N. Cabrera

One-stage and chromogenic FVIII:C assay discrepancy in mild haemophilia A and the relationship with the mutation and bleeding phenotype

Summary.  The discrepancy of the levels of factor VIII activity (FVIII:C) by different assays in some mild and moderate haemophilic A patients has been long known. Specific mutations affecting FVIII:C discrepancies have been described. No consensus exit as to which method most accurately represents the FVIII cofactor function in vivo and which has a better correlation with the haemorrhagic clinical expression. We studied 163 mild A haemophiliacs, and detected discrepancies in 20% of the patients, most of whom presented higher levels of FVIII:C with the one‐stage assay. In nine families, the FVIII mutation was found, while three showed mutations not previously described (Leu1978Phe and Ser1791Pro associated with higher levels of FVIII:C by one‐stage method; Arg1639His in a patient with low level of FVIII:C by the one‐stage, but normal, chromogenic assay). Assessing the level of FVIII:C by different methods could help to learn the possible haemorrhagic expressions of patients.

Inhibitors in haemophilia A: current management and open issues.

Summary.  The incidence of inhibitors in haemophilia A is 21–33%. The development of inhibitors to factor VIII (FVIII) is one of the most serious complications in haemophilia therapy and is an important challenge in haemophilia care. The main short‐term objective of the treatment of haemophilic patients with inhibitors is to control bleeding episodes, and the long‐term one is to eradicate the inhibitor by means of immune tolerance induction (ITI).The choice of treatment for bleeding in inhibitor patients is dictated by the current inhibitor titre, the severity of the bleed and the previous anamnesic response to FVIII. In low responder inhibitor patients the best treatment is large doses of concentrates of FVIII to attain haemostatic levels of the factor infused. The same approach can also be considered in high responders who have a temporarily low inhibitor level and major haemorrhage. High responders patients with high inhibitors titre or with minor haemorrhage must be treated with bypassing agents, such as FEIBA (factor VIII inhibitor bypassing activity) or recombinant activated FVII (rVIIa); there is no agreement which of both agents should be chosen in the different clinical situations. Only in patients waiting to start ITI treatment the rFVIIa use is clearly recommended, in order to avoide an anamnesic responce. In case of failure with this agents, extracorporeal immunoadsortion may be considered.

Comparison of a new chemiluminescent immunoassay for von Willebrand factor activity with the ristocetin cofactor‐induced platelet agglutination method

Measuring von Willebrand factor (VWF) activity is essential for the diagnosis of von Willebrand disease (VWD). The VWF activity is usually assessed based on measurement of the ristocetin cofactor (VWF:RCo). However, that test is technically challenging and has high intra‐ and inter‐assay variabilities. A new automated chemiluminescent immunoassay VWF activity has recently become commercially available (HemosIL AcuStar von Willebrand Factor Ristocetin Cofactor Activity). The main objective of this study was to evaluate this new method and to compare it with the VWF:RCo assay as the reference method. We studied 91 samples, 18 healthy volunteers samples and 73 samples from patients (VWF:RCo level <50 IU dL−1): 29 type 1 VWD, 13 type 2A, 5 type 2B, 5 type 2M, 3 type 2N, 5 type 3, 4 type 3 under treatment, 5 type 3 carriers and 4 samples with other pathologies. The HemosIL AcuStar VWF:RCo assay was 96% sensitive and 100% specific for detecting VWF abnormalities. The good analytical performance, and the sensitivity and specificity of HemosIL AcuStar VWF:RCo to detect VWF deficiency renders it a suitable method for VWD screening.

Severe and moderate hemophilia A: identification of 38 new genetic alterations

This report describes new mutations of F8 in patients with severe and moderate hemophilia A. Hemophilia A is an X-linked recessive disorder caused by a lack or decrease of factor VIII activity. Its socio-economic impact is high given its high bleeding expression and treatment cost. Our aim was to establish the mutation of each patient to improve family management. A total of 116 unrelated families with severe and moderate hemophilia A were involved. Non-carriers of intron 22 and intron 1 rearrangements were included in F8 gene screening. Intron 1 and 22 inversion frequencies were 3% and 52.5% respectively. Putative mutations were identified in all the families; 38 were new. The cumulative inhibitor incidence was 22%. Approximately half the families carry non-recurrent mutations, which were unique in around one third. Harmful effects for mutations predicting null alleles are expected. Missense mutation consequences are not easily predictable, despite the help of some bio-informatics tools.

Molecular and clinical profile of von Willebrand disease in Spain (PCM–EVW–ES): Proposal for a new diagnostic paradigm

The diagnosis of von Willebrand disease (VWD) remains difficult in a significant proportion of patients. A Spanish multicentre study investigated a cohort of 556 patients from 330 families who were analysed centrally. VWD was confirmed in 480. Next generation sequencing (NGS) of the whole coding VWF was carried out in all recruited patients, compared with the phenotype, and a final diagnosis established. A total of 238 different VWF mutations were found, 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 463 were found to have VWF mutation/s. A good phenotypic/genotypic association was estimated in 96.5% of the patients. One hundred seventy-four patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred sixteen patients presented with mutations that had previously been associated with increased von Willebrand factor (VWF) clearance. RIPA unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 70 patients. The NGS study facilitated an appropriate classification in 63 of them. The remaining seven patients presented with a VWF novel mutation pending further investigation. In five patients with a type 3 and two with a type 2A or 2B phenotype with no mutation, an acquired von Willebrand syndrome (AVWS) was suspected/confirmed. These data seem to support NGS as a first line efficient and faster paradigm in VWD diagnosis.

First application of MLPA method in severe von Willebrand disease. Confirmation of a new large VWF gene deletion and identification of heterozygous carriers

exposure on the erythrocyte membrane. Medicines, such as rebabirin and a methyldopa, have been shown to increase PS exposure, however, these agents were not prescribed to any of the subjects in this study (Haynes et al, 2008; Qadri et al, 2009). On the contrary, prior to the study, some of our patients with polycythaemia vera received hydroxycarbamide or aspirin, which are reported to decrease PS exposure (Haynes et al, 2008; Qadri et al, 2009). Iron deficiency is also reported to elevate PS exposure on the erythrocyte membrane (Kemmpe et al, 2006). In the current study, patients in the polycythaemia vera group showed a significantly lower MCV (76 ± 3 fl) compared to the secondary polycythaemia and healthy subject groups. As a detailed examination on the presence of iron deficiency was not included in our study, the possible association of iron deficiency to these results need to be further determined (Pavic et al, 2003). In conclusion, we found that PS exposure on the erythrocyte membrane was significantly increased in patients with polycythaemia vera. This finding may serve as a key to revealing the mechanism of the increased risk of thrombosis in patients with polycythaemia vera.

Identification of deletion carriers in hemophilia B: quantitative real-time polymerase chain reaction or multiple ligation probe amplification

Gross deletions in the F9 gene are easily detected by routinely sequencing hemophilia B-affected men. Nevertheless, a carrier diagnosis proves difficult as the presence of a normal allele does not recognize the partial or complete loss of the F9 gene and may be challenging if no DNA sample from affected men is available. This work aimed to identify hemophilia carriers in 2 families in which gross deletions of the F9 gene could be expected. The indirect genetic study was not conclusive, and sequencing did not show genetic defects in family 1. A real-time polymerase chain reaction (RT-PCR) assay using SYBR Green revealed the deletion of a copy of exon 8 in 3 women, whereas the multiple ligation-dependent probe amplification (MLPA) assay showed the deletion of a copy of exons 7 and 8 in these 3 women. These studies enabled us not only to rule out a pregnant woman as a carrier but also to confirm a complete deletion of the gene in the patient from family 2 and the heterozygous state of his mother. The advantages that the MLPA method offers are the identification of a multiple exon deletion in the same assay and commonly used technology. The RT-PCR technology used involves standardizing and analyzing each exon independently.

Inhibitor development in one patient and laboratory discrepancies in several families with both mild haemophilia and Arg531Cys mutation

Summary.  Certain mutations in mild haemophilia A have been associated with a greater risk of inhibitor development, especially when associated with intense treatment. We present a patient with both mild haemophilia A and Arg531Cys mutation, which developed lowtitre inhibitors and was not seen to be related to the intense substitute treatment. The inhibitor has a greater effect on the exogenous factor VIII, permiting an adequate response to treatment with desmopressin. A discrepancy exists in the factor VIII activity in this our patient and in the haemophiliacs of another two families with the same mutation when determination is performed with one‐stage or chromogenic method.

Inhibitor development after switching of FVIII concentrate in multitransfused patients with severe haemophilia A.

Switching between different therapeutic FVIII concentrate types has been postulated as a possible cause of inhibitor development in patient with haemophilia A. In this single‐centre, retrospective study, the incidence, titre and duration of inhibitor development in multitransfused patients, defined as patients with more than 150 exposure days (ED), were analysed from January 1970 to December 2007 in relation to ED and the number of switches between different products. Inhibitor titre was assessed by Bethesda assay (before 1998) or Nijmegen assay (after 1998). Medical records of 167 patients were screened, of which 97 patients met the inclusion criteria. Fourteen products of plasmatic origin (different purities) and five recombinant (three generations) were used. Nine patients (9%) developed inhibitors, all transient, low‐titre (1.41 ± 0.54 BU) after 323 ± 287 ED in average. Seventeen patients had no product switches of which four patients (23%) developed inhibitors (97 ED in average), whereas 13 patients (77%) did not (ED: 230). Fifty patients switched between plasmatic products only (median: 10 changes) of which five patients (10%) developed inhibitors (ED: 503), whereas 45 patients did not (ED: 932). Five patients switched between recombinant products only (seven changes) of which no patient developed inhibitors (748 ED). Twenty‐five patients switched between plasmatic and recombinant products (13 changes) of which no patient developed inhibitors (ED: 1654). No statistically significant differences between patient groups were observed. Neither the number of different FVIII products administered nor the switching of products influenced the incidence of inhibitor in multitransfused patients.

Founder haplotype associated with the factor VIII Asp1241Glu polymorphism in a cohort of mild hemophilia A patients.

1 Hendriks D, Scharpe S, van-Sande M, Lommaert MP. Characterization of a carboxypeptidase in human serum distinct from carboxypeptidase N. J Clin Chem Clin Biochem 1989; 27: 277–85. 2 Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem 1991; 266: 21833–8. 3 Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI, a thrombin-activatable fibrinolysis inhibitor. J Biol Chem 1995; 270: 14477–84. 4 BoffaMB,WangW, Bajzar L, NesheimME. Plasma and recombinant thrombin-activatable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulindependent activation, thermal stability, and enzymatic properties. J Biol Chem 1998; 273: 2127–35. 5 Schneider M, Boffa MB, Stewart R, Rahman M, Koschinsky M, Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme. J Biol Chem 2002; 277: 1021–30. 6 Marx PF, Hackeng TM, Dawson PE, Griffin JH, Meijers JC, Bouma BN. Inactivation of active thrombin-activatable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage. J Biol Chem 2000; 275: 12410–5. 7 Boffa MB, Bell R, Stevens WK, Nesheim ME. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activatable fibrinolysis inhibitor. J Biol Chem 2000; 275: 12868–78. 8 Ceresa E, van de Borne K, PeetersM, Lijnen HR, Declerck PJ, Gils A. Generation of a stable activated thrombin activatable fibrinolysis inhibitor variant. J Biol Chem 2006; 281: 15878–83. 9 Knecht W, Willemse J, Stenhamre H, Andersson M, Berntsson P, Furebring C, Harrysson A, Hager AC, Wissing BM, Hendriks D, Cronet P. Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis. FEBS J 2006; 273: 778–92. 10 CeresaE,PeetersM,DeclerckPJ,GilsA.Announcing a TAFIamutant with a 180-fold increased half-life and concomitantly a strongly increased antifibrinolytic potential. J Thromb Haemost 2007; 00: 418–20. 11 HillmayerK,MacoveiA,PauwelsD,CompernolleG,DeclerckPJ,Gils A. Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI) – a comparative study assessing the biological equivalence of rat, murine and human TAFI. J Thromb Haemost 2006; 4: 2470–7. 12 Kato T, Akatsu H, Sato T, Matsuo S, Yamamoto T, Campbell W, Hotta N, Okada N, Okada H. Molecular cloning and partial characterization of rat procarboxypeptidase R and carboxypeptidase N. Microbiol Immunol 2000; 44: 719–28. 13 Ceresa E, De Maeyer M, Jonckheer A, Peeters M, Engelborghs Y, Declerck PJ, Gils A. Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability. J Thromb Haemost 2007; 5: 2105–12. 14 Walker JB, Bajzar L. The intrinsic threshold of the fibrinolytic system is modulated by basic carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-activatable fibrinolysis inhibitor is masked by its instability. J Biol Chem 2004; 279: 27896– 904.