Bilirubin Interference in Plasma Amino Acid Analysis by Ion Exchange Chromatography.

Objective: To evaluate the effect of bilirubin interference on plasma amino acid analysis by Ion Exchange Chromatography (IEC).

Study Design: Cross-sectional (method validation) study.

Place And Duration Of Study: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi from August 2016 to July 2017.

Methodology: Twenty non-icteric samples from paediatric patients were collected in lithium heparin tubes and analysed for amino acids on IEC-based Biochrome® 30+ Analyzer (Harvard Biosciences UK). Baseline bilirubin levels were noted. Samples were spiked with neonatal bilirubin standard with concentration of 488.4 mol/L (Spinreact®-Spain) at final concentrations of 50, 150 and 230 mol/L and re-analysed for amino acids at these three concentrations.

Results: Among the 20 selected patients with normal amino acid profiles, 12 (60%) were males. Majority (55%) were in age group of 1-5 years. Significant difference was observed for Arginine (p = 0.01), Histidine (p = 0.001), Isoleucine (p = 0.01), Leucine (p = 0.007), Lysine (p = 0.005), Ornithine (p = 0.03) and Phenylalanine (p = 0.02). Mean rank of these amino acids showed decreasing trend with the increase of bilirubin concentration, and pronounced interference was identified at bilirubin level of 50 mol/L. No difference was observed for alanine, citrulline, glutamic acid, glycine, methionine, proline, threonine, tyrosine, asparagine, aspartic acid, cystine, valine and tryptophan.

Conclusion: Bilirubin significantly interferes with certain amino acid levels when analysis is carried out by ion exchange chromatography. A close follow-up of such patients with other biochemical tests and a repeat amino acid analysis, after jaundice is settled, is recommended to confidently rule out any possible inherited metabolic disorder in these patients.