Elizabeth Duncan

Familial discrepancy between the one‐stage and two‐stage factor VIII methods in a subgroup of patients with haemophilia A

A higher result for plasma factor VIII:C measured by the one‐stage as compared with the twostage method has been described in some patients with haemophilia A or with von Willebrands disorder. We used both methods to measure FVIII:C in 95 patients with haemophilia A. The results were equivalent in all 21 patients with severe haemophilia (16 families) and in 45 of the patients with mild or moderate haemophilia (18 families). However, the results were discrepant (FVIII: C by one‐stage assay 2‐7‐fold higher than by two‐stage assay) in the other 29 patients with mild or moderate haemophilia (12 other families). For each patient with discrepant FVIII: C results the classification was the same for all other affected members of his family. In some families with haemophilia A the gene defect leads to a discrepancy between the one‐stage and twostage FVIII: C results and may be more widespread than previously recognized.

Identification of factor inhibitors by diagnostic haemostasis laboratories. A large multi-centre evaluation

We have assessed the proficiency of diagnostic haemostasis facilities to correctly identify coagulation factor abnormalities and inhibitors. Forty-two laboratories participating in the external Quality Assurance Program (QAP) conducted by the RCPA agreed to participate and were each sent a set of eight samples (each 3 x 1 ml) for evaluation. They were asked to blind test these samples for the presence or absence of inhibitors, and where identified, to perform further analysis (including specific inhibitor analysis). In order to make the exercise more challenging, in addition to true factor inhibitors, samples were provided that reflected potential pre-analytical variables that might arise and complicate inhibitor detection or lead to false inhibitor identification. In brief, the sample set comprised a true high level factor (F) V inhibitor, a true moderate level FVIII inhibitor (but sample was defibrinogenated), a true lupus anticoagulant (LA), a normal (but slightly aged) plasma sample, a normal serum sample, a normal EDTA sample, an oral anticoagulant/vitamin K deficiency sample, and a gross heparin ( approximately 10 U/ml) contaminated sample. Sixty-three percent of participants correctly identified the true FV inhibitor as such, although the reported range varied greatly [10 to >250 Bethesda units (BU/ml)] and 46% correctly identified the true FVIII inhibitor, despite the complication of the sample presentation, although the reported range also varied (7 to 64 BU/ml). Some laboratories either failed to identify the inhibitor present, or misidentified the inhibitor type. The LA, the oral anticoagulant/vitamin K deficiency, the normal serum sample, and the normal (aged) sample were also correctly identified by most laboratories, as was the absence of specific factor inhibitors in these samples. However, a small subset of laboratories incorrectly identified the presence of specific factor inhibitors in some of these samples. The heparin sample was also correctly identified by most (68%) laboratories. In contrast, the normal EDTA sample was misidentified as a FV and/or FVIII inhibitor by most (68%) laboratories, and only one laboratory correctly identified this as an EDTA sample. Thus, we conclude that although laboratories are able, in most cases, to identify the presence of true factor inhibitors, there is a large variation in identified inhibitor levels and there are also some significant errors in identification (i.e. false negatives and misidentifications). In addition, there is a significant false positive error rate where some laboratories will identify the presence of specific factor inhibitors where no such inhibitor exists (i.e. false positives).

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.

Rituximab‐induced long‐term remission in patients with refractory acquired hemophilia

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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.

Investigations from External Quality Assurance Programs Reveal a High Degree of Variation in the Laboratory Identification of Coagulation Factor Inhibitors

The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titers during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either with the original or the Nijmegen-modified Bethesda assay. In addition, several circumstances can arise in which the laboratory may test samples that potentially reflect false identification of factor inhibitors. These include lupus anticoagulants and other events generally related to preanalytical variables, including incorrect sample presentations. This article reviews each of these elements, largely from the perspective of cross-laboratory studies undertaken within the framework of external quality assurance (EQA), a peer-laboratory process that aims to assess the ongoing performance of groups of similar laboratories. This review details the experience of the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and it also reflects on the experience of other EQA organizations. Our analysis reveals a wide variety of test practice among inhibitor testing laboratories, a wide variation in detected inhibitor levels in cross-tested samples, and substantial evidence of false-positive and false-negative detection of factor inhibitors. These findings hold some significance for the clinical management of patients affected by these inhibitors. There is still much need for standardization and improvement in factor inhibitor detection, and we hope that this report provides a basis for future improvements in this area.

Twelve years of experience of acquired hemophilia A: trials and tribulations in South Australia.

Acquired hemophilia A (AH) is a rare and serious acquired bleeding disorder where prompt and correct diagnosis is crucial, and immune suppression is often required for factor VIII (FVIII) autoantibody eradication. The acquired FVIII deficiency usually manifests as bruises and bleeding, and treatment such as FVIII has limited efficacy because of the neutralizing FVIII inhibitor. Expensive bypassing agents such as recombinant activated factor VII (rFVIIa) may be required to treat clinically significant bleeding. This report summarizes the experience related to AH from a large Australian hemophilia center based in South Australia. We identified 25 patients retrospectively over 12 years (1997 to 2008) and reviewed diagnostic features, treatment for bleeds and to eradicate the autoantibody, treatment response, and survival outcomes. The incidence in South Australia was 1.20 cases per million/year with a median age of 78 years with an approximately equivalent sex ratio (12 males versus 13 females); median FVIII and inhibitor titer were 2.5 IU/dL and 11.0 BU/mL, respectively. Twenty-four patients were evaluated further. Thirteen patients (54%) required hemostatic agents, and rFVIIa was used in seven for major bleeds, of which four were limb or life threatening. Eighteen patients were treated by hematologists with immune suppression, and combination steroid and azathioprine was used most commonly to eradicate autoantibody; 15 of these 18 achieved remission (i.e., 83% response rate). Two patients had persistent low-titer inhibitor when treatments were withdrawn, and one died of a fatal bleed shortly after starting treatment. One had spontaneous remission. Five patients (33%) relapsed, three in less than 6 months after starting treatment; all were retreated successfully. Rituximab was used in six patients for high-titer inhibitor, second relapse, two life-threatening bleeds, underlying lymphoma, and steroid intolerance, respectively. Overall mortality was 25% ( N = 6), five of whom were not treated. Advanced age and lack of treatment were predictive of poor survival outcomes. The very elderly (>75 years of age) may warrant a different treatment modality such as rituximab, which is potentially more tolerable and efficacious.

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.