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Reliable FT4 measurement is critical to assess thyroid function and diagnose and treat thyroid disorders. The Partnership for the Accurate Testing of Hormones (PATH) categorizes FT4 as a biomarker in high need for standardization, and currently high inter-assay variability restricts the interpretation of FT4 results in patient care to assay-specific reference intervals. The CDC CSP has partnered with the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and PATH to create a standardization program for FT4 to improve accuracy, reliability, and comparability of current methods and thus to improve diagnosis, treatment and prevention of thyroidal illnesses. Currently, there are no serum FT4 reference materials available to assess the accuracy and reliability of FT4 assays. CDC has developed an accurate and sensitive reference measurement procedure (RMP) to create commutable serum reference materials with FT4 target values. Assay manufacturers, research and clinical laboratories can use these reference materials to assess their calibration, certify the analytical performance of their measurements, and monitor performance over time by collaborating with CDC CSP. The CDC FT4 RMP uses equilibrium dialysis (ED) with LC-MS/MS based on an internationally recognized ED procedure[1] followed by solid-phase and solvent extractions. Certified primary reference material IRMM468 was used to prepare calibrators. Chromatographic separation is achieved with a gradient of methanol and water with 0.1% formic acid. FT4 is quantified using positive electrospray ionization in positive mode. The intra- and inter-day imprecision of the CDC RMP are 3.0% and 1.1%. A comparison among FT4 RMPs resulted in a +2.5% bias for the CDC RMP to the mean for all labs. The CDC RMP measurement range was 3.02-258 pmol/L and thus suitable for analysis of hypo- and hyperthyroid patients. The CDC FT4 RMP demonstrates good accuracy and precision, and can be used as a viable accuracy base to which routine methods can be compared. An initial comparison study of a commercially available FT4 immunoassay (IA) and the CDC RMP with 24 samples (7.98-109 pmol/L) indicated a mean bias of -37.7%, further indicating a need for standardization. Findings from an IFCC study suggest that alignment of IA measurements to a FT4 RMP can improve comparability and would allow for a uniform reference interval for FT4.[2] CDC CSP established a new standardization program for FT4 to address the needs of the community and assist with improving test comparability and reliability.1. Clin. Chem. Lab. Med. 2011, 49: 1275-81. 2. Clin. Chem. 2017, 63: 1642-52.

Free thyroxine (FT4) measurements are critical in the diagnosis, classification, and treatment of thyroid diseases. It is estimated that about 18 million FT4 tests are requested in the USA per year annually. In clinical laboratories, most of FT4 assays are performed by using immunoassays (I As). However, the significant bias of IAs and large variation between laboratories have been reported. The reference measurement procedures (RMPs) of FT4based on equilibrium dialysis (ED) - liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been established and recognized by the clinical chemistry community. However, the FT4 RMP is relatively low throughput and labor-intensive. Also, an aliquot of 1 mL sample is required for an RMP. A routine FT4 assay high-throughput procedure that is based on ED LC-MS/MS and utilized less sample volume will allow to conduct large biomonitoring studies and establish FT4 levels in US population. In the described method, FT4 in 150 uL of serum was separated from protein-bound T4 at 37.0 oC in 96-well Micro-ED Teflon devices from HTDialysis. The ED conditions suggested by CLSI C45-A guideline were followed. A volume of 150 uL dialysate samples with FT4 was obtained after the ED step. FT4 in the dialysate was purified by extractions before LC-MS/MS analysis. Chromatographic separation of T4 from the sample matrix is achieved on a C18 UPLC column with a gradient of methanol and water with 0.1% formic acid. Quantification of FT4 was performed by using selective reaction monitoring in positive electrospray ionization mode. The IRMM-468 certified primary reference material (JRC, Belgium) of T4 is used for the calibration curves. FT4 concentrations reached equilibrium after 4 hours under current dialysis conditions, which was also observed in the RMP setting. The developed routine FT4 assay 96-well ED system is within 5% bias from the FT4 RMP based on preliminary method comparison study using human serum. The studies to further characterize the FT4 routine method performance are ongoing. In summary, we are developing an analytical method based on a 96-well ED-LC-MS/MS system for the measurements of FT4 in serum. By comparison with RMP, the described method significantly improve throughput and reduce sample volume, which will fulfill the requirement of FT4 routine assay in clinical laboratories and allow for its use in the large biomonitoring studies and other activities in the research and public health settings.