Monitoring of von Willebrand factor inhibitors in patients with type 3 von Willebrand disease using a quantitative assay.

BACKGROUND: Antibodies inhibiting von Willebrand factor (VWF) develop in a subset of patients with type 3 von Willebrand disease (VWD3) and may be detected by their inhibition of ristocetin cofactor activity (VWF:RCo). Some also inhibit factor VIII activity (VIII:C). AIM: To describe monitoring of ten VWD3 patients for VWF inhibitors using a quantitative assay. METHODS: VWF inhibitor was measured by comparing VWF:RCo activity of a mix of patient and pooled normal plasma (PNP) with a mix of buffer and PNP, using agglutination of fixed normal platelets in microtiter plates or lyophilized platelets in an aggregometer. VIII:C inhibitor was measured by Bethesda assay. Preanalytical heat treatment of patient plasma was used during treatment episodes. RESULTS: Four of 10 patients monitored developed VWF inhibitors, two detected during bleeding episodes refractory to treatment and two on routine screening. Data from the first five patients were used to establish an arbitrary unit, VWU, defined as the amount of inhibitor per millilitre of patient plasma inactivating 25% of the activity of 1 mL of PNP. In three of four patients, both VWF:RCo and VIIII:C were inhibited at some time points, although VIII:C inhibition sometimes disappeared. In one patient, no VIII:C inhibition was seen. Two patients remained inhibitor positive more than 15 years after inhibitor detection, one became negative following immune tolerance induction, and one was deceased. CONCLUSIONS: VWF inhibitors can be quantitatively monitored in VWD3 patients. Preanalytical heat treatment may be required for their detection post infusion.

Genetic causes of haemophilia in women and girls.

Women and girls reported as "haemophilic females" may have complex genetic causes for their haemophilia phenotype. In addition, women and girls may have excessive bleeding requiring treatment simply because they are heterozygous for haemophilia alleles. While severe and moderate haemophilia are rare in females, 16% of patients with mild haemophilia A and almost one-quarter of those with mild haemophilia B seen in U.S. haemophilia treatment centres are women and girls. A phenotypic female with a low level of factor VIII or factor IX may be classified into one of the following categories of causality: homozygosity (two identical haemophilia alleles), compound heterozygosity (two different haemophilia alleles), hemizygosity (one haemophilia allele and no normal allele), heterozygosity (one haemophilia allele and one normal allele), genetic causes other than haemophilia and non-genetic causes. Studies required for classification may include coagulation parameters, F8 or F9 sequencing, F8 inversion testing, multiplex ligation-dependent probe amplification, karyotyping and X chromosome inactivation studies performed on the patient and parents. Women and girls who are homozygous, compound heterozygous or hemizygous clearly have haemophilia, as they do not have a normal allele. Heterozygous women and girls with factor levels below the haemostatic range also meet the definitions used for haemophilia treatment.

Evaluation of anti-factor VIII antibody levels in patients with haemophilia A receiving immune tolerance induction therapy or bypassing agents.

INTRODUCTION: Bleeding episodes in patients who have haemophilia A (HA), a hereditary bleeding disorder caused by a deficiency in factor VIII (FVIII), are treated or prophylactically prevented with infusions of exogenous FVIII. Neutralizing antibodies, referred to as inhibitors, against infusion products are a major complication experienced by up to 30% of patients who have severe HA. Bypassing agents (BPA), a class of therapeutics given to patients who have inhibitors, bypass the need for FVIII in the coagulation cascade, and long-term inhibitor eradication is accomplished using immune tolerance induction therapy (ITI). Data examining the antibody levels in patients receiving BPA and ITI are limited. AIM: Measure anti-FVIII antibody levels in specimens from patients receiving ITI or BPA in order to evaluate the anti-FVIII antibody response in those patients. METHODS: Specimens were tested using the CDC-modified Nijmegen-Bethesda assay (NBA) and the CDC fluorescence immunoassay (FLI) for anti-FVIII IgG1 and IgG4 . RESULTS: NBA-negative specimens from patients undergoing ITI or receiving BPAs have a higher frequency of anti-FVIII IgG4 positivity compared with the previously published level for NBA-negative HA patients. Analysis of anti-FVIII antibody levels in serial samples from patients undergoing ITI reveals that antibodies can persist even after the patient's NBA result falls into the negative range. CONCLUSIONS: Measurement of anti-FVIII antibodies may be a useful means to better contextualize NBA results in specimens from patients receiving BPA or ITI. In addition, assessment of anti-FVIII antibody levels has the potential to improve inhibitor surveillance and clinical decision-making related to the progress of ITI.

Women and girls with haemophilia receiving care at specialized haemophilia treatment centres in the United States.

INTRODUCTION: Females may have haemophilia with the same factor VIII (FVIII) or factor IX (FIX) levels as affected males. Characterization of females with haemophilia would be useful for health care planning to meet their unique needs. Federally-funded haemophilia treatment centres (HTCs) in the United States contribute data on all individuals with bleeding disorders receiving care to the Population Profile (HTC PP) component of the Community Counts Public Health Surveillance of Bleeding Disorders project. AIMS: To estimate the number of females with haemophilia receiving care at HTCs in the United States and compare their characteristics with those of males with haemophilia. METHODS: HTC PP data collected on people receiving care at an HTC from January 2012 through September 2020 with haemophilia A and B were evaluated by sex for demographic and clinical characteristics. RESULTS: A factor level < 40% was reported for 23,196 males (97.8%) and 1667 females (47.6%) attending HTCs; 51 (.48%) severe, 79 (1.4%) moderate, and 1537 (17.9%) mild haemophilia patients were female. Females were older, more often White, and less often non-Hispanic than males. Females were less likely to have history of HIV or HCV infection, even among those with severe disease, but twice as likely to have infection status unknown. Females with mild haemophilia were more often uninsured than males. CONCLUSIONS: Females with severe or moderate haemophilia are uncommon, even in specialized care centres; however, almost one in five patients with mild haemophilia was female, indicating needs for specialized care based on factor level and history for affected females.

Occurrence rates of von Willebrand disease among people receiving care in specialized treatment centres in the United States.

INTRODUCTION: In the network of U.S. comprehensive haemophilia treatment centres (HTCs), von Willebrand disease (VWD) is the most common bleeding disorder other than haemophilia. Estimates of the size and characteristics of the VWD population receiving treatment are useful for healthcare planning. AIM: Estimate the prevalence and incidence of VWD among males and females receiving care at U.S. HTCs (HTC-treated prevalence and incidence). METHODS: During the period 2012-2019, de-identified surveillance data were collected on all VWD patients who visited an HTC including year of birth, sex, race, Hispanic ethnicity, VWD type, and laboratory findings and used to calculate period HTC-treated prevalence by VWD type and sex. Data from patients born 1995-1999 were used to estimate HTC-treated incidence rates. RESULTS: During the period, 24,238 patients with a diagnosis of VWD attended HTCs; for 23,479 (96.9%), VWD type was reported or could be assigned. Age-adjusted HTC-treated prevalence was 8.6 cases/100,000 (7.2/100,000 for Type 1, 1.2/100,000 for Type 2 and 1.7/million for Type 3) and was twice as high in women as men (4.8 vs. 2.4 cases/100,000) for Type 1 and similar by sex for Type 2 and Type 3. HTC-treated Type 1 incidence increased over the period, averaging nearly threefold higher for women than men (26.2 vs. 9.9/100,000 live births). Sex differences were less for Type 2 (2.2 vs. 1.4 cases/100,000 births) and slight in Type 3. CONCLUSION: Prevalence and incidence of HTC-treated VWD differ by sex and type and are likely strongly influenced by differences in rates of diagnosis.

Occurrence rates of haemophilia among males in the United States based on surveillance conducted in specialized haemophilia treatment centres.

INTRODUCTION: Estimates of the size and characteristics of the US haemophilia population are needed for healthcare planning and resource needs assessment. A network of comprehensive haemophilia treatment centres (HTCs) located throughout the United States receives federal support for diagnosis and management of haemophilia and other rare bleeding disorders. AIM: Estimate the incidence and prevalence of haemophilia among US males using the HTC network. METHODS: During the period 2012-2018, de-identified surveillance data were collected on all males who visited an HTC that included year of birth, gender, race, Hispanic ethnicity, residence zip code, haemophilia type and severity. Data from all patients were used to calculate period prevalence by haemophilia type, severity and state of residence. Data from a subset of patients born 1995-2014 were used to estimate incidence rates over the 20-year period. RESULTS: During the period, 21 748 males with haemophilia visited the HTCs resulting in an age-adjusted prevalence of 15.7 cases per 100 000 males (12 for haemophilia A and 3.7 for haemophilia B). Prevalence was higher among whites (15.1) than blacks (12.4) or Hispanics of either race (12.4). State-specific prevalence varied from 1.6 to 23.3 cases per 100 000. Based on 9587 males born during the index period, the average haemophilia incidence was 1 case per 4334 live male births. CONCLUSION: Based on these data, we estimate that there are between 29 761 and 32 985 males with haemophilia living in the United States today, the majority of whom receive comprehensive care in specialized clinical centres.

Game, set, match for factor VIII mismatch?

In this issue of Blood, Gunasekera et al provide evidence that the high rate of factor VIII (FVIII) inhibitors seen in black hemophilia A (HA) patients is not due to a mismatch between the structure of treatment products and FVIII genotypes common in blacks.

Impact of inhibitors on hemophilia A mortality in the United States.

The previously published mortality studies are limited in hemophilia populations but suggest that there is no increased risk of mortality in factor VIII inhibitor patients. This retrospective study analyzed surveillance data collected on 7,386 males with severe hemophilia A over a 13-year period to assess the association between a current inhibitor and death. During the study period, 432 participants died, among whom 48 were patients with an inhibitor. Clinical characteristics most strongly associated with death were increased number of reported bleeds, signs of liver disease, infection with either HIV or HCV, and the presence of inhibitor. Patients who underwent successful tolerization were not considered inhibitor patients in our analysis. In a multivariable analysis, the odds of death were 70% higher among patients with a current inhibitor compared to those without an inhibitor (P < 0.01). Deaths among patients with inhibitors were much more likely to be attributed to bleeding complications than those among patients without an inhibitor (42 vs. 12%, P < 0.0001). We conclude that males with severe hemophilia A and a current inhibitor are at increased risk of death.

Characteristics of hemophilia patients with factor VIII inhibitors detected by prospective screening.

Characteristics of inhibitors identified by prospective screening may differ from those detected clinically. In a prospective study at 17 hemophilia centers with central inhibitor measurement by Nijmegen-Bethesda assay, 23 (2.8%) of 824 hemophilia A patients had new inhibitors detected: nine high-titer inhibitors (HTI: 7 ≥ 5.0 NBU plus 2 of 2.6 and 3.4 NBU at immune tolerance induction initiation) and 14 low-titer inhibitors (LTI: 0.5-1.9 NBU). HTI occurred at an earlier age (median 2 years, range 1-18, vs. median 11 years, range 2-61, P = 0.016). Both HTI (22%) and LTI (43%) occurred in non-severe patients. All HTI, but only 64% of LTI, were found to be FVIII-specific by chromogenic Bethesda assay or fluorescence immunoassay (FLI), indicating a high rate of false-positive LTI. Repeat specimens confirmed all HTI, 7/9 LTI, and 7/7 FVIII-specific LTI. FLI results were similar between HTI and FVIII-specific LTI; all included IgG1 and IgG4 subclasses. A comparable prospective study conducted from 1975 to 1979 at 13 U.S. centers found 31 (2.4%) new inhibitors among 1,306 patients. In both studies, one-third of inhibitors occurred in non-severe patients and one-quarter after 150 exposure days (ED). Significant differences were seen in the age at which inhibitors occurred (median 16 years in the older study vs. 5 years currently, P = 0.024) and in ED before inhibitor development, 10% in the older study and 43% currently study occurring within 20 ED, suggesting a temporal change in inhibitor development. Prospective screening detects inhibitors in patients of all severities, ages, and ED. Some LTI, however, are false positives.

Gender, race and diet affect platelet function tests in normal subjects, contributing to a high rate of abnormal results.

To assess sources of variability in platelet function tests in normal subjects, 64 healthy young adults were tested on 2-6 occasions at 2 week intervals using four methods: platelet aggregation (AGG) in platelet-rich plasma (PRP) in the Bio/Data PAP-4 Aggregometer (BD) and Chrono-Log Lumi-Aggregometer (CL); and AGG in whole blood (WB) in the CL and Multiplate Platelet Function Analyser (MP), with ATP release (REL) in CL-PRP and CL-WB. Food and medication exposures were recorded prospectively for 2 weeks prior to each blood draw. At least one AGG abnormality was seen in 21% of 81 drug-free specimens with CL-PRP, 15% with CL-WB, 13% with BD-PRP and 6% with MP-WB, increasing with inclusion of REL to 28% for CL-PRP and 30% for CL-WB. Epinephrine AGG and REL were significantly reduced in males (P < 0·0001). Ristocetin AGG and collagen and thrombin REL were significantly reduced in Blacks (P < 0·0001). One-third of specimens drawn following flavonoid-rich food exposures had aberrant results, compared to 8·5% of specimens without such exposures (P = 0·0035). PRP tests had less intra-individual variation than WB tests. Gender, race, diet and test system affected results of platelet function testing in healthy subjects, suggesting caution when interpreting the results of platelet function testing in patients.

F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review and meta-analysis.

This systematic review was designed to provide more precise effect estimates of inhibitor development for the various types of F8 gene mutations in patients with severe hemophilia A. The primary outcome was inhibitor development and the secondary outcome was high-titer-inhibitor development. A systematic literature search was performed to include cohort studies published in peer-reviewed journals with data on inhibitor incidences in the various F8 gene mutation types and a mutation detection rate of at least 80%. Pooled odds ratios (ORs) of inhibitor development for different types of F8 gene mutations were calculated with intron 22 inversion as the reference. Data were included from 30 studies on 5383 patients, including 1029 inhibitor patients. The inhibitor risk in large deletions and nonsense mutations was higher than in intron 22 inversions (pooled OR = 3.6, 95% confidence interval [95% CI], 2.3-5.7 and OR = 1.4, 95% CI, 1.1-1.8, respectively), the risk in intron 1 inversions and splice-site mutations was equal (pooled OR = 0.9; 95% CI, 0.6-1.5 and OR = 1.0; 95% CI, 0.6-1.5), and the risk in small deletions/insertions and missense mutations was lower (pooled OR = 0.5; 95% CI, 0.4-0.6 and OR = 0.3; 95% CI, 0.2-0.4, respectively). The relative risks for developing high titer inhibitors were similar.

Mutation analysis of a cohort of US patients with hemophilia B.

Hemophilia B (HB) is a disorder resulting from genetic mutations in the Factor 9 gene (F9). Genotyping of HB patients is important for genetic counseling and patient management. Here we report a study of mutations identified in a large sample of HB patients in the US. Patients were enrolled through an inhibitor surveillance study at 17 hemophilia treatment centers. A total of 87 unique mutations were identified from 225 of the 226 patients, including deletions, insertions, and point mutations. Point mutations were distributed throughout the F9 gene and were found in 86% of the patients. Of these mutations, 24 were recurrent in the population, and 3 of them (c.316G>A, c.1025C>T, and c.1328T>A) accounted for 84 patients (37.1%). Haplotype analysis revealed that the high recurrence arose from a founder effect. The severity of HB was found to correlate with the type of mutation. Inhibitors developed only in severe cases with large deletions and nonsense mutations. None of the mild or moderate patients developed inhibitors. Our results provide a resource describing F9 mutations in US HB patients and confirm previous findings that patients bearing large deletions and nonsense mutations are at high risk of developing inhibitors.

National surveillance for hemophilia inhibitors in the United States: Summary report of an expert meeting.

On March 12, 2012, the Centers for Disease Control and Prevention (CDC) held a meeting of its partners in hemophilia treatment, community-based organizations, industry, and government to review data and discuss implementation issues relevant to planned United States (U.S.) national inhibitor surveillance. Issues discussed included the current status of inhibitor surveillance in the United Kingdom (UK) and the US, the results of a US inhibitor surveillance feasibility study, proposed national surveillance schemes, laboratory testing and reporting issues and potential opportunities for future inhibitor-related research. It was concluded that implementation of a national program of inhibitor surveillance using standardized testing through an established public health registry along with patient and care provider education and targeted research provide the best opportunity to inform efforts to develop and evaluate effective prevention strategies.

Females with FVIII and FIX deficiency have reduced joint range of motion.

Little is known about rates of joint bleeding among females with FVIII/FIX deficiency or hemophilia carriers. In a cross-sectional study, we tested the hypothesis that females with FVIII or FIX deficiency enrolled in the Universal Data Collection (UDC) project had a reduced mean overall joint range of motion (ROM) compared with historic controls from the Normal Joint Study. Demographics, clinical characteristics, and joint ROM measurements on 303 females without a bleeding disorder and 148 females with FVIII and FIX deficiency, respectively, between the ages of 2-69 years and a body mass index (BMI) ≤ 35 were compared. Multivariate linear regression was performed with the overall joint ROM (sum of the right and left ROM measurements of five joints) as the dependent variable and FVIII or FIX activity as the independent variable adjusting for age, race, BMI, and number of joint bleeds reported over the last 6 months. As FVIII and FIX activity decreased, the mean overall joint ROM became reduced and in most cases was significantly lower than that of the controls regardless of age and clinical hemophilia severity. Further investigation of reduced joint ROM as evidence of subclinical joint bleeding in females with FVIII and FIX deficiency is warranted.

High factor VIII, von Willebrand factor, and fibrinogen levels and risk of venous thromboembolism in blacks and whites.

Venous thromboembolism (VTE) affects more than 300,000 people in the United States each year. However, it has been estimated that current diagnostic testing fails to identify prothrombotic risk in 50% of VTE patients. This article examines the relationship between levels of the pro-coagulant proteins factor VIII (FVIII), von Willebrand factor (VWF), and fibrinogen and risk of VTE in order to assess the impact of these novel risk factors. Data were collected from patients enrolled in the matched case-control Genetic Attributes and Thrombosis Epidemiology study. Crude and adjusted conditional logistic regression models were used to assess the impact of FVIII, VWF, and fibrinogen on risk of VTE. Before adjustment for independent predictors of VTE risk, high levels of FVIII, VWF, and fibrinogen were significantly associated with increased risk of VTE in both Blacks and Whites. After adjustment for ABO type, factor VII levels, hypertension, renal disease, recent surgery, diabetes, annual household income, alcohol use, and the other proteins of interest (FVIII, VWF, and/or fibrinogen), high FVIII and VWF levels were associated with increased risk of VTE in Blacks (OR: 1.97 [1.01-3.84] and 3.39 [1.58-7.27], respectively). High FVIII only was significantly associated with risk of VTE in Whites (OR: 2.35 [1.16-4.75]). Future research into the inclusion of these protein levels in risk models for VTE could help identify persons at highest risk.

The CDC Hemophilia A Mutation Project (CHAMP) mutation list: a new online resource.

Genotyping efforts in hemophilia A (HA) populations in many countries have identified large numbers of unique mutations in the Factor VIII gene (F8). To assist HA researchers conducting genotyping analyses, we have developed a listing of F8 mutations including those listed in existing locus-specific databases as well as those identified in patient populations and reported in the literature. Each mutation was reviewed and uniquely identified using Human Genome Variation Society (HGVS) nomenclature standards for coding DNA and predicted protein changes as well as traditional nomenclature based on the mature, processed protein. Listings also include the associated hemophilia severity classified by International Society of Thrombosis and Haemostasis (ISTH) criteria, associations of the mutations with inhibitors, and reference information. The mutation list currently contains 2,537 unique mutations known to cause HA. HA severity caused by the mutation is available for 2,022 mutations (80%) and information on inhibitors is available for 1,816 mutations (72%). The CDC Hemophilia A Mutation Project (CHAMP) Mutation List is available at http://www.cdc.gov/hemophiliamutations for download and search and will be updated quarterly based on periodic literature reviews and submitted reports.

Bleeding Disorders in Women and Girls: State of the Science and CDC Collaborative Programs.

Women and girls with bleeding disorders experience abnormal and excessive bleeding that can negatively impact their overall health and quality of life. In this report, we provide an overview of the biology, types, clinical care, and state of the science related to bleeding disorders in girls and women and describe Centers for Disease Control and Prevention (CDC) activities related to (1) surveillance of bleeding disorders in women; (2) scientific review, research, and collaboration to inform health care gaps in identifying and caring for women with bleeding disorders; and (3) development of health promotion and education programs to bring awareness about bleeding disorders to both women and girls in the population at large and various health care providers who care for women. Findings generated from surveillance and research activities inform the development of new public health programs aimed at improving diagnostic and health care services and empowering women with bleeding disorders with the knowledge they need to navigate a complex health care system with the need for specialty care services. Additional work is needed to improve provider awareness and understanding of the unique needs of women and girls with bleeding disorders to achieve appropriate care and treatment and ensure optimal outcomes and quality of life.