title: Development of an automated extraction procedure for detection of human papillomavirus DNA in liquid based cytology samples.
abstract: Liquid based cytology samples are being used increasingly to improve cervical screening and have the advantage that residual cell suspension is available for other tests such as human papillomavirus (HPV) detection. However, as the transport medium is optimised for downstream cytology, problems can be experienced during extraction of nucleic acid. This study aimed to develop a robust protocol for automated extraction of HPV DNA from cervical, liquid based cytology samples using a high throughput robotic system. Considerable modification of existing clinical extraction protocols for swab specimens, together with optimisation of required sample input volume was required to reduce sample blockage during the extraction to acceptable levels. The blockage rate and optimal processing volume was assessed by extracting a fixed volume (1/4) of re-suspended material from the centrifuged pellets of 10, 5 and 1 ml aliquots of 200 specimens. Analysis revealed 17.5% blockage with specimens originating from 10 ml aliquots; 3% with 5 ml and no blockage with 1 ml aliquots of the same samples. A 3% blockage level is acceptable for an automatic well clearance procedure to be followed. HPV testing of the extracts by real-time PCR showed a 1.5% loss of sensitivity in extracts originating from 1 ml aliquots as compared with 5 ml aliquots with a consequent loss of detectable HPV genotypes after reverse hybridisation. In short, 5 ml of liquid based cytology specimen is recommended for nucleic acid extraction, to allow optimal detection of HPV types in clinical samples while retaining maximum efficiency of the robotic extraction procedure.
authors.affiliation: Regional Clinical Virology Laboratory, Lothian University Hospitals NHS Trust, The Royal Infirmary of Edinburgh, Little France, 51 Little France Crescent, Edinburgh EH16 4SA, UK. katiecushieri@hotmail.com
authors.forename: Kate S
authors.initials: KS
authors.lastname: Cuschieri
authors.forename: A Louise
authors.initials: AL
authors.lastname: Seagar
authors.forename: Catherine
authors.initials: C
authors.lastname: Moore
authors.forename: Gaye
authors.initials: G
authors.lastname: Gilkison
authors.forename: Janet
authors.initials: J
authors.lastname: Kornegay
authors.forename: Heather A
authors.initials: HA
authors.lastname: Cubie
mesh_terms: D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D012371:Robotics; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral