B-369 Ensuring Quality of Free Hormone Measurements Through Detection of Membrane Leakage in Equilibrium Dialysis

Abstract

Hormones circulate in the body either bound to carrier proteins or unbound (free). The unbound fraction is considered the biologically active form and of greater clinical relevance for disease diagnosis than total hormone levels. Equilibrium dialysis (ED) of serum samples is considered the gold standard procedure for separating free hormones from their bound counterparts. However, membrane leakage during ED can lead to misleadingly elevated free hormone values due to contamination of free hormone in dialysate by bound ones, thereby compromising the diagnostic utility of free hormone biomarkers. In the current study, we used a free serum thyroxine (FT4) assay based on ED coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) as an example to explore practical approaches for detecting membrane leakage, with the aim of enhancing the assay quality for free hormone measurement. A commercially available micro-ED plate with dialysis membrane of 8 kDa molecular weight cut-off was employed to separate FT4 from bound T4. FT4 extraction from the dialysate was performed using a 96-well C18 solid-phase extraction (SPE) plate, followed by quantification via LC-MS/MS in positive ion mode. Certified primary reference material was utilized for FT4 assay calibration. To verify ED membrane leakage, each serum sample was measured in duplicate. Leakage was determined using two different approaches: First, if two FT4 replicate measurements differed by more than 25%, the dialysate was classified as contaminated. Second, the contaminated dialysates were crystallized with a-Cyano-4-hydroxycinnamic acid (CHCA) and subsequently analyzed using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS) to identify macromolecule biomarkers present in the contaminated samples. By comparing duplicate measurements of the same samples, we identified approximately 4% of dialysate samples exhibiting membrane leakage. MALDI-MS analysis indicated that human albumin (molecular weight: 66.5 kDa) was the predominant protein present in samples with leakage during ED, while it was absent in the non-contaminated samples. The sensitivity of MALDI-MS is adequate to detect 1:50,000 (v/v) serum contamination in the dialysate. MALDI-MS offers a sensitive, high-throughput, and cost-effective method for detecting ED membrane leakage, thereby improving the quality of free hormone assays, including FT4 assay. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.