Assay-specific reference intervals and evaluation of key preanalytical factors for C-terminal agrin fragment measured...

Growing interest surrounds circulating biomarkers for assessing neuromuscular integrity and muscle health. C-terminal agrin fragment (CAF) has emerged as a promising marker associated with sarcopenia, ageing, and renal dysfunction. Limited reference data and insufficient characterisation of preanalytical factors restrict clinical application.

This study established a reference interval for CAF in lithium-heparin (Li-heparin) plasma and investigated key preanalytical variables influencing CAF measurements using the Abcam ab216945 ELISA. Plasma samples from 120 healthy adult blood donors were used to determine reference intervals. CAF concentrations were measured using a commercial ELISA. Stability was assessed at room temperature, during storage at -20 °C, after freeze-thaw cycles, and after delayed centrifugation. Matrix comparison between Li-heparin and K-EDTA plasma was performed in 28 paired samples.

The overall non-parametric reference interval for CAF in Li-heparin plasma was 2917.5-5877.0 pg/mL (median 4260.0 pg/mL). A weak positive association between CAF concentration and age was observed, but no sex-related difference. CAF remained stable for up to seven days at room temperature, up to three freeze-thaw cycles and up to at least five hours of delayed centrifugation. Storage at -20 °C was acceptable up to 30 days only and showed increased individual variability already present after 14 days. Matrix comparison demonstrated an average bias of 14.6% between Li-heparin and K-EDTA plasma.

CAF demonstrates acceptable stability in Li-heparin plasma under standard laboratory conditions. The study establishes a CAF reference interval in Li-heparin and identifies matrix-dependent differences, supporting improved analytical standardisation and interpretation of CAF measurements.