Best practices in mitigating the risk of biotin interference with laboratory testing.

Dietary biotin intake does not typically result in blood biotin concentrations that exceed interference thresholds for in vitro diagnostic tests. However, recent trends of high-dose biotin supplements and clinical trials of very high biotin doses for patients with multiple sclerosis have increased concerns about biotin interference with immunoassays. Estimates of the prevalence of high biotin intake vary, and patients may be unaware that they are taking biotin. Since 2016, 92 cases of suspected biotin interference have been reported to the US Food and Drug Administration. Immunoassays at greatest risk from biotin interference include thyroid and reproductive hormones, cardiac, and immunosuppressive drug tests. Several case studies have highlighted the challenge of biotin interference with thyroid hormone assays and the potential misdiagnosis of Graves' disease. Biotin interference should be suspected when immunoassay test results are inconsistent with clinical information; a clinically relevant biotin interference happens when the blood biotin concentration is high and the assay is sensitive to biotin. We propose a best practice workflow for laboratory scientists to evaluate discrepant immunoassay results, comprising: (1) serial dilution; (2) retesting after biotin clearance and/or repeat testing on an alternate platform; and (3) confirmation of the presence of biotin using depletion protocols or direct measurement of biotin concentrations. Efforts to increase awareness and avoid patient misdiagnosis should focus on improving guidance from manufacturers and educating patients, healthcare professionals, and laboratory staff. Best practice guidance for laboratory staff and healthcare professionals would also provide much-needed information on the prevention, detection, and management of biotin interference.

Experimental HBsAg/anti-HBs complex assay for prediction of HBeAg loss in chronic hepatitis B patients treated with pegylated interferon and adefovir.

BACKGROUND: We studied whether hepatitis B surface antigen (HBsAg)/anti-HBs immune complex levels in chronic hepatitis B (CHB) patients receiving antiviral therapy could be used as a response marker at baseline (BL) or early during treatment to predict treatment outcome. METHODS: An experimental array-based assay (immunological multi-parameter chip technology [IMPACT]; Roche Diagnostics, Penzberg, Germany) served to determine HBsAg, anti-HBs and complex levels. We tested a panel of serum samples of 40 hepatitis B e antigen (HBeAg)-positive and 44 HBeAg-negative patients who received pegylated interferon and adefovir for 48 weeks. RESULTS: HBsAg loss occurred in 4 of 40 HBeAg-positive and 4 of 44 HBeAg-negative patients. A total of 14 of 40 HBeAg-positive patients lost HBeAg and 12 of them formed anti-HBe. At BL, complexes were present in 83 (99%) patients, whereas free anti-HBs levels were detectable in 5 patients. Complex levels at BL and week 12 were higher in HBeAg-positive patients with HBeAg loss, compared to patients who retained HBeAg (P=0.002 and P=0.005, respectively). Receiver operating characteristic analysis for HBeAg loss in HBeAg-positive patients at BL and week 12 showed area-under-the-curve values of 0.79 (P=0.002) and 0.82 (P=0.003) for complex levels. We found no correlation in either HBeAg-positive or -negative patients between complex levels and HBsAg loss. CONCLUSIONS: We demonstrated for the first time that before and during treatment HBsAg/anti-HBs immune complex levels can predict HBeAg loss in HBeAg-positive CHB patients treated with pegylated interferon and adefovir. Complexes were present in almost all patients at BL and were higher in patients who lost HBeAg. In conclusion, determining HBsAg/anti-HBs immune complex levels before and early during treatment could aid in selecting CHB patients with an optimal chance to achieve HBeAg loss.

The frequency of anti-infliximab antibodies in patients with rheumatoid arthritis treated in routine care and the associations with adverse drug reactions and treatment failure.

OBJECTIVES: To investigate the frequency of anti-infliximab antibodies in patients with RA and the associations with adverse drug reactions and treatment failure. METHODS: Based on the DANBIO registry, patients with RA who initiated treatment with infliximab at Hvidovre Hospital between 2000 and 2008 and had available serum samples were identified. The patients were followed for 52 weeks. Anti-infliximab antibodies were determined prior to infusion at baseline and during follow-up (weeks 2, 6, 14 and 52 or at withdrawal) using the IMPACT indirect assay (Roche Diagnostics) and merged with clinical data prospectively registered in the DANBIO registry. RESULTS: A total of 218 patients with RA were included (80% females, median age 56 years, disease duration 10 years, 65% RF positive, median DAS28 = 5.0). During the 52-week follow-up, 28 patients (13%) withdrew due to adverse events and 50 (23%) due to treatment failure. Antibodies were detected in 118 patients (54%) during follow-up. Patients with detectable anti-infliximab antibodies after 6 weeks had an increased risk of adverse drug reactions [hazard ratio (HR) = 5.06, 95% CI 2.36, 10.84; P < 0.0001] compared with patients without anti-infliximab antibodies. Similar results were observed in patients with anti-infliximab antibodies after 14 weeks (HR = 3.30, 95% CI 1.56, 6.99; P = 0.0009). Patients with detectable anti-infliximab antibodies during the 52-week follow-up were less likely to achieve sustained minimal disease activity and remission. CONCLUSION: Early anti-infliximab antibody formation increased the risk of adverse drug reactions, including infusion reactions. Anti-infliximab antibody formation during the 52-week follow-up decreased the likelihood of minimal disease activity and remission in patients with RA treated in routine care.

Novel multiplex technology for diagnostic characterization of rheumatoid arthritis.

INTRODUCTION: The aim of this study was to develop a clinical-grade, automated, multiplex system for the differential diagnosis and molecular stratification of rheumatoid arthritis (RA). METHODS: We profiled autoantibodies, cytokines, and bone-turnover products in sera from 120 patients with a diagnosis of RA of < 6 months' duration, as well as in sera from 27 patients with ankylosing spondylitis, 28 patients with psoriatic arthritis, and 25 healthy individuals. We used a commercial bead assay to measure cytokine levels and developed an array assay based on novel multiplex technology (Immunological Multi-Parameter Chip Technology) to evaluate autoantibody reactivities and bone-turnover markers. Data were analyzed by Significance Analysis of Microarrays and hierarchical clustering software. RESULTS: We developed a highly reproducible, automated, multiplex biomarker assay that can reliably distinguish between RA patients and healthy individuals or patients with other inflammatory arthritides. Identification of distinct biomarker signatures enabled molecular stratification of early-stage RA into clinically relevant subtypes. In this initial study, multiplex measurement of a subset of the differentiating biomarkers provided high sensitivity and specificity in the diagnostic discrimination of RA: Use of 3 biomarkers yielded a sensitivity of 84.2% and a specificity of 93.8%, and use of 4 biomarkers a sensitivity of 59.2% and a specificity of 96.3%. CONCLUSIONS: The multiplex biomarker assay described herein has the potential to diagnose RA with greater sensitivity and specificity than do current clinical tests. Its ability to stratify RA patients in an automated and reproducible manner paves the way for the development of assays that can guide RA therapy.

New automated multiplex assay for bone turnover markers in osteoporosis.

BACKGROUND: Serum C-terminal cross-linked telopeptide of type I collagen (CTX-I), N-terminal propeptide of type I collagen (PINP), and osteocalcin (OC) are among the most sensitive bone turnover markers for evaluating osteoporosis. Each marker is currently measured individually by manual or automated immunoassays that are time consuming and require substantial sample volume. We evaluated the performance characteristics of a novel, fully automated, protein-array chip system that allows the simultaneous measurement of CTX-I, PINP, OC, and intact parathyroid hormone (PTH) in 20 microL of serum. METHODS: We measured CTX-I, PINP, OC, and PTH using multiplex and corresponding automated single assays in 157 healthy premenopausal women, 74 healthy men, and 56 postmenopausal osteoporotic women before and 6 months after treatment with oral ibandronate (150 mg/month). RESULTS: Within- and between-run CVs of the multiplex assay were similar to those of single measurement assays (<10% for all markers), whereas the limit of quantification was lower, except for OC. Multiplex values highly correlated (r > 0.93, P < 0.0001 for all markers) with the corresponding single assays, and measured concentrations were comparable. After 6 months of ibandronate, CTX-I, PINP, and OC decreased by a median of 48%, 63%, and 52%, respectively (P < 0.0001 for all 3 markers), magnitudes similar to those of the corresponding single assays. CONCLUSIONS: The automated protein-array chip demonstrated similar analytical precision, improved analytical sensitivity, and comparable measured concentrations to those of single assays. The multiplex assay should be useful for assessing bone metabolism in large clinical studies, particularly when sample volume is limited.