Stability of 12 T-helper cell-associated cytokines in human serum under different pre-analytical conditions

Abstract

Cytokines are soluble and readily analyzed signaling molecules which reveal vital cues about the state of the immune system. As such, they serve in diagnosis and monitoring of immune-related disorders, where strictly controlled handling of the samples including storage and freeze/thawing procedures are required. In basic research and clinical trials, human serum samples can be left for long-term storage before processing. Storage space is commonly limited in scientific laboratories, which require storage of fewer but larger aliquots of patient serum samples. There are also practical limitations to the number of analytes to be processed at the same time. Further, new findings and technological progress might prompt analysis of hitherto unconsidered or undetectable molecules. Repeated freeze/thawing of serum samples is therefore a likely scenario, raising the question of the stability of the measured analytes under such conditions. To address this question, we subjected serum samples with spiked-in T-helper cell associated cytokines to several cycles of freeze/thawing under different conditions, including storage at −20 °C or −80 °C and thawing at 4 °C, 22 °C, and 37 °C, respectively. The concentration of TNF-α, IL-4, IL-17F, and IL-22 decreased after storage at room temperature for 4 h before freezing. Generally, storage at −20 °C resulted in reduced cytokine concentrations. This contrasts storage at −80 °C, which gave stable analyte concentrations; unaffected by repeated freeze/thaw cycles. The study presented here highlights the need for sentinel samples with known cytokine concentrations as internal control for the freeze/thaw process.

Introduction

Cytokines are pleiotropic signaling molecules with the capabilities to promote or limit inflammatory reactions. Cytokines can act in an autocrine, paracrine, or endocrine fashion [1]. Although first identified in lymphocytes, a wide range of cells, including epithelial cells, have the capacity to produce cytokines in response to both pathological and physiological cues [2]. These soluble and blood-circulating factors are thus important for diagnostic purposes as for example in sepsis and several autoimmune disorders [3], [4], [5].

The in vivo half-life of cytokines is relatively short and usually in the range of minutes [6], [7]. Some cytokines such as interleukin(IL)-6 and TNF-α have been found unstable in unprocessed whole blood [8], [9], while others, such as IL-8, and IFN-γ are stable for hours or days [8], [10]. Despite these differences in stability, reference levels have been established [11].

For diagnostic purposes, samples are usually processed in a timely fashion and, above all, they are only manipulated once. In basic research and clinical trials, human serum samples can be left for long-term storage, usually until the whole study is completed and all samples can be simultaneously analyzed to avoid batch effects. Technical constraints however might impose a limit on the number of analytes that can be measured at a given time. Additionally, scientific questions may evolve over time prompting an evaluation of previously analyzed samples for a new interesting analyte. In such cases, each sample should be stored in single use aliquots. However, for most laboratories, this can be extremely challenging, since a large number of tubes and sufficient storage capacities would be required. A more realistic use of storage capacity is to store fewer but larger aliquots. This in turn requires repeated freeze/thawing of a sample, raising concern about the stability of measured cytokines.

Here, we address the question of cytokine stability by subjecting cytokine spiked-in serum samples to several cycles of freeze/thawing under different conditions including storage at −20 °C or −80 °C and thawing at 4 °C, 22 °C, and 37 °C.