Effects of blood sample handling procedures on measurable interleukin 6 in plasma and serum

Abstract Introduction Interleukin‐6(IL‐6) measurement is used as a biomarker in medical diagnosis, therapy, and prognosis in various diseases. However, several pre‐analytical factors may yield a false IL‐6 result. In this study, we set out to investigate the effects of corrected blood sample handling procedures on measurable IL‐6. Method EDTA plasma and serum samples were collected from 45 healthy individuals. The participants were divided into three groups to perform different handling procedures. Different centrifugal timing, storage temperature, and time were executed on the samples. The changed trends of IL‐6 levels were analyzed. Results At baseline, while the paired plasma and serum IL‐6 values had a good correlation, the plasma levels were higher than serum. In general, the unseparated EDTA plasma kept steady with time. With the increase in storage temperature and time, a more pronounced rise in unseparated serum IL‐6 was observed. Nevertheless, the samples in Group 3 which centrifuged and separated immediately kept stable after a different temperature and longtime storage. Conclusion Sample types, centrifugal timing, storage temperature, and time may affect the IL‐6 levels. A standard blood sample handling procedure should be performed to ensure the accuracy and stability of IL‐6 values.

in instrument technology have improved the analytical accuracy of cytokines enormously. However, lacking standardized handling procedures in analysis influence the biomarker role of a cytokine.
Major errors of the entire diagnostic process are generated within the extra-analytical phases. 7 Especially, the pre-analytical phase may be the source of error in studying cytokines. 8 Pre-analytical variables for the test include blood collection tubes, specimen storage temperature and time, plasma or serum separation, and pretreatment steps. Studies have shown pre-analytical handling steps can influence sample quality and analytical results. 9,10 Non-standardization of pre-analytical procedures may result in inconsistent laboratory results and misleading clinical judgment, thus limiting the application prospects of the assay.
The fact that half-life of IL-6 is short, so that the degradation and production may occur simultaneously during storage under unseparated condition. In addition, coagulation processes affect inflammatory activity, resulting in up-regulation of proinflammatory cytokines in vitro. 11 Usually, the pre-analytical periods are varied for many causes. It is not negligible that immune responses, which affected by environmental temperature and storage time, are still ongoing during the period.
Interleukin-6 is measured through electrochemiluminescence method in our hospital. However, the change of IL-6 under unseparated condition is poorly understood. Therefore, we examined the effects on concentrations of IL-6 in serum or plasma under different storage conditions in the present study. This study of the pre-analytical precautions helps to carry out the correct measurement of this specific marker in clinical laboratory.

| Collection and processing of whole blood
Whole-blood samples from 45 health subjects were collected into vacutainer serum tubes (tubes with clot activator) and plasma tubes (tubes with K 2 EDTA; Guangzhou Improve Medical Instruments Co., Ltd.). Immediately after drawing, tubes were gently inverted 10 times. Subsequently, samples were aliquoted into several cryotubes.
All study participants were divided into three groups and analyzed further with reference to Figure 1. All samples were centrifuged at 1760 ×g for 15 minutes, but centrifugal timing selection was different in three groups. One aliquot from each sample that had been centrifuged and test immediately after blood collection was seen as baseline. The remaining aliquots divided into subgroups and performed different handling procedures. Group 1 were centrifuged and then stored at 4°C room temperature (RT), or 37°C for different periods, while Group 2 was centrifuged after the same storage.
Group 3 was centrifuged and then separated immediately before the longtime storage.

| Statistical analysis
Results are expressed as median, as IL-6 from partial healthy participants were lower than the lower limit of detection. The results below the lowest point of the linear range of the standard curve were assigned detection limit itself and retained for analysis. 12,13 Serum and plasma of baseline IL-6 in one individual were compared using Wilcoxon's signed-rank test and Spearman's rank correlation.
The Friedman test was used to evaluate changes in serum or plasma IL-6 levels over time. A probability (P) ≤0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism v5.00 software (GraphPad software).

| RE SULTS
The median concentrations of IL-6 in baseline were shown in Table 1. IL-6 levels were identified to be significantly different F I G U R E 1 Pre-analytical settings. Grouping and handling procedures are shown between different types of sample, while the levels in plasma were significantly higher compared with serum (P＜0.001). In addition, significant correlation was found in the blood between serum and plasma (P＜0.0001; Figure 2). The concentration of IL-6 did not change after the plasma or serum was separated immediately from whole blood with a longtime storage at the same temperature (Table 2).

| D ISCUSS I ON
IL-6 is a useful diagnostic marker in immune relative diseases.
Following the improvement of detection method, the analytical errors of IL-6 do not influence laboratory diagnostics any more due to the use of ECLIA. However, several pre-analytical variables, such as specimen types, different temperatures, storage length, or even the centrifugal timing, may still affect the stability of IL-6 concentrations. Especially, different changes of cytokines may occur during the pre-analytical period of routine blood samples, from bloods collected to transported and processed.
The potential pre-analytical variables was evaluated in this study.
To be closer to the daily working state, specimens in Group 1 and Group 2 were not isolated from blood cells in our study. The trends of TA B L E 1 Baseline IL-6 levels of Group 1 and Group 2  The effect of EDTA is to prevent the blood from clotting in vitro, which is distinct from the coagulant. Through sequestering the calcium ions, EDTA stabilizes the whole blood in fluid form. 14 Our study on IL-6 is consistent with the reported that EDTA and cold storage can maintain the stability of cytokines. 14 However, others indicate that it is not suitable for certain cytokines. 15 Many studies have shown that cytokine concentrations between different specimen types are different. Some studies demonstrate that basal concentrations are higher in plasma. Some studies, however, are not. [16][17][18][19] These differences may be attributed to the different sample processing procedures, such as freezing or different testing methods.
There is a dynamic balance between the degradation and production of cytokines in vitro. On the one hand, the half-life of cytokines is relatively short, and on the other hand, various stimuli lead to the continuous secretion of cytokines by the blood cells. 20 If one of them is inhibited or promoted, the cytokine level produces a significant change. Nowadays, coagulation has been confirmed to be one of the factors that can induce cytokine release. There is an extensive cross talk between hemostasis and inflammation. [21][22][23] Inflammation can be modulated by the components of coagulation pathway. Several studies have demonstrated that FVIIa, FXa, and thrombin can active blood cells to produce IL-6. [24][25][26] During the blood drawn in serum tubes, the initiation of coagulation pathways is accompanied by the activation of blood cells. We hypothesize that this is a major factor in serum IL-6 elevation.
As biological reaction requires proper temperature and sufficient time, these may be additional potential influencing factors. In this study, with the storage temperature and length increased, the serum IL-6 increased obviously. After the same storage period, the IL-6 elevated more significantly at 37℃ comparing to at RT. Besides, at the same temperature(RT or 37℃), the IL-6 elevated higher after a longer storage time.
Our study shows that serum IL-6 increased by more than nine times after storage at 37°C for 8 hours. This range of increase is unacceptable in laboratory analysis. Shockingly, in previous studies, the serum IL-6 levels can elevate higher, even hundreds of times, after a longtime storage at RT or warmer. 15,27 Actually, higher and earlier elevated may be found in diseased objects due to the bidirectional relation between inflammation and coagulation. This topic requires a further study.
In practical work, samples usually have no timely detection or even no immediate centrifugation due to various reasons. The centrifugal timing selection was therefore considered in this study to investigate whether it influences IL-6 change trend. Centrifugation may be another factor to activate immune cells, while the change ratios of serum IL-6 were higher slightly in pre-centrifugal. However, similar conditions were not found in plasma samples, while EDTA maybe inhibit certain immune reactions. We also measured the IL-6 in timely separated plasma and serum, and there are no different of values between storage specimens and baseline. In other words, this confirms the production of cytokines from blood cells in unseparated specimens.
There are potential limitations in this study. First, each subgroup consists of different participants due to the inconvenient in collecting too many blood samples on one person. Second, the sample size is small in each subgroup. Thus, prospective study of a large sample size is needed for validation.

| CON CLUS ION
Based on the results of this study, factors that affect changes in cytokine levels are various. Incorrect sample handling procedures may result in false results. In general, unseparated EDTA plasma can keep IL-6 steady within 24 hours. In not refrigerated conditions, unseparated serum is recommended to measure IL-6 as soon as possible.
Otherwise, the serum separation should be done.