Annette E. Bowyer

Corn trypsin inhibitor in fluorogenic thrombin-generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram parameters.

The fluorogenic calibrated automated thrombin-generation assay is influenced by contact pathway activation in platelet-rich and platelet-poor plasma. This influence lessens with increasing tissue factor (TF) concentrations and is inhibited by corn trypsin inhibitor (CTI). CTI is expensive and at what TF concentration its influence becomes irrelevant is unclear. Spiking of factor VIII (FVIII)-depleted plasma with FVIII, in samples without CTI, shows a plateau of thrombin generation at low normal FVIII levels. Given the association with thrombosis at high levels, a continuing increase in thrombin generation would be expected. We studied the effect of CTI on this relation by spiking experiments up to 4.8 IU/ml at 1 pmol/l TF and compared the influence of CTI at 1 and 5 pmol/l in platelet-poor plasma. CTI significantly influences thrombin generation in platelet-poor plasma at 1 pmol/l TF (difference of means for endogenous thrombin potential of 232.5 nmol/l per min, P < 0.0001) and peak of 48 nmol/l (P < 0.0001)) but not at 5 pmol/l. Spiking experiments without CTI confirm the hyperbolic relation between FVIII coagulant activity (FVIII:C) and endogenous thrombin potential with a plateau at 0.70–1.40 IU/ml. With CTI, a near-linear response up to 1.0 IU/ml was found with a plateau at 2.4–4.8 IU/ml. For peak thrombin, no plateau was reached with CTI. The present study confirms and extends previous data on CTI and the relationship between FVIII:C and thrombin generation. CTI is not necessary at 5 pmol/l TF, and thrombin generation remains dependent on FVIII:C up to 4.8 IU/ml at 1 pmol/l with CTI. Higher levels than previously thought may be needed to normalize thrombin generation.

Wide variation in thrombin generation in patients with atrial fibrillation and therapeutic International Normalized Ratio is not due to inflammation.

Atrial fibrillation (AF) is a common cardiac arrhythmia with a 5–20% annual risk of stroke. Warfarin reduces this risk by at least 60%. Despite adequate anticoagulation within the target International Normalized Ratio (INR) range of 2·0–3·0, some patients still experience thrombotic and bleeding events. It is now possible to assess the intensity of anticoagulation with automated thrombin generation (TG) tests, such as the calibrated automated thrombogram (CAT). These tests were compared and an inverse relationship was found between the INR and CAT in 143 elderly AF patients. There was equally good correlation between the concentration of factors II, VII, IX and X and the INR and TG parameters. The peak thrombin was most strongly associated with the concentration of prothrombin fragment 1 + 2 in plasma. There was wide variability in TG parameters in patients with identical INR values, sometimes up to a fourfold difference. This TG variability in individuals with the same INR is not due to inflammation, at least when the latter is measured as the concentration of factor VIII coagulant activity, von Willebrand factor antigen, high sensitivity C‐reactive protein and fibrinogen. It was concluded that, although the TG and INR were closely correlated there was wide variability in peak thrombin and endogenous thrombin potential in patients within the INR therapeutic range, the cause of which remains unclear.

Calibrated automated thrombin generation and modified thromboelastometry in haemophilia A

INTRODUCTION Global coagulation tests may have a better relation with phenotype in haemophilia than traditional coagulation tests. These include the Calibrated Automated Thrombin generation assay (CAT) and modified thromboelastometry using low tissue factor triggering. Both have shown marked variability in thrombin generation and clot formation profiles respectively despite similar FVIII:C levels and have been suggested as means to monitor treatment. Data with modified thromboelastometry are largely limited to severe and moderate haemophiliacs. CAT measurements in haemophilia are generally performed at low TF concentrations (1 pM) because of a higher sensitivity for the intrinsic pathway at this concentration but is also sensitive for FVIII at higher concentrations (5 pM) and this has the advantage that inhibition of contact factor activation can be avoided. No formal comparison of both TF concentrations has been reported and the data on modified thromboelastometry in mild haemophilia are limited. METHODS In this study we compared thrombin generation at 1 and 5 pM in 57 haemophilia patients without exposure to treatment and 41 patients after treatment. We also assessed the sensitivity of thromboelastometry for haemophilia A in 29 patients. RESULTS AND CONCLUSION We found that CAT discriminates well between normal individuals and haemophilia patients; also FVIII:C correlates well with the ETP/peak. We found no clear advantages of measurements at 1 compared to 5 pM but found increased variation over time at 1 pM. The sensitivity of modified thromboelastometry for haemophilia A was less than CAT with abnormal measurements largely limited to severe and moderate patients. Larger studies correlating both methods with clinical outcome are required.

The responsiveness of different APTT reagents to mild factor VIII, IX and XI deficiencies.

Introduction:  The sensitivity of APTT reagents to deficiencies of factors VIII, IX, XI and XII varies because of their composition. The APTT is used as a screening test for these factors, and a deficiency should manifest with a prolongation to the APTT, which may trigger the need for specific factor assays to be performed.

The effect of tissue factor concentration on calibrated automated thrombography in the presence of inhibitor bypass agents

Thrombin generation has been suggested as a method to monitor treatment with factor eight inhibitor bypassing activity (FEIBA®) or recombinant FVIIa (rFVIIa). The sensitivity of the assay for individual coagulation factors is dependent on the tissue factor (TF) concentration. An inverse relation between the rFVIIa concentration needed to shorten the clotting time and TF concentration has been shown but the data on thrombin generation are inconsistent. Information on TF concentration in measurements with FEIBA® is limited. We studied the influence of TF concentration (1 and 5 pm) on thrombin generation through spiking experiments with rFVIIa and/or FEIBA® in the plasma of severe haemophilia A patients and after four and three treatment episodes, respectively, using the calibrated automated thrombin generation assay (CAT) in platelet poor plasma. Spiking with FEIBA® showed a linear relation with the endogenous thrombin potential (ETP)/peak at 1 pm but substrate depletion at 5 pm. Spiking with rFVIIa showed a near linear dose–response relation with the ETP/peak at 1 pm but only a shortening of the initiation phase at 5 pm. Similar effects were present in post‐treatment samples. FEIBA® acted synergistically with rFVIIa. This suggest a role for CAT in monitoring inhibitor bypass treatment but low TF concentrations are required to avoid substrate depletion with FEIBA® and to demonstrate the effect of rFVIIa.

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual’s genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.

p.Tyr365Cys change in factor VIII: haemophilia A, but not as we know it

Haemophilia A is caused by a reduction in clotting factor VIII (FVIII). FVIII coagulant activity (FVIII:C) can be measured by three methods; the one‐stage activated partial thromboplastin time‐based clotting assay, the two‐stage Xa generation‐based clotting assay and the chromogenic Xa generation‐based assay. The FVIII:C of most patients with haemophilia A are concordant regardless of the assay method employed. Up to a third of patients show assay discrepancy, usually with the two‐stage and chromogenic assays being much lower than the one‐stage assay. Very rarely, patients have been reported with lower one‐stage compared to two‐stage and chromogenic assays, but here we report that the mutation p.Tyr365Cys accounts for most of these patients and, at least in the UK, is not rare. We have identified this mutation in 23 different families. Affected male index individuals had a lower mean one‐stage FVIII:C of 27 iu/dl compared to two‐stage FVIII:C mean of 77 iu/dl. Affected individuals had minimal or absent bleeding symptoms and when these were present they were usually in patients with another co‐inherited bleeding disorder. Affected individuals often had surgery without the need to correct the one‐stage FVIII:C.

A rapid, automated VWF ristocetin cofactor activity assay improves reliability in the diagnosis of Von Willebrand disease

INTRODUCTION The effective diagnosis and monitoring of Von Willebrand Disease (VWD) requires an accurate assessment of ristocetin co-factor activity (VWF:RCo). Current methodologies include automated platelet aggregometry and manual visual agglutination both of which are laborious to perform and notoriously subject to a high degree of inter and intra assay variation. METHODS AND MATERIALS We have evaluated an automated VWF:RCo assay (BC Von Willebrand Reagent, Siemens, Marberg, Germany) for use on the Sysmex CS2100i analyser (Milton Keynes, UK) and retrospectively compared the results with an in-house manual visual agglutination assay and VWF antigen (Siemens) in normal subjects and in 53 patients with various types of VWD and 23 patients following VWF therapeutic treatment. RESULTS The intra and interassay CV was improved with the automated assay (2.3% and 3.8% respectively) compared to 7% with the manual VWF:RCo assay. Good correlation was found between the two assays (r=0.91) in 53 patients with VWD. The mean manual VWF:RCo was 0.25IU/ml and mean automated VWF:RCo was 0.27IU/ml. A comparable increase in VWF:RCo following treatment, mostly with Desmopressin, was found in 13 patients with type 1 VWD (mean 3.9 fold increase with manual VWF:RCo and 3.1 fold with the automated VWF:RCo). In 13 patients with type 2 or 3 VWD following treatment mostly with concentrate , a higher increase was found with the automated VWF:RCo assay than the manual assay (mean 11.9 fold manually and mean 20.3 automated). CONCLUSION The automated VWF:RCo assay shows enhanced precision and analysis time in this difficult and time consuming laboratory test and its introduction should greatly improve the reliability of VWF testing.

The investigation of a prolonged APTT with specific clotting factor assays is unnecessary if an APTT with Actin FS is normal

Introduction:  An isolated prolongation to the activated partial thromboplastin time (APTT) can be caused by the presence of the lupus anticoagulant or an intrinsic or contact factor deficiency, of which only deficiencies of factors VIII, IX or XI are associated with bleeding. Our local protocol states that further investigation of a prolonged APTT by specific assays of FVIII, FIX and FXI should only be undertaken where the APTT with one reagent (Synthasil) is more than 3 s prolonged, and further investigation by an APTT with a second reagent (Actin FS) is also prolonged, unless there is a history of bleeding in the patient, in which case assays are indicated irrespective of the APTT.

The coagulation laboratory monitoring of Afstyla single-chain FVIII concentrate

The laboratory monitoring of extended halflife (EHL) concentrates containing modified FVIII molecules for the treatment of haemophilia has revealed differences between onestage and chromogenic assays as well as when using different APTT reagents in the onestage assay for measurement of some products. Specifically, the source of contact activator in the APTT reagent can influence the sensitivity to EHL products. Onestage factor VIII (FVIII) assay results in the presence of some EHL products are unaffected by the contact activator in the APTT reagent,1,2 whilst others can only be accurately monitored when using reagents containing Silica and not ellagic acid, or vice versa.3 A minority can only be monitored by a very limited panel of APTT reagents or the chromogenic assay.4 The use of a productspecific standard for one B domain deleted FVIII, the Refacto AF laboratory standard, has been successfully used in Europe for over a decade, to remove assay methodology variations; more recently, this approach has been demonstrated to reduce assay variations between onestage and chromogenic FIX assays in a recombinant FIX product.5 However, productspecific standards for EHL products are not currently available for routine use. Smaller laboratories may not have the capacity to perform onestage assays with unfamiliar reagents or indeed chromogenic assays, not least because of the need for full method validation required by laboratory accreditation agencies. In order to accurately measure EHL or modified FVIII or FIX in the laboratory, the treatment administered to the patient must be communicated to the laboratory. This may not be easy in some larger laboratories processing large numbers of samples from multiple centres. Laboratory assay data using different reagent sets should ideally be provided by the manufacturer for any product containing modified and/or EHL FVIII products to ensure that appropriate methods are used. A novel singlechain, recombinant FVIII (SCrFVIII) concentrate, lonoctocog alfa (Afstyla®; CSL Behring King of Prussia, PA), has recently been licensed for the treatment of haemophilia A. This is a B domaintruncated rFVIII molecule with covalently linked light and heavy chains.6 The manufacturer recommends that either a chromogenic (FVIII:CR) or onestage assay (FVIII:C1) should be used to monitor FVIII levels, but that a conversion factor of 2 be applied to all FVIII:C1 results regardless of APTT reagent.7 The aim of this study was therefore to assess whether a universal conversion factor for the FVIII:C1 is indeed suitable for a range of APTT reagents used in the routine monitoring of SCrFVIII. SCrFVIII was reconstituted in manufacturer supplied water then serially diluted, using the labelled potency, to 2.390.02 IU/mL in severe haemophilia A plasma containing <0.01 IU/mL FVIII and a normal concentration of VWF (George King Biomedical, Overland Park, KS, USA). FVIII:C1 was assayed at three dilutions with Synthasil, APTT SP and Synthafax (all from Werfen Barcelona, Spain), Actin FSL, Actin FS and Pathromtin SL (all from Siemens, Marburg, Germany) using Owren’s Veronal buffer as a diluent. Interim results were calculated from individual calibration curves constructed against standard human plasma (SHP; Siemens) which encompassed the range of potencies being assessed. FVIII:CR was measured by the Hyphen Biomed assay. Assays were performed on Sysmex CS5100i instrumentation Kobe, Japan. All FVIII:C1 assay results were then multiplied by a factor of 2 to obtain the final result as recommended by the Afstyla manufacturer. The percentage difference between labelled potency and final result was calculated using the formula (final resultlabelled potency)/labelled potency × 100%. At higher FVIII:C1, above 0.60 IU/mL, up to 23.3% difference was demonstrated between labelled potency and converted FVIII:C1. A spread of 43.3% was seen between highest and lowest results at the same potency with different APTT reagents (Table 1). The greatest variation was at the 0.02 IU/mL dilution which was twofold to fourfold higher than the labelled potency with all APTT reagents (Figure 1).