Heparin-dependent Antibodies Research Articles

Generation and pathogenicity of autoantibodies associated to thrombosis and hemostasis

Many acquired bleeding and thrombotic complications are provoked by autoantibodies to blood coagulation factors, or to hemostasis inhibitors and regulatory proteins. If occurrence of those antibodies remains rare or ultra-rare, affected patients are not always well-identified and associated pathologies are not always understood. Today, autoantigens tend to be better characterized. New available methods allow investigating structural changes of body components, responsible for auto-immunization. This renders it possible to develop laboratory assays for detecting autoantibodies and estimating their blood concentration. This review analyzes the major autoantibodies reported to be associated with hemorrhagic or thrombotic pathologies and their possible inducing causes when known. Pathogenicity is strongly patient- and context-dependent and is related to autoantibodies’ concentration, avidity, and capacity to bind to autoantigen structures in-vivo, misdirecting the immune system to the own body’s cells or organs. Identification of autoantigens allows for developing laboratory methods for testing autoantibodies and following their evolution kinetics. In-vitro investigations concern functional assays, to evaluate autoantibody’s capacity to inhibit physiological activities, or autoantigen-capture-based assays to detect autoantibodies, like with enzyme-linked immuno-sorbent assay (ELISA) methods. Exploring patients with autoimmune complications remains difficult as few specific assays are available. They mainly concern diseases with the highest incidence, like anti-phospholipid antibodies, lupus anticoagulants, or heparin-dependent antibodies. The present understanding suggests that antibodies to ubiquitous components, like phospholipids or polysaccharides, are actually targeted to proteins with a strong affinity binding to those components: Autoantibodies are not directed to phospholipids, but to phospholipid-binding proteins, and heparin-dependent antibodies are not directed to anticoagulant polysaccharides, but to platelet factor 4. Most pathogenic autoantibodies are of immunoglobulin G (IgG) isotype, but in some cases, IgM or IgA isotypes can be involved. Identification and characterization of autoantibodies associated to hemorrhagic or thrombotic pathologies remains complex at the laboratory level, although they are of high relevance for the right management of concerned patients.

Thrombotic anti-PF4 immune disorders: HIT, VITT, and beyond.

Antibodies against the chemokine platelet factor 4 (PF4) occur often, but only those that activate platelets induce severe prothrombotic disorders with associated thrombocytopenia. Heparin-induced thrombocytopenia (HIT) is the prototypic anti-PF4 disorder, mediated by strong activation of platelets through their FcγIIa (immunoglobulin G [IgG]) receptors (FcγRIIa). Concomitant pancellular activation (monocytes, neutrophils, endothelium) triggers thromboinflammation with a high risk for venous and arterial thrombosis. The classic concept of HIT is that anti-PF4/heparin IgG, recognizing antigen sites on (cationic) PF4 that form in the presence of (anionic) heparin, constitute the heparin-dependent antibodies that cause HIT. Accordingly, HIT is managed by anticoagulation with a nonheparin anticoagulant. In 2021, adenovirus vector COVID-19 vaccines triggered the rare adverse effect "vaccine-induced immune thrombotic thrombocytopenia" (VITT), also caused by anti-PF4 IgG. VITT is a predominantly heparin-independent platelet-activating disorder that requires both therapeutic-dose anticoagulation and inhibition of FcγRIIa-mediated platelet activation by high-dose intravenous immunoglobulin (IVIG). HIT and VITT antibodies bind to different epitopes on PF4; new immunoassays can differentiate between these distinct HIT-like and VITT-like antibodies. These studies indicate that (1) severe, atypical presentations of HIT ("autoimmune HIT") are associated with both HIT-like (heparin-dependent) and VITT-like (heparin-independent) anti-PF4 antibodies; (2) in some patients with severe acute (and sometimes chronic, recurrent) thrombosis, VITT-like antibodies can be identified independent of proximate heparin exposure or vaccination. We propose to classify anti-PF4 antibodies as type 1 (nonpathogenic, non- platelet activating), type 2 (heparin dependent, platelet activating), and type 3 (heparin independent, platelet activating). A key concept is that type 3 antibodies (autoimmune HIT, VITT) require anticoagulation plus an adjunct treatment, namely high-dose IVIG, to deescalate the severe anti-PF4 IgG-mediated hypercoagulability state.

Standardization of HIT Diagnostic Assays, with K070, a chimeric human-mouse antibody, mimicking heparin dependent pathogenic antibodies

This study reports the K070 monoclonal antibody, a human-mouse recombinant chimeric monoclonal antibody, IgG1 isotype, targeted to Heparin and PF4 complexes, which mimics Heparin Induced Thrombocytopenia. This antibody has a strong reactivity to Heparin-PF4 complexes, and a much weaker one to PF4 alone. When plasmas spiked with this antibody at concentrations ≥ 0.5 µg/ml are tested at the standard immunoassay dilution, a dose dependent reactivity is obtained. This opens the perspective to use this material to calibrate immunoassays for a quantitative measurement of heparin dependent antibodies, when the disease is suspected. Furthermore, when plasmas are spiked at a higher concentration, from 20 to 200 µg/ml, they test positive in functional assays, like Platelet Aggregation/Activation Tests or Flow Cytometry methods, in presence of a low (0.1-1.0 IU/ml) but not a high (10-100 IU/ml) heparin concentration. In functional assays K070 reactivity level depends on the platelet rich plasmas used. In Platelet Aggregometry Tests some platelet rich plasmas are already positive when tested with plasmas supplemented with 20 µg/ml, whilst others require 100 to 200 µg/ml. At this latter concentration, all tested plasmas generate a strong positive responses with functional assays. K070 is then a useful antibody for calibration of immunoassays and standardization of functional methods used to diagnose Heparin Induced Thrombocytopenia.

Heparin-Independent and Heparin-Dependent Anti-CXCL4 Antibodies Have a Reciprocal Expression in a Systemic Sclerosis Patients' Cohort.

Systemic sclerosis (SSc) is a chronic disease characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an early SSc biomarker that predicts worse disease outcome. We previously reported that CXCL4 is an autoantigen in SSc, and anti-CXCL4 antibodies correlated with IFN-I and were more abundant in patients with lung fibrosis. However, it is unclear whether antibodies to CXCL4 in SSc are only directed to CXCL4 or recognize complexes formed by CXCL4 and heparin. Here, by analyzing an SSc cohort, we addressed the occurrence of circulating heparin-dependent VS heparin-independent anti-CXCL4 antibodies and their relationship with a few disease parameters. We found that heparin-dependent, like the heparin-independent antibodies, are higher in SSc as compared to healthy donors; they are detectable in 24% and 30% of the SSc patients, respectively, and appear inversely correlated and mutually exclusive. Like the heparin-independent antibodies, heparin-dependent antibodies correlated with digital ulcers. However, in contrast to heparin-independent antibodies, heparin-dependent antibodies did not correlate with IFN-I, but were largely expressed in patients with pulmonary arterial hypertension. This pilot study indicates that heparin-dependent antibodies are worth studying in larger SSc cohorts to address whether they discriminate SSc sub-groups with different pathological characteristics and outcomes.

Incidence and severity of thrombocytopenia associated with use of intravascular microaxial ventricular assist devices for treatment of cardiogenic shock.

The use of temporary mechanical circulatory support (MCS) for patients with refractory cardiogenic shock has increased over the past decade. Impella devices (intravascular microaxial ventricular assist devices [VADs]) have become common MCS options but reportedly cause thrombocytopenia. Limited published data regarding the incidence or severity of microaxial VAD-associated thrombocytopenia exists. The goal of this study was to determine the incidence, timing, and severity of thrombocytopenia in a microaxial VAD population. A retrospective multicenter review of electronic medical records identified all patients implanted with microaxial VAD at three US academic teaching hospitals between June 2015 and August 2017. Patients were excluded for short-term procedural microaxial VAD use during percutaneous coronary intervention. Sixty-four patients underwent microaxial VAD insertion (95% for left-sided support) during the observed time period. Support was in place for a median duration of 5.2 (interquartile range [IQR]: 2.4-10.0) days. Within 7 days postinsertion, 98.5% of patients developed thrombocytopenia (platelet count <150,000/μl) and 81.3% of patients experienced a>50% platelet decrease. Average platelet count nadir was 68,200/μl or 63.9% from baseline occurring on median day 3.8 (IQR: 2.4-5.4). Twenty-four patients (38.1%) were tested for heparin-induced thrombocytopeniaby the heparin-dependent antibody (HDA) test. All HDAs were either negative or had serotonin release assay negative confirmation. Postdevice removal, platelet counts returned to baseline or >150,000/μl in 63% of patients by Day 5. Microaxial VAD-associatedthrombocytopenia is common. Practitioners should consider this when evaluating supported patients for other causes of thrombocytopenia. Platelet counts return to preimplantation levels within days of device removal.

Vaccine induced thrombotic thrombocytopenia: development and reactivity of anti-platelet factor 4 antibodies and immune pathogenic mechanisms

In ultrarare cases, patients vaccinated with DNA adenovirus vector vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), develop a vaccine-induced immune thrombotic thrombocytopenia (VITT), with a high incidence of fatal cases. The causative agent is the development of platelet factor 4 (PF4)-dependent antibodies that resemble heparin-induced thrombocytopenia (HIT) complication, although many differences can be noticed in clinical presentation, antibody reactivity, involved epitopes on the PF4 protein, and pathological mechanisms. From the literature review, and the experience of HIT and testing a few plasmas from patients with VITT, this review analyzes the possible mechanisms, which show the strong immunoglobulin G (IgG) antibody reactivity to PF4 alone, in the absence of heparin, and to a lesser extend to stoichiometric complexes of PF4 and heparin (H-PF4). In addition, much lower heparin concentrations are required for inhibiting antibody binding to PF4. These concentrations are much lower than those required for disrupting the stoichiometric H-PF4 complexes. This confirms that IgG antibodies responsible for HIT bind preferentially to PF4, to epitopes that are readily masked by low concentrations of heparin. These antibodies are at a much higher concentration than the current ones observed for HIT, keeping a strong reactivity even for plasma dilutions as high as 1/500 to 1/5,000, whilst the current dilution for testing heparin-dependent antibodies in HIT is 1/100. Although VITT anti-PF4 antibodies can be detected with the current anti-H-PF4 enzyme-linked immunosorbent assays (ELISAs) designed for HIT, some assays have low sensitivity or are unreactive, like lateral immunofiltration methods or chemiluminescent automated assays. The preferred method should concern the use of capture assays using PF4 coated solid surfaces. This report proposes that the immune response is only targeted to the binding domain of PF4 with the hexons present on the adenovirus vector, through an epitope spreading mechanism, without any exposure of neo-epitopes on PF4 protein.

Platelet-activating anti-PF4 disorders: An overview

Platelet factor 4 (PF4) is a highly cationic tetrameric protein that can be targeted by platelet-activating anti-PF4 antibodies of immunoglobulin G (IgG) class. Certain features of PF4, including its multivalent nature (duplicate antigen sites per tetramer), the ability of many PF4 tetramers to undergo close approximation through charge neutralization, and the dimeric binding of IgG molecules, results in formation of IgG-containing immune complexes in situ on platelets, neutrophils, and monocytes, resulting in Fcγ receptor-mediated pancellular activation that also activates hemostasis (potential for disseminated intravascular coagulation). This review discusses 4 anti-PF4 disorders: classic heparin-induced thrombocytopenia ([HIT]; triggered by heparin and certain other polyanionic pharmaceuticals, featuring predominantly heparin-dependent antibodies), autoimmune HIT (aHIT; severe subtype of HIT that features both heparin-dependent and heparin-independent platelet-activating antibodies), and spontaneous HIT (non-heparin triggers such as knee replacement surgery and infection; predominantly heparin-independent platelet-activating antibodies). Most recently, a novel fourth anti-PF4 disorder, vaccine-induced immune thrombotic thrombocytopenia (VITT), was identified as an ultrarare complication of adenovirus vector vaccines. VITT is characterized by thrombocytopenia, disseminated intravascular coagulation, a high frequency of thrombosis—including in unusual sites (cerebral veins, splanchnic veins)—and highly pathogenic anti-PF4 antibodies with heparin-independent platelet-activating properties.

Successful treatment of thrombotic thrombocytopenia with cerebral sinus venous thrombosis following Ad26.COV2.S vaccination

Successful treatment of thrombotic thrombocytopenia with cerebral sinus venous thrombosis following Ad26.COV2.S vaccination

Pathophysiology and Diagnosis of Drug-Induced Immune Thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) is a life-threatening clinical syndrome that is under-recognized and difficult to diagnose. Many drugs can cause immune-mediated thrombocytopenia, but the most commonly implicated are abciximab, carbamazepine, ceftriaxone, eptifibatide, heparin, ibuprofen, mirtazapine, oxaliplatin, penicillin, quinine, quinidine, rifampicin, suramin, tirofiban, trimethoprim-sulfamethoxazole, and vancomycin. Several different mechanisms have been identified in typical DITP, which is most commonly characterized by severe thrombocytopenia due to clearance and/or destruction of platelets sensitized by a drug-dependent antibody. Patients with typical DITP usually bleed when symptomatic, and biological confirmation of the diagnosis is often difficult because detection of drug-dependent antibodies (DDabs) in the patient’s serum or plasma is frequently not possible. This is in contrast to heparin-induced thrombocytopenia (HIT), which is a particular DITP caused in most cases by heparin-dependent antibodies specific for platelet factor 4, which can strongly activate platelets in vitro and in vivo, explaining why affected patients usually have thrombotic complications but do not bleed. In addition, laboratory tests are readily available to diagnose HIT, unlike the methods used to detect DDabs associated with other DITP that are mostly reserved for laboratories specialized in platelet immunology.

Utility and Pitfalls of Stepwise Laboratory Testing for Heparin Induced Thrombocytopenia: Retrospective Review from an Academic Medical Center

Introduction: Heparin induced thrombocytopenia (HIT) occurs in up to 5% of adults exposed to heparin due to formation of heparin-dependent antibodies to the heparin/platelet factor 4 (PF4) complex. Patients develop thrombocytopenia and are at risk for severe thrombotic complications. Mortality rates can be as high as 20%, but are often much lower with prompt recognition, cessation of heparin, and treatment with alternative anticoagulants. However, these alternative medications are often both labor-intensive drips and expensive, highlighting the need for quick decision making in such challenging cases. The classic workup of HIT involves assessing clinical suspicion (often using the "4T score" on a scale of 0-8), followed by lab testing for detection of the heparin-dependent antibody and then a functional assay to confirm pathogenicity of the antibody. Our institution uses an in-house rapid Particle ImmunoFiltration Assay (PIFA), which is a same-day test and reported as "positive" or "negative." Other testing available includes send-out tests for Enzyme-Linked Immunoassay (ELISA) to IgG, A, and M of the PF4 antibody as well as the C-14 Serotonin Release Assay (SRA) functional assay. The goal of this retrospective review is to analyze the utility of an algorithmic approach to laboratory testing in aiding the rapidity of diagnosis of HIT without missing possible critical cases. Methods: This was a single institutional study at a large urban academic medical center. We reviewed inpatient charts from 2015-2018 of patients who had any lab testing for HIT. As per institutional guidelines, first-line testing is recommended by using the in-house same-day testing PIFA. If positive, a sample is automatically reflexed and sent-out for PF4 ELISA testing and SRA. Furthermore, clinicians are able to directly order ELISA and SRA testing at their medical discretion. Our analysis looked at those patients with at least two different "HIT-related" laboratory tests to best analyze the concordance and discordance rates of the above testing to assess for sensitivity, specificity, and overall accuracy of using a stepwise testing approach. We used a cutoff value of &gt;0.4 optical density (OD) for ELISA testing, and &gt;20% release at low-dose heparin concentration for SRA testing. Results: There were 118 patients who had at least two different HIT-related laboratory tests sent. 91 patients had both PIFA and ELISA testing, with 37/79 (47%) positive concordance rate and 6/12 (50%) negative concordance rate, for a sensitivity of 86% and specificity of 13%. 3 patients with positive PIFA also had positive SRA, and there were 2 patients with negative PIFA with positive SRA testing (see attached Table). When comparing ELISA testing to SRA, 4/41 (10%) had concordant positive testing, while no patient with a negative ELISA test had a positive SRA (28 concordant negative cases). Overall, of 94 SRA tests run, 5 were positive, of which 2/5 had negative PIFA and 0/4 had negative ELISA testing. Conclusions: While PIFA testing had a high sensitivity compared to ELISA, the overall accuracy compared to ELISA was low, while ELISA testing was 100% sensitive in this analysis. Furthermore, there was still a risk of missing cases of HIT using PIFA testing alone. In both cases of positive SRA with a negative PIFA, patients had a high 4T score of &gt;6, consistent with a high clinical suspicion for HIT. We conclude that PIFA testing is not equivalent to ELISA testing, and that use of a laboratory "algorithm only" approach would be inappropriate in the diagnosis of HIT. Our results highlight the importance of using both clinical scoring systems and appropriate lab testing together in the workup and diagnosis of HIT. Disclosures No relevant conflicts of interest to declare.

How I treat patients with a history of heparin-induced thrombocytopenia

AbstractHeparin-induced thrombocytopenia (HIT) is a relatively common prothrombotic adverse drug reaction of unusual pathogenesis that features platelet-activating immunoglobulin G antibodies. The HIT immune response is remarkably transient, with heparin-dependent antibodies no longer detectable 40 to 100 days (median) after an episode of HIT, depending on the assay performed. Moreover, the minimum interval from an immunizing heparin exposure to the development of HIT is 5 days irrespective of the patient's previous heparin exposure status or history of HIT. This means that short-term heparin reexposure can be safely performed if platelet-activating antibodies are no longer detectable at reexposure baseline and is recommended when heparin is the clear anticoagulant of choice, such as for cardiac or vascular surgery. The risk of recurrent HIT 1 to 2 weeks after heparin reexposure is ∼2% to 5% and is attributable to formation of delayed-onset (or autoimmune-like) HIT antibodies that activate platelets even in the absence of pharmacologic heparin. Some studies suggest that longer-term heparin reexposure (eg, for chronic hemodialysis) may also be reasonable. However, for other antithrombotic indications that involve patients with a history of HIT (eg, treatment of venous thromboembolism or acute coronary syndrome), preference should be given to non-heparin agents such as fondaparinux, danaparoid, argatroban, bivalirudin, or one of the new direct-acting oral anticoagulants as appropriate.

Characterization of Human and Murine Anti-Protamine/Heparin Antibodies

Protamine/heparin (PRT/H) antibodies (Abs) are a newly described class of heparin-dependent antibodies found in ~25% of patients exposed to protamine and heparin during cardiopulmonary bypass surgery (CPB). Although recent studies show that PRT/H Abs have several serologic properties similar to platelet factor 4 (PF4)/heparin Abs, the clinical significance of PRT/H Abs is unknown. To understand their clinical significance, we undertook studies to characterize the biologic effects of PRT/H Abs in vitro. Using a previously described murine monoclonal antibody to PRT/H complexes (IgG3 isotype, ADA) and patient-derived PRT/H Abs, we examined antibody cross-reactivity with histones and nuclear proteins as well as functional effects of PRT/H Abs on neutrophil activation. Using a commercial ANA immunofluorescence assay (ImmuGlow Hep-2 Cells Anti-nuclear Antibody IFA kit), we first examined cross-reactivity of anti-PRT/H on nuclear proteins. As seen in Figure 1, both ADA and patient-derived PRT/H Abs (depicted as α-PRT/H (+) in Figure 1D) showed significant binding to Hep-2 cells. In contrast, no reactivity was seen when Hep-2 cells were incubated with plasma from CPB patients who were seronegative for PRT/H antibodies (depicted as α-PRT/H (-) in Figure 1D) or with IgG3 isotype (data not shown). To confirm that PRT/H Abs were binding to nuclear antigens, we examined the cross-reactivity of monoclonal and polyclonal anti-PRT/H on individual nuclear binding proteins, including Single Stranded Binding Protein, RO-52, JO-1, (Sigma; St. Louis, MO, USA) and nucleosomes (New England Biolabs; UK) by ELISA. As shown in Table 1, ADA showed significantly higher binding to all nuclear antigens as compared to isotype control. Polyclonal PRT/H Abs from CPB patients also showed increased binding to nuclear antigens relative to control plasma, but did not achieve statistical significance. To determine if PRT/H Abs activate neutrophils, we isolated neutrophils by gradient centrifugation and incubated cells with 100 ug/mL of ADA or isotype in the presence or absence of antigen (PRT 31 ug/mL + H4 U/mL) and measured release of myeloperoxidase (MPO). As shown in Figure 2, ADA alone or isotype control showed minimal MPO release. In the presence of PRT or PRT/H, ADA, but not isotype control, showed significant MPO release. Taken together, these studies demonstrate that PRT/H Abs cross-react with nuclear antigens and can trigger neutrophil activation. These findings suggest that PRT/H Abs cross-react with a closely related class of antigens (histones) and enhance inflammation through cross-reactivity with nuclear antigens and/or through functional effects on neutrophil activation.Table 1AntibodySSBPRO52JO-1nucleosomesADA v. Isotype1.36 ± 0.06 v. 0.13 ± 0.011.76 ± 0.06 v. 0.08 ± 0.011.64 ± 0.03 v. 0.09 ± 0.010.73 ± 0.03 v. 0.07 ± 0.01Polyclonal PRT/H Abs v. control plasma1.01 ± 0.23 v. 0.54 ± 0.051.15 ± 0.21 v. 0.72 ± 0.020.92 ± 0.16 v. 0.49 ± 0.040.81 ± 0.14 v. 0.75 ± 0.3 [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GW26-e4636 Impacts of Anticoagulation with Bivalirudin versus Low Molecular Weight Heparin on Thrombopenia during IABP Therapy

Comparing the impacts of low molecular weight heparin and bivalirudin on blood platelet counts as well as the level of antibody of platelet factor 4 heparin-dependent antibodies (H-PF4 IgG) during IABP therapy. This pilot study was designed to totally enroll 60 patients of AMI complicated cardiac

A Multiplex Flow Cytometry Bead Assay For Simultaneous Detection Of IgG, IgA, IgM Heparin-Dependent Antibodies

Heparin-Induced Thrombocytopenia (HIT) is an adverse reaction to heparin, in which affected patients produce platelet activating antibodies specific for complexes of platelet-factor 4 (PF4) and heparin (HPF4) resulting in a prothrombotic condition that in severe cases can be life-threatening. HIT is not rare, since as many as 600K people/year are affected. It is therefore important that a quick and accurate diagnosis of HIT be made, which requires both compatible clinical findings and serologic evidence of a heparin dependent antibody. First line testing consists of immunologic assays that detect IgG, IgA and IgM antibodies targeting PF4 in a complex with heparin or another polyanion. The most widely used assay is the PF4 ELISA, in which HPF4 bound to the wells of a microtiter plate is the target antigen. The ELISA has high sensitivity (near 100%) for antibody detection but a low positive predictive value for HIT diagnosis. Therefore, samples giving positive ELISA results are often retested using a functional assay like the serotonin release assay (SRA) that is more specific for HIT diagnosis. However, the SRA is difficult to perform and interpret and is not widely available. Further, the ELISA is time-consuming, costly, and most versions cannot distinguish IgG, IgA, IgM (IgG/A/M) antibodies. Knowledge of the antibody isotype can be useful when ELISA and SRA results are discrepant. For instance, strong ELISA results due to clinically insignificant IgM antibodies would not be detected by SRA, and additional testing to confirm IgM antibody isotype can lead to a delay in HIT diagnosis, and in some cases continuation of high cost treatments with direct thrombin inhibitors (DTI). Moreover, there is evidence that some IgA and IgM antibodies may cause HIT.Despite the high sensitivity of the ELISA and high specificity of the SRA, the shortcomings mentioned for these tests highlight the need for improved methods to support a diagnosis of HIT. We report development of a multiplex flow cytometry bead assay (MFBA) for simultaneous detection of IgG/A/M heparin-dependent antibodies that allows higher throughput and has sensitivity and specificity comparable to the PF4 ELISA.For the MFBA, polystyrene beads, to which chemically depolymerized heparin is covalently linked are incubated with purified platelet PF4 to produce HPF4-bead targets. Lyophilized HPF4 beads are stable for at least 3 months. HPF4-beads are incubated with serum, washed, and incubated with anti-human -IgG, -IgA, and –IgM, each with different fluorescent labels for detection of HIT antibodies by flow cytometry. A total of 164 patient samples could be tested by a single technologist in 8 hours. Serum samples from 300 patients with suspected HIT and previously tested by SRA were tested by both MFBA and an in-house PF4 ELISA including high dose heparin (100 U/L) confirmation step. Studies were approved by institutional IRB. MFBA results showed good sensitivity (87% IgG, 63% IgA, 36% IgM) for IgG, good specificity (96% IgG, 91% IgA, 93% IgM) for all isotypes when compared to the ELISA, and good sensitivity/specificity (93%/91%) compared to SRA. The area under the curve (AUC) calculated for the receiver operating characteristic graphs compared to the SRA were excellent (0.9-1.0) for both MFBA (AUC = 0.95) and PF4 ELISA (AUC = 0.98). All samples positive by MFBA inhibited with high dose heparin suggesting that this extra step is not required, and 3 samples that tested IgG positive in both MFBA and SRA gave indeterminate results (not inhibited with high dose heparin) in ELISA.Overall, these data show that the MFBA has accuracy, sensitivity, and specificity for detection of IgG antibodies comparable to the PF4 ELISA and the “gold standard” SRA with the additional advantage of simultaneously distinguishing IgG/A/M antibody isotypes, which will lead to reduced confirmation testing. Sensitivity of MFBA for detection of IgA and IgM antibodies was significantly lower compared to ELISA, but this reflects the assay design intended to increase specificity of the MFBA for these two isotypes. In addition to its multiplex capabilities, the MFBA allows for higher sample throughput and shortened turn-around times that will contribute to overall improved patient care. Disclosures:No relevant conflicts of interest to declare.

Correlation Of Heparin Confirmatory Test (HCT) With Optical Density (OD) and Serotonin Release Assay (SRA) In The Diagnosis Of Heparin Induced Thrombocytopenia (HIT)

Introduction Heparin-induced thrombocytopenia (HIT) is a potentially fatal, thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin. Accurate and rapid diagnosis with prompt commencement of therapy are imperative as delays in treatment are associated with an increasing risk of thrombosis, amputation, or death. On the flip side, initiation of therapy with direct thrombin inhibitors without laboratory confirmation carries a significant risk of bleeding. Two types of laboratory tests are available for detection of these antibodies: a widely available immunoassay (ELISA), which is very sensitive to the presence of anti-heparin/PF4 antibodies, but is less specific to the clinical syndrome of HIT because of detection of non-pathological antibodies. The Serotonin Release Assay (SRA) is a functional assay that is now considered the gold standard for confirmatory diagnosis of HIT due to its high specificity. However, the downside of SRA is the cost involved, limited availability and a turnaround time of 5-7 days. As such, a heparin confirmatory test (HCT) with excess heparin has been in use since mid 2011 on positive ELISA samples in our laboratory to improve test specificity. This test is more cost and time efficient, with a turnover time of no more than 48 hours. As noted in prior studies, inhibition of a positive ELISA result by 50% or more in the presence of excess heparin is considered confirmatory of heparin-dependent antibodies. Likewise a negative confirmatory test is defined as a decrease of 50% or less in antibody binding in the presence of heparin. Aim a) Correlation of Heparin Confirmatory test (HCT) with strength of HIT ELISA, vis-à-vis optical density (OD) of 0.4 - 0.99 and OD of &gt;/= 1.0. b) Correlation of HCT results with SRA, to see if the latter can be replaced by the heparin confirmatory test. Patients and Methods A retrospective chart review of adult patients hospitalized at our institution with suspected HIT from July 2011 until January 2013 was done. There were 101 such patients. All patients who had a positive HIT ELISA, then had HCT as per our standard lab practice, with an SRA test done for diagnosis/confirmation of HIT, as per standard clinical practice. Historically, the major strength of SRA assay is its specificity. The optical density on HIT ELISA and SRA results were then compared with the Heparin Confirmatory test to establish clinical significance. Results Of the 101 patients tested for HIT ELISA, 49 were positive. HCT and SRA were performed on all 49 samples, 1 out of which was reported as indeterminate. Hence 48 samples were used for primary analysis, comparing HCT to the OD as well as the SRA results. Out of 48 patients, 6 had positive SRA with Heparin inhibition of &gt;50% (sensitivity 6/6 = 100%). Remaining 42 patients had negative SRA, 7 out of which had Heparin inhibition of &lt;50% (specificity 7/42 = 16.6%). All 7 patients with a negative HCT had a negative SRA, making the negative predictive value of the HCT 100%; however positive predictive value was only 14.6% (6/41). There was no correlation between the OD and Heparin Confirmation test. Conclusions Although there is data suggesting that there might be some value to the Heparin Confirmation test, we were unable to show a significant correlation between HCT and OD or between HCT and SRA. The prospect of having a cost effective and rapid assay for laboratory confirmation of HIT will always be a relevant need. We feel that a larger, prospective study should be conducted to definitively assess the relationship between HCT and SRA. Disclosures: No relevant conflicts of interest to declare.

Incidence of antibodies to protamine sulfate/heparin complexes in cardiac surgery patients and impact on platelet activation and clinical outcome

A new ELISA (Zymutest HIA®), based on incubation of diluted plasma with protamine/heparin (PRT/H) complexes without and with platelet factor 4 (PF4) provided by a platelet lysate, was used to detect heparin-dependent antibodies in a cohort of 232 cardiac surgery (CS) patients and in 47 patients with heparin-induced thrombocytopenia (HIT). Significant binding of IgG/A/M to PRT/H complexes was demonstrated in 59 CS patients (25.4%), with similar absorbances whether platelet lysate was added to the plasma or not, and significant reactivity to PF4/H in 29 of them. Antibodies to PRT or heparin alone were present in 15 and two of these patients, respectively. Importantly, antibodies to PRT/H were detected in only three of the 47 HIT patients, who had also undergone recent CS. The Zymutest HIA® was positive in another 41 CS patients (17%), but only or mainly when their plasma was tested with platelet lysate, with significant levels of antibodies to PF4/H in 40 of them without detectable reactivity to PRT or heparin alone. Slight antibody binding to PRT/H complexes was also measured in six of these 41 patients. Therefore, a total of 35 CS patients exhibited dual antibody reactivity towards PRT/H and PF4/H complexes. Serotonin release assay performed with PRT alone was positive in 17 CS patients with antibodies to PRT/H, but all had normal platelet count evolution without thrombosis postoperatively. In conclusion, antibodies to PRT/H are frequently present in CS patients postoperatively (25.4%), and can activate platelets in vitro, but their clinical impact remains questionable.

Prevalence and Risk of Preexisting Heparin-Induced Thrombocytopenia Antibodies in Patients With Acute VTE

Some patients with acute VTE who may previously have been exposed to heparin products have unrecognized antibodies implicated in heparin-induced thrombocytopenia (HIT). Antibody prevalence and patient consequences upon exposure to heparin, low-molecular-weight heparin, and fondaparinux are uncertain. In this secondary analysis, we tested patients in the Matisse VTE studies at study entry for heparin-dependent antibodies and further tested patients with enzyme-linked immunosorbent assay (ELISA)-positive results for platelet-activating antibodies. We compared the risk of HIT (> 50% fall in platelet count, heparin-dependent antibodies, no contradicting features) between patients treated with heparin (either unfractionated or low molecular weight [enoxaparin]) vs those who received fondaparinux. Comparison groups for thrombocytopenia occurrence comprised patients with ELISA-positive, platelet-activating, antibody-positive results; ELISA-positive, but platelet-activating antibody-negative results; and randomly selected antibody-negative results. A total of 127 of 3,994 patients (3.2%) had ELISA-positive results at baseline, but only 14 (0.4%; 95% CI, 0.2%-0.6%) had platelet-activating antibodies. Among these 14, four treated with unfractionated or low-molecular-weight heparin developed HIT compared with zero of 10 fondaparinux-treated patients (OR, 95; 95% CI, 8-1,123; P < .001). This frequency (four of four, 100%) significantly differed from that of both heparin-treated patients whose results were ELISA positive but platelet-activating antibody negative and from heparin-treated antibody-negative control subjects (zero of 15 and zero of 27, respectively; P < .001 for both). Of patients with VTE, 0.4% had pathologic platelet-activating heparin-dependent antibodies rather than the 3.2% detected by the recommended cutoff of the commercial ELISA. Among study patients with acute VTE who had platelet-activating antibodies, treatment with fondaparinux reduced the risk of precipitating rapid-onset HIT.

The occurrence of antibodies to heparin-platelet factor 4 in cardiac and thoracic surgical patients receiving desirudin or heparin for postoperative venous thrombosis prophylaxis

Introduction This randomized, exploratory study compared the incidence of heparin-dependent antibodies associated with subcutaneous (SC) desirudin or heparin given for deep-vein thrombosis prophylaxis following cardiac and thoracic surgery. Materials and Methods Adult patients scheduled for elective cardiac or thoracic surgery received desirudin 15 mg SC twice daily or unfractionated heparin 5000 units SC thrice daily. Duration of thrombosis prophylaxis was determined by the treating physician. Primary outcome measure was the incidence of new antibody formation directed against platelet factor 4 (PF4)/heparin complex. Secondary outcomes included bleeding and thrombotic complications. Blood was tested for anti-PF4/heparin antibodies at baseline, after surgery prior to study drug administration, postdrug day (PDD) 2, PDD 7, and at 1 month. Doppler studies were done before discharge. Results Of 120 patients, 61 received desirudin, 59 received heparin. New PF4/heparin antibodies occurred in 10.2% and 13.6% of desirudin- and heparin-treated patients, respectively. Among desirudin patients with no heparin exposure, none (0/36) developed PF4/heparin antibodies versus 17.1% with heparin exposure. Incidence of deep venous thrombosis was 4.9% and 3.4% in the desirudin and heparin groups, respectively. Two heparin-group patients developed pulmonary embolism. Two patients per group had bleeding events; no patients required re-exploration for bleeding complications. Median chest tube output was similar with desirudin (900 mL) and heparin (692 mL) as was blood transfusion requirements of more than 2 units (5/61, desirudin; 2/59 heparin). Conclusions The incidence of thrombotic events was low in both groups. There were no safety concerns, and desirudin was not associated with anti-PF4/heparin antibodies.

Persistence of heparin-dependent antiplatelet antibody

Heparin-induced thrombocytopenia is an immune-mediated response that has been associated with bleeding and thromboembolic complications. We test all cardiac surgery patients who experience thrombocytopenia, resistance to heparin, or have complications of increased bleeding or thrombosis with platelet aggregation assay for heparin-dependent antiplatelet antibody (HAAb). Due to the morbidity and mortality associated with reexposure to heparin in this population, we wished to investigate the length of time patients remain positive for HAAb postoperatively. Over a 10-year period (1982–1992), 97 patients tested positive for HAAb following open heart surgery. Of 66 survivors, 43 were available for follow-up and were contacted for repeat HAAb testing. Patients were retested from 1 to 126 months (median 17) postoperatively. Eleven of 43 patients (26%) remained positive for HAAb from 3 months to 12.5 years after initial diagnosis. Conversion from a positive to negative HAAb as early as 4 weeks was also observed. We conclude that the time to conversion to a negative HAAb test is highly variable. All patients who test positive for HAAb should have repeat HAAb testing before reexposure to heparin regardless of the time interval.

Influence of Protein Tyrosine Phosphatase Polymorphisms on the Development of Antibodies to Platelet Factor 4 and the Risk of Heparin-Induced Thrombocytopenia.

AbstractAbstract 3683 Introduction.Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FcgRIIa receptors. The reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT involves both B and T cells, and protein tyrosine kinases (PTKs) and phosphatases (PTPs) are crucial for regulating antigen receptor-induced lymphocyte activation. Moreover, some PTPs such as CD148 and low-molecular-weight PTP (LMW-PTP) could also have a critical role in platelet activation. Dysregulation of the equilibrium between PTK and PTP function could therefore have pathologic consequences and influence the pathogenesis of HIT. Aim of the study.To investigate an association between polymorphisms affecting genes encoding 4 different PTPs i.e. CD45 (PTPRC), CD148 (PTPRJ), LYP (PTPN22) and LMW-PTP (ACP1) and the development of heparin-dependent antibodies to PF4 and HIT. Patients and methods.A cohort of 89 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay) and two control groups were studied. The first control group (Abneg) consisted of 179 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 160 patients who had also undergone cardiac surgery with CPB and heparin, who had all developed significant levels of PF4-specific antibodies but without HIT. Genotypes of PTPRC 77C/G (rs17612648), PTPN22 1858C/T (rs2476601), PTPRJ 2965 C/G (rs4752904) and PTPRJ 1176 A/C (rs1566734) were studied by a PCR-HRM method using the LightCycler 480 (Roche). In addition, the ACP1 A, B, C alleles were defined by combining the analysis of T/C transition at codon 43 of exon 3 (rs11553742) and T/C transition at codon 41 of exon 4 (rs11553746). Results.The frequency of PTPRC 77G and PTPN22 1858T alleles was not different in HIT patients and controls, whether they had developed antibodies to PF4 or not. The third PTP gene analyzed was ACP1, in which three alleles (A, B and C) were previously associated with the synthesis of distinct active LMW-PTP isoforms exhibiting different catalytic properties. The percentage of subjects in our study carrying the AC, BB and BC genotypes was significantly higher in the HIT and the Abpos groups than in patients without antibodies to PF4 after CPB (Abneg). In addition, the ACP1 A allele was less frequent in patients with antibodies to PF4, whether they had developed HIT (25%) or not (27.5% in Abpos controls), than in Abneg subjects (37%). The AC, BB and BC genotypes (associated in Caucasians with the highest LMW-PTP enzyme activity) therefore appeared to increase the risk of antibody formation in heparin-treated patients (OR 1.8; 95% CI 1.2–2.6, p=0.004 after comparing Abpos + HIT vs. Abneg). We also evaluated 2 SNPs affecting PTPRJ encoding CD148. No significant difference was found concerning the 2965 C/G polymorphism, but the frequency of PTPRJ 1176 AC and CC genotypes was significantly lower in the HIT (17%) than in the Abneg and Abpos groups (35%, p=0.003 and 29.5%, p=0.041, respectively). The C allele therefore appeared to provide a significant protection from the risk of HIT (OR 0.52; 95%CI 0.29–0.94, p=0.041) in patients with antibodies to PF4. Discussion-Conclusion.Recent studies have demonstrated that CD148 is a positive regulator of platelet activation by maintaining a pool of active SFKs in platelets. This non-synonym PTPRJ 1176 A/C SNP is associated with a Q276P substitution inducing a torsional stress of a fibronectin domain that is critical for the activity of CD148 and may influence the pathogenic effects of HIT Abs. This study supports the hypothesis that PTPs such as LMW-PTP and CD148 influence the immune response to heparin and the risk of HIT in patients with antibodies to PF4. Disclosures:No relevant conflicts of interest to declare.