EDTA Improves Stability of Whole Blood C-Peptide and Insulin to Over 24 Hours at Room Temperature

Timothy J Mcdonald,John O’Connor,Nicola J Pullan,Beatrice A Knight,Roy W A Peake,Beverley M Shields,Andrew T Hattersley,Mandy H Perry,Bin He

doi:10.1371/journal.pone.0042084

Abstract

IntroductionC-peptide and insulin measurements in blood provide useful information regarding endogenous insulin secretion. Conflicting evidence on sample stability and handling procedures continue to limit the widespread clinical use of these tests. We assessed the factors that altered the stability of insulin and C-peptide in blood.MethodsWe investigated the impact of preservative type, time to centrifugation, storage conditions and duration of storage on the stability of C-peptide and insulin on three different analytical platforms.ResultsC-peptide was stable for at least 24 hours at room temperature in both centrifuged and whole blood collected in K+-EDTA and serum gel tubes, with the exception of whole blood serum gel, which decreased to 78% of baseline at 24 hours, (p = 0.008). Insulin was stable at room temperature for 24 hours in both centrifuged and whole blood collected in K+-EDTA tubes. In contrast insulin levels decreased in serum gel tubes both centrifuged and whole blood (66% of baseline, p = 0.01 and 76% of baseline p = 0.01, by 24 hours respectively). C-peptide and insulin remained stable after 6 freeze-thaw cycles.ConclusionsThe stability of C-peptide and insulin in whole blood K+-EDTA tubes negates the need to conform to strict sample handling procedures for these assays, greatly increasing their clinical utility.

Highlights

C-peptide and insulin measurements in blood provide useful information regarding endogenous insulin secretion
Despite the established clinical application of blood C-peptide and insulin measurement, their widespread clinical use is limited by practical restrictions associated with sample collection: It is considered that both insulin and C-peptide concentrations rapidly decrease as a result of protease activity present in blood
The combined results of the 24 hours stability study for all three analysers are shown in Insulin levels did not drop below 90% of baseline over 24 hours at room temperature, for samples stored both as centrifuged and whole blood K+-EDTA (94% of baseline, p = 0.02 and 92%, p = 0.01 of baseline) (Figure 2A)

Summary

Introduction

C-peptide and insulin measurements in blood provide useful information regarding endogenous insulin secretion. Despite the established clinical application of blood C-peptide and insulin measurement, their widespread clinical use is limited by practical restrictions associated with sample collection: It is considered that both insulin and C-peptide concentrations rapidly decrease as a result of protease activity present in blood. Delayed separation of whole blood collected in EDTA plasma results in a dramatic decrease in activity to approximately 10% of baseline concentration after 24 hours and 70% after just 3 hours at room temperature [12]. Other studies suggest insulin is stable for between 4–5 hours in un-separated whole blood collected in both serum gel and plasma tubes at room temperature [13,14]. The reason for the variation in reported stability is unclear, but it has been suggested that this could reflect differences between assays [11]

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