Johns, Gretchen S.

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.

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.

Aortic stenosis (AS) requires repeat assessments to optimally time intervention. The utility of laboratory tests to complement clinical and hemodynamic assessments has not been established. We assessed clinical, echocardiographic, and laboratory data in 76 patients with AS or heart valve replacement (HVR), age 77 years, 37% women. Laboratory assessments included von Willebrand factor (VWF) antigen, activity, (Act/Ag, normal > 0.8), VWF multimer distribution (abnormal=loss of highest molecular weight multimers=Mult+), brain natriuretic peptide (BNP, abnormal > 155 pg/ml=BNP+), and platelet function analyzer 100 collagen-ADP closure time (PFA, normal < 121 seconds). Table: Coefficients of correlation, r, and F statistic. Chi-square, and probability. Correlation of BNP to mean gradient in patients with GFR>60 remained poor, r=0.29, while for PFA r = 0.65. Median (IQR) for PFA and BNP for mild, moderate, and severe AS and HVR are shown below. VWF multimers were abnormal in 3/14 mild AS, 6/14 moderate AS, 22/25 severe AS and 4/23 HVR patients. The severity of AS was most strongly associated with VWF multimer abnormality and prolonged PFA closure time, weakly with BNP, and not with VWF Act/Ag. Conclusion: Among various laboratory measures, PFA and VWF multimers are strongly associated with AS severity, and may merit assessment as complementary diagnostic and prognostic biomarkers in AS.

Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a critical strategy to overcome the COVID-19 pandemic. Multiple SARS-CoV-2 vaccines have been developed in a rapid timeframe to combat the pandemic. While generally safe and effective, rare cases of venous thromboembolism (VTE) have been reported after two adenovirus-based vaccines, the AstraZeneca ChAdOx1 nCoV-19 vaccine and the Janssen Ad.26.COV2.S vaccine, as well as after the Pfizer-BioNTech BNT162b2 mRNA vaccine. Here, we present the case of a patient who developed acute pulmonary emboli (PE) shortly after his second dose of the Moderna mRNA-1273 SARS-CoV-2 vaccine. We report the results of an extensive thrombophilia workup that was normal except for the identification of positive lupus anticoagulant (LA) signals. It is our goal to contribute to the body of knowledge regarding SARS-CoV-2 vaccines and encourage vaccine adverse event reporting so that clinicians can have a full appreciation and awareness of the possible adverse events related to these critical vaccines.

Abstract BACKGROUND: Glanzmann's thrombasthenia (GT) is an inherited platelet disorder (IPD) that leads to clinically significant bleeding. Platelets from patients with GT can show qualitative or quantitative defects of platelet membrane glycoprotein (GP) IIb/IIIa complex. Most GT are caused by autosomal recessive genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa, respectively) with rare cases showing an autosomal dominant pattern. Accurate diagnosis of GT requires a constellation of both phenotypic and genetic studies. Here we report a unique case of autosomal dominant GT resulted from thorough phenotypic and genetic studies. PATIENTS AND METHODS: A 19 year-old woman was recently evaluated for life-long history of easy bruising and severe menorrhagia that was only partially responsive to medroxyprogesterone. She has severe thrombocytopenia first recognized shortly after birth with a platelet count around 20x109/L. Platelet size was normal. She was presumed to have immune-mediated thrombocytopenia (ITP) since early infancy, but lacking responsiveness to the conventional treatments of ITP including immunomodulation, splenectomy and thrombopoietin receptor agonists. Family history was negative for a bleeding diathesis. Both von Willebrand factor antigen and activity were within normal range. Bone marrow aspirate and biopsy with associated chromosome studies were all normal. Platelet surface glycoprotein assessment by flow cytometry using antibodies to GP IIb, IIIa, Ia, Ib-a, VI, IX, and whole exome sequencing (WES) utilizing a predefined list of 53 clinically significant genes* related to genetically IPDs were performed. Due to thrombocytopenia platelet aggregation studies could not be performed. METHODS/RESULTS: Platelet surface expression of GPIIb (CD41) and GPIIIa (CD61) were markedly decreased suggestive of a variant of GT (Figure). WES performed with Illumina HiSeq 2500 sequencing system by using Agilent SureSelelct CRE kit V1 to capture and target the exonic regions showed presence of a heterozygous mutation in ITGB3 gene (c2213T>G; p.Leu738Arg). By in silico prediction modeling, this mutation is likely to be pathogenic and results in the substitution of hydrophobic leucine with hydrophilic arginine in the transmembrane domain of the β3 subunit of alpha IIb/beta 3 integrin (αIIbβ3), the platelet receptor that binds to fibrinogen. Interestingly, GPVI is also decreased which may be associated with GPIIb and GPIIIa deficiency or due to accelerated shedding since no GPVI mutation was identified. CONCLUSION: We describe a patient with GT associated with a novel heterozygous autosomal dominant mutation in ITGB3 gene with substitution of leucine with arginine (c2213T>G; p.Leu738Arg). This deletion caused a low expression of αIIbβ3 integrin on her platelets surface and severe thrombocytopenia. This case underscores the importance of an integrated phenotyping and genotyping approach to render a definitive diagnosis of an IPD. *Platelet exome gene list: ABCG5, ABCG8, ACBD5, ACTN1, ANKRD26, ANO6, AP3B1, BLOC1S3, BLOC1S6, CYCS, DTNBP1, ETV6, FLI1, FLNA, GATA1, GFI1B, GP1BA, GP1BB, GP6, GP9, HOXA11, HPS1, HPS3, HPS4, HPS5, HPS6, ITGA2B, ITGB3, LYST, MASTL, MPL, MYH9, NBEAL2, ORAI1, PSRX1, P2RY12, PLA2G4A, PRKACG, RASGRP2, RBM8A, RUNX1, STIM1, STX11, STXBP2, TBXA2R, TBXAS1, THPO, TUBB1, UNC13D, VIPAS39, VPS33B, VPS45, WAS Figure Figure. Disclosures No relevant conflicts of interest to declare.