Heikal, Nahla

Abstract Background Laboratory identification of lupus anticoagulant (LAC) is critical for diagnosing antiphospholipid antibody syndrome, a common cause of unprovoked thromboembolic events and pregnancy morbidity. Commonly used clot-based assays for LAC include the dilute Russell’s viper venom time (DRVVT) and activated partial thromboplastin time (APTT), but these are susceptible to anticoagulant interferences. Despite standardization efforts, LAC interpretation remains challenging due to pre- and post-analytical complexity and limited expertise. This study explored the use of deep neural networks (DNN) to facilitate LAC diagnosis. Methods Retrospective data for LAC profiling with 13 features performed at Mayo Clinic between 2021 and 2022 was collected. The profiles of 7,202 patients were randomly divided into training (n=4608), validation (n=1153), and test cohorts (n=1441). Diagnostic outputs including LAC positivity (determined by DRVVT or APTT) and the presence of warfarin (WAR) and heparin (HEP), were confirmed by expert assessment. Three DNN architectures were developed and evaluated (Figure 1): (1) four single output DNN models with a domain knowledge driven selection of input features, (2) four single output DNN models using the same 13 inputs across all models, and (3) a multi-output DNN model. Model performance was evaluated using the F1 score and the index of balanced accuracy (IBA) for each output label. Results Architecture 1 achieved high diagnostic accuracy with IBA values of 0.989-1.000 for all four outputs and F1 scores of 0.945-0.991. Architecture 2 exhibited F1 scores of 0.902-0.973 and IBA values of 0.935-0.992. Architecture 3 achieved F1 scores of 0.933-0.967 and IBA values of 0.941-0.983. Conclusions All three architectures accurately classify LAC and common anticoagulation effects. The comparable performance of architectures 1 and 2 suggests that manual feature extraction is unnecessary. Given its relative simplicity and versatility, architecture 3 is the preferred choice for implementation in a clinical laboratory to standardize LAC diagnosis.

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 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.

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.

AbstractHospitalized patients with COVID‐19 infection frequently have coagulopathy resembling disseminated intravascular coagulation (DIC). An elevation of D‐dimer level is associated with a poor prognosis; however, the role of other fibrin degradation products, such as soluble fibrin monomers (SFMC), is not known.The objective of the study was to investigate the frequency and prognostic role of elevated SFMC in patients with COVID‐19. In this retrospective cohort study, patients hospitalized between April 1, 2020 and December 14, 2020 at Mayo Clinic with COVID‐19 infection who underwent DIC panel testing were identified. Results of laboratory tests and outcomes (thrombosis and death) within 40 days of testing were obtained via medical record review.Of 108 patients, D‐dimer was elevated in 82 (75.9%) patients. Of those with elevated D‐dimer, SFMC was elevated in 19/82 (23%) patients. There were 16 thrombotic events and 16 deaths during the 40‐day follow‐up. The incidence of overt‐DIC was 4.6%. In univariate analysis, D‐dimer ≥5 x highest upper limit normal (ULN) and elevated SFMC were each associated with higher 40‐day mortality. However, when used in combination with D‐dimer ≥5 x highest ULN, an elevated SFMC provided no further mortality predictive value.Compared to 75.9% of patients with elevated D‐dimers, of those tested, only 23% had elevated SFMC. These results support the hypothesis that elevated D‐dimer in COVID‐19 infection is a direct consequence of endothelial damage and not overt‐DIC.

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&lt;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 Background: Heparin induced thrombocytopenia (HIT) is a rare immune mediated complication that is triggered in a subset of patients temporal to therapeutic heparin exposure. Laboratory testing is based on screening for the presence of serum anti-PF4 antibodies using sensitive solid-phase immunoassays. If antibodies are detected, functional testing to demonstrate the platelet activating properties and heparin dependence of these immune complexes is then performed. Previous studies have reported possible clinical utility in identifying non heparin dependent platelet activating antibodies using a buffer control with zero heparin in the serotonin release functional assay (SRA). These reports suggested a correlation between reactivity in the zero heparin buffer control and pathogenicity of HIT antibodies which may define a subtype of HIT, referred to as autoimmune HIT. We aimed to investigate the utility of zero heparin buffer control as a part of an inhouse validation study of a mass spectrometry-coupled SRA (Mayo-SRA). Methods: Three hundred archived serum samples were tested using anti-PF4 IgG antibody enzyme-linked immunosorbent assay (ELISA; Immucor Diagnostics, GA, USA). SRA was preformed on all samples using Mayo-SRA and reference 14C SRA methods. Zero heparin control buffer was included in the Mayo-SRA assay. Serotonin release &gt;20% in the low dose heparin (0.1U/mL, LDH) and ELISA optical density (OD) &gt;0.4 were considered positive. Drug interference studies were performed by spiking known SRA-positive samples with increasing concentrations of unfractionated heparin (UFH), low molecular weight heparin (LMWH) or fondaparinux in the LDH and zero heparin SRA. The clinical 4T score was calculated retrospectively calculated for all patients. Results: Of the 300 tested samples, 57 were anti-PF4 ELISA positive. 33 of the 57 samples were positive using the reference 14C SRA method. Whereas 43 samples were positive by Mayo-SRA assay. Three additional samples were positive by Mayo-SRA, but negative by both screening anti-PF4 ELISA and the reference SRA method (Fig- 1A). Lastly, 13 samples were anti-PF4 ELISA positive, but SRA negative by both methods in comparison. Interestingly, 44 of 46 (95%) samples interpreted positive by LDH were also interpreted positive (serotonin release &gt;20%) under zero heparin conditions. These included the 3 samples that were positive by Mayo-SRA but negative by both screening anti-PF4 ELISA and the reference SRA. The overall % serotonin release using zero heparin control was significantly lower (P= 0.003, paired Student T-test) compared to LDH (Fig-1B). In addition, zero heparin followed a similar pattern as LDH, with highest levels at ELISA OD units &gt;2 (Fig-1C). Strikingly, drug interference studies showed artifactual serotonin release in the zero heparin reaction, which was not detected in the absence of spiked drugs. For UFH, serotonin release in zero heparin control occurred at very low spiked concentrations, ≥0.063 U/mL. LMWH and fondaparinux spiking experiments also displayed similar zero heparin serotonin release patterns (Fig-1D). Of note, none of these interferences were detected in UFH spiked SRA-negative samples (data not shown). Conclusion: Contrary to prior results suggesting that less than 50% of LDH SRA positive samples are also positive in the zero heparin SRA, our results show high zero heparin SRA positivity rate of &gt;95%. Zero heparin SRA showed a pattern with highest levels at ELISA OD units &gt;2 suggesting that reactivity in this condition is a function of antibody strength rather than a qualitative difference (i.e. "autoimmune" HIT antibodies vs "non-autoimmune" HIT antibodies). In addition, contamination of patient sera with small amounts of remnant heparin can significantly impact platelet activation in the zero heparin SRA test. Thus, zero heparin SRA positive results may be artifactual and represent residual heparin contained in the patient sample. Figure legend: Fig-1A. Scatter plots of SRA positive samples grouped by 4T scores. Red dots are Mayo-SRA only positive samples. Fig-1B and Fig-1C. Scatter plots of % serotonin release of Mayo-SRA positive samples at LDH (circles) vs. zero heparin buffers (squares), and grouped by ELISA OD values, respectively. Fig-1D. % Serotonin release of known SRA-positive samples spiked with increasing concentrations of UFH, LMWH, or fondaparinux at LDH (white) or zero heparin buffers (red). Figure 1 Figure 1. Disclosures Pruthi: CSL Behring: Honoraria; Genentech: Honoraria; Bayer Healthcare AG: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.