Evaluation of the use of a heparin dose-response test in dogs to determine the optimal heparin dose during intravascular procedures and assessment of the in vitro heparin response in healthy dogs.

BACKGROUND: No guidelines for administering and monitoring anticoagulants intraprocedurally are currently available in dogs, despite the prevalence of procedures necessitating systemic anticoagulation with heparin. OBJECTIVES: To evaluate an activated clotting time (ACT)-based heparin dose-response (HDR) test to predict the individual required heparin dose in dogs during intravascular procedures, and to investigate both the in vitro heparin - ACT and in vitro heparin - factor anti-Xa activity (anti-Xa) relationships in dogs. METHODS: Blood was collected from eight healthy beagles undergoing a cardiac procedure and utilised to establish baseline ACT and for in vitro evaluation. Subsequently, 100 IU/kg heparin was administered intravenously (IV) and ACT was remeasured (HDR test). The required heparin dose for an ACT target response ≥300 s was calculated for each individual and ACT was remeasured after administration of this dose. For in vitro testing, a serial heparin blood dilution (0-0.5-1-2-4 international unit (IU)/mL) was prepared and ACT and anti-Xa were determined using whole blood and frozen plasma, respectively. RESULTS: The HDR test overestimated the required heparin dose in 3/7 dogs. In vitro, ACT and anti-Xa increased significantly with increasing blood heparin concentration. Heparin - ACT was nonlinear in 4/8 dogs at heparin concentrations >2 IU/mL, whereas heparin - anti-Xa remained linear throughout the tested range. CONCLUSIONS: The HDR test poorly estimated the required heparin dose in dogs. This is most likely attributed to a nonlinear heparin - ACT relationship, as observed in vitro. Anti-Xa is a promising alternative for ACT; however, unavailability as a point-of-care test and lack of in vivo target values restrict its current use.

Identification of high thrombotic risk triple-positive antiphospholipid syndrome patients is dependent on anti-cardiolipin and anti-ß2glycoprotein I antibody detection assays.

Essentials Triple-positivity is associated with a high risk for a first thrombotic event and recurrence. Identification of triple-positives is dependent on the solid phase assay used. In triple-positivity, IgM only adds value in thrombotic risk stratification together with IgG. Thrombotic risk in triple-positive patients with IgM only, depends on the platform. ABSTRACT: Background The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPL). Triple-positivity (i.e. positivity for lupus anticoagulant [LAC], anti-cardiolipin [aCL] and anti-ß2glycoprotein I [aß2GPI] antibodies) is associated with a high thrombotic risk. Objectives We investigated the variability in triple-positivity detection by measuring the same samples with four commercially available solid phase assays. In addition, the added clinical value of aPL in LAC-positive patients was investigated, as well as the association of IgM triple-positivity and thrombosis. Patients/Methods We included 851 patients from seven European medical centers. Anti-CL and aß2GPI IgG/IgM antibodies were determined by four platforms: BioPlex® 2200, ImmunoCap® EliA, ACL AcuStar® and QUANTA Lite ELISA® . Results Triple-positivity detection by solid phase assays varied, ranging from 89 up to 118 in thrombotic APS patients (n = 258), of which 86 were detected independent of the platform. Lupus anticoagulant positivity resulted in an odds ratio (OR) for thrombosis of 3.4; triple-positivity (irrespective of the isotype) increased the OR from 4.3 up to 5.2, dependent on the platform. Triple-positivity solely for the IgM isotype did not increase the OR for thrombosis compared with LAC positivity. The highest OR for thrombosis was reached for positivity for IgG and IgM aß2GPI and aCL (8.6 up to 28.9). Conclusions Triple-positivity proved to be highly associated with thrombosis, but identification is assay dependent. Within triple-positivity, IgM antibodies only have an added clinical value in patients positive for IgG antibodies.

Pre-analytical stability of coagulation parameters in plasma stored at room temperature.

INTRODUCTION: Haemostasis testing is influenced by many pre-analytical variables, such as storage time and temperature, which can affect the stability of coagulation factors and influence the results of coagulation assays. We investigated the stability of haemostasis tests after storage of aliquoted plasma at RT, including the variability of measurement principle and reagent used for determination. METHODS: Blood samples from 20 healthy volunteers were obtained, processed to PPP and aliquoted. Aliquots were stored at RT for 0 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours and 48 hours. PT, aPTT, fibrinogen, D-Dimers and coagulation factors (FII, FV, FVII, FX, FVIII, FIX, FXI, FXII) were determined by STA-R Max® and ACL-TOP® . VWF:Ag and vWF:RCo were determined by AcuStar® . Clinically relevant changes, compared to the initial measurement, were denoted as a percentage change of > 10% according to the 99% CI. RESULTS: For both analysers, a clinically relevant change of > 10% was observed for FV after 2 hours, FVIII after 4 hours and for aPTT, FII, FVII, FX and FXII after 48 hours of storage at RT. Statistically significant, but no clinically relevant differences were observed after 48-hours storage for PT, fibrinogen and FIX. D-Dimers, FXI, vWF:Ag and vWF:RCo were found stable up to 48 hours at RT. CONCLUSION: Overall, compared to the limits given by the current CLSI guidelines, for most coagulation parameters investigated in this study a longer storage period could be accepted. Time intervals for FVIII and FV dosage were shorter than recommended by the CLSI guidelines. For PT determination, our findings were consistent.

Variability of cut-off values for the detection of lupus anticoagulants: results of an international multicenter multiplatform study.

Essentials Between-lab variations of cut-off values in lupus anticoagulant detection are unknown. Cut-off values were calculated in 11 labs each testing plasma from 120 donors with 3 platforms. Major variation was observed even within the same platform. Cut-off values determined in different labs are not interchangeable. SUMMARY: Background Cut-off values for interpretation of lupus anticoagulant (LA) detection are poorly investigated. Aims (i) To assess whether results from healthy donors were normally distributed and (ii) the between-laboratories differences in cut-off values for screening, mixing and LA confirmation when calculated as 99th or 95th centiles, and (iii) to assess their impact on the detection rate for LA. Methods Each of 11 laboratories using one of the three widely used commercial platforms for LA detection was asked to collect plasmas from 120 healthy donors and to perform screening, mixing and LA confirmation with two methods (activated partial thromboplastin time [APTT] and dilute Russell viper venom [dRVV]). A common set of LA-positive or LA-negative freeze-dried plasmas was used to assess the LA detection rate. Results were centralized (Milano) for statistical analysis. Results and conclusions (i) Clotting times or ratios for healthy subjects were not normally distributed in the majority of cases. The take-home message is that cut-off values should be determined preferably by the non-parametric method based on centiles. (ii) There were relatively large inter-laboratory cut-off variations even within the same platform and the variability was marginally attenuated when results were expressed as ratios (test-to-normal pooled plasma). The take-home message is that cut-off values should be determined locally. (iii) There were differences between cut-off values calculated as 99th or 95th centiles that translate into a different LA detection rate (the lower the centile the greater the detection rate). The take-home message is that cut-off values determined as the 95th centile allow a better LA detection rate.

Influence of apixaban on commonly used coagulation assays: results from the Belgian national External Quality Assessment Scheme.

INTRODUCTION: The Belgian national External Quality Assessment Scheme performed a survey to assess the effect of the direct oral anticoagulant apixaban on the coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin as performed with a large number of reagent/instrument combinations. METHODS: Four lyophilized plasma samples spiked with apixaban (0, 41, 94 and 225 ng/mL) were sent to the 195 Belgian and Luxembourg clinical laboratories performing coagulation testing. RESULTS: PT and aPTT were barely influenced at the concentrations tested. At 225 ng/mL apixaban, PT and aPTT clotting times were only 1.15 times longer than at 0 ng/mL. Among PT reagents, RecombiPlasTin 2G® showed a slightly higher sensitivity with 225 ng/mL apixaban prolonging the PT clotting time 1.3-fold. Among aPTT reagents, there was no appreciable difference in sensitivity. Fibrinogen results were unaffected by the presence of apixaban, but antithrombin activity was considerably overestimated when measured with a FXa-based assay. At 225 ng/mL apixaban, the median percentage increase in antithrombin level was 31% when measured with the Liquid Antithrombin® reagent and 44% with the Innovance Antithrombin® reagent. CONCLUSION: Our data provide clinical laboratories with useful information on the impact of apixaban on their routine coagulation assays.

Protein modelling to understand FGB mutations leading to congenital hypofibrinogenaemia.

INTRODUCTION: Congenital hypofibrinogenaemia is a quantitative fibrinogen disorder characterized by proportionally decreased levels of functional and antigenic fibrinogen. Mutations accounting for quantitative fibrinogen disorders are relatively frequent in the conserved COOH-terminal globular domains of the γ and Bß chains. The latter mutations are of particular interest since the Bß-chain is considered the rate-limiting chain in the hepatic production of the fibrinogen hexamer. AIM: The aim of this study was to study the molecular pattern of four patients with congenital hypofibrinogenaemia. METHODS: Four novel fibrinogen Bß-chain mutations leading to congenital hypofibrinogenaemia were identified in four women with heterogeneous symptoms. The human fibrinogen beta chain precursor protein sequence (P02675) was obtained from the UniProt database. The resulting models were analysed using swisspdbviewer 4.1.0. RESULTS: Three patients were heterozygous for different missense mutations located in the highly conserved ß nodule: c.882G>C:Arg294Ser (Arg264Ser), c.1298G>T:Trp433Leu (Trp403Leu) and c.1329C>G:Asn443Lys (Asn413Lys). Modelling analyses predicted major structural modifications likely to result in impaired fibrinogen secretion. One patient was heterozygous for an intron 7 donor splice mutation (c.1244 + 1G>A), leading to the complete abolishment of the donor site. CONCLUSIONS: Protein modelling of new causative mutations and comparison of molecular, biochemical and clinical data continue to yield valuable information on the development and course of fibrinogen disorders as well as on the choice of the most appropriate treatments.

A multicenter study to assess the reproducibility of antiphospholipid antibody results produced by an automated system.

Essentials Inter-lab variation studies for antiphospholipid antibodies (aPL) with the same assay are lacking. We carried out an assessment of repeatability and reproducibility of an automated aPL assay. High intra-center repeatability for anticardiolipin and aß2 GPI makes duplicate testing unnecessary. Inter-lab reproducibility was high except for aß2GPI IgG. SUMMARY: Background Inter-assay variability is a well-known problem in antiphospholipid antibody testing, because of the lack of standardization. Inter-laboratory reproducibility for the same assay is similarly important. Objectives Testing repeatability and reproducibility of HemosIL® AcuStar for anticardiolipin (aCL) and antiß2-glycoprotein I antibodies (aß2GPI) IgG and IgM. Patients/Methods In this observational study, out of 420 samples from the thrombophilia centers of Ghent and Geneva, 100 samples were randomly selected and successively analyzed in three centers: Ghent (C1, in duplicate for repeatability evaluation), Geneva (C2) and Frankfurt (C3). Results Results from 99 samples were available, including 25 from patients with antiphospholipid syndrome (APS) and 74 from non-APS patients. The intra-center repeatability expressed as intra-class correlation coefficient (ICC) was higher than 0.99 for each parameter. Differences between two measurements rarely exceeded 1 U mL-1 for values below 100 U mL-1 , except for aß2GPI IgG, where differences varied from -4 to 4 U mL-1 . The inter-center ICCs were higher than 0.99, except for aCL IgM (ICC = 0.961). These ICCs remained high even when considering values below 100 U mL-1 (0.943, 0.964 and 0.977 for aCL IgG, aCL gM and aß2GPI IgM, respectively), except for aß2GPI IgG (ICC = 0.652). Qualitative comparison showed less than 5% discordant classification between centers, with somewhat more discordant results for aß2GPI IgG. Conclusions In terms of discriminating properties, the HemosIL® AcuStar has excellent intra-center repeatability and a good inter-center reproducibility for aCL IgG, aCL IgM and aß2GPI IgM. Some concern may arise for aß2GPI IgG.

Role of anti-domain 1-ß2 glycoprotein I antibodies in the diagnosis and risk stratification of antiphospholipid syndrome.

UNLABELLED: Essentials Antibodies to domain 1 of ß2 glycoprotein I (aD1) are a subset of antiphospholipid antibodies. We evaluated the added diagnostic value of an automated aD1 assay in antiphospholipid syndrome. AD1 IgG correctly classifies patients at risk for thrombosis. Agreement between aD1 and aß2GPI IgG is high, limiting the added value of aD1 in our setting. Click to hear Professor de Groot's perspective on new mechanistic understanding in antiphospholipid syndrome SUMMARY: Background Laboratory diagnosis of antiphospholipid syndrome (APS) includes lupus anticoagulant (LAC), anticardiolipin (aCL) or anti-ß2 glycoprotein I (aß2 GPI) antibodies. Antibodies targeting domain 1 of ß2 GPI (aD1) constitute a pathogenic subset of autoantibodies. Objectives In this cohort study, we determined the clinical performance characteristics, additional diagnostic value and the contribution to APS risk stratification of an automated aD1 assay. Patients/Methods LAC, aCL, aß2 GPI and aD1 IgG were measured in 101 APS patients, 123 patients with autoimmune disorders, 82 diseased controls and 120 healthy controls. aD1 antibodies were detected by QUANTA Flash(®) Beta2GPI-Domain 1 chemiluminescence immunoassay. Results With a cut-off value of 20.0 CU, the aD1 IgG assay identifies APS patients in a clinically affected patient cohort with a sensitivity of 53.5% and specificity of 98.8%. It implied a high odds ratio (OR) for clinical events (OR, 17.0; 95% confidence interval [CI], 7.1-40.5). aD1 IgG did not add diagnostic value to the formal aPL panel because aß2 GPI IgG was nearly as specific but more sensitive for APS (sensitivity 56.4%) with a higher OR for clinical events (36.2; 95% CI, 11.1-117.9). High aD1 titers identify triple-positive patients and patients with thrombosis in a ß2 GPI-dependent LAC-positive population. Agreement between aD1 IgG and aß2 GPI IgG was high (positive and negative agreement 91.7% and 98.4%, respectively). Conclusion Detection of aD1 IgG correctly classifies patients at risk of thrombosis. However, the contribution of aD1 IgG to APS diagnosis and risk stratification depends upon the solid phase assays used for aCL and aß2 GPI detection.

IgG/IgM antiphospholipid antibodies present in the classification criteria for the antiphospholipid syndrome: a critical review of their association with thrombosis.

UNLABELLED: Essentials The clinical value of IgM antibodies in thrombotic antiphospholipid syndrome (APS) is debated. By review of literature, we reconsidered the clinical value of IgM antibodies in thrombotic APS. More significant correlations with thrombosis were found for the IgG compared to IgM isotype. Unavailability of paired IgG/IgM results hampers evaluating the added value of IgM positivity. Click to hear Dr de Groot's perspective on antiphospholipid syndrome SUMMARY: Background Despite the update of the classification criteria for the antiphospholipid syndrome (APS), difficulties persist in the identification of patients at risk for thrombosis. Current guidelines include assays detecting IgG/IgM anti-ß2 -glycoprotein I and anti-cardiolipin antibodies, although the relevance of IgM antibodies has been debated. Objectives Through a review of the literature from 2001 to 2014, we aimed to formally establish the thrombotic risk stratification potential of IgM as compared with IgG anti-phospholipid antibodies (aPLs). Patients/methods One thousand two hundred and twenty-eight articles were selected by a computer-assisted search of the literature. Of the 177 studies that met our inclusion criteria, the clinical value of IgG/IgM aPLs was established through analysis of odds ratios for thrombosis or percentage of positives in the thrombotic population. Results/conclusions We clearly found more significant correlations with thrombosis for the IgG than for the IgM isotype. Nonetheless, in a minority of studies, significant associations with thrombosis were found for IgM but not IgG antibodies. The unavailability of paired results of IgG and IgM for each separate patient hampers evaluation of the added value of isolated IgM positivity. To fully take advantage of results obtained by future studies, we strongly encourage scientists to provide all studied information per patient. We planned a large multicenter study to investigate clinical associations of isolated/combined positivity for criteria/non-criteria aPLs. Importantly, because of the presence of non-pathogenic aPLs, quantitative assays are characterized by a high false-positivity rate. Optimization of functional assays, such as thrombin generation measuring the whole scheme of coagulation, may help to reduce APS-related morbidity and mortality.

Mixing studies in lupus anticoagulant testing are required at least in some type of samples.

BACKGROUND: According to the ISTH guidelines for lupus anticoagulant (LAC) testing, the second step in the three-step procedure (screening, mixing, and confirmation) is the mixing test, which improves the discrimination between the presence of an inhibitor and coagulation factor deficiencies such as those occurring in patients receiving vitamin K antagonists (VKAs). OBJECTIVES: From a retrospective analysis of dilute Russell viper venom (dRVVT) results, we evaluated the impact of the mixing test result on the interpretation of LAC positivity. METHODS: We interpreted the dRVVT clotting times with and without taking into account the results of the mixing test in a patient population with prolonged screening test (n = 267) with special attention to the patients receiving VKAs. RESULTS AND CONCLUSIONS: The number of samples classified as 'LAC positive' differed substantially depending on the method of interpretation; 170 and 235 of 267 samples were classified as LAC positive with the three- and two-step procedure, respectively. Discrepancy between the two-step (without mixing step) and the three-step procedure was due to not including a mixing test and was more pronounced in the VKA patient population. Screen/confirm ratios carried out on a 1:1 mix of patient and normal pooled plasma (NPP) gave a lower incidence of 59 of 267. We advise continuing to perform mixing test to avoid false-positives. In patients with discrepant results between the two- and three-step dRVVT interpretation, mainly observed in VKA-treated patients, we advise retesting of the patients preferable beyond the period of anticoagulant therapy and additional testing for anti-beta2GPI and/or anti-cardiolipin antibodies.

Lupus anticoagulant-hypoprothrombinemia syndrome: report of two cases and review of the literature.

Lupus anticoagulant-hypoprothrombinemia syndrome (LA-HPS) is a rare acquired disorder caused by prothrombin antibodies. The disease is most common in the pediatric age group (<16 years), and more prevalent in women. There are well-established clinical diseases associated with LA-HPS, most notably systemic lupus erythematosus (SLE) and viral infections. The clinical manifestation of LA-HPS varies greatly in severity and it may cause severe life-threatening bleeding diathesis. LA-HPS is to be suspected when a patient presents with bleeding and a prolonged activated partial thromboplastin and prothrombin time, in combination with a lupus anticoagulant. The diagnosis is confirmed in the laboratory by identification of reduced prothrombin levels. There are no standardized recommendations for treatment of the hemorrhage associated with the syndrome; corticosteroids are used as first-line treatment. This review summarizes what is currently known about the pathogenesis, clinical features, diagnosis, treatment and prognosis of LA-HPS, and presents two case reports.

Refinement of the cutoff values of the HemosIL AcuStar assay for the detection of anticardiolipin and anti-beta2 glycoprotein-1 antibodies.

BACKGROUND: The HemosIL AcuStar antiphospholipid assay (Instrumentation Laboratory, Bedford, MA, USA) is a fully automated assay using chemiluminescent technology for the detection of anticardiolipin and anti-beta2 glycoprotein-1 antibodies. This assay showed excellent agreement between results of different laboratories. The cutoff values to define positivity were calculated in 250 healthy blood bank donors but were associated with large confidence intervals (CIs). OBJECTIVE: The objective of this study was to more precisely determine the cutoff values of the HemosIL AcuStar antiphospholipid assay by increasing the number of healthy blood bank donors through a multicenter study and by applying a normalization procedure of the distribution of each antibody. METHODS: Five laboratories participated to this study, allowing the inclusion of 626 samples. We used a Box-Cox power transformation method to normalize the distribution and calculate the 99th percentile and the corresponding 95%CI for each antibody. RESULTS: The revised cutoff values were overall lower than those initially calculated with more stringent CIs and yielded a 4.2% increase in sensitivity with a 2.7% decrease in specificity regarding thrombotic events or obstetric complications. CONCLUSIONS: We provide refined cutoff values for the detection of anticardiolipin and anti-beta2 glycoprotein-1 antibodies with the HemosIL AcuStar Antiphospholipid assay that should be preferred for routine use.