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Eric P Dixon,
Petter Grønn,
Lorraine M King,
Heather Passineau,
Hema Doobay,
Hanne Skomedal,
Jalil Hariri,
Shauna N Hay,
Charlotte A Brown, Timothy J Fischer,
Douglas P Malinowski
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ABSTRACT: Several commercial HPV ancillary tests are available for detection of E6/E7 RNA. It is not clear how storage of a cervical Pap affects the analytical and clinical performance of the PreTect™ HPV-Proofer assay. To investigate the qualitative performance of RNA extracted from BD SurePath™ liquid-based cytology (LBC) specimens for the detection of human papillomavirus (HPV) E6/E7 mRNA using the PreTect™ HPV-Proofer assay, studies including stability, reproducibility, residual specimen analysis, and storage medium comparison assays were performed. Cervical cytology specimens were collected and stored in BD SurePath™ LBC preservative fluid and/or PreTect™ Transport Media. RNA was isolated using the RecoverAll™ Total Nucleic Acid Isolation kit and RNA integrity was evaluated in the PreTect™ HPV-Proofer assay. The performance of RNA isolated from cervical cells collected and stored in BD SurePath™ LBC preservative fluid or PreTect™ Transport Media was also evaluated through a storage medium comparison study. The RNA was found to be stable for a minimum of 21 days when stored at ambient temperature and displayed high reproducibility with the mean percentage reproducibility ranging from 90.5% to 100% for the HPV types detected by the PreTect™ HPV-Proofer assay. The prevalence rate of HPV types in this study cohort was consistent with published reports. A 93.7% first pass acceptance rate was demonstrated across all cytology grades. The positive human U1 snRNP specific A protein (U1A) and HPV rate for BD SurePath™ LBC and PreTect™ Transport Media specimens was statistically equivalent for both normal and abnormal specimens. This data support the use of RNA isolated from BD SurePath™ LBC for ancillary HPV testing and demonstrates the feasibility of using BD SurePath™ preservative fluid as a specimen type with the PreTect™ HPV-Proofer assay.
Journal of virological methods 07/2012; 185(2):199-203. · 2.13 Impact Factor
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ABSTRACT: The AMPLICOR HPV Test has been validated for use with cervical cells collected in liquid-based preservative fluids, such as BD SurePath. It is currently recommended, however, that residual BD SurePath samples be stored at 4 degrees C prior to testing.
The aim of this study was to demonstrate that DNA isolated from SurePath cervical cytology specimens and stored at ambient temperature was also compatible with the AMPLICOR HPV Test.
DNA was extracted using the AmpliLute Media Sample Preparation Kit. Amplification and detection of HPV was performed both as directed by the manufacturer and with minor protocol modifications.
Cervical specimens collected in SurePath preservative fluid remained stable for testing with the AMPLICOR HPV Test for at least 21 days. The performance of DNA extracted from specimens stored at room temperature was equivalent to DNA extracted from specimens stored at 4 degrees C. The beta-globin internal control was detected in all of the 146 residual SurePath cervical cytology specimens tested using the AMPLICOR HPV Test, and high-risk HPV was detected in 46.2% (18/39) of ASCUS cases, in 63.3% (19/30) of LSIL cytology specimens, and 92.3% (24/26) of HSIL cases. Concordance of AMPLICOR HPV Test results with Hybrid Capture II was 83.9%.
The AMPLICOR HPV Test can be successfully and reproducibly performed from DNA isolated from residual SurePath cervical cytology specimens stored at ambient temperature for at least 21 days. This provides clinical laboratories flexible storage conditions for residual SurePath cytology specimens.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 03/2010; 48(1):31-5. · 3.12 Impact Factor
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ABSTRACT: Molecular diagnostic adjuncts could improve the specificity of cervical cancer screening. Since persistent infection with human papillomavirus (HPV) is found in virtually 100% of cervical cancer cases, testing for markers of HPV integration may have a role in identifying underlying high-grade lesions in patients with low-grade cytologic abnormalities. Several proteins associated with the cell cycle are known to be affected by HPV integration into the host's DNA. Immunocytochemical identification of these upregulated proteins can assist in the identification of small numbers of pre-neoplastic or neoplastic cells in routine cytologic sampling.
Methods in molecular biology (Clifton, N.J.) 02/2009; 511:297-310.
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ABSTRACT: This study was performed to demonstrate that RNA isolated from cell lines and cervical cytology specimens stored in SurePath preservative fluid would be functional in real-time RT-PCR assays. RNA was isolated from cervical cell lines or cytology samples stored in SurePath preservative at room temperature for 2-5 weeks using five commercially available RNA purification kits, three of which contain proteinases. The quality of the RNA was assessed by real time RT-PCR amplification of GAPDH, GUSB, U1A, HPV 16 and 18 E6 mRNAs. RNA was isolated successfully from cells that were stored in SurePath preservative fluid with only the three protocols that contained proteinases. GAPDH was amplified in 98-100% of the samples, GUSB in 90-98%, and the least abundant transcript, U1A, was amplified in 81-96% of the samples. HPV 16 and 18 E6 transcripts were detected in 56% of high grade, 39% of low grade and 2% of normal samples, with a concordance between DNA genotype and E6 mRNA expression of 97%. We demonstrated that RNA can be extracted from cervical cell lines and cytology specimens stored in BD SurePath preservative fluid with three different procedures that all contain proteinases. This RNA is suitable for real-time RT-PCR applications.
Journal of Virological Methods 01/2009; 156(1-2):138-44. · 2.01 Impact Factor
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ABSTRACT: Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.
Journal of Virological Methods 09/2008; 154(1-2):220-2. · 2.01 Impact Factor
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[show abstract]
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ABSTRACT: Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath™ liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect™ HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath™ cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.
Journal of Virological Methods. 154:220-222.
