Variation in baseline factor VIII concentration in a retrospective cohort of mild/moderate hemophilia A patients carrying identical F8 mutations.

Essentials Factor VIII levels vary in mild and moderate hemophilia A (MHA) patients with the same mutation. We aimed to estimate the variation and determinants of factor VIII levels among MHA patients. Age and genotype explain 59% of the observed inter-individual variation in factor VIII levels. Intra-individual variation accounted for 45% of the variation in the three largest mutation groups. SUMMARY: Background The bleeding phenotype in patients with mild/moderate hemophilia A (MHA) is inversely associated with the residual plasma concentration of factor VIII (FVIII:C). Within a group of patients with the same F8 missense mutation, baseline FVIII:C may vary, because, in healthy individuals, von Willebrand factor (VWF) levels, ABO blood group and age are also known to influence baseline FVIII:C. Our understanding of the pathophysiologic process of the causative genetic event leading to reduced baseline FVIII:C in MHA patients is still limited. Objectives To estimate the variation and determinants of baseline FVIII:C among MHA patients with the same F8 missense mutation. Methods Three hundred and forty-six patients carrying mutations that were present in at least 10 patients in the cohort were selected from the INSIGHT and the RISE studies, which are cohort studies including data of 3534 MHA patients from Europe, Canada, and Australia. Baseline FVIII:C was measured with a one-stage clotting assay. We used Levene's test, univariate and multivariate linear regression, and mixed-model analyses. Results For 59% of patients, the observed variation in baseline FVIII:C was explained by age and genotype. Compared to FVIII:C in patients with Arg612Cys, FVIII:C was significantly different in patients with eight other F8 missense mutations. Intra-individual variation explained 45% of the observed variance in baseline FVIII:C among patients with the same mutation. Conclusion Our results indicate that baseline FVIII:C levels are not exclusively determined by F8 genotype in MHA patients. Insights into other factors may provide potential novel targets for the treatment of MHA.

Clinical phenotype in genetically confirmed von Willebrand disease type 2N patients reflects a haemophilia A phenotype.

INTRODUCTION: Von Willebrand disease (VWD) type 2N is characterized by a defective binding of factor VIII (FVIII) to von Willebrand factor (VWF) resulting in diminished plasma FVIII levels and a clinical phenotype mimicking mild haemophilia A. Several mutations in the FVIII binding site of VWF have been reported. AIM: This study aims to examine the effect of genotype on clinical phenotype in a cohort of VWD 2N patients. METHODS: Patients with at least one genetically confirmed 2N mutation were selected retrospectively from a cohort of patients with suspected VWD. Clinical and laboratory phenotypes including bleeding scores (BS) were obtained and analysed. RESULTS: Forty-two VWD 2N patients with a mean age of 44 years were included. Eleven patients were homozygous or compound heterozygous (genetically confirmed group) and 31 patients were heterozygously affected (carriers group). Statistically significant differences between genetically confirmed VWD 2N patients and carriers were found in FVIII activity, VWF antigen levels, VWF-FVIII binding capacity, FVIII/VWF antigen ratio (all P<0.001), VWF-ristocetin activity (p=0.001) and VWF collagen binding (P = 0.002). Median BS was 6 in genetically confirmed VWD 2N patients compared with 3 in carriers (P = 0.047). Haemarthrosis, muscle haematomas and postpartum haemorrhage were only reported in genetically confirmed 2N patients. CONCLUSION: Phenotypic analysis showed that all laboratory parameters are lower in genetically confirmed VWD 2N patients compared with heterozygous 2N carriers. The clinical phenotype in genetically confirmed VWD 2N patients is comparable to mild haemophilia A patients and more severe than heterozygous 2N carriers.

X-linked sideroblastic anaemia due to ALAS2 mutations in the Netherlands: a disease in disguise.

BACKGROUND: X-linked sideroblastic anaemia (XLSA; OMIM#300751) is the most common inherited form of sideroblastic anaemia and is associated with several mutations in the erythroid specific 5-aminolevulinate synthase gene (ALAS2). This gene encodes for aminolevulinic acid synthase 2 (ALAS2), the catalytic enzyme involved in the first en rate-limiting step of haem biosynthesis.1-3 The disorder is characterised by mostly mild hypochromic microcytic anaemia with bone marrow ring sideroblasts. Even untransfused patients with mild or no anaemia are at risk for severe systemic iron overload due to ineffective erythropoiesis. To date, 61 different ALAS2 mutations have been reported in 120 families with XLSA. Descriptions of molecularly confirmed case series from the Netherlands, however, are lacking. METHODS: We reviewed age of presentation, clinical and biochemical features, ALAS₋2 defects and treatment characteristics of 15 Dutch patients from 11 unrelated families diagnosed with XLSA. RESULTS AND CONCLUSIONS: In one family a novel pathogenic c.1412G>A (p.Cys471Tyr) mutation was found. All other families shared the previously described c.1355G>A (p.Arg452His) mutation. Haplotype analysis in seven probands with the p.Arg452His mutation strongly suggests that six of them were ancestrally related. Nevertheless, their phenotype was very different. Our patients illustrate the phenotypical heterogeneity in the presentation of XLSA patients, the effectiveness of treatment regimens and the various pitfalls associated with the diagnosis, follow-up and treatment of the disease. A timely diagnosis avoids unnecessary investigations and allows adequate treatment that can prevent systemic iron load with subsequent severe life-threatening complications. Therefore, we suggest considering XLSA in both male and female patients with unexplained iron overload and÷or (mild) microcytic anaemia, also at older age.

Bone cysts in patients with afibrinogenaemia: a literature review and two new cases.

Afibrinogenaemia is an autosomal recessive disease with an estimated prevalence of approximately one in a million. The most common symptoms of afibrinogenaemia are umbilical cord bleeding, bleeding into skin, mouth, muscles, gastrointestinal and genitourinary tracts and the central nervous system. Other recognized complications include; haemarthroses, spontaneous splenic rupture, epistaxis, menorrhagia, recurrent abortion and venous and arterial thromboembolism. Bone cysts have also been described as a rare complication of afibrinogenaemia. The aim of this study was to conduct a systematic literature review, summarize the reported cases and to report two new cases. Three electronic databases were searched for relevant publications: PubMed, Medline and EMBASE. The following search criteria were used: '(bone cysts OR intraosseous haematoma OR intraosseous haemorrhage) AND (afibrinogenaemia OR fibrinogen deficiency)'. The reference lists of the selected papers were searched for more relevant literature. In total, eight patients had bone cysts as complication of afibrinogenaemia and six of them suffered from pain in their extremities. Bone cysts were primarily located in the vicinity of the cortex or trabeculae in the diaphysis of the long bones, especially in the femora, tibiae and humeri. Some were regressive, probably due to reactive bone remodelling. A number of cysts were filled with serosanguinous fluid. It might be useful to check for bone cysts when patients with congenital afibrinogenaemia complain of 'rheumatic' pains in their extremities. Whole body magnetic resonance imaging is the diagnostic imaging technique of choice. Recurrent episodes of pain, but not radiological deterioration, appear to benefit from prophylactic therapy with fibrinogen concentrate.

Impact of von Willebrand disease on health-related quality of life in a pediatric population.

BACKGROUND: Von Willebrand disease (VWD) is the most frequent inherited bleeding disorder. Whether VWD is associated with health-related quality of life (HR-QoL) in children is unknown. OBJECTIVES: This nationwide cross-sectional study measured HR-QoL in children with moderate or severe VWD. Our primary aim was to compare HR-QoL of VWD patients with that of reference populations. Additionally, we studied the impact of bleeding phenotype and VWD type on HR-QoL. METHODS: HR-QoL was assessed with the Infant/Toddler QoL Questionnaire (0-5 years) and Child Health Questionnaire (6-15 years), and compared with reference population scores. Multivariate analysis was used to evaluate the influence of type of VWD and bleeding phenotype on HR-QoL scores. RESULTS: Preschool children (0-5 years, n = 46) with VWD had lower HR-QoL scores for general health perceptions and parental time than reference populations. School children (6-15 years, n = 87) with VWD had lower scores for physical functioning, role functioning - emotional/behavioral, general health perceptions, and physical summary. Type of VWD was associated with HR-QoL in school children for bodily pain, general health perceptions, parental emotion, family activities, and physical summary. Scores of children with type 3 VWD were, on average, 15 points lower than those of the reference population on the above-mentioned scales. A more severe bleeding phenotype was associated with a lower score on 11/15 physical, emotional and social scales. CONCLUSION: HR-QoL is lower in VWD children than in reference populations, in particular in school children. The negative impact of VWD is sensitive to type of VWD and bleeding phenotype; as well as physical scales, emotional and social scales are affected.

School-aged children after the end of successful treatment of non-central nervous system cancer: longitudinal assessment of health-related quality of life, anxiety and coping.

The aim of the study was to investigate: (1) health-related quality of life (HRQoL) and anxiety in school-aged cancer survivors during the first 4 years of continuous remission after the end of treatment; and (2) correlations of disease-related coping with HRQoL and anxiety. A total of 76 survivors aged 8-15 years completed questionnaires about HRQoL, anxiety and disease-related cognitive coping at one to five measurement occasions. Their HRQoL was compared with norm data, 2 months (n = 49) and 1 year (n = 41), 2 years (n = 41), 3 years (n = 42) and 4 years (n = 27) after treatment. Through longitudinal mixed models analyses it was investigated to what extent disease-related cognitive coping was associated with HRQoL and anxiety over time, independent of the impact of demographic and medical variables. Survivors reported worse Motor Functioning (HRQoL) 2 months after the end of treatment, but from 1 year after treatment they did no longer differ from the norm population. Lower levels of anxiety were associated with male gender, being more optimistic about the further course of the disease (predictive control) and less searching for information about the disease (interpretative control). Stronger reliance on the physician (vicarious control) was associated with better mental HRQoL. As a group, survivors regained good HRQoL from 1 year after treatment. Monitoring and screening survivors are necessary to be able to trace the survivors at risk of worse HRQoL.

Participation and risk-taking behaviour in sports in children with haemophilia.

The aim of this study was to investigate participation in sports activities and risk-taking behaviour in children with haemophilia and the relationship to personal and health related factors. Ninety-nine children (mean age 12.6 years) completed questionnaires regarding participation in sports and physical education, medication, health related quality of life, and perceived motor competence. Furthermore, weight, height, active range of motion, pain, and muscle strength were assessed. Based on a risk exposure factor (REF) we defined subgroups with low, medium, and high risk when participating in sport. Most children participate in sport five times a week (mean 140 min per week), and little absence during school sports was reported. In general, prophylaxis was not tailored to sport activities. Boys with haemophilia preferred other sports than their Dutch contemporaries. The top-5 being soccer, swimming, tennis, gymnastics, and cardio-fitness for the former; soccer, gymnastics, tennis, hockey, and swimming for the latter. Significant differences between the low risk group and both other groups were found for sport intensity, total energy expenditure (EE) and average risk factor (ARF), however the medium and high-risk groups did not differ in ARF. REF and sport participation increased associated with increasing interest in athletic and motor activities. No significant differences were found between REF groups regarding age, Z-BMI, Z-AROM, Z-Muscle force, and the presence of painful joints. As in normal peers motivation to participate in sport depends upon the enthusiasm and interest, in children with haemophilia choice of sports differs, probably related to sport advice.

[Rituximab instead of splenectomy in 4 children with chronic or refractory autoimmune haemolytic anaemia]. / Rituximab in plaats van splenectomie bij 4 kinderen met chronische of refractaire auto-immune hemolytische anemie.

4 children, a boy aged 10 years and 3 girls aged 3, 3, and 16 years, suffering from chronic or refractory autoimmune haemolytic anaemia (AIHA), who were dependent on high doses of steroids and were refractory to immunosuppressants, were treated with rituximab at a dose of 375 mg/m2 once a week for 3 or 4 weeks as an alternative to splenectomy. Rituximab is a monoclonal anti-CD20 antibody that prevents the production ofautoantibodies by selective destruction of B-lymphocytes. Haemoglobin levels increased and the parameters of chronic haemolysis (reticulocyte count, lactate dehydrogenase activity, bilirubin concentration) decreased to normal values. 3 patients were taken off corticosteroids completely; 1 of these was also no longer dependent on blood transfusions. Circulating B-lymphocytes were absent for 6 to 15 months after the treatment and the rituximab was well-tolerated. During the treatment, immunoglobulins were substituted and infectious complications were not seen. Rituximab was valuable in the treatment of chronic or refractory AIHA and eliminated the need for splenectomy. 1 patient did not respond to rituximab.

Cell cycle kinetics in normal human skin by in vivo administration of iododeoxyuridine and application of a differentiation marker--implications for cell cycle kinetics in psoriatic skin.

Renewal of epidermal cells is a highly coordinated process in which terminal differentiation balances the proliferative rate in the germinative compartment. Exact quantitative data on cell cycle parameters of normal human epidermis are fragmentary, and do not allow firm conclusions on issues such as cell cycle time, duration of the various cell cycle phases, and the pool sizes of the different cellular populations. As part of a study on bone marrow cell cycle kinetics, 14 lymphoma patients were infused with the thymidine analogue iododeoxyuridine (IdUrd). This provided us with the unique opportunity to study the cell cycle kinetics in normal epidermis obtained from these patients. Single epidermal cell suspensions were prepared from skin, stained with propidium iodide (PI) for relative DNA content, and simultaneously labeled with an anti-IdUrd antibody to detect DNA-synthesizing cells. In parallel samples suprabasal cells were analyzed by labeling with an anti-cytokeratin 10 antibody. Analysis was performed using bivariate flow cytometry. The results showed that 3.5% of the total epidermal cell population was in S-phase. An S-phase duration of 9.7 +/- 0.6 h and a cytokeratin 10-positive pool size of 59.6 +/- 4.6% were obtained. The duration of the G1-phase and the G2M-phase were calculated to be 7.6 +/- 2.0 h and 11.1 +/- 2.0 h, respectively. From these data a total cell cycle time can be calculated of 28.4 h. Combining this data with previous findings we were able to determine a similar cell cycle time of 27.8 h, and pool sizes of the epidermal cells: 30% quiescent (resting, G0) and 10% cycling cells. The implications of these findings for the interpretation of deviations in growth control as found in hyperproliferative skin diseases (e.g. psoriasis) are discussed.

Cell cycle kinetics of hematopoiesis before and after in vivo administration of GM-CSF in refractory anemia: evidence for a shortening of the granulocyte release time.

GM-CSF administration to patients with refractory anemia (RA) induces an increase in neutrophils and eosinophils. We studied cell kinetic mechanisms underlying this observation using clonogenic assays and in vivo iododeoxyuridine labeling of bone marrow cells. Cell cycle kinetics were studied in three patients before and during GM-CSF administration (two daily subcutaneous injections of 54 or 108 micrograms). No consistent effect on the relative number of bone marrow CFU-GM was noticed. The DNA synthesis time and potential doubling time of low-density bone marrow cells remained essentially the same. A slight decrease (1.5-3.7%) in labeling index was found, originating from the myelo(-mono)cytic lineage. In all three patients the release time of labeled granulocytes from the bone marrow into the peripheral blood was shortened (before GM-CSF treatment 5-7 days and during GM-CSF 3-4 days). Cell cycle kinetics of CD34+ cells were studied in order to obtain kinetic information on immature precursor and progenitor cells. The DNA synthesis time of the CD34+ cells was shortened during GM-CSF therapy, resulting in a shorter potential doubling time. GM-CSF administration to patients with RA results in a rise in granulocytes that might be due partly to an accelerated release of granulocytes from the bone marrow compartment into the circulating blood and partly to an increased proliferative activity of the immature precursor and progenitor cells.

Proliferation patterns in acute myeloid leukemia: leukemic clonogenic growth and in vivo cell cycle kinetics.

In a prospective study of 33 newly diagnosed patients with acute myeloid leukemia (AML), we analyzed the relationship of proliferation parameters with clinical parameters, response to induction therapy, and survival. The median follow-up was 26 months. The proliferative capacity of the leukemic progenitor cells was studied using colony-forming assays (number of colony-forming units, growth pattern, and spontaneous clonogenic growth capacity). The cell kinetic parameters of the bone marrow blasts were determined by in vivo labeling with iododeoxyuridine and subsequent flow cytometry: labeling index (LI), DNA synthesis time (Ts), potential doubling time. No or only weak relationships were observed between the experimental and clinical parameters such as age, sex, % blasts, white blood cell count, FAB subtype, cytogenetics, and % CD 34+ cells. This suggests that clonogenic growth and cell cycle kinetics of bone marrow blasts are independent cell biologic properties of AML. No association between the proliferation parameters and induction response rate was noticed. Analysis of the overall survival and event-free survival revealed trends to longer survival rates in patients with a below-median LI (< or = 7.6%) and below-median Ts value (< or = 14.3 h). These trends were more pronounced in the group of de novo AML (n = 23), where the prolonged event-free survival in patients with below-median Ts reached statistical significance (p = 0.02). None of the other parameters appeared significantly correlated with survival, although there was a trend to longer survival rates in patients who had no spontaneous clonogenic growth capacity (p = 0.13). In conclusion, proliferation parameters in leukemic cells provide additional information on the cell biologic characteristics of AML, and these parameters may have prognostic value for response and duration of survival in AML.

Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry.

Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S-phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.

Effect of doxorubicin exposure on cell-cycle kinetics of human leukemia cells studied by bivariate flow cytometric measurement of 5-iodo-2-deoxyuridine incorporation and DNA content.

Cell kinetics of two human leukemic cell lines, Molt-4 and K562, following a 2-h exposure to doxorubicin, were studied. DNA flow cytometry provided static information that for both cell lines a dose-dependent accumulation occurred at the G2 + M compartment that disappeared in time. Kinetic information was provided by time-monitoring cells labeled with 5-iodo-2-deoxyuridine (IdUrd) by two-parameter flow cytometry, analyzing the IdUrd label and the DNA content. The cell-cycle time (Tc) of exponentially growing Molt-4 cells was determined to be 20 h. Twenty-four hours after a 2-h exposure to 0.25 micrograms/ml doxorubicin, the Tc had increased to 23 h; following exposure to 1.0 micrograms/ml, it increased to 33 h. Cell kinetics of K562 cells following doxorubicin exposure were monitored in time up to 4 days. The average Tc of exponentially growing K562 cells was determined to be 24.7 h. Twenty-four hours following 2-h exposure to 0.25 or 0.5 micrograms/ml doxorubicin, the Tc were determined to be 28 and 32 h, respectively. After an additional 2 days, the Tc were both determined to be 24 h. The dose-dependent, reversible cell-cycle delay that persisted at least 48 h should be taken into account as an additional mode for decrease of a (tumor) cell population doubling time after exposure to doxorubicin.

Simultaneous measurement of DNA content and cell-surface immunofluorescence of human bone marrow cells using a single laser flow cytometer.

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell-surface immunofluorescence (s-IF). Low density (less than 1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T-lymphoid (CD2), B-lymphoid (CD19), erythroid (anti-glycophorin-A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse label was used as second step. Unfixed, MoAb-labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single-laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s-IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s-IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S-phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%).(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation.

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.