Biotin interference in routine clinical immunoassays

Abstract

Background: Laboratory examinations play a crucial role in medical diagnostics and treatment, necessitating the identification of interference factors to ensure accurate results. Biotin, a common dietary supplement, can interfere with immunoassays utilizing biotin-streptavidin interactions. Studies have documented biotin's significant impact on thyroid function tests and various immunoassays, prompting the need for effective mitigation strategies.

Methods: Samples were collected from various clinical departments and analyzed for biotin levels. Biotin interference was evaluated using both old and new Elecsys reagents in assays for thyroglobulin (TG), alpha-fetoprotein (AFP), anti-thyroglobulin (ATG), and free thyroxine (FT4). Biotin spike-in and depletion tests were conducted to assess interference mitigation methods. Additionally, the biotin tolerance of Roche and Abbott immunoassay systems was compared.

Results: Biotin levels were measured in 78 participants from different clinical departments: health management center (n = 13), emergency department (n = 21), intensive care unit (n = 12), gynecology department(n = 3), and hemodialysis department (n = 29). Patients undergoing hemodialysis and those in the intensive care unit (ICU) demonstrated significantly elevated biotin levels (mean = 3.282 ng/mL and 3.212 ng/mL, respectively) in comparison to other patient groups (p < 0.05), likely attributable to the intake of biotin-containing supplements. Biotin levels >500 ng/mL caused a 20 % change in assay values, resulting in false-low results for TG and AFP and false-high results for ATG and FT4 with older Elecsys reagents. Setting a 10 % change as the threshold, the newer Elecsys reagents demonstrated improved resistance against biotin interference, tolerating concentrations of 1000 ng/mL to 3000 ng/mL depending on the specific tests, consistent with the Roche package inserts. We employed a biotin depletion method that effectively restored assay accuracy for older reagents, generally resulting in less than a 10 % change when biotin levels were below 400 ng/mL. However, this depletion method was unnecessary with the newer reagents due to their increased biotin tolerance. Comparing the Roche and Abbott systems revealed significant differences in biotin tolerance. The Abbott system demonstrated greater resilience to biotin interference, while the Roche system showed biotin interference in assays for carcinoembryonic antigen, cancer antigen 125, cancer antigen 153, cancer antigen 19-9, with changes exceeding 30 % at 500 ng/mL of biotin.

Conclusions: Our study highlights the high prevalence of elevated biotin levels in hemodialysis and ICU patients, serving as a critical reference for clinical result interpretation. We confirm that Roche's newer reagents exhibit enhanced biotin tolerance, consistent with the manufacturer's claims, and demonstrate that biotin depletion effectively restores assay accuracy. These findings provide valuable methodological guidance for mitigating biotin interference in clinical immunoassays.

Keywords: Biotin depletion; Biotin tolerance; Elecsys; Hemodialysis; ICU; Immunoassay interference.

Fig. 1

Biotin analysis of human peripheral blood. Collected samples (n = 78) were determined biotin level by ELISA. (A) The different colors represent samples from different sources. Blue from health management center (n = 13), red from hemodialysis department (n = 29), green from emergency department (n = 21), violet from ICU (n = 12), orange from gynecology department (n = 3). (B) Ten hemodialysis patients were randomly selected, and blood samples were collected both before and after hemodialysis to compare changes in biotin levels. These data are plotted as mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. The Student's t-test was used in calculating p-value as detailed in the Supplementary Tables S1–2.

Fig. 2

Select clinical samples with low, medium, and high range of TG, ATG, AFP, and FT4 for pooling. Add 0 to 1500 ng/mL of biotin to the pooled serum with low, medium, and high concentrations, respectively. After aliquoting, test the samples using both the new Elecsys reagent and the old Elecsys reagent on the Cobas e602 instrument. Blue-spot indicates the old generation reagent, red-spot indicates the new generation reagent. (A–C) stand for low, medium, high range of pooled TG serum. (D–F) stand for low, medium, high range of pooled ATG serum. (G–I) stand for low, medium, high range of pooled FT4 serum. (J–L) stand for low, medium, high range of pooled AFP serum. The data are plotted as mean ± SD in triplicate or quadruplicate. The p-value was evaluated by Kruskal-Wallis test as detailed in the Supplementary Table S3.

Fig. 3

Select clinical samples with medium levels of TG, AFP, FT4, and ATG, and add 0 to 1500 ng/mL of biotin. Aliquot 350 μL of the biotin-added samples and use 20 μL of streptavidin-magnetic beads to remove the biotin and test the conditional samples (with and without biotin removal) with both the new and old reagents on the Cobas e602 instrument. The blue-line indicates the biotin-added group and red-line indicates the biotin-depleted group. (A) and (B) represent the biotin tolerance of the old and new TG reagents respectively. (C) and (D) represent tolerance of the old and new AFP reagents respectively. (E) and (F) represent tolerance of the old and new Free T4 reagents respectively. (G) and (H) represent tolerance of the old and new ATG reagents respectively. ∗p < 0.05, the Student's t-test was used in calculating p-value as detailed in the Supplementary Table S4.

Fig. 4

Comparison of biotin tolerance test between Roche and Abbott assays. Pooled serum spiked with 0–3000 ng/mL biotin were analyzed using Roche Cobas and Abbott Architect immunoassays. The red line indicates the results from the Abbott Architect, while the blue line represents the results from the Roche Cobas. (A) CEA (B) CA125 (C) CA153 (D) AFP (E) CA199 (F) FPSA (G) TPSA (H) ATG (I) FT4 (J) TSH. The onboard reagents for CEA, CA125, CA153, and CA199 belonged to the older reagents, while AFP, FPSA, TPSA, ATG, and FT4 belonged to the newer reagents. Data are plotted as mean ± SD in triplicates. The percent change of Roche and Abbott immunoassays are calculated and detailed in Table 3, Table 4 respectively.