The association of serum immunoglobulin and complement levels and liver fibrosis and inflammation stage in patients with chronic hepatitis B

The utility of measurement of serum immunoglobulin and complement in chronic hepatitis B (CHB) patients remains controversial. This study aimed to investigate the association of serum immunoglobulin and complement levels and liver fibrosis and inflammation stage in CHB patients. A total of 687 patients with CHB who underwent liver biopsy were enrolled. Serum immunoglobulin and complement were measured before liver biopsy, and liver pathological results were recorded. Associations of serum immunoglobulin and complement levels and liver fibrosis and inflammation stage were analysed. C3, C4, IgG and IgG1 had statistically significant differences among different fibrosis and different inflammation groups. Both C3 and C4 negatively correlated with fibrosis and inflammation stage, but IgG and IgG1 showed opposite results. C3, C4, IgG and IgG1 had statistical significance to predict ≥S2, ≥S3 and S4, and also had statistical significance to predict ≥G2, ≥G3 and G4. The area under curve (AUC) of the combination of C3, C4 and IgG (C3 + C4 + IgG) for predicting ≥S2, ≥S3 and S4 was 0.640 (95% CI: 0.603, 0.676), 0.674 (95% CI: 0.638, 0.709) and 0.744 (95% CI: 0.710, 0.776), respectively. The AUC of C3 + C4 + IgG for predicting ≥G2, ≥G3 and G4 was 0.723 (95% CI: 0.688, 0.756), 0.674 (95% CI: 0.638, 0.709) and 0.771 (95% CI: 0.738, 0.802), respectively. C3, C4, IgG and IgG1 are correlated with liver fibrosis and inflammation stage in CHB patients. C3, C4, IgG and IgG1 have diagnostic value for liver fibrosis and inflammation. C3 + C4 + IgG may improve diagnostic accuracy.

infected with HBV. 3 Thus, it is important to monitor the changes of liver and to prevent HCC development in high-risk patients with advanced fibrosis. Liver biopsy is the golden standard for determining the liver fibrosis and inflammation stage. However, the utility of liver biopsy is limited by several defects and to explore alternative indicators is essential.
Serum biomarkers are noninvasive and valuable for the management of CHB. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are most commonly used indicators to reflect liver inflammation. AST-to-platelet (PLT) ratio index (APRI), fibrosis index based on the four factors (FIB-4), gamma-glutamyl transpeptidase (GGT)-to-PLT ratio (GPR) and other formulas are proposed to predict liver fibrosis. 4,5 However, serum immunoglobulin and complement levels are less often used to reflect liver inflammation and fibrosis stage. As known, they are usually used in autoimmune diseases. As for liver diseases, serum immunoglobulin is often thought to be elevated and serum complement is often thought to be reduced. The association between serum immunoglobulin and complement and pathological features in patients with CHB is rarely reported. Also, few prior studies have previously analysed serum immunoglobulin G (IgG) subclass levels fully in patients with CHB. The clinical significance of serum immunoglobulin and complement in CHB remains controversial.
There is an urgent requirement for exploring the association between serum immunoglobulin and complement and pathological features in patients with CHB. Therefore, to provide more evidence for the use of serum immunoglobulin and complement in patients with CHB, the present study retrospectively collected and analysed the clinical data of CHB patients who received liver biopsy and serum immunoglobulin and complement detection in recent years.

| Study population
This study was a retrospective study. Between January 2017 and December 2021, 687 patients with CHB who were hospitalized for liver biopsy were consecutively recruited in the First Affiliated Hospital of Xiamen University. All patients were hospitalized for one or more following reasons: elevated ALT (>40 U/L), fatigue, inappetence, abdominal distension or right upper abdominal discomfort. They did not receive antiviral treatment within a month before liver biopsy, and they were willing to undergo liver biopsy for further treatment. All patients had positive HBsAg result for more than 6 months, and there was no evidence of concurrent other hepatitis virus infection, and no clear evidence of drug-induced liver disease, alcoholic liver disease, autoimmune liver disease, severe nonalcoholic fatty liver disease and other liver diseases. In addition, there was no clear evidence of autoimmune diseases and haematological diseases. Patients with age <18 years or > 60 years, cancer or alpha foetal protein (AFP) >200 ng/mL and incomplete clinical data were excluded. The study complied with the Helsinki Declaration and were approved by the Ethics Committee of the First Affiliated Hospital of Xiamen University.

| Liver biopsy and laboratory test
Liver biopsies were performed using 16-G biopsy needles under the guidance of ultrasound. The pathological features of liver tissue including fibrosis and inflammation stage were evaluated by two independent pathologists in the Department of Pathology. When the pathologists differed in their assessments, an agreement was reached by discussion. The grading of fibrosis and inflammation was based on Scheuer scoring system: significant fibrosis was defined as greater than or equal to S2 (≥S2), severe fibrosis was defined as greater than or equal to S3 (≥S3) and cirrhosis was defined as equal to S4. 6 Serum levels of C3, C4, IgG, IgA, IgM, IgG1, IgG2, IgG3, IgG4,

| Patient characteristics
According to the inclusion and exclusion, a total of 687 CHB patients were eventually enrolled, including 475 male patients and 212 female patients. The flowchart of patient selection was showed in Figure 1. Average age was 34.97 years. HBeAg-positive rate was 59.53%. According to Scheuer scoring system, 192 patients were classified as S0-1, 301 as S2, 137 as S3 and 57 as S4, and 97 patients were classified as G0-1, 313 as G2, 228 as G3 and 49 as G4.

| Serum immunoglobulin and complement levels
As shown in Table 1 and among different inflammation groups (p < .05). Overall, C3 and C4 decreased with the severity of fibrosis and inflammation.
However, IgG and IgG1 increased with the severity of fibrosis and inflammation.
Furthermore, the proportions of patients whose C3, C4, IgG and IgG1 were out of reference range were calculated ( Table 2). For all patients, the proportion of C3 and C4 below the lower limit of reference value is 39.59% and 4.22%, respectively, and the proportion of IgG and IgG1 above the upper limit of reference value is 47.02% and 37.86%, respectively. The proportion of C3 and C4 below the lower limit of reference value and the proportion of IgG and IgG1 above the upper limit of reference value showed statistical difference among different fibrosis groups (p < .05) and different inflammation groups (p < .05). In addition, the proportion of C3 and C4 below the lower limit of reference value increased with the severity of fibrosis and inflammation, and the proportion of IgG and IgG1 above the upper limit of reference value also increased with the severity of fibrosis and inflammation.

| Correlation analysis
As shown in Table 3, both C3 and C4 were negatively correlated with the liver fibrosis stage, liver inflammation stage, log 10

| ROC analysis
To evaluate the accuracy for predicting liver pathological features, ROC analyses were performed. As shown in Table 4 Figure 2, and the ROC curves of IgG1 were showed in Figure 3.

| DISCUSS ION
With the development of laboratory medicine, serum immunoglobulin and complement levels can be easily detected. More and more patients receive serum immunoglobulin and complement detection.
To better understand the relationship between serum immunoglobulin and complement and liver fibrosis and inflammation stage in CHB patients based on clinical data is meaningful. The present study reviewed consecutively the clinical data of CHB patients who underwent liver biopsy in recent years. Strict inclusion and exclusion criteria were established to reduce the interference of confounding factors. Patients with AFP >200 ng/mL were excluded since they had high risk of liver cancer. Some patients had previously received irregular antiviral therapy, but they did not receive antiviral treatment recently. Whether antiviral treatment had an effect on the level of immunoglobulin and complement had not yet been fully elucidated. All things considered, patients who received antiviral treatment within a month before liver biopsy were excluded in this study. In addition, even though immune fixation electrophoresis was not performed for all CHB patients to exclude haematological diseases, blood analysis was performed by automatic blood analyser for all CHB patients. If a review rule was triggered, a blood smear was made to look for abnormal cells. Finally, 687 CHB patients were enrolled. The prevalence of significant fibrosis, severe fibrosis, and cirrhosis were consistent with the previous meta analysis. 7 The results revealed that serum immunoglobulin and complement were selectively correlated with liver fibrosis and inflammation stage in CHB patients.
The complement system plays an important role in both host innate immune defence and inflammatory progress of human diseases. [8][9][10][11][12][13] It is thought to be involved in the pathogenesis of multiple liver disorders. 14 C3 and C4 were the two most important components of the complement system. In the present study, both C3 and C4 had statistic differences among different fibrosis groups and among different inflammation groups. Also, correlation analysis revealed both C3 and C4 negatively correlated with fibrosis stage and inflammation stage. Additionally, the proportion of C3 and C4 below the lower limit of reference value increased with the severity of fibrosis and inflammation. Zhu C and his colleagues found that the levels of C3 and C4 in the patients with CHB were significantly lower than healthy individuals, and they concluded that HBV could inhibit the expression of C3 and C4 in vitro and in vivo. 15 As known, liver is the major site for complement synthesis, accounting for up to 90% of the fluid-phase complement proteins. 16 Chinese population might be more suitable.
Hypergammaglobulinaemia is a common finding (67%) in patients with cirrhosis. 19 As reported, immunoglobulins exerted a direct effect on hepatic fibrogenesis. 20 In the present study, only IgG had statistic differences among different fibrosis groups and among different inflammation groups. Also, correlation analysis revealed that IgG positively correlated with fibrosis stage and inflammation stage, IgM positively correlated with inflammation stage with small correlation coefficient (r = .084) and IgA did not correlated with fibrosis stage and inflammation stage. Consistent with the previous study, there appeared to be an association between serum IgG and extent of hepatic fibrosis in CHB patients. 21 As reported, IgG stimulated the proliferation of hepatic stellate cells and the expression of smooth muscle alpha-actin. 20 Watt K's data indicated a strong association between serum immunoglobulin levels (IgA, IgG and total immunoglobulin) and hepatic fibrosis in patients with hepatitis C virus (HCV) infection. 22 It suggested that serum immunoglobulins were selectively correlated with different types of liver diseases.
Thus, compared with serum IgA and IgM, serum IgG might be more important for CHB patients. Also, correlation analysis revealed that IgG correlated with disease severity parameters, such as ALB, ALT, AST, GGT, RDW and PT, and the proportion of IgG above the upper limit of reference value increased with the severity of fibrosis and inflammation. Therefore, IgG was of significance in the management of CHB patients, and continuous monitoring might be more meaningful.

F I G U R E 3
Receiver operating characteristic curves of IgG1 were constructed to predict liver pathological features. (A) for predicting significant fibrosis, (B) for predicting sever fibrosis, (C) for predicting cirrhosis, (D) for predicting ≥G2, (E) for predicting ≥G3, (F) for predicting G.

CO N FLI C T O F I NTE R E S T S TATE M E NT
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.