Evaluating Diagnostic Algorithms for Heparin-Induced Thrombocytopenia using Two Combined Automated Rapid Immunoassays.

Heparin-induced thrombocytopenia (HIT) is an autoimmune disorder caused by antibodies against platelet factor 4 (PF4) and heparin complexes. Rapid immunoassays (IAs) for detection of these antibodies mark a milestone in HIT diagnosis, despite a higher false-positive rate compared with functional platelet-activation assays. However, combining different rapid IAs may help to improve their diagnostic specificity. Here, we compared the individual performance of the latex immunoturbidimetric assay (LIA; HemosIL HIT-Ab [PF4-H]; sensitivity 91.7%, specificity 68.4%) and chemiluminescence immunoassay (CLIA; HemosIL AcuStarHIT-Ab [PF4-H]; sensitivity 92.4%, specificity 85.8%) with their combined performance using two unique diagnostic algorithms in a single prospective cohort of suspected HIT patients. Using the simultaneous algorithm adapted from Warkentin et al, the combined LIA-CLIA had a sensitivity of 99.0% and specificity of 64.3%. The sequential algorithm adapted from Rittener-Ruff et al was applied in two theoretical scenarios to reflect real-world circumstances in diagnostic laboratories where access to clinical information is limited: (1) assuming all patients had an intermediate 4Ts score and (2) assuming all patients had a high 4Ts score. This algorithm correctly predicted HIT in 94.5% (high 4Ts) and 96.0% (intermediate 4Ts) and excluded HIT in 82.6% (high 4Ts) and 80.1% (intermediate 4Ts) of patients in either scenario, respectively. Although both combined algorithms improved diagnostic performance of individual IAs, the simultaneous algorithm showed fewer false predictions (7.9%) than the sequential algorithm (intermediate 4Ts: 37.6% and high 4Ts: 41.5%) and proved more practical as it does not rely on physician evaluations. Our findings highlight the importance of accounting for clinician and interlaboratory variability when evaluating diagnostic tests for HIT.

Vaccine-Induced Immune Thrombotic Thrombocytopenia: Clinicopathologic Features and New Perspectives on Anti-PF4 Antibody-Mediated Disorders.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet severe adverse complication first identified during the global vaccination effort against SARS-CoV-2 infection, predominantly observed following administration of the ChAdOx1-S (Oxford-AstraZeneca) and Ad26.CoV2.S (Johnson & Johnson/Janssen) adenoviral vector-based vaccines. Unlike other anti-platelet factor 4 (PF4) antibody-mediated disorders, such as heparin-induced thrombocytopenia (HIT), VITT arises with the development of platelet-activating anti-PF4 antibodies 4-42 days post-vaccination, typically featuring thrombocytopenia and thrombosis at unusual sites. AIM: To explore the unique properties, pathogenic mechanisms, and long-term persistence of VITT antibodies in patients, in comparison with other anti-PF4 antibody-mediated disorders. DISCUSSION: This review highlights the complexity of VITT as it differs in antibody behavior and clinical presentation from other anti-PF4-mediated disorders, including the high incidence rate of cerebral venous sinus thrombosis (CVST) and the persistence of anti-PF4 antibodies, necessitating a re-evaluation of long-term patient care strategies. The nature of VITT antibodies and the underlying mechanisms triggering their production remain largely unknown. CONCLUSION: The rise in awareness and subsequent prompt recognition of VITT is paramount in reducing mortality. As vaccination campaigns continue, understanding the role of adenoviral vector-based vaccines in VITT antibody production is crucial, not only for its immediate clinical implications, but also for developing safer vaccines in the future.

Antibodies against platelet factor 4 and the risk of cerebral venous sinus thrombosis in patients with vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against SARS-CoV-2. This syndrome is caused by antibodies against platelet factor 4 (PF4; CXCL4) that lead to platelet activation and is characterized by thrombocytopenia and thrombosis in unusual locations, including cerebral venous sinus thrombosis (CVST). VITT can be classified based on anti-PF4 antibodies properties in vitro: those that require PF4 to activate platelets (PF4-dependent) and those that can activate platelets without additional PF4 (PF4-independent) in the serotonin release assay. OBJECTIVES: We aim to characterize the relationship of VITT platelet-activating profiles with CVST. METHODS: We conducted a retrospective cohort study involving patients with confirmed VITT who were tested between March and June 2021. Data were collected with an anonymized form and cases were identified as VITT with high clinical suspicion according to platelet activation assays. Anti-PF4 antibody binding regions on PF4 were further characterized with alanine scanning mutagenesis. RESULTS: Of the patients with confirmed VITT (n = 39), 17 (43.6%) had PF4-dependent antibodies and 22 (56.4%) had PF4-independent antibodies. CVST occurred almost exclusively in PF4-independent patients (11 of 22 vs 1 of 17; P < .05). Additionally, PF4-independent antibodies bound to 2 distinct epitopes on PF4, the heparin-binding region and a site typical for heparin-induced thrombocytopenia antibodies, whereas PF4-dependent antibodies bound to only the heparin-binding region. CONCLUSION: These findings suggest that VITT antibodies that cause PF4-independent platelet activation represent a unique subset of patients more likely to be associated with CVST, possibly due to the 2 different types of anti-PF4 antibodies.

Unique features of vaccine-induced immune thrombotic thrombocytopenia; a new anti-platelet factor 4 antibody-mediated disorder.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a highly prothrombotic disorder caused by anti-PF4 antibodies that activate platelets and neutrophils, leading to thrombosis. Heparin-induced thrombocytopenia (HIT) is a related anti-PF4 mediated disorder, with similar pathophysiology and clinical manifestations but different triggers (i.e., heparin vs adenoviral vector vaccine). Clinically, both HIT and VITT typically present with thrombocytopenia and thrombosis, although the risk of thrombosis is significantly higher in VITT, and the thromboses occur in unusual anatomical sites (e.g., cerebral venous sinus thrombosis and hepatic vein thrombosis). The diagnostic accuracy of available laboratory testing differs between HIT and VITT; for VITT, ELISAs have better specificity compared to HIT and platelet activation assays require the addition of PF4. Treatment of VITT and HIT is anticoagulation non-heparin anticoagulants; however, heparin may be considered for VITT if no other option is available.

Vaccine-induced immune thrombotic thrombocytopenia: Updates in pathobiology and diagnosis.

Coronavirus disease 2019 (COVID-19) is a viral respiratory infection caused by the severe acute respiratory syndrome virus (SARS-CoV-2). Vaccines that protect against SARS-CoV-2 infection have been widely employed to reduce the incidence of symptomatic and severe disease. However, adenovirus-based SARS-CoV-2 vaccines can cause a rare, thrombotic disorder termed vaccine-induced immune thrombotic thrombocytopenia (VITT). VITT often develops in the first 5 to 30 days following vaccination and is characterized by thrombocytopenia and thrombosis in unusual locations (e.g., cerebral venous sinus thrombosis). The diagnosis is confirmed by testing for anti-PF4 antibodies, as these antibodies are capable of platelet activation without any cofactor. It can be clinically challenging to differentiate VITT from a similar disorder called heparin-induced thrombocytopenia (HIT), since heparin is commonly used in hospitalized patients. VITT and HIT have similar pathobiology and clinical manifestations but important differences in testing including the need for PF4-enhanced functional assays and the poor reliability of rapid immunoassays for the detection of anti-platelet factor 4 (PF4) antibodies. In this review we summarize the epidemiology of VITT; highlight similarities and differences between HIT and VITT; and provide an update on the clinical diagnosis of VITT.

The clinical and laboratory diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse syndrome occurring 5 to 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 patients in Canada with a suspected diagnosis of VITT between April and July 2021. The performance characteristics of various diagnostic laboratory tests were evaluated against the platelet factor 4 (PF4)-14C-serotonin release assay (SRA) including a commercial anti-PF4/heparin immunoglobulin G (IgG)/IgA/IgM 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-positive based on a positive PF4-SRA result and 113 (72.4%) were VITT-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 F4-SRA is unavailable.