Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

The Centers for Disease Control and Prevention (CDC) Nijmegen‐Bethesda Assay (NBA)1 is a modification of traditional methods2., 3. for measurement of factor VIII (FVIII) and factor IX (FIX) inhibitors that includes a 30‐minute preanalytical heat treatment (PHT) step to remove endogenous and infused FVIII or FIX. Specimens for inhibitor tests using PHT thus do not require the stringent conditions needed to maintain clotting factors during shipping and storage, as we have previously documented by split‐sample analysis showing that results of the CDC‐modified NBA on specimens shipped cold correlated well with those of frozen specimens.1

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.

The United States Hemophilia Treatment Centers Network (USHTCN) is made up of 145 haemophilia treatment centres (HTCs) located throughout the United States with funding from the Health Resources and Service Administration and the Centers for Disease Control and Prevention (CDC) to facilitate monitoring of health care and outcomes among people with bleeding disorders. Inherited bleeding disorders most often treated at US HTCs are hemophilia1, 2 and von Willebrand disease (VWD)3; however, these centres also provide care for people with a variety of other disorders, including deficiencies of coagulation factors other than factor VIII and IX, platelet disorders, fibrinolytic defects, and connective tissue disorders with an associated bleeding phenotype. Community Counts,4 a combined effort of the USHTCN, the American Thrombosis and Hemostasis Network (ATHN), and the CDC to gather and share information about common health issues, medical complications, and causes of death of people with bleeding disorders, includes a component known as the HTC Population Profile (HTC PP), for which limited data on all persons receiving care at HTCs have been collected since 2012. We have recently reported numbers and characteristics of males and females with hemophilia1, 2 and VWD3 treated at HTCs using HTC PP data. The aim of this study was to use data from the HTC PP to estimate the number of people with rare bleeding disorders other than haemophilia and VWD receiving care at HTCs in the United States.

Hemophilia B (HB) is a bleeding disorder caused by deficiency of or defect in blood coagulation factor IX (FIX) inherited in an X-linked manner. It results from one of over 1000 known pathogenic variants in the FIX gene, F9; missense and frameshift changes predominate. Although primarily males are affected with HB, heterozygous females may have excessive bleeding due to random or non-random X chromosome inactivation; in addition, homozygous, compound heterozygous, and hemizygous females have been reported. Somatic and germinal mosaicism for F9 variants has been observed. Development of antibodies to FIX treatment products (inhibitors) is rare and related to the type of causative variant present. Treatment is with products produced by recombinant DNA technology, and gene therapy is in clinical trials. Genetic counseling with up-to-date information is warranted for heterozygotes, potential heterozygotes, and men and women affected with HB.

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.

We previously described in this journal a modified Nijmegen-Bethesda assay (NBA) for factor VIII (FVIII) inhibitors in hemophilia A (HA) that uses preanalytical heat inactivation of infused or endogenous FVIII to allow inhibitor measurement postinfusion1 and compared that assay with a chromogenic Bethesda assay (CBA) that is identical except for use of an FVIII chromogenic substrate assay (CSA) rather than a one-stage assay (OSA) as the endpoint for inhibitor detection.2 Our primary focus was on use of the CBA as a confirmatory test for low positive NBA results. Introduction of the non-FVIII treatment product emicizumab (Hemlibra) has brought increased interest in inhibitor assays using CSA because emicizumab interferes with the OSA and thus with Bethesda assays for FVIII inhibitors using the OSA.3-5 CSA for FVIII that use bovine factor X (FX) are insensitive to emicizumab,5 and a CBA using such CSA has been successfully used for inhibitor testing in its presence.6, 7 Clinical laboratories providing inhibitor testing have the option of maintaining two inhibitor assays and choosing the correct one for each patient depending on the product used or switching to a CBA to accommodate testing on all patients. Clinical adoption of a new assay methodology requires demonstration that the new method is equivalent to the old. Recent reexamination of the dataset of paired NBA and CBA results from our original paper2 revealed differences that may influence such comparisons and that, if not considered, could hinder validation of the CBA for clinical use.

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.

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.

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.

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 IgG(1) and IgG(4) . RESULTS: NBA-negative specimens from patients undergoing ITI or receiving BPAs have a higher frequency of anti-FVIII IgG(4) 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.

Development of antibodies interfering with the function of factor VIII (FVIII) replacement products is one of the most significant complications in the treatment of hemophilia A (HA). Laboratory testing for such antibodies, called inhibitors, is an important part of hemophilia care and is conducted both to identify the cause of treatment failure and as routine screening to detect early antibody appearance. Treatment of HA patients who develop inhibitors is often carried out by giving repeated doses of FVIII to induce immune tolerance and allow use of FVIII or by use of by-passing agents that act by facilitating coagulation without the need for FVIII. The newest by-passing product, emicizumab (Hemlibra®), is a bispecific antibody that mimics the function of FVIII by bringing factor IXa and factor X (FX) together to produce the Xa complex.1,2 Emicizumab, which is long acting and given subcutaneously, is now widely available for use in patients both with and without inhibitors to avoid frequent use of intravenous FVIII replacement.

PROBLEM/CONDITION: Hemophilia is an X-linked genetic disorder that primarily affects males and results in deficiencies in blood-clotting proteins. Hemophilia A is a deficiency in factor VIII, and hemophilia B is a deficiency in factor IX. Approximately one in 5,000 males are born with hemophilia, and hemophilia A is about four times as common as hemophilia B. Both disorders are characterized by spontaneous internal bleeding and excessive bleeding after injuries or surgery. Hemophilia can lead to repeated bleeding into the joints and associated chronic joint disease, neurologic damage, damage to other organ systems, and death. Although no precise national U.S. prevalence estimates for hemophilia exist because of the difficulty identifying cases among persons who receive care from various types of health care providers, two previous state-based studies estimated hemophilia prevalence at 13.4 and 19.4 per 100,000 males. In addition, these studies showed that 67% and 82% of persons with hemophilia received care in a federally funded hemophilia treatment center (HTC), and 86% and 94% of those with the most severe cases of hemophilia (i.e., those with the lowest levels of clotting factor activity in the circulating blood) received care in a federally funded HTC. As of January 2020, the United States had 144 HTCs. PERIOD COVERED: 1998-2019. DESCRIPTION OF THE SYSTEM: Surveillance for hemophilia, which is a complex, chronic condition, is challenging because of its low prevalence, the difficulty in ascertaining cases uniformly, and the challenges in routinely characterizing and tracking associated health complications. Over time, two systems involving many stakeholders have been used to conduct ongoing hemophilia surveillance. During 1998-2011, CDC and the HTCs collaborated to establish the Universal Data Collection (UDC) surveillance system. The purposes of the UDC surveillance system were to monitor human immunodeficiency virus (HIV) and bloodborne viral hepatitis in persons with hemophilia, thereby tracking blood safety, and to track the prevalence of and trends in complications associated with hemophilia. HTC staff collected clinical data and blood specimens from UDC participants and submitted them to CDC. CDC tested specimens for viral hepatitis and HIV. In 2011, the UDC surveillance system was replaced by a new hemophilia surveillance system called Community Counts. CDC and the HTCs established Community Counts to expand laboratory testing and the collection of clinical data to better identify and track emerging health issues in persons with hemophilia. RESULTS: This report is the first comprehensive summary of CDC's hemophilia surveillance program, which comprises both UDC and Community Counts. Data generated from these surveillance systems have been used in the development of public health and clinical guidelines and practices to improve the safety of U.S. blood products and either prevent hemophilia-related complications or identify complications early. Several factors have played a role in the effectiveness of the UDC and Community Counts systems, including 1) a stable data collection design that was developed and is continually reviewed in close partnership with HTC regional leaders and providers to ensure surveillance activities are focused on maximizing the scientific and clinical impact; 2) flexibility to respond to emerging health priorities through periodic updates to data collection elements and special studies; 3) high data quality for many clinical indicators and state-of-the-art laboratory testing methods for hemophilia treatment product inhibitors (developed and refined in part based on CDC research); 4) timely data and specimen collection and submission, laboratory specimen testing, analysis, and reporting; and 5) the largest and most representative sample of persons with hemophilia in the United States and one of the largest and most comprehensive data collection systems on hemophilia worldwide. INTERPRETATION: CDC has successfully developed, implemented, and maintained a surveillance system for hemophilia. The program can serve as an example of how to conduct surveillance for a complex chronic disease by involving stakeholders, improving and building new infrastructure, expanding data collection (e.g., new diagnostic assays), providing testing guidance, establishing a registry with specimen collection, and integrating laboratory findings in clinical practice for the individual patient. PUBLIC HEALTH ACTION: Hemophilia is associated with substantial lifelong morbidity, excess premature deaths, and extensive health care needs throughout life. Through monitoring data from Community Counts, CDC will continue to characterize the benefits and adverse events associated with existing or new hemophilia treatment products, thereby contributing to maximizing the health and longevity of persons with hemophilia.

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.

Detection of low-titer factor VIII (FVIII) or factor IX (FIX) inhibitors can be difficult in the presence of endogenous or therapeutic factor.1 Pre-analytic heat treatment (PHT) of patient plasma specimens at 56⁰C for 30 minutes followed by centrifugation prior to measurement of the inhibitor titer is important to eliminate interference caused by endogenous or therapeutic factor in the specimen and to avoid the need for a washout period.1 PHT has been previously shown to enable more accurate determination of the inhibitor titer without removing antibodies in the context of traditional factor replacement products.2

INTRODUCTION: Factor IX:C (FIX:C) levels vary in hemophilia B carriers even in pedigrees with a unifying genetic defect. Analyzing the balance between pro-and anticoagulants might increase our understanding of carriers' bleeding potential. AIM: In this research study, we evaluated bleeding scores (BS) and a novel mathematical model of thrombin generation (TG) in Amish FIX:C deficient carriers and controls. METHODS: Blood samples and BS were obtained from post-menarchal females, including 59 carriers and 57 controls from the same extended pedigree. Factors II, V, VII, VIII, IX, X, antithrombin, tissue factor pathway inhibitor and protein C were assayed to generate mathematical models of TG in response to 5pM tissue factor (TF) and for TF+thrombomodulin. BS was based on a modification of the MCMDM-1VWD scoring system. RESULTS: Carriers had a lower mean FIX:C (68% vs. 119%), von Willebrand factor antigen (108 vs.133) and Tissue activatable fibrinolysis inhibitor (103 vs. 111) compared to controls; both groups had a similar mean BS. Carriers demonstrated significantly lower TG parameters on both mathematical models compared to controls. Carriers with FIX:C</=50% had lower TG curves than those >50% but similar BS. CONCLUSION: Thrombin generation showed significant differences between carriers and controls, between low (</=50%) and high (>50%) FIX:C carriers, and specifically in the TF+thrombomodulin model, between high FIX:C carriers and controls, although the BS were not different.

The Nijmegen-Bethesda assay (NBA) is the gold standard for measurement of factor VIII (FVIII) inhibitors in haemophilia A patients.1 Modification of the traditional NBA2 to use a chromogenic measurement of FVIII as the endpoint is necessary for measurement of FVIII inhibitors in the presence of heparin, lupus anticoagulants, or by-passing agents such as emicizumab, due to their interference in clot-based assays.3–5 Parallel testing has shown this modification to produce similar results to the NBA in the absence of these interfering substances.6 In the clot-based NBA, substitution of imidazole-buffered bovine serum albumin (IB-BSA) for FVIII-deficient plasma (FVIIIDP) as diluent in control mixtures and specimen dilutions has been shown to produce equivalent results when the threshold for positivity was slightly adjusted.7 This study aims to evaluate a similar substitution in the chromogenic Bethesda assay (CBA) and to describe the performance characteristics of this modified assay.

INTRODUCTION: Accurate diagnosis of an inhibitor, a neutralizing antibody to infused factor VIII (FVIII), is essential for appropriate management of haemophilia A (HA). Low-titre inhibitors may be difficult to diagnose due to high rates of false-positive inhibitor results in that range. Transient low-titre inhibitors and false-positive inhibitors may be due to the presence of a lupus anticoagulant (LA) or other non-specific antibodies. Fluorescence immunoassay (FLI) to detect antibodies to FVIII is a sensitive method to identify inhibitors in HA. Evaluations of antibody profiles by various groups have demonstrated that haemophilic inhibitors detected by Nijmegen-Bethesda (NBA) and chromogenic Bethesda (CBA) assays correlate with positivity for anti-FVIII immunoglobulin (Ig) G1 and G4. AIM: This study sought to determine whether FLI could distinguish false-positive FVIII inhibitor results related to LAs from clinically relevant FVIII inhibitors in HA patients. METHODS: Samples from haemophilic and non-haemophilic subjects were tested for LA, specific FVIII inhibitors by NBA and CBA, and anti-FVIII immunoglobulin profiles by FLI. RESULTS: No samples from LA-positive non-haemophilic subjects were positive by FLI for anti-FVIII IgG4. Conversely, 91% of NBA-positive samples from haemophilia subjects were positive for anti-FVIII IgG4. Two of 11 haemophilia subjects had samples negative for anti-FVIII IgG4 and CBA, which likely represented LA rather than FVIII inhibitor presence. CONCLUSIONS: Assessment of anti-FVIII profiles along with the CBA may be useful to distinguish a clinically relevant low-titre FVIII inhibitor from a transient LA in HA patients.

The Nijmegen-Bethesda assay (NBA), considered the “gold standard” for measurement of factor VIII (FVIII) inhibitors in haemophilia A,1 introduced two modifications to the traditional Bethesda assay (BA) for stabilization during the 2-hour incubation at 37°C: (i) buffering of normal pooled plasma (NPP) in the test and control mixtures with imidazole and (ii) substitution of FVIII-deficient plasma (FVIIIDP) for imidazole buffer (IB) in the control mixture and for specimen predilution.2 The NBA has not been widely adopted in the United States, because of the increased cost incurred by use of FVIIIDP rather than buffer and the lack of FDA-approved commercial reagents.3 Surveys of North American coagulation laboratories have shown that only 20% use the NBA, 70% use buffered NPP in a “hybrid” of the NBA and BA, and one-third use diluents other than those recommended in published methods.3 This lack of methodological uniformity may partially account for poor interlaboratory reproducibility, a well-known problem with FVIII inhibitor testing.3

INTRODUCTION: The use of pre-analytical heat treatment (PHT) with the Nijmegen-Bethesda assay (NBA) for inhibitors to factor VIII (FVIII) can remove/destroy infused or endogenous FVIII from patient plasma samples, allowing testing of recently infused patients with haemophilia. Two PHT methods have been described as follows: heating to 56 degrees C for 30 minutes and heating to 58 degrees C for 90 minutes. Data examining the effects of PHT on anti-FVIII IgG4 , the antibodies known to correlate most closely with the presence of FVIII inhibitors, are limited. AIM: To assess the effect of PHT on the levels of detectable anti-FVIII IgG4 . METHODS: Nijmegen-Bethesda assay-positive specimens were incubated at 56, 58 or 60 degrees C for 90 minutes, and anti-FVIII IgG4 was measured by fluorescence immunoassay (FLI) at 30-minute intervals. The effects of PHT on the ability of recombinant FVIII (rFVIII) to inhibit detection of patient antibodies by FLI was also examined to assess the stability of rFVIII under the various PHT conditions tested. RESULTS: Levels of anti-FVIII IgG4 showed little change following incubations at 56 degrees C (mean 101% of original value at 30 minutes and 100% at 60 minutes) but decreased upon exposure to 58 degrees C (mean 85% at 30 minutes and 66% at 60 minutes). In addition, heating to 56 degrees C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII. CONCLUSION: The optimal temperature for PHT in the FVIII NBA is 56 degrees C. Higher temperatures may lead to loss of inhibitory antibodies.

Inhibitors are antibodies directed against haemophilia treatment products which interfere with their function. Factor VIII (FVIII) inhibitors in haemophilia A and factor IX (FIX) inhibitors in haemophilia B are significant clinically when they require a change in a patient's treatment regimen. Their persistence may increase morbidity and mortality. Multiple laboratory tests are now available for detecting and understanding inhibitors in haemophilia. Inhibitors are traditionally measured by their interference in clotting or chromogenic factor assays. They may also be detected using immunologic assays, such as enzyme-linked immunosorbent assay or fluorescence immunoassay. Anti-FVIII or anti-FIX antibodies of IgG4 subclass best correlate with the presence of functional inhibitors. Improvements in inhibitor measurement have been recently introduced. Preanalytical heat treatment of patient specimens allows testing of patients without delaying treatment. Use of chromogenic and immunologic assays may aid in identification of false-positive results, which are frequent among low-titre inhibitors. Validated reagent substitutions can be used to reduce assay cost. New methods for defining assay positivity and reporting low-titre inhibitors have been suggested. Challenges remain in the areas of quality control, assay standardization, monitoring of patients undergoing immune tolerance induction therapy and testing in the presence of modified and novel treatment products.

BACKGROUND: The Bethesda assay (BA) for measurement of factor VIII (FVIII) inhibitors called for quantitation of positive inhibitors using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, recommending use of "more sensitive methods" for samples with RA closer to 100%. The Nijmegen modification (NBA) changed the reagents used but not these calculations. Some specimens negative by NBA have been shown to have FVIII antibodies detectable by sensitive immunologic methods. OBJECTIVE: To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytical heat inactivation to liberate bound anti-FVIII antibodies. METHODS: Specimens with known inhibitors were tested by CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). RESULTS: Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). Using 4 statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. CONCLUSIONS: The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (<0.5) NBU. The sharp increase in IgG4 frequency between 0.4-0.5 NBU validates the established threshold for positivity of ≥0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds This article is protected by copyright. All rights reserved.

BACKGROUND: Hemophilia B (HB) is an inherited bleeding disorder caused by the absence or dysfunction of coagulation factor IX (FIX). A subset of patients who have HB develop neutralizing alloantibodies (inhibitors) against FIX following infusion therapy. HB prevalence and the proportion of patients who develop inhibitors are much lower than that of hemophilia A (HA), which makes studies of inhibitors in patients with HB challenging due to the limited availability of samples. As a result, there is a knowledge gap regarding HB inhibitors. OBJECTIVE: Evaluate the largest group of inhibitor positive HB patients studied to date to assess the relationship between anti-FIX antibody profiles and inhibitor formation METHODS: A fluorescence immunoassay (FLI) was used to detect anti-FIX antibodies in plasma samples from 37 patients with HB RESULTS: Assessments of antibody profiles showed that anti-FIX IgG1-4 , IgA, and IgE were detected significantly more often in patients with a positive Nijmegen-Bethesda Assay (NBA). All NBA-positive samples were positive for IgG4 . Anti-FIX IgG4 demonstrated a strong correlation with the NBA, while correlations were significant, yet more moderate, for anti-FIX IgG1-2 and IgA CONCLUSIONS: The anti-FIX antibody profile in HB patients who develop inhibitors is diverse and correlates well with the NBA across immunoglobulin (sub)class, and anti-FIX IgG4 is particularly relevant to functional inhibition. The anti-FIX FLI may serve as a useful tool to confirm the presence of antibodies in patients who have low positive NBA results and to more clearly define, predict, and treat alloantibody formation against FIX.

In this issue, Montalvão and colleagues [1] present important information on the value of heat treatment of specimens for inhibitor measurement in hemophilia A patients undergoing immune tolerance induction therapy (ITI). Inhibitors, antibodies directed against infused treatment products, are the most significant complication of hemophilia therapy today, occurring in up to 30% of severe patients during their initial exposures to factor VIII (FVIII) replacement. Risk of inhibitor development persists through the lifespan, with a second peak of inhibitors occurring later in life [2], and patients with all severities of hemophilia A develop inhibitors [3]. Annual testing is recommended for all patients, with more frequent monitoring for those at highest risk [4]. In addition to clinical use of the inhibitor test in detection of new inhibitors, selection of therapy, and monitoring for successful inhibitor eradication, this test is also the key endpoint measurement for evaluation of product safety, assessment of population trends, and studies of inhibitor risk factors. We have previously introduced modifications to traditional inhibitor tests to improve their usefulness for these purposes by minimizing false negative and false positive results [5,6]. | The first standardized method for measuring hemophilic inhibitors, the Bethesda assay, was described in 1975 [7], with reagent modifications to improve its performance added in 1995 [8], producing what has come to be called the Nijmegen–Bethesda assay. In the past 20 years, however, the usefulness of these assays has been compromised by changes in hemophilia treatment. Patients receiving prophylactic therapy, by design, have factor present at all times and must refrain from treating to “wash out” their factor prior to undergoing traditional inhibitor tests. Presence of factor in the test specimen may result in failure to detect low titer inhibitors and produce a false negative result [5]. Patients undergoing ITI present similar problems due to frequent FVIII infusions.

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.

PURPOSE: To characterize patients with inhibitors identified by prospective screening. FINDINGS: 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: 9 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 1975-9 at 13 U.S. centers found 31 (2.3%) new inhibitors among 1360 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 in the current study, p=0.024) and in ED prior to inhibitor development, 10% in the older study and 43% in the current study occurring within 0-20 ED. CONCLUSIONS: Prospective screening detects Inhibitors in patients of all severities, ages, and ED. Some LTI, however, are false positives.

In June 2012, an academic haematologist in a tertiary health facility notified the Centers for Disease Control and Prevention (CDC) of new onset inhibitors during hospitalization in four previously treated people with haemophilia (PWH). The four patients were considered at low risk for this complication. Inhibitor onset occurred among patients hospitalized during a 14-month period, whereas no inhibitor cases had occurred among inpatients during the previous 12-month period. All four cases had received a recombinant clotting factor concentrate (CFC) from a single manufacturer, raising concern about a possible product-related issue. The situation was reported to the North Carolina Department of Health and Human Services who advised that an investigation be carried out and requested assistance from CDC. | This report summarizes the results of the investigation conducted with the following specific aims: (i) to determine whether the cases represented an increase in the baseline inhibitor incidence among inpatients with haemophilia at the index facility; (ii) to assess whether the cases were at greater risk for an inhibitor than other inpatients with haemophilia as a result of personal, clinical or treatment factors; and (iii) to evaluate the possible influence of changes in hospital practices related to CFC use during the period.

BACKGROUND: Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in Factor VIII (FVIII). von Willebrand disease (VWD) is characterized by a quantitative or qualitative defect in von Willebrand Factor (VWF). Patients with VWD with severely low VWF or VWD Type 2N (VWD2N), a VWD subtype distinguished by defective VWF binding to FVIII, may have reduced FVIII levels secondary to their VWD. These patients superficially resemble patients with HA, and pose a potential for misdiagnosis. OBJECTIVES: Investigate the unexplained cause of bleeding in HA patients without known FVIII mutations by assessing plasma VWF antigen (VWF:Ag), FVIII binding capacities, and VWF genotypes. PATIENTS/METHODS: Thirty-seven of 1027 patients with HA studied as part of the Hemophilia Inhibitor Research Study lacked identifiable F8 mutations. These patients (cases) and 73 patients with identified F8 mutations (controls) were evaluated for VWF:Ag, patient's VWF capacity to bind FVIII (VWF:FVIIIB), and VWF sequence. RESULTS: Four cases had VWF:Ag <3 IU/dL and VWF mutations consistent with Type3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type1 VWD (VWD1) (n=5 cases and 1 control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n=1 case). One control had VWF:Ag <30 IU/dl, and seven patients (4 cases and 3 controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB. CONCLUSIONS: These data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy.

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.

BACKGROUND: The development of neutralizing antibodies, referred to as inhibitors, against factor VIII (FVIII) is a major complication associated with FVIII infusion therapy for the treatment of hemophilia A (HA). Previous studies have shown that a subset of HA patients and a low percentage of healthy individuals harbor non-neutralizing anti-FVIII antibodies that do not elicit the clinical manifestations associated with inhibitor development. OBJECTIVE: Assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes. METHODS: A fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients. RESULTS: Assessments of antibody profiles showed that the presence of anti-FVIII IgG1 , IgG2 , or IgG4 correlated qualitatively and quantitatively with the presence of a FVIII inhibitor as reported by the Nijmegen-Bethesda assay (NBA). Forty-eight patients with a negative inhibitor history contributed serial samples to the study, including seven patients who had negative NBA titers initially and later converted to NBA-positive. The FLI detected anti-FVIII IgG1 in five of those seven patients prior to their conversion to NBA-positive. Five of 15 serial-sample patients who had a negative inhibitor history and a positive anti-FVIII IgG1 later developed an inhibitor, compared to 2 of 33 patients with a negative inhibitor history without anti-FVIII IgG1 . CONCLUSIONS: These data provide a rationale for future studies designed both to monitor the dynamics of anti-FVIII antibody profiles in HA patients as a potential predictor of future inhibitor development and to assess the value of the anti-FVIII FLI as a supplement to traditional inhibitor testing.