8408 Comparison of Free Thyroxine (fT4) Reference Ranges in Pregnancy: Evaluanting a Novel Equilibrium Dialysis Coupled with Liquid Cromatography Tandem Mass Spectrometry Analytical Method Versus a Standard Routine Immunoassay
Authors: Biscolla R M , Lebkuchen A , Cardozo K H , Carvalho V M , Justa M T , Vieira J G , Chiamolera M I 
Published: October 2024

Abstract
Disclosure: R.M. Biscolla: Employee; Self; Fleury Medicina e Saude. A. Lebkuchen: Employee; Self; Fleury Medicina e Saude. K.H. Cardozo: Employee; Self; Fleury Medicina e Saude. V.M. Carvalho: Employee; Self; Fleury Medicina e Saude. M.T. Justa: Employee; Self; Fleury Medicina e Saude. J.G. Vieira: Owner/Co-Owner; Self; Fleury Medicina e Saude. M.I. Chiamolera: Employee; Self; Fleury Medicina e Saude.
There is a difficulty in assessing thyroid function during the gestational period and one of the most important reasons is the changes in TBG during pregnancy, resulting from the increase in estrogen, lead to interference in the dosages of free thyroxine (fT4) measured by commercial competitive immunoassays, posing limitations on the accuracy and sensitivity of existing assessment methods. Therefore, it is necessary to establish specific reference values for each trimester and for the different methods, so the analysis of fT4 results could be more confidently used in clinical practice.Objective: This study aimed to describe a new direct method for measuring free thyroxine (fT4) utilizing equilibrium dialysis (ED) coupled with liquid chromatography tandem mass spectrometry (LC–MS/MS). Additionally, the performance of this method was compared to a commercial immunoassay, and trimester-specific normal reference ranges for fT4 in pregnancy were determined. Methods: We developed a novel ED LC–MS/MS analytical method employing 96-well dialysis plates for measuring fT4 that demonstrated an adequate sensitivity, precision, and linearity. A total of 291 samples from pregnant women were analyzed, the gestational trimester was determined using ultrasound. Additionally, serum fT4 was assessed using an immunoassay with Beckman Coulter kits to compare the two methods. Results: The lower limit of detection (LLOQ), and upper limit of detection (ULOQ) of the new method were respectively calculated to be 0.4 ng/dL and 20.8 ng/dL. The comparison between the two methods showed median FT4 concentrations of 0.7 ng/dL (range, 0.5–1.1 ng/dL) for the immunoassay and 1.1 ng/dL (0.6–2.1 ng/dL) for the ED LC–MS/MS assay (paired t test, p &lt; 0.0001). A significant correlation between methods was observed (Spearman’s r= 0.4898, p &lt; 0.0001). fT4 reference values for ED LC–MS/MS (2.5–97.5 percentiles) were 0.9–1.9 ng/dL, 0.7–1.4 ng/dL, and 0.7–1.5 ng/dL for the first, second, and third trimesters, respectively, whereas median TSH levels were 1.7 mIU/L (0.05–4.0 mIU/L), 1.7 mIU/L (0.44–3.7 mIU/L), and 1.9 mIU/L (0.28–3.5 mIU/L) for the first, second, and third trimesters, respectively.Conclusions: The study with a recent developed LC-MS/MS fT4 method pointed to the differences with an immunoassay method and underscores the importance of establishing trimester- and method- specific normal reference ranges in pregnancy.
Presentation: 6/3/2024