title: Development of optimal liquid based cytology sample processing methods for HPV testing: minimising the 'inadequate' test result.
abstract: Incorporation of HPV testing into cervical screening is anticipated and robust methods for DNA extraction from liquid based cytology (LBC) samples are required. This study compared QIAamp extraction with Proteinase K digestion and developed methods to address DNA extraction failure (β-globin PCR negative) from clinical specimens. Proteinase K and QIAamp extraction methods in paired LBC samples were comparable with adequate DNA retrieved from 93.3% of clinical specimens. An HPV prevalence cohort (n=10,000) found 7% (n=676) LBC samples tested negative for β-globin, and were classified as inadequate. This 'failure' rate is unsuitable for population screening, particularly as the sampling method is intrusive. 379/676 samples were assessed to determine the cause of test failure. Re-testing confirmed adequate DNA in 21.6% of the original extracts; re-extraction from stored material identified 56.2% samples contained adequate material; dilution to overcome sample inhibition (1:10) resolved 51.7% cases in original extracts and 28% in new extracts. A standardised approach to HPV testing with an optimal DNA concentration input rather than standard volume input is recommended. Samples failing initial DNA extraction should be repeat extracted and assessed for sample inhibition to reduce the 7% of HPV tests being reported as inadequate and reduce the need for retesting of those women to <1%.
authors.affiliation: HPV Research Group, Department of Obstetrics and Gynaecology, School of Medicine, Heath Park, Cardiff University, Cardiff CF14 4XN, United Kingdom. peevorrj@cardiff.ac.uk
authors.forename: R
authors.initials: R
authors.lastname: Peevor
authors.forename: J
authors.initials: J
authors.lastname: Jones
authors.forename: A N
authors.initials: AN
authors.lastname: Fiander
authors.forename: S
authors.initials: S
authors.lastname: Hibbitts
mesh_terms: D000328:Adult; D000368:Aged; D003584:Cytological Techniques / Q000379:methods*; D004279:DNA, Viral / Q000302:isolation & purification; D005188:False Negative Reactions; D005260:Female; D006801:Humans; D008875:Middle Aged; D027383:Papillomaviridae / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis*; D013048:Specimen Handling / Q000379:methods*; D014773:Virology / Q000379:methods*
chemical_list: D004279:DNA, Viral