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Review 1: "Anti-PF4 levels of patients with VITT do not reduce 4 months following AZD1222 vaccination"

This paper claims that, although anti-PF4 antibody levels remain high in VITT patients months after follow-up, it is not associated with increased platelet activation Reviewers found it timely and reliable but in need of minor revisions on its methodology and discussion.

Published onSep 14, 2021
Review 1: "Anti-PF4 levels of patients with VITT do not reduce 4 months following AZD1222 vaccination"
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key-enterThis Pub is a Review of
Anti-PF4 levels of patients with VITT do not reduce 4 months following AZD1222 vaccination

AbstractBackgroundAnti-Platelet Factor 4 (PF4) IgG antibodies that activate platelets via FcγRIIa have been shown to be an important part of the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT). There is now extensive literature on its presentation and initial management. There is no literature however on what happens to these patients following discharge.MethodsWe collected clinical data and samples from seven patients presenting with VITT and followed them up for 82-145 days. We also collected clinical samples from them at last follow-up. Testing for anti-PF4/heparin antibodies was done using an anti-PF4/heparin enzymatic immunoassay. Flow Cytometry was used to look at FcγRIIa levels on patient platelets. Light Transmission Aggregometry with patient serum and healthy donor / patient platelets was used to analyse platelet responsiveness, in the presence and absence of PF4.FindingsAll patients were discharged on direct oral anticoagulants. Two patients remain completely symptom free, three have ongoing headaches, two have residual neurological deficits. Two patients developed mild thrombocytopenia and worsening headache (but without cerebral venous sinus thrombosis) and were retreated, one of these with rituximab. All patients, except the one treated with rituximab, had similar anti-PF4 antibody titres at 80-120 days to their levels at diagnosis. Platelets from patients at follow-up had normal levels of FcγRIIa and had normal responses to thrombin and collagen-related-peptide. Patient serum from diagnosis strongly activated healthy donor platelets in the presence of PF4. Serum from follow-up was much weaker at stimulating platelets, even in the presence of PF4.InterpretationThis study shows that despite similar PF4 antibody titres at diagnosis and during follow-up, there are further differences in patient serum, that are not apparent from currently used testing, that result in lower levels of platelet activation during the follow-up period. Further understanding of these factors are important in order to assess duration of anticoagulation for these patients.FundingThis work was supported by an Accelerator Grant (AA/18/2/34218) from the British Heart Foundation (BHF) and by a National Institute for Health Research (NIHR) grant.Key pointsPF4 antibody titres do not reduce up to 4-months post ChAdOx1 nCoV-19 in patients with VITTDespite similar PF4 antibody titres, diagnostic serum is more potent at activating platelets in the presence of PF4 than follow-up serum.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.




This preprint manuscript described a clinical consequence of VITT and a persistent high titer of anti-PF4 following vaccine-induced thrombocytopenia. This is an important topic. The findings contribute to a broader research understanding. The authors found that despite the persistent high titer of anti-PF4, platelet activation was much weaker with patients’ serum at the diagnostic time than at the follow-up time. This finding could indicate other potential mechanisms of VITT other than the presence of anti-PF4 antibodies.

Main comments

The manuscripts described a case series of clinical sequelae of 7 patients following the diagnosis of vaccine-induced thrombotic thrombocytopenia (VITT) after AZD1222. In this case series, the researchers observed recurrent thrombocytopenia and persistent symptoms requiring either rituximab, IVIG, or corticosteroid. The authors also performed anti-PF4 antibody and platelet aggregation using the patients’ serums and platelets at the time of follow-up (>80 days) since the date of vaccination. A persistently high titer of the anti-PF4 antibody was observed in all patients, except for one who received retreatment with rituximab. Though persistently high titer anti-PF4 antibody was demonstrated, platelet activation with the patient’s serum was much weaker with the follow-up serum than with the serum at the time of diagnosis. Since data on clinical sequelae of VITT is lacking, this manuscript describes important clinical data and is of interest in the field. The methods are sound, and the laboratory was performed on standard procedure. The demonstration of persistently high anti-PF4 titer but negative on functional assay at the follow-up time is a major finding which warrants further research in this area. The manuscript could be published with minor revisions. Few comments are as below.

1. The limitation of a robust conclusion of the findings is the small number of samples used to perform platelet activation. Only 3 samples were available. Whether these samples can represent most VITT cases needs to be elucidated.

2. Anti-PF4 antibody in autoimmune HIT can persist for several months while functional assay has become negative. Therefore, the findings could represent natural history, like that of autoimmune HIT. The author discussed other manners besides a high PF4- antibody titer alone at the time of diagnosis that could cause VITT.

3. The author demonstrated that the levels of FcγRIIa on patients’ platelets at the follow-up time were comparable to platelets in healthy control. Therefore, the data of FcγRIIa of patients’ platelet at the diagnostic time is potentially informative about whether or not the platelets contributed to the occurrence of VITT.

4. From supplementary figure 1, diagnostic serum of patient 4 caused robust aggregation on its platelets. However, there was no robust aggregation when PF4 was added. In addition, while inpatients 6 and 7 demonstrated no spontaneous platelet activation, robust aggregation occurred when PF4 was added. Are there possible explanations for these findings?

5. The results and the figures should be matched together. The referral of figure 2 and the mention of patient 2 in the result section should be clarified.

  1. Similar findings on the persistence of PF4 were published in NEJM (DOI: 10.1056/NEJMc2112760). The reference should be cited.

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