PSEUDO-HYPERTHYROXINAEMIA UNCOMMON CAUSE OF IMMUNOASSAY INTERFERENCE DUE TO BIOTIN THERAPY Nilika G Wijeratne1, James CG Doery1,2, Zhong X Lu1,2 1 Department of Pathology, Southern Health, Clayton Victoria; 2 Department of Medicine, Monash University, Clayton, Victoria It is well recognized that immunoassays are subjected to a number of interferences giving abnormal results which may lead to unnecessary investigations and treatment. Here we present our experience on a rare case of interference in immunoassays that use biotin-streptavidin mechanisms in their assay design. Selected analytes after ingestion of 30 mg biotin DISCUSSION Biotin acts as a coenzyme in a number of one-carbon transfer reactions, eg: in gluconeogenesis and lipogenesis. It is widely distributed in food especially in milk and egg. Although not very common, high dose of biotin (≥ 10mg/day) is used therapeutically in some metabolic disorders. CASE Baby WN, 7 days old One of non-identical twins Born with features of liver failure and lactic acidosis due to mitochondrial disorder. 2nd twin healthy. A THYROID FUNCTION RESULTS Done on Beckman Coulter DxI In clinical chemistry, many immunoassays apply biotin and streptavidin in the assay principle to increase the sensitivity of the assay. Presence of high levels of biotin in patient’s sample can cause negative or positive interference depending on the assay format. Analyte Age: 1 week 1 month Ref range FT3 24.9* 28.9* 3.8-6.0 pmol/L FT4 >77.75** 12-30 pmol/L TSH 3.75 7.3* 0.3-5.0 mU/L B Beckman competitive assay Competitive assays Excess biotin competes with biotinylated analog for the binding sites on streptavidin resulting in low signal and apparently high concentration of the analyte. Eg: Beckman: FT3, FT4 Roche E170: DHEAS, Oestradiol, Testosterone LABORATORY ACTIONS TFTs repeated after treatment with Scantibodies - No change in results. Reanalyzed on different assay formats: C Analyte On Advia Centaur (At 1 week) On Abbott Architect (At 1 month) FT3 2.8 pmol/L 3.48 pmol/L FT4 12.8 pmol/L 16.3 pmol/L TSH 2.8 mU/L 6.0 mU/L D Beckman sandwich assay Further inquiry revealed the baby was being treated with biotin 10 mg eight hourly since day 2 of life. Possibility of biotin interference was suspected in Beckman FT3/FT4 assays due to the use of biotin- Streptavidin in its assay format. Biotin was then discontinued and TFTs measured one week later (Beckman Coulter DXI): FT3 4.5 pmol/L FT4 16.3 pmol/L TSH 7.2 mU/L Sandwich assays Excess biotin displaces biotinylated antibodies resulting in apparently low concentration of the analyte. Eg: Beckman: Thyroglobulin Roche E170: Ferritin E FURTHER EXPERIMENT One of the authors ingested 30 mg of biotin. Blood samples were collected at 0, 1, 2, 4, 8 and 25 hrs following ingestion. Serum samples were tested on Beckman for FT4 & FT3 (A) and on Roche E170 for oestradiol (B), DHEAS (C) and testosterone (D). All of these use competitive immunoassay principle with biotin-streptavidin and the results were falsely high. These specimens were also tested on E170 for ferritin (E) and on Beckman for thyroglobulin (F). Both use sandwich assay principle with biotin-streptavidin and the results were falsely low, especially 2-4 hrs post ingestion of biotin. CONCLUSION Understanding assay formats is essential for troubleshooting possible interferences. Therapeutic biotin and possibly over the counter biotin- containing supplements may cause seriously misleading results in assays using biotin-streptavidin mechanisms. F Acknowledgements Dr. David Deam, Healthscope Pathology, for Advia Centaur assays Santo Greco, Melbourne Pathology, for Abbott Architect & Roche E170 assays Moira Rooney, Beckman Coulter, for assay schematics