Heparin-induced Thrombocytopenia Antibodies Research Articles

The use of 1E12, a monoclonal anti-platelet factor 4 antibody, to improve the diagnosis of vaccine-induced immune thrombotic thrombocytopenia

BackgroundVaccine-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. ObjectivesWe assessed the usefulness of competitive anti-PF4 enzyme immunoassays (EIAs) in this context. MethodsThe 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. ResultsA 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. ConclusionA simple competitive anti-PF4 EIA with 1E12 could help confirm VITT diagnosis and distinguish it from HIT in patients when both diagnoses are possible.

Abstract 1032: Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies

Abstract Background: CLDN18.2, the isoform 2 of CLDN18, is physiologically confined to gastric mucosa tight junctions, the epitopes of which would be exposed on the cancer cell surface upon malignant transformation and is highly expressed in a significant proportion of cancers, e.g., gastric and pancreatic adenocarcinomas, which makes it a promising drug target for cancer therapy. Currently, multiple drug modalities, such as monoclonal antibodies, ADCs, and CARTs targeting CLDN18.2 are being evaluated in clinical trials. A substantial correlation exists between the expression of CLDN18.2 and the efficacy of the these therapies. However, to date, no companion diagnostic (CDx) antibodies that are specific to CLDN18.2 have been approved. Therefore, it is important to promptly develop an anti-CLDN18.2 CDx antibody with a high specificity, sensitivity, and suitability for immunohistochemistry (IHC). In this study, we present the discovery and validation of a novel, highly sensitive IHC antibody that selectively identifies CLDN18.2. Methods: 3 CLDN18.2 specific peptide fragments from extracellular domain of human CLDN18.2 were conjugated with KLH. Antibodies were discovered by immunizing BALB/c and SJL mice with these peptides-KLH conjugates. Screening and selection process was relied on positive ELISA results against the immunogen, as well as FACS and Acumen Explorer Microplate staining of CLDN18.2-experssing cells. Hit antibodies were evaluated for IHC on formalin-fixed paraffin-embedded CHOK1 cells expressing full-length CLDN18.2 or CLDN18.1. The antibodies were further validated on human gastric cancer tissue samples to establish the analytical sensitivity and specificity of the IHC assay. Results: The mAb clone 43F11 exhibited binding to a fusion polypeptide comprising the first extracellular loop of human CLDN18.2 (aa32-53) at a half maximal effective concentration (EC50) of 0.212 nM. 43F11 showed positive cell surface IHC staining on CLDN18.2-experssing cells after fixation but demonstrated no staining on CLDN18.1-experssing cells. The IHC test method was established by utilizing clinical tumor tissues and PDX samples that had predetermined expression levels of CLDN18.2. The 43F11 antibody accurately identified the expression level of CLDN18.2 in the IHC assay, with a minimum concentration of 0.25 μg/mL. When compared to the commercial CLDN18.2 IHC antibody EPR19202, 43F11 demonstrated greater sensitivity, allowing for positive staining on cancer tissues that have significantly lower levels of CLDN18.2 expression. Conclusion: 43F11 exhibits specific recognition of human CLDN18.2 in IHC assays, while showing no reactivity towards CLDN18.1. It demonstrated superior sensitivity compared to the benchmark antibody. These data suggest that 43F11 has the capacity to serve as an effective diagnostic tool for identifying patients who positively express CLDN18.2. Citation Format: Jishun Chen, Suya Bai, Yu Bai, Zaoshun Hu, Peng Chen, Jay Mei, Bo Shan, Bing Hou. Development of a novel companion diagnostic immunohistochemistry antibody for claudin 18.2-targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1032.

Analysis of hospital length of stay and cost savings with an in-house heparin-induced thrombocytopenia antibody assay at a midsized institution.

Analysis of laboratory value often lacks assessment of the laboratory's impact on quality of care. In this study, we aimed to determine the impact of bringing a heparin-induced thrombocytopenia (HIT) antibody assay in-house on a quality metric-patient hospital length of stay (LOS)-and assess any associated cost savings. A retrospective review of patient visits with a HIT antibody assay over a 7-year period determined the mean LOS in send-out vs in-house HIT antibody assay cohorts as well as cohorts of positive and negative results. Our systemwide mean LOS and metrics of acuity were analyzed. We performed a financial analysis of estimated cost savings. We found a mean LOS reduction of 3.97 days in the in-house cohort, with no evidence of a systemwide LOS decrease or a decline in patient acuity. This reduction was largely driven by a reduction in LOS among patients with a negative assay result. We found an estimated total cost savings of $3.9 million and an estimated mean savings per patient of $7,305, despite escalating health care costs over time. We demonstrated a reduction in LOS following the introduction of an in-house HIT antibody assay that cannot be attributed to either systemwide initiatives or reduced patient acuity and was driven largely by patients with negative assays. This reduction was associated with significant estimated cost savings.

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.

When HIT Testing Hurts- the Financial and Clinical Repercussions of Heparin Induced Thrombocytopenia Testing: A Single Center Analysis

Introduction: Thrombocytopenia is a common inpatient hematologic concern. Most often, thrombocytopenia is a multifactorial phenomenon, without one clear cause. Heparin Induced Thrombocytopenia (HIT) is a rare but serious hematologic consequence of exposure to heparin. In many hospitalized patients, heparin is routinely used as venous thromboembolism (VTE) prophylaxis or for therapeutic anticoagulation. The incidence of HIT is approximately 0.5-1.0% in patients exposed to unfractionated heparin (UFH). Though rare, there seems to be over-testing of HIT antibody in the inpatient setting. As such, there is potential for high cost with low yield and false positives that alter patient management, resulting in prolonged hospital stays and further complications in patient care. Methods: This was a retrospective, observational study reviewing the indications, results, and patient characteristics of cases in which HIT testing was ordered. Indications for testing and influence of results on management were determined based on clinical documentation. The tests of interest included HIT antibody testing and Seratonin Release Assay (SRA). We analyzed how many times a HIT antibody was positive with a negative result in the SRA. We also determined whether there was an increased length of stay in patients who had HIT testing ordered. The study population included patients who were admitted to University of Florida Shands Medical Center, an academic institution in Jacksonville, FL in 2022. All patients were >18 years of age. Results: 190 patients were identified, for whom 190 HIT antibody tests were ordered and 75 SRAs. All patients who had a SRA performed had exposure to low dose heparin or therapeutic high dose heparin. Out of 190 patients, HIT testing was positive in 30 patients (15.8%). SRA testing was performed on 75 patients despite only 30 positive HIT tests. The true positives were 10 patients who had positive SRA confirmatory testing out of 190 patients with suspected HIT. Based on the results of confirmatory testing, the false positive fraction of HIT testing was 66%. When analyzing the 190 patients who underwent HIT testing, the average length of stay was 14 days. On average, a hospital stay in the US is 5.5 days and evaluation of HIT results in 8.5 additional days of hospital admission at our institution. The prolonged length of stay for HIT testing is likely impacted by awaiting SRA results along with other factors. Conclusion: This analysis examines the relative over-testing for HIT at a single institution. Compared with the known incidence of positive HIT tests, there is a significant number of positive HIT antibody tests at our hospital. The data further reveals a high probability of a false positive HIT antibody test results in the hospitalized patient, possibly due to immune reactivity. Of 190 patients suspected with HIT, only 10 patients had a positive confirmatory SRA, indicating that only 5% of suspected patients truly had HIT. This demonstrates that 95% of the time HIT assays ordered did not provide clinically useful information. From the time work up was initiated to the time of discharge, the average stay length was 14 days in a patient suspected with HIT. Cost burden of HIT testing was evaluated. Each HIT panel is $153.83 and each confirmatory SRA test is approximately $250, at a commercial laboratory. Taken together, the total average cost per patient with suspected HIT is $413.83 in diagnostic testing. Prolonged hospital stay contributes to approximately $25,000 increase in hospital stay per patient being evaluated for HIT. This does not include the cost of further work up or interventions. Changing to an alternate anticoagulant such as Argatroban increased costs by nearly $1,500 per case and this number increased further when adding the daily coagulation panel required while on this drug. In other studies, this cost has been estimated to add $90,000 to the hospital stay. Prolonged hospital stays are also associated with unintended incidents such as GI bleeding leading to blood transfusions, invasive testing (such as endoscopy/colonoscopy) and increased morbidity to the patient. We will use this information to implement a tool in our Electronic Medical Record (EMR) that will require answering the 4T score in order to establish high pre-test probability, prior to ordering HIT antibody testing at the University of Florida in Jacksonville.

Anti-PF4/Heparin Monoclonal Antibodies Induce Thrombus Formation in an Ex Vivo Endothelialized Microfluidic System

Introduction: Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin. Antibodies against platelet factor 4 (PF4) or a heparin/PF4 complex have been reported to activate platelets, neutrophils and possibly endothelial cells, resulting in thrombocytopenia, hypercoagulability and thromboembolic complications. While the impact of anti-PF4/heparin antibodies on platelet, neutrophil and monocyte activation has been extensively studied, the role of endothelial cells in HIT-associated thrombosis remains underexplored. In particular, the interactions between HIT-induced procoagulancy and endothelial cells under dynamic conditions is not completely analyzed. In this report, we investigated how HIT antibodies induce thrombosis in an endothelialized microfluidic system. Methods: Fibrinogen microfluidic channels were coated with confluent monolayer of human umbilical vein endothelial cells (HUVECs). Cells were primed with low-dose TNF-α to induce a sub-thrombotic endothelial activation, prior to perfusion of whole blood. Recalcified unstimulated or Thrombin Receptor Activator Peptide 6 (TRAP-6) activated whole blood was perfused at a venous flow rate. HIT-thrombosis model was established and tested with monoclonal anti-PF4/heparin antibodies in whole blood. In brief, whole blood was pre-incubated with unfractionated heparin (UFH, 0.2 IU/mL and 100 IU/mL). Next, anti-PF4/heparin antibodies were introduced to the whole blood mixture and incubated for 30 minutes. Whole blood was recalcified and perfused over resting or primed endothelial cells at a venous shear stress. Thrombus formation was recorded over time. Results: The endothelialized microfluidic model successfully captures sub-thrombotic conditions under venous shear. Activated platelets induced a procoagulant shift and three-dimensional thrombi. We applied our thrombosis model to investigate thrombus formation induced by a HIT mimicking monoclonal antibody. We show that the heparin-dependent platelet activation predicts the thrombotic response in the microfluidic system: HIT antibodies in absence of heparin did not exert a pro-thrombotic effect on activated endothelial cells. Co-incubation with 0.2 IU/mL UFH induced thrombosis, embolization of thrombus material and channel occlusion only when endothelial cells were primed with TNF-α. The pro-thrombotic effect of HIT antibodies was fully reversed at the super-therapeutic dose of 100 IU/mL UFH. Conclusions: HIT antibodies induce thrombosis and embolization in a system emulating physiological environment. For the first time, we present a comprehensive thrombosis model that incorporates the established thrombogenicity of HIT-mimicking antibodies. We show that our thrombosis model incorporates both endothelial- and blood-based control of thrombosis. Primed endothelial cells and antibody-induced procoagulancy trigger thrombosis in a concerted fashion, allowing investigation of the underlying mechanisms and possible preclinical evaluation of potential inhibitors of HIT-thrombosis. Figure 1, upper panel: Maximal thrombus surface area coverage of TNF-α primed endothelial cells, perfused with unstimulated, TRAP-6 activated or HIT-like IgG/heparin challenged whole blood. HIT-like IgG did not increase thrombus formation in absence of heparin. Addition of low-dose heparin exerted a strong pro-thrombotic effect, that was fully reversible with a super-therapeutic concentration of heparin. Lower panel: representative images of thrombi formed by HIT-like IgG in absence and under low or high-dose heparin. Platelets: green, Calcein-AM. Scale bar: 200 µm.

Low Inter-Rater Reliability of Calculating the 4T's Score for Heparin Induced Thrombocytopenia

Introduction Heparin induced thrombocytopenia (HIT) is an immune-mediated drug reaction that can cause thromboembolism in the setting of thrombocytopenia following heparin exposure. The “4T's score” has been validated to determine the pre-test probability of HIT and to assist in decision making around ordering testing for HIT. The 4T's scoring system requires an individual clinician to determine each component of the score. The objective of our study is to investigate the inter-rater reliability of calculating the 4T's score among clinicians. Methods Through retrospective query of Northwestern Enterprise Data Warehouse, we identified patients who had a HIT antibody (Ab) test ordered between 10/2019 and 10/2022 after implementation of a clinical decision support (CDS) tool that asked clinicians to calculate a 4T's score as part of HIT PF-4 Ab orders. From this cohort, an independent clinician randomly selected 15 patients. Four raters, including an attending hematologist, a hospital medicine attending, a hematology/oncology fellow, and an internal medicine resident, performed manual chart review of the randomly selected subjects to calculate a 4T's score. Data collected for each case included individual components of the 4T's score and overall 4T's score from each rater. In addition, we compared raters' scores to the 4T's score entered by the ordering clinician for the patient. We then categorized the numerical scores into validated pre-test probability categories: 0-3 as low, 4-5 as intermediate, and ≥6 as high. Inter-rater reliability for the categories was calculated using the Fleiss kappa statistic. Our study was approved by the Northwestern University Institutional Review Board. Results Of 15 cases selected, 5 each were scored as low, intermediate, and high probability 4T's scores by the ordering clinician (associated with the HIT Ab order). The overall agreement between the score categories for the 5 clinicians (4 raters and the ordering clinician) was 50.7%, with a Fleiss Kappa statistic of 0.26 (95% CI [0.07-0.45]), indicating poor inter-rater reliability (Figure 1). Excluding the ordering clinician, the overall agreement in score category between the 4 raters was 58.9%, with a Fleiss Kappa statistic of 0.38 (95% CI [0.14-0.62]). The same 4T's score pre-test probability category was calculated by the 4 raters in 5 cases, with only one case in which all four raters calculated the same numerical 4T's score. There was one case in which all 5 clinicians calculated the same 4T's score probability category, but none with the same numerical 4T's score. The hematology/oncology fellow had highest inter-rater agreement with the original clinical (Kappa 0.30 [-0.09-0.69]), whereas the internal medicine attending and resident had the lowest (both with Kappa 0.00 [-0.37-0.37]). Of the 15 4T's scores, 12/15 ordered by the hematology attending, 5/15 by the internal medicine attending, 12/15 by the hematology/oncology fellow, and 11/15 by the internal medicine resident were lower than those calculated by the original ordering clinicians. Conclusion Our study demonstrates poor inter-rater reliability of HIT 4T's score calculation, across levels of training and specialty. Importantly, poor inter-rater reliability was seen across 4T's categories, which has implications for clinical management of patients undergoing evaluation for HIT. This suggests that different strategies are necessary to help clinicians better use the 4Ts score.

Pharmacological analysis and remote monitoring of a patient with skin ulcers caused by anticoagulation

A woman in her 76 s has valvular heart disease, atrial fibrillation, and myelodysplastic syndrome. Warfarin bridged with low molecular weight heparin (LMWH), was treated for anticoagulation therapy in this patient. Five days later, The patient informed the anticoagulant management personnel from the APP of abnormal coagulation, thrombocytopenia, and ulcers, as well as pain in the left forearm pinhole, by Atrial fibrillation management mobile app software “Guidance on alfalfa anticoagulation”. Following the diagnosis of warfarin-induced skin ulceration, the patient received intravenous vitamin K and fresh frozen plasma (FFP) to reverse the effects of warfarin. After 3 days of effective treatment, the patient 's forearm ulceration improved and healed soon. This case highlights the importance of telemedicine in patients with anticoagulant therapy. And warfarin should be used with caution in patients with HIT, HIT antibodies detection should be requested in patients presenting withthrombocytopenia after using of heparin. We suggest that medical staff should provide the remote medical and remote drug monitoring for patients with chronic diseases and those taking high-risk drugs.

ELISA-Negative Platelet-Activating Anti-PF4 Antibodies Can Cause HIT and Monoclonal Gammopathy of Thrombotic Significance (MGTS)

We identified a patient with a recently described syndrome, monoclonal gammopathy of thrombotic significance (MGTS; Blood. 2023 Apr 6;141(14):1772-1776), and three additional patients with high 4Ts scores who were consistently negative in the PF4-polyanion HIT ELISA (Lifecodes PF4 IgG, Immucor). The MGTS patient, with a monoclonal IgG kappa, had recurrent thrombosis, and transient mild thrombocytopenia (nadir in the low 100s) over a period of years, and was noted to have a HIT ELISA optical density (OD) of 0.117. The three HIT patients experienced thrombocytopenia and thrombosis with ELISA ODs of 0.37, 0.18 and 0.20. All patients were positive in platelet-activation (functional) testing demonstrating PF4 dependence and inhibited with both high concentrations of heparin (100U/mL) and the FcγRIIa blocking monoclonal antibody (mAb) IV.3, consistent with the presence of anti-PF4 antibodies in the patient samples ( data not shown). Because the serologic profile of these patients suggested a novel HIT subtype characterized by negative HIT ELISA results, we undertook a functional platelet assay screen of 500 consecutive HIT ELISA-negative patients tested in our diagnostic laboratory. Research studies were approved by the Institutional Review Board of Mayo Clinic. Of the 500 patients tested, 20 (4.0%) stimulated platelet activation in the PF4-dependent P-selectin Expression Assay (PEA ≥19%; data not shown). To exclude non-specific activation that can be induced by HLA antibodies, additional testing was performed to evaluate whether platelet activation was PF4-dependent using buffer or PF4-treated platelets. Three patients demonstrated PF4-dependent activation that was inhibited by high heparin concentrations and mAb IV.3, as would be expected for HIT antibodies (ELISA-negative HIT, EN-HIT1-3; Figure 1). On retrospective review, all three patients thus identified had clinical histories and courses consistent with high clinical probability for HIT. To develop tools to study this new class of antibodies, monoclonal HIT antibodies were generated in mice after PF4/heparin immunizations and screened using a novel approach that included simultaneous testing of hybridoma supernatants in the PEA and PF4-polyanion ELISA. This strategy was designed to enabled us to identify mAbs that activated platelets (in the PEA), but that did not bind antigenic targets in the HIT ELISA. A novel mAb 431.4 that was developed using this approach, and the well-characterized HIT-like mAb KKO were evaluated for their abilities to both bind PF4-polyanion targets in ELISA, and to activate PF4-treated platelets in the PEA (Figure 2). In significant contrast to KKO, mAb 431.4 did not bind PF4/polyanion complexes, however, like KKO, strongly activated platelets in a PF4-dependent manner. Activation induced by both KKO and mAb 431.4 was inhibited by high heparin concentrations, as would be expected for HIT antibodies. In vivo murine studies using this novel mAb are pending at this time. In summary, the non-canonical diagnostic profile of HIT ELISA-negative but platelet activation-positive antibodies were found at a relatively high incidence in HIT-suspected patients (0.6%; 3 of 500 ELISA-negative patients). These findings have important diagnostic/management implications and suggest a re-evaluation of ASH's HIT management guidelines as noted in recommendations 2.7 and 2.8 of that document (“In patients with an intermediate (Recommendation 2.7) or high (Recommendation 2.8)-probability 4Ts score and a negative immunoassay, the ASH guideline panel recommends discontinuation of the non-heparin anticoagulant and resumption of heparin, if indicated ”; Blood Adv (2018) 2 (22): 3360-3392). Figure 1. A subset of ELISA-negative HIT-suspected patients harbor anti-PF4 platelet-activating antibodies. Three ELISA-negative (EN) HIT patient samples from a screen of 500 samples demonstrate PF4-dependent platelet activation and inhibition with high heparin concentration and mAb IV.3 incubation. PBS, phosphate buffered saline; IC, isotype control; HDH- high dose heparin (100U/mL). Figure 2. Murine mAb 431.4 serologically mimics pathogenic ELISA-negative HIT/MGTS antibodies. ELISA and PEA testing was performed using the “HIT-like” mAb KKO, novel mAb 431.4, or an isotype control (IC) antibody. PVS-polyvinyl sulfonate (polyanion used in the Immucor IgG ELISA assay).

3Ts Score, an Attempt for Less Testing in Thrombocytopenia Patients with Suspected Heparin Induced Thrombocytopenia (HIT): Retrospective Analysis in a University Hospital Setting

Background: Heparin-induced thrombocytopenia (HIT) is a prothrombotic and potentially fatal adverse drug reaction mediated by platelet-activating antibodies against multimolecular complexes of platelet factor 4 (PF4) and heparin. The 4Ts is a pretest scoring system for HIT that was developed to improve and standardize clinical diagnosis. The strength of the 4Ts score lies in the negative predictive value of a low pretest probability score. Those with a 4T score ≥4 is considered to have moderate to high probability of HIT and should undergo confirmatory laboratory test. Delays in treatment are associated with risk of thrombosis. Immediate cessation of heparin and initiation of a non-heparin anticoagulant is therefore standard of care. Method: This is a single institutional retrospective study. We reviewed the charts of 219 patients with thrombocytopenia with suspected HIT to evaluate the appropriateness of ordering the HIT testing based on the pretest probability of the 4Ts scoring system. All patients had enzyme linked immunosorbent assay (ELISA) test for HIT antibodies (the screening test). We studied tests that were sent from 03\16\2021 through 07\03\2023. The serotonin release assay (SRA) which is the confirmatory test for HIT was only sent for patients who had a positive ELISA test. 4T scores were calculated retrospectively using chart's information available to clinicians at the time of the HIT tests were sent. Results: Out of the 219 patients, 80(36.5%), 120(54.7%) and 19(8.6%) patients had a low, intermediate and high pretest probability respectively. The HIT ELISA test was positive in 33 (15.7%) patients and 196 (84.9%) patients had negative results. HIT ELISA had 100% and 93.5% sensitivity and specificity respectively while the positive and negative predicative values were 60% and 100% respectively. A positive SRA test was reported in 23 patients (10.5% of the total study population). Seventy-nine patients (36% of our total sample) scored 4 points which represents intermediate pretest probability. Out of these 79 patients only 6 (11.3%) patients had a positive ELISA test, and 5 (6.3%) patients had a positive SRA test. Aiming to minimize the HIT over testing we examined the increase in the cutoff point of the intermediate pretest probability from 4 points to 5 points which resulted in significant increase in the sensitivity and positive predictive value of the intermediate pretest probability (75.4% and 21% versus 39% and 12.9% respectively). However, the sensitivity dropped from 90% to 65%. The 4th part of 4Ts score gives points based on the probability of HIT versus other pathology as a cause of the thrombocytopenia. Most of the patients with suspected HIT will score one point out of the 4th part of 4Ts score which makes this part of the score is less helpful in distinguishing between patients with suspected HIT and will result in higher overall pretest probability and more testing. We studied the 3Ts score (the 4Ts scoring system after elimination of the 4th part) effect on the intermediate pretest probability. The cutoff points for the 3Ts score are 1-3, 4-5 and 6 for low, intermediate and high pretest probability respectively. The 3Ts score model resulted in significant increase in the specificity and the positive predictive value (76% and 21.7% versus 39.2% and 12.9% respectively) however the sensitivity dropped from 90% to 65%. The average turnaround time for HIT ELISA test and the SRA test was 2.3 days and 4.4 days respectively. Fifteen patients (21% out the patient had low pretest probability on the 4Ts score), 41 patients (59% of the patient who had an intermediate pretest probability on the 4Ts score) and 13 patients (18.8% of the patients who had high pretest probability on the 4Ts score) started on non-heparinoid anticoagulation meanwhile awaiting on the HIT testing result. Conclusion: Over testing of HIT is common secondary to underuse of 4Ts scoring system. Multicomponent educational interventions to increase the awareness and utility of the 4Ts score in addition to implantation of clinical decision support based on HIT computerized score will help to reduce unnecessary diagnostic testing of HIT in patients with low pretest probability. Increasing the cutoff point of the intermediate pretest probability or applying the 3Ts score model resulted in significant increase in the specificity and the positive predictive value of the 4Ts scoring system on expense of the scoring system sensitivity.

Improving Judicious Use of Heparin-Induced Thrombocytopenia Testing Through Electronic Health Record-Based Intervention.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated drug reaction that can cause thromboembolism in the setting of thrombocytopenia. An enzyme-linked immunosorbent assay (ELISA)-based assay to screen for HIT antibodies (HAb) is available but has relatively low specificity and a correspondingly high false positive rate. The 4Ts score has been validated to determine the pretest probability of HIT. The authors hypothesized that an electronic health record (EHR)-based clinical decision support (CDS) tool incorporating the 4Ts score would reduce the volume of HAb orders. After implementing a CDS tool into the EHR, the researchers retrospectively evaluated the impact from November 2019 to October 2021, compared to a preintervention period (January to October 2019). The primary outcome was average tests per month. Secondary outcomes included rates of tests ordered per total inpatient encounters and proportion of HAb sent despite low 4Ts score in the postintervention study period. Of 1,833 HAb sent during the study period, 1,217 occurred in the postintervention period. In the postintervention period compared with the preintervention period, the average orders per month was 50.5 (standard deviation [SD] 9.7) vs. 61.6 (SD 7.2) (p = 0.003), and the order incidence rate was 8.0 per 1,000 patient encounters postintervention vs. 9.2 per 1,000 patient encounters preintervention (rate ratio [RR] 0.87, 95% confidence interval [CI] 0.79-0.96, p = 0.002). Postintervention, 252 (20.7%) had a 4Ts score calculated as low probability, 759 (62.4%) as intermediate probability, and 131 (10.8%) as high probability, and 75 had no associated 4Ts score. Implementation of a simple CDS tool reduced the rate of HAb orders, reducing unnecessary HAb testing.

Cost-Effectiveness and Return on Investment Analysis of an In-house HemosIL Heparin-Induced Thrombocytopenia Antibody Assay at a Mid-Sized Institution.

Laboratories face the challenge of providing quality patient care while managing costs and turnaround times (TATs). To this end, we brought the heparin-induced thrombocytopenia (HIT) antibody test in-house with the goal of reducing costs and the time to diagnosis. To determine the cost-effectiveness and return on investment of our in-house HIT antibody test by comparing it to send-out assays with TATs of 2, 3, or 4 days. We performed a retrospective chart review of all patients with a HIT antibody assay and analysis of laboratory financial records. Analysis included the percentage of patients receiving alternative treatment, cost of treatment, startup costs of bringing the test in-house, and average TAT of the in-house test. We found significant reductions in the cost of treatment for patients and the overall cost to the health care system. The in-house assay became cost-effective at between 8 and 20 tests, with a return on investment of up to 298%. Bringing the HIT antibody assay in-house becomes cost-effective at a very low test volume with excellent return on investment. This novel analysis can provide a framework for other laboratory medicine professionals to analyze the benefits of bringing this and other assays in-house.

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

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

Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

AbstractHeparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4–polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4–polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

Platelet factor 4 triggers thrombo-inflammation by bridging innate and adaptive immunity.

Platelet factor 4 (PF4, synonym: CXCL4) is an evolutionary old chemokine with proposed roles in hemostasis and antimicrobial defense. In addition, PF4 has attracted considerable attention as a crucial mediator of one of the most prothrombotic adverse drug effects affecting blood cells, heparin-induced thrombocytopenia (HIT). Interest in PF4 substantially increased in 2021 when it was identified as the target antigen in the life-threatening adverse effect, vaccine-induced immune thrombotic thrombocytopenia (VITT). We address the concept that a major biological function of PF4-a strongly cationic chemokine-is to bind to negatively-charged prokaryotic microorganisms, resulting in structural changes in PF4 that trigger a danger signal recognized by the adaptive immune system. Application of biophysical tools has provided substantial insights into the molecular mechanisms by which PF4 becomes immunogenic, providing insights into a new mechanism of autoimmunity. Binding of autoantibodies with high affinity induces conformational change(s) in the endogenous protein, which are then recognized as foreign antigen, as exemplified by the prothrombotic disorders, autoimmune HIT and VITT. The final part of our review summarizes current assays for HIT and VITT, explaining how structural aspects of anti-PF4 pathobiology relate to assay design and performance characteristics. Currently, functional (platelet activation) assays using washed platelets detect HIT antibodies when heparin is added, and VITT antibodies when PF4 is added. Solid-phase PF4-dependent immunoassays using microtiter plates are sensitive for both HIT and VITT antibodies, while rapid immunoassays, in which the PF4/heparin antigen is coated on beads, are sensitive and specific for HIT, but not for VITT antibodies.

Analysis of heparin-induced thrombocytopenia diagnostic and management strategies in individuals with inconclusive antibody optical densities.

Heparin-induced thrombocytopenia (HIT) is an uncommon but serious complication of exposure to heparin. Antibody optical densities (ODs) used to diagnose HIT exceeding 2 are highly suggestive of disease, whereas ODs less than 0.5 often 'rule out' HIT. Variation in the clinical care of patients with inconclusive ODs between 0.5 and 2 is likely. This single-centre, retrospective analysis evaluates the diagnosis, management and outcomes of those with antibody ODs between 0.5 and 2. We queried our institution's Healthcare Enterprise Repository for Ontological Narration (HERON) database to identify individuals with antibody ODs between 0.5 and 2. Chart review was completed to calculate 4T scores, corroborate diagnosis codes with documented information in our electronic health record (EHR) and evaluate the diagnosis, management and outcomes of these individuals. These data were evaluated using descriptive and univariate statistics. Among individuals evaluated for HIT between November 2007 and July 2020, we identified 302 individuals with ODs between 0.5 and 2. Serotonin release assays (SRAs) were assessed in 55% (165/302) and were positive in 12% (20/165). In those with available data, 96% with low 4T scores had negative SRAs and 4% had positive SRAs. As 4T scores and antibody ODs proportionally increased, SRA positivity also increased. Clinical management varied widely; however, 4T scoring remains a valuable assessment in this cohort. In those with HIT antibody ODs between 0.5 and 2, true positives were uncommon, and their clinical management varied widely. Fortunately, 4T scoring is a useful prognostic tool that improves the diagnosis and management among those with inconclusive HIT.

Heparin-Induced Thrombocytopenia in Chronic Hemodialysis Patients.

Heparin-induced thrombocytopenia (HIT) is a disorder originating from exposure to heparin and has a spectrum of presentation ranging from asymptomatic positive antibodies to thrombotic complications. When symptomatic, it is associated with morbidity and mortality. The incidence of HIT in the ESRD population is yet to be defined. End-stage renal disease (ESRD) patients are at particular risk due to constant exposure to heparin. The main treatment of HIT is to avoid heparin and pursue alternative anticoagulants. Since 1 of the main advantages of heparin in ESRD patients is the ease of its use due to non-renal clearance, the use of alternative anticoagulants poses yet another challenge for this population due to cost, availability, and adverse effects on ESRD patients. Argatroban seems like the best alternative to heparin in hemodialysis (HD) patients due to its liver clearance. Despite having limited studies in HIT, direct oral anticoagulants (DOACs) were added as a potential treatment for HIT, with apixaban favored in kidney dysfunction as it is the least dependent on kidney clearance. Other treatment modalities exist but are still being studied in ESRD patients. The presence of HIT antibodies is not always associated with clinical syndrome, and some studies suggested that heparin antibodies are transient, and the reintroduction of heparin is still being evaluated as a treatment option. Hence, HIT is a challenging diagnosis in ESRD patients, a population that has frequent exposure to anticoagulants, and a risk/benefit ratio should be weighed between the risk of progression to symptomatic HIT and the benefit of switching to a non-heparin anticoagulant bearing in mind the difficulties associated with the latter.

High-frequency Contactless Sensor for the Detection of Heparin-Induced Thrombocytopenia Antibodies via Platelet Aggregation.

Heparin-induced thrombocytopenia (HIT), a severe autoimmune disorder, occurs in patients undergoing heparin therapy. The presence of platelet-activating antibodies against platelet factor 4/Heparin in the blood confirms patients suffering from HIT. The most widely used methods for HIT diagnosis are immunoassays but the results only suit to rule out HIT as the assays provide only around 50% specificity. To confirm HIT, samples with positive results in immunoassays are retested in functional assays (&gt;98% specificity) that track platelet-activating antibodies via platelet aggregation. However, the protocols in functional assays are either time-consuming (due to the requirement of the detection of serotonin release) or require highly trained staff for the visualization of platelets. Here, we applied a cheap and easy-to-use contactless sensor, which employs high-frequency microwaves to detect the changes in the resonant frequency caused by platelet aggregation/activation. Analysis of change in conductivity and permittivity allowed us to distinguish between HIT-like (KKO) and non-HIT-like (RTO) antibodies. KKO caused a stronger reduction of conductivity of platelet samples than RTO. Our results imply that the high-frequency contactless sensor can be a promising approach for the development of a better and easier method for the detection of HIT.

"Autoimmune HIT", a Potential Diagnostic Artifact Due to Remnant Heparin in HIT Samples, Is Not Associated with More Severe Outcomes

In recent years, there has been increasing use of the term "autoimmune" heparin-induced thrombocytopenia (HIT) to describe patients with HIT antibodies that activate platelets in the absence of exogenous heparin (i.e., the "zero heparin" serotonin release assay, SRA), with the proposed implication that such patients present with more severe phenotypes. Platelet activation in the SRA in the absence of exogenous heparin is likely a real phenomenon in rare/infrequent patients who did not receive proximate heparin therapy (Spontaneous HIT) and in patients who develop HIT after heparin treatment has ceased (delayed-onset HIT). However, in other patients deemed to have "autoimmune" HIT based on reactivity in the "zero heparin" SRA, we hypothesized that platelet reactivity in this SRA test system might result from remnant heparin in the patient's diagnostic blood sample. To further complicate the interpretation of autoimmune HIT testing, there is a lack of rigorous data demonstrating a correlation between this diagnosis and disease severity. This study was undertaken to evaluate the technical validity and clinical relevance of an autoimmune HIT diagnosis. First, thirty "real-world" diagnostic HIT patient samples were subjected to anti-Xa testing. Remarkably, fourteen of the 30 samples (47%) had detectable heparin, with a range of 0.11-0.55 IU/mL for thirteen samples, while one sample had heparin <0.1 IU/mL (data not shown due to Abstract 2-figure panel limit). Next, to determine the extent to which low concentrations of unfractionated or low molecular weight heparin spiked into patient samples impact reactivity in the SRA, spiked samples were tested in the "conventional" low dose heparin (0.1 IU/ml) SRA and the "zero heparin" SRA (autoimmune HIT test). Fig 1A demonstrates that even ultra-low concentrations of unfractionated heparin (0.06 IU/mL) spiked into the sample facilitate serotonin release in the "zero heparin" SRA. On the other hand, the conventional SRA was not impacted by heparin spiked into samples unless concentrations were 5 IU/mL or higher, several-fold greater than what would be seen in patient samples. Similarly, LMWH induced platelet reactivity in the zero heparin SRA at concentrations between 0.25-1.0 IU/mL, while there was no effect on the conventional SRA at these concentrations (data not shown). As proof-of-concept that low levels of heparin mediate reactivity in a zero-heparin functional test, a HIT patient sample devoid of heparin was spiked with 0.05 IU/ml of heparin and evaluated with and without digestion with heparinase I. Heparin-spiked samples induced platelet activation in a zero-heparin functional assay which was abrogated upon heparinase I digestion (data not shown). Together, these results are consistent with the hypothesis that at least some cases of "autoimmune HIT" are labeled as such due to residual heparin present in patient samples rather than due to the presence of a qualitatively different ("autoimmune") HIT antibody. To evaluate the clinical relevance of an "autoimmune" HIT diagnosis, platelet nadir and the presence of thrombosis were interrogated as markers of disease severity in a cohort of 48 HIT patient samples with unknown heparin levels. Thirty-six patients were positive in the zero heparin SRA ("Autoimmune HIT" cohort; >50% serotonin release), while 12 were negative ("Classical HIT" cohort; positive in the conventional SRA, but <50% serotonin release in the zero heparin SRA). Fig 1B demonstrates that there was no difference in platelet nadir between the two groups (p=0.562); further, there were no differences between these groups in rates of confirmed thrombosis/skin necrosis and acute system reactions (p=0.324; data not shown). In summary, "autoimmune HIT" diagnostic testing (zero-heparin SRA) should not be performed until samples are confirmed to be devoid of heparin, and rigorous large-scale studies are needed to evaluate whether results of validly-conducted autoimmune HIT tests correlate with disease severity. Figure 1. (A) Low concentrations of spiked heparin in a HIT patient sample facilitates platelet activation in the "autoimmune" HIT test (zero-heparin SRA; red line) but does not affect the conventional SRA (black line). (B) Platelet nadir was similar between "autoimmune" (n=36) and conventional HIT (n=12) cohorts. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Profiling of Heparin-Induced Thrombocytopenia Antibodies in Thai People Who Received ChAdOx1 Ncov-19 Vaccination

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but fatal complication characterized by unusual site thrombosis followed by thrombocytopenia after the ChAdOx1 nCoV-19 vaccine. Heparin-induced thrombocytopenia antibodies or anti-platelet factor 4 (anti-PF4) antibodies have been identified as pathogenic antibodies in almost all patients. Data on anti-PF4 antibodies in Asian countries was still scarce. Objectives: To determine the prevalence of anti-PF4 antibodies in the Thai population after administering the ChAdOx1 vaccine and the effect of the ChAdOx1 vaccine on anti-PF4 antibodies. Methods: This study was a prospective cohort study. Healthy adults with aged ≥ 18 years were enrolled. Participants with thrombocytopenia (< 150 x 109/L), anticoagulant use, an immunocompromised state, or a recent major surgery were excluded. Blood samples were collected before and 4-6 weeks after the first dose of the ChAdOx1 vaccine. Participants with detectable anti-PF4 antibodies after the first dose of the ChAdOx1 vaccine were evaluated for clinical thrombosis, platelets, and D-dimer. They were also scheduled for the third blood sample to repeat anti-PF4 antibodies after 3 months of the second dose of the ChAdOx1 vaccine. Anti-PF4 antibodies were identified using Zymutest® HIA, IgG ELISA (Hyphen Biomed, Neuville-sur-Oise, France). We defined the negative cutoff optical density (OD) value was ≤ 0.3. The OD values of > 0.3 to 0.5, > 0.5 were assigned as weakly positive, and positive, respectively. Results: A total of 396 participants were enrolled with a median age of 50 years (range 18-87). Of which, 65.91% were female and 8% were aspirin users. Twelve participants had detectable anti-PF4 antibodies after the first dose of the ChAdOx1 vaccine, given the prevalence of 3.03% (95% confidence interval [CI], 1.58-5.23), which was 3 participants (0.76%) in the weakly positive group. Ten of twelve participants (83.33%) had detectable anti-PF4 antibodies before the first dose of the ChAdOx1 vaccine. For participants with detectable anti-PF4 antibodies, only one participant (8.33%) had D-dimer greater than 500 ng/ml. There was no participant who had platelet counts less than 150x109/L. There was no significant difference of OD value in participants with detectable antibodies among pre-vaccination, 4-6 weeks after the first dose, and 3-month after the second dose of vaccine (Figure 1.). There were no participants with signs and symptoms of thrombosis after 1-month after both first and second dose of the ChAdOx1 vaccine. Conclusion: The prevalence of pathogenic anti-PF4 antibodies and VITT after the ChAdOx1 vaccine was low in Thai people. There was minimal effect of the ChAdOx1 vaccine on the OD value of anti-PF4 antibodies, indicating that anti-PF4 antibodies should be tested only when VITT is suspected. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal