Clinical Detection of Peripheral Blood Natural Killer Cell Activity

Natural Killer (NK) cells are specific immune cells in human immune system. They have a quick effect and can exert a cytotoxic function without prior sen-sitization, and they show great application potential in cell-based immunotherapy, anti-infection in vivo. NK cell activity in peripheral blood can be used as one of the biomarkers of immune function response. It has a great positive guiding significance for the clinical prognosis of tumor patients, the prevention of cancer and anti-aging. The clinical detection strategies of NK cell activity in circulation mainly grouped into five types: methyl thiazolyl tetrazolium colorimetric, lactate dehydrogenase release, radionuclide labeling, flow cytometry and NK Vue cytokine release method. It has played an important role in different stages of clinical application development. This paper will make a comparative review of the above-mentioned detection strategies for the NK cell activity.


Introduction
Natural killer cells were first discovered in peripheral blood to recognize and effectively kill cancer cells in patients with leukemia [1]. Unlike T cells and B cells, NK cells do not require antigen stimulation when they play a killing effect and secrete anti-inflammatory factors. But these three kinds of cells are considered to be the effector cells of the human body's specific immunity, and are the main functional cells for monitoring malignant diseases in the body. NK cells account for about 10% to 15% of peripheral blood lymphocytes [2]. Circulating NK cells in the blood have a shorter survival time, with a half-life of only 7 -10 days [3]. In clinical application, flow cytometry is used to detect the expression of NK cell surface marker CD3-CD56+, and the expression rate is greater than 60%, which can be used in clinical practice. According to the difference in the expression density of CD56 molecules on the surface of NK cells, NK cells are divided into two subgroups, namely CD3-CD16+CD56 dim and CD3-CD16-CD56 bright . The CD3-CD16+CD56 dim subgroup accounts for more than 90% of NK cells in the peripheral blood, and the main role in the blood is cell killing, such as tumor cells, virus-infected cells and senescent cells and other abnormal cells [4] [5] [6], expressing Interleukin-12 receptor (IL-2R) with high affinity, and the CD3-CD16-CD56 bright subgroup only accounts for 10% of peripheral blood NK cells. The cells of this subgroup are mainly immunomodulatory. CD3-CD16-CD56 bright NK cells synthesize and secrete a variety of pro-inflammatory factors (interferon-γ, tumor necrosis factor-α, etc.), and express high-affinity IL-2 receptors [7] [8]. However, mature NK cells in the blood of experimental mice express CD27 and CD11b, and can be divided into four types according to the density of their expression on the cell surface, namely CD11 blow CD27 low , CD11 blow CD27 high , CD11b high CD27 high , and CD11b high CD27 low [9].
Researchers have found that when the number of NK cells and the activity of NK cells in the peripheral blood of the elderly (age ≥ 80 years) are higher, the mortality rate is lower, the life time is longer, and some age-related diseases can be avoided. This phenomenon is more obvious in centenarians [10] [11] [12]. More and more evidences show that the activity and killing function of NK cells in cancer patients are significantly lower than those in normal people, such as breast cancer [13], lung cancer [14] [15] [16], ovarian cancer [17], colorectal cancer [18] [19], prostate cancer [20] [21], etc., and the peripheral blood NK cells of tumor patients will experience NK cell phenotype and functional exhaustion due to the presence of the tumor microenvironment (TME) [22] [23] [24], which cannot play the better role of NK cells. Lin et al. [25] believe that the level of NK cell activity can provide an assessment of the disease state and is related to the mechanism of evading immune surveillance. Therefore, detecting the activity of peripheral blood NK cells is particularly important for evaluating the immune function of cancer patients. There are five main types of detection of NK cell activity in clinical use, namely, tetramethylazazole blue colorimetric method, lactate dehydrogenase release method, radionuclide labeling method, flow cytometry method and NK Vue cytokine release method. These detection methods have their own advantages and disadvantages, and have played an important role in different periods of clinical application development. A comparative review of these detection methods is now made as below.

Tetramethylazo Blue Colorimetric Method
The Methyl Thiazolyl Tetrazolium (MTT) colorimetric method is also called the NK cells. The larger the OD value, the higher the activity of NK cells. As the most classic method of detecting cell activity, it is widely used in scientific research. However, the MTT method also has some drawbacks. For example, the co-cultivation time of effector cells and target cells is relatively long, and the growth rates of the two different types of cells are different, which easily causes a non-specific increase in NK cell activity, which ultimately affects the experimental results [26]. And because formazan is insoluble in water, it can only be detected after adding DMSO to dissolve it. This greatly increases the workload in clinical applications, and the repeatability of the entire experimental process is poor, which will cause a certain deviation in the accuracy of the experimental results. DMSO has certain toxicity. Once inhaled after volatilization, it will cause nausea, headache and dizziness, which is not conducive to the health of experimenters, so this method is not suitable for clinical testing.

Lactate Dehydrogenase Release Method
Lactic dehydrogenase (LDH) release method is one of the in vitro cytotoxicity detection methods of NK cells. The principle of this method is that LDH exists in the cytoplasm of living cells and will be released when the cell membrane is damaged. It catalyzes the formation of pyruvate from lactic acid, and reacts with tetra-salts to form formazan. Finally, the OD value is also detected by a microplate reader. This value is positively correlated with the activity of NK cells in vitro. The LDH release method has the advantages of sensitivity, simple operation, and rapidity, but there are many factors that affect the final result. For example, there is no standard and unified detection procedure between the detection platforms, and the detection results are not well repeatable. Guo Jiqiang et al. [27] compared flow cytometry and LDH method to detect the activity of NK cells in vitro, and found that after Annexin-V-FITC/PI double staining, flow cytometry can distinguish each type well. Cells, while the LDH method cannot achieve this function, and the LDH detection method has poor accuracy and relatively poor stability. At present, this method is no longer suitable for detection in clinical practice.

Radionuclide Labeling Method
There are three commonly used radionuclide labels, namely 51Cr, 125I-Udr and 3H-TdR [28]. Sodium chromate (Na251CrO4) can enter the cytoplasm through These three labeling methods have the possibility of radioactive contamination.
Therefore, the radionuclide labeling method is no longer available used in scientific research and clinical practice.

Flow Cytometry
In recent years, more and more NK cell activity detection methods have been developed based on flow cytometry. Zhang et al. [29] selected K562 as the target cell, constructed a stably expressed enhanced green fluorescent protein in vitro, and labeled the target cell with Annexin V-PE/7AAD apoptotic staining. The target cells were co-cultured for 4 hours with an effective target ratio of 10:1.
Cytometry to detect the apoptotic ratio of target cells is the NK cell activity, which proves that the method has good repeatability and stability. Chuang et al.

NK Vue Cytokine Release Method
The NK Vue cytokine release method is a kit specially developed by South Korea NK MAX for detecting the activity of peripheral blood NK cells in vitro. The basic principle is to use the human recombinant protein Promaca to stimulate NK cells in peripheral blood in vitro to make them secrete a large amount of IFN-γ, ELISA method to detect the content of IFN-γ, with this value to reflect the activity of human peripheral blood NK cells [18]. Jobin G et al. [19] used this method to detect and analyze the relationship between NK cell activity and high-risk co- cer. Subjects with NK cell activity were 10 times higher. Barkin J et al. [21] found that this method was used to detect NK cell activity, and the results showed that subjects with low NK cell activity values were more likely to have positive results in prostate cancer biopsy. Tae BS et al. [34] also proved that the NK cell activity   [38], proton pump inhibitors [39], long-term smoking [40], drinking [41] and foods rich in zinc and selenium [42] [43], etc., will temporarily decrease or increase the value of NK cell activity high. Therefore, the clinical application of this method needs to be further verified, and the influence of more exclusionary factors needs to be considered.

Discussion
NK cell activity can directly reflect the functional state of human immune cells, and the immune functional state has a very strong correlation with the occurrence and development of cancer. Malignant tumors in the human body [20] [44] [45], stress and lack of sleep [35] [36], pregnancy [46] [47], autoimmune diseases [48], the activity of NK cells in the peripheral blood will be continuously low, so it is particularly important to detect the activity of peripheral blood NK cells in vitro.
The clinically used detection of NK cell activity in peripheral blood must have good repeatability, operability and stability of results, and be able to be widely

Conclusion
Although flow cytometry is currently used in clinical applications to detect NK cell activity, and different fluorescent dyes are added to label effective target cells, there may be differences between the experimental results, which should be worthy of attention. The emerging detection method-NK Vue cytokine release method is still in the initial stage of clinical application, and the accuracy of the detection method needs further clinical verification.