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This document is a letter to the editor discussing a French field study on the cross-reactivity of human anti-FVIII antibodies to porcine rFVIII. The study aimed to validate the modified Nijmegen method used in French laboratories for the titration of anti-porcine FVIII inhibitors. The study compared the stability of rpFVIII in different dilution media and assessed the cross-reactivity of plasma samples from patients with AHA and congenital hemophilia A with inhibitors. The results showed a decrease in rpFVIII activity after incubation, but no statistical difference between dilution media. The study provides valuable information for the laboratory assessment of rpFVIII in the treatment of AHA. Another study discussed in the letter found that cross-reactivity with rpFVIII was detected in 26% of AHA samples and 75% of PWA samples, but high cross-reactivity above 20 BU/mL was rare and not observed in the AHA population. The study confirmed the feasibility of using imidazole buffer to detect anti-rpFVIII antibodies. [Extracted from the article]

Introduction Factor XIII ( FXIII) deficiency is a rare hemorrhagic disorder whose early diagnosis is crucial for appropriate treatment and prophylactic supplementation in cases of severe deficiency. International guidelines recommend a quantitative FXIII activity assay as first-line screening test. FXIII antigen measurement may be performed to establish the subtype of FXIII deficiency ( FXIIID) when activity is decreased. Methods The aim of this multicenter study was to evaluate the analytical and diagnostic levels of performance of a new latex immunoassay, K-Assay® FXIII reagent from Stago, for first-line measurement of FXIII antigen. Results were compared to those obtained with the Berichrom® FXIII chromogenic assay for measurement of FXIII activity. Of the 147 patient plasma samples, 138 were selected for analysis. Results The accuracy was very good, with intercenter reproducibility close to 7%. Five groups were defined on FXIII activity level (<5% ( n = 5), 5%-30% ( n = 23), 30%-60% ( n = 17), 60%-120% ( n = 69), above 120% ( n = 24)), without statistical differences between activity and antigen levels ( P value >0.05). Correlation of the K-Assay® with the Berichrom® FXIII activity results was excellent (r = 0.919). Good agreement was established by the Bland and Altman method, with a bias of +9.4% on all samples, and of −1.4% for FXIII levels lower than 30%. One patient with afibrinogenemia showed low levels of Berichrom® FXIII activity but normal antigen level and clot solubility as expected. Conclusions The measurement of FXIII antigen using the K-Assay® is a reliable first-line tool for detection of FXIII deficiency when an activity assay is not available. [ABSTRACT FROM AUTHOR]

The platelet function analyser ( PFA-100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor ( VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in 'real-world' situations. We have chosen to study the reliability of PFA-100 for screening VWF ristocetin cofactor ( VWF: RCo) deficiency. We retrospectively analysed the results ( n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA-Epi, PFA- ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA-100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in 'real-world' conditions. The negative predictive value ( NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA-100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique. [ABSTRACT FROM AUTHOR]

Introduction Reliable measurement of FVIII inhibitor is critical in the follow-up of haemophilia A patients. We performed a multicentre study to evaluate whether the presence of von Willebrand factor ( VWF) in FVIII-deficient plasma ( FVIII- DP) influences FVIII inhibitor titres. Methods Six French haematology laboratories participated in this study. Three samples with varying FVIII inhibitor titres (1, 5 and 15 BU/mL) and one sample without any detectable FVIII inhibitor were tested using four different procedures for FVIII inhibitor assay. The Nijmegen method and a modified assay with imidazole were performed using FVIII- DP with and without VWF in the control mixture and as substrate plasma in the FVIII one stage assay ( OSA). Each mixture (reference and test) was incubated for two hours at 37 °C with buffered normal pool plasma. Results Higher inhibitor titres were measured in 5 and 15 BU/mL samples when assays were performed with the Nijmegen method and FVIII- DP without VWF. When samples were diluted in imidazole buffer, similar inhibitor titres, close to expected values, were measured whether VWF was present in the FVIII- DP or not. The data obtained were also more accurate when residual FVIII activity levels between 40% and 60% were used to calculate inhibitor titres, despite a linear type I reaction kinetics. Conclusion These results support the hypothesis that reliable FVIII inhibitor titres can be measured without the use of FVIII- DP containing VWF when an imidazole-modified assay is used. [ABSTRACT FROM AUTHOR]

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population. [ABSTRACT FROM AUTHOR]

Summary. Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL−1 of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity. [ABSTRACT FROM AUTHOR]

Summary. Factor VIII coagulant (FVIII:C) levels measured in patients receiving ReFacto® (B-domain-deleted recombinant FVIII) using chromogenic substrate assay (CSA) and one-stage clotting assay (OSA) have frequently shown discrepancies, and the use of the ReFacto Laboratory Standard (RLS) has therefore been recommended to minimize these differences. The potency of ReFacto AF®, the albumin-free successor of ReFacto®, is determined using CSA for the titration of vials, and a new standard (RLS-AF) was developed to measure its biological efficacy using OSA. This multicentre study therefore evaluated the efficacy of this new RLS in minimizing differences between OSA and CSA when measuring FVIII:C levels in plasma. Mock plasma samples were prepared by diluting ReFacto AF® in FVIII-deficient plasma to obtain four concentrations ranging from 15 to 90 IU dL−1. FVIII:C levels were then measured in six laboratories on four separate days using three different procedures, i.e. OSA with a plasma standard (PS) as reference, OSA with RLS-AF and CSA with PS. The inter-centre standard deviation ranged from 1.4 to 5.5 IU dL−1. However, FVIII:C levels measured with OSA were closer to the expected values when RLS-AF was used. In addition, the uncertainty of measurement, reflecting the inter-method discrepancy was greatly reduced when RLS-AF was employed in OSA (15%) in place of PS (33%). This study demonstrates that the OSA performed with RLS-AF to establish calibration curves provides a valuable alternative to CSA to measure FVIII:C in ReFacto-AF-treated patients. [ABSTRACT FROM AUTHOR]

The article offers information on a study conducted by the authors on discrepancies between chromogenic substrate assay and one stage assay in patients who are treated with coagulation factor VIII. Topics discussed include evaluation of the ReFacto antihemophilic factor in clinical practice, testing of plasma samples of the patients, and mentions the need for guidelines to be followed for postinfusion monitoring and pharmacokinetic assessment of coagulation factor FVIII concentrates.

Factor V ( FV) inhibitor arises rarely after using fresh frozen plasma ( FFP) to treat inherited FV deficiency and is often a real therapeutic challenge. Here, we report a patient with a severe FV deficiency who developed such an inhibitor and was then treated with recombinant activated FVII (r FVIIa) and platelet concentrates ( PC). Monitoring was assessed by thrombin generation assay ( TGA). PC were more effective than r FVIIa in treating bleeding, but there was no correlation between the TGA results and clinical efficacy. [ABSTRACT FROM AUTHOR]

Constitutional thrombocytopenias are rare disorders, often difficult to discriminate from acquired thrombocytopenias. More than 80 genes have been described as being at the origin of these diseases. Among them, several variants of the glycoprotein Ib platelet subunit alpha (GP1BA) and glycoprotein Ib platelet subunit beta (GP1BB) genes, coding for the GpIb-IX-V glycoprotein complex, have been reported in the literature. The study reported here aimed at describing newly identified monoallelic anomalies affecting the GP1BA and GP1BB genes on a clinical, biological and molecular level. In a cohort of nine patients with macrothrombocytopenia, eight heterozygous variants of the GP1BA or GP1BB genes were identified. Five of them had never been described in the heterozygous state. Computer modelling disclosed structure/function relationships of these five variants., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

A rapid lateral flow immunoassay (LFIA) (STic Expert(®) HIT), recently developed for the diagnosis of heparin-induced thrombocytopenia (HIT), was evaluated in a prospective multicentre cohort of 334 consecutive patients. The risk of HIT was estimated by the 4Ts score as low, intermediate and high in 28·7%, 61·7% and 9·6% of patients, respectively. Definite HIT was diagnosed in 40 patients (12·0%) with positive results on both enzyme-linked immunosorbent assay (Asserachrom(®) HPIA IgG) and serotonin release assay. The inter-reader reproducibility of results obtained was excellent (kappa ratio > 0·9). The negative predictive value of LFIA with plasma samples was 99·6% with a negative likelihood ratio (LR) of 0·03, and was comparable to those of the particle gel immunoassay (H/PF4-PaGIA(®) ) performed in 124 cases. Positive predictive value and positive LR were 44·4% and 5·87, respectively, and the results were similar for serum samples. The probability of HIT in intermediate risk patients decreased from 11·2% to 0·4% when the LFIA result was negative and increased to 42·5% when it was positive. In conclusion, the STic Expert(®) HIT combined with the 4Ts score is a reliable tool to rule out the diagnosis of HIT., (© 2014 John Wiley & Sons Ltd.)

We report two patients who developed acquired factor V (FV) inhibitors not related to exposure to bovine thrombin. Associated conditions were found in one patient (surgery, antibiotic administration) but none in the other one. Bleeding complications occurred only in the patient with idiopathic FV inhibitor, leading to packed red cell infusion. Laboratory findings showed the presence of specific FV inhibitors with titers of 5.5 and 5 Bethesda units, respectively. These two patients received high-dose intravenous immunoglobulin and FV levels normalized within a few days with a concomitant disappearance of FV inhibitors., (Copyright 2003 Wiley-Liss, Inc.)

Increased expression of tissue factor (TF) procoagulant activity by blood monocytes and tissue macrophages is implicated in a number of thrombotic disorders, as well as in fibrin deposition associated with inflammatory lesions and immunological diseases. We found that interleukin 4 (IL-4), a T lymphocyte-derived cytokine known to regulate a number of monocyte functions, inhibited the production of TF by monocytes in response to endotoxin and phorbol myristate acetate (PMA) in vitro. IL-4 had a concentration-dependent inhibitory effect on functional TF procoagulant activity (PCA) and reduced the binding of an anti-TF antibody, as assessed by flow cytometry analysis. Moreover, IL-4 reduced LPS- and PMA-induced TF mRNA levels. TF mRNA stability was not modified by IL-4 after the arrest of transcription by actinomycin D. We thus conclude that mRNA suppression is mediated by an effect occurring at the transcriptional level. Our results also show that the suppressive effect of IL-4 is independent of an increase in the intracellular concentration of cyclic AMP, another established inhibitor of TF production. Locally produced IL-4 might thus contribute to limiting the consequences of monocyte activation.