Comparison of three indirect methods for verification and validation of reference intervals at eight medical laboratories: a European multicenter study

Objectives: Indirect methods for the indirect estimation of reference intervals are increasingly being used, especially for validation of reference intervals, as they can be applied to routine patient data. In this study, we compare three statistically different indirect methods for the verification and validation of reference intervals in eight laboratories distributed throughout Europe.Methods: The RefLim method is a fast and simple approach which calculates the reference intervals by extrapolating the theoretical 95 % of non-pathological values from the central linear part of a quantile-quantile plot. The TML method estimates a smoothed kernel density function for the distribution of the mixed data, for which it is assumed that the ''central'' part of the distribution represents the healthy population. The refineR utilizes an inverse modelling approach. This algorithm identifies a model that best explains the observed data before transforming the data with the Box-Cox transformation.Results: We show that the different indirect methods each have their advantages but can also lead to inaccurate or ambiguous results depending on the approximation of the mathematical model to real-world data. A combination of different methodologies can improve the informative value and thus the reliability of results.Conclusions: Based on routine measurements of four enzymes alkaline phosphatase (ALP), total amylase (AMY), cholinesterase (CHE) and gamma-glutamyl transferase (GGT) in adult women and men, we demonstrate that some reference limits taken from the literature need to be adapted to the laboratory's particular local and population characteristics.