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

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.

Qualitative and quantitative modifications of von Willebrand factor in patients with essential thrombocythemia and controlled platelet count.

BACKGROUND: Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleeding in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. OBJECTIVES: We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. PATIENTS/METHODS: Sixty-nine ET patients (M = 29; median age, 62 [48-70] years; platelets, 432 [337-620] × 10(3)  µL(-1) ), 69 matched controls and 10 subjects with reactive thrombocytosis (RT) were studied. VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoproteinV (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. RESULTS: In ET patients, VWF:Ag was increased by 31 ± 13% vs. controls (P < 0.01), without dependence of blood groups, while VWF:act was reduced by 21 ± 12% vs. controls and by 50 ± 24% vs. RT (P < 0.01). The VWF:act/VWF:Ag ratios in ET were reduced by 35 ± 17% vs. controls and RT patients (P < 0.001) and significantly associated with: immature or total platelet counts, GC, GpV and TXB2 . In multivariable analysis, only GC inversely predicted ET patients' VWF:act/VWF:Ag ratios (ß = -0.42, P = 0.01). By electrophoresis analyses, high-molecular-weight VWF multimers were variably reduced with atypical cleavage bands in ET only. VWF:propeptide, ADAMTS-13 and elastase levels were normal in ET patients. Platelet-associated ADAM-10 and ADAM-17 hydrolyzed VWFm in vitro, showing patterns similar to those in ET samples. CONCLUSIONS: In ET patients with controlled platelet counts, the VWF:act/VWF:Ag ratio is decreased and predicted by GC, a product of platelet activation. ADAM-10 and/or ADAM-17 might be involved. In vivo platelet activation, which characterizes ET, might contribute to disease-specific VWF alterations.

In vivo prostacyclin biosynthesis and effects of different aspirin regimens in patients with essential thrombocythaemia.

Essential thrombocythaemia (ET) is characterised by enhanced platelet generation and thrombosis. Once daily (od) aspirin incompletely inhibits platelet thromboxane (TX)A2 production in ET. A twice daily (bid) dosing is necessary to fully inhibit TXA2. Whether this dosing regimen affects in vivo prostacyclin (PGI2) biosynthesis is unknown. PGI2 biosynthesis was characterised in 50 ET patients on enteric-coated (EC) aspirin 100 mg od, by measuring its urinary metabolite, 2,3-dinor-6-keto-PGF1α (PGI-M). Moreover, in a crossover study 22 patients poorly responsive to standard aspirin based on serum TXB2 levels (≥4 ng/ml) were randomised to different seven-day aspirin regimens: EC aspirin 100 mg od, 100 mg bid, 200 mg od, or plain aspirin 100 mg od. PGI-M measured 24 hours after the last aspirin intake (EC, 100 mg od) was similar in patients and healthy subjects both on (n=10) and off (n=30) aspirin. PGI-M was unrelated to in vivo TXA2 biosynthesis, and not affected by EC aspirin 100 mg bid or 200 mg od as compared to EC 100 mg od. PGI2 biosynthesis in aspirin-treated ET patients appears unrelated to TXA2 biosynthesis, and not affected by an improved aspirin regimen, demonstrating its vascular safety for future trials.

The spectrum of factor XI deficiency in Italy.

Factor XI (FXI) deficiency is a rare inherited bleeding disorder invariably caused by mutations in the FXI gene. The disorder is rather frequent in Ashkenazi Jews, in whom around 98% of the abnormal alleles is represented by Glu117X and Phe283Leu mutations. A wide heterogeneity of causative mutations has been previously reported in a few FXI deficient patients from Italy. In this article, we enlarge the knowledge on the genetic background of FXI deficiency in Italy. Over 4 years, 22 index cases, eight with severe deficiency and 14 with partial deficiency, have been evaluated. A total of 21 different mutations in 30 disease-associated alleles were identified, 10 of which were novel. Among them, a novel Asp556Gly dysfunctional mutation was also identified. Glu117X was also detected, as previously reported from other patients in Italy, while again Phe283Leu was not identified. A total of 34 heterozygous relatives were also identified. Bleeding tendency was present in very few cases, being inconsistently related to the severity of FXI deficiency in plasma. In conclusion, at variance with other populations, no single major founder effect is present in Italian patients with FXI deficiency.

Efficacy and safety during formulation switch of a pasteurized VWF/FVIII concentrate: results from an Italian prospective observational study in patients with von Willebrand disease.

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate(®) P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate(®) P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93-99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate(®) P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate(®) P was at least as effective and well-tolerated as the previous formulation.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

Effects of secondary prophylaxis started in adolescent and adult haemophiliacs.

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.

Molecular characterization of five Italian families with inherited severe factor XIII deficiency.

Factor XIII (FXIII) deficiency is a very rare (1:2 000 000) severe autosomal recessive bleeding disorder, mostly due to mutations in the coagulation FXIII A-subunit gene. We have studied the molecular basis of FXIII deficiency in five unrelated Italian families. The coding region, intron-exon boundaries and 5'- and 3'-untranslated regions of the FXIII gene encoding the A subunit were amplified and directly sequenced. Candidate mutations were identified in all the patients. Three novel mutations occurred in three patients. These include a novel homozygous deletion of two base pairs (bp) in exon 14 (c.2002-2003 DelCT). This deletion causes a frameshift from Leu667 and the formation of a stop codon at amino acid position 681. The second patient presents a novel homozygous (c.2126 G>A) transition in exon 15, predicting a Ser708Asn in Barrel 2 domain. The third patient is compound heterozygote for two missense mutations, a previously reported Arg260His substitution, and a novel transition in exon 4 (c.560 C>T) predicting a Pro186Leu in the core domain. The remaining two patients have two previously reported mutations: a 4-bp homozygous deletion in exon 11 (c.1392-1395 Del AATT), previously reported to occur in the Vicenza Area, and a homozygous nonsense mutation in exon 8 (c.979 C>T) predicting an Arg326X in the core domain. The novel mutations occurred at amino acid residues highly conserved among different species (pig, monkey, mouse and dog) and were not detected in 110 normal alleles. Structural analysis shows that Pro186Leu mutation leads to the replacement of the rigid proline pyrrolidine ring by the larger and more flexible leucine side chain and Ser708Asn may probably disrupt the hydrogen bond with Ala291.

Allogeneic bone marrow transplantation for Fanconi anemia.

Five patients (age range 7-14 years) received allogeneic bone marrow transplantation (BMT) for Fanconi anemia (FA). All patients showed progressive pancytopenia associated with congenital malformations. Diagnosis was confirmed by studies of cellular hypersensitivity to the clastogenic effect of the DNA crosslinking agent diepoxybutane. The conditioning regimen consisted of low dose cyclophosphamide (5 mg/kg x 4) and fractionated total body irradiation (167 cGy x 3). For graft-versus-host disease prophylaxis one patient was given cyclosporin alone while the remaining four patients received a combination of cyclosporin and two doses of methotrexate. Marrow was given unmanipulated from HLA-identical siblings. All patients are alive 18-67 months after grafting with Karnofsky scores of 100% and normal hemopoiesis of donor origin. Modifications in transplant protocols such as those here described have resulted in a decreased risk of severe transplant-related complications. These results confirm that BMT is a curative therapy in FA patients and should be considered as a first choice treatment if an HLA-identical donor is available.

AIDS-related complex treated by antiviral drugs and allogeneic bone marrow transplantation following conditioning protocol with busulphan, cyclophosphamide and cyclosporin.

A 26-year-old man with AIDS-related complex (ARC) was treated with high-dose busulphan and cyclophosphamide, followed by allogeneic bone marrow transplantation. For 3 months before transplantation he received a combination of four drugs considered active against human immunodeficiency virus (HIV) to reduce the viral burden: zidovudine, acyloguanosine, fusidic acid and phenylidantoin. Although in reduced doses in coincidence with marrow engraftment, zidovudine therapy was scheduled after transplantation in order to protect donor cells from infection with HIV. Engraftment rapidly occurred and was documented by cytogenetic analyses. The post-transplant course was characterized by severe acute GvHD with irreversible hepatorenal failure. The patient died on day 48 after transplantation. Polymerase chain reaction analyses for detecting HIV DNA showed the persistence of positivity at day +30 and +45 after transplantation. Antibodies to specific HIV proteins evaluated with Western blot testing also persisted at days +21 and +35 after transplantation. Circulating immunocomplexes disappeared on day +31, and an increase in the CD4/CD8 ratio occurred. The short survival of the patient, affected by chronic hepatitis too, does not allow final conclusions about the role of BMT in HIV disease.

Evaluation of four different methods for platelet freezing. In vitro and in vivo studies.

This report describes our experience with various techniques for the freezing of platelet-rich plasma, removed from the final product after leukapheresis procedures performed on 14 hematological patients. A total of 194 platelet units were frozen for subsequent autologous transfusion, by the following four methods: (1) 6% dimethyl sulfoxide (DMSO); (2) a combination of 5% DMSO/6% hydroxyethyl starch; (3) 3% glycerol; (4) 5% glycerol/4% glucose. Each technique was evaluated by measuring the percentage of platelet recovery, malondialdehyde (MDA) production, and lactate dehydrogenase release. To investigate the safety and therapeutic effectiveness of the previously frozen platelets, in vivo comparison of four platelet freezing methods was made in 8 thrombocytopenic patients, using corrected platelet increment (CCI), determined at 24 h. Our in vitro results indicate that the cryopreservation with 6% DMSO, without controlled cooling rate, provides significantly (p less than 0.05) greater platelet recovery (75%) as compared to other systems. The decrease of MDA production and the increase in plasma lactate measured after the thawing process was less in the DMSO-frozen units than in the other platelet units. When platelets, cryopreserved by this method, were subsequently transfused into patients, a significantly better CCI (greater than 5,000/microliters) was obtained. In our series, 6 patients were entirely supported with frozen autologous platelets. It appears from this study that a better understanding of the physical and biochemical events occurring during the freezing process will improve platelet cryopreservation, allowing a more systematic use of frozen platelets in the support of thrombocytopenic patients.

Density gradient separation of hematopoietic stem cells in autologous bone marrow transplantation.

Recently, the principles of density gradient cell separation have been transferred to the marrow fractionation, and the Ficoll technique by using a COBE 2991 blood cell processor has been developed and widely employed as well. This method is particularly useful in view of a chemical antineoplastic purging intended for autologous marrow transplantation. Forty marrows, which derived from patients suffering with leukemia and lymphoma, were fractionated with Ficoll on a COBE machine and in vitro purged with Mafosfamide at a dose of 50 micrograms/ml/1 x 10e7 MN cells. The density gradient separation enables to reduce the initial volume to 10%, the contaminating RBC to less than 1%, the total nucleated cells to 25% (greater than 80% of MNC) sparing about 80% of the CFU-GM. After purging, the surviving hemopoietic progenitor cells were 2.5%. The clinical effects of the fractionated purged cells were studied in 11 autotransplanted patients and compared with 14 transplants performed with untreated buffy-coat marrow derived cells. Ficoll cells produced less adverse effects at the time of reinfusion, while, as expected, the time of hematopoietic recovery was delayed in these patients (mafosfamide treated cells). These results confirm the usefulness of the gradient density cell separation to reduce the side effects of the DMSO and to make reliable the Mafosfamide purging manoeuvre, preventing the interference of contaminating RBC aldehyde dehydrogenase.

Detection of bacterial contamination in bone marrow graft.

A rapid method of determining bacterial contamination was used in a series of fifty-five bone marrow harvests (20 allogeneic and 35 autologous transplantations). The microbiological culture assays executed on bone marrow samples soon after the harvest and before the manipulation, showed a positivity only in the autologous bone marrow group. Another positivity was revealed after the freezing process of bone marrows for autologous transplantation. Two samples in the bone marrow group submitted to purging were positive after treatment and freezing process. In this study, the increase of bacterial contamination frequency seemed to be related to extensive handlings, such as pharmacological in vitro purging and freezing procedures.

Comparison of five methods for concentrating progenitor cells in human marrow transplantation.

Enrichment of bone marrow (BM) aspirates is an important prerequisite prior to in vitro treatment or cryopreservation. In this regard, we have analyzed the results obtained on 190 BM processed by the following 5 techniques: HES sedimentation with centrifugation; COBE 2991 blood cell processor; Ficoll/hypaque (F/H) gradient centrifugation; Continuous flow cell separator (CS 3000 Fenwal); Semicontinuous blood cell separator (Dideco T 90). Each procedure was evaluated by measuring the recovery of nucleated marrow cells (NC), mononuclear cells (MNC), committed progenitor cells (CFU-GM), the reduction of BM volume and the removal of red blood cells (RBC) and polymorphonuclear cells (PMN). The results of this comparative study show that F/H gradient on a COBE 2991 cell washer provides the most efficient system for purifying a MNC fraction (89% recovery) from unwanted cells (RBC less than 2% and PMN less than 2%) in a very small volume (98% reduction) with a good recovery of CFU-GM (80%).

A preliminary survey of Italian experience on bone marrow harvesting, processing and manipulation. The Italian Cooperative Study Group on Cell Manipulation in Hematology.

The following report describes the initial stage of the activity of the Italian Cooperative Study group on cellular manipulation in hematology consisting of a retrospective evaluation of data regarding bone marrow (BM) harvesting processing, and proceedings from 20 Italian Centers. Two thousand, three hundred and eighty-four BM have considered: 1073 were performed for autografts and 1311 for allografts. A cohort of adverse effects in marrow harvesting were reported by 7 Centers out of 20, including one death caused by tracheomalacia during the anesthesia. Twelve Centers used blood separators as a mean for marrow processing. Eight Centers used the RBC removal technique in major donor/recipient AB0 incompatibility. Ficol-Hipaque gradient was employed in 7 Centers. T-depletion were accomplished with monoclonal antibodies in 7 Centers and elutriation in two Centers. Fifteen Centers provided to purge the residual tumour cells with chemicals (11), immunological (4) and chemo-physical means (1).