Jones, Curtis G.

ABSTRACT Background Platelet factor 4‐polyanion enzyme‐linked immunosorbent assays (ELISAs) are considered highly sensitive for diagnosing heparin‐induced thrombocytopenia (HIT), such that current practice guidelines recommend use of ELISA‐negative results to exclude HIT. Once HIT is ruled out, alternative, non‐heparin‐based anticoagulant treatments are ceased, and heparin reintroduction frequently occurs. Methods Antigen‐based and PF4‐dependent functional testing were used to study PF4/polyvinylsulfonate ELISA‐negative platelet‐activating antibodies in HIT‐suspected patients. Results Three patients with clinical presentations consistent with HIT tested negative in an ELISA using PF4‐polyvinylsulfonate (PF4/PVS), an antigenic target very commonly used for HIT antibody screening. All three patients demonstrated PF4‐dependent platelet activation in functional testing that was sensitive to blockade of platelet FcγRIIa receptors and inhibited by high concentrations of heparin, consistent with pathogenic HIT antibodies. Functional testing‐based screening of 500 ELISA‐negative patients identified three additional patients whose sera activated platelets in a PF4‐ and FcγRIIa‐dependent manner and had clinical histories consistent with HIT. Five of the six ELISA‐negative HIT patients were re‐exposed to heparin, which precipitated a decrease in platelet counts in all re‐exposed patients, and one patient developed a new thrombus. Conclusions Recognition of ELISA‐negative HIT is critical to avoid harm due to the cessation of alternative anticoagulation therapy and re‐exposure of these patients to heparin.

Introduction Intravenous Immunoglobulin G (IVIg) is a pooled antibody Immunoglobulin G product that is used for a large and ever-growing number of medical indications. It is also known to be associated with an increased risk of thromboembolic complications such that the Food and drug administration (FDA) has mandated a “black box” warning alerting physicians to this risk. Particular lots of IVIg preparations have been recalled due to a higher-than-expected rate of thromboembolic adverse events (“Evaluation of the Risk of Thromboembolic Events After Immunoglobulin Administration, 2006-2012”. Sentinel Product Assessment, US FDA, 7/13/2017). While a number of mechanisms have been proposed to explain the thromboembolic risk, platelet activation induced by small amounts of aggregated IgGs in IVIg preparations remains the leading hypothesis. Currently, testing of IVIg lots specifically looking at their ability to induce platelet activation is not routinely performed due to challenges with acquisition of fresh platelets for testing, and lack of availability of technically simple platelet activation endpoints. Recent breakthroughs in platelet storage technology have demonstrated that platelet viability can be preserved for long periods (months-years) by controlled-rate cryopreservation which also allows platelets to retain FcγRIIa-mediated signaling (FcγRIIa is the IgG receptor on platelets). This technology has been successfully leveraged for the detection of platelet-activating heparin-induced thrombocytopenia (HIT) and Vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies (Blood, 140 (25) 2722-2729, 2022). Goals of the study We hypothesized that long-term stored cryopreserved platelets could be used to detect platelet-activating IVIg preparations using a technically simple ELISA endpoint that measures levels of thrombospondin-1 (TSP-1), a platelet granule protein, released upon platelet activation. If successful, this tool could help de-risk IVIg from the standpoint of thrombotic adverse reactions. Methods Aggregated IgG was prepared by heat treatment of IVIg (Pel-Freez- Rogers, AR) as previously described (Immunol. Lett. (15) 311-316, 1987). Platelets were cryopreserved as previously described (Blood, 140 (25) 2722-2729, 2022) with minor modifications. Cryopreserved platelets were thawed and treated with heat-aggregated or untreated IVIg at concentrations ranging from 19 to 75mcg/mL. After incubation, supernatants were collected by centrifugation and were assayed for TSP-1 by an antigen capture ELISA which has been shown previously to correlate with platelet activation. Results 10-month-old “off-the-shelf” cryopreserved platelets treated with buffer control displayed minimal platelet activation as measured by the TSP1 ELISA (Optical density, OD: 0.21). Similarly, platelets treated with IVIg (non-aggregated IgG) at concentrations ranging from 19 to 75mcg/mL displayed little-to-no platelet activation in the TSP1 ELISA (OD: 0.22-0.29). In contrast, cryopreserved platelets treated with heat aggregated IVIg demonstrated marked platelet activation in the TSP1 ELISA with ODs in the 0.60-0.82 range, correlating with increasing concentrations of heat-aggregated IVIg in the reaction. Conclusions De-risking IVIg from a prothrombotic standpoint has been challenging due to the need for fresh platelets for testing and the lack of technically simple means to assess platelet activation. Here, we leveraged breakthrough platelet storage technology coupled to a technically simple ELISA endpoint that allowed for rapid and easy identification of platelet activating forms of IVIg. This easy-to-use self-contained test kit could be implemented as a quality control/patient safety tool to de-risk IVIg preparations from the standpoint of platelet activation-mediated thrombosis. Future studies aim to test a large number of IVIg lots, including “problem” lots associated with thromboembolic events to assess the incidence of platelet activating IgG molecules in IVIg preparations. Acknowledgments This work was supported in part by a grant from the National Institutes of Health (C.G.J.: HL147734).

Monoclonal gammopathy of thrombotic significance (MGTS) is a recently recognized thrombophilia characterized by the production of a monoclonal antibody that binds to platelet factor 4 (PF4) and stimulates platelet activation (Blood. 2022 Jul 7;140(1):73-77). Typical signs of MGTS include a history of recurrent thrombosis, persistent thrombocytopenia, and positive laboratory results in PF4/polyanion enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA) testing, as in the related thrombotic disorder heparin-induced thrombocytopenia (HIT). Here, we present a case of MGTS, presenting with transient thrombocytopenia coincident with thrombotic episodes and negative test results in SRA and PF4/polyanion-based testing platforms (ELISAs and automated assays). A 56-year-old male patient initially presented with acute left lower extremity deep vein thrombosis (DVT) and a pulmonary embolism. Following the administration of warfarin, the patient experienced a second DVT four months later and was transitioned to a factor Xa inhibitor. Fifty-two months later, the patient developed occlusion of the splenic and superior mesenteric veins despite additional anticoagulation therapy. Further testing led to a diagnosis of an IgG2 kappa monoclonal gammopathy of undetermined significance (MGUS). Eleven months later, the patient again suffered breakthrough thrombosis, specifically radial artery thrombosis, and a thrombotic cerebrovascular accident, leading to the addition of aspirin to the patient's treatment regimen. Notably, where detailed blood counts were available in the patient's clinical history, the patient experienced only transient thrombocytopenia coinciding with his two most recent thrombotic episodes and has had platelet counts in the normal range in the four-year period since his last thrombotic event. After initiating dual anticoagulant/antiplatelet therapy, the patient has not developed recurrent thrombocytopenia or new thrombotic events. Antigen-based and functional testing for HIT antibodies (ELISA, SRA, and automated immunoassays) were negative in this patient, unlike in the previously described index case of MGTS, where serological testing mimicked classical HIT serology (i.e., HIT ELISA and SRA positivity). The sample was also non-reactive in a vaccine-induced immune thrombotic thrombocytopenia (VITT)-specific ELISA employing uncomplexed PF4 targets, as previously described (Am J Hematol. 2022 May;97(5):519-526). Conversely, the patient's antibody consistently stimulated platelet activation in a PF4- and IgG-dependent manner in functional testing using PF4-treated platelets. This included PF4-treated platelets that were freshly isolated and used in the PF4-dependent P-selectin expression assay (PEA) and cryopreserved platelets used in the thrombospondin-1 release assay. Interestingly, consistently positive PEA results have been documented from four samples over the last four years, during which the patient had normal platelet counts. To further characterize the patient's IgG2 MGUS antibody, the patient's monoclonal was immuno-enriched. Patient sera and the immuno-enriched MGUS were then compared by mass spectrometry, which generated identical mass profiles. After confirming the effective enrichment of the patient's MGUS by mass spectrometry, the antibody was de novo sequenced or “reverse-engineered” to obtain the antibody's amino acid sequence. The antibody was then expressed and tested in ELISA-based and functional platelet assays. Importantly, the recombinant antibody replicated the PF4-dependent platelet activation observed with patient serum and did not bind PF4/polyanion targets in ELISA testing. Together, these data strongly support the identification of a new class of ELISA- and SRA-negative anti-PF4 monoclonal antibodies that cause MGTS. This case illustrates the diagnostic challenges in MGTS. MGTS patients may not exhibit persistent thrombocytopenia, and diagnostic tests for detecting HIT antibodies may be ineffective in accurately diagnosing this new thrombophilia. This case emphasizes the importance of reviewing platelet counts in the peri-thrombotic period to recognize transient thrombocytopenia and establishes the utility of PF4-enhanced functional assays to detect pathogenic anti-PF4 antibodies, even in settings where classical HIT testing (i.e., ELISA/SRA) is negative.

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).

AbstractHeparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

AbstractHeparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Abstract Background: Platelet-activating anti-PF4 antibodies that cause thrombocytopenia and thrombosis are implicated in Heparin-induced thrombocytopenia (HIT) and, more recently, in a newly recognized entity, vaccine-induced immune thrombotic thrombocytopenia (VITT). Current frontline PF4-Polyanion ELISAs utilize simple techniques but are highly non-specific. Functional gold-standard assays such as the serotonin-release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have limited availability due to technical complexity and the requirement for freshly-drawn normal donor platelets which results in significant delays in diagnosis. Thus, there is an unmet need for near-patient, functional assays for HIT/VITT diagnosis. Aim: To develop a highly sensitive and specific functional assay to detect platelet-activating antibodies associated with HIT and VITT using long-term stored platelets. The assay should be technically simple, avoiding current functional HIT testing endpoints such as aggregometry, radioactivity measurement or flow cytometry. Methods: Platelets were subjected to controlled-rate freezing in a trehalose-based buffer. Cryopreserved platelets were then thawed after variable storage periods, incubated with platelet factor 4 (PF4), and sera from HIT or VITT suspected patients. Thrombospondin-1 (TSP1), a protein highly expressed in platelet α-granules, was quantified from activated platelet supernatants in an ELISA-based assay as a measure of platelet activation. Results: In initial studies, p-selectin expression was induced by platelet-activating HIT antibodies using cryopreserved platelets and the TSP1-release assay (TRA) detected HIT antibody-mediated PF4-dependent cryopreserved platelet activation across 20 different platelet lots (data not shown). A representative study using platelets stored for 11 months is shown in Figure 1A. A cohort of thirty-six HIT-suspected patient sera, half with PF4-dependent platelet-activating antibodies (PEA+) and without (PEA-), were used to evaluate the diagnostic accuracy of the TRA (Figure 1B). All 18 PEA+ samples activated platelets strongly with a mean TSP1 concentration of 147 µg/mL measured in the supernatant compared to a mean of 43 µg/mL obtained with non-activating samples. TSP1 release in a subgroup of 7 PEA+/SRA+/ELISA+ and 4 PEA-/SRA-/ELISA+ samples that were matched for ELISA Optical Density was examined (Figure 1C). Results demonstrated that even strong ELISA+ samples that were non-activating (PEA-/SRA-) did not induce platelet activation in the TRA, confirming high specificity of the assay. The TRA was tested for its ability to detect platelet-activating antibodies in four patients who developed VITT after the Janssen vaccine (Ad26.COV2.S). All samples induced high levels of TSP1 release from cryopreserved platelets (Figure 1D). Conclusions: Our results demonstrate that cryopreserved platelets stored for several months are viable and capable of degranulation upon activation. Further, we demonstrate that measurement of TSP-1 released from cryopreserved, PF4-treated platelets is highly accurate for detecting platelet-activating HIT and VITT anti-PF4 antibodies. Specificity and sensitivity were both 100% even with samples that were strongly false-positive in the HIT ELISA. Coupling cryopreserved platelet activation to a simple ELISA endpoint, as demonstrated here, precludes the need for fresh platelets and complex testing techniques. Thus, the TRA has the potential to transform the diagnostic testing paradigm in HIT and VITT by making near-patient in-hospital functional testing available for rapid and accurate diagnosis. Figure Legend: Figure 1. TSP-1 quantification of (A) 3 HIT patient samples (closed circles) relative to 3 normal control samples (open circles) after 11-months of frozen platelet storage. (B) Thirty-six HIT-suspected patient samples: 18 PEA+ samples (closed circles), and 18 PEA- samples (open squares). Data are compared using two-tailed, unpaired Student's t test. **** P<0.0001. (C) Eleven high optical density matched HIT ELISA patient sera: 7 PEA+/SRA+ samples (closed circles) and 4 PEA-/SRA- samples (open squares). (D) TSP-1 quantification of VITT patient sera. NC-normal control. Figure 1 Figure 1. Disclosures Pruthi: Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare AG: Honoraria; Genentech: Honoraria; CSL Behring: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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

Abstract Introduction: Heparin-induced Thrombocytopenia (HIT) is a potentially life and limb threatening pro-thrombotic syndrome triggered by antibodies to platelet factor 4 (PF4)/polyanion complexes. Commonly used screening tests such as the PF4/Heparin ELISA (PF4 ELISA), while easy to perform in the near-patient setting, are highly non-specific. In contrast, more accurate tests such as the serotonin release assay (SRA) are technically complex, require radioactivity and are performed in only a handful of reference laboratories. These diagnostic challenges lead to both unnecessary anticoagulant use as well as avoidable cases of severe bleeding. Recent reports suggest that a relatively simple three-hour flow cytometry-based test that uses PF4-treated normal platelets, the PEA (PF4-dependent p-selectin Expression Assay), is at least as accurate as the SRA for identifying patients who have HIT (Thromb Haemost. 2015 Nov 25;114(6):1322-3; Chest. 2016 Sep;150(3):506-15; Chest. 2017 Oct;152(4):e77-e80). Here we describe results of a large prospective study designed to compare the accuracy of the SRA and PEA for HIT diagnosis in a clinical setting. Methods: The PEA and SRA were performed on serum samples obtained from adult patients (≥18 yrs.) from 2 large tertiary care academic hospitals from May 2016 to Sep 2017. All inpatients for whom PF4 ELISA testing was ordered were included in the study. The PEA was performed with platelets pretreated with PF4 at 3 concentrations: 30mcg/mL (PEA30), 10mcg/mL (PEA10) and 3mcg/mL (PEA3). Sera were batch-tested in both assays (SRA and PEA) using the same target platelets. The central laboratory was blinded to clinical history and to the results of the PF4 ELISA performed by participating hospitals at the time of diagnosis. Both centers performed IgG-specific PF4 ELISAs and utilized manufacturer-recommended cut-offs for positivity. Predefined "more-stringent" and "less stringent" criteria based on clinical findings (4Ts score) and results of PF4 ELISA testing were used to define the likelihood of a patient having HIT (Table 1A). Receiver Operating Curve (ROC) analysis was performed and Area Under the Curve (AUC) of each assay was calculated to assess their diagnostic accuracy. Since high test specificity was desired, positive cut-offs that maximized the sum of sensitivity and specificity in a specificity range of ≥90% and ≥95% were derived. To evaluate non-inferiority of the PEA to the SRA, we assessed whether the lower limit of the one-sided 95% confidence interval of the ΔAUC (PEA-SRA) was higher than a non-inferiority margin of 5% and 10%. Results: A total of 440 patient samples were tested with both assays. Seventeen and 28 patients were classified as having true HIT based on the use of the more- and less-stringent criteria, respectively. When classified by more-stringent criteria, the AUC was numerically greater for each of the three PEA tests than for the SRA (Table 1B). All tests had excellent accuracy (AUC>0.90, "excellent" by the Traditional academic point system). Using less-stringent criteria, the PEA30 had the highest AUC estimate followed by the SRA (Table 1C). Positive cut-offs generated at test specificities of ≥90% and ≥95% using data classified by more stringent criteria are shown in Table 1D. PEA3 was non-inferior to the SRA at a 5% non-inferiority margin, and all PEA tests were non-inferior at the 10% margin using the more stringent classification for true HIT (Fig 1A). In the less-stringently classified data set, the PEA30 was non-inferior to the SRA at a 10% margin (Fig 1B). Conclusions: The PEA, a 3-hour, non-radioactive functional HIT assay demonstrated high diagnostic accuracy and was non-inferior to the SRA using commonly accepted non-inferiority margins for diagnostic testing (5-10%). The technical advantages of the PEA over the SRA may encourage its widespread adoption as a rapid-turnaround, accurate and near-patient diagnostic for patients with suspected HIT. Disclosures Jones: BloodCenter of Wisconsin: Patents & Royalties. Curtis:Ionis Pharmaceuticals: Consultancy. Bougie:BloodCenter of Wisconsin: Patents & Royalties. Aster:BloodCenter of Wisconsin: Patents & Royalties. Garcia:Shingoi: Consultancy; Retham Technologies LLC: Consultancy; Incyte: Research Funding; Daiichi Sankyo: Research Funding; Bristol Meyers Squibb: Consultancy; Portola: Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy; Boehringer Ingelheim: Consultancy. Padmanabhan:BloodCenter of Wisconsin: Patents & Royalties: Patent/royalties for HIT diagnostic testing; Veralox Therapeutics: Other: Advisory Board; Retham Technologies LLC: Equity Ownership, Other: Retham seeks to develop HITDx(TM), a new assay for HIT diagnosis; TerumoBCT: Consultancy; GE Healthcare: Other: Material support for Clinical Quality Improvement Initiative; Janssen Pharmaceuticals: Consultancy.