INTRODUCTION:Observational studies provide a signal that phosphodiesterase-5 inhibitors such as sildenafil are associated with lower mortality and ischaemic stroke during durable left ventricular assist device (LVAD) support. This study aims to determine the causal effects of sildenafil on platelet activation and circulating mediators of vascular remodelling during LVAD support. METHODS:We conducted a double-blind, randomized, placebo-controlled study to determine the effect of sildenafil on platelet activation and circulating mediators of vascular remodelling. Stable participants on LVAD support were assigned to sildenafil or placebo every 8 h for a 15-day period. Three primary endpoints were percent change in platelet activation by collagen, thromboxane (Tx) A2, and adenosine diphosphate (ADP). Secondary endpoints included changes in circulating mediators of vascular remodelling. RESULTS:Twenty participants were randomized. On day 15, those on sildenafil had lower collagen (-41%, -60 to -24, vs. -7%, -18 to 79, P = .038), but not TxA2 (-24%, -45 to -14 vs. 3%, -15 to 87, P = .112), and ADP (-5%, -70 to 25 vs. 31%, -29 to 242, P = .122) induced platelet activation compared to placebo. Endothelin-1 changed by -30% (-53 to 4) in the sildenafil group vs. -3% (-18 to 22) with placebo (P = .041). Angiopoietin (ang)-2 changed by -5% (-11 to -4) with sildenafil compared to 3% (-5 to 15) with placebo (P = .039), while ang-1 increased by 24% (17-54) with sildenafil and was -4% (-19 to -2) with placebo (P = .001), leading to a change in the ang-2/ang-1 ratio by -23% (-45 to -15) with sildenafil compared to 8% (-3 to 24) with placebo (P = .001). Hs-CRP and fibrinogen were unchanged between the groups. CONCLUSION:Fifteen days of sildenafil exposure modifies platelet activation and lowers mediators of vascular remodelling on LVAD support.

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.

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.

The ADAMTS13 activity assay is considered a diagnostic test for confirmation of clinical diagnosis of thrombotic thrombocytopenia purpura (TTP). Nonetheless, most common methodologies involve ELISA-based assays that are manual and time consuming, limiting the availability and turnaround time (TAT). A faster, semi-automated assay in a coagulation analyzer can help expedite cases and improve the TAT. Objective: To validate the ADAMTS13 activity and inhibitor assay in the Ceveron-s100 coagulation analyzer (DiaPharma) in comparison with the ELISA-like assay (ACTIFLUOR™ ADAMTS13 Activity Kit from BioMedica Diagnostics). Both methods are based on Fluorescence resonance energy transfer (FRET), thus allowing a direct comparison of the TAT. The inter-, intra-assay precision, accuracy, linearity, interferences and verification of the reference range in the Ceveron were validated in comparison to the ELISA-like assay. The TAT was monitored before and after go-live with the Ceveron. 28 samples were tested for the ADAMTS13 activity and 29 for the inhibitor assay to establish accuracy and performance. Results were compared between the original ELISA results and each individual Ceveron analyzer. The correlation between the two methods was good (R = 0.95, and slope = 0.9 ± 0.3). The inter- and intra-assay precision for both instruments achieved <10% CV. To verify the linearity, we ran several calibrator dilutions from the ACTIFLUOR™ ADAMTS13 Activity Kit, achieving an R2 greater than 0.990 when both results from both Ceverons were compared to the ELISA results. To establish the limit of detection, heat-inactivated Normal Pooled Plasma was tested 10 times on the analyzer. The limit of detection was < 1% activity (mean ±3SD). We verified the thresholds for interferences and validated a 1:5 dilution for hemolysis of 3+ and bilirubin >10 mg/dl. To verify the reference range, 25 different normal donor samples were tested in the Ceveron instruments resulting in an activity mean ±2SD of 54-133%. 29 samples were tested for the inhibitor assay in the Ceveron compared to the original results from the ELISA. 94% of samples had a CV less than 20%. We monitored the TAT one month before go-live using the ELISA-like assay and it compared to the first month after go-live with the Ceverons. The average TAT improved to 12.3 hrs. from 34 hrs. and the median improved to 3.7 hrs. compared to 25.7 hrs. the month prior. In conclusion the semi-automated Ceveron ADAMTS13 activity is comparable to the ELISA-like FRET assay. The Ceveron offers several advantages including decreased sample preparation, minimal cleaning and maintenance, lot-stable monthly calibration curve, individual sample testing allowing for fast TAT with potential 24/7 availability. Lastly a cost-effective reflex algorithm was established wherein only samples with a low ADAMTS13 activity are sequentially tested for an inhibitor.

The ADAMTS13 activity assay is considered a diagnostic test for confirmation of clinical diagnosis of thrombotic thrombocytopenia purpura (TTP). Nonetheless, most common methodologies involve ELISA-based assays that are manual and time consuming, limiting the availability and turnaround time (TAT). A faster, semi-automated assay in a coagulation analyzer can help expedite cases and improve the TAT. To validate the ADAMTS13 activity and inhibitor assay in the Ceveron-s100 coagulation analyzer (DiaPharma) in comparison with the ELISA-like assay (ACTIFLUOR™ ADAMTS13 Activity Kit from BioMedicaDiagnostics). Both methods are based on Fluorescence resonance energy transfer (FRET), thus allowing a direct comparison of the TAT. The Inter-, intra-assay precision, accuracy, linearity, interferences and verification of the reference range in the Ceveron were validated in comparison to the ELISA-like assay. The TAT was monitored before and after go-live with the Ceveron. 28 samples were tested for the ADAMTS13 activity and 29 for the inhibitor assay to establish accuracy and performance. Results were compared between the original ELISA results and each individual Ceveron analyzer. The correlation between the two methods was good (R ≥ 0.95, and slope = 0.9 ± 0.3). The inter- and intra-assay precision for both instruments achieved <10% CV. To verify the linearity, we ran several calibrator dilutions from the ACTIFLUOR™ ADAMTS13 Activity Kit, achieving an R2 greater than 0.990 when both results from both Ceverons were compared to the ELISA results. To establish the limit of detection, heat-inactivated Normal Pooled Plasma was tested 10 times on the analyzer. The limit of detection was < 1% activity (mean ±3SD). We verified the thresholds for interferences and validated a 1:5 dilution for hemolysis of 3+ and bilirubin >10 mg/dl. To verify the reference range, 25 different normal donor samples were tested in the Ceveron instruments resulting in an activity mean ±2SD of 54-133%. 29 samples were tested for the inhibitor assay in the Ceveron compared to the original results from the Elisa. 94% of samples had a CV less than 20%. We monitored the TAT one month before go-live with the ELISA-like assay compared to the first 3 weeks since go-live with the Ceverons up to date. The average TAT improved from 34 hrs. to 12 hrs. and a median of 3.6 hrs. compared to 25.7 hrs. The quickest results resulted within 79 mins compared to 221 mins before go-live. the semiautomated Ceveron ADAMTS13 activity is comparable to the ELISA-like FRET assay. The Ceveron offers several advantages including decreased sample preparation, minimal cleaning and maintenance, lot-stable monthly calibration curve, individual sample testing allowing for fast TAT with potential 24/7 availability. Lastly a cost-effective reflex algorithm was established wherein only samples with a low ADAMTS13 activity are sequentially tested for an inhibitor.

BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-β-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.

Abstract Lupus anticoagulant (LA) is one laboratory criterion for diagnosing antiphospholipid antibody syndrome (APS), a condition associated with hypercoagulability, pregnancy complications, and death. LA diagnosis requires a complex panel, often performed at a specialized laboratory. Ruling out false positive or transient LAs requires ≥ two positive tests ≥ 12 weeks apart. Most LA tests are negative, with small subsets resulting positive or indeterminate. Indeterminates leave clinicians with limited understanding of appropriate clinical follow-up. Thus, the International Society on Thrombosis and Hemostasis discourages weak resulting nomenclature such as “indeterminate.” Our study used artificial intelligence to predict LA indeterminate results as positive or negative, identifying patients requiring further testing. This IRB approved study reviewed nearly 4000 LA tests performed from 2019-2023. Parameters included CBC, thromboelastography, renal/hepatic function assays, coagulation tests, antiphospholipid antibodies and LA tests. LA test components included screening tests (APTT, Dilute Russel Viper Venom Test (DRVVT) screen), mixing studies (1:1 APTT mix, DRVVT mix), phospholipid-dependent confirmation (DRVVT, hexagonal phase, platelet neutralization), and factor VIII. A positive LA test fulfilled 4 criteria: at least one positive screening test, mixing study and phospholipid-dependent assay and factor VIII inhibitor exclusion. An LA panel meeting 2-3 criteria resulted indeterminate. Studies meeting <2 criteria were negative. This analysis was performed using Machine Intelligence Learning Optimizer (MILO) platform which displays the performance of multiple models. MILO was trained with two classifications: positive and negative cases. Data was split into training (100 training positives; 100 negatives) and testing (primary and secondary validation) sets (100 positives; 1388 negatives). LA panels affected by drug interactions (9.4%) or containing incomplete datasets (21%) were excluded. Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) determined model efficacy. 2903 negative (84%), 318 positive (9.1%), and 246 indeterminate (7.1%) LA results were included from 3467 patients. Only 84 of the 246 indeterminate patients (34%) were retested. Of these, 28 remained indeterminate, 43 became negative and 13 resulted positive. The best performing MILO models were the K-nearest neighbor (KNN) and logistic regression. The ability of these models to determine which indeterminate cases would become positive or negative was evaluated. KNN displayed the highest specificity (0.88) and PPV (0.57). All models had a high NPV and specificity due to substantive exposure to negative cases for the training and testing. Exclusion of some parameters did not improve the performance of the models. However, even the best model (various KNN models) had low sensitivity and PPV due to a small pool of positive training/testing cases and a much smaller group of indeterminate LA with subsequent positive results. Creating models accurately predicting indeterminate cases that may become positive enables appropriate clinical follow-up. Thus, improving model accuracy will require a larger sample size of positive indeterminates with follow-up.

Abstract Background Protein S deficiency is a hereditary thrombophilia which occurs in three forms: Type I (quantitative), type II (qualitative) and type III (qualitative due to a gain of function). Literature suggests that of these, type II is the rarest and most difficult to diagnose. Type II protein S deficiency is only detectable by assaying protein S activity with a functional, clot-based test. However, this test is susceptible to interference and has a lower specificity when compared to antigen assays used to detect free and total protein S levels. Subsequently, the Choosing Wisely Guidelines released by the ABIM Foundation recommends against using protein S activity assays entirely. Instead, they suggest using only free protein S antigen assays. Although the rationale is to avoid false positive results from the protein S activity assay, it ultimately may result in a failure to detect type II protein S deficiency. Objective This study, performed at a tertiary medical center in the Midwestern region of the United States, aims to determine the proportion of false positives in functional assays as well as the percent of true type II protein S deficiency cases within this testing population. Methods All cases with abnormal protein S function within the institution were evaluated from July 2018 through July 2023 and classified as acquired or hereditary deficiencies. Cases were classified as acquired if there was evidence of liver disease, vitamin K deficiency, ongoing thrombosis, consumption, disseminated intravascular coagulopathy, or pregnancy. Possible hereditary deficiency cases were classified as type I, type II and type III: Type I if all assays were low, type III if both active and free protein S assays were low with normal total protein S, and type II if only protein S activity was low. The cutoff defined for normal total and free protein S levels was greater than 70% activity, and the low protein S activity cutoff was defined as less than 50%. Patients with low protein S activity but normal immunologic protein S and an elevated factor VIII were considered falsely low values. Chart review will be performed for remaining cases with low protein S activity to rule out confounders such as vitamin K deficiency, warfarin use, acute phase reaction, or pregnancy. Results From a total of 8805 panels performed to assess for possible thrombophilia, 1580 (18%) were found to have decreased protein S activity. Of these cases, 599 (38%) were considered falsely decreased due to elevated factor VIII levels. 322 cases (3.7%) were considered possible hereditary deficiencies: 217 type III (2.5%), 27 type II (0.3%), and 80 type I (0.9%). Discussion Our data indicates the prevalence of subtypes may be differently distributed than previously believed, which may call for current guidelines to be reevaluated.