Summary
The ristocetin cofactor activity assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity but remains difficult to perform, and the coefficient of variation ...of the method is high (about 20–30%). This study evaluated and compared the performance for measuring the VWF activity of two newly commercialised assays VWF:Ac Innovance (VWF:Ac) and VWF:RCo Acustar (VWF:RCo Acu) with the reference VWF:RCo aggregation in 123 pathological plasma samples. The correlation and concordance between both new tests (VWF:RCo-Acu and VWF:Ac) and the reference VWF:RCo were good. The results of the VWF activity to VWF antigen ratio were also comparable whatever the method for the classification of VWF deficiency in all patients. Our results showed that both new tests could replace the “gold standard” VWF:RCo in aggregometry with several benefits: they are fully automated, easier and faster to perform, better adapted to emergency situations if necessary.
Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the ...flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.
Inherited factor VII (FVII) deficiency is a rare autosomal disorder characterized by a weak relationship between FVII activity (FVII:C) and operative bleeding risk. We report a retrospective study of ...17 patients with a FVII:C below 0·1 IU/ml, in whom surgery was performed without any replacement therapy. Clinical and biological data were analysed to establish predictive criteria for bleeding tendency. We found that systematic preoperative replacement therapy may not be necessary for ‘minor’ surgical procedures, for patients suffering from inherited FVII deficiency, unless the clinical history includes severe haemorrhagic symptoms such as haemarthrosis, severe haematomas (even of soft tissue) or abundant epistaxis.
Intracranial haemorrhage (ICH) is known to be a severe although uncommon complication of haemophilia. A national survey has been conducted in France in order to collect information about ICHs which ...occurred in haemophiliacs between 1991 and 2001 and to propose recommendations for the diagnostic and treatment of ICH. Within this period, 123 episodes of ICH were recorded from 106 patients. Two‐thirds of ICH concerned patients with severe haemophilia. Half of the cases occurred in patients under 15 years of age, 67.2% of which were post‐traumatic. Ten cases occurred in neonates with three fatal outcomes. Overall mortality was high (21.9%) suggesting that availability of clotting factor concentrates has not improved the prognosis of this event. Morbidity was also high with 60% of long‐term sequelae. The following parameters have been identified as prognostic factors for death: thrombocytopenia, HCV infection, intraventricular or intraparenchymatous haemorrhage. A delay in diagnosis was mentioned in 43.3% of cases, often related to the lack of recognition of the initial symptoms, which may be very common (apathy, tearfulness in young children and headache in elder patients). Delayed replacement therapy was recorded in 37.2% of cases. Emergency units initially dealt with half of these patients. Information concerning recognition and management of these episodes, not only in severe haemophilia, but also in moderate and mild forms, should be regularly supplied to paediatricians in maternity and physicians from emergency units, as well as to patients and their relatives.
Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have ...been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the γ-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor–like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.
Summary
The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and ...processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbα found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbα were transfected into CHO cells expressing GPIbβ and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPIbαΔLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient’s platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbαΔLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbα leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOαΔLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOαβIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOαΔLeuβIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbα is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbα in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.
Factor VII (FVII) is a plasma glycoprotein that plays a key role in the initiation of blood coagulation cascade. Inherited FVII deficiency is a rare autosomal recessive disorder with a wide ...heterogeneous clinical pattern. The severe form may be associated with intracranial haemorrhages occurring closely to birth with a high mortality rate. In the present article, we report two novel cases of neonatal intracerebral bleeding associated with FVII activity levels below 1% of normal. FVII genotyping investigations revealed particular genotypes including the deleterious Cys135Arg mutation and a novel Ser52Stop nonsense mutation at the homozygous state. Both mutations, through different mechanisms, are expected to be inconsistent with the production of functional FVII. These putative mechanisms are discussed through a review of the literature on phenotypic and genotypic characteristics of cerebral haemorrhages in severe inherited FVII deficiency.
Summary
A double blind randomized cross-over multi-center study has been conducted to compare the pharmacokinetic and coagulation activation markers of high-purity factor IX concentrate subjected to ...both solvent/ detergent (SD) treatment and 15 nm-filtration (FIX-SD-15) with the licensed product subjected only to solvent-detergent (FIX-SD). This filtration process allows the elimination of small particles, such as non-enveloped viruses (i.e., hepatitis A and parvovirus B19). Eleven severe hemophilia B patients (FIX coagulant activity <2 IU/dl) received one infusion of 60 IU/kg of FIX-SD and one infusion of 60 IU/kg of FIX-SD-15 at least at 10 days interval. Blood samples were obtained before and at various time up to 72 h after infusion. The decay curves of factor IX (FIX:C and FIX:Ag) were evaluated by a model independent method. Bioequivalence was found between the two concentrates using the Schuirmann test. The mean FIX:C and FIX:Ag recovery of FIX-SD-15 was 1.08 and 0.89 IU/dl/IU/kg respectively with a mean half-life of 33.3 h for FIX:C and 25.6 h for FIX:Ag. Six months after initial enrollment, pharmacokinetic parameters were similar in the 7 patients tested. There was no significant variation of prothrombin fragment 1+2 and thrombin-antithrombin complexes measured up to 6 h after infusion, indicating that there was no activation process after administration of FIX. In conclusion, these data demonstrate that the introduction of a 15 nm filtration does not alter the pharmacokinetic profile of a well characterized SD FIX concentrate while providing additional viral safety.
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