key: cord-0901433-hbl19aak
authors: Bissola, Anna-Lise; Daka, Mercy; Arnold, Donald M; Smith, James W.; Moore, Jane C.; Clare, Rumi; Ivetic, Nikola; Kelton, John G; Nazy, Ishac
title: The clinical and laboratory diagnosis of vaccine-induced immune thrombotic thrombocytopenia
date: 2022-05-25
journal: Blood Adv
DOI: 10.1182/bloodadvances.2022007766
sha: 5fac09e60ec0b5eafeaffa5987f5cf288130905c
doc_id: 901433
cord_uid: hbl19aak

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse syndrome occurring 5-30 days after adenoviral vector COVID-19 vaccination. Therefore, a practical evaluation of clinical assessments and laboratory testing for VITT is needed to prevent significant adverse outcomes as the global use of adenoviral vector vaccines continues. We received the clinical information and blood samples of 156 patient samples with a suspected diagnosis of VITT between April to July 2021 in Canada. The performance characteristics of various diagnostic laboratory tests were evaluated against the PF4-SRA including a commercial anti-PF4/heparin IgG/A/M enzyme immunoassay (EIA, PF4 Enhanced; Immucor), in-house IgG-specific anti-PF4 and anti-PF4/heparin-EIAs, the standard SRA, and the PF4/heparin-SRA. Of those, 43 (27.6%) had serologically confirmed VITT based on a positive PF4-SRA result and 113 (72.4%) were negative. The commercial anti-PF4/heparin EIA, the in-house anti-PF4-EIA, and anti-PF4/heparin-EIA were positive for all 43 VITT-confirmed samples (100% sensitivity) with a few false-positive results (mean specificity 95.6%). These immunoassays had specificities of 95.6% [95% confidence interval (CI) 90.0-98.6], 96.5% (95% CI 91.2-99.0), and 97.4% (95% CI 92.4-99.5), respectively. Functional tests, including the standard SRA and PF4/heparin-SRA, had high specificities (100%), but poor sensitivities for VITT [16.7% (95% CI 7.0-31.4); and 46.2% (95% CI 26.6-66.6), respectively]. These findings suggest EIA assays that can directly detect antibodies to PF4 or PF4/heparin have excellent performance characteristics and may be useful as a diagnostic test if the PF4-SRA is unavailable.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse event associated with vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the ChAdOx1 nCoV-19 (Oxford-AstraZeneca; AZ) and the Ad26.COV2.S vaccine (Johnson & Johnson; J&J). [1] [2] [3] [4] VITT is characterized by moderate to severe thrombocytopenia and arterial and/or venous thrombosis 5 -30 days post vaccination with unusual manifestations, such as cerebral venous sinus thrombosis. [1] [2] [3] 5 Recent updates on the approximate risk of VITT after an AZ or J&J vaccine are reportedly 1/90,000 6 and 1/260,000 7 , respectively; or roughly 1/100,000 for both. Although the suspension of adenoviral vector vaccines for COVID-19 has led to a decline in VITT across Canada and other parts of the world, many low-and middle-income countries continue to administer these vaccines because of their stability and favourable storage conditions. As VITT remains a global health problem, a fulsome description of the diagnostic accuracy of clinical and laboratory evaluations for VITT is needed to uphold vaccine safety worldwide.

The immunological mechanism of VITT resembles an adverse reaction to the common anticoagulant heparin, which is known as heparin-induced thrombocytopenia (HIT). 8, 9 HIT develops when immune complexes formed of immunoglobulin (IgG) antibodies and platelet factor 4 (PF4)/heparin interact with platelet surface FcγRIIa receptors, triggering platelet activation. 10 Similarly, VITT is caused by platelet activating anti-PF4 IgG antibodies, but without previous exposure to heparin. 11, 12 We recently confirmed the presence of highly reactive anti-PF4 antibodies in patients with VITT that, like HIT, 13 bind to conserved sites on PF4 and cause platelet activation and thrombosis. 14 VITT and HIT are diagnosed using similar laboratory tests due to their similar pathophysiologies. 15 These include enzyme immunoassays (EIAs) to detect anti-PF4 antibodies followed by functional assays that confirm their platelet-activating ability. Initial experience using the standard SRA for VITT diagnosis showed a decrease in antibody-mediated platelet activation in the presence of heparin thereby producing frequent false-negative results. 5 It was then discovered that the sensitivity of platelet functional assays, including but not limited to the 14 C-serotonin release assay (SRA), heparin-induced platelet activation assay (HIPA), and pselection expression assay (PEA), 16 could be increased with the addition of exogenous PF4 rather than heparin to the test system. 5, 15 Therefore, PF4-enhanced platelet functional assays are currently considered the most accurate diagnostic assays for VITT. 5, 15, 16 From March 2021 to January 2022, over three million Canadians received the AZ COVID-19 vaccine. 17 The prompt recognition of clinical signs that might indicate VITT is important to prevent severe morbidity and mortality. 8, 18 Clinicians may be prone to overcalling VITT (as is the case with HIT) 19 or under calling VITT, as the signs of thrombocytopenia and thrombosis are fairly non-specific. We evaluated the clinical diagnostic criteria for VITT using a practical approach based on comprehensive cohort of patients with suspected VITT in Canada.

We also describe the performance characteristics of various HIT laboratory assays for the diagnosis of VITT.

Blood samples used for this study were obtained from patients whose 

Testing for IgG/A/M anti-PF4/heparin antibodies was performed using a commercially available EIA (LIFECODES PF4 Enhanced assay; Immucor GTI Diagnostics, Waukesha, WI, USA [positive optical density (OD) 405nm ≥ 0.4]) as per the manufacturer's instructions. 21 Human recombinant PF4 used in our immunoassays, as well as our platelet functional assay, were purified in-house as previously described. 13 Subsequently, an in-house IgG-specific anti-PF4-EIA and anti-PF4/heparin-EIA were performed on all samples as previously described. 21 Briefly, patient sera were incubated in 96-well Maxisorp plates (Thermo Fisher BioTek 800TS microplate reader at 405 nm to determine the presence of anti-PF4 or anti-PF4/heparin antibodies in patient sera (positive OD 405nm ≥ 0.45).

All samples were tested for platelet activation using the PF4-SRA with increasing concentrations of exogenous PF4 (0, 25, 50, 100 μg/mL) (positive ≥ 20% 14 C-serotonin release). 20, 22 Blood samples were also tested for platelet activation in the standard SRA in the presence of therapeutic (0.1-0.3 U/mL) and high (100 U/mL) doses of unfractionated heparin (Pfizer, New York, NY, USA) without exogenous PF4, as previously described. 23 Platelet activation was also assessed in a PF4/heparin-SRA, which tests blood samples in the presence of 0.5 U/mL unfractionated heparin and 10 μg/mL recombinant PF4. 24 Each assay was performed with an anti-human CD32 Fc receptor-blocking monoclonal antibody (IV.3) to confirm FcγRIIa involvement in platelet activation.

GraphPad Prism version 9.1.2 for Mac OS software (GraphPad Software, Inc) was used to create all graphs and perform statistical analysis. Performance characteristics, including sensitivity, specificity, and confidence intervals, were calculated using Microsoft Excel for Mac version 15.37. Differences between data sets were tested for statistical significance using an unpaired t-test or a testing for significant difference in medians, using R package coin. P-values are reported as two-tailed where P < 0.05 was considered statistically significant.

We received samples for diagnostic testing from 156 patients across Canada with clinically suspected VITT from April to July 2021. All patients included in this study had received at least one dose of an adenoviral vector or mRNA COVD-19 vaccine (Table 1) .

Presenting clinical signs and symptoms were severe headaches ( 

Serological testing was carried out on all referred samples using the commercial anti- A breakdown of antibody reactivity between VITT-positive and VITT-negative patients in all three EIAs can be found in Figure 1 . Based on our evaluation compared to PF4-SRA results, the commercial anti-PF4-enhanced IgG/A/M-EIA had a sensitivity of 100% and a specificity of 95.6%, the in-house IgG-specific anti-PF4-EIA had a sensitivity of 100% and specificity of 96.5%, and the in-house IgG-specific anti-PF4/heparin-EIA had a sensitivity of 100% and specificity of 97.4% (Table 3 ). The positive predictive value (PPV) and negative predictive value (NPV) were also calculated for each assay ( Table 3 ). The commercial Immucor anti-PF4-enhanced IgG/A/M-EIA had a PPV of 93.5% and a NPV of 100%, the in-house IgGspecific anti-PF4-EIA had a PPV of 89.6% and NPV of 100%, and the in-house IgG specific anti-PF4/heparin-EIA had a PPV of 91.5% and NPV of 100%.

Patient samples were also tested in the standard SRA 25 activation, we determined that the standard SRA had a sensitivity of only 16.7% but had a specificity of 100% with a PPV of 100% and NPV of 74.3% (Table 3) .

To further assess the diagnostic capacity of different platelet activating functional assays for platelet activation, a subset of VITT-positive samples (n=26), and VITT-negative samples (n=7) were tested in the PF4/heparin-SRA, which measures activation in the presence of therapeutic heparin (0.5 U/mL) with the addition of exogenous PF4 (10 µg/mL). 23, 24 The sensitivity of the PF4/heparin-SRA assay was 46.2% and the specificity was 100% based on their performance compared to the PF4-SRA (Table 3 ). We also determined that this assay had a PPV of 100% and a NPV of 33.3% (Table 3 ).

As the reference laboratory for VITT testing in Canada, we report the efficacy of clinical diagnosis of VITT and the performance characteristics of various laboratory assays. We mostly included thrombocytopenia with thrombosis, but also thrombocytopenia alone and thrombosis alone. Similar findings were also noted in previous studies 7, 22, 28 and suggest that thrombocytopenia precedes thrombosis in VITT (termed "pre-VITT" 28 ), similar to the clinical profile seen in HIT patients. 12 CVST, a rare manifestation of a cerebrovascular disorder, was among one of the most common thrombotic manifestations in the VITT-positive patient cohort and occurred in a similar frequency to both DVT and PE. However, we found that the mere presence of CVST is insufficient to diagnose VITT as it occurred in only one third of VITTpositive patients and was also observed in some VITT-negative patients.

We found that 72.4% of the suspected patients in our study with a clinical presentation of thrombosis and/or thrombocytopenia did not have serologically confirmed VITT, but instead had hematological complications post-vaccination that were not caused by platelet activating anti-PF4 antibodies. The causes of thrombosis and/or thrombocytopenia in these patients is currently unknown and the possible role of adenoviral vector vaccines in causing these complications requires further investigations. While thrombocytopenia and/or thrombosis in VITT-positive patients occurred more significantly than in VITT-negative patients, it was still observed frequently in both cohorts, making it difficult to use these as clinical criteria to implicate or exclude VITT. In agreement with previous reports, 18 we also found that although CVST was present in both the VITT-negative and VITT-positive cohorts, CVST manifested more often in patients positive for VITT. In this study, VITT-negative patients were clinically suspected of VITT because they had experienced some symptoms of thrombosis and/or thrombocytopenia following vaccination. However, many of these patients did not meet the defining criteria as outlined in the Brighton Collaboration (BC) case definition, 22, 29 indicative of a high clinical suspicion of VITT. Previously, our group showed that the majority of suspected HIT cases are not HIT (86.5%; false-positive or true-negative) and laboratory confirmation is essential to minimize overdiagnosis and overtreatment. 19 Similarly, we found that the use of clinical criteria to identify VITT frequently led to disease overcall in our study. Unlike previous retrospective studies on VITT, 22, 30 we found that most referred patients suspected of VITT with hematological abnormalities were negative for anti-PF4 antibodies. Indeed, thrombocytopenia and/or thrombosis often presents in many clinical conditions likely coincidental with COVID-19 vaccination and serological testing for VITT was required to confirm a diagnosis in these patients. 30, 23 All referred patient samples were tested for VITT in three EIAs, which revealed positive Using a subset of our VITT samples, we also compared the standard SRA and the PF4/heparin-SRA to the PF4-SRA. We found these assays have a reduced diagnostic sensitivity for VITT compared to what is previously reported in HIT, possibly due to the competition between heparin and VITT antibodies for the heparin binding site on PF4. 14,23 However, some patients could have received therapy without our knowledge before specimen procurement, such as intravenous immunoglobulin (IVIg), which can inhibit platelet activation in functional assays 5 and may have an unknown influence on our study. Although neither the standard SRA nor the PF4/heparin-SRA appear to be suitable tests for VITT diagnosis based on our analysis, the PF4/heparin-SRA had a higher sensitivity than the standard SRA, which is consistent with a previous report on this assay involving VITT. 31 We suspect the difference in reactivity in the PF4/heparin-SRA could be attributed to the addition of exogenous PF4 and heparin in this assay, similar to the PF4-SRA, while the standard-SRA is performed only with exogenous heparin.

One limitation of this study was incomplete or absent data concerning referred patients, This work presents an assessment of the clinical and laboratory diagnosis of VITT using a large cohort of referred patients from across Canada, confirming previous retrospective investigations of VITT. 7, 8, 22 However, our study is the first to our knowledge that examined VITT diagnosis from a practical perspective using real-world data. This revealed that a clinical suspicion of VITT far exceeds its expected frequency based on BC criteria. Therefore, clinical evaluations alone are insufficient to distinguish between patients with VITT and those with thrombocytopenia and thrombosis syndrome (TTS) unrelated to anti-PF4 antibodies in an uncontrolled hospital setting. We also found EIAs have excellent sensitivity and specificity for 

The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author (Dr. Ishac Nazy, nazyi@mcmaster.ca) on reasonable request. 

Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination

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A spontaneous prothrombotic disorder resembling heparin-induced thrombocytopenia

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A comparative study of platelet factor 4-enhanced platelet activation assays for the diagnosis of heparin-induced thrombocytopenia

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Most cases of Thrombosis and Thrombocytopenia Syndrome (TTS) post ChAdOx-1 nCov-19 are Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT)

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PF4-Dependent Immunoassays in Patients with Vaccine-Induced Immune Thrombotic Thrombocytopenia: Results of an Interlaboratory Comparison

Figure 1. Results of three immunoassays for VITT antibody detection

PF4-SRA, ≥ 20% 14 C-serotonin release) and VITT-negative patients (n=113; PF4-SRA, ≤ 20% 14 C-serotonin release). An OD 405nm ≥ 0.40 is considered positive for anti-PF4 and anti-PF4

shown as a red dotted line. An OD 405nm ≥ 0.45 is considered positive for anti-PF4 and anti-PF4/heparin antibodies in the in-house IgG specific PF4/heparin-EIA and PF4-EIA

We thank Milena Hadzi-Tosev for her contribution to statistical analyses. Funding