Application of Immunohistochemistry to Monitor the CD3+T Cell Subset Counts in Patients with Sepsis

[Objective]: To evaluate the application signicance of Immunohistochemistry for monitoring peripheral blood CD3 + T cell subset (CD3+/CD3 + CD4+/CD3 + CD8+) counts in patients with sepsis. [Methods]: Two peripheral blood samples of 117 patients with sepsis on the rst day of admission (D1) and 20 healthy control subjects were collected, and two peripheral blood samples of 20 patients with sepsis on the fourth day of admission (D4) were randomly collected and used to detect the lymphocyte counts of routine blood tests and CD3 + T cell subset count by Immunohistochemistry; the lymphocyte count levels between the sepsis group and the healthy control group were compared, and the correlation between the two in the same group were analyzed. [Results]:lymphocyte counts by routine blood tests and CD3 + T cell subset counts of patients with sepsis were signicantly lower than those in healthy control subjects (P < 0.01). In the surviving group, the mean values of D4 CD3 + T cell subset counts increased signicantly compared with D1, while the nonsurviving group did not rebound signicantly; There was a signicant positive correlation between lymphocyte counts by routine blood tests and CD3 + T lymphocyte subset counts in patients with sepsis and the healthy control subjects. (P < 0.01). [Conclusion]: Detection of CD3 + T cell subset counts by immunohistochemical method can reect the cellular immune status of patients at a given time, thus it can be used as one of the immune monitoring methods in patients with sepsis.

[Methods]: Two peripheral blood samples of 117 patients with sepsis on the rst day of admission (D1) and 20 healthy control subjects were collected, and two peripheral blood samples of 20 patients with sepsis on the fourth day of admission (D4) were randomly collected and used to detect the lymphocyte counts of routine blood tests and CD3 + T cell subset count by Immunohistochemistry; the lymphocyte count levels between the sepsis group and the healthy control group were compared, and the correlation between the two in the same group were analyzed.
[Results]:lymphocyte counts by routine blood tests and CD3 + T cell subset counts of patients with sepsis were signi cantly lower than those in healthy control subjects (P < 0.01). In the surviving group, the mean values of D4 CD3 + T cell subset counts increased signi cantly compared with D1, while the nonsurviving group did not rebound signi cantly; There was a signi cant positive correlation between lymphocyte counts by routine blood tests and CD3 + T lymphocyte subset counts in patients with sepsis and the healthy control subjects. (P < 0.01).
[Conclusion]: Detection of CD3 + T cell subset counts by immunohistochemical method can re ect the cellular immune status of patients at a given time, thus it can be used as one of the immune monitoring methods in patients with sepsis.

Background
Sepsis is a life-threatening organ dysfunction syndrome caused by multiple pathogenic microorganism infection, which is also the main cause of death occurring in intensive care units (ICU). Recently, the incidence of sepsis has been increasing annually. The number of sepsis patients worldwide exceeds 19 million each year, of which 6 million patients die with a case fatality rate over 25%. Particularly, the death rate of septic shock patients is as high as 45%~ 50% [1] . Essentially, sepsis is an in ammatory response beyond control. At the initial stage, it is called systemic in ammatory response syndrome (SIRS), characterized by the release of pro-in ammatory mediators [2] . Subsequently, anti-in ammatory mediators are released by SIRS for counter-regulation, which is called compensatory anti-in ammatory response syndrome (CARS). However, the immune disorder mechanism of patients with sepsis is caused by the unbalanced regulation of pro-in ammation and anti-in ammation. What is worse, immunosuppression is observed in many cases, and can last up to 28 days according to some studies [3][4][5][6] . Through the autopsy of patients who died from sepsis, it is observed that a considerable number of their spleen CD4 + T cells, CD8 + T cells, and HLA-DR cells cannot be found. [7][8] In addition to the absolute change in the number of lymphocytes, sepsis can also render signi cant changes in the effector function of lymphocytes. For example, compared with survivors, the memory/effect CD8+/CD45 + T lymphocyte subsets of patients who died from sepsis has showed a signi cant reduction in interferon synthesis [9] . Sustained immunosuppression is the main cause of death in the late stage of sepsis [10] . Therefore, the evaluation of the immune status of patients with sepsis is of great signi cance. The dynamic changes of CD3 + CD4 + and CD3 + CD8 + T cells can effectively re ect their immune status. In this study, immunohistochemistry is employed to detect the CD3 + T cell subset counts to assess the immune status of patients. Immunohistochemistry has been employed as a routine tissue detection method to this day, and it is a powerful tool for pathological tissue identi cation. However, there is no relevant report on its application in detecting immune cells in blood either. International Guidelines for Management of Sepsis and Septic Shock: 2016). The exclusive criteria include: 1) age <18 years; 2) patients diagnosed with hematological or immune diseases who received cytotoxic drugs or hormone treatments within the last six months. The control group consists of 20 healthy volunteers.

Instruments and reagents
Blood routine examination equipment: the blood analysis of Sysmex XN-2800 with its original reagents; immunohistochemistrical detection equipment for CD3+T cell subsets: automatic visual cell meter of SemiBio-CytoCounter series and slides for cell detection of SemiBio-CD series (CD3/4/8) by Shanghai SemiBio Technology Co., Ltd.

Detection method
Two peripheral blood samples of patients with sepsis on the rst day of admission (D1) and those of healthy volunteers were selected. Then, they were sent to the laboratory and pathology department for lymphocyte counts and CD3+T cell subset counts in the routine blood examination respectively. A correlation comparison was conducted based on the results of the two groups. On the fourth day of admission (D4), lymphocyte counts and CD3+T cell subset counts were carried out in the routine blood examination of 20 sepsis cases, and a trend analysis was conducted accordingly.

Data collection
The laboratory data and result data of patients were collected according to inclusive and exclusive criteria. The day of admission was de ned as day 0 of data collection and day 1 of the following 24 hours. The de nition of lymphopenia in blood routine examination was that the absolute lymphocyte Page 4/14 counts were less than 1.1×10^9/L (1100/μl), the normal value oor of the institution. Severe lymphopenia was de ned as an absolute lymphocyte count of less than 0.6×10^9/L (600/μl), which was half of the normal value oor. The reference range of automatic visual cell meter of SemiBio-CytoCounter series on CD3+T cell subset counts was: 770/μl CD3+T cell 2860/μl, 500/μl CD3+CD4+T cell 1440/μl, 238/ μl<CD3+CD8+T cells<1250/μl.

Statistical methods
SPSS 25.0 was employed to analyze the data statistically. The measurement data were in accordance with normal distribution and expressed as mean ± standard deviation. The paired t-test was performed for comparison between groups, and the difference was statistically signi cant (P<0.05). The correlation test was determined by Pearson's simple correlation coe cient, and the difference was statistically signi cant (P<0.05).

Results
Among 117 patients diagnosed with sepsis or septic shock, four patients were diagnosed with a tumor, one patient with systemic lupus erythematosus, and one patient with pemphigus, and were excluded from the study. Finally, 111 patients were then enrolled. The mortality rate was 22.5%; the average APACHE score (± standard error) was 23.5 ± 0.7; and the average SOFA score (± standard error) was 8.4 ± 0.3. The clinical characteristics of the patients are shown in Table 1: was found between men and women. However, a signi cant difference was noticed in the APACHE score and that of the non-surviving group was higher than the surviving group, with the difference statistically signi cant (P < 0.01). There was no signi cant difference in the SOFA comparison (P > 0.05). The focal infection was mainly found in the lungs and urinary system.
2.1 Results of lymphocyte counts and immunohistochemistry of CD3 + T cell subset counts in routine blood examination Two peripheral blood samples of 111 patients with sepsis were collected on the rst day (D1) in the ICU.
Then, lymphocyte counts and CD3 + T cell subset counts in routine blood examination were carried out. Likewise, counting was also conducted amongst 20 healthy control subjects. Subsequently, a comparative analysis between the two groups was achieved: the lymphocyte counts and CD3 + T cell subset counts in blood routine examination of the sepsis group were signi cantly lower than those of the healthy control group. The difference was statistically signi cant (P < 0.01). Remarkably, the average of CD3 + CD4 + T cells in the subset was lower than the lower limit of normal (500/µl < CD3 + CD4 + T cells < 1440/µl). The comparison between the sepsis group and healthy group showed that only the average of CD3 + CD4 + T cells in the surviving group was slightly lower than the lower limit of normal, while the average level of CD3+, CD3 + CD4+, and CD3 + CD8 + T cells in the non-surviving group was lower than the lower limit of normal. There were signi cant differences between the two (P < 0.01). There was no signi cant difference in the comparison of sepsis groups. However, the average of D3+, CD3 + CD4+, CD3 + CD8 + T cells in the non-surviving group was lower than that of the surviving group, while the lymphocyte counts were slightly higher than that of the surviving group. The test results are shown in Table 2: Data are mean ± standard error of the mean;P values were determined by bivariate statistical analysis.

Correlation analysis of lymphocyte counts by routine blood tests and CD3 + T cell subset counts by Immunohistochemistry
There was a signi cant positive correlation between lymphocyte counts by routine blood tests and CD3 + T cell subset counts by Immunohistochemistry in patients with sepsis compared to those in the healthy control group. The correlation scatter diagrams between lymphocyte counts by routine blood tests and CD3 + T cell subset counts by Immunohistochemistry are shown in Fig. 1 and Fig. 2: 2.3 Data of lymphocyte counts by routine blood tests and CD3 + T cell subset counts on D1 and D4 were collected from 20 patients with sepsis and then analyzed: 17 out of 20 patients with sepsis survived 28 days and 3 died within 28 days; in the surviving group, the mean values of lymphocyte counts by routine blood tests on D4 CD3 + T cell subset counts increased signi cantly compared with D1, in which the mean values of CD3 + CD8 + T cells and CD3 + T cells increased to the normal range and the mean value of CD3 + CD4 + T cells was also close to the lower limit of normal value; in contrast, the nonsurviving group did not rebound signi cantly on day 4 compared with day 1, and their lymphocyte counts by routine blood tests and CD3 + T cell subset counts on day 4 were below the lower limit of normal values. Test results are shown in Table 3: Data are mean ± standard error of the mean; P values were determined by bivariate statistical analysis.
Statistical analysis could not be conducted due to the small amount of data. The trend graphs are shown in Fig. 3.

Discussion
In the early stage, sepsis is characterized by excessive in ammation and is often referred to as a "cytokine storm", whereas later, patients with sepsis usually develop immunosuppression [11][12] . Immunosuppression is featured by apoptosis of T lymphocytes, decreased expression and dysfunction of HLA-DR while CD3 + CD4 + T cells are mainly involved in the regulation of immune reactions and CD3 CD8 T Cells are essential in cytotoxic reactions [13][14][15] . Apoptosis of CD3 CD8 T Cells eventually leads to innate and adaptive immune destruction, which is also regarded as an important cause of death in the later stages of sepsis [16] . In this study, lymphocyte counts by routine blood tests and CD3+, CD3 + CD4+, CD3 + CD8 + T cell counts in sepsis group on D1 were signi cantly lower than those in healthy control group; however, in the sepsis subgroup study, only the mean of CD3 + CD4 + T-cell were slightly below the lower limit of normal value in the surviving group, while the mean of CD3+, CD3 + CD4+, and CD3 + CD8 + T-cell levels were all below the lower limit of normal in the nonsurviving group, and the initial criticality was higher in the nonsurviving group in terms of severity, mainly in the APACHE II score, which was distinctly higher than that of the survivor group and was statistically signi cant (P < 0.01).The data of this study suggest that T lymphocyte apoptosis and immune function inhibition exist initially in patients with sepsis, while immunosuppression appears to be more prominent in the nonsurviving group. Severe immunosuppression is closely related to the mortality of septic patients, which is also consistent with the relevant studies at home and abroad. Therefore, it seems that the detection of CD3+, CD3 + CD4+, CD3 + CD8 + T cells by Immunohistochemistry can be used as a method to detect the immune status of septic patients.
Recent studies have demonstrated the potential value of absolute lymphocyte counts in predicting secondary infection and increased risk of sepsis-induced immunosuppression on day 4 in patients con rmed with sepsis, and the risk of persistent immunosuppression will occur if low values persist on day 4 [17][18] . Moreover, it has also been reported that dynamic routine lymphocyte counts can also be used as an immune monitoring method for sepsis, but there are also immature lymphocytes and nonfunctional lymphocytes in routine lymphocyte counts, so the evaluation of immune status by this method is unsound; from the analysis of lymphocyte counts on day 1 in 111 septic patients in this study, it can be seen that the mean CD3+, CD3 + CD4+, CD3 + CD8 + T cell counts in the nonsurviving group were signi cantly lower than those in the survival group, while the routine lymphocyte counts were higher than that in the survival group, which seems to con rm the limitations of routine lymphocyte counts for immune evaluation. The routine lymphocyte and CD3+, CD3 + CD4+, CD3 + CD8 + T cell counts of 20 septic patients with peripheral blood collected on day 1 and 4 were compared, and it was found that the cell counts on day 4 in the surviving group had signi cantly increased compared with those on day 1 and basically returned to the normal range. While the nonsurviving group did not rebound signi cantly on day 4 and continued to be below the lower limit of normal, which was in line with the ndings of existing studies. Therefore, the detection of CD3+, CD3 + CD4+, CD3 + CD8 + T cell counts by Immunohistochemistry can be used as an immune monitoring method for septic patients.
At present, ow cytometry remains a classical detection technology for immune cytology. However, the cost for overall equipment of ow cytometry is about 500,000 US dollars. Thus it cannot be popularized in many primary medical institutions. And the detection cost is also high at $77.8 per test, which is not cost-effective to allow repeated dynamic monitoring. In this study, compared with ow cytometry, the classical cell detection method, Immunohistochemistry used for detecting CD3 + T-cell subsets subjects has certain advantages as its cost in our hospital was US $60.1/time, and the cost for overall equipment was only US $34,000, which would favour promotion in primary hospitals. Meanwhile, CD3 + T-cell subsets detected by Immunohistochemistry in this study showed a statistically signi cant positive correlation with the lymphocyte count in blood routine, and its dynamic monitoring index and trend are consistent with domestic and international studies. Therefore, it is feasible to assess the cellular immunity of patients by Immunohistochemistry testing. During the treatment of the disease, subsequent visits can also be used to assess the recovery of cellular immune function, determine the prognosis of the disease, guide clinical treatment, and save test costs. In hospitals without ow cytometry, the CD3 + T-cell subsets can be measured by Immunohistochemistry to assess the status of the body's immune function and re ect the severity of the disease.
There are still shortcomings in this study. The continuous collection of sample size of patients with sepsis on day 1 and day 4 is too small to be statistically analyzed and the classical ow cytometry test group was not added when correlation analysis was conducted. Therefore, the detection accuracy remains to be further improved. In the future, it will be necessary to increase the sample size and compare the correlation between the two with ow cytometry to provide a more accurate control.

Conclusion
Detection of CD3 + T cell subset counts by Immunohistochemistry can re ect the cellular immune status of patients at the time, and can thus be used as one of the immune monitoring methods in patients with sepsis.

Declarations
Availability of data and materials The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was conducted in accordance with the principles of the Declaration of Helsinki, and the study protocol was approved by the ethics committee of Huizhou Third People's hospital. Because of the retrospective nature of the study, the ethics committee of Huizhou Third People's hospital believes that the patient's informed consent can be waived.
However, due to the lack of control, we recruited 20 healthy volunteers. We have obtained written informed consent from all healthy volunteers. All of the procedures were performed in accordance with the relevant policies in China. Figure 1 Correlation between lymphocyte counts by routine blood tests and CD3+T cell subset counts by Immunohistochemistry in healthy control subjects: There was a statistically signi cant correlation between them, P < 0.01.

Figure 2
Correlation between lymphocyte counts by routine blood tests and CD3+T cell subset counts in the sepsis group: there was statistically signi cant correlation between lymphocyte counts by routine blood tests and CD3+T