Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC

Objectives: The aim of our study was a) to optimize assays for measurement of total (T-) and pancreatic (P-)amylase at 37°C based on the principle recommended by the IFCC at 30°C, b) to evaluate the analytical performance of these assays in a multicentric study and c) to establish reference intervals for serum and urine for either method. Methods: Optimized conditions for 37°C were elaborated with regard to substrate concentration, pH, inorganic additives and glucosidase activity. The cleavage pattern of the EPS substrate was studied by HPLC. Liquid ready-to-use reagents for T- and P-amylase were provided to six European laboratories. Results: The assays showed good performance characteristics (median intraassay CVs 1.0% for T- and 1.3% for P-amylase, median interassay CVs 3.0% for either assay, dynamic range 15-fold URL for T- and 30-fold for P-amylase), high correlation with the previous EPS methods (r > 0.996, slope 0.43, intercept < 5 U/L) in serum, heparin plasma and urine and good analytical specificity of the P-amylase assay (residual S-amylase activity 2.4%). Serum reference ranges were found to be 28 to 100 U/L for T- and 13 to 53 U/L for P-amylase (n = 775); URLs in urine were estimated as 490 U/L or 280 U/g creatinine for males and 450 U/L or 380 U/g creatinine for females with total amylase. Conclusion: We believe that these assays based on the 30°C IFCC recommendation represent a further improvement in amylase methodology at 37°C and merit broad application in clinical routine.