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Case Reports
. 2021 Jun 3;384(22):2092-2101.
doi: 10.1056/NEJMoa2104840. Epub 2021 Apr 9.

Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination

Andreas Greinacher  1 , Thomas Thiele  1 , Theodore E Warkentin  1 , Karin Weisser  1 , Paul A Kyrle  1 , Sabine Eichinger  1
Affiliations
Case Reports

Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination

Andreas Greinacher et al. N Engl J Med. .

Abstract

Background: Several cases of unusual thrombotic events and thrombocytopenia have developed after vaccination with the recombinant adenoviral vector encoding the spike protein antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (ChAdOx1 nCov-19, AstraZeneca). More data were needed on the pathogenesis of this unusual clotting disorder.

Methods: We assessed the clinical and laboratory features of 11 patients in Germany and Austria in whom thrombosis or thrombocytopenia had developed after vaccination with ChAdOx1 nCov-19. We used a standard enzyme-linked immunosorbent assay to detect platelet factor 4 (PF4)-heparin antibodies and a modified (PF4-enhanced) platelet-activation test to detect platelet-activating antibodies under various reaction conditions. Included in this testing were samples from patients who had blood samples referred for investigation of vaccine-associated thrombotic events, with 28 testing positive on a screening PF4-heparin immunoassay.

Results: Of the 11 original patients, 9 were women, with a median age of 36 years (range, 22 to 49). Beginning 5 to 16 days after vaccination, the patients presented with one or more thrombotic events, with the exception of 1 patient, who presented with fatal intracranial hemorrhage. Of the patients with one or more thrombotic events, 9 had cerebral venous thrombosis, 3 had splanchnic-vein thrombosis, 3 had pulmonary embolism, and 4 had other thromboses; of these patients, 6 died. Five patients had disseminated intravascular coagulation. None of the patients had received heparin before symptom onset. All 28 patients who tested positive for antibodies against PF4-heparin tested positive on the platelet-activation assay in the presence of PF4 independent of heparin. Platelet activation was inhibited by high levels of heparin, Fc receptor-blocking monoclonal antibody, and immune globulin (10 mg per milliliter). Additional studies with PF4 or PF4-heparin affinity purified antibodies in 2 patients confirmed PF4-dependent platelet activation.

Conclusions: Vaccination with ChAdOx1 nCov-19 can result in the rare development of immune thrombotic thrombocytopenia mediated by platelet-activating antibodies against PF4, which clinically mimics autoimmune heparin-induced thrombocytopenia. (Funded by the German Research Foundation.).

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Figures

Figure 1
Figure 1. Reactivity of Patient Serum on Platelet-Activation Assays and Immunoassays.
Panel A shows the results of platelet-activation assays in serum samples obtained from the first 4 patients with vaccine-induced immune thrombotic thrombocytopenia (VITT) who were assessed in the study. The four colors in each experiment indicate the results obtained in the four samples; values are expressed as means, with 𝙸 bars indicating standard errors. The platelet-activation assay is performed by adding 20 μl of patient serum to 75 μl of washed platelets per well of a microtiter plate that contains the other reagents as indicated. Reactivity is expressed semiquantitatively as reaction time, with a shorter reaction time indicating stronger platelet-activating levels. A reaction time of more than 30 minutes indicates background or clinically insignificant reactivity. The asterisk indicates the reactivity of the outlier serum, which was strongly positive on subsequent retesting along with platelets of other volunteers in the presence of platelet factor 4 (PF4). Panel B shows the results of platelet-activation assays in serum samples obtained from an additional 24 patients with clinical VITT. The reactivity pattern resembles that observed in the 4 patients who were initially investigated. The serum caused variable platelet activation in the presence of buffer, which for most samples was inhibited in the presence of low-molecular-weight heparin but was strongly enhanced in the presence of PF4; in contrast, high levels of unfractionated heparin inhibited the reaction in all but one serum sample. Panel C shows the results of PF4–heparin and PF4 immunoassays of serum obtained from patients with VITT (including all 28 samples represented in Panels A and B) that showed PF4-dependent platelet activation. The results, which were obtained with the use of a microplate reader with a 450-nm filter, include all 28 PF4–heparin enzyme-linked immunosorbent assay (ELISA) experiments (with the addition of 100 IU per milliliter of heparin in 19 experiments) and the results of 10 PF4 ELISA experiments. The cutoff for a negative result is 0.50 optical-density units. LMWH denotes low-molecular-weight heparin, UFH unfractionated heparin, and IVIG intravenous immune globulin.
Figure 2
Figure 2. Potential Diagnostic and Therapeutic Strategies for Management of Suspected Vaccine-Induced Immune Thrombotic Thrombocytopenia.
Shown is a decision tree for the evaluation and treatment of patients who have symptoms of thrombocytopenia or thrombosis within 20 days after receiving the ChAdOx1 nCov-19 vaccine and who have had no heparin exposure. The diagnostic and therapeutic strategies in such patients differ from those in patients with autoimmune heparin-induced thrombocytopenia (HIT). DIC denotes disseminated intravascular coagulation, INR international normalized ratio, PF4 platelet factor 4, and PTT partial thromboplastin time.
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