Change of circulating lymphocyte subsets is related to disease activity and secondary infection in children with primary nephrotic syndrome—a retrospective study

Introduction

Primary nephrotic syndrome (PNS) is a common chronic illness in childhood (1). Clinically, PNS is a renal syndrome characterized by intense proteinuria, hypoalbuminemia, edema, and hyperlipidemia caused by damage to podocytes of the glomerular filtration barrier (2).

Immunosuppressive therapy can lead to temporary or persistent remissions. Currently, with the increased incidence of steroid-resistant patients, second-line immunosuppressive therapies for remission induction have been recommended, such as cyclophosphamide (CYC), mycophenolate mofetil (MMF), and rituximab (RTX) (3).

Effective treatment in different stages of the disease partly depends on dynamic monitoring of immune functioning. In 1974, Shalhoub et al. (4) reported that PNS was a disorder of T-cell homeostasis by inducing lymphocyte-derived permeability factors, which interfered with the expression and function of key podocyte proteins to causing proteinuria. Some studies have demonstrated that corticosteroid medication influences the distribution of lymphocyte subpopulations in childhood PNS (5,6). The T-cell subsets in PNS include Th1/Th2, the CD4/CD8 ratio, NK cells, T_reg populations, and cytokine disruption (7).
Furthermore, T_reg cells, a group of inhibitory T cells, are correlated with the occurrence of proteinuria in patients with PNS (8). NK cells are considered indicators of prognosis in PNS. This evidence indicates that the lymphocyte subpopulations may play a crucial role in the pathogenic progression of PNS (9,10). However, the changes in peripheral lymphocyte subpopulations in children with PNS were also seldom seen.

More than 60% of steroid-sensitive patients experience relapses after being treated with glucocorticoids. Studies have found that glucocorticoids medication influence the distribution of lymphocyte subpopulations in PNS children (5,6). These changes in the distribution of lymphocyte subpopulations are associated with the occurrence of infection in children with PNS, which has become the leading cause of relapse, and some pediatric patients may even develop the end-stage renal disease. Therefore, it is critically important to seek some biomarkers for monitoring immune functions and preventing further infections in pediatric PNS patients.

With the development of precise detection techniques, an increasing number of lymphocyte subpopulations have been discovered. However, the present clinical application of molecular markers, such as CD4⁺ T cells, has not met the needs of patients. Therefore, a detailed analysis of T cell subpopulations and their vital functions in childhood patients with PNS is necessary.

In the present study, we aimed to compare the distribution of peripheral blood lymphocyte subpopulations in the active, partial remission, and complete remission stages of PNS patients to those of healthy controls. Furthermore, we aimed to explore the relationship between lymphocyte subsets and infectious complications to develop effective and promising biomarkers for diagnosing, monitoring progression, and predicting infection throughout PNS. We present the following article in accordance with the STARD reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-22-581/rc).

Methods

Patients and study design

This study is a single-center observational, retrospective study conducted at the Nanfang Hospital, Southern Medical University (Guangzhou, China). Data of all patients aged 18 years or younger, who visited Nanfang Hospital and were diagnosed with PNS from September 2019 to April 2021 were extracted from the Hospital Information System (HIS) and retrospectively analyzed. Data on peripheral lymphocyte subpopulation from healthy controls who intended to do peripheral blood stem cell donation was collected. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Nanfang Hospital Ethics Committee board (No. NFEC-2022-076) and individual consent for this retrospective analysis was waived.

Enrollment criteria included: (I) age <18 years old, (II) first PNS episode or diagnosed PNS.

The exclusion criteria were as follows: (I) infantile or hereditary nephrotic syndrome; (II) secondary nephrotic syndrome; (III) hepatitis B virus associated glomerulone nephritis, Henoch-Schonlein Purpura and lupus nephritis (IV) function of heart, lung, and liver abnormal seriously.

According to the Improving Global Outcomes (KDIGO) guidelines for kidney disease in 2012 (11), PNS patients were divided into three groups according to their serum biochemical tests: an active group (n=40), a partial remission group (n=30), and a complete remission group (n=19). Nineteen children who intended to do peripheral blood stem cell donation were selected as the control group. The active PNS group (A) was defined by a low serum albumin concentration (<2.5 g/dL) and a high urinary protein excretion (>40 mg/m² per hour) with high cholesterol levels. The partial remission (PR) group was according to a serum albumin concentration (<3.5 g/dL) and a urinary protein excretion of <20 mg/m² per hour. While the PNS in the complete remission (CR) group was defined as no proteinuria using the colorimetric qualitative test and a urinary protein/creatinine ratio of <0.2 on a random urine sample.

PNS patients with infection symptoms were divided into a co-infection group, others were divided into a non-infection group.

Sample size: the sample size was estimated based on the prior reported study (6).

Blood samples and clinical data

Venous blood specimens were collected in an EDTA-Na₂ anticoagulant tube, and serum was obtained using a coagulation tube. All the tests were completed within 24 hours after the specimens were received. Clinical data such as age, gender, clinical diagnosis, infectious disease status, and biochemical tests that were related to PNS were recorded. For the PNS patients, 24-hour urine protein (UTP), serum albumin (ALB), total serum cholesterol (CHOL), and serum creatinine (CRE) were collected as the biochemical tests.

Reagents

Multitest^TM 6-color TBNK (Cat:662995), anti-CD4 (Cat:340133, 341115), anti-CD25 (Cat:340938), anti-CD3 (Cat:340663), anti-CD8 (Cat:335805), anti-CD28 (Cat:348047), anti-CD45RA (Cat:347723), and hemolysin for flow cytometry were purchased from BD Biosciences, USA. Flow tubes and FACS Lysing Solution were also purchased from BD Biosciences, USA. PE-anti-CD127 (Cat: P010034-B) and APC-anti-CD45RO (Cat: P010028-C) were bought from Jiangxi CELGENE Biotechnology Corporation, China.

Flow cytometry

The percentage and absolute counts of lymphocyte subpopulations, including CD3⁺T cells, CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells, Regulatory T cells, cytotoxic T cells, Inhibitory T cells, Naive CD4⁺ T cells, and Effector memory CD4⁺ T cells were analyzed on BD Canto II flow cytometry (FCM), according to standard operating procedure. The results were analyzed by BD FACS Diva software v8.02 (Becton, Dickinson and Company, USA).

Statistical analysis

The statistical analysis was conducted by GraphPad Prism 8.0 (GraphPad Software, USA) and SPSS 20.0 (IBM, USA). Results of the data in each group are displayed as means ± standard deviations (mean ± SD). Multiple comparisons were performed with an analysis of variance (ANOVA) and Kruskal–Wallis test followed by Tuckey or Dunn’s post-test for sequential pairwise comparisons. The independent samples t-test was used for data with a normal distribution. For data with a non-normal distribution, the Mann-Whitney U test was used for the comparisons. A correlation analysis was conducted using Spearman’s rank correlation test. Logistic regression was used to analyze the risk factors of PNS with infectious diseases. All statistical tests were two-sided and a P value <0.05 indicated a statistically significant difference.

Results

Patient characteristics

According to the curative effect, all PNS patients were divided into three groups: an active group (A), a partial remission (PR) group, and a complete remission (CR) group. This study included 89 PNS patients (A: mean age 9.5 years, 31 males, 9 females, range: 4–14 years; PR: mean age 9.9 years, 23 males, 7 females, range: 5–15 years; CR: mean age 8.1 years, 16 males, 3 females, range: 4–15 years) and 19 healthy controls (mean age: 9.3 years, 10 males, 9 females, range: 3–16 years) (Table 1). A total of 33 PNS patients had infections, of which 23 had upper respiratory tract infection (URTI), five had pneumonia, and there was one case each of acute bronchitis, EB virus infection, virus rash, urinary system infection, and skin and fungal infection (Table 2).

No significant difference in lymphocyte subpopulations between different genders

The following lymphocyte subpopulations were studied in all participants: total T lymphocytes (CD3⁺), CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, total B lymphocytes (CD3⁻CD19⁺), natural killer (NK) lymphocytes (CD3⁻CD16⁺CD56⁺), regulatory T lymphocytes (T_reg, CD3⁺CD4⁺CD25⁺CD127^Low), cytotoxic T lymphocytes (T_C, CD8⁺CD28⁺), suppressive T lymphocytes (T_S, CD8⁺CD28⁻), naive CD4⁺ T lymphocytes (CD4⁺CD45RA⁺), and effector memory T lymphocytes (CD4⁺CD45RO⁺). As shown in Table 3, the differences in lymphocyte subpopulations between genders were not statistically significant (P>0.05).

Alteration in lymphocyte subpopulations between PNS patients and healthy controls

A fluorescence-activated cell sorting (FACS) analysis revealed that all groups of patients with PNS had a higher percentage of total lymphocytes and CD3⁺CD8⁺ T cell counts (Figure 1A,1B). There was no significant difference in CD4⁺ T cell percentage or counts in PNS patients and healthy controls (Figure 1C,1D). Meanwhile, the percentage and counts of CD8⁺ T cell in PNS patients were significantly increased, which caused a decrease in CD4/CD8 ratio (Figure 1E-1G). Moreover, there was no significant difference in T_reg cell percentage or counts in PNS patients and healthy controls (Figure 1H,1I). The percentage and the counts of NK cells were significantly decreased in PNS patients compared to healthy controls (Figure 2A,2B). There was no significant difference in B lymphocyte percentage or counts in PNS patients compared to healthy controls (Figure 2C,2D). Interestingly, the percentage and counts of T_C cell were significantly increased in PNS patients compared to healthy controls (Figure 3A,3B), but there was no significant difference in T_S cell percentage or counts (Figure 3C,3D), indicating that the activation of PNS was related to an increase in T_C cells. In addition, T_C/T_S ratios were significantly increased in PNS patients compared to healthy controls because of the increase in T_C cell (Figure 3E).

Figure 1 Comparative analysis of T cell subsets (total lymphocytes, CD4⁺ T cells, CD8⁺ T cells, T_reg, and CD4/CD8 ratio) between PNS patients and healthy control. (A) Percentage of total lymphocytes of PNS patients and healthy controls; (B) total lymphocyte counts of PNS patients and healthy controls; (C) percentage of CD4⁺ T cells of PNS patients and healthy controls; (D) CD4⁺ T cell counts of PNS patients and healthy controls; (E) percentage of CD8⁺ T cells of PNS patients and healthy controls; (F) CD8⁺ T cell counts of PNS patients and healthy controls; (G) CD4/CD8 ratio of PNS patients and healthy controls; (H) percentage of T_reg of PNS patients and healthy controls; (I) T_reg cell counts of PNS patients and healthy controls. **, P<0.01. A, active group; PR, partial remission group; CR, complete remission group; HC, healthy control. PNS, primary nephrotic syndrome.

Figure 2 Differences in NK cells and B cells between PNS patients and healthy controls. (A) Percentage of NK cells in PNS patients and healthy controls; (B) NK cell counts of PNS patients and healthy controls; (C) percentage of B cells in PNS patients and healthy controls; (D) B cell count of PNS patients and healthy controls. *, P<0.05, **, P<0.01. A, active group; PR, partial remission group; CR, complete remission group; HC, healthy control. NK, natural killer; PNS, primary nephrotic syndrome.

Figure 3 Differences in T_C cells, T_S cells, and the T_C/T_S ratio between PNS patients and healthy controls. (A) Percentage of T_C cells in PNS patients and healthy controls; (B) T_C cell counts of PNS patients and healthy controls; (C) percentage of T_S cells in PNS patients and healthy controls; (D) T_S cell counts of PNS patients and healthy control; (E) T_C/T_S ratio of PNS patients and healthy controls. *, P<0.05, **, P<0.01. A, active group; PR, partial remission group; CR, complete remission group; HC, healthy control. PNS, primary nephrotic syndrome.

There was no significant difference in naive T cell percentage and counts (Figure 4A,4B), but the effector memory CD4⁺ T cell percentage and counts were significantly increased in PNS patients in the active and partial remission groups compared to healthy controls (Figure 4C,4D). However, the ratio of naive CD4⁺ T cells/effector memory CD4⁺ T cells was significantly increased in PNS patients in the complete remission group compared to the active and partial remission groups (Figure 4E). This result suggested that the counts and ratio of naive CD4⁺ T cells and effector memory CD4⁺ T cells may be important indicators for judging the progression and remission of PNS in children.

Figure 4 Differences in naive CD4⁺ T cells, effector memory CD4⁺ T cells, and the naive CD4⁺ T cells/effector memory CD4⁺ T cells ratio between PNS patients and healthy controls. (A) Percentage of naive CD4⁺ T cells in PNS patients and healthy control; (B) naive CD4⁺ T cell counts of PNS patients and healthy controls; (C) percentage of effector memory CD4⁺ T cells in PNS patients and healthy controls; (D) effector memory CD4⁺ T cell counts of PNS patients and healthy controls; (E) naive CD4⁺ T cells/effector memory CD4⁺ T cells ratio of PNS patients and healthy controls. *, P<0.05, **, P<0.01. A, active group; PR, partial remission group; CR, complete remission group; HC, healthy control. PNS, primary nephrotic syndrome.

Analysis of risk factors for infection in children with PNS

To evaluate the risk factors for infection in children with PNS, we compared the lymphocyte subpopulation percentage and counts in children without PNS infection and those with co-infections. As is shown in Figure 5, the CD4⁺ T cell percentage and counts, CD4/CD8 ratios, percentage of naive CD4⁺ T cell counts, and T_reg cell counts were significantly lower in patients with infection, which indicated that the reduction of CD4⁺ T cells, naive CD4⁺ T cells, and the CD4/CD8 ratios were related to PNS with infection. There was no significant difference between the other lymphocyte subpopulations of co-infection and non-infection (Figure S1). Using the receiver operating curve (ROC) method, curves were plotted to identify the cutoff values that best predicted PNS with infection (Figure 6A-6D, and Figure S2A,S2B). We found that lower CD4⁺ T cell, naive CD4⁺ T cell, and T_reg cell percentages and counts and the CD4/CD8 ratio reached statistical significance in predicting PNS with infection (Table 4). Accordingly, lower levels of these lymphocyte subpopulations in the peripheral blood were associated with significantly higher infection rates than subgroups with high levels (percentage of CD4⁺ T cells: 47.27% vs. 18.28%, P<0.01; CD4⁺ T cell counts: 52.38% vs. 20.00%, P<0.01; CD4/CD8 ratio: 52.38% vs. 22.22%, P<0.01; naive CD4⁺ T cell counts: 61.90% vs. 20.93%, P<0.001; T_reg cell counts: 58.10% vs. 17.40%, P<0.001; (Figure 6E-6H and Figure S2C,S2D). Among them, T_reg cell counts were highly accurate in predicting PNS with infection (P=0.001). The cutoff value derived from the ROC analyses (the Youden index) for the T_reg cell counts and the AUC value was 82.5 and 0.730, respectively, and yielded a sensitivity and specificity of 69.2% and 74.5%, respectively (Table 4). Subsequently, we used logistic regression analysis to determine the independent clinical factors predicting PNS with infection. Patients were divided into two groups according to the cutoff value. The results showed that lower CD4⁺ T cell, T_reg cell, and naive CD4⁺ T cell percentages and counts and the CD4/CD8 ratio were independent risk factors for PNS with infection (Table 5). Patients with lower T_reg cell counts [<82.5 cells/µL, P<0.001, OR =6.577 (range, 2.315–18.684)] had a significantly higher risk of infection than those with higher counts (Table 5), which could be used as a predictor of infection in children with PNS.

Figure 5 Risk factors for infection in children with primary nephrotic syndrome. (A) Percentage of CD4⁺ T cells in infectious and non-infectious PNS patients; (B) CD4⁺ T cell counts of infectious and non-infectious PNS patients; (C) CD4/CD8 ratio of infectious and non-infectious PNS patients; (D) percentage of CD4⁺CD45RA⁺ T cells in infectious and non-infectious PNS patients; (E) CD4⁺CD45RA⁺ T cell counts of infectious and non-infectious PNS patients; (F) T_reg cell counts of infectious and non-infectious PNS patients. *, P<0.05, **, P<0.01. PNS, primary nephrotic syndrome.

Figure 6 The ROC curve analysis of lymphocyte subpopulations predicts infection in children with primary nephrotic syndrome. (A) ROC curves for the correlation of CD4⁺ T cells with infection in children with primary nephrotic syndrome. (B) ROC curves for the correlation of the CD4/CD8 ratio with infection in children with primary nephrotic syndrome. (C) ROC curves for the correlation of CD4⁺CD45RA⁺ T cells with infection in children with primary nephrotic syndrome. (D) ROC curves for the correlation of T_reg cells with infection in children with primary nephrotic syndrome. (E) Comparison of the frequency of infection in PNS patients with different CD4⁺ T cell counts. (F) Comparison of the frequency of infection in PNS patients with different CD4/CD8 ratio levels. (G) Comparison of the frequency of infection in PNS patients with different CD4⁺CD45RA⁺ T cell levels. (H) Comparison of the frequency of infection in PNS patients with different T_reg cell levels. The cutoff value between high and low levels was determined using the Youden index. Fisher’s test was used to compare the variables. **, P<0.01, ***, P<0.001. ROC, receiver operating characteristic; PNS, primary nephrotic syndrome.

Discussion

The role of the immune system in PNS has been investigated in relevant clinical and experimental studies (12).
However, the evidence for potential biomarkers for patients with PNS is scarce. The present study comprehensively analyzed the relationship between lymphocyte subsets and disease activity, and the prediction of infection in childhood PNS. Moreover, our results showed that an upregulated expression of CD8⁺ cells was significantly positively correlated with PNS-, dominated by T_C cells. Secondly, the immune response of CD4⁺CD45⁺ T cells may also vary in the different PNS disease phases, with an increased level of CD4⁺CD45RO⁺ T cells, a decreased CD4⁺CD45RA⁺/CD4⁺CD45RO⁺ ratio in disease activity, and downregulation of CD4⁺CD45RA⁺ T cells in patients with predicted infection, which has never been reported previously. Thirdly, a previous study demonstrated that the CD4⁺ cell count was more predictive of overall infection than the total lymphocyte count (13). Our results indicated that CD4⁺T cell subpopulations (T_reg cell counts and CD4⁺CD45RA⁺ T cell counts) have higher predictive values than the CD4⁺T cell count for infection in PNS.

A disease state biomarker depicts the intensity of the disease process, while a disease stage biomarker depicts how far the process has progressed. Thus, we conducted lymphocyte subpopulation investigations between groups with three clinically distinct progressions (active, partial remission, and complete remission) and a healthy group. For example, CD8⁺ T cells may injure the kidneys through the production of chemokines and the recruitment by antigen capture (10). Furthermore, CD8⁺ T lymphocytes contain two functionally distinct subsets: T_C cells and T_S cells. Tc cells produce IFN-γ and conduct cytotoxic activities, while Ts cells restrain the Th1 responses (14).

NK cells may play a vital role in the pathogenesis and the progression of PNS disease when followed by immunogenic challenges (such as vaccine immunizations, allergens, or viral infections). In our study, we found that some laboratory indicators may play an important role in the PNS, such as an increase in T_C cell counts, an elevated ratio of T_c/T_s cells, and a decline in the percentage of NK cells. More recently, the role of B cell depletion in inducing disease remission has become evident (15). A previous report suggested that the effectiveness of anti-CD20 monoclonal antibodies, such as rituximab, for refractory PNS strongly implicates B cells in the pathogenesis of PNS (16). However, our findings were inconsistent with previous reports. We found no statistical significance in the level of B cells over the disease course. The reason may be due to the insufficient sample size in this study. Currently, the immune mechanisms underlying the effect of B cell depletion are unclear and are worthy of further investigation.

Lama et al. observed a significant decrease in CD4⁺ cells in short and long remissions in PNS children compared with relapsed patients (17). In the last decade, one potential target of T cells on the podocyte has been found in pediatric patients during disease relapse (18). In metastatic colorectal cancer (mCRC) patients, CD4⁺ T cell levels and the ratio of CD4/CD8 were shown to be potential independent biomarkers for the objective response rate and overall survival (19). Notably, no statistically significant difference was found in CD4⁺ T cell counts between our groups in this study. Therefore, given the controversy about the function of CD4⁺ T cells, our group conducted further exploration of the peculiar subgroup of CD4⁺ T cells. CD4⁺ T cells can be divided into mutually exclusive subsets based on the expression of the common leukocyte antigen CD45⁺ isoforms, namely CD45RA⁺ and CD45RO⁺ (20). CD45RA⁺ cells differentiate into CD45⁺RO⁺ “memory” cells, defined as “naïve” lymphocytes. Upon antigenic stimulation, the CD45RA⁺ phenotype is switched to the CD45RO⁺ phenotype. A previous article indicated that the proportion of CD4⁺CD45RO⁺ T cells and CD4⁺CD45RA⁺ T cells was significantly higher in patients with steroid-resistant nephrotic syndrome than in controls (21). Similarly, we found that the percentage and CD4⁺CD45RO⁺ T cell counts in the active and partial remission stages were significantly higher than in healthy controls. The active phase of the disease plays a crucial role in treatment. In recent retrospective studies (22,23), MMF and corticosteroids were shown to be effective in treating active disease. Moreover, a previous study suggested that TFH1, TFH2, and TFH17 are indicators of the active state (24). However, knowledge of the clinical usefulness of biomarkers is limited, and the relevant indicators of active disease deserve further investigation. Meanwhile, the CD4⁺CD45⁺ cells responded differently in different phases of PNS in this study. Furthermore, our finding that the CD4⁺CD45RA⁺/CD4⁺CD45RO⁺ ratio was reduced significantly in the active group compared with the partial and complete remission groups was unexpected. These findings demonstrated that the CD4⁺CD45RA⁺/CD4⁺CD45RO⁺ ratio might be associated with disease activity.

Nephrotic syndrome in children requires additional treatment to prevent complications, particularly serious infections (25). Based on different types of pathology in children and adults, different types of infections were documented. The most common adult infections are hepatitis B and C (26), whereas children more commonly experience URTIs (27). Consistent with the above conclusion, our study indicated that approximately 70% of children suffered from URTI. Several studies have reported that the detection of lymphocyte subsets is significantly related to infection prediction (4,28). As shown in the results, the percentage and the counts of CD4⁺ T cells, CD4/CD8 ratio, the percentage and the counts of CD4⁺CD45RA⁺ T cells, and T_reg cell counts were significantly lower in patients with infections. Tregoning et al. reported that T cells play a critical role in defending neonates against respiratory syncytial virus infection (29). The results also showed that lower T_reg cell counts were an important risk factor for infection in children with PNS. Previous research has demonstrated that impaired T_reg cell function may elucidate the mechanism for patients with minimal in-change nephrotic syndrome and regulatory T cells induce remission of the disease (30). In addition, we found that T_reg cell counts and CD4⁺CD45RA⁺ T cell counts were both independent risk factors of overall infection.

The current study has some limitations. Firstly, selection bias cannot be excluded due to the retrospective nature of the study. For instance, early glucocorticoid exposure for the pretreatment samples ensured that all pretreatment samples reflected only the disease state at presentation, whereas the post-treatment samples reflected the cumulative effects of glucocorticoid treatment on the disease. Secondly, regulatory T cell secretions may have a role to play in regulating atopic reactions and humoral immunity via their cytokines.

Additionally, the clinical sample size was relatively small and should be increased in future studies to confirm our current findings.

Conclusions

Measuring peripheral blood lymphocyte subpopulations by flow cytometric analysis aids in monitoring disease progression and predicting infection. This study indicated that CD8⁺ T cells, dominated by T_C cells, were associated with the occurrence of disease. Moreover, the CD4⁺ T cell counts, CD4/CD8 ratio, CD4⁺CD45RA⁺ T cells, and T_reg cell counts were dependent factors in the PNS. The CD4⁺CD45RA⁺/CD4⁺CD45RO⁺ ratio is an indicator of the active state in PNS. In addition, the T_reg cell counts and CD4⁺CD45RO⁺ T cell counts have higher predictive values than the CD4⁺ T cell counts for overall infection. However, additional studies are required for more detailed typing of lymphocyte subgroups in childhood PNS.

Acknowledgments

The author would like to thank the support from all colleagues at Nanfang Hospital of Southern Medical University.

Funding: This research was supported by the Medical Research Fund of Guangdong Province (No. A202228) and the Clinical Research Program of Southern Medical University (No. 2021CR006).

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-22-581/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-22-581/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-22-581/coif). All authors report that this work was supported by the Medical Research Fund of Guangdong Province (No. A202228) and the Clinical Research Program of Southern Medical University (No. 2021CR006). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Nanfang Hospital Ethics Committee board (No. NFEC-2022-076) and individual consent for this retrospective analysis was waived.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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