A multicentre assessment of contemporary laboratory assays for heparin induced thrombocytopenia.

Heparin induced thrombocytopenia (HIT) is a rare but potentially fatal complication of heparin therapy. In some patients, HIT causes platelet activation and thrombosis (sometimes abbreviated HITT), which leads to adverse clinical sequalae ('pathological HIT'). The likelihood of HIT is initially assessed clinically, typically using a scoring system, of which the 4T score is that most utilised. Subsequent laboratory testing to confirm or exclude HIT facilitates exclusion or diagnosis and management. The current investigation comprises a multicentre (n=9) assessment of contemporary laboratory testing for HIT, as performed over the past 1-3 years in each site and comprising testing of over 1200 samples. The primary laboratory test used by study participants (n=8) comprised a chemiluminescence procedure (HIT-IgG(PF4-H)) performed on an AcuStar instrument. Additional immunological testing performed by study sites included lateral flow (STiC, Stago), enzyme linked immunosorbent assay (ELISA), Asserachrom (HPIA IgG), PaGIA (BioRad), plus functional assays, primarily serotonin release assay (SRA) or platelet aggregation methods. The chemiluminescence procedure yielded a highly sensitive screening method for identifying functional HIT, given high area under the curve (AUC, generally ≥0.9) in a receiver operator characteristic (ROC) analysis against SRA as gold standard. ELISA testing resulted in lower ROC AUC scores (<0.8) and higher levels of false positives. Although there is clear association with the likelihood of HIT, the 4T score had less utility than literature suggests, and was comparable to a previous study reported by some of the authors.

A multicenter laboratory assessment of a new automated chemiluminescent assay for ADAMTS13 activity.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal disorder caused by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency. Prompt identification/exclusion of TTP can thus be facilitated by rapid ADAMTS13 testing. The most commonly utilized (enzyme-linked immunosorbent assay [ELISA]-based) assay takes several hours to perform and so does not generally permit rapid testing. OBJECTIVES: To evaluate the utility of a new automated test for ADAMTS13 activity, the HemosIL AcuStar ADAMTS13 Activity assay, based on chemiluminescence and able to be performed on an ACL AcuStar instrument within 33 minutes. PATIENTS/METHODS: This multicenter (n = 8) assessment included testing of more than 700 test samples, with similar numbers of prospective (n = 348) and retrospective (n = 385) samples. The main comparator was the Technozym ADAMTS13 Activity ELISA. We also assessed comparative performance for detection of ADAMTS13 inhibitors using a Bethesda assay. RESULTS: Overall, the chemiluminescent assay yielded similar results to the comparator ELISA, albeit with slight negative bias. ADAMTS13 inhibitor detection was also comparable, albeit with slight positive bias with the AcuStar assay. Assay precision was similar with both assays, and we also verified assay normal reference ranges. CONCLUSIONS: The HemosIL AcuStar ADAMTS13 Activity assay provided results rapidly, which were largely comparable with the Technozym ADAMTS13 Activity ELISA assay, albeit lower on average. Conversely, inhibitor levels tended to be identified at a higher level on average. Thus, the HemosIL AcuStar ADAMTS13 Activity assay provides a fast and accurate means to quantitate plasma levels of ADAMTS13 for TTP/ADAMTS13 identification/exclusion, and potentially also for other applications.

Relationship of waist and hip circumference with coagulation and fibrinolysis in postmenopausal women.

The contribution of obesity to the occurrence of cardiovascular events may not be wholly related to its influence on traditional risk factors. Coagulation and fibrinolysis may also influence cardiovascular risk, but the relationship of adiposity with these processes is unclear. The aim of the present study was to investigate the relationships of BMI (body mass index), waist circumference, hip circumference and WHR (waist-to-hip ratio) with VIIc (factor VII activity), plasma markers of thrombin generation [F1+2 (prothrombin fragment 1+2)], fibrin formation [SF (soluble fibrin)] and fibrin turnover (D-dimer), and PAI-1 (plasminogen activator inhibitor-1; a marker of fibrinolytic inhibitory capacity). The study cohort was 80 healthy postmenopausal women who were not diabetic, current smokers or taking hormone therapy and who had a fasting sample of blood collected. VIIc, F1+2, SF and PAI-1 were all positively correlated with BMI, waist circumference and WHR, whereas D-dimer was positively correlated with waist circumference and WHR, but not BMI. WHR was the strongest correlate of all the markers except for PAI-1, which was most closely related to BMI. Hip circumference became a negative correlate of F1+2 and D-dimer after adjusting for waist circumference. The relationships of WHR with F1+2 and SF, but not with VIIc and D-dimer, were independent of traditional risk factors. The positive association between waist circumference and markers of thrombin generation, fibrin production and fibrin turnover suggests that abdominal adiposity may contribute to atherothrombosis by activating intravascular coagulation. In contrast, a larger hip circumference appears to have a protective affect against coagulation activation.

Comparison of effects of pravastatin and hormone therapy on soluble P-selectin and platelet P-selectin expression in postmenopausal hypercholesterolemic women.

OBJECTIVE: Recent trials have suggested an adverse early effect on cardiovascular risk of hormone therapy (HT) in postmenopausal women, an effect which could be due to an increase in arterial thrombosis via platelet activation. We examined the effect of HT on platelet surface expression of P-selectin, a marker of platelet activation, and plasma levels of soluble P-selectin, also believed to be a marker of platelet activation, and compared these effects with pravastatin, a drug proven to reduce cardiovascular events and reported to decrease both platelet and soluble P-selectin. METHODS: Surface expression of platelet P-selectin, soluble P-selectin and fasting lipids were measured at baseline and 6 months in a randomized, double-blind study of postmenopausal hypercholesterolemic women comparing low-dose combined HT (1mg estradiol + 0.5 mg norethisterone acetate; n = 26) with pravastatin (n = 24). RESULTS: After adjusting for baseline levels, HT and pravastatin produced similar reductions in soluble P-selectin (p < 0.0001 for both). The percentage of platelets expressing P-selectin was also reduced by pravastatin (p = 0.025), but there was a trend to an increase in platelet P-selectin expression with HT (p = 0.13), and a significant difference between pravastatin and HT in the changes in platelet P-selectin (p < 0.002). No relationship was evident between changes in soluble or platelet P-selectin and changes in lipids with either treatment. CONCLUSIONS: In postmenopausal hypercholesterolemic women, both pravastatin and HT reduced soluble P-selectin levels, but only pravastatin reduced P-selectin expression on the surface of platelets. An implication of these findings is that the reduction in soluble P-selectin by HT may occur by a non-platelet related mechanism.

Dietary soy containing phytoestrogens does not activate the hemostatic system in postmenopausal women.

The soybean is rich in isoflavone phytoestrogens, which are ligands for estrogen receptors, but it is unknown whether soy/phytoestrogens have similar procoagulant effects to estrogen. In this randomized double-blind trial, 40 healthy postmenopausal women of age 50-75 yr received soy protein isolate (40 g soy protein, 118 mg isoflavones) (n = 19) or casein placebo (n = 21). Plasma markers of coagulation, fibrinolysis, and endothelial dysfunction were measured at baseline and 3 months. The baseline characteristics of the two groups were similar. Compared with casein placebo, soy decreased triglycerides (P < 0.005) and low-density lipoprotein/high-density lipoprotein ratio (P < 0.001) and increased lipoprotein (a) (P < 0.05). Activity of coagulation factor VII (VIIc) decreased similarly in both groups (P < 0.005). Prothrombin fragments 1 + 2 (a marker of thrombin generation) decreased in the soy group (P < 0.005), but the change was not different from the casein group. There was no effect of soy on soluble fibrin (a marker of fibrin production), plasminogen activator inhibitor-1 (a marker of fibrinolytic inhibitory potential), D-dimer (a marker of fibrin turnover), or von Willebrand factor (a marker of endothelial damage). In conclusion, the results of the current study do not support biologically significant estrogenic effects of soy/phytoestrogens on coagulation, fibrinolysis, or endothelial function.

The effect of dabigatran on the activated partial thromboplastin time and thrombin time as determined by the Hemoclot thrombin inhibitor assay in patient plasma samples.

Dabigatran is an oral direct thrombin inhibitor that does not require routine laboratory monitoring. However, an assessment of its anticoagulant effect in certain clinical settings is desirable. We examined the relationship between dabigatran levels, as determined by the Hemoclot thrombin inhibitor assay (HTI), the thrombin time (TT) and the activated partial thromboplastin time (aPTT) using different reagents. We describe these parameters with the clinical outcomes of patients receiving dabigatran. Seventy-five plasma samples from 47 patients were analysed. The HTI assay was established to measure dabigatran level. aPTTs were performed using TriniCLOT aPTT S reagent (TC) and three additional aPTT reagents. From linear regression lines, we established the aPTT ranges corresponding to the therapeutic drug levels for dabigatran (90-180 ng/ml). The aPTT demonstrated a modest correlation with the dabigatran level (r= 0.80) but the correlation became less reliable at higher dabigatran levels. The therapeutic aPTT ranges for reagents were clinically and statistically different compared with our reference reagent (46-54 s (TC) vs 51-60 s (SP), 54-64 s (SS) and 61-71 s (Actin FS) (p<0.05)). The TT was sensitive to the presence of dabigatran with a level of 60 ng/ml resulting in a TT > 300 s. In conclusion, the aPTT demonstrated a modest correlation with the dabigatran level and was less responsive with supra-therapeutic levels. aPTT reagents differed in their responsiveness, suggesting individual laboratories must determine their own therapeutic range for their aPTT reagent. The TT is too sensitive to quantify dabigatran levels, but a normal TT suggests minimal or no plasma dabigatran.

Factor XIII assays.

Factor XIII (FXIII) deficiency is a rare cause of bleeding and pregnancy loss that is easily treated with plasma products. Reliable assays for FXIII are necessary not only for the diagnosis of deficiency state but also to guide prophylaxis and replacement therapy in patients during times of increased risk. Diagnostic tests for FXIII activity whilst not technically demanding have a number of pitfalls including limited sensitivity and overestimation of activity at the lower end. Despite these shortcomings the performance of these assays is adequate for the diagnosis and management of the majority of patients with clinically significant deficiency.

Chronic threshold testing of implantable cardioverter defibrillators does not increase coagulation activity or platelet activation.

It is unknown if the brief periods of circulatory statis occurring with induction of VF during DFT testing in patients with an ICD activate blood coagulation processes. To address this question, coagulation activity and platelet activation were measured in peripheral venous blood samples obtained from 12 patients undergoing DFT testing under general anesthesia, 3 (n = 11) or 6 months (n = 1) after ICD implantation for recurrent ventricular arrhythmias. Five patients were anticoagulated with warfarin and two to six episodes of VF (median five) were induced per patient. Blood samples were drawn at baseline, after each DFT test and on the following morning. Coagulation activity was assessed by measuring prothrombin fragment 1 + 2 (F1 + 2) (a marker of thrombin generation), soluble fibrin (a marker of fibrin production), and D-dimer (a breakdown product of cross-linked fibrin). Platelet activation was evaluated by measuring the expression P-selectin on the platelet surface using flow cytometry. No significant changes in F1 + 2, soluble fibrin, D-dimer, or P-selectin expression occurred during DFT testing, or between baseline and morning after samples. Moreover, results were unaffected by warfarin. These findings suggest that chronic threshold testing of implantable cardioverter defibrillators is not associated with activation of blood coagulation processes.