Diagnosis of inherited von Willebrand disease: comparison of two methodologies and analysis of the discrepancies.

Diagnostics of von Willebrand disease (VWD) includes assessment of factor VIII (FVIII) coagulant activity, von Willebrand factor (VWF) antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), and more specific tests as multimeric and genetic analyses are necessary for the correct VWD classification. The ACL AcuStar analyzer introduces chemiluminescence (CL) technology in detection of VWD with automated VWF:Ag and VWF:RCo assays. Compare VWF:Ag-ELISA and VWF:RCo by aggregometry conventional assays with new CL VWF:Ag-IL and VWF:RCo-IL assays, investigate the ability to make accurate VWD diagnosis and concordance with multimeric and genetic analyses. 146 patients with congenital VWD (51 Type 1; 34 Type 2A; 16 Type 2B; 31 Type 2M; 5 Type 2N; 9 Type 3) and 30 healthy normal subjects were included. A comparison was made between CL and conventional methods. Diagnostic evaluation included: VWF:RCo/VWF:Ag ratio, multimeric distribution (sodium dodecyl sulfate [SDS]-agarose gel) of VWF and genetic analysis in 110 of 146 patients. CL and conventional methods revealed good correlation. Kappa test agreement diagnosis was >0.8. CL diagnostic sensitivity was 100% and specificity 97%. Multimeric and genetic analysis were of help in clarifying 13 discrepancies of diagnosis between methods, of which six discrepancies were explained by lack of conventional methods' sensibility. CL methodology can detect VWD and discriminate between type 1, 3 and variant forms and offers an automated, faster, sensitive and less cumbersome method when compared to conventional assays, in particular VWF:RCo by aggregometry. In some cases, even with all phenotype and genetic analyses, discrepancies exist in the classification of VWD.

Alloantibody from a patient with severe von Willebrand disease inhibits von Willebrand factor-FVIII interaction.

The aim of this study was to analyze the ability of an alloantibody from a patient with severe von Willebrand disease (vWD) to interfere with the vWF domain for FVIII, to inhibit factor VIII (FVIII), and to compare it with a rabbit polyclonal antibody. The vWF domain for binding to FVIII was assayed by a method previously described but using recombinant FVIII (r-FVIII, Kogenate), which contains no vWF, instead of Hemofil M (HM). Rabbit or human antibodies towards FVIII (FVIII-Ab) were analyzed using microtiter wells with immobilized r-FVIII through a monoclonal anti-FVIII antibody and an ELISA method. IgG from plasma of a patient with hemophilia A and FVIII inhibitor was used as a positive control. Normal human and rabbit IgGs were included as negative controls. Human vWD alloantibody IgG and the rabbit anti-vWF antibody IgG reacted with immobilized normal vWF, inhibiting its binding to r-FVIII in a dose-dependent manner, which suggests that it is specific. Normal human IgG fraction, as well as nonspecific rabbit IgG, did not interfere with this binding at all. The monoclonal antibody used in this assay to immobilize vWF did not alter this interaction at all. Human vWD alloantibody IgG and the rabbit antibody against vWF showed a partial inhibitory activity to plasma FVIII as well as r-FVIII. The inhibition reached a plateau with residual FVIII activity. FVIII-Ab were not detected in human alloantibody or in rabbit antibody preparations. In contrast, hemophiliac FVIII inhibitor showed FVIII-AB. This human vWD alloantibody behaves like polyclonal heterologous antibodies, and their inhibition of FVIII seems to be nonspecific due to a steric hindrance mechanism provided that both have no FVIII antibodies.

Antibodies to factor VIII in plasma of patients with hemophilia A and normal subjects.

Non-neutralizing factor VIII (FVIII) antibodies (FVIII-Ab) in hemophilia A may be associated with an abnormal clinical response to FVIII concentrates. Patients with FVIII inhibitors may develop noncoagulation FVIII-Ab after the induction of immunotolerance. Natural FVIII-Ab may be detected in the plasma of some healthy subjects. The aim of this study was to analyze the presence of FVIII-Ab in the plasma of 53 normal blood donors and 124 patients with hemophilia A (18 patients had a previous history of FVIII inhibitor, but only 12 had inhibitor at the moment this study was performed). FVIIII inhibitor was measured using the Bethesda method. FVIII-Ab were analyzed by a specific ELISA assay using purified FVIII from a monoclonal concentrate and a standard plasma containing 26 Bethesda units (BU) of FVIII inhibitor. Purified FVIII was used to coat wells of a microtiter plate and was incubated with dilutions of plasma to be tested. Bound human IgG FVIII-Ab were detected by incubation with polyclonal sheep anti.human IgG alkaline phosphatase conjugate, and the OD405 was quantitated. A linear fit was obtained (by plotting FVIII-Ab positivity [OD 405nm] versus BU titer) when serial dilutions of this standard inhibitor plasma, containing titers of 0.5 BU or higher, were used. Four different levels of FVIII-Ab positivity [OD 405nm] were distinguished in this assay: Negative levels (-) were obtained with dilutions of the standard inhibitor containing < 0.5 BU. Mild levels (+) were obtained with dilutions of 0.5-5 BU. Moderate levels (+2) were obtained for dilutions ranging from 5-25 BU. Maximum positivity (+3) was obtained for dilutions of titers > 25 BU. FVIII-Ab positivity was detected in eight of the normal subjects (15%): three were found to be moderately positive (+2) and five mildly positive (+). No inhibitory activity was detectable when whole plasma was used. All the hemophilic patients with a presence of FVIII inhibitor at the time of the study were found to be positive for FVIII-Ab. In addition, the level of positivity correlated with the corresponding BU. Four of the six patients who had a history of inhibitory were negative and two positive. Twenty additional patients (16.12%) in whom no inhibitory activity was detected were found to be positive for FVIII-Ab: 16 + and four +2. The mean age of patients with FVII-Ab positivity was significantly higher than that of patients of the FVIII-Ab negative group (p < 0.005). In conclusion, FVIII-Ab positivity in patients with hemophilia A was 17.7% higher than the level of positivity detected by an inhibitory assay. We propose that this method for FVIII-Ab analysis could be used for patients with hemophilia A, at least to complement the functional inhibitor assay. FVIII recovery or half-life should be assessed in patients who test positive for FVIII-Ab and who show no evidence of inhibitor.