Splinter, Noah P.

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.

Abstract Introduction: Monoclonal gammopathy of thrombotic significance (MGTS) is a recently recognized thrombophilia where monoclonal platelet factor 4 (PF4)-specific IgG antibodies initiate a chronic prothrombotic state characterized by recurrent and potentially life-threatening thrombosis. Studies from large data sets demonstrate that patients with monoclonal gammopathy of undetermined significance (MGUS) have a heightened risk of experiencing thromboembolic events without a precise explanatory mechanism. The prevalence of MGTS in MGUS-related thrombosis is unknown; however, the recent identification of MGTS antibodies as a cause of thrombosis suggests a potentially causative mechanism. Emerging evidence indicates that detecting pathogenic MGTS antibodies presents diagnostic challenges due to the insensitivity of FDA-approved diagnostic assays adopted from HIT (HIT ELISAs) and gold-standard functional tests that use heparin-treated platelets, such as the serotonin release assay (SRA). Only PF4-enhanced functional assays, including the PF4-dependent P-selectin expression assay (PEA) and PF4-SRA, have proven sensitive for detecting MGTS antibodies. In this study, the PEA was utilized to better define the incidence of pathogenic anti-PF4 antibodies among a cohort of MGUS patients with a history of thrombosis. Methods: A search of the Mayo Clinic Dysproteinemia Bank was conducted to identify two cohorts of IgG MGUS patients, one with venous thromboembolism-associated diagnosis code(s) and the other without. A diagnosis of multiple myeloma was exclusionary. Each serum sample was assessed using the PEA. Mayo Clinic's Institutional Review Board approved the research study. Results: Among the 134 patients studied in the thrombosis cohort, 17 (13%) stimulated platelet activation in the PEA (positive cut-off ≥19%), some weakly. Of these, four patients (Patients 1-4) were confirmed to have PF4-dependent platelet-activating antibodies inhibitable with high concentrations of heparin and sensitive to blockade of the IgG receptor on platelets, FcGRIIa. The thrombotic complications experienced included recurrent deep venous thrombosis (DVT) over several years (Patient 1), a single DVT (Patient 2), a thrombotic cerebrovascular accident (CVA) (Patient 3), and a series of thrombotic events, including DVTs, left atrial thrombosis, pulmonary embolism, and acute limb ischemia (Patient 4). Notably, longitudinal samples spanning twelve years (Patient 1), six years (Patient 2), three years (Patient 3), and on a single sample date (Patient 4) were confirmed positive by PEA. The prolonged persistence of platelet-activating anti-PF4 antibodies in Patients 1-3 supported a diagnosis of MGTS in these patients. Strikingly, all four patients tested negative in two FDA-approved HIT ELISAs (Immucor PF4 IgG and Zymutest HIA IgG). SRA was negative for Patients 1 and 2, weakly positive for Patient 3 (33%), and positive for Patient 4 (72%). Of the 166 patients in the MGUS minus thrombosis cohort, one patient demonstrated a non-heparin induced HIT-like antibody that was HIT ELISA (Optical Density >1.0) and PEA-positive, and inhibitable using high concentrations of heparin and FcGRIIa blockade. No follow-up samples were available to assess antibody longevity. The patient had no history of thrombosis, and testing demonstrated that the anti-PF4 antibody was not synonymous with the MGUS antibody.Conclusions: Among a cohort of 134 MGUS patients with thrombosis, the prevalence of MGTS was found to be 2.2% (3 patients with persistent platelet-activating anti-PF4 antibodies). The identification of anti-PF4 antibodies among MGUS patients provides a mechanism contributing to the increased thrombotic risk associated with monoclonal gammopathies. Antibody negativity in HIT ELISA and variable positivity in the SRA highlight the nonclassical serology associated with anti-PF4 antibodies in MGTS and support performing PF4-enhanced functional testing. In addition, some patients appear to produce coincidental (non-monoclonal) platelet-activating anti-PF4 antibodies without evidence of thrombosis, although the longevity of such antibodies could not be examined in the one patient where it was identified. These findings highlight the need for larger, prospective MGUS studies to validate these observations, further define the interplay between monoclonal gammopathies and thrombotic risk, and to better understand the long-term outcomes of patients with MGTS.

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/polyvinyl sulfonate ELISA-negative platelet-activating antibodies in HIT-suspected patients and mice immunized with PF4/heparin. 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 detection. 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 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. To advance the study of ELISA-negative HIT antibodies, mice were immunized with PF4/heparin, and functional and antigenic assays were simultaneously used to successfully identify an ELISA-negative, PF4-dependent platelet-activating murine monoclonal antibody that recapitulated the serological characteristics of ELISA-negative HIT patients. 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: Ticagrelor is a widely used reversible platelet adenosine 5'-diphosphate (ADP) P2Y12 receptor inhibitor used in patients with acute coronary syndrome and coronary interventions. Ticagrelor treated patients may be concomitantly exposed to heparin treatment for prophylaxis/treatment of thrombosis, and heparin induced thrombocytopenia (HIT), a thrombotic thrombocytopenic adverse event may be suspected in such patients. Objective: Here we describe the serological profile of a patient on ticagrelor treatment and strong clinical suspicion of HIT who presented with negative serotonin release assay (SRA) results. A follow up systematic study on the impact of ticagrelor on this gold standard HIT assay is presented. Methods: Remnants from 23 patient samples submitted for clinical SRA testing (5- SRA negative; 6- “low positive” [20-50%]; 5- “moderate positive” [>50 to <90%]; 7- “strong positive” [>90%]) were spiked with ticagrelor in concentrations of 100, 250, 500, and 1000 ng/mL before SRA re-testing. Testing was performed using heat inactivated serum, with low (0.1 units/mL) or high (100 units/mL) heparin and washed platelets (Leger, THSNA 2022). A positive SRA result was defined as ≥20% serotonin release with the low heparin condition and >50% inhibition of serotonin release with high concentration of heparin. A subset of SRA positive patient samples that tested falsely negative upon ticagrelor addition were subsequently incubated with activated charcoal and retested in the SRA to determine if ticagrelor interference was reversible using this methodology. Results: The index patient on ticagrelor had a high 4Ts score, ELISA optical density >1.0 but tested negative in the SRA. Re-testing of this patient with an activated charcoal treated sample demonstrated a striking change in SRA to a strong positive result (91% serotonin release). Upon testing of ticagrelor-spiked samples in the SRA negative patient cohort, as expected, the drug had no effect on results, with all 5 samples persistently staying SRA negative. Of the 6 patients that had low positive SRA results, all converted to SRA negative with ticagrelor concentrations >500 ng/mL. One of these patients became SRA negative at 100 ng/mL and 3 at 250 ng/mL. Of the 5 patients with moderately positive SRA results, 2 converted to SRA negative with ticagrelor concentrations >100 ng/mL, and 4 of 5 patients became negative with spiking at 500 ng/mL. Finally, of the 7 patients with strong positive SRA results, 1 became falsely negative in the SRA at a ticagrelor concentration of 1000 ng/mL. Activated charcoal was incubated with 6 samples that demonstrated negative SRA results with drug concentration of 500 ng/mL. Two samples reverted to SRA positive, while 4 samples remained persistently negative. Conclusion: In preliminary studies, ticagrelor has been shown to interfere with the heparin induced platelet activation (HIPA) assay but its impact on the SRA has been relatively unexplored. Here, we demonstrate that patients on ticagrelor may present with spuriously false HIT functional assay testing by the SRA, particularly if the HIT antibody is not strongly platelet activating. Activated charcoal treatment was only able to reverse drug interference in a subset of samples, even at drug concentrations below peak levels recorded in patients (approx. 800ng/mL). Given these findings, we recommend de-emphasizing the utility of the SRA in ticagrelor treated patients with reliance more on criteria such as pre-test probability of HIT (e.g. 4Ts score), and ELISA results in order to facilitate an accurate diagnosis of HIT.

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe thrombotic thrombocytopenia that predominantly occurs after adenoviral vector-based vaccines. VITT patients produce anti-platelet factor 4 (PF4) antibodies with restricted clonality (i.e., mono/oligoclonal) with lambda light chains. Long-term follow-up studies in VITT show a disconnect in the persistence of anti-PF4 antibodies, whereby ELISA-positive antibodies persist significantly longer than platelet-activating antibodies. The mechanistic basis of this is unclear and could be explained by a couple of hypotheses: (1) Epitope spreading where, with time, the specificity of platelet-activating anti-PF4 antibodies may “spread” to recognize only PF4 complexed to heparin vs. (2) Persistence of clonally restricted platelet-activating anti-PF4 antibodies which decrease in titers with time such that their levels are insufficient to crosslink platelet FcGRIIa and induce platelet activation. Methods: Acute, convalescent patient sera and affinity-purified anti-PF4 antibodies were evaluated in antigen-based, functional (platelet-activation-based), and mass spectrometry studies as previously described (Blood, 2022 Jul 7;140(1):73-77). The median time to last follow-up was 243 days after the acute episode (range: 114-664 days). Results: Follow-up samples were obtained from 6 VITT patients after Ad26.COV2.S vaccination. At last follow-up, anti-PF4 IgG antibodies reactive in PF4/polyanion ELISA were detected in the sera of 5/6 VITT patients. However, only one of these samples stimulated platelet activation in the PF4-dependent P-selectin expression assay (PEA; 199 days after acute presentation). Affinity-purified anti-PF4 antibodies were detected by ELISA in all follow-up samples and three of the six affinity-purified anti-PF4 antibody samples activated platelets in the PEA. Four of the six VITT patients demonstrated a monoclonal anti-PF4 antibody, while one each had a biclonal and triclonal profile. Five VITT patients produced IgG lambda light chain antibodies, while one produced an IgG kappa. All six VITT patients demonstrated synonymous anti-PF4 antibody profiles when comparing acute and follow-up antibody repertoires, without evidence of VITT patients producing new anti-PF4 antibodies after the acute episode. In all cases, levels of anti-PF4 antibodies were significantly lower in convalescent samples relative to acute samples. Given this data on the persistence of clonally-restricted VITT antibodies, samples from another VITT patient with thrombocytopenia (100-150,000/uL) persisting for more than three years were similarly evaluated. This patient, who experienced severe thrombosis after ChAdOx1 nCoV19 vaccination, demonstrated persistent ELISA and PEA positivity even three years after symptom onset but had not experienced breakthrough thrombosis while on anticoagulation therapy. Serum protein electrophoresis (SPEP) did not detect a monoclonal gammopathy. Anti-PF4 antibodies immuno-enriched from a sample obtained >2 years after acute presentation demonstrated a monoclonal profile. While non-availability of an acute sample for anti-PF4 antibody characterization precluded a firm determination of whether this “Long VITT” case represented a subset of monoclonal gammopathy of thrombotic significance (MGTS; Blood 2023 Apr 6;141(14):1772-1776; New Eng J Med 2024 Aug 8), the chronicity of thrombocytopenia, presence of a monoclonal anti-PF4 antibody and persistently positive anti-PF4 serology supports this diagnosis. Conclusions. Clonally-restricted VITT antibodies persist for months-years with no evidence for the production of novel anti-PF4 antibodies after the acute event. Decreased abundance of anti-PF4 antibodies leads to negative functional results with follow up samples. Some platelet-activating antibodies can persist for very long periods (>3 years) and mediate persistent thrombocytopenia, representing an SPEP-negative MGTS-like state. This study also demonstrates the first example of a VITT antibody with a kappa light chain. The requirement for anticoagulation in patients demonstrating ELISA-positive/functional test-negative results is not clear, but the persistence of mono/oligoclonal anti-PF4 antibodies raises the possibility for the upregulation of pathogenic antibody production in proinflammatory settings.

Introduction. Platelet-activating anti-Platelet factor 4 (PF4) antibodies mediate a number of thrombotic thrombocytopenic syndromes, including heparin-induced thrombocytopenia (HIT), and more recently recognized conditions, vaccine-induced immune thrombotic thrombocytopenia (VITT), adenovirus-associated thrombotic thrombocytopenia, and monoclonal gammopathy of thrombotic significance (MGTS). In MGTS (Blood. 2023 Apr 6;141(14):1772-1776), a monoclonal IgG antibody to PF4 mediates a chronic prothrombotic state characterized by recurrent thrombosis. More recent evidence suggests that MGTS may present with negative results in HIT enzyme-linked immunosorbent assay (ELISA) and serotonin release assay (SRA), and positivity only in PF4-dependent functional assays such as the PF4-dependent P-selectin expression assay (PEA; submitted). The presence of MGUS in patients has long been associated with high risks of thrombotic disease (Blood. 2010 Jun 17;115(24):4991-8), but the mechanism by which they predispose to thrombosis has been relatively unexplored. In this study, we evaluated a cohort of MGUS patients who experience thrombosis for the presence of platelet-activating anti-PF4 antibodies. M ethods. An administrative database search was performed of Mayo Clinic records to identify patients who had a diagnosis of MGUS, and thrombosis. Research samples from 134 MGUS patients with IgG monoclonal protein and thrombosis were obtained from Mayo Clinic's dysproteinemia biobank and subjected to PF4 antigen-based and functional screening. Research studies were approved by the Institutional Review Board of Mayo Clinic. Results. Of the 134 patients, 17 (12.7%) activated platelets in the PEA (≥19%). To exclude non-specific activation (e.g. due to HLA antibodies), secondary PEA testing was performed to assess whether platelet activation was mediated by a HIT-like antibody (i.e., PF4- and IgG-dependent, and inhibitable with high concentrations of heparin). Four patients, hereafter referred to as patients 1-4, were identified as having a HIT-like antibody, as demonstrated by PF4-dependent platelet activation that could be inhibited with high concentrations of heparin (100 U/mL), and blockade of the platelet IgG receptor, FcγRIIa. Patients 1 and 2 had persistent, platelet-activating anti-PF4 antibodies based on PEA testing of multiple samples obtained several years apart. Patient 1 had a recurrent, multi-year history of deep venous thrombosis (DVT), while patient 2 had one DVT episode. Patient 3 demonstrated a HIT antibody-like profile in the PEA in two of three samples available for testing and had a history of thrombotic cerebrovascular accident (CVA). Patient 4 demonstrated a HIT-like platelet-activating antibody in only one of four samples tested, and consistent with this finding, had an MGUS identified on clinical testing at only one time point (“small” IgG lambda, not quantified), 3 months prior to the platelet-activating sample. This patient experienced multiple thrombotic events including DVTs, left atrial thrombus and embolic CVA, pulmonary embolism and acute limb ischemia. All patients tested negative in two FDA-approved antigen-based HIT assays tested (Immucor PF4 IgG and Zymutest HIA IgG), similar to a recent MGTS case (submitted). SRA was negative in both Patients 1 & 2, weakly positive in Patient 3 (33%, with high CV), and positive (72%) in Patient 4. Conclusions. Definitive diagnosis of patients with MGTS provides insight into a mechanism of “unexplained” thrombosis and may offer additional treatment pathways for these patients with recurrent thrombosis (New Eng J of Med, 2024 Aug 8). Serologic testing in two patients correlated strongly with observations of thrombosis, and the study confirmed recent findings that anti-PF4 antibodies may present with unusual serology that is neither HIT- nor VITT-like (with negative results in both ELISA and SRA). The incidence of confirmed/suspected MGTS was 3% (4/134) in this population of MGUS patients with thrombosis. Prospective studies are needed to confirm these observations.

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).