significant difference by Wilcoxon test, and correlation greater than or equal to 0.90 in comparison to baseline in all tests for FVIII and FIX inhibitors, except for the FIX NBA, which showed a concordance of 97% from a single FIX specimen that tested at 0.1 NBU before and 0.3 NBU after storage refrigerated for 1 week. Specimens kept at room temperature (15– 25°C) for 1 week showed 100% concordance, no significant difference by Wilcoxon test, and correlation greater than 0.9 in comparison to baseline in all tests for FVIII and FIX inhibitors.

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.

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.Miller C.H. Platt S.J. Rice A.S. Kelly F. Soucie J.M. The Hemophilia Inhibitor Research Study InvestigatorsValidation of Nijmegen‐Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance.J Thromb Haemost. 2012; 10: 1055-1061Crossref PubMed Scopus (84) Google Scholar 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.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar 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.Adamkewicz J.I. Chen D.C. Paz‐Priel I. Effects and interferences of emicizumab, a humanised bispecific antibody mimicking activated factor VIII cofactor function, on coagulation assays.Thromb Haemost. 2019; 119: 1084-1093Crossref PubMed Scopus (71) Google Scholar, 4.Müller J. Pekrul I. Pötzsch B. Berning B. Oldenburg J. Spannagl M. Laboratory monitoring in emicizumab‐treated persons with hemophilia A.Thromb Haemost. 2019; 119: 1384-1393Crossref PubMed Scopus (47) Google Scholar, 5.Bowyer A. Kitchen S. Maclean R. Effects of emicizumab on APTT, one‐stage and chromogenic assays of factor VIII in artificially spiked plasma and in samples from haemophilia A patients with inhibitors.Haemophilia. 2020; 26: 536-542Crossref PubMed Scopus (26) Google Scholar CSA for FVIII that use bovine factor X (FX) are insensitive to emicizumab,5.Bowyer A. Kitchen S. Maclean R. Effects of emicizumab on APTT, one‐stage and chromogenic assays of factor VIII in artificially spiked plasma and in samples from haemophilia A patients with inhibitors.Haemophilia. 2020; 26: 536-542Crossref PubMed Scopus (26) Google Scholar and a CBA using such CSA has been successfully used for inhibitor testing in its presence.6.Adamkewicz J.I. Schmitt C. Asikanius E. Factor VIII (FVIII) inhibitor testing using a validated chromogenic Bethesda assay (CBA) in HAVEN 1 (BH29884), a phase 3 trial of emicizumab in persons with hemophilia A (PwHA) with inhibitors.RPTH. 2017; 1: 724-725Google Scholar, 7.Miller C.H. Boylan B. Payne A.B. Driggers J. Bean C.J. Validation of the chromogenic Bethesda assay for factor VIII inhibitors in hemophilia A patients receiving emicizumab.Int J Lab Hematol. 2021; 43Crossref PubMed Scopus (7) Google Scholar 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.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar revealed differences that may influence such comparisons and that, if not considered, could hinder validation of the CBA for clinical use. The results reexamined were from 1005 specimens collected from subjects with congenital HA enrolled in the Hemophilia Inhibitor Research Study between 2006 and 2012 at 17 US hemophilia treatment centers with previous inhibitor history collected from the enrolling sites.2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar Briefly, the US Centers for Disease Control and Prevention–modified NBA was performed using an FVIII OSA and included heating of patient plasma to 56°C for 30 min and centrifugation before testing.1.Miller C.H. Platt S.J. Rice A.S. Kelly F. Soucie J.M. The Hemophilia Inhibitor Research Study InvestigatorsValidation of Nijmegen‐Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance.J Thromb Haemost. 2012; 10: 1055-1061Crossref PubMed Scopus (84) Google Scholar The threshold for positivity was set at ≥0.5 Nijmegen‐Bethesda units (NBU) based on distributions of results on patients with positive and negative history of inhibitor1.Miller C.H. Platt S.J. Rice A.S. Kelly F. Soucie J.M. The Hemophilia Inhibitor Research Study InvestigatorsValidation of Nijmegen‐Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance.J Thromb Haemost. 2012; 10: 1055-1061Crossref PubMed Scopus (84) Google Scholar and validated by the frequency of positivity for anti‐FVIII antibodies.8.Miller C.H. Boylan B. Shapiro A.D. Lentz S.R. Wicklund B.M. The Hemophilia Inhibitor Research Study InvestigatorsLimit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.J Thromb Haemost. 2017; 15: 1971-1976Crossref PubMed Scopus (15) Google Scholar The CBA was performed by the NBA method, except that FVIII activity was measured using a bovine CSA (Siemens Factor VIII Chromogenic Assay, Siemens, Marburg, Germany).2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar Antibodies binding to FVIII were measured by a fluorescence immunoassay detecting both immunoglobulin (Ig)G and IgM in a subset of 268 specimens.2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar Results were expressed as median fluorescence intensity. The threshold for positivity was set at two standard deviations above the mean median fluorescence intensity of the results obtained for 56 healthy subjects. For this analysis, comparisons between CBA and NBA results were made by nonparametric methods, using the Wilcoxon matched‐pairs signed‐rank test and the Spearman correlation coefficient (r), and by chi‐squared test, with significance set at p < .05, using GraphPad Prism 8.3 (GraphPad Software Inc.). In our previous paper using these data, we reported excellent correlation between paired NBA and CBA results for specimens with ≥2.0 NBU (n = 42; r = .98) and discrepancies between the two assays for positive specimens in the 0.5–1.9 NBU range.2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar We have now found differences in the group with negative titers (<0.5 NBU, n = 883), as well. In that group, the NBA median was 0.1 (interquartile range 0–0.1), and the CBA median was 0 (interquartile range 0–0; p < .0001). As illustrated in Figure 1A, the CBA and NBA distributions were significantly different (p < .0001). The CBA produced a much larger number of zero Bethesda unit results than the NBA (73.9% vs. 40.4%) with smaller proportions for the CBA than the NBA in the remainder of the negative range (17.4% vs. 47.5%) and in the range of 0.5–1.9 (4.0% vs. 8.0%). Results were similar with the two methods in low‐positive (2.0–4.9) specimens at 1.8% vs. 1.2% and high‐positive (≥5.0) specimens at 2.9% vs. 3.0%. There appears to be a previously unreported shift toward lower CBA results among NBA‐negative specimens. Using the CBA, 84.7% of 746 specimens from patients with negative history of inhibitor had zero CBU, whereas only 48.3% had zero NBU. The observed differences do not alter our previous conclusions that both NBA‐negative specimens and NBA‐positive specimens with ≥2.0 NBU are classified correctly by the CBA, with only those with 0.5–1.9 NBU showing classification changes2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar; however, these differences need to be considered when attempting to establish equivalence between the two assays for clinical purposes and might be taken to indicate that the CBA is less sensitive than the NBA. We have recently reported, however, that the limit of detection for the CBA is 0.1,7.Miller C.H. Boylan B. Payne A.B. Driggers J. Bean C.J. Validation of the chromogenic Bethesda assay for factor VIII inhibitors in hemophilia A patients receiving emicizumab.Int J Lab Hematol. 2021; 43Crossref PubMed Scopus (7) Google Scholar which is lower than the 0.2 calculated for the NBA,8.Miller C.H. Boylan B. Shapiro A.D. Lentz S.R. Wicklund B.M. The Hemophilia Inhibitor Research Study InvestigatorsLimit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.J Thromb Haemost. 2017; 15: 1971-1976Crossref PubMed Scopus (15) Google Scholar indicating that the CBA is slightly more sensitive than the NBA, as we originally showed in dilution studies.2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar Among specimens tested for specific anti‐FVIII antibodies, significantly more CBA‐positive specimens were antibody‐positive than NBA‐positive specimens (50/51, 98.0% vs. 83/99, 83.8%; p = .012), with the single CBA‐positive specimen not showing antibody positivity positive in both NBA and CBA. These results for the CBA in patients receiving replacement therapy are similar to our recent findings in patients receiving emicizumab, which showed 97.6% of 250 CBA‐positive specimens to be positive for anti‐FVIII IgG4 antibodies,7.Miller C.H. Boylan B. Payne A.B. Driggers J. Bean C.J. Validation of the chromogenic Bethesda assay for factor VIII inhibitors in hemophilia A patients receiving emicizumab.Int J Lab Hematol. 2021; 43Crossref PubMed Scopus (7) Google Scholar the most reliable antibody subclass to predict that a functional inhibitor is present, as reviewed.9.Miller C.H. Laboratory testing for factor VIII and IX inhibitors in haemophilia: a review.Haemophilia. 2018; 24: 186-197Crossref PubMed Scopus (31) Google Scholar Thus, the lower results in the CBA are not due to lower sensitivity but to greater accuracy at detecting truly negative specimens. The CBA is thought to give fewer false‐positive results because lupus anticoagulants, heparin, or nonspecific inhibitors of coagulation affect the OSA but not the CSA, as reviewed.9.Miller C.H. Laboratory testing for factor VIII and IX inhibitors in haemophilia: a review.Haemophilia. 2018; 24: 186-197Crossref PubMed Scopus (31) Google Scholar We also examined two other characteristics of the CBA in this dataset. Our previous suggestion that specimens in the range of 0.5–1.9 NBU had a high frequency of false‐positive results and should be confirmed by testing in the CBA and measurement of anti‐FVIII antibodies was based on the finding that the frequency of positivity for anti‐FVIII antibodies was significantly lower among specimens with 0.5–1.9 NBU than among those with ≥2.0 NBU (75.4% vs. 97.4%; p = .004).2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar A similar analysis for the CBA shows antibody positivity rates in those ranges to be similar at 24/24 (100%) and 26/27 (96.3%), respectively (p > .99). Additional antibody testing for confirmation, therefore, is not necessary when the CBA is used as the primary test in patients receiving traditional therapy, as we have shown in those receiving emicizumab.7.Miller C.H. Boylan B. Payne A.B. Driggers J. Bean C.J. Validation of the chromogenic Bethesda assay for factor VIII inhibitors in hemophilia A patients receiving emicizumab.Int J Lab Hematol. 2021; 43Crossref PubMed Scopus (7) Google Scholar We also examined the threshold for positivity of the CBA, which was previously assumed to be the same as for the NBA.2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar CBA results plotted by history of inhibitor (Figure 1B) were very similar to the NBA results previously reported.1.Miller C.H. Platt S.J. Rice A.S. Kelly F. Soucie J.M. The Hemophilia Inhibitor Research Study InvestigatorsValidation of Nijmegen‐Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance.J Thromb Haemost. 2012; 10: 1055-1061Crossref PubMed Scopus (84) Google Scholar Among 746 negative‐history specimens, 745 (99.9%) were below 0.5 CBU; one specimen differed in classification between the two methods with 0.4 NBU and 0.6 CBU. For the 204 positive‐history specimens, the distribution was bimodal, as expected because of resolved or treated inhibitors, and similar to that of the NBA, both showing a break at 0.4. Thus, a threshold for positivity of ≥0.5 appears to be appropriate for the CBA in non‐emicizumab specimens, as we have recently confirmed for specimens from patients receiving emicizumab.7.Miller C.H. Boylan B. Payne A.B. Driggers J. Bean C.J. Validation of the chromogenic Bethesda assay for factor VIII inhibitors in hemophilia A patients receiving emicizumab.Int J Lab Hematol. 2021; 43Crossref PubMed Scopus (7) Google Scholar The current analysis identified differences among negative results not previously reported, which affect statistical comparison of results but do not affect classification of specimens as positive or negative. It also confirmed that a threshold for positivity of ≥0.5 is appropriate for the CBA, as previously shown for the NBA,2.Miller C.H. Rice A.S. Boylan B. et al.Comparison of clot‐based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.J Thromb Haemost. 2013; 11: 1300-1309Crossref PubMed Scopus (51) Google Scholar, 8.Miller C.H. Boylan B. Shapiro A.D. Lentz S.R. Wicklund B.M. The Hemophilia Inhibitor Research Study InvestigatorsLimit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.J Thromb Haemost. 2017; 15: 1971-1976Crossref PubMed Scopus (15) Google Scholar when using the US Centers for Disease Control and Prevention–modified methods including preanalytical heat inactivation of patient plasma in patients not receiving emicizumab. The higher rate of antibody positivity among CBA‐positive specimens allows a greater degree of confidence in low‐titer positive results when tests are performed with the CBA than with the NBA and eliminates the need for additional testing methods to confirm NBA results. In spite of the slight differences observed, the similarity of the CBA to the NBA should allow its use with confidence in patients treated with traditional products as well as those receiving emicizumab. Dr. Miller and Mr. Boylan state that they have no real or potential conflicting interests. Connie H. Miller planned the study, analyzed results, and wrote the manuscript. Brian Boylan performed research, analyzed results, and wrote the manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. The work was supported by the CDC Foundation through grants from Pfizer Pharmaceuticals and Baxter Healthcare. The authors wish to thank the Hemophilia Inhibitor Research Study coordinators and patients and Fiona Bethea, CDC study coordinator.CDC FoundationPfizer PharmaceuticalsBaxter Healthcare

International Journal of Laboratory HematologyVolume 43, Issue 2 p. e84-e86 LETTER TO THE EDITOR Validation of the chromogenic Bethesda assay for factor VIII inhibitors in hemophilia a patients receiving Emicizumab Connie H. Miller, Corresponding Author Connie H. Miller cmiller2@cdc.gov orcid.org/0000-0002-3989-7973 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USA Correspondence Connie H. Miller, Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, 1600 Clifton Road MS D02, Atlanta, GA 30329, USA. Email: cmiller2@cdc.govSearch for more papers by this authorBrian Boylan, Brian Boylan orcid.org/0000-0003-3930-4565 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this authorAmanda B. Payne, Amanda B. Payne orcid.org/0000-0003-1027-7639 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this authorJennifer Driggers, Jennifer Driggers Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this authorChristopher J. Bean, Christopher J. Bean orcid.org/0000-0001-7433-9769 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this author Connie H. Miller, Corresponding Author Connie H. Miller cmiller2@cdc.gov orcid.org/0000-0002-3989-7973 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USA Correspondence Connie H. Miller, Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, 1600 Clifton Road MS D02, Atlanta, GA 30329, USA. Email: cmiller2@cdc.govSearch for more papers by this authorBrian Boylan, Brian Boylan orcid.org/0000-0003-3930-4565 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this authorAmanda B. Payne, Amanda B. Payne orcid.org/0000-0003-1027-7639 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this authorJennifer Driggers, Jennifer Driggers Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this authorChristopher J. Bean, Christopher J. Bean orcid.org/0000-0001-7433-9769 Division of Blood Disorders, Centers for Disease Control and Prevention, National Center on Birth Defects and Developmental Disabilities, Atlanta, GA, USASearch for more papers by this author First published: 10 November 2020 https://doi.org/10.1111/ijlh.13384Citations: 6Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume43, Issue2April 2021Pages e84-e86 RelatedInformation

HaemophiliaVolume 25, Issue 5 p. e342-e344 LETTER TO THE EDITOR Reagent substitution in the chromogenic Bethesda assay for factor VIII inhibitors Amanda B. Payne, Corresponding Author Amanda B. Payne bvx2@cdc.gov orcid.org/0000-0003-1027-7639 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USA Correspondence Amanda B. Payne, Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USA. Email: bvx2@cdc.govSearch for more papers by this authorConnie H. Miller, Connie H. Miller orcid.org/0000-0002-3989-7973 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorDorothy Ellingsen, Dorothy Ellingsen Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorJennifer Driggers, Jennifer Driggers Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorBrian Boylan, Brian Boylan orcid.org/0000-0003-3930-4565 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorChristopher J. Bean, Christopher J. Bean orcid.org/0000-0001-7433-9769 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this author Amanda B. Payne, Corresponding Author Amanda B. Payne bvx2@cdc.gov orcid.org/0000-0003-1027-7639 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USA Correspondence Amanda B. Payne, Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USA. Email: bvx2@cdc.govSearch for more papers by this authorConnie H. Miller, Connie H. Miller orcid.org/0000-0002-3989-7973 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorDorothy Ellingsen, Dorothy Ellingsen Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorJennifer Driggers, Jennifer Driggers Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorBrian Boylan, Brian Boylan orcid.org/0000-0003-3930-4565 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this authorChristopher J. Bean, Christopher J. Bean orcid.org/0000-0001-7433-9769 Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA, USASearch for more papers by this author First published: 29 July 2019 https://doi.org/10.1111/hae.13827Citations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume25, Issue5September 2019Pages e342-e344 RelatedInformation

IntroductionThe 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°C for 30 minutes and heating to 58°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.AimTo assess the effect of PHT on the levels of detectable anti‐FVIII IgG4.MethodsNijmegen‐Bethesda assay‐positive specimens were incubated at 56, 58 or 60°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.ResultsLevels of anti‐FVIII IgG4 showed little change following incubations at 56°C (mean 101% of original value at 30 minutes and 100% at 60 minutes) but decreased upon exposure to 58°C (mean 85% at 30 minutes and 66% at 60 minutes). In addition, heating to 56°C effectively decreased the ability of rFVIII to block antibody binding compared to unheated rFVIII.ConclusionThe optimal temperature for PHT in the FVIII NBA is 56°C. Higher temperatures may lead to loss of inhibitory antibodies.

D. Coffin1 C. Herr1 J. O’Hara2 S. Diop3 R. Hollingsworth4 A. Srivastava5 D. Lillicrap6 H. M. van den Berg7 A. Iorio8 G. F. Pierce1 1World Federation of Hemophilia, Montreal, Canada 2HCD Economics, Manchester, UK 3Cheikh Anta Diop University, Dakar, Senegal 4Medical Data Solutions and Services (MDSAS), Manchester, UK 5Christian Medical College, Vellore, India 6Queen’s University, Kingston, Canada 7University Medical Center, Utrecht, The Netherlands 8McMaster University, Hamilton, Canada

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.

Background: The incidence and prevalence of inhibitors among hemophilia patients in the U.S. has not been determined. The Centers for Disease Control and Prevention (CDC) has established a national inhibitor surveillance program called the Community Counts Registry for Bleeding Disorders Surveillance, through a collaboration with the American Thrombosis and Hemostasis Network and the U.S. Hemophilia Treatment Center (HTC) Network, to collect data and specimens to monitor inhibitors.

Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA. SUMMARY:Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with 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 preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four 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 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.

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 after infusion therapy. HB prevalence and the proportion of patients who develop inhibitors are much lower than those for 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 patients with inhibitor-positive HB studied to date to assess the relationship between anti-FIX antibody profiles and inhibitor formation.Methods A fluorescence immunoassay was used to detect anti-FIX antibodies in plasma samples from 37 patients with HB.Results Assessments of antibody profiles showed that anti-FIX IgG(1-4), IgA, and IgE were detected significantly more often in patients with a positive Nijmegen-Bethesda assay (NBA). All NBA-positive samples were positive for IgG(4). Anti-FIX IgG(4) demonstrated a strong correlation with the NBA, while correlations were significant, yet more moderate, for anti-FIX IgG(1-2) 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 IgG(4) is particularly relevant to functional inhibition. The anti-FIX fluorescence immunoassay 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.

BackgroundHemophilia 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.ObjectivesTo 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/MethodsThirty-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, a patient's VWF capacity to bind FVIII (VWF:FVIIIB) and VWF sequence.ResultsFour cases had VWF:Ag<3 IUdL(-1) and VWF mutations consistent with Type 3 VWD. Six cases and one control were heterozygous for mutations previously reported to cause Type 1 VWD (VWD1) (n=five cases and one control) or predicted to be deleterious by Polyphen2 and SIFT prediction tools (n=1 case). One control had VWF:Ag<30IUdL(-1) and seven patients (four cases and three controls), including two cases who were heterozygous for a known VWD2N mutation, had reduced VWF:FVIIIB.ConclusionsThese data emphasize that some patients diagnosed with HA require VWF assessments in order to achieve a comprehensive diagnosis and an optimal treatment strategy.

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 IgG 1 and IgG 4 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. Am. J. Hematol. 90:871–876, 2015. © 2015 Wiley Periodicals, Inc.

BackgroundThe development of neutralizing antibodies, referred to as inhibitors, against factorVIII is a major complication associated with FVIII infusion therapy for the treatment of hemophiliaA (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.ObjectiveTo assess HA patients' anti-FVIII antibody profiles as potential predictors of clinical outcomes.MethodsA fluorescence immunoassay (FLI) was used to detect anti-FVIII antibodies in 491 samples from 371 HA patients.ResultsAssessments of antibody profiles showed that the presence of anti-FVIII IgG(1), IgG(2) or IgG(4) correlated qualitatively and quantitatively with the presence of an FVIII inhibitor as determined with 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 being NBA-positive. The FLI detected anti-FVIII IgG(1) 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 had anti-FVIII IgG(1) later developed an inhibitor, as compared with two of 33 patients with a negative inhibitor history without anti-FVIII IgG(1).ConclusionsThese 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.

Among the extracellular cues that cells continuously sample and respond to, adhesion is among the most potent, eliciting a wide range of cell biological processes, including but not limited to changes in cytosolic calcium, protein and lipid phosphorylation, cytoskeletal architecture, gene transcription, and cell migration. Members of the integrin family, composed of 24 ab heterodimer transmembrane receptors, are particularly adept at transmitting adhesion-initiated signals into the cell in a process sometimes referred to as outside-in signaling. The platelet-specific integrin aIIbb3 (CD41/CD61, glycoprotein [GP] IIb-IIIa in the platelet literature) is among the best-studied members of the integrin family, and is thought to be particularly responsive to intracellular and extracellular stimuli. Thus, binding of adhesive ligands to aIIbb3 has been shown to result in dramatic conformational changes within the aIIbb3 extracellular domain that, in a process likely involving ligand-binding–induced swing out of the b3 subunit hybrid domain and separation of integrin a and b subunit stalk domains, become propagated across the plasma membrane to the cytoplasmic face of the integrin. Binding of multivalent ligands like fibrinogen, fibronectin, and von Willebrand factor additionally results in clustering of integrin receptors. These events converge to initiate a series of biochemical and cell biological events at the cytosolic face of the integrin that ultimately lead to changes in cytoskeletal architecture, granule secretion, and further integrin activation that together serve to stabilize the platelet-platelet interactions that occur at sites of a growing thrombus. Outside-in signaling, therefore, contributes importantly to both thrombosis and hemostasis. An extensive number of adaptor proteins, kinases, and phosphatases have been found to participate in aIIbb3-mediated, adhesion-initiated signaling. In some cases, ligand binding induces association of the heterotrimeric guanine nucleotide-binding protein (G protein), Ga13, with the b3 cytoplasmic domain, 6 where it activates integrin-associated Src-family kinases (SFKs) that go on to phosphorylate and activate a plethora of molecular targets. Of these, 2 tyrosine residues that reside within the immunoreceptor tyrosine-based activation motif (ITAM) of the cytoplasmic domain of Fc receptor g-chain IIa (FcgRIIa) have been identified as SFK targets that, once phosphorylated, create a docking site for the tandem Src homology 2 (SH2) domains of the tyrosine kinase Syk. Activated Syk, in turn, goes on to amplify multiple pathways involved in integrin and platelet activation.

Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety and assessment of population trends.Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA) and a novel fluorescence immunoassay (FLI).Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU) <0.5 and positive on 42/42 specimens (100%) with NBU 2.0 and 43/80 specimens (53.8%) with NBU 0.5-1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0-1.9 NBU specimens and 43.1% and 50.0% of 0.5-0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, five (23.8%) with 0.7-1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5-1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%).Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5-1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays.