Experience with central venous access devices (CVADs) in the Canadian hemophilia primary prophylaxis study (CHPS).

INTRODUCTION: Haemophilia A treatment with factor VIII concentrates requires frequent venipunctures; a central venous access device (CVAD) may be required to facilitate reliable venous access, especially in young children. While CVADs provide reliable venous access, complications such as infection and thrombosis may occur. AIM: The aim of this study was to assess CVAD use in the Canadian Hemophilia Primary Prophylaxis Study (CHPS), a single-arm, multi-centre prospective study whereby factor use is tailored to individual prophylactic need. METHODS: Participants received a tailored, escalating dose, prophylaxis regimen of increasing frequency of FVIII infusions: step-1: 50 IU kg(-1) once weekly; step-2: 30 IU kg(-1) twice weekly; and step-3: 25 IU kg(-1) on alternate days, according to their level of bleeding. CVAD insertion was at the discretion of the local health care team. Details regarding CVAD use during this protocol were analysed. RESULTS: Fifty six boys were enrolled, 21 required 25 CVADs due to difficult venous access. CVADs were inserted at a median age of 1.3 years (range: 0.6-2.1) and were removed at a median age of 8.7 years (range 6.3-11.8). Six participants experienced non-life threatening CVAD-complications, the most frequent being device malfunction requiring CVAD replacement (n = 4). Two boys were shown to have CVAD-associated thrombosis detected on routine imaging; one required removal due to infusion difficulties and the other was asymptomatic and did not require device removal. No CVAD-related infections were documented. CONCLUSION: Our study shows that the CHPS tailored prophylaxis regimen is associated with a decreased requirement for CVADs and with few device-related complications.

Laboratory testing for platelet function disorders.

Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

von Willebrand disease and platelet disorders.

The diagnosis and management of bleeding disorders is made difficult by the complexity and variety of disorders, clinical symptoms and bleeding type and severity. von Willebrand disease (VWD) and platelet disorders are disorders of primary haemostasis and together represent the most common inherited bleeding disorders. In this article, we describe the diagnosis of VWD and platelet disorders and the treatment options for VWD.

Musculoskeletal health of subjects with hemophilia A treated with tailored prophylaxis: Canadian Hemophilia Primary Prophylaxis (CHPS) Study.

BACKGROUND: Full-dose prophylaxis is very effective at minimizing joint damage but is costly. Tailored prophylaxis has been proposed as a way of reducing costs while still protecting joints. OBJECTIVE: To report detailed findings in index joints of 56 subjects with severe hemophilia A entered into the Canadian Hemophilia Prophylaxis Study, and treated with tailored prophylaxis, after 13 years. METHODS: Boys with severe hemophilia A (< 2% factor) and normal joints were enrolled between the ages of 1 and 2.5 years. Initial treatment consisted of once-weekly factor infusions, with the frequency escalating in a stepwise fashion when breakthrough bleeding occurred. During the first 5 years, subjects were examined every 3 months using the modified Colorado Physical Evaluation (PE) scale; subsequently, every 6 months. The Childhood Health Assessment Questionnaire (CHAQ) was administered at each visit. RESULTS: Median age at study entry was 19 months (range 12-30 months); median follow-up was 92 months (range 2-156). The median PE score was 2, 3 and 3 at ages 3, 6 and 10 years. Persistent findings were related to swelling, muscle atrophy and loss of range of motion. The median score for each of these items (for the six index joints) was 0 at ages 3, 6 and 10 years. The median overall CHAQ score was 0 at ages 3, 6 and 10 years, indicating excellent function. CONCLUSIONS: Canadian boys treated with tailored primary prophylaxis exhibit minimal joint change on physical examination and minimal functional disability.

The development of bone mineral lateralization in the arms.

UNLABELLED: Bone mineral content (BMC) is known to be greater in the dominant arm after the age of 8 years. We studied a group of children and found that BMC sidedness gradually increased up to the age of 6 years and then remained stable into late adolescence. INTRODUCTION: Bone mineral content (BMC) exhibits sidedness in the arms after the age of 8 years, but it is not known whether BMC is greater in the dominant arm from birth or whether lateralization develops in early childhood. To address this, we examined bone mineral status in relation to handedness and age. METHODS: Subjects (N = 158) were children recently initiating glucocorticoids for underlying disease (leukemia 43 %, rheumatic conditions 39 %, nephrotic syndrome 18 %). Handedness was determined by questionnaire and BMC by dual-energy X-ray absorptiometry. RESULTS: Median age was 7.2 years (range, 1.5 to 17.0 years), 49 % was male, and the spine BMD Z-score was -0.9 (SD, 1.3). By linear regression, BMC sidedness in the arms was significantly related to age (r = 0.294, p = 0.0005). Breakpoint analysis revealed two lines with a knot at 6.0 years (95 % CI, 4.5-7.5 years). The formula for the first line was: dominant:nondominant arm BMC ratio = 0.029 × age [in years] + 0.850 (r = 0.323, p = 0.017). The slope of the second line was not different from 0 (p = 0.332), while the slopes for the two lines were significantly different (p = 0.027). CONCLUSIONS: These results show that arm BMC sidedness in this patient group develops up to age 6 years and then remains stable into late adolescence. This temporal profile is consistent with mechanical stimulation of the skeleton in response to asymmetrical muscle use as handedness becomes manifest.

Magnetic resonance imaging and joint outcomes in boys with severe hemophilia A treated with tailored primary prophylaxis in Canada.

BACKGROUND/OBJECTIVES: Tailored primary prophylaxis (TPP) is a reduced-intensity treatment program for hemophiliacs with the goal of preventing arthropathy. Our primary aim was to evaluate the joint outcomes of treated subjects using magnetic resonance imaging (MRI) and physical examination as outcome measures. METHODS: Ankles, elbows and knees (index joints) of 24 subjects (median [range] age at start of therapy, 1.6 [1-2.5] years) with severe hemophilia A enrolled in the Canadian Hemophilia Primary Prophylaxis Study (CHPS) were examined by MRI at a median age of 8.8 years (range 6.2-11.5 years). Subjects were treated with TPP using a recombinant factor VIII concentrate, starting once weekly and escalating in frequency and dose according to frequency of bleeding. RESULTS: Osteochondral changes (cartilage loss/subchondral bone damage) were detected in 9% (13/140) of the index joints and 50% (12/24) of study subjects. Osteochondral changes were restricted to joints with a history of clinically reported joint bleeding. Soft tissue changes were detected in 31% (20/65) of index joints with no history of clinically reported bleeding (ankles 75% (12/16); elbows 19% (6/32); and knees 12% (2/17)). In these apparently 'bleed free' index joints hemosiderin deposition was detected by MRI in 26% (17/65) of joints (ankles 63% (10/16); elbows 16% (5/32), and knees 12% (2/17)). CONCLUSION: TPP did not completely avoid the development of MRI-detected structural joint changes in hemophilic boys in this prospective study. A longer period of follow-up is required for assessment of the longitudinal course of these early changes in hemophilic arthropathy, detected using a sensitive imaging technique (MRI).

Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred.

The hereditary stomatocytoses are a group of heterogeneous conditions associated with chronic red cell hemolysis for which the causative genetic mutations are not known. We investigated 137 members of a large Canadian kindred with phenotypic findings consistent with hereditary xerocytosis, one of the most common stomatocytosis syndromes. The objectives of this study were to characterize the clinical hallmarks of the hemolytic process, and to define the chromosomal region carrying the disease locus. The mode of inheritance was autosomal dominant. Affected family members had a well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Quantitative erythrocyte morphologic evaluation revealed increased schistocytes, target cells, reticulocytes, and eccentrocytes in affected individuals; stomatocytes were not increased. Genetic linkage analysis confirmed the localization of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million base pair interval containing 51 known or predicted genes.

Inherited disorders of platelet function and challenges to diagnosis of mucocutaneous bleeding.

SUMMARY: Platelets play a pivotal role in the arrest of bleeding at sites of vascular injury. Following endothelial damage, they respond rapidly by adhesion to subendothelial matrix proteins resulting in platelet activation, spreading, aggregation, secretion and recruitment of additional platelets to form the primary haemostatic plug. This mass provides a surface for thrombin generation and fibrin mesh formation that stabilizes the clot. Careful study of patients with inherited platelet disorders and, subsequently, of informative animal models, has identified structural platelet abnormalities that have enhanced our understanding of platelet function. The investigations of rare, but severe, inherited platelet disorders have led us to the discovery of causative molecular defects. One of the most informative is the rare autosomal recessive disorder Glanzmann thrombasthenia, caused by defect or deficiency in the platelet integrin alphaIIbbeta3, resulting in absent platelet aggregation and a significant clinical bleeding diathesis. Our new challenge is to understand the mechanisms underlying more common, but less well-defined, mucocutaneous bleeding (MCB) disorders. Present diagnostic testing for platelet function disorders and von Willebrand's Disease often fails to identify the cause of bleeding in individuals with inherited MCB.

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells.

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.

Tailored prophylaxis in severe hemophilia A: interim results from the first 5 years of the Canadian Hemophilia Primary Prophylaxis Study.

BACKGROUND: Prophylactic treatment for severe hemophilia A is likely to be more effective than treatment when bleeding occurs, however, prophylaxis is costly. We studied an inception cohort of 25 boys using a tailored prophylaxis approach to see if clotting factor use could be reduced with acceptable outcomes. METHODS: Ten Canadian centers enrolled subjects in this 5-year study. Children were followed every 3 months at a comprehensive care hemophilia clinic. They were initially treated with once-weekly clotting factor; the frequency was escalated in a stepwise fashion if unacceptable bleeding occurred. Bleeding frequency, target joint development, physiotherapy and radiographic outcomes, as well as resource utilization, were determined prospectively. RESULTS: The median follow-up time was 4.1 years (total 96.9 person-years). The median time to escalate to twice-weekly therapy was 3.42 years (lower 95% confidence limit 2.05 years). Nine subjects developed target joints at a rate of 0.09 per person-year. There was an average of 1.2 joint bleeds per person-year. The cohort consumed on average 3656 IU kg(-1)year(-1) of factor (F) VIII. Ten subjects required central venous catheters (three while on study); no complications of these devices were seen. One subject developed a transient FVIII inhibitor. End-of-study joint examination scores--both clinically and radiographically--were normal or near-normal. CONCLUSIONS: Most boys with severe hemophilia A will probably have little bleeding and good joint function with tailored prophylaxis, while infusing less FVIII than usually required for traditional prophylaxis.

A survey of factor prophylaxis in the Canadian haemophilia A population.

High-dose factor prophylaxis, defined as the infusion of 25-40 factor (F) VIII International Units (IU) kg bodyweight (bw)(-1)> or = x 3 per week, started at age 1-2 years of life in boys with severe haemophilia A prevents the development of significant bleed-related arthropathy. However, programmes of prophylaxis are very expensive and venous access is a challenge. To ascertain patterns of prophylaxis in Canada during the period of a global shortage of recombinant FVIII concentrate a survey was conducted in 2001. The response rate was 83% and the survey identified 247 inhibitor-negative haemophilia A cases receiving prophylaxis, defined as the regular administration of FVIII at least once weekly, from 14 Canadian haemophilia treatment centres. The median age of the group identified was 13 years (range: 1-65) and 95% of cases had severe haemophilia A defined by a circulating factor level of <1%. The median FVIII infusion dose was 26 (range: 16-33) IU kg(-1); infusions were administered > or = x 3 per week in 67% of cases. High-dose factor prophylaxis was used most frequently in boys <5 years of age (23 of 28 cases, 82%) as compared with 56% (56 of 100), 66% (40 of 61) and 62% (36 of 58) of males ages 5-12, 13-18 and >18 years. Prophylaxis accounted for 50% of the annual Canadian FVIII consumption and was a major driving force in the 10% increase (=19.3 million FVIII IU) in the FVIII consumption in Canada in the 4-year period 1999-2003. Given the economic implications of increased use of prophylaxis prospective studies are warranted to better define optimal prophylaxis regimens in the haemophilia A population.

Home management of haemophilia.

The demonstrated benefits of home care for haemophilia include improved quality of life, less pain and disability, fewer hospitalizations, and less time lost from work or school. Although reduced mortality has not been demonstrated, the substantial increase in longevity since the early 1980s correlates with the introduction of home treatment and prophylaxis programmes. These programmes must be designed and monitored by haemophilia treatment centres (HTC), which are staffed with professionals with broad and complementary expertise in the disease and its complications. In return, patients and their families must be willing to accept the reciprocal responsibilities that come from administering blood products or their recombinant equivalents at home. Patients with inhibitors to factors VIII or IX pose special challenges, but these complications do not obviate participation in home care programmes. Home care was an essential prerequisite to the introduction of effective prophylactic factor replacement therapy. Prophylaxis offers significant improvements in quality of life, but requires a substantial commitment. The use of implantable venous access devices can eliminate some of the difficulty and discomfort of peripheral venous access in small children, but brings additional risks. The future holds the promise of factor concentrates for home use that have longer half-lives, or can be administered by alternate routes. Knowledge of patient genotypes may allow treatments tailored to avoid complications such as inhibitor development. Gene therapy trials, which are currently ongoing, will ultimately lead to gene-based treatments as a complement to traditional protein-based therapy.

Incorporation of map kinases into the platelet cytoskeleton.

Erk1 (p44) and erk2 (p42) mitogen-activated protein (MAP) kinases are activated in agonist-stimulated platelets, although their role(s) in the activation process is unknown. In the present study, erk1, erk2 and the phosphorylated forms of both enzymes became associated with the contractile cytoskeleton in thrombin-stimulated platelets. Enzyme incorporation was accompanied by an increase in MAP kinase activity in the cytoskeleton, which was inhibited by PD98059. Pretreatment of the platelets with the arginine-glycine-aspartic acid-serine (RGDS) polypeptide enhanced both the cytoskeletal association and the enzyme activity, but cytochalasin D had no significant effect. Platelets from a patient with Glanzmann's thrombasthenia lack the alpha(IIb)beta(3) integrin and form only a rudimentary cytoskeleton, however, this cytoskeleton is enriched with both erk1 and erk2. These data suggest either that MAP kinases play a role in cytoskeletal rearrangement or that the cytoskeleton act as a frame to align MAP kinases with substrates in a highly integrated signal transduction pathway.

CD63 associates with the alphaIIb beta3 integrin-CD9 complex on the surface of activated platelets.

The tetraspanins are integral membrane proteins expressed on cell surface and granular membranes of hematopoietic cells and have been identified in multi-molecular complexes with specific integrins. In resting platelets, CD63, a member of the tetraspanin superfamily, is present in dense granule and lysosomal membranes and, following platelet activation, translocates to the plasma membrane. In the present study, platelet activation by thrombin leads to incorporation of CD63 into the Triton-insoluble actin cytoskeletal fraction. This incorporation was inhibited by preincubation of platelets with RGDS or EGTA and did not occur in platelets from a patient with Glanzmann's thrombasthenia, suggesting that it was dependent upon alphaIIbbeta3. In activated platelets, the anti-CD63 MoAb, D545, co-immunoprecipitated CD63 with other surface-labeled proteins, including alphaIIbbeta3 and another tetraspanin, CD9. The association of CD63 with CD9 and alphaIIbbeta3, was not inhibited by preincubation of platelets with RGDS or EGTA. D545 did not inhibit the adhesion of activated platelets to purified extracellular matrix proteins, but significantly decreased adhesion of thrombin-activated platelets to neutrophils in a rosetting assay. D545 also caused disaggregation of platelets stimulated by ADP, but had no effect on aggregation induced by other agonists. These results are consistent with the proposal that CD63 becomes part of an alphaIIbbeta3-CD9-CD63 integrin-tetraspanin complex in activated platelets--an association that may modulate the function of alphaIIbbeta3-dependent interaction with other cells such as neutrophils.

Evaluation of primary hemostasis in neonates with a new in vitro platelet function analyzer.

With the use of the PFA-100 platelet function analyzer to evaluate primary hemostasis in whole blood, measured as closure time (CT), neonates had shorter CTs than members of an adult control group. Multivariate analysis of measures that contribute to primary hemostasis showed that higher hematocrits and increased ristocetin cofactor activity were the best correlates for CTs of cord blood. These 2 factors may also enhance primary hemostasis in vivo and compensate for the impaired platelet function of the newborn.

BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.

Outcome of pediatric thromboembolic disease: a report from the Canadian Childhood Thrombophilia Registry.

The outcome for children with deep vein thrombosis (DVT) and pulmonary embolism (PE) is unknown. An understanding of morbidity and mortality of DVT/PE is crucial to the development of rational treatment protocols. The Canadian Childhood Thrombophilia Registry has followed 405 children aged 1 mo to 18 y with DVT/PE for a mean of 2.86 y (range, 2 wk to 6 y) to assess outcome. The all-cause mortality was 65 of 405 children (16%). Mortality directly attributable to DVT/PE occurred in nine children (2.2%), all of whom had central venous line-associated thrombosis. Morbidity was substantial, with 33 children (8.1%) having recurrent thrombosis, and 50 children (12.4%) having postphlebitic syndrome. Recurrent thrombosis and postphlebitic syndrome were more common in older children, although deaths occurred equally in all age groups. The incidence of recurrent thrombosis and postphlebitic syndrome are likely underestimated because of difficulties in diagnosis, especially in younger children. The significant mortality and morbidity found in our study supports the need for international multicenter randomized clinical trials to determine optimal prophylactic and therapeutic treatment for children with DVT/PE.

The ribonucleotide reductase R2 gene is a non-transcribed target of c-Myc-induced genomic instability.

The c-Myc oncoprotein is highly expressed in malignant cells of many cell types, but the mechanism by which it contributes to the transformation process is not fully understood. Here, we show for the first time that constitutive or activated overexpression of the c-myc gene in cultured mouse B lymphocytes is followed by chromosomal and extrachromosomal amplification as well as rearrangement of the ribonucleotide reductase R2 gene locus. Electron micrographs and fluorescent in situ hybridization (FISH) demonstrate the c-Myc-dependent generation of extrachromosomal elements, some of which contain R2 sequences. However, unlike other genes that have been shown to be targets of c-Myc-dependent genomic instability, amplification of the R2 gene is not associated with alterations in R2 mRNA or protein expression. These data suggest that c-Myc-dependent genomic instability involves a greater number of genes than previously anticipated, but not all of the genes that are amplified in this system are transcriptionally upregulated.