A retrospective study to describe the incidence of moderate to severe allergic reactions to factor IX in subjects with haemophilia B.

Using a patient chart review process, we conducted a retrospective study to describe the frequency of allergic reactions in individuals with haemophilia B receiving factor IX (FIX) replacement therapy. The number of allergic reactions in individuals receiving a recombinant FIX (rFIX) product (BeneFix(®)) was then compared with the number of reactions in patients receiving plasma-derived FIX (pdFIX) products. Of the 180 subjects in the study, 163 received rFIX, 88 received pdFIX; 71 received both product types. A total of seven (3.89%) subjects had a moderate or severe allergic reaction to a FIX product (95% confidence interval [CI], 1.06-6.71%). Among those receiving rFIX, four subjects (2.45%) had an allergic reaction (95% CI, 0.08-4.83%). Of individuals taking pdFIX products, three (3.41%) developed an allergic reaction (95% CI, 0-7.20%). It was noted that three (1.84%) of those taking rFIX developed an inhibitor to FIX (95% CI, 0-3.90%), while four (4.55%) of those receiving a pdFIX product developed an inhibitor (95% CI, 0.19-8.90%). Inhibitor development was frequently associated with allergic reaction. These results provide evidence that there is no difference in the frequency of allergic reactions or inhibitor development in individuals receiving rFIX compared with those receiving pdFIX concentrates. The current study and a previous study of similar design have now compared the rate of allergic reactions associated with rFIX and pdFIX concentrates has now been compared in a total of 414 subjects; this represents the largest collection of data to date on this rare complication of haemophilia B therapy.

Sites of initial bleeding episodes, mode of delivery and age of diagnosis in babies with haemophilia diagnosed before the age of 2 years: a report from The Centers for Disease Control and Prevention's (CDC) Universal Data Collection (UDC) project.

Lack of detailed natural history and outcomes data for neonates and toddlers with haemophilia hampers the provision of optimal management of the disorder. We report an analysis of prospective data collected from 580 neonates and toddlers aged 0-2 years with haemophilia enrolled in the Universal Data Collection (UDC) surveillance project of the Centers for Disease Control and Prevention (CDC). This study focuses on a cohort of babies with haemophilia whose diagnosis was established before the age of two. The mode of delivery, type and severity of haemophilia, onset and timing of haemorrhages, site(s) of bleeding, provision of prophylaxis with coagulation factor replacement therapy, and the role played by the federally funded Haemophilia Treatment Centers (HTC) in the management of these infants with haemophilia were evaluated. Seventy-five per cent of haemophilic infants were diagnosed early, in the first month of life, especially those with a family history or whose mothers were known carriers; infants of maternal carriers were more likely to be delivered by C-section. Involvement of an HTC prior to delivery resulted in avoidance of the use of assisted deliveries with vacuum and forceps. Bleeding from the circumcision site was the most common haemorrhagic complication, followed by intra- and extra-cranial haemorrhages and bleeding from heel stick blood sampling. Eight per cent of the infants were administered factor concentrate within 24 h of birth; more than half were treated to prevent bleeding. This study highlights the significant rate and the sites of initial bleeding unique to very young children with haemophilia and underscores the need for research to identify optimal evidence-based recommendations for their management.

ReFacto and Advate: a single-dose, randomized, two-period crossover pharmacokinetics study in subjects with haemophilia A.

ReFacto is a recombinant B-domain-deleted, monoclonal antibody-purified, solvent-detergent-treated factor VIII (BDDrFVIII) with no albumin added to the final formulation. Although ReFacto has been shown to be bioequivalent to a plasma-derived FVIII product (Hemophil-M) in a randomized, crossover pharmacokinetic (PK) study, the comparability of ReFacto with the full-length (complete sequence) recombinant FVIII (FLrFVIII, Advate) product has not been previously examined in this manner. The primary objective of this study was to compare the PKs of ReFacto with those of Advate in patients with severe haemophilia A. This was a third-party unblinded, randomized, multicentre, two-period crossover PKs study of ReFacto and Advate in subjects with severe haemophilia A (FVIII:C < or =1%). Blood samples were collected over a 48-h period after i.v. administration of each of the FVIII products. FVIII:C was determined using the chromogenic substrate assay (CSA) in a central laboratory. The plasma FVIII:C PK parameters of ReFacto and Advate were determined using non-compartmental analysis. Bioequivalence was assessed on maximum plasma concentration (C(max)) and the area under the plasma concentration vs. time curves (AUCs) using an anova. The two products were judged to be equivalent if the 90% confidence limits of the ratio of the geometric mean values of C(max) and AUCs fell within the interval of 80-125%. Results from this PKs comparison of two different rFVIII products, using chromogenic substrate assay to measure FVIII:C, showed that ReFacto and Advate are bioequivalent to each other.

Complications of central venous access devices in paediatric haemophilia patients.

We conducted a retrospective survey of our experience with central venous access devices (CVADs) implanted in children with haemophilia seen at the Vanderbilt Hemostasis-Thrombosis Clinic from 1986 to 2000. Following discussion with parents on the merits and risks associated with the use of CVADs for immune tolerance induction or factor prophylaxis, catheters were inserted under sterile technique in the operating room. One nurse provided demonstration and teaching about catheter care and access. Thirty central venous catheters were inserted in 22 children. Our survey revealed that the two most common complications associated with central venous catheters were bacteraemia and thrombosis. We found a sepsis rate of 0.30/1000 catheter-days or one episode of bacteraemia for every 3346 days of catheter use. The thrombosis rate of our cohort was 0.13/1000 catheter-days or one episode of thrombosis for every 7529 days of catheter use. Uncomplicated venous access is essential in children with severe haemophilia who require prophylaxis or immune tolerance induction. While infection was the most common complication observed in our series, we experienced a lower overall infection rate than several reported series. Catheter thrombosis and subsequent obstruction may occur as a result of intraluminal fibrin deposits. We conclude that the use of implantable central venous catheters is an effective method for accessing children with haemophilia. We accept that the benefits of CVADs in the treatment of paediatric haemophilia patients outweigh the previously documented risks. Future prospective studies should be designed to define all associated risks and to determine effective strategies to reduce them.

Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine.

PURPOSE: To assess thiopurine S-methyltransferase (TPMT) phenotype and genotype in patients who were intolerant to treatment with mercaptopurine (MP) or azathioprine (AZA), and to evaluate their clinical management. PATIENTS AND METHODS: TPMT phenotype and thiopurine metabolism were assessed in all patients referred between 1994 and 1999 for evaluation of excessive toxicity while receiving MP or AZA. TPMT activity was measured by radiochemical analysis, TPMT genotype was determined by mutation-specific polymerase chain reaction restriction fragment length polymorphism analyses for the TPMT*2, *3A, *3B, and *3C alleles, and thiopurine metabolites were measured by high-performance liquid chromatography. RESULTS: Of 23 patients evaluated, six had TPMT deficiency (activity < 5 U/mL of packed RBCs [pRBCs]; homozygous mutant), nine had intermediate TPMT activity (5 to 13 U/mL of pRBCs; heterozygotes), and eight had high TPMT activity (> 13.5 U/mL of pRBCs; homozygous wildtype). The 65.2% frequency of TPMT-deficient and heterozygous individuals among these toxic patients is significantly greater than the expected 10% frequency in the general population (P <.001, chi(2)). TPMT phenotype and genotype were concordant in all TPMT-deficient and all homozygous-wildtype patients, whereas five patients with heterozygous phenotypes did not have a TPMT mutation detected. Before thiopurine dosage adjustments, TPMT-deficient patients experienced more frequent hospitalization, more platelet transfusions, and more missed doses of chemotherapy. Hematologic toxicity occurred in more than 90% of patients, whereas hepatotoxicity occurred in six patients (26%). Both patients who presented with only hepatic toxicity had a homozygous-wildtype TPMT phenotype. After adjustment of thiopurine dosages, the TPMT-deficient and heterozygous patients tolerated therapy without acute toxicity. CONCLUSION: There is a significant (> six-fold) overrepresentation of TPMT deficiency or heterozygosity among patients developing dose-limiting hematopoietic toxicity from therapy containing thiopurines. However, with appropriate dosage adjustments, TPMT-deficient and heterozygous patients can be treated with thiopurines, without acute dose-limiting toxicity.

Familial thrombophilia associated with fibrinogen paris V: Dusart syndrome.

We report on a family with a history of venous thromboembolism associated with fibrinogen Paris V (fibrinogen Aalpha-Arg554-->Cys). Ten members experienced thrombotic events, including 4 with fatal pulmonary emboli. Pulmonary embolism was the presenting feature in 4. Those with the mutation and a history of thrombosis had somewhat higher fibrinogen concentrations than those with the mutation and no thrombosis (294 +/- 70 mg/dL vs 217 +/- 37 mg/dL, respectively). The Paris V mutation consistently caused a prolongation of the reptilase time, and fibrin clots containing the abnormal fibrinogen were more translucent than normal clots. Given the early onset of symptoms and the initial presentation with pulmonary embolism in some family members, it was justifiable to offer prophylactic anticoagulation with warfarin to carriers of the mutation. Fibrinogen Paris V has now been reported in 4 apparently unrelated families, indicating that it is a relatively common cause of dysfibrinogenemia-associated thrombosis.

High-dose cytosine arabinoside and etoposide: an effective regimen without anthracyclines for refractory childhood acute non-lymphocytic leukemia.

The purpose of this report is to describe the tolerability and activity of the combination of high-dose cytosine arabinoside (Ara-C) given at the maximum tolerated dose of 36 g/m2, together with high doses of etoposide in relapsed and refractory childhood acute leukemias. Eighteen children with relapsed or refractory acute leukemia were treated with Ara-C 3 g/m2 every 12 h on days 1-6, followed by etoposide 400 mg/m2 on days 7-9 (HDAC/VP-16). Eight children with refractory disease received HDAC/VP-16 as salvage induction therapy after failing conventional induction regimens; four of five refractory ANLL patients (80%) had a complete response (CR) after HDAC/VP-16 therapy. Ten patients received HDAC/VP-16 as post-remission intensification therapy; five patients (four ANLL, one relapsed ALL) remain in second CR at 56, 26, 9, 5 and 2 months. Toxicities were primarily hematologic and dermatologic. Seven patients (39%) developed bacterial or fungal infections; four patients developed grade 3 or 4 acral erythema. No patient died of therapy-related toxicity. The combination of 36 g/m2 cytosine arabinoside and 1200 mg/m2 etoposide is an effective regimen for children with relapsed or refractory acute nonlymphocytic leukemia, with tolerable toxicities; the absence of anthracyclines makes this regimen suitable for patients who have previously received maximal doses of anthracyclines or who have evidence of cardiac dysfunction. Further evaluation of this regimen in acute nonlymphocytic leukemia is presently being investigated.

Stress and incidence of bleeding in children and adolescents with hemophilia.

OBJECTIVE: To examine the relationship of stress and incidence of bleeding in boys with hemophilia. STUDY DESIGN: We conducted a 6-month longitudinal study of 97 subjects (ages 4 to 16 years) from six hemophilia centers. Diaries recorded bleeding episodes (including site and history of previous trauma) and both child and parent daily stress. Parent and child stressful life event measures were obtained monthly. Socioeconomic data and clotting factor level were determined at enrollment. Logistic regression models examined the influence of recent stress on likelihood of bleeding on each day, controlling for factor level and socioeconomic data. We also determined associations of aggregated previous month's events with bleeding likelihood in the succeeding month. RESULTS: Fifty-eight percent of study participants had severe hemophilia. The sample population averaged nine bleeding episodes per 6 months; of these; two thirds of bleeding incidents occurred into joints and 44% after injury. Factor level strongly predicted bleeding incidence (p < 0.0001). Increased parent stress was associated with increased bleeding in general (odds ratio = 1.37, p < 0.003) and with injury (odds ratio = 1.65, p < 0.001), but not bleeding into joints. Similar findings followed parent reports of positive life events. Increased parent negative life events in 1 month were associated with increased bleeding in the succeeding month (p < 0.05). CONCLUSION: Short- and long-term parental stress may lead to increased bleeding incidence in hemophilia, although factor level much more strongly predicts bleeding.

Analysis of factor VIII gene inversion mutations in 166 unrelated haemophilia A families: frequency and utility in genetic counselling.

Haemophilia A is an X-linked recessive bleeding disorder of variable severity that is caused by a deficiency of coagulation factor VIII (FVIII). The disease results from mutations in the FVIII gene which are heterogenous both in type and position within the gene. Recently, however, inversion mutations were found to be common to patients with severe disease (Lakich et al., 1993). These mutations result from intrachromosomal recombinations between DNA sequences in the A gene (located in intron 22 of the FVIII gene) and one of two A genes upstream to the FVIII gene. To determine the frequency of these inversions we performed Southern blot analysis on banked DNA from 166 consecutive, unrelated haemophilia A families previously referred for carrier or prenatal testing. In 57/166 (34%) families an inversion or other unique mutation was detected. The distal and proximal A genes lying upstream to the FVIII gene were involved in 79% and 18% of the mutations, respectively, but in 3% of the families the sequences involved in the mutation have not been identified. In 20/38 (53%) families with severe disease a mutation was detected. Interestingly, the relative risk of developing inhibitors in patients with FVIII gene inversions or other 3° mutations detected by this assay, as compared to patients with no detectable mutation by this assay, was 3.8. In families for which a mutation is detected, direct DNA testing is an accurate and inexpensive alternative to linkage analysis for prenatal or haemophilia A carrier testing.

A prospective study of patterns of bleeding in boys with haemophilia.

To describe the patterns of bleeding and clotting factor concentrate use in boys with haemophilia over a 6-month period, daily diary records of bleeding, factor use, levels of physical activity, chore performance and school attendance were collected from parents of 96 males between 4 and 17 years of age with haemophilia A or B followed at six comprehensive haemophilia treatment centres in Massachusetts, Rhode Island and Tennessee. 14 243 person days were available for analysis. The sample cohort averaged approximately nine bleeding episodes (1.5 per months), almost two-thirds of which were haemarthroses. 44% of bleeds were associated with injury and the average duration was 1.4 days. New bleeding episodes were significantly more likely to begin on weekdays (Monday-Thursday) than on weekends (Friday-Sunday). Boys with more severe disease had significantly more bleeding episodes and a higher frequency of haemarthroses. Boys with the most severe disease were also more likely to have joints involved when they bled and to have more spontaneous bleedings without apparent preceding trauma. Bleeding was associated with increased school absence, decreased levels of physical activity and decreased rates of household task performance. Relatively high rates of bleeding associated with trauma suggest the need for preventive interventions.

Long-term granulocyte-macrophage colony-stimulating factor and immunosuppression in the treatment of acquired severe aplastic anemia.

PURPOSE: Seven children with newly diagnosed acquired severe aplastic anemia (SAA) were treated with a combination of long-term granulocyte-macrophage colony-stimulating factor (GM-CSF) and immunosuppression to assess the potential for GM-CSF to induce sustained neutrophil recovery, reduce the incidence of infection, and enhance the therapeutic efficacy of immunosuppression. METHODS: Patients received a 14-day course of i.v. antithymocyte globulin 15 mg/kg/day with oral prednisone 1 mg/kg/day, long-term daily oral cyclosporine A 10 mg/kg/day, and long-term daily s.c. GM-CSF 5 micrograms/kg/day. RESULTS: All seven children recovered an absolute neutrophil count of > 1.0 x 10(9)/L within 3.5 months of diagnosis (mean 60 days). Of the six children followed throughout their entire illness (follow-up 10-27 months), five are platelet and red cell transfusion independent (three off-therapy, two on tapering therapy) and one continues on therapy with a diminishing transfusion requirement. Compared with seven children treated previously with immunosuppression alone, children who received GM-CSF spent fewer days in the hospital and were less likely to develop infection. CONCLUSIONS: The addition of GM-CSF to immunosuppressive therapy appears to be beneficial in the treatment of children with acquired SAA with GM-CSF stimulating granulopoiesis. The children are better protected from infectious complications while immunosuppressive agents achieve full therapeutic potential.

Cross-platform hypermedia examinations on the Web.

The authors developed a multiple-choice medical testing system delivered using the World Wide Web. It evolved from an older, single-platform, locally-developed computer-based examination. The old system offered a number of advantages over traditional paper-based examinations, such as digital graphics and quicker, easier scoring. The new system builds on these advantages with its true cross-platform design and the addition of hypertext learning responses. The benefits of this system will increase as more medical educational resources migrate to the Web. Faculty and student feedback has been positive. The authors encourage other institutions to experiment with Web-based teaching materials, including examinations.

Why does the human factor IX gene have a G + C content of 40%?

The factor IX gene has a G + C content of approximately 40% in all mammalian species examined. In human factor IX, C----T and G----A transitions at the dinucleotide CpG are elevated at least 24-fold relative to other transitions. Can the G + C content be explained solely by this hot spot of mutation? Using our mathematical model, we show that the elevation of mutation at CpG cannot alone lower the G + C content below 45%. To search for other hot spots of mutation that might contribute to the reduction of G + C content, we assessed the relative rates of base substitution in our sample of 160 families with hemophilia B. Seventeen independent single-base substitutions are reported herein for a total of 96 independent point mutations in our sample. The following conclusions emerge from the analysis of our data and, where appropriate, the data of others: (1) Transversions at CpG are elevated an estimated 7.7-fold relative to other transversions. (2) The mutation rates at non-CpG dinucleotides are remarkably uniform; none of the observed rates are either more than twofold above the median for transitions or more than threefold above the median for transversions. (3) The pattern of recent mutation is compatible with the pattern during mammalian evolution that has maintained the G + C content of the factor IX gene at approximately 40%.

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.

Characterization of a thrombin cleavage site mutation (Arg 1689 to Cys) in the factor VIII gene of two unrelated patients with cross-reacting material-positive hemophilia A.

The molecular defect responsible for moderate and severe hemophilia A has been identified for two unrelated patients with the CRM-positive form of this disorder (factor VIII activity of 0.02 and 0.05 U/mL with factor VIII antigen of 0.87 and 2.20 U/mL). In both cases, the immunopurified dysfunctional factor VIII protein is abnormal, in that the 80 Kd light chain is not cleaved by thrombin at arginine-1689. The basis for this failure was identified by polymerase chain reaction amplification of exon 14 of the variant factor VIII genes and direct sequencing of the amplified products. In both cases, a single base substitution (C to T) was identified that produces an arginine to cysteine substitution at amino acid residue 1689. These data identify the molecular defects of the two identical factor VIII variant proteins. The dysfunctional factor VIII has been designated "Factor VIII-East Hartford," the residence of the patient in whom the defect was first identified.