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Development and Validation of a Preanalytic Procedure for Performing the cobas HPV Test in SurePath Preservative Fluid

2016; Elsevier BV; Volume: 19; Issue: 2 Linguagem: Inglês

10.1016/j.jmoldx.2016.10.003

1943-7811

Mark D. Krevolin, David C. Hardy, Jim Pane, Shagufta Aslam, Catherine Behrens,

Hepatitis B Virus Studies

The formation of chemical cross-links between nucleic acids and proteins in formalin-containing media presents challenges for human papillomavirus (HPV) testing of cervical samples collected in SurePath Preservative Fluid. A preanalytic process involving addition of a nucleophilic buffer and heating the sample to 120°C was developed to reverse the effects of cross-linking and improve nucleic acid accessibility for the cobas HPV Test in SurePath. Cycle threshold (CT) values for cobas HPV detection were evaluated over time and various temperatures, and mean CT differences between pretreated and both untreated SurePath samples and those collected in PreservCyt were assessed. Without pretreatment, low viral levels (1 × limit of detection) of HPV were no longer detectable by 7 days. For prospectively collected specimens, mean (95% CI) CT differences between pretreated and untreated samples indicated enhanced HPV DNA recovery in all categories of treated samples: −2.58 (−3.16 to −2.01), −2.63 (−3.62 to −1.64), and −3.39 (−4.95 to −1.82), respectively, for other 12 high-risk HPV types, HPV16, and HPV18. Furthermore, mean (95% CI) CT differences of pretreated SurePath samples were comparable to simultaneously collected PreservCyt samples: −0.48 (−0.98 to 0.02) and −0.23 (−0.93 to 0.46), respectively, for HPV16 and HPV18; a borderline significant difference [−0.35 (−0.57 to −0.13)] was observed for other 12 high-risk HPV types. This preanalytic procedure therefore ensures a validated, safe, and accurate method for cobas HPV testing in SurePath. The formation of chemical cross-links between nucleic acids and proteins in formalin-containing media presents challenges for human papillomavirus (HPV) testing of cervical samples collected in SurePath Preservative Fluid. A preanalytic process involving addition of a nucleophilic buffer and heating the sample to 120°C was developed to reverse the effects of cross-linking and improve nucleic acid accessibility for the cobas HPV Test in SurePath. Cycle threshold (CT) values for cobas HPV detection were evaluated over time and various temperatures, and mean CT differences between pretreated and both untreated SurePath samples and those collected in PreservCyt were assessed. Without pretreatment, low viral levels (1 × limit of detection) of HPV were no longer detectable by 7 days. For prospectively collected specimens, mean (95% CI) CT differences between pretreated and untreated samples indicated enhanced HPV DNA recovery in all categories of treated samples: −2.58 (−3.16 to −2.01), −2.63 (−3.62 to −1.64), and −3.39 (−4.95 to −1.82), respectively, for other 12 high-risk HPV types, HPV16, and HPV18. Furthermore, mean (95% CI) CT differences of pretreated SurePath samples were comparable to simultaneously collected PreservCyt samples: −0.48 (−0.98 to 0.02) and −0.23 (−0.93 to 0.46), respectively, for HPV16 and HPV18; a borderline significant difference [−0.35 (−0.57 to −0.13)] was observed for other 12 high-risk HPV types. This preanalytic procedure therefore ensures a validated, safe, and accurate method for cobas HPV testing in SurePath. Most cervical cancer cases and deaths can be prevented through early detection of precancerous changes in the cervix. Cytology has been central to cervical cancer screening programs for >50 years and has contributed to the 70% decline in rates of cervical cancer in the developed world.1Saslow D. Runowicz C.D. Solomon D. Moscicki A.B. Smith R.A. Eyre H.J. Cohen C. American Cancer SocietyAmerican Cancer Society guideline for the early detection of cervical neoplasia and cancer.CA Cancer J Clin. 2002; 52: 342-362Crossref PubMed Scopus (804) Google Scholar Human papillomavirus (HPV) is now recognized as a single, necessary cause of cancer of the cervix and has been isolated from the tissue of nearly all cervical cancer cases.2Walboomers J.M. Jacobs M.V. Manos M.M. Bosch F.X. Kummer J.A. Shah K.V. Snijders P.J. Peto J. Meijer C.J. Muñoz N. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol. 1999; 189: 12-19Crossref PubMed Scopus (7025) Google Scholar Thirteen HPV genotypes are classified as carcinogenic or high risk (HR): 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. An additional genotype (66) is classified as possibly carcinogenic.3Bouvard V. Bean R. Straif K. Grosse Y. Secretan B. El Ghissassi F. Benbrahim-Tallaa L. Guha N. Freeman C. Galichet L. Cogliano V. WHO International Agency for Research on Cancer Monograph Working GroupA review of human carcinogens—part B: biological agents.Lancet Oncol. 2009; 10: 321-322Abstract Full Text Full Text PDF PubMed Google Scholar Therefore, tests that detect infection with these HR HPV genotypes are now being used increasingly in cervical cancer screening programs. The 2007 Consensus Guidelines for the Management of Women with Abnormal Cervical Cancer Screening Tests recognized the benefit of using a combination of cervical cytology, tests for HR HPV infection, and type-specific HPV16/18 testing for women undergoing screening for cervical cancer.4Wright Jr., T.C. Massad L.S. Dunton C.J. Spitzer M. Wilkinson E.J. Solomon D. 2006 American Society for Colposcopy and Cervical Pathology-Sponsored Consensus Conference2006 Consensus guidelines for the management of women with abnormal cervical cancer screening tests.Am J Obstet Gynecol. 2007; 197: 346-355Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar Current guidelines in the United States now recommend the combination of cytology and HR HPV testing (cotesting) as the preferred method of screening, with HPV16/18 genotype–specific testing an added option to triage women with negative cytology to colposcopy.5Saslow D. Solomon D. Lawson H.W. Killackey M. Kulasingam S.L. Cain J.M. Garcia F.A. Moriarty A.T. Waxman A.G. Wilbur D.C. Wentzensen N. Downs Jr., L.S. Spitzer M. Moscicki A.B. Franco E.L. Stoler M.H. Schiffman M. Castle P.E. Myers E.R. Chelmow D. Herzig A. Kim J.J. Kinney W. Herschel W.L. Waldman J. American Cancer Society, American Society for Colposcopy and Cervical Pathology, and American Society for Clinical Pathology screening guidelines for the prevention and early detection of cervical cancer.CA Cancer J Clin. 2012; 62: 147-172Crossref PubMed Scopus (937) Google Scholar In 2014, the Federal Drug Administration approved the use of a HR HPV test as the first-line primary screen for cervical cancer and by 2015 US professional societies issued interim guidance that supports HPV primary screening as an option.6Huh W.K. Ault K.A. Chelmow D. Davey D.D. Goulart R.A. Garcia F.A. Kinney W.K. Massad L.S. Mayeaux E.J. Saslow D. Schiffman M. Wentzensen N. Lawson H.W. Einstein M.H. Use of primary high-risk human papillomavirus testing for cervical cancer screening: interim clinical guidance.Gynecol Oncol. 2015; 166: 178-182Abstract Full Text Full Text PDF Scopus (326) Google Scholar, 7American College of Obstetricians and GynecologistsClinical Management Guidelines for Obstetricians and Gynecologists: Cervical cancer screening and prevention. American College of Obstetricians and Gynecologists, Washington, DCOctober 2016Google Scholar SurePath Preservative Fluid (SPPF; formerly AutoCyte; BD TriPath, Burlington, NC) was approved by the Federal Drug Administration in 1999 for use as a collection medium for liquid-based cytology of cervical specimens. Because of decreased cost and a lower percentage of cytology slides read as unsatisfactory (College of American Pathologists, CYP.07600 statistical records, http://www.cap.org/apps/docs/proficiency_testing/CYP07600.pdf, last accessed April 27, 2016), the use of SPPF has been adopted by a high number of laboratories. This change has implications for HPV testing because molecular testing is generally performed on the same liquid medium sample as the cytology specimen. However, until recently, none of the current Federal Drug Administration–approved HPV tests were approved for cervical samples collected in SPPF. To accommodate HR HPV testing in SPPF, many laboratories conducted their own internal validation for testing in SPPF, and in effect used an off-label testing method. This approach to molecular testing in SPPF may, however, raise patient safety issues. It has been documented for nearly a decade that there are limitations in the recovery of nucleic acids from SPPF.8Powell N. Smith K. Fiander A. Recovery of human papillomavirus nucleic acids from liquid-based cytology media.J Virol Methods. 2006; 137: 58-62Crossref PubMed Scopus (45) Google Scholar, 9Horvath C.A. Boulet G. Sahebali S. Depuydt C. Vermeulen T. Vanden Broeck D. Vereecken A. Bogers J. Effects of fixation on RNA integrity in a liquid-based cervical cytology setting.J Clin Pathol. 2008; 61: 132-137Crossref PubMed Scopus (19) Google Scholar For DNA, the reduction in recovery was observed to be up to 1000-fold in cell-spiked samples in SPPF.9Horvath C.A. Boulet G. Sahebali S. Depuydt C. Vermeulen T. Vanden Broeck D. Vereecken A. Bogers J. Effects of fixation on RNA integrity in a liquid-based cervical cytology setting.J Clin Pathol. 2008; 61: 132-137Crossref PubMed Scopus (19) Google Scholar Recent reports of false-negative HPV results in clinical specimens collected in SPPF have also raised safety concerns.10Naryshkin S. Austin R.M. Limitations of widely used high-risk human papillomavirus laboratory-developed testing in cervical cancer screening.Drug Healthc Patient Saf. 2012; 4: 167-172Crossref PubMed Google Scholar Although the exact formulation for SPPF is proprietary, the Medical Safety Data Sheet from the manufacturer states that formaldehyde (formalin) is present in the medium. As early as the 1950s, it had been shown that formaldehyde induces cross-linkages between protein and nucleic acids.11Fraenkel-Conrat H. Reaction of nucleic acid with formaldehyde.Biochim Biophys Acta. 1954; 15: 307-309Crossref PubMed Scopus (87) Google Scholar Awareness of this chemical reactivity and its potential to cause poorer recovery of HPV DNA from SPPF led to the development of a preanalytic treatment in which a nucleophilic reagent is added to the sample and then heated to ensure release of trapped DNA from SPPF. In July 2016, the Federal Drug Administration granted approval for the cobas HPV Test to be performed in samples collected in SPPF, provided that they are subjected to the preanalytic treatment to reverse the effects of cross-linking and maximize DNA accessibility. The description of this preanalytic procedure and the supporting analytic data will be presented in this study. In addition, comparisons of the cycle threshold (CT) values generated with the cobas HPV Test between pretreated and untreated clinical samples collected in SPPF and between pretreated SPPF and PreservCyt (PC) samples will also be described. The cobas HPV Test is a fully automated real-time PCR DNA assay that qualitatively detects the presence of 14 HR HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) in cervical specimens. Results are simultaneously reported as positive or negative for the pooled 12 HR HPV types (HPV 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) from channel 1 and HPV16 and HPV18 genotypes read individually from channels 2 and 3, respectively. The CT values determined to be the cutoff for positive results are 40.0, 40.5, and 40.0 for channels 1, 2, and 3, respectively. A fourth channel detects the human β-globin gene as a control that serves to confirm that cellular DNA is present or to identify the presence of an inhibitory substance.12Rao A. Young S. Erlich H. Boyle S. Krevolin M. Sun R. Apple R. Behrens C. Development and characterization of the cobas human papillomavirus test.J Clin Microbiol. 2013; 51: 1478-1484Crossref PubMed Scopus (55) Google Scholar For the analytic studies, all cervical specimens were procured through clinics using appropriate patient consent and institutional review board approval. Cervical specimens collected in SPPF were maintained at 2°C to 8°C during shipping, storage, and processing. For the comparison of clinical samples collected in SPPF (untreated and pretreated) and PC, specimens were collected during year 3 of the Addressing The Need for Advanced HPV Diagnostics (ATHENA) study follow-up, as described previously.13Wright Jr., T.C. Stoler M.H. Behrens C.M. Apple R. Derion T. Wright T.L. The ATHENA human papillomavirus study: design, methods, and baseline results.Am J Obstet Gynecol. 2012; 206: 46e1-46e11Abstract Full Text Full Text PDF Scopus (183) Google Scholar Briefly, women aged ≥21 years presenting for routine screening were enrolled and received both cytology and HPV testing performed in PC medium (ThinPrep; Hologic, Bedford, MA). Those who screened positive for either cytology (aged ≥21 years) or HR HPV (aged ≥25 years) were referred for baseline colposcopy and biopsy. The follow-up phase of the ATHENA study included those women who had been referred to colposcopy and were found not to have high-grade cervical disease (cervical epithelial neoplasia grade 2 or greater) on biopsy. During this longitudinal phase, women were seen annually for cytology and HPV testing and were referred to colposcopy if they had abnormal cytology. As in the baseline phase, those having cervical epithelial neoplasia grade less than 2 on colposcopic biopsy continued to be followed up; at year 3, an exit colposcopy was performed on all consenting women. All women presenting at year 3 were also offered the option of cocollection with a second cervical sample in SPPF. The ATHENA study protocol was approved by institutional review boards of all study sites, and written informed consent was obtained. Cervical specimens collected in SPPF were subjected to the preanalytic process involving addition of the cobas sample preparation buffer (CSPB; Roche Molecular Systems, Pleasanton, CA) and heating, described in detail as follows. The formulation for CSPB contains 0.4% SDS and 0.09% sodium azide buffered with Tris-HCl and is stable for 24 months when stored at 2°C to 30°C. The following steps were performed in a biological hood to minimize any cross contamination between samples. CSPB (0.5 mL) was pipetted into 13-mL round-based tubes (Sarstedt P/N 60.541.122 or equivalent). After vortexing each SPPF specimen for 10 seconds, 0.5 mL of SPPF specimen was transferred into the 13-mL round-based tube containing CSPB. Pipette tips were changed for each specimen. Tubes were capped and vortexed for 1 second before being transferred to a heating unit, allowing up to 48 tubes to be processed per batch [120-V digital heater block (catalog number 12621-096; VWR, Radnor, PA)]. Samples were heated to 120°C for 20 minutes, removed to a collection rack, and cooled at ambient temperature for 10 minutes. Each tube was vortexed again for 5 seconds and transferred to 24 position cobas 4800 specimen racks to be processed on the cobas 4800 System; HPV testing was performed within 2 hours of the pretreatment procedure. Twenty archived cervical specimens collected in SPPF underwent HPV testing (without the preanalytic treatment) on the cobas 4800 System, followed by storage at 2°C to 8°C for 18 to 24 months. After 2°C to 8°C storage, the SPPF specimens were tested for HPV with and without pretreatment with CSPB/heating and these results were compared to the initial HPV test results obtained when the specimens were originally collected. All preanalytic treated samples were processed on the cobas 4800 System within 2 hours of undergoing the pretreatment process. Twenty-five HPV-negative SurePath clinical specimens were pooled for a total volume of 225 mL. A single HPV16 positive specimen was diluted into the HPV negative pool to produce a CT of 38 to 39, representing a low positive HPV16 result (approximately 1 × limit of detection). All materials were stored at 2°C to 8°C until the start of incubation. Aliquots (12 mL each in SurePath primary vials) were incubated at 25°C, 27°C, and 30°C subsequent to day 0 testing. Testing was done at days 3, 7, 10, and 14. At each time point, four vials were removed for each storage temperature to produce 10 × 1-mL replicates from each vial. A total of 40 replicates were tested for each temperature at each time point with and without preanalytic treatment before performing the cobas 4800 HPV test, and all preanalytic treated samples were tested within 2 hours of the pretreatment procedure. From the year 3 follow-up of the ATHENA study, 3975 cervical samples collected in both PC and SPPF were eligible for analysis. Samples arrived at the initial ATHENA testing laboratories between 1 and 7 days after collection. For the SPPF pretreated and untreated sample comparison, residual samples were stored at 2°C to 8°C until they were shipped back to the Roche laboratory, where they were again stored at 2°C to 8°C for 1 to 14 months before being tested there. For a subset of 80 ATHENA specimens, selected from a total of 986 samples where a CT value was detected, the mean CT difference between treated and untreated SurePath samples was compared for each channel using a paired t-test. For the SPPF pretreated versus PC specimen comparison, cobas HPV testing was performed in real time (generally within 1 week of arrival) at the four ATHENA laboratories and the CT values of the entire year 3 ATHENA cohort (n = 3975) were evaluated. Only paired samples for which a CT value was detected in both PC and SPPF were included in the analysis; the mean CT difference of the 986 test results was compared for each channel using a paired t-test. For primary HPV DNA recovery from SurePath specimens at 2°C to 8°C and 32°C, 19 HPV-positive pools were prepared, each from four individual HPV-positive SPPF cervical specimens. Half of each of the 19 pools was incubated at 2°C to 8°C and 32°C. Aliquots from each of the 19 pools at each temperature were tested for HPV DNA with the cobas HPV test using the pretreatment process at day 0 and months 1, 3, 6, and 9. For recovery of preanalytic treated HPV16/HPV18 samples stored at 2°C to 8°C and 32°C before HPV testing, a pool of HPV-negative SurePath specimens was prepared and spiked with HeLa (HPV18) and SiHa (HPV16) cell lines (cultured and quantified at Roche Molecular Systems, Pleasanton, CA) to approximately 5 × limit of detection. The HPV spiked pool was aliquoted into 150 individual secondary tubes at 0.5 mL each. All secondary tubes were incubated at 32°C for 7 days to induce cross-links. All tubes were then subjected to the preanalytic treatment, followed by stability testing at 2°C to 8°C and 32°C storage. Every condition was tested with 15 replicates for each time point. In all cases, SPPF specimens stored for 18 to 24 months at 2°C to 8°C showed significant losses of recoverable HPV DNA without pretreating before testing. Samples that underwent the pretreatment process were within 1.3 CT of the initial results (mean value of all specimens tested) (Figure 1). With no pretreatment, the retest results showed a mean delay of 4.9 CT values, indicating that retesting without pretreatment failed to detect from 90% to >99% of the HPV DNA originally detected in those samples. In addition, it was observed that several of the SPPF specimens with positive results detected among all three channels before storage at 2°C to 8°C were found to test negative after storage without pretreatment: 2 of 12 for other 12 HR HPV types, 1 of 13 for HPV16, and 2 of 9 for HPV18. All of these negative results, however, reverted back to positive when retested after undergoing the pretreatment process (Figure 1). As would be expected, samples where the initial positive results had CT values closest to the cutoff were the most likely to convert from a positive to negative result when not undergoing the pretreatment. Testing of replicate low HPV16-positive samples stored for 3, 7, 10, and 14 days demonstrated that by day 7 a significant number of untreated samples were negative by PCR (Table 1). Among 40 replicates, the number of samples testing positive for HPV16 was as follows: 32 of 40 (P = 0.0053), 32 of 40 (P = 0.0053), and 31 of 40 (P = 0.0024) for 25°C, 27°C, and 30°C, respectively. When the preanalytic treatment was used, the low positive HPV16 pool remained stable up until day 14 for all storage temperatures, as evidenced by the positive hit rate: 40 of 40 and 40 of 40 at 25°C and 30°C, respectively (data for 27°C not available because of a processing error) (Table 1).Table 1Stability of cobas HPV Test Results for HPV16 in SurePath SpecimensTime pointTemperature, °CTotal validPositiveNegativeHit rate, %P value∗P values based on Fisher exact test.Without preanalytic treatment Day 0NA40400100NA Day 32540391981.00002740391981.00003040364900.1156 Day 72540328800.00532740328800.00533040319780.0024 Day 1025402911730.000427402911730.00043040231758 37 had not been previously evaluated. In the current study, specimens with CT values closer to the assay cutoff (38 to 39) were investigated and a decrease in signal by 7 days is observed if pretreatment is not performed in these samples with presumed low viral loads. We also demonstrate that SPPF specimens that are subjected to the preanalytic procedure can be stored for extended periods at ambient or refrigerated temperatures before cobas HPV testing and also that samples refrigerated for up to 9 months can then be successfully pretreated and undergo testing. This information regarding sample stability will allow flexibility in laboratory workflows and also can accommodate a variety of research protocols. Several studies have been published to date evaluating the ability to detect HPV in SPPF, and most reach the apparent conclusion that HPV DNA testing in samples collected in SPPF without any pretreatment yields results that are valid and/or are comparable to those in PC medium.14Hardie A. Moore C. Patrick J. Cuschieri K. Graham C. Beadling C. Ellis K. Frew V. Cubie H.A. High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage.Cytopathology. 2009; 20: 235-241Crossref PubMed Scopus (14) Google Scholar, 15Gilbert L. Oates E. Ratnam S. Stability of cervical specimens in SurePath medium for human papillomavirus testing with the Roche cobas 4800 assay.J Clin Microbiol. 2013; 51: 3412-3414Crossref PubMed Scopus (11) Google Scholar, 16Kubik M.J. Permenter T. Saremian J. Specimen age stability for human papilloma virus DNA testing using BD SurePath.Lab Med. 2015; 46: 51-54Crossref PubMed Scopus (6) Google Scholar, 17Dixon E.P. Lenz K.L. Doobay H. Brown C.A. Malinowski D.P. Fischer T.J. Recovery of DNA from BD SurePath cytology specimens and compatibility with the Roche AMPLICOR human papillomavirus (HPV) test.J Clin Virol. 2010; 48: 31-35Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 18Zhou F. Pulinthanathu R. Elgert P. Cangiarella J. Simsir A. Sensitivity of high-risk HPV Hybrid Capture II (hrHPV hc2) test using SurePath™ specimens in the prediction of cervical high-grade squamous lesions.Diagn Cytopathol. 2015; 43: 381-387Crossref PubMed Scopus (6) Google Scholar, 19Zhou F.H. Hu S.Y. Bian J.J. Liu B. Peck R.B. Bao Y.P. Pan Q.J. Frappart L. Sellors J. Qiao Y.L. Comparison of ThinPrep and SurePath liquid-based cytology and subsequent human papillomavirus DNA testing in China.Cancer Cytopathol. 2011; 119: 387-394Crossref PubMed Scopus (30) Google Scholar, 20Siddigi A. Spataro M. McIntire H. Akhtar I. Baliga M. Flowers R. Lin E. Guo M. Hybrid capture 2 human papillomavirus DNA testing for women with atypical squamous cells of undetermined significance Papanicolaou results in SurePath and ThinPrep specimens.Cancer. 2009; 117: 318-325PubMed Google Scholar However, most of these studies were retrospective, performed on low numbers of residual samples and from populations with relatively high viral loads. A more focused review, therefore, raises issues that question the stability of SPPF samples under varying conditions. In a study assessing Amplicor and Digene hc2 results from residual SPPF samples, Hardie et al14Hardie A. Moore C. Patrick J. Cuschieri K. Graham C. Beadling C. Ellis K. Frew V. Cubie H.A. High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage.Cytopathology. 2009; 20: 235-241Crossref PubMed Scopus (14) Google Scholar demonstrate concordance between samples stored at ambient and refrigerated temperatures when using a population enriched with abnormal samples with a high HPV prevalence. When the analysis was confined to samples with normal cytology and a presumed lower viral load, a higher concordance was seen in the SPPF samples refrigerated versus those held at ambient temperature. Likewise, Gilbert et al15Gilbert L. Oates E. Ratnam S. Stability of cervical specimens in SurePath medium for human papillomavirus testing with the Roche cobas 4800 assay.J Clin Microbiol. 2013; 51: 3412-3414Crossref PubMed Scopus (11) Google Scholar compared cobas HPV test results in ambient versus refrigerated pooled samples collected in SPPF from a colposcopy referral population, and also treated a proportion of the ambient samples with the same preanalytic procedure reported herein. Overall, they saw high concordance of results across all groups. However, it is likely that higher viral loads in the sample populations that were used in both these studies masked any decrease in HPV detection, even in the presence of some formalin-induced cross-linkage that occurred at ambient temperature. Such studies can therefore yield falsely reassuring results on the stability of samples collected in SPPF. In contrast, Kubik et al16Kubik M.J. Permenter T. Saremian J. Specimen age stability for human papilloma virus DNA testing using BD SurePath.Lab Med. 2015; 46: 51-54Crossref PubMed Scopus (6) Google Scholar performed cobas HPV testing on samples with atypical cells of undetermined significance cytology results collected in SPPF and retested after ambient storage for 21 days; although limited by a small sample size, they found a 16% false-negative rate. In summary, HR HPV testing has become a critical component of cervical cancer screening. As new assays and variations of methods are introduced, careful validation must be performed to ensure that these results guide appropriate clinical management. We describe herein the challenges of nucleic acid testing in SPPF and propose a preanalytic procedure that obviates concern over the age of the specimen, the temperature to which it is subjected and/or stored, or the viral load present. Removing these restrictions will no doubt enhance the reliability of performing HPV testing in cervical samples collected in SPPF. Download .pdf (.14 MB) Help with pdf files Supplemental Figure S1Primary SurePath specimen stability at 2°C to 8°C and 32°C with preanalytic treatment for other high-risk HPV specimens (A and B), HPV16 specimens (C and D), and HPV18 specimens (E and F). Nineteen HPV-positive pools were prepared, each from four individual HPV-positive SurePath Preservative Fluid cervical specimens. Half of each of the 19 pools was incubated at 2°C to 8°C and 32°C. Aliquots from each of the 19 pools at each temperature were tested for HPV DNA with the cobas HPV test using the pretreatment process at day 0 and months 1, 3, 6, and 9. The dashed arrows represent cutoff values for positive results, and were determined to be 40.0, 40.5, and 40.0 for channels 1, 2, and 3, respectively. Pos, position. Download .docx (.03 MB) Help with docx files Supplemental Table S1