SUBCLASSIFYING LOW GRADE SQUAMOUS INTRAEPITHELIAL LESION AND ATYPICAL SQUAMOUS CELLS OF UNDETERMINED SIGNIFICANCE BY HUMAN PAPILLOMA VIRUS TYPE USING POLYMERASE CHAIN REACTION AND DOT BLOT HYBRIDIZATION
LO YF, CHAN ETY, CHUNG LP, COLLINS RJ.
Histopathology Department, Hospital Pathology Service, Queen Mary Hospital, Hong Kong
The majority of abnormalities detected by cervical smear belongs to low grade squamous intraepithelial lesions (LSIL) or atypical squamous cells of undetermined significance (ASCUS), groups of the Bethesda classification. These lesions are thought to progress to more significant pathology such as high grade squamous intraepithelial lesions (HSIL) or squamous cell carcinoma if they are associated with the high risk types of human papilloma virus (HPV16, 18, 33). The present PCR based method was developed as a screening test for HPV, and dot blot hybridization was used to determine the sub-types.
Materials adherent on the spatula used for cervical scraping were sent in absolute alcohol for analysis. Extracted DNA was amplified using primers specific to the HPV genome. HPV +ve samples were identified after agarose electrophoresis and ethidium bromide staining. The HPV +ve PCR products were dot blotted to nylon membrane and cross-linked by UV light. HPV sub-type specific, digoxigenin labeled probes were then used to hybridize and identify the high or low risk viruses with the aid of a colorimetric protocol.
CaSki, Hela and SiHa cell lines were used as controls. The method also proved to be applicable to formalin fixed paraffin embedded tissues. 300 clinical samples have been tested and the method has proved to be both reliable and useful.
Copyright 1997 Hong Kong Medical Technology Association .
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