Multi-center Evaluation Of Gen.2 ECL Assay In The Detection Of Hepatitis B Surface Antigen

1. BackgroudAccording to WHO statistics, Hepatitis B virus (Abbreviate:HBV) infection is a global health problem that two billion people have been infected worldwide. It is even much more serious in China. So what we should do is to improve the detection ability of HBV and give early treatment. It is a significant case to maintain chineses'health.HBV has strong capacity resistant to abominable environment. Hepatitis B surface antigen(Abbreviate:HBsAg) is one of the first serum markers to appear during the course of HBV infection and can be detected 2 to 8 weeks before biochemical evidence of liver dysfunction and the onset of jaundice. HBsAg is cleared within a few months in self-limiting illness. So HBsAg is the established serological marker used routinely for the diagnosis of acute or chronic HBV infection, the screening of blood or organ donors, and the surveillance of persons at risk of acquiring or transmitting. But HBV antigenic variation occurs frequently, especially S gene mutations has been detected in vaccinated children, in liver transplant recipients receiving Anti-HBs immunoprophylaxis, and in chronic carriers. The "a" determinant is the double-loop of HBV surface structure located from position 124 to 147 of HBsAg. It exists in all HBV genotype, also be the main antibody combined site of commercial HBsAg detecting assay. HBV variants with mutations on the "a"determinant have been identified following vaccination. Natural variation and mutation can induce HBsAg conformational changes, also account for the false-negative results in immunoassays. Due to the above factors, more improvements are done to decrease the interference of false-negative results caused by mutation. Routine techniques used by Chinese laboratories are in the process of changing from ELISA to ELCA/ECL. Meantime, newly developed HBsAg commercial assays exist, showing a performance increase in terms of specificity and sensitivity. Meantime, so-called generation 2 HBsAg detecting assay has been launched by Roche daignaostics, Germany. It is essential to know clearly and evaluate these commercial assays in Chinese clinical laboratories due to their high costs. Applying rationally can make largest profit on medical resource. 2. ObjectivesEvaluate the performance of Gen.2 ECL assay detecting HBsAg in conjuction with its clinical value.3. Contents1) Performance of Gen.2 ECL HBsAg immunoassay detecting HBVmutants forms.2) A view of clinical application with Gen.2 ECL HBsAg immunoassay via prevalent test in Shanghai Area.4. Methods1) The portocol is designed to evaluate newly launched Gen.2 ECL immunoassay comparing to another two HBsAg commercial immunoassays (Abbott ELCA and Kehua ELISA) via 8 hospitals. First, collecting 212 patient samples to know the accordance and precision. Secondly, analyze the HBsAg mutant detection capabilities of Gen.2 ECL immunoassay in comparison to Abbott ELCA and Kehua ELISA assay by detecting 13 recombinant mutants and 3 natural mutants;.Thirdly,8 seroconversion panels are used to investigate the sensitivity of these 3 assays. At last, account the sensitivity, specificity, concordance rate, ROC curve of Gen.2 ECL immunoassay in parallel with Abbott assay known as the golden standard from 1916 routine samples. My aid is to get the conculsion through above experiments:·Which one is well designed to anti-mutants interference;·Which one is optimized to respond to the HBV conversion;·Which one can help to screening hepatitis disease earlier, shortening the mean time of diagnostic window.2) Analyze 950 health checkup samples by Gen.2 ECL immunoassay from Shanghai XX hospital, have a close look at clinical application of this newly launched assay.5. ResultsThe study showed nearly 100% coincidence results to 212 positive serum sample among Roche Gen.2 ECL assay, Abbott ELCA (99.25% includes one negative result) and kehua ELISA assay (100%). Generally these 3 assays had consistent correlation trend which means the higher results of Roche, the higher abbott/Kehua did. The phenomena appeared in low-result segment particularly. Roche Gen.2 assay could detect 98.1% mutant forms (totally 13 recombinant mutants and 3 natural mutants) as Abbott ELCA assay got 87.6% and Kehua got only 19.3%. So Roche Gen.2 ECL assay had a higher precision than Abbott and Kehua. Also Kehua ELISA assay showed large vacancy in detecting HBV mutants. In 5 seroconversion panels test, Roche Gen.2 ECL assay could catch most stages of hepatitis B disease while Kehua gave poor performance:The day-since-first-HBsAg-positive-bleed of Roche was 162 days,78 days earlier versus Kehua; median was 19 days,20 days earlier versus Kehua; the sensitivity was statistically significant better for Roche HBsAg assay versus Kehua(wilcoxon, P=0.043<0.05).8 seroconversion panels were detected nearly the same with Roche versus Abbott. The day-since-first-HBsAg-positive-bleed of Roche was 179 days,15 days later versus Abbott; median was 16 days while Abbott is 15.5days; the sensitivity was not statistically significant better between Roche HBsAg assay versus Abbott(wilcoxon, P=0.553>0.05). A sensitivity index (number of samples not detected devided by the total panel number investigated, f-neg/panel) was:Roche Gen.20.49, Abbott ELCA 0.55, Kehua ELISA 0.90 (Index closer to "0" showed better clinical sensitivity). Calculating 1916 routine outpatient sample results, got the data of Gen.2 ECL HBsAg assay:sensitivity 99.7%, false-negative rate 0.3%, specificity 99.0%, false-positive rate 1.0%, PPV 98.5%, NPV 99.8%, ROC curve 0.994, SD 0.002,95% confidence range from 0.990 to 0.998.Randomly enrolled 950 healthcheck samples from a Shanghai hospital, then detected them by Roche Gen.2 ECL HBsAg immunoassay (Male/Female rate: 1.081:1; Age ranged from 19-88 years old, distributes on 48.06±12.85 years old). HBsAg positive rate was 8.32%, male positive rate was 9.02% while female was 7.21%, ranged between 45±11 years old. There was no statistically significance on Gender (X2=0.973, P value=0.324>0.05) and age (X2=5.556, P value=0.592> 0.05).According to above studies, Roche Gen.2 ECL HBsAg immunoassay presented great performance on both sensitivity and specificity. It took obvious advantage comparing to Kehua assay specially in detecting HBV mutants. It also helped to screening hepatitis disease earlier, shortening the mean time of diagnostic window. Investigation revealed no obvious false-positive rates by using Roche Gen.2 assay in clinical experiment. So we could get the conclusion that Roche Gen.2 ECL HBsAg immunoassay is a proper choice for clinical laboratory.