A flow cytometry detection at a protein level of human papillomavirus

Abstract

Abstract Introduction: Human papillomavirus (HPV) is one of the most common sexually transmitted infections and a necessary cause of cervical cancer. Fortunately about 90% of HPV infections clear within two years without treatment. Only in a small amount of the women infected with oncogenic types of HPV the infection will progress to high-grade lesions or even cervical cancer in the absence of treatment. During persistent infections the viral genomes often integrate into the host chromosome, which results as an over expression of the viral proteins E6 and E7. These oncoproteins induce immortalization and malignant transformation of cells conferring a certain growth advantage to the infected cells. Method: A total of 100 women undergoing routine cervical cytology samples joined this cohort study. A Thin Prep Pap Test sample was taken following consent for study participation and was tested for HPV. Cytological screening of the samples was done by trained cytologists. After sample collection, a 1 ml aliquot was removed and prepared for flow cytometric analysis and the rest of the sample was sent for PCR analysis. The samples were run on a Beckman Coulter FC500 with a 488 nm argon laser, with forward (FS) and side (SS) scatter. The gating was fixed for all samples. The instrument was set to measure counts collected from the whole volume of the samples under investigation Results: In the total of 100 cases, 4 specimens were characterized as unsatisfactory. For the rest 96 specimens, 82 specimens were found to be positive for the HPV virus and 14 negative. In the total of 100 cases the analysis of flow cytometry and PCR detection gives the same results in most cases. The cervical cells found in the liquid specimen are resolved in distinct populations using forward and side scatter of a flow cytometer. All samples in the study were analyzed using previously established gates for the presence of endocervical, ectocervical and polymorphonuclear cells. Each population was determined and the percentage of cells included was counted. The total time of analysis depended on the density of each specimen. Conclusion: HPV analysis by flow cytometry takes advantage of the fact that those oncogenic genotypes over express E6 and E7 mRNA following integration of HPV into genomic DNA. If the percentage is over 2% the result is positive, and if is lower is a negative result. With a negative result the risk of having a cervical cancer in the future is almost null. It can identify persistent HPV infections that can progress to high-grade lesions, detects the oncogenic activity, analyzing cervical cells that over express oncoproteins E6 and E7, and can be used at any time of woman life because there is no low age limit. The specificity and positive predictive value was higher than PCR analysis. This might be very useful in screening young women, a cohort in which the prevalence of HPV DNA positivity may be as high as 20%.