key: cord-1020528-djiagf4u
authors: Favaloro, Emmanuel J.
title: Laboratory testing for suspected COVID‐19 vaccine–induced (immune) thrombotic thrombocytopenia
date: 2021-06-17
journal: Int J Lab Hematol
DOI: 10.1111/ijlh.13629
sha: ba5245827b0fb184d048858f5fdfdf363f4f89a1
doc_id: 1020528
cord_uid: djiagf4u

COVID‐19 (coronavirus disease 2019) represents a pandemic, and several vaccines have been produced to prevent infection and/or severe sequelae associated with SARS‐CoV‐2 (severe acute respiratory syndrome coronavirus 2) infection. There have been several reports of infrequent post vaccine associated thrombotic events, in particular for adenovirus‐based vaccines. These have variously been termed VIPIT (vaccine‐induced prothrombotic immune thrombocytopenia), VITT (vaccine‐induced [immune] thrombotic thrombocytopenia), VATT (vaccine‐associated [immune] thrombotic thrombocytopenia), and TTS (thrombosis with thrombocytopenia syndrome). In this report, the laboratory test processes, as utilised to assess suspected VITT, are reviewed. In published reports to date, there are notable similarities and divergences in testing approaches, potentially leading to identification of slightly disparate patient cohorts. The key to appropriate identification/exclusion of VITT, and potential differentiation from heparin‐induced thrombocytopenia with thrombosis (HITT), is identification of potentially differential test patterns. In summary, testing typically comprises platelet counts, D‐dimer, fibrinogen, and various immunological and functional assays for platelet factor 4 (PF4) antibodies. In suspected VITT, there is a generally highly elevated level of D‐dimer, thrombocytopenia, and PF4 antibodies can be identified by ELISA‐based assays, but not by other immunological assays typically positive in HITT. In addition, in some functional platelet activation assays, standard doses of heparin have been identified to inhibit activation in suspected VITT, but they tend to augment activation in HITT. Conversely, it is also important to not over‐diagnose VITT, given that not all cases of thrombosis post vaccination will have an immune basis and not all PF4‐ELISA positive patients will be VITT.

primary hemostasis (ie, platelets, von Willebrand factor, endothelium), secondary hemostasis and fibrinolysis. [8] [9] [10] [11] [12] In addition, thromboses may arise from disturbances in immune response, creating cytokine disturbance (so-called 'cytokine storm'), according to immunothrombosis-/endotheliitis-type mechanisms. 2 Perhaps unsurprisingly, then, several autoimmune events have also been associated with COVID-19, including presence of antiphospholipid antibodies. [13] [14] [15] Of greater relevance, however, are reports of platelet factor 4/heparin (PF4/H) antibodies being present in COVID-19 patients, as recently reviewed in this journal. 16 In brief, PF4/H antibodies can be observed in COVID-19 patients, and they may occur at higher incidence than in historical non-COVID-19 cohorts. However, the situation is complex, since not all PF4/H antibodies may lead to platelet activation, and not all identified antibodies are heparin-dependent.

Thus, such antibodies may or may not identify the condition called heparin-induced thrombocytopenia (HIT), and not all such occurrences are associated with thrombosis (or 'HITT'). Quite recently, a 'HIT-like' syndrome has also been reported in patients who have been vaccinated against COVID-19. The current review looks at this 'thrombotic condition' with a focus on laboratory testing. It is also important to recognise the potential to over-diagnosis of this disorder, if based solely on laboratory parameters.

This is a narrative review. The PubMed database (https://pubmed. ncbi.nlm.nih.gov) was searched as required for both background information as well as specific papers related to post vaccinationrelated thrombosis. For the latter, the author primarily used various search terms and most notably "((Vaccine induced immune thrombotic thrombocytopenia) OR (vaccine associated thrombotic thrombocytopenia)) OR (thrombosis with thrombocytopenia syndrome) OR (Vaccine Induced Prothrombotic Immune Thrombocytopenia))". An initial search performed on 8th May, 2021, identified 2236 publications, which was reduced to 66 after including only publications published in 2021, which represents the year in which the first cases of post COVID-19 vaccination-associated events was published in preprint form by the German group of Greinacher et al. 17 The search was updated to be current of 27th May, 2021, when a total of 76 publications were identified, which included the recent associated review. 16 These publications were further screened by title and abstract to remove non-relevant publications and to identify additional relevant content.

The condition, as reported by different groups, has been given several different names, as summarised in Table 1 . In the initial report, 17 the German researchers coined the condition "VIPIT", for "vaccineinduced prothrombotic immune thrombocytopenia". In their subsequent publication, 18 the same workers instead used the term "VITT"

for "vaccine-induced immune thrombotic thrombocytopenia".

Interestingly, a separate group of Norwegian workers, also publishing in the same issue of the journal, also used the term "VITT", 19 suggesting some "harmonisation" of terminology among the separate groups, perhaps facilitated by editorial guidance. A third series of patients was reported by a UK group, 20 in the same journal, and the TA B L E 1 Terms used to describe post COVID-19 vaccine associated thrombosis with thrombocytopenia publication also used the termed "VITT", perhaps for consistency.

The term "VITT" also links to a separate, perhaps pathophysiologically related, condition termed "HITT", for "Heparin-induced thrombocytopenia with thrombosis".

Nevertheless, a few other terms have alternatively been proposed by others. One potentially useful term is "VATT", for "vaccineassociated (immune) thrombotic thrombocytopenia". This term is supported by some researchers concerned with use of the term "induced", where a clear pathological link to the vaccine is not apparent.

As a broader term excluding "immune", the term can also potentially capture other thrombotic events associated with both thrombocytopenia and vaccine use, but where an immune relationship is unclear.

As an alternative to VATT, and for similar purpose, some researchers may instead use the term "suspected VITT" for "suspected vaccineinduced thrombotic thrombocytopenia". Finally, the term "TTS", for "thrombosis with thrombocytopenia syndrome" has also been used and also thrombotic thrombocytopenia purpura (TTP).

For the purpose of "harmonisation", this review will use the term "suspected VITT", similar to most publications to date, but qualified with "suspected" since it is not always clear from the literature and in clinical practice if a post-vaccination thrombotic event is truly induced by the vaccine or can be proven to have an immune basis.

The term also suits the utility within the context of laboratory testing for the condition in typical clinical practice, as here the search is for "suspected VITT", and most cases investigated for post-vaccine thrombosis will not prove to be VITT, just as most cases investigated for HITT, will not prove to be HITT.

A summarised review of the published case series and case studies identified by the Medline search is presented in [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] The uncertainty in total case numbers is because there may be duplication of cases in at least four publications. 17, 18, 20, 24 Most reports, in keeping with early reports, have been in women (in total, n = 69, vs 21 males).

It is interesting, nonetheless, that the different publications include both similarities and differences in testing approaches and case definitions. Part of this relates to publication date, given this was, and remains, an emerging situation. The original non-peer-reviewed

German publication 17 presented on 9 cases, predominantly young women, with a high fatality rate (~50%), who presented thrombotic symptoms some 4-16 days post the AstraZeneca ChAdOx1 nCoV-19 vaccine (AZD1222; 'AZ vaccine'). The most striking laboratory findings reported in the manuscript was the presence of thrombocytopenia in all patients, with a platelet count ranging from 9-100 × 10 9 /L, and all patients that were tested (4/4) being positive for PF4 antibodies by both ELISA (enzyme-linked immunosorbent assay) and functional platelet activation (modified 'HIPA') assay.

Notably, D-dimer levels and fibrinogen were not reported in this paper. The follow up paper by the German group was published in the New England Journal of Medicine (NEJM), 18 and described 11

cases, presumably including the 9 cases in the preprint publication. 20 They separated the 43 patients into "probable" VITT (n = 27), "possible" VITT (n = 7) and "unlikely" VITT (n = 9) and assessed six different ELISA assays as well as four different rapid assays. They were able to calculate sensitivity and specificity and compare these with previously published data for HITT. All ELISA assays had high sensitivity and "specificity" for VITT, although there was some variability, and it should be clarified here that data (especially 'specificity') are biased according to the study population. None of the rapid assays had any sensitivity to VITT, apart from low sensitivity using PAGIA (particle gel immunoassay).

Vayne et al 25 reported a French study of 9 cases of suspected VITT in which they concluded 7/9 to be likely VITT post AZ vaccine. Findings were broadly similar to other case series, with age range 24-73 years, all with thrombocytopenia (platelet counts 9-61 × 10 9 /L) and 5/7 with fibrinogen <2 g/L. D-dimer was highly raised in all cases (>4 to 105 mg/L). PF4 antibodies were positive in all 7 patients when using ELISA, but negative when using both CLIA and lateral flow STiC (Stago) methods. Standard SRA was positive in 5/7 cases, but all 7/7 were positive using an SRA modified by addition of PF4.

Althaus et al 26 Table 2 F I G U R E 2 Summary of reported ELISA OD readings for PF4 antibodies according to the literature using case series cited in Table 2 , excluding the study of Platton et al 24 

Early reports identified mostly young women, but later reports have indicated both males and females across a wide age group. It appears that earlier reports, in particular with the AZ vaccine, may simply have been reporting on the predominant cohort being vaccinated at that time, being mostly (young) female healthcare workers. Thus, there may not be a gender or age restriction in regard to suspected VITT. The ages of all cases reported to date is shown in Figure 1 .

The date of onset of symptoms or hospitalisation (depending on the study reporting) ranged from 3-25 days post vaccination (Table 2, Figure 1 ). Case fatality was very high in the first few reports but seems to be falling with the newer case series descriptions, perhaps due to better understanding of the disease progression, its prompter recognition and earlier and better initiation of treatment.

Of particular relevance to this review, however, are the laboratory test findings. The most striking initial test findings are thrombocytopenia and highly elevated D-dimer levels (Table 2, Figure 1 ).

Overall platelet counts ranged from 5-127 × 10 9 /L, with worsen- Hyphen methods, respectively. The potentially noteworthy fact is that the ODs reported for the Stago assay were a little lower than those of other ELISA assays in the initial case series (Figure 2 ), possibly linked to the assay method or assay substrate incubation times. However, in the Platton et al 24 study (Figure 3 ), this did not appear to be the case.

It is important to always note the potential for bias due to the type of reported study. In particular, case reports and small cases series, comprising some the current literature ( in normal individuals. 26, 33, 34 In any case, potential selection bias in the literature always needs to be considered in any evaluation of suspected VITT, or perhaps for any vaccine associated "TTS".

Thus, whether the literature is describing the same condition in all patients remains to be clarified.

It is not possible to be entirely sure of the true incidence of suspected VITT, given limited publications. Notably, a high relative proportion of cases based on the vaccinated population was reported in the Norwegian study, 19 37 The EMA estimates the incidence of "TTS" at around 1 in every 100 000 vaccinated people. 38 In Australia, the latest report from the TGA identifies 24 cases of 'TTS' from approximately 2.1 million administered doses of the AZ vaccine, which would also estimate the incidence at around 1 in every 88 000 vaccinated people. 39 after a 30 minutes incubation, the reaction is stopped, and color is read on a plate-reader. Finally, the Hyphen Biomed Zymutest HIA IgG kit ('for the detection and quantitation of heparin dependent F I G U R E 3 Summary of reported ELISA OD readings for PF4 antibodies and results of rapid assays, according to the study of Platton et al 24 For rapid assays, CLIA and LIA results given in U/mL; for PAGIA, the numbers refer to the grade of response, and, for STiC, a value of 0 represents negative and a value of 1.5 represents positive. The horizontal lines indicate the negative/positive cut-off values antibodies of the IgG isotype') has microwells coated with unfractionated heparin, "biologically available, saturated, and then stabilized". After addition of diluted plasma or serum, supplemented with a kit provided platelet lysate, any heparin-dependent antibody in the added plasma/serum is expected to bind any complexes formed on the immobilised heparin. After 1 hour incubation and washing steps, goat anti-human IgG antibodies coupled with horse radish peroxidase (HRP) were added. After an additional 1hr incubation, with additional washing, TMB is added; after exactly 5-minutes incubation, the reaction is stopped with acid, and color read on a plate-reader.

There are other potential commercial PF4-based ELISA assays, possibly using alternate methods, and it is not known if all such ELISA assays will detect the PF4 antibodies so far identified in patients. 

Here there was some divergence in the publications in regard to assays used for assessing platelet activation. In Greinacher et al, 17 4/9 cases were tested, and all showed platelet activation in a modified HIPA assay. Importantly, reactivity could be inhibited termed the underlying disease mechanism the "vaccine-induced covid-19 mimicry" syndrome (VIC19 M syndrome)". Another interesting fact is that the effect of heparin in the assays is used to identify suspected VITT, potentially inhibiting ELISA OD and some platelet activation tests, at both low and high concentration, although this was not evident in all cases. Nevertheless, this is in contrast with HIT, where low doses of heparin would be expected to augment reactivity, and only a high dose to inhibit. These features also provide potential clues to the differential identification of HIT vs suspected VITT and should be further assessed in future studies. On the other hand, not all positive ELISA findings will necessarily identify pathogenic antibodies. It is well known in HIT studies that around 50% of HIT detected immunologically by ELISA will not cause platelet activation. 50 That the majority of ELISA positive cases, apart from the US study, also show platelet activation may also reflect some selection bias, with these patients being highly clinically suspected to have VITT.

A broader investigation of cases will likely find more cases positive by ELISA but negative by functional assay, as partly suggested by more recent studies. However, in several other ways, 'VITT' is dissimilar to HITT, particularly the markedly elevated D-dimer and the heparin independence being potentially critical to their differentiation.

Finally, not all thrombocytopenia post vaccination will be "VITT".

For example, there have been several cases of apparent secondary immune thrombocytopenia (ITP) after SARS-CoV-2 vaccination with both the Pfizer and Moderna vaccines. 51

The opinions expressed in this review are those of the author and do not necessarily reflect the opinion of NSW Health Pathology.

The author has no competing interests.

This is a review of the existing literature; no new data were created. All data shown are condensed/summarized from the reviewed literature.

Emmanuel J. Favaloro https://orcid.org/0000-0002-2103-1661

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How to cite this article: Favaloro EJ. Laboratory testing for suspected COVID-19 vaccine-induced (immune) thrombotic thrombocytopenia