Paolo Radossi

Effects of secondary prophylaxis started in adolescent and adult haemophiliacs

Summary.  While primary prophylaxis is a well‐established and recommended method of care delivery for children with severe haemophilia, fewer studies have documented the benefits of secondary prophylaxis started in adolescence or adulthood. To evaluate the role of secondary prophylaxis started in adolescent and adult severe haemophiliacs, a retrospective observational cohort study was conducted in 10 Italian Centres that investigated 84 haemophiliacs who had bled frequently and had thus switched from on‐demand to prophylactic treatment during adolescence (n = 30) or adulthood (n = 54). The consumption of clotting factor concentrates, the orthopaedic and radiological scores, quality of life and disease‐related morbidity were compared before and after starting secondary prophylaxis. Prophylaxis reduced the mean annual number of total and joint bleeds (35.8 vs. 4.2 and 32.4 vs. 3.3; P < 0.01) and of days lost from work/school (34.6 vs. 3.0, P < 0.01). A statistically significant reduction in the orthopaedic score was observed during prophylaxis in adolescents, but not in the whole cohort. Patients used more factor concentrates with corresponding higher costs on prophylaxis, but experienced a better quality of life. With respect to on‐demand treatment, higher factor consumption and cost of secondary prophylaxis were balanced by marked clinical benefits and greater well‐being in this cohort of adolescent/adult haemophiliacs.

Comparison of the rates of joint arthroplasty in patients with severe factor VIII and IX deficiency: an index of different clinical severity of the 2 coagulation disorders.

Data from the Italian Hemophilia Centres were collected to perform a retrospective survey of joint arthroplasty in patients with severe hemophilia. Twenty-nine of 49 hemophilia centers reported that 328 of the 347 operations were carried out in 253 patients with severe hemophilia A (HA) and 19 in 15 patients with severe hemophilia B (HB). When results were normalized to the whole Italian hemophilia population (1770 severe HA and 319 severe HB), patients with HA had a 3-fold higher risk of undergoing joint arthroplasty (odds ratio [OR], 3.38; 95% confidence interval [CI], 1.97-5.77; P < .001). These results were confirmed after adjustment for age, HIV, hepatitis C virus (HCV), and inhibitor in a Cox regression model (HR, 2.65; 95% CI, 1.62-4.33; P < .001). The survival analysis of time to joint arthroplasty in the subset of patients with severe HA was not affected by the severity of factor VIII (FVIII) gene mutations. A systematic review of literature articles reporting joint arthroplasties in HA and HB showed that the proportion of HA patients who had undergone arthroplasties was higher than that of HB patients, in agreement with the findings in our Italian cohort. These data suggest that the 2 inherited coagulation disorders have a different severity of clinical phenotype.

High incidence of anti‐FVIII antibodies against non‐coagulant epitopes in haemophilia A patients: a possible role for the half‐life of transfused FVIII

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post‐transfusion antibodies may also arise against non‐coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII‐induced antibodies by use of an enzyme‐linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non‐inhibitory antibodies was evaluated in three patients; although half‐life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti‐FVIII antibodies by ELISA when routinely assessing haemophilia A patients.

Detection of B-Cell Monoclonality in Fine Needle Aspiration by PCR Analysis

Cytologic examination of fine-needle aspiration (FNA) sometimes fails to diagnose the malignant nature of B-cell proliferations. In this study we analyzed the Ig gene rearrangement of 49 FNA samples by polymerase chain reaction (PCR) in order to evaluate whether molecular analyses can improve the accuracy of FNA. Twenty-six patients had non-Hodgkins lymphoma, 11 had reactive lymphoid diseases, 5 had chronic inflammation and 7 had carcinoma. A semi-nested PCR was performed using an oligoprimer specific for consensus sequences of the V regions (FR3A) and two oligoprimers derived from conserved sequences of the J regions (LJH and VLJH). Histologic examination always followed the molecular and cytologic analysis. The sensitivity of PCR and FNA morphological examination in detecting a neoplastic pattern was 92% and 78%, respectively. When samples were considered inadequate for cytologic examination, PCR always reached a diagnosis consistent with the histologic features. Our results demonstrate that PCR analysis of FNA specimens is a reliable and sensitive method capable of enhancing the diagnostic accuracy of cytologic examination.

F8 gene mutation profile and ITT response in a cohort of Italian haemophilia A patients with inhibitors

Summary.  Anti factor VIII (FVIII) antibodies represent the main complication of replacement therapy in severe cases of haemophilia and most patients with inhibitor have gross gene rearrangements or point mutations that hamper the production of normal circulating FVIII. In this study we have investigated 82 haemophilia A patients with inhibitors. Seventy six were severe, three were moderate and three were mild. We screened the patients for the causative mutations using long range PCR for the recurrent intron 22 inversion (invint22), multiplex PCR for intron 1 inversion (invint1) and conformation sensitive gel electrophoresis followed by DNA sequencing for all other mutation types in the F8 gene. Diverse genetic defects were detected in the severe cases (with a predominance of severe molecular defects): F8 gene inversions, large deletions and non‐sense mutations account for 71% of the mutations. Only missense and splicing mutations were identified in the non‐severe patients and we confirmed that the presence of inhibitors correlates well with the presence of severe mutations, but a proportion of severe patients develops inhibitors despite the presence of diverse less severe mutations. When we have analysed the subgroup of patients who underwent immunetolerance, we have found that F8 gene large deletions are likely to be a high risk factor also for immunetolerance therapy unresponsiveness, while no clear evidence has been demonstrated for other mutation types.

Homozygous patients with APC resistance may remain paucisymptomatic or asymptomatic during oral contraception.

The effect of oral contraceptive therapy was studied in five patients with homozygous activated protein C resistance. Patients with this congenital abnormality, in contrast to those with antithrombin, protein C or protein S deficiencies, showed only a mild thrombotic tendency. In fact, only two of six observations (one patient took the pill on two separate occasions many years apart) showed deep vein thrombosis. No patient had pulmonary embolism. Two additional patients had a superficial vein thrombosis of the legs. In two instances, a superficial vein thrombosis and a deep vein thrombosis, concomitant risk factors were present (immobilization and surgery for an ovarian cyst, respectively). However, compared with heterozygous for the same abnormality, the symptomatic homozygous patients with APC resistance appeared to develop thrombosis after a shorter period of oral contraception.

IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII

The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein.

Prevalence of anti-FVIII antibodies in severe haemophilia A patients with inversion of intron 22

The aim of our study was to investigate whether haemophilia A patients with inversion of intron 22 are at high risk for non‐inhibitory anti‐FVIII antibodies development detected by ELISA. It is known that patients with severe forms of haemophilia A are more likely to develop anti‐FVIII antibodies. The incidence of inhibitory anti‐FVIII antibodies in patients with factor VIII gene inversion has been extensively evaluated, but if this defect has to be considered a predisposing factor is still debatable. Non‐inhibitory anti‐FVIII antibodies are attracting interest, due to the potential influence on FVIII half‐life. Our data show that FVIII gene inversion was a major predisposing factor for anti‐FVIII antibodies development.

Total ankle replacement for end-stage arthropathy in patients with haemophilia

Dear Editor, In Haemophilia November 2006 van der Heide [1] and colleagues reported on the feasibility of a total ankle prosthesis (TAP) in two patients with coagulation disorders. Even though it is a recognized fact that the ankle is one of the most affected joints, in haemophiliacs prostheses are uncommon, at least in this group of patients, being considered to be an innovative surgical approach. It is well known that from an early age the ankle joint is frequently affected by bleeding in haemophiliacs and standard haemarthrosis treatment, including replacement therapy administered as primary prophylaxis or on-demand, is not always able to prevent joint damage. Physical therapy, footwear modification or orthoses may be useful but when chronic haemophilic synovitis occurs and recurrent haemarthroses become unresponsive to substitution therapy, synoviorthesis is to be considered using intra-articular injections of radioactive material or chemical substances [2]. These procedures, although effective in reducing the frequency of bleeding, are not able to alter the natural history of a degenerative arthropathy. When advanced ankle arthropathy develops arthrodesis is considered the standard treatment for pain relief [3] but total ankle replacement may also be considered for persistent pain and/or motion improvement [4]. As only limited reports have been published on this subject to date it is hoped that the following description of surgical intervention performed on two of our patients affected by severe haemophilia A (HA) will be helpful. Patient 1 was a 30-year-old male with severe HA and chronic HCV infection. The World Federation of Haemophilia joint score developed by Pettersson and Gilbert was used as the assessment instrument, according to this scale the total ankle score was 8 at the time of orthopaedic evaluation. Surgery was indicated because of limited motion and severe pain but in the year 2000 the patient, being worried about the possibility of further movement loss, refused ankle fusion. In the past he had been identified as being low responder anti-factor VIII (FVIII) inhibitor (max. titre 2 BU), before the surgery in question, however, the inhibitor was not detected. At the beginning of the operation, performed under general anaesthesia, replacement therapy was started with a bolus infusion of 100 IU kg of high-purity plasma-derived FVIII concentrates (Emoclot D.I., Kedrion, Italy) to obtain 100% plasma level. The prosthesis was implanted according to the instructions given by manufacturer, all the components were inserted without cement. Substitution treatment was continued by bolus injections every 12 h and the total amount of FVIII concentrates was 50 000 IU (684 IU Kg). PTT (partial thromboplastin time) and plasma FVIII levels were measured daily and concentrate dosage was adjusted in order to obtain >70% plasma activity. After the surgery the ankle was placed in a splint for 30 days. The postoperative period was uneventful, morphine was given to control the pain, no drainage was applied, there were no haemorrhages and the substitution treatment lasted a total of 10 days. This patient has had a 7-year followup, during which time he has had painless ankle movement and improved walking ability. There have not been any radiographic signs of prosthetic loosening or infection. A STAR prothesis cementless was used; Figs 1 and 2 show postoperative radiographs of this patient. Patient 2 was a 43-year-old male with severe HA, co-infected with HCV and HIV. HIV infection was kept under control by using highly Correspondence: Paolo Radossi, Transfusion Service, Haemophilia and Regional Blood Diseases Centre, Italy. Tel.: +39 0423 732336; fax: +39 0423 732337; e-mail: radossi.p@alice.it

Correlation between FIX genotype and pharmacokinetics of Nonacog alpha according to a multicentre Italian study

Pharmacokinetic (PK) studies on recombinant FIX concentrate, Nonacog alpha, were conducted with different sampling time designs which gave rise to not complete and homogenous outcomes. In addition, patients FIX genotype/PK relationship has never been investigated.