A-098 Evaluation of an in-house cholinesterase assay for serum, plasma, and whole blood

Abstract Background There are two major types of cholinesterase (CHE). Acetylcholinesterase, found in erythrocytes, is responsible for prompt hydrolysis of acetylcholine. Pseudocholinesterase (butyrylcholinesterase), in plasma, has broad activity, including the hydrolysis of certain surgical anesthetics. Analysis of both types of CHE is useful in evaluating insecticide poisoning and pseudocholinesterase deficiency. Recent replacement by the manufacturer of an acetylthiocholine-based CHE assay, with a butyrylthiocholine-based assay, removed the ability to measure acetylcholinesterase in erythrocytes, which is clinically important in the evaluation of chronic insecticide exposure. The purpose of this study was to validate an in-house manufactured acetylthiocholine-based assay for use on the Roche c501 analyzer for CHE tests in serum, plasma, and whole blood (WB). Method Assay reagents were formulated to be consistent with concentrations used in the discontinued manufacturer assay. Chemicals were purchased from Sigma-Aldrich Inc. Studies included method comparison, linearity, imprecision, analytical sensitivity, freeze/thaw stability, sample carryover, and reference intervals (RI). Method comparison was performed using the in-house manufactured reagent compared to the discontinued commercially available reagent, both on the Roche cobas c501 (45 serum and plasma specimens and 47 WB samples). Linearity was evaluated for both serum and WB by diluting a high sample pool with saline to create a set of 6 samples, tested in duplicate. Imprecision was assessed by analyzing a commercial control (1815.1 U/L), a serum pool control (5056.8 U/L) and a WB pool control (1179.6 U/L). Single-use aliquots were tested once daily, for 5 days, 4 replicates per level, per run. Limit of detection (LOD) was assessed by measuring 10 replicates of saline and 3 replicates of a low serum patient pool. A combined serum and plasma RI was established using a total of 147 self-reported healthy subjects (84 serum and 63 plasma specimens; 78 females and 69 males; age range 18-68 years old). The current WB RI (24-37 U/g Hb) was verified using 20 fresh-drawn self-reported healthy donors. All data were analyzed using EP Evaluator. Results Method comparison yielded y=0.966x+39.0, R=0.999 by serum and plasma specimens, and y=1.043x-106.3, R=0.93 by WB specimens. Linearity by serum yielded a slope of 1.01, intercept of 56.3, recovery range was 100.0-104.9% up to 7206 U/L. Linearity by WB yielded a slope of 1.00, intercept of 46.4, recovery range was 98.1-106.5% up to 7194 U/L. Precision studies observed total %CVs =3.8%, for all three controls. Reagent onboard stability was evaluated as part of the precision study and found to be acceptable for up two weeks. The LOD of 20 U/L was verified. Freeze/thaw stability studies using serum resulted with CHE activities changing less than ±4% after 2 freeze/thaw cycles. Sample carryover was not observed on the c501. The nonparametric RI for CHE (serum and plasma combined) was determined to be 2339-5959 U/L. The current RI for WB (24-37 U/g Hb) was verified. Conclusions: The present study found the in-house manufactured CHE reagent for use on the Roche c501 analyzer to be acceptable for clinical use. The in-house CHE reagent exhibited comparable performance to the discontinued commercially available reagents.