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Introduction: Anti-β2-glycoprotein I (β2GPI)/human leukocyte antigen (HLA)-DR antibodies may be a risk factor for recurrent pregnancy loss (RPL). The therapeutic modality for women with RPL and anti-β2GPI/HLA-DR antibody positivity has not been evaluated. This prospective, multicenter, observational study aimed to assess whether low-dose aspirin (LDA) and/or heparin therapies improve pregnancy outcomes in women with RPL who tested positive for anti-β2GPI/HLA-DR antibodies., Methods: Between August 2019 and December 2021, 462 women with RPL underwent anti-β2GPI/HLA-DR antibody measurements and risk assessments for RPL. Each attending physician decided the treatment modality for women with RPL who tested positive for anti-β2GPI/HLA-DR antibodies, and their pregnancy outcomes were followed up until December 2023. Finally, 47 pregnancies in 47 women with RPL and anti-β2GPI/HLA-DR antibody positivity were included in the analysis and were divided into two groups regarding whether they were treated with LDA and/or unfractionated heparin (UFH) (LDA/UFH group, n = 39) or with neither of them (non-LDA/non-UFH group, n = 8). The rates of live birth and pregnancy complications (i.e., preeclampsia and preterm delivery before 34 gestational weeks due to placental insufficiency) were compared between the two groups., Results: The live birth rate in the LDA/UFH group was higher than that in the non-LDA/non-UFH group (87.2% vs 50.0%, p = 0.03). The pregnancy complication rate in the LDA/UFH group was significantly lower than that in the non-LDA/non-UFH group (5.9% vs 50.0%, p = 0.048). Among 21 women who tested positive for anti-β2GPI/HLA-DR antibodies and had no other risk factors for RPL, the live birth rate in the LDA/UFH group (n = 14) was much higher than that in the non-LDA/non-UFH group (n = 7) (92.9% vs 42.9%, p = 0.03)., Discussion: This study, for the first time, demonstrated that LDA and/or UFH therapies are effective in improving pregnancy outcomes in women with RPL and aβ2GPI/HLA-DR antibody positivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tanimura, Saito, Tsuda, Ono, Deguchi, Nagamatsu, Fujii, Nakatsuka, Kobashi, Arase and Yamada.)

Background: Heparin-induced thrombocytopenia (HIT) is a serious adverse effect of heparin treatment caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Accurate diagnosis of HIT is essential but remains challenging. The aim of our study was to explore the performance of our optimized diagnostic laboratory algorithm, based on Chemiluminescence (CliA) and ELISA immunoassays, on suspected HIT patients. The study compared the prevalence of HIT diagnosis in A.O. Mauriziano with the literature., Methods: 163 consecutive patients were investigated for suspected HIT with CliA HemosIL Acustar HIT-IgG, Werfen. HIT was ruled out in all patients with CliA <0.13 U/mL. All patients with CliA >0.13 U/mL were further investigated with Zymutest-HIA anti-PF4 IgG ELISA immunoassay. In these patients, HIT was ruled out on the combination of CliA between 0.13 and 1.0 U/mL followed by ELISA assay <0.300 OD. HIT was ruled in patients whose plasma tested positive or doubtful with CliA and positive with ELISA immunoassay and confirmed positive with a platelet aggregation test (PAT). Suspicion of HIT was revealed with clinical 4Ts score or recent suggestive anamnestic history., Results: Our diagnostic algorithm ruled out HIT diagnosis in 144/163 patients (88%) and predicted a positive PAT in 5/19 (26%) of CliA positive (4/5) or ELISA positive and CliA doubtful (1/5) patients., Conclusions: Our prevalence was 3.1%, comparable with the literature. The approach combining 2 quantitative immunoassays' (CliA and ELISA) results and 4Ts score probability was able to rule out the diagnosis within 1 h in 66% of patients with suspected HIT and within 24 h in 88% of patients. In the remaining 12% of cases, management decisions have to be based on individualized judgment while awaiting functional confirming results (48-72 h)., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Heparin-induced thrombocytopenia (HIT) is an antibody-mediated immune response against complexes of heparin and platelet factor 4 (PF4). The electrostatic interaction between heparin and PF4 is critical for the anti-PF4/heparin antibody response seen in HIT. The binding of metal cations to heparin induces conformational changes and charge neutralization of the heparin molecule, and cation-heparin binding can modulate the specificity and affinity for heparin-binding partners. However, the effects of metal cation binding to heparin in the context of anti-PF4/heparin antibody response have not been determined. Here, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to quantify 16 metal cations in patient plasma and tested for correlation with anti-PF4/heparin IgG levels and platelet count after clinical suspicion of HIT in a cohort of heparin-treated patients. The average age of the cohort (n = 32) was 60.53 (SD = 14.31) years old, had a mean anti-PF4/heparin antibody optical density [OD 405 ] of 0.93 (SD = 1.21) units, and was primarily female (n = 23). Patients with positive anti-PF4/heparin antibody test results (OD 405  ≥ 0.5 units) were younger, had increased weight and BMI, and were more likely to have a positive serotonin release assay (SRA) result compared to antibody-negative patients. We observed statistical differences between antibody-positive and -negative groups for sodium and aluminum and significant correlations of anti-PF4/heparin antibody levels with sodium and silver. While differences in sodium concentrations were associated with antibody-positive status and correlated with antibody levels, no replication was performed. Additional studies are warranted to confirm our observed association, including in vitro binding studies and larger observational cohorts., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Heparin-induced thrombocytopenia (HIT) is an immune-mediated condition characterized by a decrease in platelet count and an increased thrombotic risk. HIT event is caused by antiplatelet factor/heparin (PF4/H) antibodies that can activate the platelets. The diagnosis of HIT is based on a clinical evaluation and laboratory results. Aim of our study was to evaluate whether the combined use of two rapid assays for diagnosis of HIT provides a diagnostic advantage over the use of a single automate assay. We extracted from the laboratory informatic system all the determinations requested for the detection of antibodies against heparin/PF4 complexes from July 2020 to June 2024 (n. 229). In our laboratory, total antibodies against heparin/PF4 complex [HemosIL HIT-Ab-(PF4-H), Instrumentation Laboratory] and IgG Ab (HemosIL AcuStar HIT-IgG, Instrumentation Laboratory) are measured simultaneously with two different instruments. Two hundred six samples tested negative for both methods, 23 samples tested positive for at least one method and nine samples tested positive for both methods. The grade of concordance between the two assays shows a weighted Kappa of 0.536 (moderate agreement). No sample tested positive only for IgG-Ab. The sensitivity of HIT-Ab-(PF4-H) was 1, whereas the specificity was 0.95. For the HIT-IgG (PF4-H) method, sensibility and specificity were 0.77 and 1, respectively. Our results suggest that performing these two tests simultaneously does not provide additional useful information in patients with suspicion of HIT. The total Ab assay seems to be sufficient, as it shows higher sensitivity although at the expense of lower specificity., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a complication of adenoviral-based vaccine against SARS-CoV-2 due to prothrombotic immunoglobulin (Ig) G antibodies to platelet factor 4 (PF4) and may be difficult to distinguish from heparin-induced thrombocytopenia (HIT) in patients treated with heparin., Objectives: We assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context., Methods: The ability of F(ab')2 fragments of 1E12, 1C12, and 2E1, 3 monoclonal anti-PF4 antibodies, to inhibit the binding of human VITT or HIT antibodies to PF4 was evaluated using EIAs. Alanine-scanning mutagenesis was performed to define the amino acids involved in the interactions between the monoclonal antibodies and PF4., Results: A strong inhibition of VITT IgG binding to PF4 was measured with 1E12 (median inhibition, 93%; n = 8), whereas it had no effect on the binding of HIT antibodies (median, 6%; n = 8). In contrast, 1C12 and 2E1 inhibited VITT (median, 74% and 76%, respectively) and HIT antibodies (median, 68% and 53%, respectively) binding to PF4. When a competitive anti-PF4 EIA was performed with 1E12 for 19 additional VITT samples, it strongly inhibited IgG binding to PF4, except for 1 patient, who had actually developed HIT according to the clinical history. Epitope mapping showed that 1E12 interacts with 5 key amino acids on PF4, of which 4 are also required for the binding of human VITT antibodies, thus explaining the competitive inhibition., Conclusion: A simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible., Competing Interests: Declaration of competing interests C.V. reports honorarium from Viatris. J.R. reports research grant from Stago. H.G. reports transport fees from Stago. F.M. reports speaker fees from Fresenius, Technoclone, and Werfen, all outside the submitted work. Y.G. reports research grant and symposium fees from Stago. C.P. reports research grant from Stago. All other authors of this paper have no conflicts of interest., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Objectives: To demonstrate treatment efficacy on composite and non-length-dependent (NLD) punch biopsy specimens from intravenous immunoglobulin (IVIG) in pure small-fiber neuropathy (SFN) with trisulfated heparin disaccharide (TS-HDS), fibroblast growth factor-3 (FGFR-3), or Plexin D1 antibodies. SFN has an increasing prevalence, and over 30% of cases may be immune-mediated. TS-HDS, FGFR-3, and Plexin D1 autoantibodies have been shown to be present in 44%-55% of cryptogenic SFN cases, suggesting an immune mechanism. Reports have shown IVIG to be effective for this condition, but some controversy exists based on length-dependent (LD) post-IVIG treatment data in a recent trial., Methods: In a retrospective review, all pure SFN cases tested for the 3 antibodies from January 2021 to May 2022 were tabulated, and patients who underwent IVIG treatment were separated and analyzed for changes in epidermal nerve fiber density (ENFD) on skin biopsy, as well as SFN-specific questionnaire and pain scores., Results: Ninety-one patients with pure SFN had antibody testing. Sixty of these (66%) were seropositive, and 31 (34%) were seronegative. Seventeen seropositive patients (13 female patients, 4 male patients, 6 FGFR-3, 2 TS-HDS, 4 Plexin D1, 2 with all 3 antibodies, 1 with FGFR-3 and Plexin D1, 1 with FGFR-3 and TS-HDS, and 1 with TS-HDS and Plexin D1) underwent IVIG treatment. Of these, 2 patients stopped treatment due to side effects, and the remaining 15 completed at least 6 months of IVIG. Of these, 12 had a post-IVIG skin biopsy, and of these, 11 (92%) had a 55.1% improved mean composite ENFD (P = 0.01). NLD-ENFD specimens improved by 42.3% (P = 0.02), and LD-ENFD specimens improved by 99.7% (P = 0.01). Composite ENFD in Plexin D1-SFN patients improved by 139% (P = 0.04). In addition, 14 patients had questionnaires pre-IVIG/post-IVIG, and average pain decreased by 2.7 (P = 0.002)., Conclusions: IVIG shows disease-modifying effect in immune SFN with novel antibodies, especially Plexin D1-SFN, as well as significantly improved pain. NLD-ENFD should be examined as well as LD-ENFD to see this effect. Further randomized controlled trials looking at NLD-ENFD as well as LD-ENFD improvement, along with pain and SFN-specific questionnaires, are needed to confirm these findings., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Heparin-induced thrombocytopenia (HIT) is rare, affecting fewer than 1 in 1500 hospital admissions. Despite the increasing adoption of new therapies in HIT, such as direct oral anticoagulants and pooled immunoglobulins, there is limited high-quality evidence to guide clinicians. Numerous uncommon presentations of HIT and HIT-like entities have recently been recognized, and a harmonized approach to their classification is required to study them better. We present the results of an international survey of opinions from experts and practitioners in the field of platelet immunology regarding the role of direct oral anticoagulants in HIT, novel definitions of subclassifications of HIT-like platelet factor 4 immune conditions (spontaneous autoimmune HIT, persistent autoimmune HIT, and treatment-refractory HIT), and the role for intravenous immunoglobulins in the treatment paradigm of HIT and these HIT-like conditions. From 102 survey responses, there was broad acceptance of rivaroxaban (74.5%) and apixaban (73.5%) even before platelet recovery, as well as for intravenous immunoglobulin in the management of spontaneous (85.6%), persistent (83.7%), and treatment-refractory HIT (87.4%). With this mandate for harmonizing terminologies and treatment approaches in special situations without robust clinical data owing to their rarity, we plan to conduct a robust survey, establish international consensus, and draft management guidelines for HIT and platelet factor 4 immune diseases in the near future., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Aim: Heparin-induced thrombocytopenia (HIT) is a rare, life-threatening, immune-mediated adverse effect of heparin administration. This study compares frequently used laboratory assays in terms of their effectiveness in HIT diagnosis. Materials & methods: Fifty patients with suspected HIT were tested by gel immunoassay and solid phase PF4/heparin antibody ELISA. On positive results, platelet activation markers P-selectin and Annexin V were assayed using flow cytometry. Results: Thirty/50 patients were negative for both immunoassays. Flow cytometry was performed in the 20 immunoassay positive patients. Platelet activation was observed in 7/20 in the presence of low heparin concentration (0.2 IU/ml). Conclusion: The results are in accordance with the currently available literature and flow cytometry seems a promising alternative in HIT laboratory investigation.

Introduction: Heparin is derived from swine and has been suggested as a possible source of HEV. To study the potential risk of HEV infection associated with heparin treatment, two groups of individuals were compared. Sera from heparinized (N=93) and non-heparinized individuals (N=111) were tested for markers of acute HEV infection and anti-HEV IgG seroprevalence., Methods: An acute HEV case was defined by the presence of anti-HEV IgM and/or HEV RNA. From the 93 heparinized individuals, one was positive for IgM and IgG anti-HEV and two were positive for HEV RNA (for both ORF3 and ORF2), and there were a total of two (2.2%) cases of current or recent HEV infection. From the 111 non-heparinized individuals, three were positive for IgM anti-HEV, one was positive for both IgM and IgG anti-HEV, and none was positive for HEV RNA, and there were a total of three (2.7%) cases of current or recent HEV infection. The difference between HEV cases in the heparinized individuals and the non-heparinized individuals was not statistically significant (2.2% vs. 2.7%; p = 0.799)., Results: Concerning IgG anti-HEV, it was detected in 32 individuals from the heparinized group and in 18 from the non-heparinized control group. A statistically significant difference was observed in the presence of anti-HEV IgG in heparinized individuals and controls (p = 0.003)., Conclusion: This study has not found any association between heparin treatment and acute HEV infection, but has shown the use of therapeutic heparin as a risk factor for IgG anti-HEV seropositivity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Intermittent hemodialysis (HD) is almost invariably performed with heparin, and thus HD patients are at risk of developing the immune-mediated adverse effect heparin-induced thrombocytopenia (HIT), caused by anti-platelet factor 4/heparin IgG, which strongly activates platelets. HIT patients develop hypercoagulability with greatly increased risk of thrombosis, both venous and arterial. Certain HIT-associated complications are more likely to develop among HD patients, including hemofilter thrombosis despite heparin, intravascular catheter and/or arteriovenous fistula-associated thrombosis, post-heparin bolus anaphylactoid/anaphylactic reactions, and thrombotic stroke and acute limb artery thrombosis (reflecting the high frequency of underlying arteriopathy in many patients with renal failure). Management of HIT in HD usually requires use of an alternative (non-heparin) anticoagulant; for example, danaparoid sodium (outside the USA) or argatroban (USA and elsewhere). Whether heparin-grafted hemodialyzers (without systemic heparin) can be used safely in acute HIT is unknown. The HIT immune response is remarkably transient and usually not retriggered by subsequent heparin administration. Accordingly, since renal failure patients often require long-term HD, there may be the opportunity-following seroreversion (loss of platelet-activating HIT antibodies)-to restart heparin for HD, a practice that appears to have a low likelihood of retriggering HIT., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders., (© 2021 by The American Society of Hematology.)

Background: The histo-blood group ABO system has been associated with adverse outcomes in COVID-19, thromboembolic diseases and Plasmodium falciparum malaria. An integral part of the severe malaria pathogenesis is rosetting, the adherence of parasite infected red blood cells (RBCs) to uninfected RBCs. Rosetting is influenced by the host's ABO blood group (Bg) and rosettes formed in BgA have previously been shown to be more resilient to disruption by heparin and shield the parasite derived surface antigens from antibodies. However, data on rosetting in weak BgA subgroups is scarce and based on investigations of relatively few donors., Methods: An improved high-throughput flow cytometric assay was employed to investigate rosetting characteristics in an extensive panel of RBC donor samples of all four major ABO Bgs, as well as low BgA expressing samples., Results: All non-O Bgs shield the parasite surface antigens from strain-specific antibodies towards P. falciparum erythrocyte membrane protein 1 (PfEMP1). A positive correlation between A-antigen levels on RBCs and rosette tightness was observed, protecting the rosettes from heparin- and antibody-mediated disruption., Conclusions: These results provide new insights into how the ABO Bg system affects the disease outcome and cautions against interpreting the results from the heterogeneous BgA phenotype as a single group in epidemiological and experimental studies., (© 2021. The Author(s).)

Background: Heparin-induced thrombocytopenia (HIT) is an immune-mediated adverse reaction to heparin. Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are routinely anticoagulated with heparin before the initiation of bypass. Heparin is contraindicated, however, in patients with acute HIT, and alternatives to routine practice are often used. While guidelines have recently been published addressing this topic 10, there remains variance between institutions in how these cases are treated. Our goal was to better delineate practice trends in the diagnosis and management of HIT patients requiring CPB., Methods: We surveyed members of the Society of Cardiovascular Anesthesiologists (SCA) and the American Society for Extracorporeal Technology (AmSECT) using an online survey tool., Results: We received 304 completed surveys (5.8% response rate), 75% completed by an anesthesiologist, and 24% by a perfusionist. The majority of respondents used clinical history and/or antibody testing (71% and 63%, respectively) to diagnose HIT. Seventy-five percent of respondents reported using an institutional protocol for HIT-CPB cases. Most respondents (89%) reported having at least 1 case in the last 3 years, with a total case experience of at least 785 cases (785 = the minimum number of cases in each case volume category × the number of respondents choosing that category). The strategy recommended in published guidelines, bivalirudin, was the most commonly reported alternative anticoagulation strategy (75%) used by respondents in HIT cases, with most (83%) using the activated clotting time (ACT) to monitor anticoagulation., Conclusions: Most responding SCA and AmSECT members reported that their institution used a protocol or guideline for HIT/CPB cases, and most guidelines directed the use of bivalirudin as an alternative anticoagulant. Various other methods such as plasmapheresis are also being used with success in this patient population. Further research, including comparison studies of alternative anticoagulant strategies, is required to elucidate the best approach to these difficult cases., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 International Anesthesia Research Society.)

COVID-19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID-19-infected patients. This has thus been identified in some publications as representing a high incidence of heparin-induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin-induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin-dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID-19-infected patients, and they may occur at higher incidence than in historical non-COVID-19-infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin-dependent, such that they do not necessarily reflect "true" HIT. Most recently, a "HIT-like" syndrome has reported in patients who have been vaccinated against COVID-19. Accordingly, much more is yet to be learnt about the insidious disease that COVID-19 represents, including autoimmune outcomes in affected patients., (© 2021 John Wiley & Sons Ltd.)

Vaccine-induced immune thrombotic thrombocytopaenia (VITT) is a rare adverse effect of COVID-19 adenoviral vector vaccines 1-3 . VITT resembles heparin-induced thrombocytopaenia (HIT) in that it is associated with platelet-activating antibodies against platelet factor 4 (PF4) 4 ; however, patients with VITT develop thrombocytopaenia and thrombosis without exposure to heparin. Here we sought to determine the binding site on PF4 of antibodies from patients with VITT. Using alanine-scanning mutagenesis 5 , we found that the binding of anti-PF4 antibodies from patients with VITT (n = 5) was restricted to eight surface amino acids on PF4, all of which were located within the heparin-binding site, and that the binding was inhibited by heparin. By contrast, antibodies from patients with HIT (n = 10) bound to amino acids that corresponded to two different sites on PF4. Biolayer interferometry experiments also revealed that VITT anti-PF4 antibodies had a stronger binding response to PF4 and PF4-heparin complexes than did HIT anti-PF4 antibodies, albeit with similar dissociation rates. Our data indicate that VITT antibodies can mimic the effect of heparin by binding to a similar site on PF4; this allows PF4 tetramers to cluster and form immune complexes, which in turn causes Fcγ receptor IIa (FcγRIIa; also known as CD32a)-dependent platelet activation. These results provide an explanation for VITT-antibody-induced platelet activation that could contribute to thrombosis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The development of vaccines to fight COVID-19 has been a remarkable medical achievement. However, this global immunization effort has been complicated by a rare vaccine-related outcome characterized by thrombocytopenia and thrombosis in association with platelet-activating anti-platelet factor 4 antibodies. In this Spotlight, we will discuss the recently described complication of vaccine-induced immune thrombotic thrombocytopenia (VITT) occurring in response to certain COVID-19 vaccines. Although information about this clinical condition is rapidly evolving, we will summarize our current understanding of VITT., (© 2021 by The American Society of Hematology.)

Importance: Cases of cerebral venous sinus thrombosis in combination with thrombocytopenia have recently been reported within 4 to 28 days of vaccination with the ChAdOx1 nCov-19 (AstraZeneca/Oxford) and Ad.26.COV2.S (Janssen/Johnson & Johnson) COVID-19 vaccines. An immune-mediated response associated with platelet factor 4/heparin antibodies has been proposed as the underlying pathomechanism., Objective: To determine the frequencies of admission thrombocytopenia, heparin-induced thrombocytopenia, and presence of platelet factor 4/heparin antibodies in patients diagnosed with cerebral venous sinus thrombosis prior to the COVID-19 pandemic., Design, Setting, and Participants: This was a descriptive analysis of a retrospective sample of consecutive patients diagnosed with cerebral venous sinus thrombosis between January 1987 and March 2018 from 7 hospitals participating in the International Cerebral Venous Sinus Thrombosis Consortium from Finland, the Netherlands, Switzerland, Sweden, Mexico, Iran, and Costa Rica. Of 952 patients, 865 with available baseline platelet count were included. In a subset of 93 patients, frozen plasma samples collected during a previous study between September 2009 and February 2016 were analyzed for the presence of platelet factor 4/heparin antibodies., Exposures: Diagnosis of cerebral venous sinus thrombosis., Main Outcomes and Measures: Frequencies of admission thrombocytopenia (platelet count <150 ×103/μL), heparin-induced thrombocytopenia (as diagnosed by the treating physician), and platelet factor 4/heparin IgG antibodies (optical density >0.4, in a subset of patients with previously collected plasma samples)., Results: Of 865 patients (median age, 40 years [interquartile range, 29-53 years], 70% women), 73 (8.4%; 95% CI, 6.8%-10.5%) had thrombocytopenia, which was mild (100-149 ×103/μL) in 52 (6.0%), moderate (50-99 ×103/μL) in 17 (2.0%), and severe (<50 ×103/μL) in 4 (0.5%). Heparin-induced thrombocytopenia with platelet factor 4/heparin antibodies was diagnosed in a single patient (0.1%; 95% CI, <0.1%-0.7%). Of the convenience sample of 93 patients with cerebral venous sinus thrombosis included in the laboratory analysis, 8 (9%) had thrombocytopenia, and none (95% CI, 0%-4%) had platelet factor 4/heparin antibodies., Conclusions and Relevance: In patients with cerebral venous sinus thrombosis prior to the COVID-19 pandemic, baseline thrombocytopenia was uncommon, and heparin-induced thrombocytopenia and platelet factor 4/heparin antibodies were rare. These findings may inform investigations of the possible association between the ChAdOx1 nCoV-19 and Ad26.COV2.S COVID-19 vaccines and cerebral venous sinus thrombosis with thrombocytopenia.

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119., (© 2021 by The American Society of Hematology.)

Introduction: The accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential to ensure adequate treatment and prevent complications. First step diagnosis test are immunoassays including enzyme-linked immunosorbent assays (ELISAs) and rapid immunoassays., Methods: Using a Bayesian approach, we prospectively evaluated the performance of the IgG PF4/polyvinylsulfonate ELISA and a chemiluminescent immunoassay (CLIA), which are specific for IgG and use the same antigenic target to detect HIT antibodies., Results: One hundred and eighty-four 184 consecutive patients with an intermediate (n = 159) or high (n = 25) clinical pretest probability of HIT based on the 4Ts score or platelet pattern were included. Both immunoassays (IAs) were performed on all 184 samples, and definite HIT was confirmed with a positive serotonin release assay in 29 patients (12.7%). The sensitivity (Ss) and negative predictive value (NPV) of ELISA were excellent (100%) allowing HIT to be excluded with good confidence when the test was negative. In addition, the Ss and NPV of the CLIA equalled 93.1% and 98.6%, respectively, as it was negative in two definite HIT. When the CLIA was negative, the post-test probability of HIT was 0.7% in case of intermediate risk. Although there was excellent agreement between CLIA and ELISA results, the quantitative values provided by the two IAs were not correlated., Conclusion: AcuStar HIT ® detects more than 90% of HIT, as do all rapid IAs, and appears to be a good tool for excluding HIT when the pretest probability is intermediate. A chemiluminescent signal higher than 10 IU/mL is highly predictive of definite HIT with a PPV of 100%., (© 2020 John Wiley & Sons Ltd.)

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT., (© 2021 by The American Society of Hematology.)

Background: Serological assays for the diagnosis of heparin-induced thrombocytopenia (HIT) detect both platelet-activating and platelet non-activating anti-heparin/platelet factor 4 (PF4) antibodies and have therefore a limited positive predictive value. Functional assays confirm the presence of platelet-activating antibodies but require platelets from healthy donors, whose response to patient serum can differ. Our aim was to investigate the correlation between the level of anti-heparin/PF4 antibodies, 4T score, and the extent of panel donor platelet activation in the functional assay., Materials and Methods: In total, 38 sera from enzyme immunoassays (ELISA) positive patients were tested against panel platelets obtained from 10 healthy, randomly selected donors, using our routine flow cytometry functional test for CD62P expression. Levels of anti-heparin/PF4 antibodies from medical and surgical patients and 4T pretest probability scores (where available) were correlated with the number of activated panel platelets., Results: Sera with low ELISA optical density (OD) values (0.4-1) activated on average 5.6, sera with intermediate ELISA OD values (>1-2.5) activated on average 7.3, and sera with high ELISA OD values (>2.5) activated on average 8.6 out of 10 panel platelets. One serum with low 4T score did not activate donor platelets, 12 sera with intermediate 4T score activated on average 6.3 donors, 8 sera with high 4T score activated on average 8.5 panel platelets., Discussion: Sera with higher ELISA OD values activated platelets from a higher number of platelet donors, independently of patient type (medical or surgical). The average number of activated panel platelets increased with rising 4T score. Results indicate that both donor platelet reactivity and quantity of anti-heparin/PF4 antibodies affect the result of the functional assay, meaning special attention is needed in platelet donor selection when testing sera with low levels of antibodies.

Heparin-induced thrombocytopenia is an immune-mediated disorder caused by antibodies that recognize complexes of platelet factor 4 and heparin. Thrombosis is a central and unpredictable feature of this syndrome. Despite optimal management, disease morbidity and mortality from thrombosis remain high. The hypercoagulable state in heparin-induced thrombocytopenia is biologically distinct from other thrombophilic disorders in that clinical complications are directly attributable to circulating ultra-large immune complexes. In some individuals, ultra-large immune complexes elicit unchecked cellular procoagulant responses that culminate in thrombosis. To date, the clinical and biologic risk factors associated with thrombotic risk in heparin-induced thrombocytopenia remain elusive. This review will summarize our current understanding of thrombosis in heparin-induced thrombocytopenia with attention to its clinical features, cellular mechanisms, and its management.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT., (© 2020 by The American Society of Hematology.)

Prompt diagnostic evaluation of suspected heparin-induced thrombocytopenia (HIT) is critical for guiding initial patient management. We assessed the performance of 3 immunoassays detecting anti-platelet factor 4 (PF4)/heparin antibodies, derived a diagnostic algorithm with a short analytical turnaround time (TAT), and prospectively validated the algorithm. Plasma samples were analyzed by Zymutest-HIA-IgG, HemosIL-AcuStar-HIT-IgG, and ID-H/PF4-PaGIA in retrospective (n = 221) and prospective (n = 305) derivation cohorts. We calculated likelihood ratios of result intervals and cutoff values with 100% negative (NPV) and positive (PPV) predictive values for a positive gold standard functional assay (heparin-induced platelet activation [HIPA]). A diagnostic algorithm was established based on the Bayesian combination of pretest probability and likelihood ratios of first- and second-line immunoassays. Cutoffs with 100% PPV for positive HIPA were >3.0 U/mL (HemosIL-AcuStar-HIT-IgG) and titer ≥16 (ID-H/PF4-PaGIA); cutoffs with 100% NPV were <0.13 U/mL and ≤1, respectively. During the prospective validation of the derived algorithm (n = 687), HemosIL-AcuStar-HIT-IgG was used as unique testing in 566 (82.4%) of 687 cases (analytical TAT, 30 minutes). In 121 (17.6%) of 687 unresolved cases, ID-H/PF4-PaGIA was used as second-line testing (additional TAT, 30 minutes). The algorithm accurately predicted HIT in 51 (7.4%) of 687 patients and excluded it in 604 (87.9%) of 687 patients, leaving only 20 (2.9%) cases unresolved. We also identified 12 (1.7%) of 687 positive predictions not confirmed by HIPA: 10 patients with probable HIT despite negative HIPA and 2 possible false-positive algorithm predictions. The combination of pretest probability with first- and second-line immunoassays for anti-PF4/heparin antibodies is accurate for ruling in or out HIT in ≥95% of cases within 60 minutes. This diagnostic approach improves initial management of patients with suspected HIT., (© 2020 by The American Society of Hematology.)

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis., (© 2020 by The American Society of Hematology.)

HIT, a prothrombotic disorder caused by heparin-dependent antibodies, is often treated with fondaparinux, usually with good outcomes. A 70-year-old female developed severe HIT (platelet count, 25 × 10 9 /L) post-glioblastoma resection during heparin thromboprophylaxis, complicated by disseminated intravascular coagulation (DIC) and symptomatic lower-limb deep-vein thrombosis (DVT). Despite therapeutic-dose fondaparinux, thrombocytopenia/hypofibrinogenemia persisted, with new symptomatic catheter-associated upper-extremity DVT. This clinical picture could be explained by autoimmune HIT (aHIT) refractory to fondaparinux or by fondaparinux cross-reactivity, so high-dose intravenous immunoglobulin (IVIG) was given (to treat possible aHIT) and fondaparinux switched to rivaroxaban, with subsequent clinical recovery. In vitro studies revealed strong fondaparinux cross-reactivity, without aHIT antibodies. Moreover, the patient's serotonin-release assay became negative post-IVIG, suggesting in-vivo inhibition of HIT antibody-induced platelet activation. Our case illustrates fondaparinux cross-reactivity in HIT manifesting as persisting thrombocytopenia, new thrombosis, and DIC, with successful rivaroxaban treatment, adding to emerging data that oral factor Xa inhibitors are efficacious for treating HIT.

Background: Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential to ensure timely treatment and prevent complications. Current diagnostic assays include enzyme-linked immunosorbent assays (ELISAs) and rapid immunoassays (RIs). RIs offer fast turnaround times but were not significantly represented in previous external proficiency testing challenges., Objectives: To use external proficiency testing to assess qualitative concordance for heparin/PF4 antibody detection., Methods: From 2013 to 2017, the External Quality Control for Assays and Tests (ECAT) Foundation distributed 10 samples internationally., Results: In total, 437 laboratories submitted 3149 results. ELISAs accounted for 1484 (47%) responses with RIs accounting for 1665 (53%) responses. RI use increased over the 5-year period. ELISAs classified 96% of both consensus positive and consensus negative samples concordantly. The coefficient of variation (CV) for positive sample optical densities (ODs) ranged from 35% to 50% when combining ELISA assay methods together. Quantitative RIs classified 97% of consensus-positive and 98% of consensus-negative samples concordantly. Qualitative RIs had a higher proportion of discordant responses and classified 88% of consensus-positive samples and 73% of consensus-negative samples concordantly. Of RIs only latex immunoassays and IgG specific chemiluminescent assays identified > 95% of samples concordantly with consensus., Conclusion: Quantitative RIs and ELISAs classify > 95% of samples concordantly. The ODs from different ELISA methods vary considerably and are not interchangeable. Qualitative RI use is increasing despite a greater proportion of discordant classifications. This includes a higher than expected number of negative classifications for consensus-positive samples among many RIs, challenging their use as "rule out" tests., (© 2019 International Society on Thrombosis and Haemostasis.)

Heparin-induced thrombocytopenia is a relatively common drug-induced immune disorder that can have life-threatening consequences for affected patients. Immune complexes consisting of heparin, platelet factor 4 (PF4), and PF4/heparin-reactive Abs are central to the pathogenesis of heparin-induced thrombocytopenia. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that play a key role in regulating immune responses, but their role in controlling PF4/heparin-specific Ab production is unknown. In the studies described in this article, we found that Foxp3-deficient mice lacking functional Treg cells spontaneously produced PF4/heparin-specific Abs. Following transplantation with bone marrow cells from Foxp3-deficient but not wild-type mice, Rag1-deficient recipients also produced PF4/heparin-specific Abs spontaneously. Adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific Abs in Foxp3-deficient mice and inhibited PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune responses mainly through releasing anti-inflammatory cytokines, such as IL-10. IL-10-deficient mice spontaneously produced PF4/heparin-specific Abs. Moreover, bone marrow chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production upon PF4/heparin complex challenge. Short-term IL-10 administration suppresses PF4/heparin-specific IgG production in wild-type mice. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific Ab production., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Heparin-induced thrombocytopenia (HIT) is an immune-mediated condition characterized by thrombocytopenia with possible arterial and/or venous thrombosis. The overall incidence of HIT is low but ranges from 0.1% to 5%. 1,2 The incidence can be as high as 3% in patients undergoing cardiac surgery. The use of unfractionated heparin (UFH) is ubiquitous in patients who undergo cardiac procedures and carries a 10-fold higher incidence of HIT over low molecular weight heparin. Patients undergoing cardiac surgery thus form a unique group that warrants specific attention to this clinicopathologic entity considering the relatively high incidence and associated morbidity and mortality with a delay in diagnosis. In this article, we will discuss 5 clinical aspects pertinent to the diagnosis and management of HIT in cardiac surgery patients and review the current literature., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: Type II heparin-induced thrombocytopenia (HIT) is mediated by formation of antibodies to platelet factor 4 (PF4)-heparin complexes. We evaluated anti-PF4-heparin-negative samples for the presence of additional anti-platelet and anti-red blood cell (RBC) antibodies using whole-cell platelet/ RBC ELISAs we developed., Methods: Seventy-three samples tested for anti-PF4-heparin by ELISA were included: 62 tested negative, 9 tested positive, and 2 had equivocal results. Plasma specimens from healthy donors were used as controls., Results: 100% (9/9) anti-PF4-positive samples had anti-platelet antibodies detected by whole-cell platelet ELISA. 42.2% (27/64) anti-PF4-heparin-negative samples were negative for anti-platelet and anti-RBC antibodies. 32.8% (21/64) negative samples showed reactivity to both platelets and RBC; 12.5% (8/64) negative samples were each reactive with either platelet or RBC ELISA, respectively. Additionally, two samples that tested equivocal by anti-PF4-heparin ELISA had antibodies to both platelets and RBC by whole-cell ELISA., Conclusions: Our study suggests that patients with thrombocytopenia testing negative for anti-PF4-heparin may still harbor antibodies to platelets. However, additional research is needed to determine the significance of these antibodies. Nevertheless, these findings may encourage clinicians to further investigate patients with possible immune-mediated etiologies of thrombocytopenia and anemia., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Essentials At low pH and low salt concentrations: Maximal conformational change of PF4 upon complexation with heparin occurs. Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating- and nonactivating PF4/H Abs in antigen tests., Background: Enzyme immunosorbent assays (EIA) are widely used to detect human antiplatelet factor 4/heparin antibodies (aPF4/H Abs) to rule out heparin-induced thrombocytopenia. EIAs cannot differentiate between clinically relevant, platelet-activating, and nonrelevant, nonplatelet-activating Abs and only ~50% of patients' sera testing positive by EIA contain antibodies that activate platelets. Recently, we have shown platelet-activating aPF4/H Abs bind more strongly to PF4/H complexes than nonplatelet-activating antibodies. Antigen-antibody interactions are known to depend on electrostatic interactions governed by pH, heat, and ionic strength. We tested whether changes in pH and ionic strength can improve the specificity of EIAs detecting aPF4/H Abs., Methods: We investigated first the conformational change of PF4 when binding to heparin under various pH and salt conditions using circular dichroism spectroscopy, and then the binding of aPF4/H Abs to PF4/H complexes by EIA., Results: Maximal conformational change of PF4 on complexation with heparin was identified at low pH and low salt concentrations. EIA tested with a large number of sera at 50 mmol/L NaCl, pH 6.0 shows a potential to increase the specificity for the detection of platelet-activating aPF4/H Abs., Conclusion: Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating and nonactivating PF4/H Abs in antigen tests., (© 2019 International Society on Thrombosis and Haemostasis.)

Introduction: Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder that occurs following the administration of heparin and is caused by antibodies to platelet factor 4 and heparin. Diagnosis of HIT is essential to guide treatment strategies using non-heparin anticoagulants and to avoid unwanted and potential fatal thromboembolic complications. This consensus statement, formulated by members of the Thrombosis and Haemostasis Society of Australia and New Zealand, provides an update on HIT pathogenesis and guidance on the diagnosis and management of patients with suspected or confirmed HIT., Main Recommendations: A 4Ts score is recommended for all patients with suspected HIT prior to laboratory testing. Further laboratory testing with a screening immunoassay or confirmatory functional assay is not recommended in individuals with a low 4Ts score. However, if there are missing or unreliable clinical data, then laboratory testing should be performed. A positive functional assay result confirms the diagnosis of HIT and should be performed to confirm a positive immunoassay result. Heparin exposure must be ceased in patients with suspected or confirmed HIT and initial treatment with a non-heparin alternative instituted. Non-heparin anticoagulants (danaparoid, argatroban, fondaparinux and bivalirudin) used to treat HIT should be given in therapeutic rather than prophylactic doses. Direct oral anticoagulants may be used in place of warfarin after patients with HIT have responded to alternative parenteral anticoagulants with platelet count recovery., Changes in Management as a Result of This Statement: These are the first Australasian recommendations for diagnosis and management of HIT, with a focus on locally available diagnostic assays and therapeutic options. The importance of examining both clinical and laboratory data in considering a diagnosis of HIT cannot be overstated., (© 2019 AMPCo Pty Ltd.)

Although some suggest anti-Xa assays should be the preferred method for monitoring intravenous unfractionated heparin therapy, which method is best is unknown owing to the lack of large randomized controlled trials correlating different assays with clinical outcomes. This article provides an overview of heparin monitoring and the pros, cons, and clinical applications of anti-Xa assays., (Copyright © 2019 Cleveland Clinic.)

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder characterized by heparin-dependent antibodies that activate platelets (PLTs) via PLT FcγIIa receptors. "Autoimmune" HIT (aHIT) indicates a HIT subset where thrombocytopenia progresses or persists despite stopping heparin; aHIT sera activate PLTs strongly even in the absence of heparin (heparin-independent PLT-activating properties). Affected patients are at risk of severe complications, including dual macro- and microvascular thrombosis leading to venous limb gangrene. High-dose intravenous immunoglobulin (IVIG) offers an approach to interrupt heparin-independent PLT-activating effects of aHIT antibodies., Case Report: A 78-year-old male who underwent cardiopulmonary bypass for aortic dissection developed aHIT, disseminated intravascular coagulation, and deep vein thrombosis; progression to venous limb gangrene occurred during partial thromboplastin time (PTT)-adjusted bivalirudin infusion (underdosing from "PTT confounding"). Thrombocytopenia recovered with high-dose IVIG, although the PLT count increase began only after the third dose of a 5-day IVIG regimen (0.4 g/kg/day × 5 days). We reviewed case reports and case series of IVIG for treating HIT, focusing on various IVIG dosing regimens used., Results: Patient serum-induced PLT activation was inhibited in vitro by IVIG in a dose-dependent fashion; inhibition of PLT activation by IVIG was much more marked in the absence of heparin versus the presence of heparin (0.2 U/mL). Our literature review indicated 1 g/kg × 2 IVIG dosing as most common for treating HIT, usually associated with rapid PLT count recovery., Conclusion: Our clinical and laboratory observations support dose-dependent efficacy of IVIG for decreasing PLT activation and thus correcting thrombocytopenia in aHIT. Our case experience and literature review suggests dosing of 1 g/kg IVIG × 2 for patients with severe aHIT., (© 2019 AABB.)

Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible., (© 2019 International Society on Thrombosis and Haemostasis.)

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies. Platelet activation assays that use "washed" platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)-based assays. Moreover, heparin-exposed patients vary considerably with respect to the risk of PF4/heparin immunization and, among antibody-positive patients, the risk of subsequent "breakthrough" of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests, and physicochemical methods to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody-platelet interaction, thus explaining differences in functional assays. To investigate a modulating risk for PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from 2 serosurveillance trials. Fibronectin levels were measured in 4 patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (vs PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes, and anti-PF4/heparin antibody-induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ < ELISA+/SRA+/HIT- ∼ ELISA+/SRA-/HIT- < ELISA-/SRA-/HIT-. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody-induced platelet activation. Reduced fibronectin levels in washed platelet assays help to explain the greater sensitivity of washed platelet (vs PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor for PF4/heparin immunization and clinical breakthrough of HIT., (© 2019 by The American Society of Hematology.)

Essentials Many patients produce antibodies but few lead to heparin-induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non-pathogenic antibodies. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti-PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. Objectives To identify PF4 amino acids essential for binding pathogenic HIT antibodies. Methods Alanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet-activating murine monoclonal anti-PF4/heparin antibody (KKO) and HIT patient sera (n = 9). Results and Conclusions We identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n = 45) and non-pathogenic antibodies (EIA-positive but not platelet activating, n = 28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4., (© 2018 International Society on Thrombosis and Haemostasis.)

A high frequency of PF4-ELISA testing in patients suspected to have heparin-induced thrombocytopenia (HIT) despite low 4T scores has been observed in multiple medical centers. Education of clinicians has been suggested to reduce inappropriate testing. We determined trends of PF4-ELISA testing in our institution after the introduction of a HIT education program for clinicians. A HIT Program was developed that included ongoing education, individual feedback, and continuous clinical audit of PF4-ELISA utilization. To assess the impact of education on PF4-ELISA testing trends, we conducted a prospective cohort review of all adult patients who had a PF4-ELISA ordered over a 3 month period (the last quarter of the academic year). 72 PF4-ELISA tests were ordered during the study period. Prospectively calculated 4T scores by investigators revealed 60 low-risk (83.3%), 9 intermediate-risk (12.5%), and 3 high-risk (4.16%). We observed divergent 4T scores with the ordering clinician calculating a higher 4T score compared to the Hematology Quality Improvement (QI) team. The majority of PF4-ELISA testing was ordered by the intensive care units (ICUs) (n = 32, 44.44%). Our study revealed that the frequency of calculation of 4T scores remains poor with the majority inappropriately performed in the ICU setting, with ordering clinicians calculating higher 4T scores than the Hematology QI team. This suggests that clinician education alone is insufficient. Introducing mandatory 4T score calculation prior to PF4-ELISA testing may not be helpful as ordering clinicians can bypass the restriction through inaccurate 4T score calculation.

Heparin-induced thrombocytopenia (HIT) type II is caused by antibody production that bind complexes between heparin and platelet factor 4 leading to platelet consumption and thrombosis. In a small subset of cases referred to as autoimmune HIT, the antibodies activate platelets even in the absence of heparin. Refractory HIT is a type of autoimmune HIT in which thrombocytopenia persists for weeks after heparin discontinuation and carries increased risk for thrombosis and more severe thrombocytopenia. We present a case of refractory HIT with cerebral venous sinus thrombosis (CVST) that was successfully treated with a change in anticoagulant alongside steroids and a second trial of intravenous immunoglobulin (IVIg).

Background: Heparin-induced thrombocytopenia (HIT) in infants is a rare disorder, and the diagnosis and management of HIT still remains challenging. Argatroban is a synthetic direct thrombin inhibitor (DTI) that is widely used for treating HIT. However, little is known about the efficacy of the activated clotting time (ACT) test in monitoring DTI treatment as an alternative to the routinely used activated partial thromboplastin time (aPTT)., Methods: Between July 2013 and January 2015, four infants were diagnosed with HIT after surgical correction of congenital anomalies. In all cases, heparin was used during cardiopulmonary bypass (CPB). Diagnosis of HIT was based on the "4 Ts" pretest clinical scoring system, and platelet factor 4 (PF4) antibody was detected using enzyme-linked immunosorbent assay. Argatroban was used in treating HIT. When argatroban was infused, anticoagulation tests (aPTT, prothrombin time [PT], thrombin time [TT], and fibrinogen) were performed every 4 to 12 hours. ACT was used in addition to monitor the anticoagulation effect of argatroban. The target ACT was 1.5 to 3.0 times the baseline. ACT was measured every 2 to 4 hours and remeasured 1 hour after each dosage adjustment., Results: Thrombocytopenia (defined as a 50% decrease in platelet count) occurred during the 3rd to 6th day postoperatively. After the diagnosis of HIT, argatroban was started immediately, and platelet counts stabilized and gradually increased. Anticoagulation effect of argatroban was successful monitored by ACT and aPTT. Poor correlation between the ACT test and aPTT test ( R  = 0.270, p  = 0.092) was noted in one patient. ACT values increased rapidly after 3 to 7 days on argatroban treatment. In most cases, low dosage of argatroban was given ranging from 0.04 to 5.00 μg/kg/min., Conclusion: Argatroban may be an effective medicine in treating HIT in infants, in a reduced dosage. The great fluctuation in argatroban dosage during the course of HIT treatment necessitates close monitoring. ACT test may be reliable and convenient for monitoring HIT treatment and may contribute to positive clinical outcomes in infants. The efficacy of argatroban and the use of ACT monitoring in the management of HIT infants needs further study., Competing Interests: None., (Georg Thieme Verlag KG Stuttgart · New York.)

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder "phenotype" (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels ( r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

Essentials FcγRIIa mediates life-threatening heparin-induced thrombocytopenia (HIT). Most anti-platelet factor (PF)4-heparin IgGs are not pathogenic so diagnosis of HIT is challenging. Dimeric rsFcγRIIa was used to quantify receptor-binding activity of anti-PF4-heparin antibodies. Dimeric rsFcγRIIa binding specifically correlated with occurrence of HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is a major and potentially fatal consequence of antibodies produced against platelet factor 4 (PF4)-heparin complexes following heparin exposure. Not all anti-PF4-heparin antibodies are pathogenic, so overdiagnosis can occur, with resulting inappropriate use of alternative anticoagulation therapies that have associated risks of bleeding. However, definitive platelet functional assays are not widely available for routine analysis. Objectives To assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies. Patients/Methods Plasma from 27 suspected HIT patients were tested for pathogenic anti-PF4-heparin antibodies by binding of a novel dimeric FcγRIIa ectodomain probe. Plasmas were also tested by the use of PF4-heparin IgG ELISA, the HemosIL AcuStar HIT IgG-specific assay, and a serotonin release assay (SRA). Results The dimeric rsFcγRIIa test produced no false positives and excluded four samples that were positive by IgG ELISA. In this small patient cohort, the novel assay correctly assigned 93% of the suspected HIT patients, with two of the HIT patients being scored as false negatives. The improved discrimination of the novel assay over the IgG ELISA, which scored four false positives, supports the mechanistic interpretation that binding of dimeric rsFcγRIIa detects pairs of closely spaced IgG antibodies in PF4-heparin immune complexes. Conclusions This study found the cell-free, function-based dimeric rsFcγRIIa assay to be convenient, simple, and potentially predictive of HIT. The assay had improved specificity over the IgG ELISA, and correlated strongly with the AcuStar HIT IgG-specific assay, warranting further evaluation of its potential to identify HIT in larger patient cohorts., (©2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)