developed a flow cytometric assay using whole blood to detect PF4-dependent platelet-activating antibodies in ChAdOx1 nCov-19 vaccinated patients. By analogy to the washed platelet test, we refer to the modified assay as the PIFPA test (PF4-induced flow cytometry-based platelet activation).

For the PIFPA test, citrated whole blood was obtained from healthy donors. Whole blood was supplied with 54U/mL (final) of hirudin (Canyon Pharmaceuticals, Switzerland; if hirudin is not available other thrombin inhibitors like PPACK can be used) and incubated with 0, 5 or Platelets were positively gated using CD61-PE. Activation was determined by granule release measured using CD62P-PE-Cy5 and given as mean fluorescence intensity (MFI) of the CD62Ppositive gated events multiplied by the percentage of gated platelets. Platelet activation for each serum was given as median activation from the four whole blood samples. Further details of test method are given in the supplementary material.

The use of whole blood and platelets from healthy donors and serum from patients was approved by the ethics board at Universitätsmedizin Greifswald and was conducted in accordance with the Declaration of Helsinki. The data supporting the findings of this study are available from the corresponding author upon reasonable request.

We tested sera from individuals who developed vaccine-induced prothrombotic immune The PIFPA test shows very comparable results to the PIPA test (Tab. 1) and can be applied to confirm or rule out platelet-activating, PF4-dependent antibodies which occur 5-16 days after ChAdOx1 nCov-19 vaccination based on available data without the need for washed platelets. The exact mechanism whereby vaccination with ChAdOx1 nCov-19 is associated with these platelet-activating anti-PF4 antibodies is still unknown. The disorder appears analogous to autoimmune HIT, where antibodies are capable of inducing strong platelet activation despite the absence of heparin, in contrast to typical HIT sera, which require the addition of heparin. Although the PIFPA seems to be specific, we cannot rule out that patients with less severe complications, e.g. isolated thrombocytopenia, which have not been identified up to now, may have more weakly-reacting antibodies induced by ChAdOx1 nCov-19 for which an additional cofactor might be required (by analogy to HIT, akin to heparin) to produce platelet activation by patient serum. The scientific community should undertake all efforts to screen for such (a) cofactor(s). In addition, laboratory tests for antibodies causing heparin-induced thrombocytopenia using washed platelets are always more sensitive and probably even more specific than assays using whole blood 13 . We therefore recommend that patients with typical clinical presentation of VITT but testing negative in the PIFPA, should be further assessed in the PIPA until more information is available.

In conclusion, we present an assay to detect strongly platelet activating antibodies after vaccination with ChAdOx1 nCov-19, which seem to be highly specific for VITT. It will help to rapidly identify the real incidence of VITT caused by platelet-activating antibodies and provide the opportunity to identify a potential cofactor inducing the antigen to which lower titer anti-PF4 antibodies bind, thereby further clarifying the pathogenesis of this severe adverse effect threatening the vaccination program in the SARS-CoV-2 pandemic.

Results of the PF4/heparin EIA and washed platelet PIPA test were compared to flow cytometrybased PIFPA test. + = test was rated positive. Results of the PF4/heparin EIA are given in brackets.

PIPA PIFPA VITT Platelet activation induced by (heat inactivated; 56°C, 30 min) sera and PF4 is shown by CD62Pexpression and given by the MFI multiplied by percentage of gated events. Data are shown for 16 samples from patients with VITT (14 sera, 2 plasmas), 10 sera from patients after vaccination with positive EIA but negative PIPA result, 10 sera from patients after vaccination with negative EIA and PIPA and 4 sera from patients with typical HIT. One data point for each serum represents median activation from four different whole blood samples. Cut-off was determined with sera of 13 unvaccinated healthy controls incubated with 5 µg/mL PF4 as the mean + 2SD. With the addition of 5 µg/mL PF4 sera from patients with VITT can be readily discriminated from vaccinated donors with no functionally relevant antibodies (p <0.0003). Statistical significance was calculated by unpaired t-test.

*=p<0.05, **= p<0.01), *** =p<0.001

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We thank Ulrike Strobel, Ricarda Raschke, Ines Warning, Carmen Freyer, Katrin Stein, Jessica Fuhrmann and Julia Klauke for excellent technical support.The study has been funded by the Deutsche Forschungsgemeinschaft (DFG, GermanResearch Foundation) -Projektnummer 374031971 -TRR 240.Author's contribution: