B-170 Age-Specific reference intervals for ethanolamine plasmalogen species in red blood cells using liquid chromatography tandem mass spectrometry

Background Plasmalogens are critical membrane structural components that are mainly generated by de novo synthesis starting in peroxisomes. Hence, patients with defects in peroxisome biogenesis (PBD) exhibit markedly reduced plasmalogen levels. Plasmalogen ratios are traditionally measured by gas chromatography-mass spectrometry (GC-MS); however, this method entails a lengthy sample extraction and derivatization and does not report concentrations of individual plasmalogen species. We have developed a robust and easy-to-implement liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify the 18 most abundant ethanolamine plasmalogen (PlsEtn) species in packed red blood cells (RBCs). However, no reference intervals have been published for individual PlsEtn. Here, we describe the establishment of age-specific reference intervals for individual PlsEtn species and their total values (16:0, 18:0, 18:1 species, and total plasmalogens). Methods Plasmalogens were extracted using methanol containing two labeled internal standards, shaking for one hour at room temperature. Chromatographic separation was performed using an Acquity Premier BEH C18 UPLC column with a binary gradient of 5 mM ammonium acetate in water:methanol (15:85) and 5 mM ammonium acetate in methanol. Analysis was performed using a XEVO TQ-XS Mass Spectrometer with Ultra-High Performance Liquid Chromatography (Waters) in multiple reaction monitoring mode. Eighteen PlsEtn species were quantified using four commercially available standards; additionally, totals were calculated for 16:0, 18:0 or 18:1 species, and for total plasmalogens. Reference intervals were established using 376 RBCs from self-reported healthy volunteers and de-identified clinical samples referred for unrelated testing (182 females and 194 males; range 0 to 88 years). Data was analyzed using the R programming language. The study was approved by the Institutional Review Board of the University of Utah. Results Initial age groups were identified using a model-based clustering algorithm followed by iterative Harris-Boyd analysis. Finally, the adjacent groups were merged if their means differed by less than 10%. Once the final age groups were partitioned, data in each individual age group were analyzed using parametric or non-parametric statistics to determine reference intervals (95%, with 90%confidence intervals). PlsEtn species displayed the lowest concentration in the first few months of life, which increased in childhood until adolescence or adulthood (depending on PlsEtn). For most species, the concentrations increased over time reaching a plateau between 18 and 48 years of age, and then starting to decrease. The total values followed the same trend, with neonates showing significantly lower values compared to other age groups. Conclusions We applied a novel statistical approach to identify age groups and determine age-specific reference intervals for 18 individual PlsEtn in RBC and their totals. Lacking previously published data, this study is critical for supporting test implementation in clinical laboratories.