FVIII Assays Research Articles

Identification and Functional Characterization of a Novel 27bp Deletion in the Macroglycopeptide-Coding Region of the GPIbα Gene Resulting in Platelet-Type Von Willebrand Disease.

Platelet-type Von Willebrand disease (PT-VWD) is a rare autosomal dominant bleeding disorder. It results from an abnormally high affinity interaction between the platelet membrane glycoprotein Ib/IX/V complex and Von Willebrand factor (VWF), leading to characteristic platelet hyperaggregability. The condition shares most of the clinical and laboratory features of type 2B VWD and the final discrimination between these two diseases requires either platelet mixing studies or a molecular genetic approach. Five members of a British family with PT-VWD were studied; three affected and two unaffected. The proposita, had a long history of bleeding with recurrent epistaxis, postoperative and dental bleeding and menorrhagia. An initial series of hematological investigations lead to a diagnosis of type 2B VWD but a significant fall in her platelet count following therapy with a VWF/FVIII concentrate resulted in the consideration of PT-VWD as the correct diagnosis. This diagnosis was subsequently confirmed with appropriate FVIII and VWF assays. Affected individuals had VWF:Ag values in the range 0.34–0.47 U/mL, FVIII 0.45–0.64 U/mL and VWF:RCo ranging from 0.18–0.42 U/mL. These subjects showed loss of the HMW VWF multimers from plasma and enhanced ristocetin-induced platelet agglutination (RIPA). Also, washed normal platelets did not show enhanced RIPA when mixed with the patient's plasma. On molecular genetic analysis of the GPIbα locus, the three affected family members were heterozygous for a 27 bp in-frame deletion (nucleotides 4374–4400). This corresponds to residues 421–429 in the macroglycopeptide region of GPIbα. This deletion was not identified in any of 50 normal controls. Flow cytometric quantitative analysis of the platelet GPIb/IX/V complex showed that the number of molecules for each of GPIb, GPIX and GPV, in each of the affected and non-affected family members fell within the normal range. Wild type (WT) GPIbα and the 27bp deletion mutant GPIba were then expressed in CHO b/IX cells. To examine the effect of the 27bp deletion on the conformation of the receptor, the cells were incubated with monoclonal antibodies that bind to different sites on GPIbα. We demonstrated that each of the three monoclonal antibodies bound WT and Mut GPIba to a similar extent and that the mutant protein was expressed at a normal level on the CHO cell surface. Finally, the effect of the 27bp deletion on VWF binding was analysed in CHO b/IX cells expressing the mutant and WT GPIbα receptor with 125I-VWF in the presence of varying ristocetin concentrations. We showed that the mutant receptor binds significantly more VWF than the WT GPIbα in the absence of ristocetin and also displays heightened sensitivity to low ristocetin concentrations compared to the WT cells. This study reports the first mutation resulting in PT-VWD that does not directly involve the VWF binding region of GPIbα. Our functional studies with the mutant receptor confirm that the deletion of 9 amino acids from the macroglycopeptide region of the protein enhances its interaction with VWF.

SSC/ISTH classification of hemophilia A: can hemophilia center laboratories achieve the new criteria?

To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL-1 were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1-29 U dL-1 and 1-33 U dL-1 respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1-5 U dL-1. The median FVIII:C for the third sample was 5.8 U dL-1 with a range of 1.5-36 U dL-1. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL-1; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.

Desmopressin (DDAVP) and factor VIII

Desmopressin (DDAVP) and factor VIII

Clinical guidelines for treating von Willebrand disease patients who are not candidates for DDAVP--a survey of European physicians.

While certain plasma-derived factor VIII/von Willebrand factor (FVIII/vWF) concentrates have proven to be quite useful in preventing or controlling bleeding in persons with von Willebrand disease who are not candidates for DDAVP, most of the information concerning dosage and effectiveness has been anecdotal. Additionally, the laboratory tests used to quantify vWF (the vWF:RCoF assay and collagen binding assay) are not well standardized. Thus, the US Food and Drug Administration (FDA) has been reluctant to grant licensed indication for use of these products in von Willebrand disease. This brief report describes a survey of non-US physicians (from major centres) who are recognized experts in haemophilia and von Willebrand disease. Twenty-four of 27 questionnaires were completed, returned and analysed. Products thought to be effective in von Willebrand disease included Haemate P, Facteur von Willebrand, Alphanate, and the UK's BPL '8Y'. In calculating dosage to stop or prevent bleeding, most aim for a certain level of both FVII and vWF:RCoF. Postoperatively, 16/24 respondents follow both of these laboratory tests once daily. Twenty-two of 24 would follow FVIII levels once daily. It is noteworthy that FVIII assay results are generally the only test results readily available to guide the respondents' clinical decisions. For treatment of significant mucous membrane bleeding, respondents often individualize dosage and monitoring, depending on the type, location and extent of bleeding. Gastrointestinal bleeding is generally treated more aggressively. Patient monitoring varies between merely looking for cessation of bleeding, to a battery of laboratory tests, including haemoglobin and haematocrit. Seven out of 24 would monitor BT as well. In addition to FVIII content, 22/24 respondents noted that they would find it helpful to have vWF:RCoF listed on the label, while 15/24 would like to know the vWF multimeric composition.

Discrepancies in potency assessment of recombinant FVIII concentrates.

Results of assays of recombinant FVIII concentrates have been reviewed over a 10-year period. Initially there was wide variability between laboratories but this was minimised by the development of standardised assay methodology, in particular the use of haemophilic plasma for pre-dilution and 1% albumin in assay buffers. Using this standardised methodology and concentrate standards, there were no major differences in potency between one-stage, two-stage and chromogenic assays on the two full-length recombinant FVIII concentrates. However, using a plasma standard, the chromogenic method gave much higher potencies than the one-stage method on the same concentrates, and this explains a similar discrepancy found in patients' post-infusion samples after injection of recombinant concentrates. It is suggested that concentrate standards be used for such post-infusion samples in order to minimise this discrepancy.

In-vitro stability of porcine factor VIII (Hyate:C(®) ).

The stability of porcine factor VIII (Hyate:C(®) ) 30 units mL(-1) , 15 units mL(-1) and 5 units mL(-1) prepared aseptically in 0.9% sodium chloride injection was studied. Solutions were stored in plastic syringes at room temperature and ambient light, and at body temperature protected from light. Samples obtained immediately after mixing and at 4, 24, 48 and 72 h were assayed for FVIII activity using a one-stage FVIII assay. Samples were considered stable if FVIII activity did not decline more than 10% compared with baseline values. Hyate:C(®) 5 units mL(-1) stored at room temperature retained FVIII activity within 90% of baseline values for at least 24 h. When stored at body temperature, FVIII activity of Hyate:C(®) 5 units mL(-1) declined to less than 90% of baseline values within 4 h. Stability of Hyate:C(®) 15 units mL(-1) and 30 units mL(-1) stored at room temperature was retained for at least 72 h. When Hyate:C(®) 15 units mL(-1) and 30 units mL(-1) were stored at body temperature, stability was retained for 24 h. Results of this study will permit further evaluation of Hyate:C(®) stability when administered by ambulatory infusion pumps.

PLASMA FVIII LEVELS MEASURED AFTER INFUSION OF RECOMBINANT F VIII (rFVIII) VARY SIGNIFICANTLY WITH DIFFERENT ASSAY METHODS - (WILL THE REAL FVIII LEVEL PLEASE STAND UP!). † 934

As part of a bioequivalence study comparing two lots of rFVIII(Kogenate®), 9 persons with hemophilia A were infused with rFVIII from each lot, and multiple plasma samples were then obtained from each subject for determination of FVIII assays over 48 hrs. Samples were assayed by 4 different methods: chromogenic, and one stage APTT method using 3 different types of activators (A)-micronized silica, ellagic acid, and kaolin. All samples were immediately centrifuged, snap frozen and stored at -70°C until assayed in duplicate. The same reference plasma standard was used throughout. Results, shown below, indicate a consistent difference in F VIII assay values, with those run by chromogenic substrate method being roughly twice as high as those run by one-stage APTT method with kaolin as A. One stage results with micronized silica or ellagic acid as A were comparable, and were consistently higher than those with kaolin. Most U.S. clinical labs use a one-stage assay, with micronized silica or ellagic acid A. Because significantly different results can be obtained depending on F VIII assay method used, multicenter studies must carefully standardize assay techniques and reagents in order to combine data for analysis. Additionally, such differences have significant cost implications (partially funded by Bayer). Figure

P75. One-stage/two-stage discrepancies in assays of FVIII concentrates???a possible role of solvent/detergent

P75. One-stage/two-stage discrepancies in assays of FVIII concentrates???a possible role of solvent/detergent

Acquired yon Willebrand's syndrome in association with a lupus‐like anticoagulant corrected by intravenous immunoglobulin

We are reporting on a 47-year-old man who presented with a prolongation of the activated partial thromboplastin time (APTT) prior to orthopedic surgery. An evaluation suggested an inhibitor when his plasma prolonged a normal control APTT upon 50:50 solution of patients with normal plasma. The platelet-neutralizing procedure (PNP), anticardiolipin antibody, and antinuclear antibody (ANA) were positive. Further studies revealed decreased von Willebrand factor ristocetin cofactor (vWF:RCoF), von Willebrand factor antigen (vWF:Ag), an inhibitor to vWF, and absent high-molecular-weight vWF multimeters. Assays of FVIII:C, FIX, and FXI were nonparallel to the standard curve. Intravenous immunoglobulin (IVIG) corrected the APTT, multimeric pattern, and FVIII:C by the 7th day postinfusion. This case demonstrates the efficacy of IVIG for acquired von Willebrand's syndrome (vWS) and also represents a unique combination of a lupus-like anticoagulant and acquired vWS in a patient without the full serological requirement for systemic lupus erythematosus (SLE). Whether patients with acquired vWS and lupus inhibitors are more or less susceptible to either a thrombotic complication or hemorrhage is not established. Prospective studies for the incidence of lupus inhibitor/antiphospholipid syndromes and vWF deficiencies are needed to assess this question.

On the Intraindividual and Gender Variability of Haemostatic Components

The intraindividual variability in terms of coagulation analyses was explored in 10-16 samples collected from each of 15 women during one menstrual cycle. For comparison, six men were sampled six times during a period of 30 days. The following variables were analysed: FVII, FVIII, FX, vWF:Ag, vWF:ristocetin cofactor, fibrinogen, antithrombin, plasminogen and anti-plasmin. The results demonstrated mean coefficients of variation ranging between 4.5 (plasminogen) and 20.7 (vWF:Ag). In certain individuals, the intraindividual variability amounted to nearly 40%, in particular in the assays of FVIII and vWF:Ag. No direct relation between these two factors and oestradiol, progesterone or testosterone levels could be observed in our individuals. The implications of these variations are discussed in terms of disease prediction and diagnosis of coagulation disorders.