Estimation of age-specific reference intervals for laboratory blood tests in children

Abstract

Introduction: Reference intervals (RI) are an important clinical assessment tool used by physicians and laboratory professionals to interpret test results. A RI is commonly defined as a range between the 2.5th and 97.5th percentile and represents the middle 95% of the reference population. The estimation of RIs is a complex process which involves defining a reference population, selecting reference individuals, collecting, and testing reference samples, and applying appropriate statistical methods.

Estimation of RIs in children is challenging for a number of reasons. There is a need for adequate representation of the general population in the reference population. Analytes are influenced by the nutrition, development, and growth of a child. Hence, RIs should reflect these age-dependent changes in the analytes as children grow older. Most laboratory tests are instrument dependent and variation between different analysers has been documented in several studies. In addition, the best statistical methods for estimating and reporting RIs varying with continuous age are still not widely agreed upon. Finally, there are no available guidelines for laboratories to validate continuous age-specific RIs for their use.

Hence, this project addressed the following four gaps: 1) the lack of appropriate and reliable continuous age-specific paediatric RIs, 2) an absence of head-to-head comparison of laboratory results on different analysers, 3) the lack of clear evidence and accompanying guidelines regarding the best statistical methods for constructing continuous age-specific paediatric RIs, and 4) the lack of guidelines for validating continuous age-specific RIs.

Methods: This research project consists of four studies. Data collected as part of the HAPPI Kids study, from children 30 days to < 18 years, were used to compare laboratory test results on different analysers and to establish and validate continuous age-specific RIs. Differences in mean test results of analytes by analyser types were investigated using mixed-effects regression analysis and by comparing maximum variation between analysers with analyte-specific allowable total error reported in the Westgard QC database. Continuous age-specific paediatric RIs were estimated using quantile regression where power variables in age were selected based on fractional polynomial regression for the mean, with modification by sex when appropriate. The continuous age-specific paediatric RIs were considered valid if more than 90% of test results from a routine laboratory were within the intervals. A systematic review was conducted to examine the range of statistical methods used over the past 25 years for the estimation of age-specific RIs and to identify trends in usage and reporting. A simulation study was conducted to evaluate and compare statistical methods for constructing continuous age-specific RIs in children under different scenarios and for different sample sizes.

Results: The variation in the mean test results across five analysers was not clinically significant for 24 out of 30 biochemistry analytes examined and common continuous age-specific RIs were established for children 30 days to 18 years. The continuous age-specific paediatric RIs were validated in a routine laboratory after initial analysis for most analytes and after secondary analysis for a few analytes.

According to the results of the systematic literature review, a wide variety of statistical methods have been used for estimating age-specific RIs in children over the past 25 years. However, there has been insufficient uptake of modern statistical methods in estimating continuous age-specific paediatric RIs for analytes that change with age. The results of the simulation study show that in comparison to the commonly used methods for estimating continuous age-specific paediatric RIs, the applied novel method that we applied performed better in most scenarios for sample sizes of 400 and over.

Discussion: The comparison of test results for common biochemistry analytes should provide evidence of lack of differences between analysers and in turn improve clinical interpretation. It is recommended that continuous age-specific RIs are used as a gold standard for interpreting test results. Laboratories should estimate or validate continuous age-specific paediatric RIs for their use by applying appropriate statistical methods based on the evidence provided in this research.