Effects of sample handling on the stability of interleukin-6 in patients with breast neoplasms

Introduction

Breast cancer is the second leading cause of cancer death among women [1]. Early diagnosis and treatment can greatly improve outcomes. Inflammation is often associated with the development and progression of breast cancer [2]. Interleukin-6 (IL-6) is a pleiotropic cytokine. IL-6 is produced by a variety of cell types, including macrophages, T cells, B cells, endothelial cells, and tumor cells [3]. IL-6 plays an important role in immune-related diseases, such as tumors [4]. IL-6 also plays an important role in breast cancer for its important role in immunity [5]. So IL-6 is considered as a multifunctional cytokine with obviously tumor-promoting effects in breast neoplasms. High IL-6 levels are correlated with poor overall survival in metastatic breast neoplasm.

Recently, IL-6 has attracted extensive interest in potential monitor and therapeutic applications, IL-6 is related with neoplasm pathogenesis [6]. The variation trend of IL-6 may reflect the progress or regression of diseases. So it’s very important to get the real value of IL-6 in patients. Blood sample handling and storage conditions may influence the real value [7]. We found IL-6 level of breast neoplasm patients had aberrance during the pre-analytical phase. Lots of studies had investigated on the factors affecting IL-6 stability in health blood sample. Yan et al. had found sample types, centrifugal time, storage temperature, and time may affect the IL‐6 levels [8, 9]. Stervbo et al. found the concentrations of TNF-α, IL-4, IL-17F, and IL-22 decreased after storage at room temperature for 4 h before freezing [10]. IL-6 has relationship to all stages of tumor pathological process, including initial, progression and metastasis [11]. In order to monitor reliable changes during the pathological process, appropriate methods should be used to get accurate IL-6 values. The real value is important for safe treatment and prevents adverse events. However, false value can lead to misdiagnosis or even improper treatment of breast neoplasms. Pre-analytical variables for the test include sample type, transportational process, and preliminary treatment, all of them are the key factors to influence the value accuracy in clinical laboratory.

In the detection procedure, a certain amount of time is required for sample preparation, especially for the process of experiment research, most of which are collected and tested together. Temporary storage of samples is a common practice in many studies. IL-6 stability in specimens (either serum or plasma) stored at routine temperatures or ultra-low temperature of neoplasms patient has not been adequately investigated [12]. Therefore, we examined the trustworthy measurement of IL-6 concentrations process in pre-analytical phase.

Materials and methods

Collection and processing of whole blood

Stage 1–4 breast neoplasms cancer patients plasma were collected. These patients were composed of 30 women with breast neoplasms, ranging between 28 and 58 years. Whole blood samples were collected before receiving chemotherapy. Direct extraction from elbow venous blood. The phlebotomy was carried out in a standardized way. Whole blood samples were collected into three tubes (tubes with lithium-heparin anticoagulant and analyzed further with reference to Figure 1). Three tubes were centrifuged at 1760×g for 15 min.

Figure 1:

Pre-analytical settings and handling procedures.

The blood collection tubes were gently inverted 10 times after the blood was drawn immediately. The sample (whole blood samples) is then divided into three tubes. All tubes had been centrifuged and test as baseline immediately. The first group tubes were test and stored at 4 °C. The other tubes were test and stored at 25 °C (be equal to room temperature). After different times same tubes had taken out for testing. Unified conditions were all homogenized (Table 1).

Table 1:

Relationship between pathological characteristics.

Category	Patients, n (total 20)	%
Age, years
<40	2	10
40–60	14	70
>60	4	20
Positive lymph nodes, n (%)
N0 0	6	30
N1 1–3	6	30
N2 ≥4	8	40
Depth of invasion
T1	7	35
T2	9	45
T3	3	15
T4	1	5
Molecular subtype
Luminal A-like	2	10
Luminal B/HER−	2	10
Luminal B/HER+	6	30
HER2	7	35
Triple-negative	3	15

Results

The concentrations of IL-6 in the control samples that centrifuge and test immediately after blood collection as a baseline, as well as 0 h concentration. The changes of the median concentrations of IL-6 when stored from 1 h to 12 h at 4 °C and 25 °C are shown in Figure 2. IL-6 levels were identified to be stable at different temperatures (Figure 2). However, significantly increased IL-6 concentration was found in healthy patients when stored at 25 °C for 24 h compared to 4 °C (Figure 2, p=0.003).

Figure 2:

The change of median concentration of IL-6 in healthy patients stored at 4 °C and 25 °C.

The changes of IL-6 levels in blood samples of patients with breast neoplasms at different storage temperatures are show in Figure 3. IL-6 levels slightly increased after stored at 4 °C for 2 h. However, significantly higher levels of IL-6 were found in plasma when stored at 4 °C for 24 h compared to baseline (0 h) (Figure 3A, p<0.001). Interestingly, though the concentrations of IL-6 did not alter in the patients group with breast neoplasms when stored at 4 °C, it increased significantly when stored at 25 °C. The median values increased more than 91-folds to baseline after stored at 25 °C for 4 h (Figure 3B). Surprisingly, the IL-6 levels increased in plasma separated from the whole blood when stored at 25 °C, but kept stable when stored at 4 °C after 24 h (Figure 4).

Figure 3:

Concentrations of IL-6 in patients with breast neoplasms stored at 4 °C (A) and 25 °C (B) at different times.

Figure 4:

The levels of IL-6 in separate plasma when stored at 4 °C and 25 °C after 24 h.

There was no significant difference compared to the control group; *p>0.05.

In this study, the levels of the inflammatory cytokines IL-6 were significantly different after stored at different temperatures (p<0.001; Figure 3A, B). Their levels in the patients group with breast neoplasms were significantly higher than in the control group, indicating that patient types, centrifugal timing, storage temperature, and time may affect the IL-6 levels.

Discussion

Breast neoplasm is a heterogeneous disease with variable biological and clinical behaviors. Tumor growth is related to the invasion, metastasis, and immune escape response of proliferating cells after the imbalance of cell proliferation [12, 13]. These processes are accompanied by changes in growth-regulating cytokines [14]. Cytokine levels can reflect clinical outcome. Inflammatory factors, as the main signal transducers in tumor microenvironment, have been gradually recognized in the development and progression of breast neoplasm [15]. Many studies have found inflammatory factors, such as increased concentrations of IL-6, TNF-α, IL-1β, IL-10, and TGF-β in patients with breast neoplasms [16]. These pro-inflammatory factors can promote tumor growth, inhibit apoptosis, induce epithelial to mesenchymal transformation, and ultimately promote the invasion and metastasis of breast neoplasm [17].

It is important to accurately monitor cytokine levels in breast neoplasm patients. Following the improvement of detection method, ECLIA assays are highly sensitive and precise due to an extremely low background of chemiluminescence. However, to our surprise, unexpected findings occurred during the test. We found that several pre-analytical variables, such as different temperatures, storage conditions and time may affect the stability of IL-6 concentration values. It is necessary to clearly understand the reasons of deviations in results and to reduce the occurrence of deviations in order to improve the level of diagnosis and treatment to avoid missed diagnosis and misdiagnosis.

The present study investigates the stability of IL-6 concentrations during different blood sample handling procedures in patients with metastatic breast neoplasm. In our study, in the healthy group the concentrations of IL-6 were stable when stored at 4 °C and 25 °C after 1 h up to 12 h (Figure 2). In the breast neoplasm group, the concentrations of IL-6 were slightly elevated before 12 h, except when stored at 4 °C. Surprisingly, IL-6 levels were found to be significantly increased from the beginning when stored at 25 °C (Figure 3). Similarly, the IL-6 concentration increased significantly after 12 h when stored at 4 °C.

Though the concentrations of IL-6 did not alter in the healthy group after 12 h and kept stable for 72 h when stored at 4 °C, it increased significantly after 12 h when stored at 25 °C (Figure 2). However, regardless of whether stored at 4 °C or at 25 °C, IL-6 concentrations increased significantly (Figure 3).

Many of the results had shown that the cytokines concentration between different sample types and different pre-analytical procedures are different [18, 19]. Kristin et al. demonstrated that trustworthy measurement of IL-1b, IL-2, IL-6, sIL-6ra, and mmp-9 relies on handling of whole blood sample at low temperature and rapid isolation of plasma and serum [20]. So different sample handling procedures, such as freezing or different test methods may lead to different results. The main reasons probably attribute to different anticoagulation activate the blood cells to release cytokines in vitro. Different sample processing procedures such as freezing or different temperature may stimulate different speed of cells produce cytokines in vitro [21, 22]. These factors should be fully considered in cytokine detection. But surprisingly, IL-6 levels rose even after serum were separated from the whole blood cells (Figure 4). This may remind us IL-6 levels may rise except for the blood cells original release. There are many other factors affecting IL-6 detection levels. It may be related to the structural changes of IL-6. The structural modification of IL-6 resulted in the increase of detection epitopes, which eventually led to the pseudo increase of IL-6 level [23]. This conjecture needs to be confirmed by further research.