N09 - Lipemia Interference in Automated Immunoassay Measurement of Plasma Phenobarbital Concentration

Background:  Phenobarbital is a commonly used antiseizure medication with a narrow therapeutic window, utilizing Therapeutic Drug Monitoring (TDM) to guide appropriate dosing. Lipemia may interfere with automated immunoassay (IA) results, potentially leading to inaccurate phenobarbital concentrations and inappropriate dose adjustments. However, this interference has not been directly evaluated against a reference method in veterinary medicine.

Objectives:  To evaluate the impact of lipemia on plasma phenobarbital concentrations measured by IA, using high-performance liquid chromatography (HPLC) as the reference method.

 Animals: Archived canine plasma samples (n=80) submitted to the Auburn University TDM service, including non-lipemic, lipemic, and simulated lipemic plasma spiked with 20% Intralipid®.

 

Methods:  Phenobarbital concentrations were measured using IA and HPLC. Percent bias was calculated as the difference between quantitated phenobarbital concentration and the reference method. Non-parametric statistics were applied due to skewed distribution. Lipemia indices were assigned using an automated analyzer, with index 4–6 representing moderate to marked lipemia.

 

Results:  HPLC demonstrated consistent specificity, accuracy, and precision across all lipemia levels. In simulated lipemic plasma, percent bias increased with lipemia severity, with the highest median bias at index 6 (34.4%, 13.6–132.5; P<0.001). In clinical samples, plasma with index 4–6—thresholds where turbidity and analytical interference are more likely—showed significantly higher median bias (23.8%, –13.9–89.9) than non-lipemic samples (1.2%, –27.1–17.1; P<0.001).

 Conclusions and Clinical Importance: Lipemia significantly interferes with IA-based phenobarbital measurements. IA results should be interpreted cautiously in lipemic patients, and confirmation with HPLC considered.