| BackgroundHepatitis B virus (HBV) infection is a global health problem, with more than 350 million people being chronically infected worldwide, especially in China, the ratio of HBV infection is 7.18% nearly. HBV is the major aetiological agent of acute and chronic liver disease, including fulminant hepatitis, cirrhosis and hepatocellular carcinoma (HCC). At present HBV serological markers (HBV-M) test was checked by Enzyme-linked immunosorbent assay (ELISA) method commonly. With the developing of science and technology, HBV-M was detected by the time-resolved fluorescence Immunoassay (TRFIA) and electrochem-iluminescence Immunoassay (ECLIA) recently. In the field of HBV-M detection, there was the rapid colloidal gold technique, radioimmunoassay technique, the chemiluminescent technology, and so on. ELISA, the big advantage is economic, spending little time and asking for lower equipment is the advantage, too. But there is some shortcoming such as NOT quantitative, the false-negative and the false-positive result. TRFIA, the advantages are that the method can measure quantitativly, sensitivity and specificity more than ELISA. But the weak points are which spend long time and demand for large specimens. ECLIA, the strong point is which spend short time, the sensitivity and specificity is very high. RIA, the advantage is high sensitivityly and economical, but the shortcoming is radioactive-contamination. Different detection method had different result; medical disputes become more and more prominent.Objective1. Preliminary study the result of HBV-M is whether true or false by compare with three detection systems. In order the lab had given a perfect report, that patient and clinical doctor can adopt a more scientific personalized program to prevent hepatitis B infection, or slow down and stop the progression of cirrhosis and HCC.2. To explore the S/CO ratios of protection significance scope of HBsAb with ELISA to HBV infection, and to provide a reference information that the result of HBsAb has protective function or not, then to decide to strengthen immunization oneself.3.To judge the accurate result of low concentration of HBsAg by Logiest equation with ELISA.Method1. Comparison the differences of HBV-M by the three detection systems with ECLIA, TRFIA, ELISA452 cases were selected to our experiments, each specimen were checked HBsAg, HBsAb, HBeAg, HBeAb, HBcAb by three detection systems with ECLIA, TRFIA, ELISA. The unit of ELISA is S/CO ratio in HBV-M, and the unit of ECLIA is mIU/ml in HBsAb, Cutoff index (COI) with the other four serologic marks in HBsAg, HBeAg, HBeAb, HBcAb, and the unit of TRFIA is ng/ml in HBsAg, mIU/ml in HBsAb, PEI U/ml in HBeAg, HBeAb, HBcAb. TRFIA and ELISA detection systems used the quality control materials to strengthen quality control by the Center for Clinical Laboratory of Ministry of Health; ECLIA detection system used the Roche supporting quality control materials to strengthen the quality control.17 discrepant samples were confirmed by HBsAg confirmation assay based on the principle of specific antibody neutralization. Briefly, samples were pre-treated with the confirmatory reagent and control reagent before running the assay. Then the discrepant samples were confirmed by PCR of fluorescence quantitative with HBV (FQ-HBV-DNA).2. The S/CO ratios value of protection significance scope of HBsAb with ELISA756 cases (male 480, female 276) were selected to our experiments, each specimen were measured HBsAb by the two detection systems with ELISA and ECLIA, and HBsAg by ECLIA detection system. The unit of ELISA is S/CO ratio, which was used as quantified result. If the S/CO≥1, we regard the result as positive. While ECLIA can be directly quantified,≥10mIU/ml judged as positive. ELISA detection system used the quality control materials of 10mIU/ml and 30mIU/ml to strengthen quality control by the Center for Clinical Laboratory of Ministry of Health, ECLIA detection system used the Roche supporting quality control materials to strengthen the quality control.3. The judgment of the gray area of HBsAg to accurate result with ELISA detection system193 specimens which the S/CO ratio is from 0.5 to 5.0 in HBsAg with ELISA were selected to our experiments. All samples were checked by ECLIA detection system in the COBAS e601 platform. And 40 randomly samples were confirmed by HBsAg confirmation assay based on the principle of specific antibody neutralization. Briefly, samples were pre-treated with the confirmatory reagent and control reagent before running the assay.92 randomly specimens were confirmed by FQ-HBV-DNA. Logiest regression were running with the Dependent factor of HBsAg by ECLIA and the Covariates of HBsAg,HBsAb,HBeAg,HBeAb and HBcAb by ELISA. Then we derived a mathematical expression.4. Statistical methods or StatisticsAll date were registered by Excel2007 software, SPSS 16.0 statistical analysis software for analysis. The Crosstabs with Chi-square Test was used to compare the positive rate of HBV-M with three detection system of ELISA, TRFIA and ECLIA. The partitions of X2 methods were used to pair wise comparison.McNemar chi-square test and Kappa test were used to the HBsAb by two detection systems with ELISA and ECLIA, and the correlation analysis using linear regression.McNemar chi-square test and Kappa test were used to the HBsAg by two detection systems with ELISA and ECLIA. Logiest regression was used to the relationship between the HBsAg by ECLIA and the HBV-M by ELISA.Significance level a= 0.05.Results1. Comparison the differences of HBV-M by the three detection systems with ECLIA, TRFIA, ELISAThere was no significant difference of positive ratio in HBsAg, HBsAb, HBeAg by the three systems of ELISA, TRFIA, ECLIA (P> 0.05). There were statistically significant of positive ratio in HBeAb, HBcAb by the three systems of ELISA, TRFIA, ECLIA (P<0.05). The highest positive ratio in HBeAb was by TRFIA system, the lowest was by ELISA system, and the middle was by ECLIA system. The highest positive ratio in HBcAb was by ECLIA system, the lowest was by ELISA system, and the middle was by TRFIA system. There were statistically significant difference of positive ratio in HBeAb between the ELISA and the TRFIA, between the ELISA and the ECLIA (P<0.0125); while there was no significant difference of positive ratio between the ECLIA and the TRFIA (P> 0.0125). There were statistically significant differences of positive ratio in HBcAb between the ELISA and the TRFIA, between the ELISA and the ECLIA, between the ECLIA and the TRFIA (P＜0.0125).175 HBsAg-positive cases and 260 HBsAg-negative cases were checked by the three systems of ELISA, TRFIA, and ECLIA. The total coincidence rate was 96.24%. From this study,5 cases (2.75%) specimen were occurred cross-contamination. And 17 inconsistency cases were confirmed by HBsAg confirmation assay and the FQ-HBV-DNA.4 cases (2.22%) were false-negative,1 patient (0.04%) was false-positive of HBsAg by ELISA system, sensitivity was 97.22%, specificity was 99.63%. And 3 cases (1.67%) were false-negative,4 cases (1.47%) were false-positive, sensitivity was 97.78%, specificity was 98.53% by TRFIA system. But 0 cases (0.00%) was false-negative,0 cases (0.00%) was false-positive, sensitivity was 100.00% and specificity was 100.00% by ECLIA system.207 HBsAb-positive cases and 193 HBsAb-negative cases were checked by the three systems of ELISA, TRFIA, ECLIA, the total coincidence rate was 88.50%.46 HBeAg-positive cases and 390 HBeAg-negative cases were checked by the three systems of ELISA, TRFIA, ECLIA, the total coincidence rate was 95.58%.167 HBeAb-positive cases and 192 HBeAb-negative cases were checked by the three systems of ELISA, TRFIA, ECLIA, the total coincidence rate was 79.42%.261 HBcAb-positive cases and 66 HBcAb-negative cases were checked by the three systems of ELISA, TRFIA, ECLIA, the total coincidence rate was 96.24%.Completed one sample of HBV-M, the ECLIA system spending the time is shortest, just 27 minutes. And completed one group of samples (90 specimens) of HBV-M, the ELISA detection system is quickest, just 55 minutes.2. The S/CO ratios value of protection significance scope of HBsAb with ELISAHBsAb of 756 cases were detected by two detection systems of ELISA and ECLIA, 349 cases were positive,337 cases were negative by ECLIA and ELISA, the total coincidence rate was 90.74%, the value is 0.815 by the Kappa test. The results of 21 cases were positive by ELISA, but the results were negative by ECLIA. And 49 cases were positive by ECLIA, but there were negative by ELISA.In the 453 HBsAg-negative samples, If the S/CO ratios below 1.0 in HBsAb with ELISA (n=98),58 cases lower than 10mIU/ml (82.7%),11 cases more than 10mIU/ml with ECLIA (17.3%), no one more than 100mIU/ml. If the S/CO ratios between 1.0-5.0(n=86),11 cases less than 10mIU/ml (12.8%),72 cases in the 10-100mIU/ml(83.7%), only 3cases more than 100mIU/ml(3.5%). If the S/CO ratios between 5.0-13.0(n=69),29 cases in the 10～100mIU/ml(42.0%), then 40 cases more than 100mIU/ml(58.0%),and no one less than 10mIU/ml. If the S/CO ratio more than 13.0(n=200),199 cases more than 100mIU/ml(99.5%).3. The judgment of the gray area of HBsAg to accurate result with ELISA detection system149 low concentrations of HBsAg positive specimens with ELISA,there were 37 negative-HBsAg by rechecked with ECLIA, the false-positive rate was 19.17%.44 HBsAg-negative specimens with ELISA, there were 16 positive-HBsAg by rechecked with ECLIA, the false-negative rate was 8.29%. The total coincidence rate was 72.54%, the value was 0.332 by the Kappa test.There were statistically significant differences(X2=8.321, P=0.005).After excluding 10 cases of neonatal specimens, the Logiest regression of 183 cases showed, the negative predictive accuracy was 86.0%, positive predictive accuracy was 98.4%, predictive accuracy for all cases was 94.5%, if P≥0.9, then the positive predictive accuracy was 100%.-2Log likelihood value was 58.224, Nagelkerke R2 is 0.848.Logiest regression equation is: (2.445x HBsAg-1.586x HBsAb+5.464xHBeAg-1.048xHBeAb-4.045xHBcAb-0.117) P=e/(1+e2.445×HBsAg-1.586×HBsAb+5.464×HBeAg-1.048×HBeAb-4.045×HBcAb-0.117)Conclusion1. There were certain false positive and false negative in detecting HBsAg by ELISA and TRFIA system. But there was no significant difference in positive rate by three kinds of systems (P> 0.05). There was no significant difference in positive rate of HBsAb and HBeAg, too (P> 0.05). And there were significantly difference in positive rates of HBeAb, HBcAb (P<0.05). HBsAb, HBeAg, HBeAb, HBcAb is had no gold standard currently, so can't distinguish who was superior.2. The S/CO ratios of protection significance scope of HBsAb with ELISA to the HBV infection is more than 13.0.3. Without rechecking the specman in HBsAg of the low concentration by ECLIA method, we can calculate the P value by Logiest regression, then as P=0.5 for the critical point, the higher P value, the greater positive predictive accuracy, the smaller P value, the greatter negative predictive accuracy.
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