Long-term stability of biochemical markers in pediatric serum specimens stored at − 80 °C: A CALIPER Substudy

Abstract

Objectives

Pediatric serum samples collected from healthy children in the CALIPER (Canadian Laboratory Initiative in Pediatric Reference Interval) project are stored at − 80 °C for various periods of time. This study aimed to determine the stability of chemistry, protein, and hormone analytes under these conditions.

Design and methods

Serum samples collected from children of 0–18 years of age attending outpatient clinics were pooled into a single pool or into age-group specific pools. Following baseline measurement, each pool was aliquoted and kept frozen at − 80 °C until analysis. Samples were analyzed for 57 biochemical markers at monthly intervals over a 10–13 month period and each aliquot was subject to one freeze–thaw cycle before analysis. The analysis was performed on VITROS® Chemistry System, COBAS INTEGRA® 400 Plus and IMMULITE® 2500. Values obtained at monthly intervals were compared to baseline measurements and examined for trends over time.

Results

A majority of analytes measured in this study showed no significant time-dependent change relative to baseline or trend over time after up to 13 months of storage. PTH showed up to 27.2% decline after 10 months of storage with most of the decline evident after 2 months. Most analytes showed variability over time, which is thought to reflect assay variability rather than changes in analyte stability.

Conclusions

The study shows stability for a majority of analytes stored in serum at − 80 °C after up to 13 months of storage. Samples do not require immediate testing for reference interval determination for the selected analytes with possible exception of PTH.

Introduction

Reference intervals are commonly used for the interpretation of patients test results. Accurate population-based reference intervals corresponding to a particular patient are not always available. This is of particular concern in pediatrics. Physiological changes associated with child development can result in changes in the reference intervals that are potentially age, gender, Tanner stage, ethnicity, and perhaps geographic location, specific. Recent reference interval literature reviews indicate that age-specific reference intervals are available only for a limited number of biomarkers/analytes, many determined on outdated methods, and predominantly on a Caucasian population using hospitalized patients [1], [2], [3], [4].

The Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) project aims to establish a comprehensive pediatric reference database with partitions for age, gender, Tanner stage, and ethnic origin for analytes found to be affected by these variables [5]. The reference intervals are being established on healthy participants recruited through a number of community outreach initiatives for key clinical chemistry, protein, and hormone analytes [6]. The transference of reference intervals between different instrument platforms will also be tested and separate reference intervals will be established for each platform if necessary. Preliminary reference intervals determined in a series of pilot projects from metabolically stable outpatient clinics are also available [7], [8], [9], [10].

A key aim of the CALIPER project has been the establishment of periodically updated biobank of samples from healthy and outpatient clinic children. In addition to the current reference interval initiatives, the biobank will also be a source of samples for future updating and validation of reference intervals for current biomarkers on novel analytical platforms and establishment of reference intervals for new biomarkers that may reach the clinical practice in the future.

One of the main issues for samples stored in the biobank is analyte stability. Stability of biochemical markers must be assured before establishment of new reference intervals. Most biomarkers targeted by the CALIPER initiative have previously been reported as stable; however with discrepancies in the literature, differences in storage conditions, sample processing or matrix [11], [12], [13], [14], [15], [16], [17]. This CALIPER sub-study was designed to experimentally assess the stability of key serum chemistry, protein and hormone biomarkers in serum samples stored at − 80 °C for up to 13 months.