Stability of specimens for use in the Centers for Disease Control and Prevention assays for factor VIII and IX inhibitors

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 Because nonspecific inhibitors, such as heparin and lupus anticoagulants, interfere with the clot-based FVIII and FIX assays used in the NBA, the CDC has adopted additional tests to confirm the presence of specific FVIII or FIX antibodies in specimens with low-positive results in the NBA.4 These include a chromogenic Bethesda assay (CBA) for FVIII inhibitors and fluorescence immunoassays (FLI) for anti-FVIII5 and anti-FIX6 IgG antibodies. The CBA can also be used for testing of specimens containing the drug emicizumab (Hemlibra) that interferes with the FVIII NBA.7, 8 We have now conducted studies to validate acceptable storage and shipping conditions for specimens to be used in the NBA, CBA, and FLI. Knowledge of specimen stability will aid care providers in collection of specimens from patients with limited access to treatment facilities and provide a basis for specimen collection protocols for clinical and public health studies. To assess anti-factor antibody stability in plasma specimens after long-term storage at −70°C, original testing results on specimens from patients who have hemophilia A or hemophilia B were compared with results obtained on aliquots from the same parent sample stored for 1 to 15 years at −70°C. Specimens were selected to represent a range of results and included 10 specimens negative by ranges established for these methods (<0.5 Nijmegen-Bethesda units [NBU]1, 9; <0.5 chromogenic Bethesda units [CBU]4, 8, 10) and 30 positive specimens (≥0.5 NBU; ≥0.5 CBU) for FVIII inhibitors, and 10 negative specimens (<0.3 NBU1, 11) and 30 positive specimens (≥0.3 NBU) for FIX inhibitors. All specimens selected had been concordantly negative or positive for anti-FVIII IgG1 and IgG4 or anti-FIX IgG4 antibodies by FLI according to reference ranges in use at the time of the original assay. Median storage time for the specimens studied was 4.6 years, including five specimens stored for 15 years. A freshly thawed aliquot of each stored specimen was tested using the same methods as in the original NBA,1 CBA,4 and FLI5 tests for FVIII inhibitors or NBA1 and FLI6 tests for FIX inhibitors. Three specimens positive for FIX inhibitors were not tested because of low specimen volume. FLI results could not be generated for five FIX specimens because of low specimen volume. To investigate potential effects of variations in temperature encountered during shipping, freshly thawed aliquots of plasma specimens stored at −70°C from hemophilia A patients and hemophilia B patients were selected to represent a range of results. Ten specimens were known negative (<0.5 NBU; <0.5 CBU) and 30 known positive (≥0.5 NBU; ≥0.5 CBU) for FVIII inhibitors according to previous testing, and 10 were known negative (<0.3 NBU) and 30 known positive (≥0.3 NBU) for FIX inhibitors according to previous testing. All specimens selected had been concordantly negative or positive for anti-FVIII IgG1 and IgG4 or anti-FIX IgG4 antibodies by FLI according to reference ranges in use at the time of the original assay. Aliquots were tested immediately after thawing (baseline) and then stored at refrigerator temperature (2–8°C) or room temperature (15–25°C) for 1 week and tested with the same methods. One specimen negative for FVIII inhibitors was not tested after storage at room temperature for 1 week because of low specimen volume. Ten and nine specimens positive for FIX inhibitors were not tested after storage at refrigerator temperature or room temperature, respectively, because of low specimen volume. Results pre- and poststorage were compared by calculating (1) concordance of the original and stored results for positivity or negativity, (2) Wilcoxon matched pairs signed rank test, and (3) Pearson correlation, using SAS v.9.4 (SAS Institute Inc.). FLI results in median fluorescence intensity were log-transformed because of the wide range of values observed. Comparison of pre and post storage measurements are shown in Table 1. Specimens stored frozen at −70°C were stable for up to 15 years, showing 100% concordance, no significant difference by Wilcoxon test, and correlation greater than 0.9 in comparison to prestorage test results across all tests for FVIII and FIX inhibitors. Specimens kept at 2 to 8°C for 1 week showed 100% concordance, no 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. PHT for factor inhibitor tests is designed to eliminate interference by endogenous or infused FVIII and FIX, leaving the more stable antibodies to react with normal plasma during incubation in the assay, as reviewed.11 We previously have shown that such antibodies, as measured by FLI, are stable upon heating to 56°C for 30 or 60 min, whereas antibody levels decrease when specimens are heated to higher temperatures.12 Results in the current study indicate that long-term storage of plasma specimens at −70°C appears to be acceptable for the inhibitor tests evaluated, observations that are likely because the analytes measured in these assays are stable under the described conditions and because the assay results are not influenced by decay of clotting factors during storage. In addition, the current analysis demonstrates that FVIII and FIX inhibitors are stable when maintained at refrigerator temperature or even at room temperature for up to 1 week for all of the tests done as part of the CDC inhibitor panel. The conditions studied apply only to plasma, which should be separated from whole blood within 4 h of collection to avoid release of cellular contents.13 Plasma shipment at ambient temperature is not recommended because of seasonal variability in shipping conditions, which may cause specimens to reach higher temperatures than those evaluated here. These findings should aid laboratories in establishing collection and storage procedures for monitoring inhibitor status in patients with hemophilia A and B and in conducting studies on stored specimens. A. B. P. developed the study design, conducted the data analysis, and drafted the manuscript. B. B. performed the laboratory experiments and edited the manuscript. G. N. performed the laboratory experiments and edited the manuscript. B. W. performed the laboratory experiments and edited the manuscript. J. D. developed the study design and edited the manuscript. C. H. M. developed the study design, conducted the data analysis, and drafted the manuscript. C. J. B. developed the study design, provided supervisory support, and edited the manuscript. The authors have no relevant conflicts of interest to disclose. The contents are those of the author(s) and do not necessarily represent the official views of, nor an endorsement, by CDC/HHS, or the U.S. Government.