Measuring FVIII activity of glycopegylated recombinant factor VIII, N8-GP, with commercially available one-stage clotting and chromogenic assay kits: a two-centre study.

INTRODUCTION: Factor VIII activity (FVIII:C) assays of samples containing glycoPEGylated recombinant FVIII such as turoctocog alfa pegol (N8-GP) can be associated with differences in FVIII recovery in vitro between various one-stage activated partial thromboplastin time (APTT)-based clotting assays and some chromogenic assays. Careful validation and qualification of specific assays and conditions is therefore necessary for the assessment of FVIII:C in samples containing modified FVIII molecules. AIM: To assess the ability of various one-stage clotting and chromogenic FVIII:C assays to measure samples containing N8-GP compared to unmodified recombinant FVIII (rFVIII) across two laboratory sites. METHODS: Factor VIII activity in severe haemophilia A (HA) plasma spiked with a range of concentrations (from low, 0.20 IU mL-1 , to high, 0.90 IU mL-1 ) of N8-GP and rFVIII, was determined at two laboratory sites using 12 commercially available one-stage clotting and chromogenic FVIII:C assays. Assays were performed using a plasma calibrator and different analysers. RESULTS: Acceptable N8-GP recovery was observed in the low to high concentration samples tested using the majority of the tested APTT reagents with only one reagent causing a significant underestimation as compared to rFVIII. For the chromogenic assays, a slight overestimation was observed with some of the kits. Variability between the two laboratory sites are likely attributable to the use of different analysers with the respective APTT reagents. CONCLUSIONS: These results highlight the need to investigate the performance of modified factor products using standard assays. The performance of different one-stage clotting assays, APTT reagents, reference calibrators and instrumentation should also be evaluated.

Measuring factor IX activity of nonacog beta pegol with commercially available one-stage clotting and chromogenic assay kits: a two-center study.

UNLABELLED: Essentials Validated assays are required to precisely measure factor IX (FIX) activity in FIX products. N9-GP and two other FIX products were assessed in various coagulation assay systems at two sites. Large variations in FIX activity measurements were observed for N9-GP using some assays. One-stage and chromogenic assays accurately measuring FIX activity for N9-GP were identified. SUMMARY: Background Measurement of factor IX activity (FIX:C) with activated partial thromboplastin time-based one-stage clotting assays is associated with a large degree of interlaboratory variation in samples containing glycoPEGylated recombinant FIX (rFIX), i.e. nonacog beta pegol (N9-GP). Validation and qualification of specific assays and conditions are necessary for the accurate assessment of FIX:C in samples containing N9-GP. Objectives To assess the accuracy of various one-stage clotting and chromogenic assays for measuring FIX:C in samples containing N9-GP as compared with samples containing rFIX or plasma-derived FIX (pdFIX) across two laboratory sites. Methods FIX:C, in severe hemophilia B plasma spiked with a range of concentrations (from very low, i.e. 0.03 IU mL(-1) , to high, i.e. 0.90 IU mL(-1) ) of N9-GP, rFIX (BeneFIX), and pdFIX (Mononine), was determined at two laboratory sites with 10 commercially available one-stage clotting assays and two chromogenic FIX:C assays. Assays were performed with a plasma calibrator and different analyzers. Results A high degree of variation in FIX:C measurement was observed for one-stage clotting assays for N9-GP as compared with rFIX or pdFIX. Acceptable N9-GP recovery was observed in the low-concentration to high-concentration samples tested with one-stage clotting assays using SynthAFax or DG Synth, or with chromogenic FIX:C assays. Similar patterns of FIX:C measurement were observed at both laboratory sites, with minor differences probably being attributable to the use of different analyzers. Conclusions These results suggest that, of the reagents tested, FIX:C in N9-GP-containing plasma samples can be most accurately measured with one-stage clotting assays using SynthAFax or DG Synth, or with chromogenic FIX:C assays.

Comparison of several von Willebrand factor (VWF) activity assays for monitoring patients undergoing treatment with VWF/FVIII concentrates: improved performance with a new modified automated method.

BACKGROUND: The ability of von Willebrand factor (VWF) to bind platelet GP Ib and promote platelet plug formation is measured in vitro using the ristocetin cofactor (VWF:RCo) assay. Automated assay systems make testing more accessible for diagnosis, but do not necessarily improve sensitivity and accuracy. OBJECTIVE: We assessed the performance of a modified automated VWF:RCo assay protocol for the Behring Coagulation System (BCS(®) ) compared to other available assay methods. METHODS: Results from different VWF:RCo assays in a number of specialized commercial and research testing laboratories were compared using plasma samples with varying VWF:RCo activities (0-1.2 IU mL(-1) ). Samples were prepared by mixing VWF concentrate or plasma standard into VWF-depleted plasma. Commercially available lyophilized standard human plasma was also studied. Emphasis was put on the low measuring range. VWF:RCo accuracy was calculated based on the expected values, whereas precision was obtained from repeated measurements. RESULTS: In the physiological concentration range, most of the automated tests resulted in acceptable accuracy, with varying reproducibility dependent on the method. However, several assays were inaccurate in the low measuring range. Only the modified BCS protocol showed acceptable accuracy over the entire measuring range with improved reproducibility. CONCLUSIONS: A modified BCS(®) VWF:RCo method can improve sensitivity and thus enhances the measuring range. Furthermore, the modified BCS(®) assay displayed good precision. This study indicates that the specific modifications - namely the combination of increased ristocetin concentration, reduced platelet content, VWF-depleted plasma as on-board diluent and a two-curve calculation mode - reduces the issues seen with current VWF:RCo activity assays.

Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays.

INTRODUCTION: Apixaban is an oral direct factor Xa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information. OBJECTIVES: To investigate the effects of apixaban on commonly used coagulation methods, and to evaluate anti-FXa assays for specific determination of the drug concentration. MATERIALS AND METHODS: Apixaban was added to plasma from healthy subjects in the concentration range 0-1000 µg L(-1) , and analyses were performed with different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, protein C, and protein S. A lupus anticoagulant assay and an APTT assay with varying phospholipid concentrations were used to study the phospholipid dependence. RESULTS: In general, apixaban showed fewer effects in vitro than have been shown for rivaroxaban, another direct FXa inhibitor. The concentration needed to double the APTT varied between 2200 and 4700 µg L(-1) , and the concentration needed to double the PT varied between 700 and 3900 µg L(-1) . The effects on antithrombin, protein C and protein S assays were dependent on the type of reagent. Apixaban did not cause false-positive lupus anticoagulant results. Chromogenic anti-FXa assays showed linear dose-response curves with apixaban. CONCLUSIONS: Therapeutic concentrations of apixaban variably affect different assay groups, and even different reagents within an assay group. The effects were much smaller than with rivaroxaban. The use of APTT and/or PT assays to screen the anticoagulant activity of apixaban cannot be recommended. A chromogenic anti-FXa assay can be used for reliable measurements of apixaban concentration.

Laboratory aspects of von Willebrand disease: test repertoire and options for activity assays and genetic analysis.

The deficiency or abnormal function of von Willebrand factor (VWF) causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder. The laboratory diagnosis of VWD can be difficult as the disease is heterogeneous and an array of assays is required to describe the phenotype. Basic classification of quantitative (type 1 and 3) and qualitative (type 2) VWD variants requires determination of VWF antigenic (VWF:Ag) levels and assaying of VWF ristocetin cofactor (VWF:RCo) activity, determining the capacity of VWF to interact with the platelet GPIb-receptor. Knowing the VWF:RCo activity is essential for identifying, subtyping and monitoring VWD, but the assay is poorly standardized and many protocols do not fulfil the clinical need in all situations. This has led to the development of novel activity assays, independent of ristocetin, with enhanced assay characteristics. Results from the first independent clinical evaluations are promising, showing that they are reliable and suitable for VWD diagnosis. The qualitative type 2 VWF deficiency can be further divided into four different subtypes (A, B, M and N) using specific assays that explore other activities or the size distribution of VWF multimers. These methods are discussed herein. However, in a number of patients it may be difficult to correctly classify the VWD phenotype and genetic analysis may provide the best option to clarify the disorder, through mutation identification.

Measuring Oral Direct Inhibitors (ODIs) of thrombin and factor Xa: A recommendation from the Subcommittee on Control of Anticoagulation of the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis.

Oral direct inhibitors (ODIs) of thrombin and factor Xa are now approved as anticoagulant drugs. The first two drugs to complete phase III clinical trials for thromboprophylaxis in orthopaedic surgery and treatment of patients with atrial fibrillation or venous thromboembolism were dabigatran and rivaroxaban. These small molecules are given at fixed dose with no requirement for monitoring as pharmacokinetic and pharmacodynamic responses are reliably predicted in patients with adequate renal function who are not taking other interacting drugs. However, there will be clinical circumstances in specific patients when measurement of the anticoagulant effect of an ODI will be required. © 2013 International Society on Thrombosis and Haemostasis.

Antibody formation and specificity in Bethesda-negative brother pairs with haemophilia A.

Antibodies directed towards non-neutralizing epitopes on the factor VIII protein (FVIII) may be detected in patients with haemophilia A. We evaluated the prevalence of non-neutralizing antibodies, in 201 inhibitor-negative brother pairs with severe haemophilia A, enrolled in the Malmö International Brother Study and the Haemophilia Inhibitor Genetics Study. To evaluate binding specificity of the antibodies, ELISA plates were coated with two recombinant full-length (FL) FVIII-products and one recombinant B-domain-deleted (BDD) product. Seventy-nine patients (39.3%) had a history of positive inhibitor titre measured by Bethesda assay, and FVIII antibodies were detected in 20 of them (25.3%). Additional 23 samples from subjects without a history of FVIII inhibitors were ELISA-positive corresponding to a frequency of non-neutralizing antibodies of 18.9%. The antibody response towards the different FVIII products was heterogenous, and was raised not only towards the non-functional B-domain but also towards both FL-rFVIII and BDD-rFVIII. In patients considered successfully treated with immune tolerance induction, 25.4% had remaining FVIII antibodies. The number of families with an antibody response in all siblings was increased when the total antibody response was taken into account, further supporting the concept of a genetic predisposition of the immune response. Further studies and careful monitoring over time are required to appreciate the immune response on the risk of inhibitor development or recurrence in the future.

Difficulties and pitfalls in the laboratory diagnosis of bleeding disorders.

von Willebrand disease (VWD) is the most common inherited bleeding disorder, but variable severity and several classification types mean that diagnosis is often not straightforward. In many countries, the assays are not readily available and/or are not well standardized. The latest methods and the basis of VWD are discussed here, together with information from the international quality assessment programme (IEQAS). Factor XIII deficiency is a rare, but important bleeding disorder, which may be missed or diagnosed late. A discussion and update on this diagnosis is considered in the final section of our review.

Effects of the oral, direct factor Xa inhibitor rivaroxaban on commonly used coagulation assays.

INTRODUCTION: Rivaroxaban is an oral direct factor Xa inhibitor developed for prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary but the dose-dependent effects on common reagents and assay procedures are largely unknown. OBJECTIVES: To investigate the effect of rivaroxaban on commonly used coagulation assays. MATERIALS AND METHODS: Rivaroxaban was added to plasma from healthy subjects in the concentration range 0-1000 µg L(-1) and analyzed using different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, fibrinogen and activated protein C (APC) resistance assays. RESULTS: At an expected peak concentration of rivaroxaban in clinical use, the APTTs were almost invariably prolonged but at lower concentrations the effect was weak. The concentration needed to double the APTT varied between 389 ± 106 and 617 ± 149 µg L(-1) for different reagents. The PT assays showed a marked degree of difference. In general, the Quick PT type assays were more sensitive compared with the Owren type PT assays. The results from antithrombin assays were dependent on the type of reagent, with the Xa-based assay being sensitive for rivaroxaban with an estimated increase of 0.09 IU mL(-1) per 100 µg L(-1) rivaroxaban. There were only minor effects on fibrinogen assays based on thrombin reagents. The APTT-based assay for APC resistance is affected in a dose-dependent manner whereas an assay based on the activation of coagulation at the prothrombinase level was unaffected. CONCLUSIONS: Different assays, and even different reagents within an assay group, display variable effects by therapeutic concentrations of rivaroxaban.

Improved performance characteristics of the von Willebrand factor ristocetin cofactor activity assay using a novel automated assay protocol.

UNLABELLED: BACKGROUND, OBJECTIVES AND METHODS: An accurate, sensitive and precise assay for reliable determination of the ristocetin cofactor activity of von Willebrand factor (VWF:RCo) in plasma and von Willebrand Factor (VWF)-containing concentrates has been evaluated. The assay is based on a commercially available automated protocol with modifications including a combination of adding additional ristocetin and the use of two calibration curves for the high and low measuring ranges. RESULTS: Addition of extra ristocetin resulted in improved measurement of VWF recoveries from various VWF-containing concentrates that were underestimated using the standard automated protocol. The modifications resulted in improved assay performance over an extended measuring range (2.00-0.03 IUmL(-1) ). Accuracy was tested using VWF deficiency plasma spiked with the 1st international standard (IS) for VWF concentrate. Seven dilutions, ranging from 1.80 to 0.05IUmL(-1) , were analyzed and resulted in measured concentrations between 80% and 100% of the assigned potency of the standard. Linearity was determined from the regression plot of the same concentrate dilutions and resulted in a correlation coefficient of 0.998. The repeatability, expressed as coefficient of variation, was 2% in the normal range (0.90IUmL(-1) ) and 8% at the level of 0.05IUmL(-1) . The corresponding reproducibility results were 2% and 15% at the normal and low measuring ranges, respectively. CONCLUSIONS: Analysis of patients with von Willebrand disease (VWD) indicates that the modified automated BCS(®) protocol has a superior discrimination power compared with the standard protocol. This is especially true in samples with low VWF, as in patients with type 3 VWD.

Considerations in the laboratory assessment of haemostasis.

SUMMARY: This review outlines a number of key issues when performing laboratory testing of homeostasis. The effect pre-analytical variables have on the reliability and consistency of screening tests is often forgotten due to a lack of understanding and awareness. This can be improved through educating healthcare professionals who are involved in taking blood for assessment. Recent advances in coagulation testing have not enabled laboratories to replace the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) screening tests with more advanced assays and they continue to play an important role with the advantage of being easily automated. However, there are many analysers on the market, each with varying sensitivity to coagulation defects and it is important to keep this in mind when interpreting results.

Minor stroke as singular manifestation of hereditary thrombotic thrombocytopenic purpura in a young man.

The authors describe a case of a 38-year-old male with minor stroke due to exacerbation of hereditary deficiency of ADAMTS 13 resulting in a chronic relapsing form of thrombotic thrombocytopenic purpura (TTP). The clue to the unusual pathogenesis was given by laboratory findings of a mild anaemia and thrombocytopenia. After two days of observation, the patient was treated with plasmapheresis resulting in normalized platelet levels and continued clinical improvement. Subsequent clinical and laboratory investigation verified the diagnosis and the patient was put on regular treatments with plasma substitution.

Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1.

Inherited deficiency of protein S constitutes an important risk factor of venous thrombosis. Many reports have demonstrated that causative mutations in the protein S gene are found only in approximately 50% of the cases with protein S deficiency. It is uncertain whether the protein S gene is causative in all cases of protein S deficiency or if other genes are involved in cases where no mutation is identified. The aim of the current study was to determine whether haplotypes of the protein S gene cosegregate with the disease phenotype in cases where no mutations have been found. Eight protein S-deficient families comprising 115 individuals where previous DNA sequencing had failed to detect any causative mutations were analyzed using four microsatellite markers in the protein S gene region. Co-segregation between microsatellite haplotypes and protein S deficiency was found in seven of the investigated families, one family being uninformative. This suggests that the causative genetic defects are located in or close to the protein S gene in a majority of such cases where no mutations have been found.

A comparative in vitro evaluation of six von Willebrand factor concentrates.

UNLABELLED: The efficacy of von Willebrand factor (VWF) concentrates for treatment of von Willebrand disease (VWD) is dependent on their content of VWF and factor VIII (FVIII). STUDY OBJECTIVES: To measure the content and quality of VWF and FVIII in six VWF concentrates: Haemate-P (Aventis Behring), Immunate (Baxter Bioscience), Koate (Bayer Corp.), 8Y (BPL), Innobrand (LFB) and Facteur Willebrand (LFB). METHODS: The VWF antigen content (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen-binding activity (VWF:CB), VWF multimers with electrophoresis and densitometry, FVIII activity and total protein content. RESULTS: Specific activity (VWF:RCo/total protein) varied considerably (4.7-129.5 IU mg(-1)). Activity measures, VWF:RCo and VWF:CB, correlated well, but we found no correlation between any of these and VWF:Ag. The content of high-molecular weight multimer (HMWM) was normal or close to normal in Haemate-P, Innobrand and Facteur Willebrand, moderately reduced in Koate and 8Y, and significantly reduced in Immunate. The HMWM content correlated significantly with the VWF:RCo/VWF:Ag ratio. Only Haemate-P, Innobrand and Facteur Willebrand had VWF:RCo/VWF:Ag ratios >0.7. We found large differences in the content of FVIII and in the FVIII/VWF:RCo ratio. Facteur Willebrand had the lowest (0.02) and Immunate the highest (6.00) ratio. CONCLUSION: Treating physicians must be aware of the large differences between different VWF concentrates and the potential clinical implications. Concentrates lacking HMWM are probably less efficient for mucosal bleedings. FVIII is most important for surgical bleedings, but concentrates with high FVIII/VWF-ratio may induce very high FVIII levels with increased risk of thrombosis. A low FVIII content may be preferable except in case of acute surgery.

Increased sensitivity to ADP-aggregation in aspirin treated patients with recurrent ischemic stroke?

AIM: Antiplatelet therapy in order to reduce the platelet aggregability is widely used to prevent recurrent stroke events. Data from several studies indicates that the inter-individual variation concerning the ability of standard doses of aspirin to inhibit platelet aggregation is substantial. The rationale of the present study was to test whether platelet aggregation in whole blood was enhanced in subjects that had suffered an ischemic stroke event under aspirin treatment. METHODS: Two groups of patients were included: 1) patients that have suffered 1 stroke event and were thereafter under continuous treatment with aspirin 75-160 mg once daily (n=17); 2) patients that have suffered at least 2 stroke events, and aspirin 75-160 mg was prescribed after the 1(st) event (n=17). Platelet aggregation was tested in whole blood with collagen (5 microg/mL and 1 microg/mL), ADP (5 microMol/L) and arachidonic acid (0.5 microg/mL). Aggregation was recorded as change in impedance and release of ATP after the addition of a luciferin-luciferase reagent. RESULTS: The inhibitory effect of aspirin tested with arachidonic acid as an agonist was complete in all the tested subjects. Aggregation induced by ADP 5 microMol/L was significantly higher in the subjects with recurrent stroke compared to those with a single stroke, when tested as impedance change. ATP release with ADP as an agonist was the same in both groups. CONCLUSION: The present study gives some indication that differences in ADP-induced aggregation, with a higher remaining aggregating ability after ASA treatment, might be of importance for the risk of stroke recurrence.

Influence of factor V Leiden on the development of neovascularisation secondary to central retinal vein occlusion.

AIMS: To investigate if the presence of factor V Leiden has an influence on the prognosis in central retinal vein occlusion (CRVO). METHODS: 166 patients with CRVO were studied retrospectively. They were tested for factor V Leiden using DNA analysis. The presence of the mutation was studied in correlation with the development of neovascular complications 1 year after the thrombotic event. RESULTS: 56 of 166 patients (34%) developed neovascular complications after 1 year. In the patients who had the studied mutation 11 of 20 (55%) had developed neovascular complications after 1 year, compared to 45 of 146 patients (31%) in the group without factor V Leiden (p=0.04). CONCLUSION: The presence of factor V Leiden seems to enhance the risk of developing neovascular complications in CRVO.