Susan E. Rodgers

In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results.

In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one‐stage assay are more than double than those by two‐stage assay. This may be due to the longer incubation times (10–12 min) in the two‐stage assay. This study aimed to determine the time course of the activation phase of the two‐stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one‐stage and two‐stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short‐ or long‐incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23–56%, mean 41%) than after 10 min (19–41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21–64%, mean 37%) than with the longer incubation times usually used (13–29%, mean 23%). These time‐course experiments have verified that the longer incubation time used in the two‐stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

High incidence of ankle arthropathy in mild and moderate haemophilia A

A clinical study of patients with mild haemophilia A to document the frequency and severity of arthropathy has not been previously published. We studied ankle arthropathy in 34 patients with mild/moderate haemophilia A. The patients were assessed for the presence and severity of pain, and by the physical and radiological scoring systems for the evaluation of haemophilic arthropathy recommended by the World Federation of Haemophilia (WFH). Of the 34 patients, 16 (47%) had ankle pain, which was of moderate to severe degree in nine patients, and associated with limitation of physical activities in 13 patients. Of 33 patients examined by radiology 17 (52%) were positive for ankle arthropathy, and of these, 16 were also positive by the physical score. The presence and severity of ankle arthropathy was more common in patients with a one-stage factor VIII level of less than or equal to 11 IU/dl. There was a significant relationship between the presence of ankle arthropathy and a history of bleeds into the ankle joint as a child. We conclude that arthropathy of the ankle in these patients is common, is often severe and disabling, and is due to episodes of bleeding into the ankle joint during childhood.

Diagnostic testing for mild hemophilia a in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays.

In recent years, there has been greater awareness among hemostasis scientists and clinicians that factor VIII coagulant activity (FVIII:C) measured in certain patients with mild hemophilia A can show different results depending on the assay system. A subgroup of mild hemophilia families have a method-related discrepancy in FVIII:C results, whereby the one-stage clotting assay (FVIII:C-1) is significantly higher than the two-stage clotting assay (FVIII:C-2) or the chromogenic assay (FVIII:C-chr). To identify such patients, the routine laboratory can use automated procedures for the FVIII:C-chr to replace the complex, manual FVIII:C-2 method. Laboratories must employ appropriate quality management to ensure accurate and precise results, especially in the abnormal range. This discrepant phenotype of hemophilia A is seen in up to 40% of mild hemophilia A cases and represents a clinically significant bleeding disorder. A small proportion of these cases have FVIII:C-1 within the normal range and risk a missed diagnosis if the FVIII:C-chr is unavailable. Other patients may be mismanaged if FVIII:C-1 gives an overestimate of FVIII:C and their bleeding risk is consequently underestimated. Affected family members in the discrepant group of patients have a limited range of FVIII (F8) gene missense mutations, causing alterations of the structure of the A1, A2, or A3 domains of FVIII. Therefore, both FVIII:C-chr and F8 gene mutation analysis are recommended to confirm the diagnosis of mild hemophilia A and assist with decisions about the patients phenotype.

Identification of von Willebrand Disease Type 2N (Normandy) in Australia A Cross-Laboratory Investigation Using Different Methods

We report on a cross-laboratory study of type 2N von Willebrand disease (vWD). We tested 101 selected plasma samples for factor VIII and factor VIII binding activity of von Willebrand factor (vWF). Of these plasma samples, 31 were cotested by 2 specialist centers using different detection procedures for vWF-factor VIII binding: there was good agreement between results obtained by chromogenic assay and enzyme-linked immunosorbent assay. In total, 8 patients with type 2N vWD were identified. The 2-stage factor VIII assay detected a deficiency of factor VIII relative to vWF antigen in all 8 patients; the 1-stage factor VIII assay detected a relative deficiency in only 3 patients. Four patients were homozygous for the most common type 2N mutation (R854Q), 3 patients were presumed to be compound heterozygotes, and in 1 patient no type 2N mutations were identified. In this study of patients from 5 specialist centers in Australia, type 2N vWD was found in 5 families. The 2-stage factor VIII assay was more useful as a screening test than the 1-stage assay, and both vWF-factor VIII binding assays were equally effective.

Classification of the kinetics of factor VIII inhibitors in haemophilia A: plasma dilution studies are more discriminatory than time-course studies

Factor VIII inhibitors have previously been classified as type I or type II using complex experiments that study the time course of inactivation of factor VIII and the effect of varying the antibody concentration. Classification may be important to better understand inhibitor behaviour in vivo. To determine the most reliable method of classifying the kinetics of factor VIII inactivation, we studied 11 patients with haemophilia A, comprising five severe, three mild and three acquired cases, and compared the classification obtained from plasma dilution studies and time‐course studies. The plasma dilution studies showed two distinctly different patterns: a steep slope with complete FVIII:C inactivation at high antibody concentrations for type I inhibitors and a FVIII:C plateau with incomplete inactivation for type II inhibitors. Six type I (four severe, one mild and one acquired) and two type II (one mild and one acquired) inhibitors were classified using either plasma samples or purified and concentrated IgG, while the remaining were undetermined owing to insufficient available plasma. In contrast, the time‐course studies could not discriminate between these groups. We recommend that plasma dilution studies be used for the classification of in vitro kinetics of factor VIII inhibitors.

Evaluation of Pre-analytical Variables in a Commercial Thrombin Generation Assay

BACKGROUND There is minimal data on the influence of pre-analytical variables on the use of calibrated automated thrombography (CAT), to measure thrombin generation. OBJECTIVES To evaluate the impact of centrifugation methods, time after collection, and contact activation inhibition on the CAT assay performed using two commercial reagents. METHODS AND RESULTS Six different methods of plasma separation were examined. Thrombin generation triggered by a 5 pM tissue factor reagent was not greatly affected by plasma separation method, with similar results obtained with all methods apart from single centrifugation and membrane filtration. Membrane filtration increased APTT and is not recommended. Extended double centrifugation at higher speed was required to minimise the impact of residual phospholipid with 1 pM tissue factor trigger, particularly with inhibition of contact activation. The effect of a delay of up to 24 hours in preparing plasma was assessed. No significant difference in results was observed among samples processed between 0.5 and 6 hours after blood collection into plastic Vacuette® tubes. The presence or absence of corn trypsin inhibitor had a significant impact on all parameters with 1 pM tissue factor trigger, with minor differences seen on Peak and ttPeak results using 5 pM tissue factor. CONCLUSIONS The impact of pre-analytical variables on thrombin generation results is dependent on the concentration of tissue factor in the trigger reagent used. Results with 1 pM tissue factor are particularly sensitive to centrifugation method and contact activation, and standardisation is required to allow large collaborative studies to be performed.

Diagnosis and management of adult patients with von Willebrand disease in South Australia.

We have analyzed the databases for von Willebrand disease (VWD) from the hemophilia center for adult patients with bleeding disorders in South Australia. We define the prevalence of types of VWD to determine the proportion of who would be treated with factor (F) VIII/von Willebrand factor (VWF) concentrate to prevent or control hemorrhage. In severe or moderately severe patients, we use plasma-derived FVIII/VWF concentrate, and for mild to severe cases, we use desmopressin plus tranexamic acid. There are 103 patients with VWF ristocetin (RCo) ≤50 IU/dL: 38 (37%) severe (VWF:RCo <10 IU/dL), 28 (27%) moderate (VWF:RCo 10 to 29 IU/dL), and 37 (36%) mild (VWF:RCo 30 to 50 IU/dL). Hence in 66 (64%), FVIII/VWF concentrate is the mainstay of treatment. The prevalence of VWD in our region according to data from our center is ~1 per 12,000. A total of 52% of patients are type 1, 44% type 2, and 5% type 3. In our experience, type 2M (45% of type 2) is much more common than types 2A and 2B (each 9% of type 2). Mutation detection is useful for identifying some subtypes of VWD.

Partial and severe factor XI deficiency in South Australia and the usefulness of factor XI mutation analysis for diagnosis.

Aims: To correlate the presence or absence of a factor XI gene mutation with factor XI activity in patients with severe or partial reduction in factor XI. Methods: Patients previously found to have reduced factor XI levels were recalled for repeat testing and factor XI genetic analysis. Also, during the 18 month study period, any routine patient found to have an isolated reduced or low normal factor XI level had factor XI genetic analysis. Results: Twenty‐two cases were studied and 11 with factor XI from <2 to 57 U/dL (reference 55–130 U/dL), were found to have a factor XI gene mutation. Gene sequencing identified 15 different mutations, with four patients found to be compound heterozygotes. One patient with no bleeding history had a novel polymorphism which family studies showed was not associated with his low factor XI. No factor XI gene abnormality was detected in 10 patients and they have either acquired causes of deficiency or factor XI levels in the lower portion of the normal range. Conclusion: Genetic analysis of the factor XI gene is important to confirm or exclude inherited causes of factor XI deficiency, especially when the reduction is mild.

Utility of the PFA-100 as a screening test of platelet function: an audit of haemostasis laboratories in Australia and New Zealand.

The PFA-100 is a relatively new laboratory instrument, first described in 1995. There have since been numerous studies assessing its utility as a screening tool for platelet dysfunction and/or von Willebrands disease (VWD). The PFA-100 displays variable sensitivity to different types of platelet disorders, as well as to antiplatelet medication (e.g. aspirin), with similar caveats for monitoring of primary haemostasis-promoting therapies in platelet dysfunction. There is therefore considerable uncertainty regarding its utility within this context, and we have accordingly performed an audit of usage among participants of the Royal College of Pathologists of Australasia Quality Assurance Program. Of 105 laboratories surveyed, 40 responded that they performed platelet function testing, with 26 (65%) further indicating they utilized the PFA-100. We report a wide variety of laboratory usage among these users, including numbers of tests performed [annual median (range) = 270 (15–6000)], sources of requests (clinical sources and localities), testing criteria and follow-up action. Most tests were completed within 4 h of collection, as recommended by the manufacturer, and most tests were performed as a replacement, or as a preliminary screen of platelet function (i.e. classical aggregation). Most abnormal findings, however, were attributed to antiplatelet medication such as aspirin.

Grandpaternal mosaicism in a family with isolated haemophilia A

About one third of cases of haemophilia A have no family history of the disorder, and 20% are thought to be due to a new mutation. In the family reported here, a 3 bp deletion was detected in DNA from the proband at the 3′ end of exon 15. Direct sequencing of genomic DNA prepared from blood and buccal cells of the grandfather revealed both normal and mutant sequences, suggesting that he is a mosaic for this mutation. This highlights the usefulness of mutation detection, both for accurate genetic counselling and to determine the origin of new mutations of haemophilia.