Chen, Dong

Abstract Objectives Fluorescence resonance energy transfer (FRET)–based ADAMTS13 activity assays are critical for the diagnosis of thrombotic thrombocytopenic purpura. However, these assays are susceptible to interference. As iodide has been suggested to interfere in laboratory testing via fluorophore quenching or promotion, we aimed to determine whether iodinated contrast (Omnipaque) interferes with the ATS-13 ADAMTS13 Activity Assay 2.0. Methods We evaluated the excitation, emission, and absorbance spectrum of Omnipaque alone and spiked in patient plasma with known ADAMTS13 activity and ADAMTS13 activity on Omnipaque alone, an abnormal control of patient plasma previously observed to display elevated baseline relative fluorescent units, and variable concentrations of patient plasma with known ADAMTS13 activity spiked with Omnipaque. Results No atypical fluorescent peaks were observed on any sample (Omnipaque alone or spiked in plasma) between 250 and 700 nm. There was no difference in the mean ADAMTS13 activity among the various concentrations of plasma spiked with Omnipaque or plasma spiked with saline. Conclusions Iodinated contrast does not appear to interfere—either via spectral overlap of the fluorophore or through physiologic inhibition of the ADAMTS13 enzyme—with ADAMTS13 activity FRET-based assays based on the findings from this in vitro analysis. Delaying sample collection for ADAMTS13 activity testing from suspected patients with thrombotic thrombocytopenic purpura following administration of iodinated radiocontrast agents is not necessary, and recent contrast administration should not yield erroneous ADAMTS13 activity results.

Abstract The A1 domain in Von Willebrand Factor (VWF) initiates coagulation through binding to platelet glycoprotein GPIbα receptors. Von Willebrand Disease (VWD)-Mutations in A1 that either impair (type 2M) or enhance (type 2B) platelet adhesion to VWF can locally destabilize and even misfold the domain. We leveraged misfolding in the gain-of-function type 2B VWD phenotype as a target, distinct from the normal conformation. Two nuclease-resistant 2′-fluoropyrimidine RNA aptamers were selected to discriminate normal A1 domains from a type 2B V1314D A1 variant in a glycosylated A1A2A3 tri-domain VWF-fragment. Two aptamers, W9 and V1, were isolated that selectively recognize, bind, and inhibit the A1–GPIbα interaction with WT A1A2A3 and V1314D A1A2A3, respectively. These aptamers were tested against their respective recombinant targets, plasma VWF, VWF concentrates, and patient plasma with the heterozygous type 2B VWD R1306W variant using clinical assays, surface plasmon resonance and inhibition assays of platelet adhesion to recombinant A1 and A1A2A3 domains under shear stress. The specificity of W9 and V1 aptamers confirms that pathological conformations of VWD Type 2B proteins are different from normal VWF. The availability of aptamers that distinguish normal plasma-derived VWF from VWD suggests potential applicability in clinical diagnosis of severe gain-of-function phenotypes.

AbstractBackgroundClot based assays used for lupus anticoagulant (LAC) detection are typically interpreted in a qualitative fashion and may not reflect LAC potency. In this cross‐sectional study, we describe a method for quantifying the LAC titer using serial (dependent) two‐fold dilutions in normal pooled plasma.MethodsSerial dilutions of 51 residual plasma samples from 50 patients were tested using the Russell's viper venom screening time (DRVVT) and activated partial thromboplastin screening time (APTT) methodologies. The measured clotting times and the corresponding dilution factors were then used to derive a four‐parameter logistic model. The LAC titer for each patient was interpolated as the sample dilution that corresponds to the upper reference interval limit of the corresponding assay.ResultsCalculated APTT and DRVVT LAC titers displayed a strong linear correlation (R2 = 0.84) between each other, but not with the degree of prolongation of the APTT/DRVVT screening time in the neat undiluted samples. Using data driven partitioning, patients could be grouped into low (<10) or high (≥10) DRVVT LAC titer. There were no significant differences in anticardiolipin (aCL) or anti‐beta 2 glycoprotein 1 (aB2GPI) antibody levels or prevalence of thromboembolic events between low and high LAC titer groups. In contrast, antiphosphatidylserine/prothrombin (aPS/PT) IgM antibody levels, but not IgG, were significantly higher in the high LAC titer group.ConclusionsThe degree of prolongation of the APTT/DRVVT screening time is not correlated with the LAC titer. Only aPS/PT IgM antibodies levels were strongly correlated with the LAC titers. Additional studies are warranted to determine clinical implications of high LAC titers.

Abstract Objectives VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic pathogenic mutation in the UBA1 (ubiquitin-like modifier activating enzyme 1) gene. Patients present with rheumatologic manifestations and cytopenias and may have an increased predisposition to myelodysplastic syndrome (MDS) and plasma cell neoplasms. Prior studies have reported on the peripheral blood and bone marrow findings in patients with VEXAS syndrome. Due to the protean clinical presentation and lack of specificity of morphologic features (eg, vacuoles in early erythroid and granulocytic precursors), an optimal screening methodology to identify these patients in a timely fashion is desirable. Methods To further evaluate and describe the salient diagnostic morphologic features in VEXAS syndrome, we carried out a comprehensive study of the largest single-institution cohort to date. Diagnostic and follow-up bone marrow biopsy specimens from 52 male patients with molecularly identified VEXAS syndrome underwent central review. Results Cytopenias were common in all cases, primarily macrocytic anemia, monocytopenia, and thrombocytopenia. Bone marrow aspirate and biopsy were often hypercellular, with an increased myeloid/erythroid ratio, granulocytic hyperplasia with left shift, erythroid left shift, and megakaryocyte hyperplasia, which exhibited a range of striking morphologic findings. Distinctly vacuolated myeloid and erythroid precursors were seen in more than 95% of cases. Conclusions Our data reveal potential novel diagnostic features, such as a high incidence of monocytopenia and distinct patterns of atypical megakaryopoiesis, that appear different from dysmegakaryopoiesis typically associated with MDS. In our experience, those findings are suggestive of VEXAS, in the appropriate clinical context.

Abstract Objectives To determine the impact of residual platelets on dilute Russell’s viper venom time (DRVVT) assay in frozen-thawed plasma submitted for lupus anticoagulant (LAC) testing. Methods We measured platelet counts in frozen-thawed samples submitted for LAC testing and evaluated the association between platelet count and the DRVVT screening time and ratios. We also spiked platelets into a LAC-positive sample to observe the effect on the DRVVT. Results Progressive increase in platelet count resulted in a statistically significant shortening of the DRVVT assay results on plasma after 1 freeze-thaw cycle. A similar effect was noted on the LAC-positive sample. Conclusions Residual platelets in plasma samples result in shortening of DRVVT assay after 1 freeze-thaw cycle. This may result in a false-negative LAC test result.

AbstractThe World Health Organization (WHO) classification system categorizes advanced systemic mastocytosis (SM‐Adv) into aggressive SM (ASM), mast cell leukemia (MCL), and SM with associated hematological neoplasm (SM‐AHN). By contrast, the International Consensus Classification (ICC) requires “immature” MC cytomorphology for the diagnosis of MCL and limits SM‐AHN to myeloid neoplasms (SM‐AMN). The current study includes 329 patients with SM‐Adv (median age 65 years, range 18–88; males 58%): WHO subcategories SM‐AHN (N = 212; 64%), ASM (N = 99; 30%), and MCL (N = 18; 6%); ICC subcategories SM‐AMN (N = 190; 64%), ASM (N = 99; 33%), and MCL (N = 9; 3%); WHO‐defined MCL with “mature” MC cytomorphology and SM‐AHN associated with lymphoid neoplasms were operationally labeled as “MCL‐mature” (N = 9) and SM‐ALN (N = 22), respectively, and distinguished from ICC‐defined MCL and SM‐AMN. Multivariable analysis that included the Mayo alliance risk factors for survival in SM (age >60 years, anemia, thrombocytopenia, increased alkaline phosphatase) revealed more accurate survival prediction with the ICC versus WHO classification order: (i) survival was significantly worse with MCL‐immature versus MCL‐mature (hazard ratio [HR] 15; p < .01), (ii) prognostic distinction between MCL and SM‐AHN/AMN was confirmed in the context of ICC (HR 9.3; p < .01) but not WHO classification order (p = .99), (iii) survival was similar between MCL‐mature and SM‐AMN (p = .18), and (iv) SM‐AMN (HR 1.7; p < .01) but not SM‐ALN (p = .37) was prognostically distinct from ASM. The current study provides evidence for the independent prognostic contribution of both the ICC system for SM‐Adv and the Mayo alliance risk factors for survival in SM.