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Background Circulating dysfunctional factor IX (FIX) might modulate distribution of infused FIX in hemophilia B (HB) patients. Recurrent substitutions at FIX activation sites (R191-R226, >300 patients) are associated with variable FIX activity and antigen (FIXag) levels. Objectives To investigate the (1) expression of a complete panel of missense mutations at FIX activation sites and (2) contribution of F9 genotypes on the FIX pharmacokinetics (PK). Methods We checked FIX activity and antigen and activity assays in plasma and after recombinant expression of FIX variants and performed an analysis of infused FIX PK parameters in patients (n = 30), mostly enrolled in the F9 Genotype and PK HB Italian Study (GePKHIS; EudraCT ID2017-003902-42). Results The variable FIXag amounts and good relation between biosynthesis and activity of multiple R191 variants results in graded moderate-to-mild severity of the R191C>L>P>H substitutions. Recombinant expression may predict the absence in the HB mutation database of the benign R191Q/W/K and R226K substitutions. Equivalent changes at R191/R226 produced higher FIXag levels for R226Q/W/P substitutions, as also observed in p.R226W female carrier plasma. Pharmacokinetics analysis in patients suggested that infused FIX Alpha distribution and Beta elimination phases positively correlated with endogenous FIXag levels. Mean residence time was particularly prolonged (79.4 h, 95% confidence interval 44.3-114.5) in patients (n = 7) with the R191/R226 substitutions, which in regression analysis were independent predictors (β coefficient 0.699, P = .004) of Beta half-life, potentially prolonged by the increasing over time ratio between endogenous and infused FIX. Conclusions FIX activity and antigen levels and specific features of the dysfunctional R191/R226 variants may exert pleiotropic effects both on HB patients' phenotypes and substitutive treatment.

BACKGROUND In older people, multiple chronic ailments lead to the intake of multiple medications (polypharmacy) that carry a number of negative consequences (adverse events, prescription and intake errors, poor adherence, higher mortality). Because ageing patients with haemophilia (PWHs) may be particularly at risk due to their pre-existing multiple comorbidities (arthropathy, liver disease), we chose to analyse the pattern of chronic drug intake in a cohort of PWHs aged 60 years or more. PATIENTS AND METHODS S + PHERA is a multicentre observational study, with the broad goal to evaluate prospectively the health status and medication intake in 102 older patients with severe haemophilia A or B compared with 204 age- and residence-matched controls chosen randomly from the same general practices of PWHs. The rate of potential drug-drug interactions (PDDI) was evaluated as a proxy of prescription appropriateness. RESULTS After excluding replacement therapies and antiviral drugs, PWHs took in average less daily drugs than controls (2.4 ± 2.5 vs 3.0 ± 2.4) and had a lower rate of polypharmacy. Moreover, their prevalence of PDDI was lower (16.7% vs 27%). CONCLUSIONS The rate of polypharmacy and the appropriateness of medications other than those for haemophilia and related comorbidities are acceptable in Italian PWHs, and better than those in their age peers without haemophilia, perhaps owing to drug tailoring and deprescribing by the specialized haemophilia centres at the time of regular visits. However, the PWHs investigated herewith were relatively young and the rate of polypharmacy and related PDDIs may become more prominent and crucial when older ages are reached, suggesting the need of continuous surveillance on prescribed drugs and the risk of drug-drug interactions.

63rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) -- DEC 11-14, 2021 -- Atlanta, GA, [No Abstract Available], Amer Soc Hematol

Hemophilia A is a rare X-linked disease that occurs as a result of a defect in the FVIII-encoding gene. The reduction or absence of plasma FVIII compromises the coagulation cascade, resulting in frequent bleeds, especially in joints or soft tissues. Currently, replacement therapy with coagulation factor concentrates is the gold standard for the treatment of FVIII deficiency. Herein, we report a case series of five hemophilia A patients treated with an extended half-life recombinant human coagulation factor, FVIII-Fc fusion protein (efmoroctocog alfa). The prophylactic regimen for each patient was individualized based on their pharmacokinetic profile. Compared to previous prophylactic treatments, most patients received a reduced weekly dose of concentrate, all underwent a reduced frequency of administration, the annualized bleeding rates (ABR) and hemophilia joint health scores (HJHS) were stable or improved. The half-life of efmoroctocog alfa and the 72-hour trough levels were higher than those observed in the A-LONG Phase III trial. In conclusion, all patients reported clinical improvements and general subjective wellbeing in the absence of significant safety concerns after switching to efmoroctocog alfa.

Objectives Inherited dysfibrinogenemia is a rare disorder, for which clinical studies related to the risk of bleeding or thrombosis and the type of causative mutation are scanty. Materials and methods We analyzed the laboratory, clinical, and genotypic features of 50 patients with inherited dysfibrinogenemia belonging to 19 unrelated families. Results In all the index cases, fibrinogen activity by Clauss method was below the normal range, while it was observed in 57.9% only by PT-derived method. In three families, hypodysfibrinogenemia was evident, associated with three novel mutations (Ter492Gln in FGB, Cys365Asp, and Leu370Phe in FGG). Three additional novel mutations were also identified (Arg114Lys in FGA, Ile131Thr and Trp234Arg in FGG). Bleeding symptoms assessed by ISTH-BAT scored at least 1 in 30% of patients and, significant bleeding symptoms were mainly present in female patients, especially associated with pregnancy. Two patients with FGB Arg44Cys suffered from venous thromboembolism, and two with FGA Arg35His had ischemic stroke at older age. Conclusions This study confirms the heterogeneity of clinical features in inherited dysfibrinogenemia, due to the wide spectrum of the causative mutations. Larger multicenter studies are needed to assess the definitive correlation of some mutations with bleeding or thrombosis.

Background An open-label phase II, multicenter clinical trial was conducted at 11 Haemophilia Centres in Italy, Romania, and Turkey, to evaluate the pharmacokinetics (PK), efficacy, and safety of high purity, plasma-derived, double virus inactivated and double nano-filtered factor IX (pd-FIX) concentrate (Kedrion FIX), EudraCT Number: 2005–006186-14. Material and Methods 16 previously treated patients (PTPs) with severe or moderately severe haemophilia B were enrolled in the study. At enrolment, 14 underwent the first PK assessment (PK I), and the second PK (PK II) assessment was performed after six months of treatment (5 on-demand and nine prophylaxis) at the end of the study. PK parameters were evaluated by Non-Compartmental Analysis (NCA), One-Compartment model (OCM), and Two-Compartment Model (TCM). Efficacy of Kedrion FIX in all 16 patients was evaluated by the number of bleeding events, and clinical response following the infusions. Periodic FIX inhibitor assays and thrombogenicity tests were scheduled throughout the study to assess the safety of the drug. Results As compared to the published data on PK of pdFIX, Kedrion FIX displayed a longer half-life (22.37–55.73 hrs), reduced clearance, and regular volume of distribution at PK I by both NCA and OCM. The comparison of outcomes of PK II with those of PK I by OCM, also showed significant changes, particularly in patients on prophylaxis, who showed some improved parameters of PK. Due to two outlier values at the end of the trial, the NCA parameters of PK I were not compared to those of PK II. Breakthrough bleeds were successfully treated with 1 or 2 infusions. No significant adverse events were observed during the study. Discussion During the six-month clinical study period, the use of Kedrion FIX resulted in a safe and effective pd-FIX concentrate with excellent PK characteristics.

The objective of this scoping review is to summarize the current use of pharmacokinetics for tailoring prophylaxis in hemophilia patients switching between clotting factor products. Patients with hemophilia may require switching of clotting factor concentrates due to a variety of factors, but there have been perceived risks associated with switching, such as inhibitor development or suboptimal protection due to inadequate dosing while titrating treatment. Studies that look at patients switching from one clotting factor concentrate to another are categorized in terms of their primary and/or secondary objectives, notably biosimilarity and comparative pharmacokinetic studies and inhibitor development studies. Research on how best to switch concentrates with respect to dosing regimen are lacking, and currently a trial-and-error approach is used for dosing the new factor concentrate. In the future, studies looking at the predictability of pharmacokinetics (PK) of a new factor concentrate based on individual PK knowledge of the original factor concentrate may offer clinical benefit by providing a safer switching approach and protocol.

Introduction Molecular characterization has shown a wide mutational spectrum underlying haemophilia A (HA) and haemophilia B (HB). Different molecular assays have allowed laboratories to perform genetic testing for F8 and F9 mutations. Aim Recently, multiplex ligation-dependent probe amplification (MLPA), a simple technique for relative quantitation of targeted genomic regions, has been introduced in HA and HB for detection of large deletions and duplications. We want to verify if MLPA might be used at the beginning of the molecular investigation. Methods We used it to test 22 patients with suspected large deletions, nine patients negative for mutation detection by other methods and finally, 45 new patients as their first screening test. Results Carrier status was also established in 28 related females and gross rearrangements were also searched for by MLPA in 19 females with reduced FVIII or FIX levels. All suspected deletions were confirmed apart from two patients. In patients with a negative screening test, MLPA revealed one large duplication, while in two patients where MLPA was used as the first screening step, an exon duplication was detected. In females with reduced FVIII or FIX, no large deletions or duplications were found. Conclusions Owing to its simplicity, MLPA seems useful at the beginning of the molecular investigation, saving all the following steps, where positive. Single exon deletion diagnosis requires caution due to the risk of misdiagnosis, but benefits of MLPA appear to overcome the pitfalls.

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44(+)/CD49c(+)/CD49e(+)/CD151(+)/CD73(+)/CD49f(-)/CD26(-) resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

Introduction 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, patient's FIX genotype/PK relationship has never been investigated. Aim Investigate how different sampling times may affect PK parameters and try to find a FIX genotype/PK relationship. Patients and Methods A cohort pharmacokinetic, Nonacog Alpha single-dose, open-label, non-comparative study was conducted in eight Comprehensive Care Haemophilia Centres in Italy. Seventeen previously treated moderate or severe haemophilia B patients were enrolled. Factors IX:C one-stage clotting assay, FIX genotype and PK analysis were centralized. Results The evaluation of PK outcomes showed a quite long half-life, smaller clearance and volume of distribution of Nonacog Alpha in comparison with the results from previously reported studies, where blood sampling was stopped too early. The relationship between PK outcomes and FIX genotype showed that small deletions displayed the higher clearance and shorter half-life, the nonsense mutations (the lower and the longer respectively), and missense mutations were in between. Conclusions It is evident that area under the curve (AUC) and other PK parameters depend from the sampling time design. In order to have a complete evaluation of clotting factors in vivo decay, blood samples must be collected until the baseline factor concentration has been achieved again. Due to the relationship between FIX genotype and clearance, tailored prophylaxis of HB patients could be partially predicted by genotyping.

Summary. Patients with haemophilia show high prevalence of hepatitis C infection but low rate of progressive liver disease when they are not co-infected with HIV. The balance between host immune system and hepatitis C virus (HCV) variability seems to play a major role in the evolution of the HCV-related disease. To address this point we have studied, in a group of selected patients with haemophilia, the composition and in some cases the evolution, of the highest variable envelope gene within the hypervariable region 1 (HVR1) of the HCV, which is the region more directly exposed to the host immune response. Five of 12 patients show a very high homogeneity of the HVR1 and four of those had severe progressive liver disease. These results seem to confirm the major role of the immunity in driving the variability of the HCV rather than the high degree of different HCV strains to which haemophiliacs have been in touch with, during their long-term replacement therapy. Our results seem in keeping with other studies on different type of patients, where a low degree of quasispecies variability has been demonstrated in relationship with the progression and the severity of their liver disease.

Synoviorthesis is the intra-articular injection of chemical or radioactive substances able to produce fibrosis of hypertrophied synovium, which has proved effective in the treatment of chronic haemophilic synovitis. Between September 1999 and October 2001 we treated 28 outpatients (25 with haemophilia A, three with haemophilia B). Our treatment was focused on pain and functional limitation of joints. A schedule was adopted to treat each joint using intra-articular rifamycin once a week, repeated five times. Patients were covered with factor replacement on demand. Oral analgesia was offered as required because of acute but transient painful inflammatory reaction. Their median age was 34 years (range 15-60 years). The indication for synoviorthesis was chronic synovitis characterized by recurrent haemarthroses, persistent pain and limited range of motion (ROM). Thirty-five joints were treated with a total of 169 injections, including six joints (20%) in patients with inhibitors. In five patients two joints were treated in the same session. Thirty procedures were completed: 24 (80%) were considered effective (as excellent or good), while six were considered insufficient (20%). Pain was reduced in 96% of cases and in 70% the ROM was improved. In our experience intra-articular infiltration with rifamycin appears to be effective in reducing joint pain and in improving the ROM. The procedure presents a low risk of bleeding, can be used for patients with inhibitors and multiple joints can be treated without any additional cost.

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.

Haemophilia A displays a broad heterogeneity of genetic defects and of clinical severity. Inhibitor development is the main complication of replacement therapy in severe cases and most patients with inhibitors have gross gene rearrangement or point mutations, which hamper the production of normal circulating factor VIII (FVIII). We have investigated three related severe haemophilia A patients, all of whom have high titre inhibitors. By using long-range polymerase chain reaction (PCR) for FVIII gene inversion, we observed an unusual pattern in these patients. We therefore decided to screen the whole FVIII gene by conformation-sensitive gel electrophoresis. A large FVIII gene deletion spanning exon 2 to exon 25 was identified and we were able to obtain a 18.5 kb PCR product, which is specific for this mutation and useful for determining the carrier state in this family. All three haemophiliacs carrying this very large gene deletion show similar clinical history and very high-titre inhibitors, supporting the observation that inhibitor development seems to be an inherited characteristic. On the basis of our observations we think that this subgroup of patients at very high risk of inhibitor development should be identified by mutation analysis whenever possible, before the beginning of replacement therapy.

A relevant aspect in the treatment of patients with hemophilia A (HA) presenting inhibitor against factor VIII (FVIII) is the different antigenicity of FVIII used for replacement therapy. The aim of the study was to assess the effect of different products, with variable von Willebrand factor (vWF) concentration, in preventing the binding of inhibitor to FVIII. The reactivity of inhibitors from plasma of 18 patients with HA versus three commercial concentrates containing different amounts of vWF was compared. The results show that increasing amounts of vWF might have a protective effect on the transfused FVIII inactivation.

Background The natural history of inhibitors in patients with haemophilia A not undergoing immune tolerance induction (ITI) is largely unknown. A recent randomized controlled trial suggests that the higher the FVIII dose used for ITI, the faster the clearance and the lower the rate of bleeding, without any difference in the rate of tolerance. We aimed at assessing the rate of spontaneous inhibitor clearance in a large cohort of patients not undergoing ITI. Methods A retrospective analysis of anti-FVIII inhibitors of long-term registry data in a single centre cohort of 524 haemophilia A patients considered for synovectomy was performed. Patients were tested for inhibitors before and 15 days after any and each surgical episode and thereafter did not undergo immune tolerance at any time. Results The cumulative incidence of inhibitors overall was 34% (180 out of 524) with the highest percentage of 39% (168 out of 434) in severe patients which represented 83% of the cohort. Among the 180 inhibitor patients: 63 had permanent inhibitors; 70 fulfilled current criteria for transient inhibitors but a third category of 47 additional patients cleared the alloantibody spontaneously in >6 months. At logistic regression, both the inhibitor titre and the gene mutation were shown to predict time to clearance. Conclusions Spontaneous clearance of inhibitors over variable time in the absence of ITI treatment was found in up to 2/3 of the cases.

SummaryTwo unrelated patients belonging to two Italian kindreds with a history of thrombotic manifestations were found to have a double heterozygous defect of factor V (F. V), namely type I quantitative F. V defect and F. V Leiden mutation. Although DNA analysis confirmed the presence of a heterozygous F. V Leiden mutation, the measurement of the responsiveness of patients plasma to addition of activated protein C (APC) gave results similar to those found in homozygous defects. It has been recently reported in a preliminary form that the coinheritance of heterozygous F. V Leiden mutation and type I quantitative F. V deficiency in three individuals belonging to the same family resulted in the so-called pseudo homozygous APC resistance with APC sensitivity ratio (APC-SR) typical of homozygous F. V Leiden mutation. In this study we report two new cases of pseudo homozygous APC resistance. Both patients experienced thrombotic manifestations. It is likely that the absence of normal F. V, instead of protecting from thrombotic risk due to heterozygous F. V Leiden mutation, increased the predisposition to thrombosis since the patients became, in fact, pseudo-homozygotes for APC resistance. DNA-analysis is the only way to genotype a patient and is strongly recommended to confirm a diagnosis of homozygous F. V Leiden mutation also in patients with the lowest values of APC-SR. It is to be hoped that no patient gets a diagnosis of homozygous F. V Leiden mutation based on the APC-resi-stance test, especially when the basal clotting tests, i.e., PT and aPTT; are borderline or slightly prolonged.

Hairy cell leukaemia (HCL) is a chronic lymphoproliferative disease of B-cell lineage. One of the peculiar immunophenotypic markers is the strong expression of the p55 chain of the interleukin-2 receptor (IL2R), recognized by anti-CD25 (or anti-Tac) monoclonal antibody. However, it is known that in rare cases CD25 may not be detectable, even when variant forms of HCL are excluded. The possibility has not been investigated that in these situations CD25 is present in the cytoplasm of the neoplastic cells. This paper describes a case in which the clinical, histological, and electron microscopic features were consistent with a typical HCL. Immunophenotype analysis showed the whole spectrum of markers of HCL, except for the expression of IL2R. The soluble form of the molecule was, however, increased in the patient's serum. Cytospin staining of the neoplastic B cells with anti-CD25 clearly demonstrated the presence of IL2R in the cytoplasm of hairy cells. When the cells were cultivated in vitro in the presence of interferon-alpha 2b, CD25 was detectable at the membrane level. These findings suggest that at least some cases of CD25-negative HCL may express cytoplasmic IL2R.

Summary. The introduction of activated recombinant factor VIIa (rFVIIa) has allowed elective surgery to be safely performed in haemophiliacs with inhibitors. The main problems associated with its use are the short half-life, necessitating frequent intravenous injections, and its very high cost. Here we describe, for the first time, the performance of total hip and knee replacements in a haemophiliac with inhibitors at the same operation. The amount of rFVIIa concentrate used (8.57 mg) was similar to that normally used for a single joint replacement. The use of continuous infusion allowed for easier administration and further contributed to the reduction in cost as it avoids the peak levels associated with bolus injections.

Unfortunately, commercial concentrates transmitted infections such as hepatitis and human immunodeficiency virus (HIV), which dramatically changed the life of the hemophiliac community [2] and are still heavily affecting patients and doctors dealing with this disease.

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

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.

Although primary prophylaxis is a well-established and widely recommended practice for the management of patients with severe hemophilia, there are limited data in the literature documenting the benefit of secondary prophylaxis initiated in adolescence or adulthood. In order to address this issue we have conducted a retrospective study among the 10 Italian Hemophilia Centers on adolescent and adult patients with severe hemophilia switched from on-demand treatment to a long term secondary prophylaxis regimen. On the whole, we collected information on 73 consecutive patients with severe hemophilia (66 hemophilia A and 7 hemophilia B). Their median age started prophylaxis was 12 (range 10–17) for 23 adolescents and 30 (17.4–72) for the 50 adults and continued for a median of years 5.8 (2–14.7) in adolescents and 4 years (1.5–12) in adults. The clinical impact of secondary prophylaxis with respect to the last year on-demand regimen before switching is showed in the following table for each group of patients. Adolescents (n=23) Adults (n=50) On-Demand Prophylaxis On-Demand Prophylaxis Values are reported as mean (standard deviation) Bleedings 32.4(24.8) 2.8 (3.2) 42.7 (24.5) 5.5 (5.2) Joint bleedings 25.6 (22.1) 1.9 (2.3) 36.4 (24.5) 4.2 (4.1) Orthopaedic score 8.4 (7.5) 3.7 (2.4) 21.6 (12.2) 17.7 (12.2) Pettersson score 5.1 (3.4) 5.0 (3.3) 14.7 (13.8) 15.2 (14.3) Work/school days lost 35.1 (34.5) 1.0 (1.5) 33.8 (20.9) 2 (0–17) Concentrate consumption U/Kg/y 2449.8 (1761.9) 3508.3(803.1) 3222.6 (2241.8) 4058.5 (952.3) The decreased joint bleeding, better orthopaedic score, less work/school days lost, delayed progression of arthropathy and improved well-being observed with secondary prophylaxis show the benefit of this therapeutic approach and appear to counterbalance its higher clotting factor consumption. In conclusion large, prospective, long-term randomized studies are needed to definitevely assess the clinical impact of this strategy in adolescent and adult hemophiliacs and to identify those groups of patients who would benefit from this therapeutic approach.

Introduction: The efficacy of factor VIII and IX concentrates administered to prevent bleeding episodes in patients with hemophilia A and B is correlated with the plasma levels measured over time after the infusion. The inter-patient variability of pharmacokinetic (PK) parameters is large, and it is difficult to assess individual PK profiles due to the need for multiple time points. This is often not feasible, particularly for pediatric patients. Population PK modeling potentially provides a practical solution to this problem. The successful modelling of PK parameters at the population level requires knowledge of disposal characteristics and relevant covariates. We performed a systematic review of the available evidence in order to identify available PK data for factor VIII and IX concentrates to facilitate the implementation of a population PK approach. Methods: We conducted a literature search in MEDLINE and EMBASE from January 1997 to May 2014, using the keywords "hemophilia" and "pharmacokinetic". We included only articles that published original PK data for factor VIII and IX concentrates in humans and published in English. Two authors independently screened the studies and extracted the relevant data. Results: We retrieved 237 unique articles published between 1998 and 2013. We excluded 185 articles that did not meet our research criteria. We included 52 articles, with a total of 1365 patients included in PK analyses. 26 articles reported PK data on factor VIII concentrates, 18 articles report PK data on factor IX concentrates, and one article reported on both factor VIII and IX concentrates. Seven articles reported pharmacokinetic data on both factor VIII and Von Willebrand factor concentrates. We extracted the following data: number of patients, type and severity of hemophilia, patient age, factor concentrate infused, dose infused, sampling data points, half-life, clearance, recovery and the model used for pharmacokinetics, and inclusion of patients undergoing surgery or with inhibitors. The main results are summarized in table 1. Conclusions: This review provides the first systematic appraisal of the methods and results of published papers in the field. The data gathered confirms the intra-patient variability of factor concentrate PK and provides useful information on which to build population based PK models. *3 FIX articles and 2 FVIII articles did not report lab test; one article reported PK data for both FIX and FVIII †11 articles reported FVIII PK data for both one-stage clotting and chromogenic assays ǂPapers reporting on long-acting FVIII and FIX were included in the review, but not summarized in the table. For this reason, not all 1365 patients are accounted for in the table §Estimate of the range of the means found in the papers Disclosures Xi: Baxter: Research Funding. Navarro-Ruan:Baxter: Research Funding. Mammen:Baxter: Research Funding. Collins:Baxter: Consultancy, Honoraria, Research Funding, Speakers Bureau; CSL: Consultancy, Honoraria, Research Funding, Speakers Bureau; NovoNordisk: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau. Neufeld:Baxter: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: data safety monitoring board, data safety monitoring board Other; Biogen IDEC: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Membership on an entity's Board of Directors or advisory committees; Pfiser: consultancy, data and safety monitoring board Other; Octapharma: Research Funding. Dunn:CSL Behring,: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Baxter: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; Pfiser: Membership on an entity's Board of Directors or advisory committees. Iorio:Baxter: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; NovoNordisk: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Pfiser: Honoraria, Research Funding.

We report a patient with myasthenic syndrome who, 2 years after diagnosis, developed an oligoclonal lymphocytosis. This disorder was sustained by both kappa+ and lambda+ CD5+ B-cell clones; over the following year, the white blood cell count increased and phenotypic characterization revealed a clear imbalance in the immunoglobulin light chain ratio (84% kappa+). Accordingly, persistence of a kappa+ B-cell clone was disclosed by molecular analysis of immunoglobulin heavy chain gene rearrangements. Our results may suggest that prolonged immune system stimulation due to an autoimmune disease can drive a benign lymphoproliferation into a B-cell neoplastic process.

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.

The therapeutic approach to hairy cell leukemia (HCL) is in some instances still debated. A variant form of HCL (HCL-V) characterized by high white cell count, splenomegaly, hypercellular and aspirable bone marrow has been described; differential diagnosis often arises with some other B-cell disorders which also show circulating hairy or villous lymphocytes. Conventional treatment for HCL is often less effective in HCL-V. In this report we describe a case with features consistent with HCL-variant treated with splenic radiotherapy. We not only obtained an hematological response but also the near total disappearance of bone marrow infiltration, compatible with a clinical complete remission. Clinical and biological implications of this phenomenon are discussed on the basis of this unexpected therapeutic result, obtained with splenic radiotherapy alone.

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.

By the introduction for routine tests of automated counters it has become easy and extremely common the finding of abnormal or near normal laboratory results. During blood donations haematocrit (Htc) and haemoglobin level (Hb) are regularly tested to avoid a blood donation in the case of anaemia as this is the most frequent cause of exclusion from the donation. However it is not rare to observe a level of Hb or Htc upper or close the normal highest limit. Starting from the 1st of January 2006 to the 31st December 2006, we have collected 11240 donations in total from 5636 regular blood donors (total donation index 1.99) and selected on the basis of Htc, limit upper 50% for man and 46% for women, 84 males and 19 females. In this cohort, during a 1846 years donation period, the average of donations for males was 22.2 while for females was 11.4. We collected randomly and used as control group 79 donors (59 males and 20 females) with normal Htc levels in the last blood donation. For the identification of the V617F (1848 G>T) mutation within JAK2 we used the method published by Jones ed al. in 2005 named amplification refractory mutation system (ARMS). Among the 84 men with high haematocrit level we have been able to identify 1 blood donor positive for this mutation. This blood donor had Htc equal to 50.6 in the last donation, while the average Htc in the last year was 51.7. These results account for a total prevalence of the JAK2 mutation in 1% of the blood donor population with elevated Ht (1 out of 103) while it is 0.6% (1 out of 183) of the entire population of blood donors in this study or 0.02% of the entire cohort appertaining to our transfusion service. Our results seem also in keeping with the few studies evaluating blood donors (Zanella et al., Transfusion 1987) or normal population (Ruggeri et al., Ann Int Med 2003). In both these studies the prevalence of MPD was around 3% which is not so far away from that found in our cohort (asymptomatic patients) but much higher than that supposed in hospital based cohorts in previous studies (100 fold increased). The hypothesis that the prolonged myeloid stimulus from phlebotomies can induce inhibition of the feed-back control as well as blood donations triggered the onset of a pre-existing latent primary myeloproliferative disease needs further studies to be confirmed.

Introduction: Immune tolerance therapy (ITT), consists of administration of daily high doses of factor VIII concentrates by intravenous infusions. It can induce tolerance to the exogenous protein and eradicate the antibody response to FVIII, which still represents the main complication of replacement therapy in haemophilia A (HA) severe cases. Methods: We have investigated 71 HA patients with inhibitors (sixty-nine severe, one moderate and one mild cases). We screened the patients for the causative mutations in the F8 gene using Long Range PCR for the intron22 inversion, multiplex-PCR for intron1 inversion or conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing for other mutation types. Results: Diverse genetic defects were detected in the severe cases, with a predominance of gross mutations: F8 gene inversions, large deletions and nonsense mutations account for 68% of the mutations, whereas in the two non severe patients specific missense mutations were identified. ITT has been attempted in 16 HR patients of this cohort but failed in 5 cases. Conclusion: We confirmed that the presence of inhibitors correlates well with the presence of null mutations, as reported by Schwaab et al (Thromb Haemost 1995). Several predictive factors for ITT outcome have been so far taken into consideration but no correlation has been made between F8 gene defect and ITT response. In our cohort 2 large deletions, 2 intron22 inversions and 1 nonsense mutations failed to respond. For the remaining 2 large deletions no ITT were attempted so no information are available. Large deletions appear to be a high risk genetic factor both for inhibitor development and for long term inhibitor persistence and ITT unresponsiveness. The identification of these patients at very high risk of inhibitor development and ITT unresponsiveness by mutation analysis should therefore be strongly recommended soon after the diagnosis, even because of the high cost of the therapy.

It has been shown that glycosaminoglycans play a role in the regulation of immune response. In particular, heparin exerts an antiproliferative and apoptotic action in different cellular systems. In this study we evaluate whether heparin can also induce a naturally occurring programmed cell death in lymphocytes from B-chronic lymphocytic leukemia (B-CLL), a neoplastic lineage where apoptosis is blocked by the expression of the proto-oncogene bc1-2. Peripheral blood lymphocytes (PBL) from 7 cases of B-CLL patients in different stages were cultured with three different heparin sodium concentrations for 4 days. Apoptosis was evaluated by agarose gel electrophoresis and by cytofluorimetric analysis. Bcl-2 expression was tested by flow cytometric analysis and immunohistochemistry on cytospin preparations. Agarose gel electrophoresis showed the characteristic DNA fragmentation pattern of apoptosis in all the cases of B-CLL stage III and IV after heparin incubation. DNA from normal and neoplastic lymphocytes cultured without heparin did not undergo spontaneous apoptosis. Cytofluorimetric analysis confirmed the agarose gel pattern and found a level of apoptosis over 50% after culture of neoplastic PBL with heparin. In these cases bcl-2 expression was found to be significantly reduced after heparin incubation when comparing to bcl-2 level before incubation. Our data adds further evidence regarding the potential role of heparin in oncogene inhibition and in apoptosis induction. In particular, the induction of apoptosis in neoplastic lymphocytes by heparin may have a role in the complicated field of interactions between the immune system and the blood vessels by glycosaminoglycans.

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-Hodgkin's 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.