MON-602 Improving The Accuracy And Reliability Of Free Thyroxine Measurements Through The CDC Clinical Standardization Programs

Reliable free thyroxine (FT4) measurements are essential for assessing thyroid function and properly diagnosing and treating thyroid disorders. Although FT4 measurements are used extensively in research and clinical practice, the accuracy and reliability of current methods prevent correct detection, treatment, and prevention of thyroid disorders in patient care, making standardization of FT4 measurements a priority. Currently, there are no serum-based reference materials commercially available for FT4. CDC Clinical Standardization Programs (CDC CSP) is working with the Committee for Standardization of Thyroid Function Tests (C-STFT) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the Partnership for the Accurate Testing for Hormones (PATH) to address these issues through development of an accurate and sensitive higher-order Reference Measurement Procedure (RMP) for FT4. The CDC Clinical Reference Laboratory has developed a FT4 method using a modified equilibrium dialysis procedure. (1) FT4 in serum was isolated from the binding proteins in 1 mL equilibrium dialysis cells for 4 hours at 37(o)C. Dialysates were spiked with internal standard (thyroxine-13C6) and further isolated with extractions prior to injection on a liquid chromatography tandem mass spectrometry (LC-MS/MS) instrument. Bracketed calibration and primary reference materials were used to determine the concentration of FT4 in serum. Chromatographic separation was achieved using a C18 reverse phase column with a gradient of water and acetonitrile with addition of 0.1% formic acid. Quantification by selective reaction monitoring (SRM) analysis was performed in the positive ion mode using electrospray ionization (ESI). Two transitions were monitored for each analyte and internal standard, and triplicate injections were used to minimize any instrument instability. The proposed RMP has been evaluated and optimized for precision, accuracy, and sensitivity. The within-day and between-day imprecision are 2.5-2.9% and 0.9-1.3%, respectively. By comparisons with the Reference Laboratory at the University of Ghent, the proposed CDC RMP reported a bias within +1.0%. Factors affecting measurement accuracy were investigated and steps were taken to maximize recovery for optimum performance of the method. FT4 was detectable in all samples and thus suitable for analysis of hypo-, eu-, and hyperthyroid patients. To ensure accurate values are reported for FT4, careful consideration is needed for all steps of sample preparation and analysis. This candidate reference method for FT4 in serum demonstrates good accuracy and precision, and as such this method can be used as a viable base for accuracy to which routine methods for FT4 can be compared. 1. Van Houcke, S.K., et. al. Clin. Chem. Lab. Med. 2011, 49, 1275-1281.