title: Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance.
abstract: BACKGROUND
  As part of a pilot U.S. inhibitor surveillance project initiated at the Centers for Disease Control and Prevention (CDC) in 2006, a centralized inhibitor measurement was instituted.
  
  
  OBJECTIVE
  To validate a modified method for inhibitor measurement suitable for surveillance of treated and untreated patients.
  
  
  RESULTS
  In all, 710 subjects with hemophilia A were enrolled; 122 had a history of inhibitor (HI). Nijmegen-Bethesda assay (NBA) results on 50 split specimens shipped on cold packs and frozen were equivalent (r=0.998). Because 55% of 228 initial specimens had factor (F)VIII activity (VIII:C) present, a heat treatment step was added. Heating specimens to 56°C for 30 min and centrifuging removed FVIII, as demonstrated by a reduction of VIII:C and FVIII antigen to <1 U dL(-1) in recently treated patients. Among specimens inhibitor-negative before heating, one of 159 with negative HI and five of 30 with positive HI rose to ≥ 0.5 Nijmegen-Bethesda units (NBU) after heating. Correlation of heated and unheated inhibitor-positive specimens was 0.94 (P=0.0001). The modified method had a coefficient of variation (CV) for a 1 NBU positive control of 10.3% and for the negative control of 9.8%. Based on results on 710 enrollment specimens, a positive CDC inhibitor was defined as ≥ 0.5 NBU. Results were similar when 643 post-enrollment specimens were included. Of 160 enrolled hemophilia B patients, two had HI. All others had NBU ≤ 0.2 at enrollment.
  
  
  CONCLUSIONS
  The CDC experience demonstrates that this modified NBA can be standardized to be within acceptable limits for clinical tests and can be used for national surveillance.
authors.affiliation: Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. cmiller2@cdc.gov
authors.forename: C H
authors.initials: CH
authors.lastname: Miller
authors.forename: S J
authors.initials: SJ
authors.lastname: Platt
authors.forename: A S
authors.initials: AS
authors.lastname: Rice
authors.forename: F
authors.initials: F
authors.lastname: Kelly
authors.forename: J M
authors.initials: JM
authors.lastname: Soucie
mesh_terms: D000906:Antibodies / Q000097:blood*; D000937:Antigen-Antibody Reactions; D016477:Artifacts; D001780:Blood Coagulation Tests / Q000592:standards; D002138:Calibration; D003029:Coagulants / Q000276:immunology* / Q000627:therapeutic use*; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000276:immunology* / Q000627:therapeutic use*; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis / Q000188:drug therapy* / Q000276:immunology; D002836:Hemophilia B / Q000097:blood / Q000175:diagnosis / Q000188:drug therapy / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D011237:Predictive Value of Tests; D011446:Prospective Studies; D011489:Protein Denaturation; D012015:Reference Standards; D015203:Reproducibility of Results; D012720:Severity of Illness Index; D013997:Time Factors; D016896:Treatment Outcome; D014481:United States
chemical_list: D000906:Antibodies; D003029:Coagulants; D005169:Factor VIII