Long-Term Stability of Human Genomic and Human Papillomavirus DNA Stored in BD SurePath and Hologic PreservCyt Liquid-Based Cytology Media

Abstract

We evaluated the effect of storage at 2 to 8°C on the stability of human genomic and human papillomavirus (HPV) DNA stored in BD SurePath and Hologic PreservCyt liquid-based cytology media. DNA retained the ability to be extracted and PCR amplified for more than 2.5 years in both medium types. Prior inability to detect DNA in archived specimens may have been due to failure of the extraction method to isolate DNA from fixed cells. Liquid-based cytology (LBC) media (BD SurePath [Becton, Dickinson and Company] and Hologic PreservCyt [Hologic Inc.]) are routinely used to prepare liquid Pap preparations for cervical cancer screening. In the United States, molecular human papillomavirus (HPV) testing out of the LBC vial is recommended (i) as a triage test for determining whether a woman with atypical squamous cells of undetermined significance (ASC-US) needs immediate colposcopic evaluation and (ii) as an adjunct to cervical cytology, "cotesting," for cervical cancer screening of women of age 30 years and older. In addition, there is a growing interest in the use of LBC media for primary HPV screening in which positive specimens could be reflexed to cytology, HPV type-specific genotyping, or another triage method, although molecular tests have yet to be approved for this intended use (1, 2). LBC media were originally developed and approved for the preservation and preparation of cells for Pap smear evaluation (3, 4). BD SurePath and Hologic PreservCyt media are both alcohol-based preservatives and are designed to fix cellular and subcellular components and aid in the cytological evaluation of the obtained smears. Fixative solutions can prove problematic for downstream molecular extraction methods because of the potential to inhibit cellular lysis, because of the potential to interfere with the proteolytic enzymes used in this process, or due to cross-linking of nucleic acids and proteins (5, 6). Some prior studies have reported poor recovery of nucleic acids from SurePath and PreservCyt media and inferred that the DNA had degraded over time during storage (7-10). SurePath medium contains a low concentration of formalin, which is known to cross-link nucleic acids and protein (11,12). Formalin treatment results in reversible cross-linking of proteins to DNA, which can render it refractory to magnetic-or silica-based purification and subsequent downstream nucleic acid biochemistry, such as PCR (13). Cross-linking can be removed using proteinase K digestion and/or heat (14-16).We have developed a simple, one-step chemical lysis method for use with the BD hrHPV-GT assay that can process 0.5 ml of either LBC specimen without prior cell harvesting or the use of lytic enzymes. The method uses a combination of heat and chemicals to lyse LBC-preserved cells directly in the media. It also efficiently reverses cross-linking effects and allows biologically active DNA to be extracted directly from the resulting lysate (17-19).The objective of the current study was to examine the effect of long-term storage of LBC specimens at 2 ...

A comparison of different human papillomavirus tests in PreservCyt versus SurePath in a referral population—PREDICTORS 4

“…A 0.5 ml aliquot of thoroughly vortexed SurePath or PreservCyt was added to 1.7 ml of a proprietary HPV diluent. A heat step was employed to ensure that exfoliated cells were lysed and the sample homogenized prior to extraction of sample DNA [4], [5].The Genera PapType Test is a semi-automated, bead-based multiplex full genotyping DNA assay for 14 high-risk HPV types (16,18,31,33,35,39,45,51,52,56,58,59,66,68) and two low-risk HPV types (6,11). The Sirocco platform (Genera Biosystems, Scoresby, Australia) was used.…”

Sensitivity of high‐risk HPVHybrid Capture II (hrHPV HC2) test using SurePathTM specimens in the prediction of cervical high‐grade squamous lesions

“…Other retrospective and prospective published studies of hrHPV HC2 testing on SurePath TM specimens with abnormal cytology have reported sensitivities ranging from 93% to 100% for CIN 2 and higher. 27, 28 Castle et al found hrHPV HC2 on ThinPrepV R specimens was unaffected by a storage time of up to 8 years at ambient conditions, though cytomorphology and DNA recovery by PCR decreased significantly. 12,[14][15][16][17][18][19][20][21] The lowest sensitivity, reported by Evans et al, 17 was not replicated in other studies; in this study, the sensitivity was 50% for LSIL, but they had only 10 cases.…”

“…Siddiqi (2009) (retrospectively) and Zhao (2011) (cross sectionally) studied hrHPV HC2 testing on both Sure-Path TM and ThinPrepV R specimens and found the falsenegative rates to be comparable. 28,30 Low false-negative rates have been reported by Ko et al, who undertook a prospective validation study using routine clinical cases handled with specific specimen storage and processing protocols that take SurePath TM limitations into consideration (6.6% for CIN 2 and 5% for CIN 3, among cases with ASCUS cytology), 11 and by Qureshi et al compared to ThinPrepV R (4% vs. 6%, P 5 0.72) using PCR as gold standard. 27, 28 Castle et al found hrHPV HC2 on ThinPrepV R specimens was unaffected by a storage time of up to 8 years at ambient conditions, though cytomorphology and DNA recovery by PCR decreased significantly.…”

“…Other studies found 76-100% sensitivity; when ASC is excluded and cytology is LSIL and above, hrHPV HC2 sensitivity is 85-100% for ThinPrepV R . 28 The potential limitations of SurePath TM , if there are any, can be ameliorated by (1) prompt testing, (2) specimen storage at lower temperatures but above freezing, 28,[30][31][32] (3) the addition of 50% ethanol 33 or Qiagen's own aliphatic amine Specimen Transport Medium 11 shortly after cytology processing, and (4) the use of DNA extraction methods that reverse cross-linking. 12,26 Some studies report a lower nucleic acid recovery rate in SurePath TM medium compared to ThinPrepV R medium, and this has been postulated to be due to a higher rate of nucleic acid degradation over time or cross-linking in SurePath TM specimens due to the presence of a low concentration of formalin in the medium.…”

Evaluation of RNA and DNA extraction from liquid‐based cytology specimens

“…In previous studies, the efficiencies of different methods for nucleic acid extraction from LBC specimens were examined. However, it was unclear how many cells were needed for detection in genetic analyses . We determined the number of cells necessary for the detection of DNA and RNA by PCR using bladder and cervical cancer cell lines.…”