Reference Materials For Human Papillomavirus Genotyping:Establishment And Application

BackgroundPersistent human papillomavirus (HPV) infection is responsible for the majority of cervical cancer and some genital warts. Many genotypes of HPV have been molecularly characterized, which were classified as high-risk (HR) types and low-risk (LR) types. HPV HR types are associated with cervical cancer and HPV-16and-52are most common HR types in China. The next most common HPV types in China are HPV-18,-33and-58. HPV LR types are associated with genital warts and HPV-6and-11are most common LR types in China. HPV genotyping is associated with screening of cervical cancers, evaluation and monitoring of HPV vaccination and epidemiological investigation. However, various assays for human papillomavirus (HPV) genotyping are currently used for cervical cancer screening, such as PCR-reverse dot hybridization (PCR-RDB), real-time PCR and so on. There were some problems for HPV genotyping, such as cross-reaction and false-negative. Standards for analytical sensitivity were not available. International Standards (IS) of HPV16and HPV18DNA were established for detection and quantification of HPV16and HPV18DNA by the WHO to make HPV genotyping comparable. For manufacturers, the IS could be used to determine the analytical sensitivity of the assays, calibrate the commercial assays and prepare the calibrators for quality control. For clinical laboratories, the IS could be used for the performance confirmation of the assays and internal quality control (IQC). Nevertheless, international standards are expensive and not enough for calibration and evaluation of commercial assays, IQC and external quality assessment (EQA). EQA is necessary for quality assurance of HPV genotyping. The homogeneity and stability of human cell lines is not satisfying for EQA, because the cells sink when dispensing in vials. Before this study, a proficiency test system for HPV genotyping is not available for standardizing and evaluating the analytical sensitivity, accuracy and cross-reaction of assay performance in China.The plasmids of HPV types16(ATCC number:45113) and18(ATCC number:45152) were purchased from the American Type Culture Collection (ATCC). The HPV genomes were sequenced and blasted to confirm the genotypes, then the plasmids were transformed and purified. Purified plasmids containing viral DNAs for HPV types were measured at260nm and280nm to estimate the purity and concentration. Then, the plasmids were diluted to a stock concentration of108copies/μL. After human placenta DNA added, the preparation was dispensed and subjected to the research of homogeneity and stabilities at37℃, room temperature,4℃and-20℃. Reference materials of HP V-16and18were detected by4real-time PCR assays. The concentration of reference materials was valued and uncertainty was calculated, according to standard curves established with ISs of HPV-16(NIBSC code:06/202,5×106IU/ampoule) and HPV-18(NIBSC code:06/206,5×106IU/ampoule). The reference materials are confirmed to be stable after storage at temperatures up to-20℃for12month,4℃for3month, room temperature for15days and37℃for7days. The concentration of HPV-16and18was valued to be (7.1±1.6)×106IU/ml and (3.5±0.9)×106IU/ml. Reference materials of HPV-16and18were established to apply for National reference materials.Similarly, samples of HP V-6、-11、-31、-33、-39、-51and-52were prepared for EQA. The plasmids of HPV-11and HPV-52were purchased from ATCC, while other plasmids were provide by City University of HongKong. Since ISs for these types were not available, plasmids of the7types were diluted according to the concentration calculated using absorbance at260nm compared with the corresponding values of HPV16and HPV18plasmids. The genotypes of plasmids were confirmed by sequencing and PCR-RDB. Then the panel of25samples was prepared for EQA, including9genotypes with different concentration, multiple infections and1negative sample. The panel contained HPV-16and18at concentration of106IU/mL.105IU/mL and104IU/mL, HPV-6、-11、-31、-33、-39.-51and-52at concentration of106GE/mL和105GE/mL,3samples of multiple infections (HPV-6、-51and-16, HPV-33、-39and-52, HPV-18and-31) and1negative sample. The panel was distributed to76clinical laboratories at room temperature. All laboratories were required to detect the samples using their routine procedures and submit the results before deadline. Results reported by participants were compared with the relevant reference results. The percentages of agreement and proficiency test (PT) results were calculated, according to different HPV genotyping assays. The samples containing106IU HPV16/mL and106IU HPV18/mL were (98.7%and96.0%, respectively) identified correctly most often. For other high-risk HPV types, about90%of data sets correctly identified samples containing106GE/mL of HPV6,-31,-33, and-52. HPV51,-11and-39in106GE/mL were correctly identified by only42.7%,55.6%and21.3%of laboratories, respectively. Eighteen of the75laboratories had false-positive results in28samples. Eighteen of the75laboratories had false-positive results in28samples. Laboratories using PCR-RDB reported false-positive results in24 samples. According to the criteria agreed upon at a WHO meeting regarding the standardization of HPV assays, a test should detect at least50IU per5μL of HPV16and HPV18and detect at least500GE per5μL of the other HPV types. And at most,1false-positive result was acceptable. Based on the criteria,9data sets were100%proficient. Our results showed that datasets for HPV-39,-51and multiple infections reported by the laboratories were not satisfactory. False-positive results were another noticeable problem in our study, which was more common in laboratories using PCR-RDB. Our results indicated that it was essential for clinical laboratories to confirm the performance of the assays, improve their operation and quality control and avoid the "contamination" of PCR products.We established reference materials of HPV-16and18DNA traceable to ISs, which was used to apply for National reference materials. Based on the panel including9genotypes, the EQA for HPV genotyping was first carried out in China. Some problems in HPV genotyping were found, which will contribute to the improvement of quality control in clinical laboratories.