Analytical and Clinical Evaluation of a Chemiluminescent Immunoassay to Detect Serum Chitinase-3-like Protein 1 in HBV-Related Liver Diseases

Serum chitinase-3-like protein 1 (CHI3L1) is a diagnostic marker for liver diseases, such as hepatocellular carcinoma (HCC). Herein, we aimed to evaluate the analytical performance of a chemiluminescent immunoassay (CLIA) for the quantitative detection of CHI3L1 and its application in hepatitis B virus (HBV)-related liver diseases. The CLIA for CHI3L1 detection presented good analytical performance, with a linear range of 1.50–2000.00 ng/mL and a detection limit of 0.98 ng/mL. To evaluate its clinical application, serum CHI3L1 levels were detected in 82 patients with chronic hepatitis B (CHB) and in 21 healthy controls. The patients with CHB and HCC had higher CHI3L1 levels than the healthy controls and the patients with CHB without HCC. However, CHI3L1 levels did not change significantly with the increase in liver fibrosis stages. The area under the receiver operating characteristic curve for the diagnosis of HBV-related HCC was 0.808, representing a moderate diagnostic value. Correlation analysis revealed a significant association between CHI3L1 and alpha-fetoprotein (AFP) levels, the fibrosis-4 (FIB-4) index, and the aspartate aminotransferase-to-platelet ratio index (APRI). In conclusion, compared with currently reported methods for CHI3L1 detection, the CLIA has a high sensitivity, a wide linear range, and an acceptable accuracy, precision, and reference intervals, making it valuable in the diagnosis of HBV-related HCC.


Introduction
Hepatitis B virus (HBV) is the most common cause of liver diseases in China, which can lead to acute and chronic hepatitis B (CHB), hepatic fbrosis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) [1].In the clinical setting, timely, accurate, and comprehensive diagnoses are critical to reducing the progression of HBV-related liver diseases.As the gold standard for the diagnosis of liver diseases, liver biopsy has some limitations in clinical application because of its invasive nature [2].Clinical symptoms, physical examination, imaging screening, and routine laboratory indicators can be insufciently specifc to detect HCC [2].Terefore, a noninvasive, safe, and specifc method would have crucial clinical application in HBV-related liver diseases.
Chitinase-3-like protein 1 (CHI3L1, YKL-40 protein) is a glycoprotein that plays a role in numerous diseases, such as arthritis, idiopathic pulmonary fbrosis, and liver diseases [3][4][5].In particular, CHI3L1 is abundantly expressed in liver tissue and is mainly involved in infammation and tissue remodeling [6].In clinical applications, studies have shown that CHI3L1 levels were signifcantly elevated in liver diseases and increased with the severity of the disease [5,7], which was also emphasized in the "Guidelines on the Management of Hepatic Encephalopathy in Cirrhosis" [8].Furthermore, serum CHI3L1 has been recommended as a noninvasive marker for liver diseases.A meta-analysis showed that serum CHI3L1 served as an excellent marker to diagnose liver fbrosis, in which the pooled diagnostic values were signifcantly higher than the clinical indicators such as FibroScan, the aspartate aminotransferase-to-platelet ratio index (APRI), and the fbrosis-4 (FIB-4) index [9].Moreover, the "Guidelines on the Prevention and Treatment in Chronic Hepatitis B" emphasized the predictive role of CHI3L1 in HBV-related liver diseases [2].Tese studies indicated that CHI3L1 could be used as a biomarker for the diagnosis, staging, and prognosis of HBV-related liver diseases.
Various analytical methods have been used to detect serum CHI3L1 [10][11][12].Te enzyme-linked immunosorbent assay (ELISA) is the most frequently used method to quantify serum CHI3L1 levels; however, its time-consuming operations have limited its clinical application [10].Later, fuorescent immunoassay methods with simple operation processes were developed.However, the results of the magnetic bead fuorescent immunoassay (MB-FIA) and the fuorescence immunochromatography assay (FICA) were not accurate enough [11,12].With the characteristics of a short determination time and high specifcity, the chemiluminescent immunoassay (CLIA) has been increasingly used in routine clinical applications [13].
Considering the important clinical value of a CLIA for the rapid and accurate quantifcation of CHI3L1, it is necessary to explore its analytical performance and clinical application.In this study, we evaluated the analytical performance of a CLIA to quantitatively detect CHI3L1 and the value of CHI3L1 in diagnosing HBV-related liver diseases.

Reagents and
Instruments.An automatic CLIA analyzer (iFlash3000-A, Shenzhen Yahui Long Biological Technology Co., Ltd., Shenzhen, China) was used to detect serum CHI3L1 levels.Te CHI3L1 assay kits (CLIA method) (lot no.20210201), supporting reagents, including three calibrators (0, 80.05, and 1070.48 ng/ml) and two calibrators at diferent concentrations from another lot (lot no.20210601; 70.90 and 864.90 ng/ml), and other materials for the instrument, were also purchased from Shenzhen Yahui Long Biological Technology Co., Ltd.Te CHI3L1 assay was a sandwich immunoassay using a direct chemiluminometric technique.According to the manufacturer's instructions, we loaded the samples, reagents, and other materials related to the CHI3L1 assay into the iFlash3000-A.At least 5 μl of the sample was measured for each determination.Ten, the automatic CLIA analyzer performed operations "incubation-washing-signal triggering" and measurement.Te CLIA assay procedure from sample addition to result acquisition was performed in less than 20 min.

Performance Verifcation
2.2.1.Linear Range.Following the Clinical and Laboratory Standards Institute's (CLSI) EP6-A guidelines, three levels of traceable calibration solution (lot no.20210201; 0, 80.05, and 1070.48 ng/mL) were analyzed in duplicate to obtain the calibration curve for CHI3L1 [14].Ten, to verify the linear range of 1.50-2000.00ng/mL, as suggested by the manufacturer's instructions, low-and high-value serum specimens were prepared.Te low-and high-value specimens had concentrations that covered the linear range as far as possible.A series of sample concentrations were tested by mixing low (L)-and high(H)-value plasma in certain proportions (5L, 4L + 1H, 3L + 2H, 2L + 3H, 1L + 4H, and 5H).Two replicates of each sample were tested.A scatter diagram and regression analysis were then performed.A linear correlation coefcient of more than 0.99 met the industryrecognized standards.

Functional Sensitivity: Limit of Detection.
Following the CLSI's EP17-A guidelines, the limit of detection (LOD) was determined from an assay of 10 replicates of the zerolevel calibration solution (lot no.20210201; 0 ng/mL) [15].Based on the calibration curve, the chemiluminescence value of the mean + 2 standard deviations (SD) at the zeroconcentration point was converted into the corresponding concentration, which was the LOD concentration.
2.2.3.Accuracy.Tis procedure followed the EP10-A3 guidelines published by the CLSI [16].In this study, the detection of two concentrations of traceable calibration solutions (lot no.20210601; 70.90 and 864.90 ng/mL) was repeated twice a day and for a total of 5 days.Te accuracy was accepted when the bias was not more than 12.5%.

Precision.
Following the CLSI's EP15-A2 guidelines, two levels of mixed fresh serum were used [17].Te intraanalysis precision was assessed by calculating the coefcient of variation (CV) (%) of each level three times per run in one day.Tese samples were analyzed three times for fve days to obtain the interanalysis comparison data.Te precision was acceptable when the CV was not greater than 10.0%.

Reference Intervals.
Following the CLSI's EP28-A3c guidelines [18], 21 specimens collected from healthy individuals were analyzed for their CHI3L1 levels.On the condition that less than two specimens exceeded the reference interval suggested by the manufacturer, the validation was accepted.

Clinical Samples.
Patients with hepatitis B were diagnosed according to the "2019 Guidelines for the Prevention and Treatment of Chronic Hepatitis B." Te exclusion criteria included the following: (1) aged <18 years; (2) lack of liver biopsy results; (3) lack of sufcient samples for the detection of CHI3L1; (4) complicated with severe diseases; and (5) pregnant or lactating women.Te selection criteria for healthy controls were mainly based on negative results for hepatitis B surface antigen (HBsAg) and normal liver function.Te sample size required more than 100 cases to meet the requirements of CLSI's EP9-A3 [19] where ULN is the upper limit of normal.
Tis study was approved by the Ethics Committee of the Guangdong Provincial Hospital of Chinese Medicine (ZE2021-084-01).Written informed consent was obtained from the patients and healthy volunteers.

Statistical Analysis.
All statistical analyses were performed using SPSS 23 (IBM Corp., Armonk, NY, USA).Normally distributed data are shown as the mean ± SD; otherwise, M (P25 ∼ P75) was used.Comparisons of multiple groups were carried out by using a one-way analysis of variance (ANOVA) followed by a Bonferroni test for normally distributed and equal variance data; otherwise, a Kruskal-Wallis test was used.Te receiver operating characteristic (ROC) curve was plotted to assess the diagnostic accuracy.We used Pearson's correlation analysis to assess whether the CHI3L1 concentration correlated with the clinical indicators.P values of <0.05 were considered statistically signifcant.

Performance Verifcation
3.1.1.Analysis of the Linear Range.Te CHI3L1 standard curve equation was y � 147.345x − 103.971,R 2 � 0.999 (Figure 1(a) and raw data in Supplementary Table 1).Te expected concentration and the experimental results are summarized in Table 1.As shown in Figure 1(b), the linear correlation coefcient was 0.9987, which was greater than 0.9900.Tese results indicated that the linear range of 1.50 ng/ mL-2000.00ng/mL stated in the reagent instructions was acceptable (Table 1 and Figure 1(b)).

Analysis of the LOD.
Te LOD concentration was determined as 0.98 ng/mL (raw data in Supplementary Table 2).Terefore, the detection range was between 0.98 and 2000.00ng/mL.In addition, several immunoassay methods for serum CHI3L1 levels are summarized in Table 2. Compared with the methods detailed in previous reports [10][11][12], CLIA presented a shorter detection time, a wider linear range, and a lower detection limit.

Analysis of Accuracy.
Te biases of the lowconcentration calibration solution and high-concentration calibration solution between the instrumental concentration and the theoretical concentration were 0.48% and 0.08%, respectively, which were both less than 12.5% (Table 3 and raw data in Supplementary Table 3).

Analysis of the Precision of the Assay.
As shown in Table 4, the intraassay CVs for levels 1 and 2 were 1.81% and 2.76%, respectively; the interassay CVs were 7.76% and 1.61%, respectively; and the total precision CVs were 7.91% and 2.88%, respectively, which were all less than the acceptable range of not more than 10.0%, indicating that the precision of the testing kit was acceptable (raw data in Supplementary Table 4).

Validation of Biological Reference Intervals.
In samples from 21 healthy individuals, the results were all <79.0 ng/mL (raw data in Supplementary Table 5).Tus, the reference interval passed the verifcation standard.

Discussion
In the present study, we evaluated a simple and rapid CLIA for the quantitative detection of serum CHI3L1, which presented good analytical performance, making it valuable for the diagnosis of HBV-related HCC.
International Journal of Analytical Chemistry Currently, CLIAs are increasingly being used in the biological analysis because of their extreme sensitivity, high specifcity, efcient simplifed detection procedures, and short assay time [13].Terefore, we evaluated the performance of a CLIA to detect serum CHI3L1 according to the CLSI guidelines.Te assay kit showed good linearity at CHI3L1 concentrations ranging from 1.50 to 2000.00 ng/ mL, with a lower LOD of 0.98 ng/mL (Tables 1 and 2).It has been reported that the upper pathophysiological concentration range of CHI3L1 can exceed 1000.00 ng/mL [21].In this case, the CLIA for CHI3L1 detection had the advantage of a wide linear range compared with other detection methods (Table 2).Its low background signal meant that the sensitivity of CLIA for CHI3L1 detection was higher than other detection technologies (Table 2).Compared with the widely applied ELISA method, the CLIA assay greatly reduces the detection time because it provides a simplifed procedure.However, the available equipment and costs of the CLIA assay should be considered in primary hospitals.In the precision assay, the intraassay, interassay, and total precision CVs were all below 10.00% (Table 4).Compared with previously reported methods [10][11][12], CLIA presented a simple operation process and shorter detection time, which was the main reason for its excellent precision and avoidance   [10] 2015 [11] 2021 [12] Present study ELISA, enzyme-linked immunosorbent assay; MB-FIA, magnetic bead fuorescent immunoassay; FICA, fuorescence immunochromatography assay; CLIA, chemiluminescence immunoassay; LOD, limit of detection; CHI3L1, chitinase-3-like protein 1. 4 International Journal of Analytical Chemistry of unpredictable variations.In addition, it has been reported that the CHI3L1 level is approximately 40 ng/mL in healthy blood serum [22], which was consistent with our data (Figure 2(a)).Notably, the CLIA method is highly automated, making it suitable for the detection of CHI3L1 in large-scale clinical samples.Te CLIA method to detect CHI3L1 represents a powerful tool to further explore the clinical value of CHI3L1.
Liver diseases are commonly caused by virus infections, especially HBV infections, in China [1].Considering the important clinical value of CHI3L1, we evaluated the CLIA for serum CHI3L1 detection in HBV-related liver diseases.From the baseline clinical characteristics, no pregnant or lactating women were found at the time of sample exclusion.
In addition, we found that HBV infection predominantly occurred in males (71/103), which might be due to their poorer lifestyle habits, such as alcohol consumption and smoking, compared with women [23].Te CHI3L1 concentrations were signifcantly higher in patients with CHB and HCC than those in the healthy controls and patients with CHB without HCC (Figure 2(a)), which was consistent with the results obtained by Jiang et al. [7] and Liang [24].
Studies have shown that CHI3L1 plays critical roles in cancer cell growth, proliferation, invasion, metastasis, angiogenesis, and immunoregulation [3,25,26].Tese features might account for the high CHI3L1 levels in HCC; therefore, the efect of CHI3L1 on the occurrence and development of HCC should be further studied.We also observed a threefold increase in the median level of CHI3L1 in patients with CHB and HCC (Figure 2(a)).Tis distinct diferential expression suggested that CHI3L1 might be highly specifc in HBV-related HCC.Indeed, compared with other clinical indicators, both AFP and CHI3L1 had a moderate diagnostic value for HBV-related HCC (Table 5).Serum AFP is a noninvasive marker commonly used in clinical laboratory screening for HCC.Meanwhile, the highest signifcant correlation was between CHI3L1 and AFP (Table 5, r � 0.694, P < 0.001).Tese results indicated that CHI3L1 has a good diagnostic value in HBV-related HCC.CHI3L1 concentrations did not change signifcantly with the increase in liver fbrosis stages, nor did the FIB-4 and APRI indices, and all three showed poor diagnostic value for liver fbrosis (Figure 2(b)).However, serum CHI3L1, FIB-4, and APRI were recommended indicators to assess liver fbrosis [9,20].
Te main reason for this discrepancy might be that the number of patients was small, especially the sample size of patients at the stage S3-S4 fbrosis (19/61).Te early stages of liver fbrosis can be reversible with treatment.Otherwise, liver fbrosis can lead to life-threatening LC or HCC [9].Terefore, it is necessary to further explore CHI3L1 for the diagnosis of liver fbrosis, including large-scale and multicenter studies, multifactor analysis, and the combined application of multiple indicators or methods.CHI3L1 can be used as a biomarker for prognostic assessment and targeted therapy in liver diseases [27,28], which also needs to be validated by using the CLIA.Considering the important clinical value of CHI3L1 in other diseases [3], the developed CLIA will be a powerful tool to further explore the clinical relevance of CHI3L1.

Conclusions
In conclusion, we validated the CLIA for the rapid determination of serum CHI3L1 levels in HBV-related liver diseases.Compared with the widely applied ELISA, the CLIA assay greatly reduces the detection time.In addition, the CLIA assay has a high sensitivity, a wide linear range, and an acceptable accuracy and precision for the quantitative detection of CHI3L1, which meets the requirements for clinical testing.Te CLIA assay for CHI3L1 detection also showed a good diagnostic performance in HBV-related HCC.Tis CLIA represents a powerful tool to further explore the clinical value of CHI3L1.

Figure 1 :
Figure 1: Analysis of the CHI3L1 calibration curve and the linear range.(a) Calibration curve for the CLIA assay to detect CHI3L1.(b) Scatter diagram of the measured concentration and the expected concentration.Each point represents the mean value of replicate measurements (n � 2).CHI3L1: chitinase-3-like protein 1.

Figure 2 :
Figure 2: Serum CHI3L1 detection in clinical samples.(a) Comparison of CHI3L1 levels among the three groups ( * * * P < 0.001).(b) Comparison of CHI3L1 levels among the diferent stages of liver fbrosis.(c) Te ROC curve for the ability of CHI3L1 to diagnose in HBV-related HCC.Te number by the dot in the graphs is the sample number.CHI3L1: chitinase-3-like protein 1; CHB: chronic hepatitis B; HCC: hepatocellular carcinoma; ROC curve: receiver operating characteristic curve; AUC: the area under the receiver operating characteristic curve.

Table 1 :
Analysis of the CHI3L1 linear range.

Table 2 :
Immunoassay methods to detect serum CHI3L1 levels.

Table 3 :
Evaluation of accuracy.

Table 5 :
Comparison of serum CHI3L1 and clinical indicators.