Evaluation of three automated chromogenic FVIII kits for the diagnosis of mild discrepant haemophilia A.

In some mild haemophilia A patients (discrepant phenotype), coagulation FVIII levels by one-stage assay (FVIII-1st) are more than double those by classical two-stage coagulation assay (FVIII-2st), and may fall within the normal range. Our aim was to assess automated two-stage chromogenic FVIII assays (FVIII-chr) for diagnosis of mild discrepant haemophilia A. Three chromogenic FVIII kits (Biophen, Coamatic and Dade-Behring) were evaluated, using recommended and extended incubation times. Samples were tested from patients with discrepant haemophilia (n = 7) and equivalent mild haemophilia (agreement between FVIII-1st and FVIII-2st, n = 4). For equivalent haemophilia, FVIII-chr were consistent with FVIII-1st and FVIII-2st for all kits at all incubation times. For discrepant haemophilia, using recommended incubation times, mean FVIII-chr using Biophen reagents was 22 IU/dl (range 13-31), with Coamatic 26 (17-34) and with Dade-Behring 41 (33-47), compared with 36 (27-44) for FVIII-1st and 8 (6-9) for FVIII-2st. FVIII-chr decreased as incubation time was increased with Biophen and Coamatic, but decreased less with Dade-Behring. FVIII-chr using the Dade-Behring kit gave similar results to FVIII-1st and is not suitable for diagnosis of mild discrepant haemophilia A. FVIII-chr by Biophen and Coamatic kits is suitable for diagnosis of these patients, especially with an extended incubation time.

New approaches in the measurement of coagulation.

In this session contributors present recent developments in laboratory tools for investigation of haemorrhagic disorders as well as their relative utility in clinical research. In an overview of B. Sørensen the present knowledge is summarized on the dynamic properties of whole blood fibrin formation as studied by changes in whole blood elasticity on a thrombelastometry system. Additionally, fibrin formation dynamics using simple APTT methods are presented. G. Castaman reviews the pathophysiology of von Willebrand's disease (VWD) and explains which tests are best used in diagnosis and subclassification of VWD accounting for recent developments. This presentation also describes the treatment technologies available today and their implications in clinical management of bleeding episodes in VWD. J. Lloyd addresses the assay discrepancy phenomenon that is found in some of our patients suffering from mild haemophilia A. Assay discrepancy most often means a much lower factor VIII:C value by a two-stage or chromogenic assay for factor VIII:C compared to the activity recorded by the one-stage clotting system for factor VIII:C. In rare cases, the opposite phenomenon exist. The presentation includes data from 16 Australian families with discrepant results. D. Varon reports on an assay for study of platelet function in whole blood under flow conditions. The equipment is described as a cone-and-plate(let) analyser in which the adhesion and aggregation of platelets onto a polystyrene surface is studied under arterial flow conditions. Basically, it is anticipated that proteins such as VWF and fibrinogen of flowing blood is attached to the polystyrene surface where they build up a thrombogenic surface. In the study of the author platelets were pre-activated with agonists and platelet deposits were determined after passage of whole blood for a pre-set time interval. Data presented suggest that the assay is sensitive to platelet numbers as well as qualitative changes in platelets themselves, and several examples of disorders characterized by enhanced as well as reduced platelet aggregating activities illustrates the sensitivity of the method.

Low detection rate of antibodies to non-functional epitopes on factor VIII in patients with hemophilia A and negative for inhibitors by Bethesda assay.

In patients with hemophilia A who have an inhibitor to factor (F)VIII measured by Bethesda assay, enzyme-linked immunosorbent assay (ELISA) can also be used to detect the inhibitor. In some studies non-inhibitory antibodies were also detected by ELISA in many patients who were negative by Bethesda assay. Our aim was to investigate whether there is a higher detection rate of FVIII antibodies by ELISA compared with Bethesda assay. We also compared outcomes using three different preparations of recombinant FVIII (rFVIII) to coat the microtiter plates for ELISA. Inhibitor detection by ELISA generally agreed with the Bethesda method. Only four of 26 patients with no clinical suspicion of an inhibitor and with no detectable inhibitor by Bethesda assay showed a non-inhibitory antibody by ELISA, and three of these were only weakly positive. Patients with severe hemophilia A and the intron 22 inversion (n = 21) did not show a higher incidence of non-inhibitory antibodies compared with those without that mutation. Finally, we found that the formulation of rFVIII has a small effect on ELISA performance, mainly in detection of low-level antibody. The results of the present study are in contrast to and fail to confirm previously published reports showing a higher incidence of non-inhibitory antibodies in hemophilia A.

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.

Assessment of in vitro-generated platelet microparticles using a modified flow cytometric strategy.

Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytometric methods for detection and quantification of PMPs has not been systemically evaluated. This study reports the optimisation of flow cytometric procedures for the detection of PMPs, the determination of limits of size detection using microbeads, and the characterisation of PMP generation by in vitro activation of platelets using collagen and adenosine 5' diphosphate (ADP). Fluorescent and plain microbeads proved useful for defining the limits of the flow cytometer in detecting PMPs. A systematic calibration of the forward scatter (FS) threshold parameter (size) of the flow cytometer using microbeads allowed for the detection of very small particles (down to 0.1 microm diameter). PMPs generated in vitro using ADP and collagen were reliably detected by flow cytometry using monoclonal antibodies (MAb) directed towards platelet surface membrane glycoproteins (Gp). The PMP events were detected in the FS low (i.e., small size events) and fluorescence (FL) high (i.e., platelet Gp MAb-labelled events) region. PMPs of different size profiles were observed for each of the agonists. Flow cytometry can be used as a tool in the assessment of PMPs. As detection of particles of this type is at the limit of resolution of flow cytometers, careful attention is required with the choice of platelet-specific MAb, isotype control, and optimisation of procedure setup and performance.

Acquired haemophilia in South Australia: a case series.

Of eight cases of acquired haemophilia presenting over an 8-year period, six received immunosuppressive treatment, five with cyclophosphamide, vincristine and prednisolone (CVP). Five patients (four on immunosuppressive treatment) entered remission, two patients died and one was lost to follow up. Initially, the remissions were only partial. The median duration until partial remission was 10 weeks (range 1-55 weeks) and until complete remission was 35 weeks (range 2-59 weeks). Partial remission may proceed to complete remission without further chemotherapy.

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.

Diagnostic importance of the two-stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954-->Leu substitution in the factor VIII A3 domain.

In some families with mild haemophilia higher results are obtained for factor VIII activity (FVII:C) determined by one-stage assay than by two-stage or chromogenic assays. Amino-acid substitutions in the A1, A2 and A3 domains of factor VIII have been described in affected individuals with this phenotype. We describe a case of mild haemophilia A in which FVIII:C measured by one-stage assay was normal at 106%. However, FVIII:C levels measured by two-stage and chromogenic assays were 18% and 35% respectively. DNA analysis revealed a novel mutation in the A3 domain of factor VIII, His1954-->Leu. In a molecular model of the FVIII A domains, His1954 is placed in close proximity to two other mutations that have previously been shown also to be associated with one-stage/two-stage discrepancies. In this patient the diagnosis of haemophilia A would be missed if only the one-stage assay was used.

Functional lymphocyte immunophenotypes observed in thalassaemia and haemophilia patients receiving current blood product preparations.

Immune abnormalities have been reported in recipients of cellular and plasma blood products. To document the effect of current transfusion practices, we performed ex vivo lymphocyte immunophenotypic studies on patients with thalassaemia major who had received multiple (leucocyte-depleted) transfusions and patients with haemophilia A and B who had received heat viral-inactivated factor concentrates. Patients with thalassaemia major showed a significant lymphocytosis, with mainly B-cell changes consistent with ongoing B-cell stimulation associated with chronic exposure to red cell antigens. Reduced T-cell IL-2Ralpha expression would be consistent with inhibition by desferrioxamine chelation therapy. In contrast, patients with haemophilia showed predominantly T-cell changes. Patients with haemophilia A showed significantly elevated activated CD8+ cytotoxic T lymphocytes whereas those with haemophilia B showed an increase in CD8+CD11adim and CD4+CD45RA+ suppressor T cells. Several of the immune abnormalities found may be due to the presence of cytokines not removed by leucocyte filtration or destroyed by factor concentrate production (e.g. TGF-beta) causing a T-helper-2-like response. The extensive lymphocyte characterization in this study has not previously been performed and has enabled a closer examination of the functional lymphocyte immunophenotypes seen in patients treated according to current transfusion practices.

ADP, adrenaline and serotonin stimulate inositol 1,4,5-trisphosphate production in human platelets.

Although adenosine diphosphate (ADP) is a well-known stimulus of platelet aggregation, it is not the generally accepted view that ADP stimulates phosphatidylinositolbisphosphate (PtdIns(4,5)P2) hydrolysis. Using a very sensitive competitive receptor binding assay for inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), we have detected Ins(1,4,5)P3 production at early ( < 10 s) time points after stimulation of human platelets by the weak agonists ADP, adrenaline and serotonin (5-hydroxytryptamine, 5-HT). When adrenaline or 5-HT was combined with ADP in the presence of aspirin, there was a significant potentiation of ADP-induced platelet aggregation, but there was no potentiation of Ins(1,4,5)P3 generation. Also, the increases in intracellular calcium (Ca2+) concentrations stimulated by ADP were not potentiated by adrenaline in the presence of aspirin. Therefore, the synergism between the purinergic and adrenergic pathways of platelet activation occurs downstream from PtdIns(4,5)P2 hydrolysis and intracellular Ca2+ mobilization, although prior to platelet aggregation.

Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand.

BACKGROUND: Inhibitory antibodies which neutralise factor VIII develop in 10-20% of individuals with inherited haemophilia A and rarely as autoantibodies in normal individuals to cause acquired haemophilia. The antibodies are directed against human factor VIII but cross-react to varying degrees with porcine factor VIII. Porcine factor VIII can be used for treatment in individuals with low cross-reactivity. AIMS: To determine the cross-reactivity of factor VIII inhibitors between human factor VIII and porcine factor VIII, in a population of patients with inherited and acquired haemophilia A. Also, to determine whether patients with inherited haemophilia and inhibitors have a higher incidence of factor VIII gene inversion in intron 22. METHODS: Samples and data sheets from 43 patients with inherited and ten with acquired haemophilia were submitted from hospitals in Australia and New Zealand. Inhibitor levels to human and porcine factor VIII were measured by the Bethesda method in 39 with inherited and nine with acquired haemophilia A. RESULTS: Of 39 patients with inherited haemophilia A, cross-reactivity was 0% in 17 patients, 1-19% in six, 20-39% in 11 and 40-80% in five. In six of nine patients with acquired haemophilia cross-reactivity was < or = 7%. In inherited severe haemophilia A, the frequency of the intron 22 inversion was not greater in 37 study patients than in 28 patients without an inhibitor. CONCLUSIONS: Many patients in Australia and New Zealand with inhibitors to human factor VIII presently show a low or absent level of cross-reactivity to porcine factor VIII. These may respond to treatment with this concentrate at least in the short term. There remains a group of patients with high cross-reactivity who will respond only to recombinant factor VIIa or prothrombin complex concentrates.

Mutations in a subgroup of patients with mild haemophilia A and a familial discrepancy between the one-stage and two-stage factor VIII:C methods.

A subgroup of patients with haemophilia A who have a familial discrepancy between the one-stage and two-stage factor VIII:C results has previously been described. These patients show factor VIII:C levels by one-stage assay that are 2-7-fold higher than their two-stage results. We have studied 10 such families and identified six different mutations in the factor VIII gene in this group. The chemical cleavage method and DNA sequencing was used to identify mutations in factor VIII gene fragments generated by reverse transcription and PCR. All available family members were tested to confirm the presence of the mutation in affected individuals. These patients were found to have one of six single point substitutions causing a missense mutation and alteration to one codon in exons 7, 11, 14 or 18. The mutations comprise three that have not previously been described (Ala284Glu. Arg698Leu. Leu1932Phe) and three that have been previously described (Ser289Leu, Arg531His, Arg698Trp). Alterations to the amino acid composition of the A1, A2 and A3 domains of factor VIII are predicted by these molecular studies. In contrast, a control group of 23 mild haemophilia families with equivalent factor VIII:C results by one-stage and two-stage assays did not have any of the above mutations. Detailed studies in seven of these latter families identified four mutations affecting the A3, C1 and C2 domains of factor VIII. These findings suggest a genetic basis to the unusual factor VIII phenotype but do not explain the mechanism of the discrepant factor VIII activity.

Detection of carriers of haemophilia A: use of bioassays and restriction fragment length polymorphisms (RFLP).

BACKGROUND: Haemophilia A is a sex-linked bleeding disorder carried by unaffected females. Currently, the two main methods used for the determination of carrier status in women from families with haemophilia A are bioassays and DNA-based assays using restriction fragment length polymorphisms (RFLP). AIM: The aim of this paper was to assess the current usefulness of these two methods. METHODS: Bioassays measured factor VIII coagulation activity by a two-stage coagulation assay and von Willebrand antigen by immunoelectrophoresis. RFLP were determined with two intragenic probes (p114 and p486) and two linked probes (St14 and DX13). Data were analysed using a Bayesian analysis to allow for all possible recombination events. We also incorporated an estimate for the risk of mosaicism into calculations in isolated haemophilia families. Both bioassays and RFLP were used to determine carrier status in 63 women, 31 from known haemophilia families and 32 from families of isolated cases. The techniques were assessed for their ability to classify the patients as normal (p < 0.2) or carrier (p > 0.7). Where applicable, intron 22 inversion was also tested. RESULTS: In the known families, six women could not be classified after bioassay, but all could be classified by RFLP. Of the 32 women from families of isolated cases, eight were unclassified by bioassay and 12 were not definitely classified using RFLP. However, RFLP was useful in determining that a recent mutation had occurred in six of the eight families in which DNA from the grandparents was available. CONCLUSION: For diagnosis of carriers of haemophilia, RFLP is the preferred method in familial haemophilia, but is less useful in isolated haemophilia.

Haemophilia A management in Victorian, New South Wales and South Australian haemophilia centres.

OBJECTIVE: To examine the management of haemophilia A in Australia and to compare it with international trends. METHODS: Six haemophilia centres treating most patients in Victoria, New South Wales and South Australia were surveyed in 1993 by means of a written questionnaire followed by an "on site" interview. RESULTS: The centres were treating 739 patients; 234 (32%) had severe haemophilia. Factor VIII inhibitors were present in 5.9% of all patients and in 19% of those with severe disease. Twenty-three per cent were human immunodeficiency virus (HIV) antibody-positive and 74% were hepatitis C virus (HCV) antibody-positive. The main treatment was "on demand" therapy for acute bleeds (average use of factor VIII: 1350 IU/kg per year for children; and 780 IU/kg per year for adults). Prophylactic therapy was used in only 17 patients, with doses of 3000-4500 IU/kg per year. One million IU was used for three patients with high titre inhibitors who had "tolerising" therapy. While most developed countries have a factor VIII supply of 2-5 IU per capita, the total supplied to the States represented 1.46 IU per capita, while use at the centres represented 1.1 IU per capita. CONCLUSION: Because supply of factor VIII is limited, use was less than half that recommended internationally. Shortage of factor VIII has compromised prophylactic therapy and virtually prevented "tolerising" therapy.

Platelet activation induced by a murine monoclonal antibody directed against a novel tetra-span antigen.

MAb 14A2.H1 identifies a novel low-abundance platelet surface antigen, PETA-3, which is a member of the tetra-span (TM4) family. This MAb brings about platelet aggregation and mediator release, which is completely inhibitable by prostaglandin E1, and partially inhibitable by aspirin and ketanserin. Platelet activation by MAb 14A2.H1 is dependent on interaction with both the platelet Fc receptor, Fc gamma RII, and the specific antigen as it was prevented by either a blocking MAb to Fc gamma RII (IV.3) or F(ab')2 fragments of 14A2.H1. The extent of platelet activation by the antibody varied considerably between donors, and is believed to reflect the polymorphism of Fc gamma RII. Subaggregating concentrations of 14A2.H1 synergized with other platelet agonists, ADP, adrenaline, collagen and serotonin, indicating signalling via a pathway distinct from these activators. Synergy was also blocked by MAb IV.3, or F(ab')2 fragments of 14A2.H1. The similar low copy number of PETA-3 and Fc gamma RII in the platelet membrane (approximately 1000/platelet), together with the dependence on Fc gamma RII for activation by MAb 14A2.H1, suggests that PETA-3 may be a component of the Fc gamma RII signal transducing complex in platelets.

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 two-stage method has been described in some patients with haemophilia A or with von Willebrand's 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 two-stage FVIII:C results and may be more widespread than previously recognized.