Liquid-based Cytology Medium Research Articles

CDKN2A/p16 evaluation in cytology specimens.

CDKN2A/p16 evaluation in cytology specimens.

Analytical and Clinical Sample Performance Characteristics of the Onclarity Assay for the Detection of Human Papillomavirus.

The objective of this study was to determine the result reproducibility and performance of the BD Onclarity human papillomavirus (HPV) assay (Onclarity) on the BD Viper LT platform using both contrived and clinical specimens. Reproducibility was assessed in BD SurePath liquid-based cytology (LBC) medium (SurePath) using contrived panels (HPV genotype 16 [HPV16] positive, HPV18 positive, or HPV45 positive) or clinical specimens (HPV16, -18, -31, -33/58, -45, or -52 positive or HPV negative). In addition, specimens from 3,879 individuals from the Onclarity trial were aliquoted prior to or following cytology processing and tested for HPV. Finally, specimens were collected using either the Cervex-Brush or Cytobrush (or Cytobrush/spatula) for comparison of HPV results. Contrived specimens showed >95% concordance with the expected results, and pooled clinical specimens had standard deviations and coefficients of variation ranging from 0.87 to 1.86 and 2.9% to 5.6%, respectively. For precytology and postcytology aliquot analyses, specimens showed >98.0% overall agreement and mean differences in cycle threshold (CT ) scores for HPV ranging from -0.07 to 0.31. Positivity rates were close between the Cervex-Brush and Cytobrush/spatula for all age groups tested. Onclarity results are reproducible and reliable, regardless of sample collection before or after cytology aliquoting. Onclarity performs well regardless of the method of specimen collection (Cervex-Brush or Cytobrush/spatula) for cervical cancer screening.

Associations among Serum Lipocalin-2 Concentration, Human Papilloma Virus and Clinical Stage of Cervical Cancer.

Background and objective: Lipocalin 2 (LCN2) has an oncogenic role in promoting tumorigenesis through enhancing tumor cell proliferation and the metastatic potential. The aim of our study was to determine whether serum LCN2 could serve as a diagnostic marker of cervical cancer (CC) and to evaluate the correlation between its serum concentration, the clinical stage of the cancer and Human Papilloma Virus HPV infections in women. Materials and methods: A total of 33 women with histologically proven cervical cancer (CC), 9 women with high- grade cervical intraepithelial neoplasia (HSIL) and 48 healthy women (NILM) were involved in the study. A concentration of LCN2 was assayed with the Magnetic LuminexR Assay multiplex kit. An HPV genotyping kit was used for the detection and differentiation of 15 high-risk (HR) HPV types in the liquid-based cytology medium (LBCM) and the tissue biopsy. Results: The majority (84.8%) of the women were infected by HPV16 in the CC group, and there was no woman with HPV16 in the control group (P < 0.01). Several types of HR HPV were found more often in the LBCM compared to in the tissue biopsy (P = 0.044). HPV16 was more frequently detected in the tissue biopsy than the LBCM (P < 0.05). The LCN2 level was higher in HPV-positive than in HPV-negative women (P = 0.029). The LCN2 concentration was significantly higher in women with stage IV than those with stage I CC (P = 0.021). Conclusions: Many HR HPV types, together with HPV16/18, can colonize the vagina and cervix, but often HPV16 alone penetrates into the tissue and causes CC. The serum LCN2 concentration was found to be associated not only with HR HPV infection, irrespective of the degree of cervical intraepithelial changes, but also with advanced clinical CC stage. LCN2 could be used to identify patients with advanced disease, who require a more aggressive treatment.

Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages

BackgroundA change of cervical cancer screening algorithms to an HPV-based screening setting is discussed in many countries, due to higher sensitivity of HPV testing compared to cytology. Reliable triage methods are, however, an essential prerequisite in such a setting to avoid overtreatment and higher screening costs.ResultsIn this study, a series of cervical scrapes collected in PreservCyt liquid-based cytology (LBC) medium from women with cervical cancer (n = 5), cervical intraepithelial neoplasia grade 1–3 (n = 74), and normal cytology (n = 201; further n = 352 collected in SureThin®) were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 using the GynTect assay and compared to cobas® HPV and CINtec Plus® biomarker results. All samples from women with cervical cancer, 61.2% of CIN3, 44.4% of CIN2 and 20.0% of CIN1 cases were scored positive for the GynTect methylation assay. In contrast, all CIN, irrespective of severity grade, and carcinomas were positive by both, CINtec Plus and cobas HPV. The specificity of GynTect for CIN3+ was 94.6% compared to 69.9% for CINtec Plus and 82.6% for cobas HPV (all HPV types) and 90.6% for cobas HPV 16/18. DNA methylation analysis of this methylation marker panel (GynTect assay) in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1/2 lesions. The detection rate among cytologically normal samples is extraordinarily low (1.5%).ConclusionGynTect shows excellent performance when using cervical scrape material collected in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to the other test systems used in this work, GynTect showed higher specificity while still detecting all cancer cases.

Stability, integrity, and recovery rate of cellular nucleic acids preserved in a new liquid-based cytology medium.

Liquid-based cytology (LBC) has replaced the conventional Papanicolaou test in cervical cancer screening. The cervical swab specimens collected in LBC media can also be used for additional analyses including high-risk HPV (HR-HPV) test, DNA methylation analysis, and HPV E6/E7 mRNA test. The stability, integrity, and recovery rate of cellular DNA and RNA after storage at different conditions were evaluated by a quantitative real-time PCR (qPCR) based HR-HPV test, reverse transcription qPCR (RT-qPCR), and agarose gel electrophoresis. Cervical swab specimens collected in a newly developed LBC medium, VersaMedium, and ThinPrep PreservCyt medium were processed on Hologic ThinPrep 5000 instrument. Cervical exfoliative cells fixed by VersaMedium exhibited good cellular morphology with intact membranes and delineated chromatin structures. Cellular DNA preserved in VersaMedium exhibited high level of stability at both room temperature and 4°C, and remained mostly intact at 4°C for up to 28 days. Cellular RNA preserved in VersaMedium maintained higher level of stability and integrity at 4°C than at room temperature. VersaMedium also showed no apparent adverse effect on the recovery rate of nucleic acids. In addition to maintaining cellular morphology, when stored at 4°C, VersaMedium preserves cellular nucleic acids and PreservCyt medium without noticeable adverse effects on the recovery rate during purification. Therefore, VersaMedium is an appropriate LBC medium for the collection and preservation of cervical swab specimens. And VersaMedium preserved cellular nucleic acids are of such high quality that they are suitable for HR-HPV qPCR test and RT-qPCR analyses.

Validation of Novaprep(®) HQ+ liquid-based cytology medium for high-risk human papillomavirus detection by hc2.

BackgroundPreanalytical conditions determine the reliability and validity of bioassays. Therefore, the analytic performances of biological tests need to be determined when preanalytical steps differ from those recommended by the manufacturer. The objective of the study was to assess the analytic performance of the hc2 test for the detection of high-risk HPV DNA from cells stored in the new Novaprep® HQ+ medium.MethodsRepeatability, reproducibility, method comparison and stability (-20 °C, +4 °C, +20 °C and +40 °C up to six months) were evaluated from HPV16 and HPV18 positive cell lines diluted in the Novaprep® HQ+ medium and the reference Specimen Transport Medium (STM). A series of cervical samples with atypical squamous cells of undetermined significance (ASC-US) cytology and stored in the Novaprep® HQ+ medium was also tested.ResultsCoefficients of variation for repeatability and reproducibility were less than 8 %. Method comparison showed perfect agreement in hc2 results when the HPV-positive cells were diluted in HQ+ and reference media. Stability experiments demonstrated that the storage conditions did not alter the hc2 test results. Furthermore, clinical samples were adequately preserved for hc2 testing.ConclusionsOverall, our data show that the new Novaprep HQ+ medium is suitable for high-risk HPV testing by hc2.

Performance of the BD CTQx and GCQx Amplified Assays on the BD Viper LT Compared With the BD Viper XTR System.

We evaluated the BD Viper LT System for detection of Chlamydia trachomatis and Neisseria gonorrhoeae using samples collected from symptomatic patients that included urine, vaginal swabs, and cervical samples in liquid-based cytology media. Results were compared with those obtained using the BD Viper XTR platform. The positive and negative percent agreements for all sample types were at least 95.8% and at least 96.4% for chlamydia and gonorrhea and at least 95.0% when both organisms were present, respectively. This medium throughput system performs well compared with a high-throughput platform and may offer smaller health care facilities the opportunity to test for these infections locally.

High frequency of genital human papillomavirus infections and related cervical dysplasia in adolescent girls in Belgium.

Human papillomavirus (HPV) infections are causally related to cervical cancer and a range of other diseases, both in adults and in minors. Information on the frequency of genital HPV infections in adolescents is sparse. The aim of this study was to gain insight into the genotype-specific distribution of HPV genotypes in patients younger than 18 years of age. This observational retrospective study included 4807 samples of patients presenting for opportunistic screening in Belgium between June 2006 and January 2012. For statistical analysis, only the first visits of patients were withheld, reducing the sample to 4180. Samples were collected in liquid-based cytology medium and analyzed using a series of genotype-specific real-time PCR reactions. Cytology was read with previous knowledge of HPV infection and scored using the Bethesda classification. The mean age was 16.9 years. Most youngsters had no complaints (88.4%), were using hormonal contraception (79.5%), and clinical examination did not show any abnormalities (96.0%). The overall HPV frequency was 15.7%, with the most frequently found types being HPV16 (16.7%), HPV51 (14.6%), HPV66 (10.4%), HPV31 (9.9%), and HPV39 (9.1%). More than one-third (39.0%) of the infected girls harbored an infection with at least two HPV genotypes. Cytological abnormalities were found in 8.2% of samples. L-SIL (4.2%) was most frequently observed, followed by ASC-US (3.6%), HSIL (0.3%), and ASC-H (0.1%). The severity of lesions worsened with increasing age. Our findings indicate that an aberrant HPV genotype profile can be found in adolescent girls; moreover, this group shows a high rate of cervical abnormalities.

Clinical Evaluation of a New Pap Test–Based Method for Screening of Chlamydia trachomatis and Neisseria gonorrhoeae Using Liquid-Based Cytology Media

The majority of chlamydial and gonococcal infections in women are asymptomatic and, if left untreated, may result in serious sequelae. Simple and accurate testing of men and women at risk for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) is the single most effective strategy for control of sexually transmitted infections. New tests using easy-to-acquire samples, such as the Papanicolaou (Pap) test, need to be validated in an effort to expand and improve detection. The objective of this study was to determine the performance of a new nucleic acid amplification test using two liquid-based cytology media for the detection of CT and GC. The study was conducted in two phases at 11 geographically diverse, high- and low-prevalence sites. Three endocervical reference swabs as well as an endocervical SurePath or PreservCyt liquid cytology specimen sampled with a broom or brush/spatula were collected in a randomized order from each subject. Reference endocervical swabs were tested with three Food and Drug Administration-approved methods and compared to two new automated tests, the CT Q Amplified DNA Assay (CTQ) and the GC Q Amplified DNA Assay (GCQ). For the SurePath phase, 1838 subjects were enrolled. The sensitivity and specificity of the CTQ assay were 95.0% and 99.7%, respectively, and the GCQ assay was 100% for both. In the PreservCyt phase, 2164 subjects were enrolled. The sensitivity and specificity of the CTQ assay were 94.1% and 99.8%, respectively, and the GCQ assay was 95.3% and 99.95%, respectively. There was no significant difference in the results. In this investigation, high sensitivity and specificity of the CTQ and GCQ assays were demonstrated for samples collected in either of two liquid-based cytology media when compared with endocervical swabs. The results were similar in both collection methods (broom or brush/spatula) and in high- and low-risk populations.

Randomized comparison of vaginal self-sampling by standard vs. dry swabs for Human papillomavirus testing

BackgroundTo evaluate if human papillomavirus (HPV) self-sampling (Self-HPV) using a dry vaginal swab is a valid alternative for HPV testing.MethodsWomen attending colposcopy clinic were recruited to collect two consecutive Self-HPV samples: a Self-HPV using a dry swab (S-DRY) and a Self-HPV using a standard wet transport medium (S-WET). These samples were analyzed for HPV using real time PCR (Roche Cobas). Participants were randomized to determine the order of the tests. Questionnaires assessing preferences and acceptability for both tests were conducted. Subsequently, women were invited for colposcopic examination; a physician collected a cervical sample (physician-sampling) with a broom-type device and placed it into a liquid-based cytology medium. Specimens were then processed for the production of cytology slides and a Hybrid Capture HPV DNA test (Qiagen) was performed from the residual liquid. Biopsies were performed if indicated. Unweighted kappa statistics (к) and McNemar tests were used to measure the agreement among the sampling methods.ResultsA total of 120 women were randomized. Overall HPV prevalence was 68.7% (95% Confidence Interval (CI) 59.3–77.2) by S-WET, 54.4% (95% CI 44.8–63.9) by S-DRY and 53.8% (95% CI 43.8–63.7) by HC. Among paired samples (S-WET and S-DRY), the overall agreement was good (85.7%; 95% CI 77.8–91.6) and the κ was substantial (0.70; 95% CI 0.57-0.70). The proportion of positive type-specific HPV agreement was also good (77.3%; 95% CI 68.2-84.9). No differences in sensitivity for cervical intraepithelial neoplasia grade one (CIN1) or worse between the two Self-HPV tests were observed. Women reported the two Self-HPV tests as highly acceptable.ConclusionSelf-HPV using dry swab transfer does not appear to compromise specimen integrity. Further study in a large screening population is needed.Trial registrationClinicalTrials.gov: NCT01316120

Development and Characterization of the cobas Human Papillomavirus Test

The cobas human papillomavirus (HPV) test, approved by the FDA in April 2011, is a fully automated assay for the detection of 14 high-risk (hr) HPV genotypes from cervical specimens collected in liquid-based cytology medium using real-time PCR amplification of the L1 gene and TaqMan probes. Results are simultaneously reported as positive or negative for the pooled 12 oncogenic HPV types (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68) from channel 1, with HPV16 and HPV18 genotypes read individually from channels 2 and 3. A fourth channel detects the human β-globin gene as a control for sample adequacy and assay inhibition. To optimize clinical sensitivity and specificity, cutoff values (cycle thresholds [C(T)]) were established for each channel based on the detection of cervical intraepithelial neoplasia grade 2 (CIN2) or greater (≥CIN2). For women aged ≥21 years with cytology results indicating atypical squamous cells of undetermined significance (ASC-US), CT values provided a sensitivity of 90% (95% confidence interval [CI], 81.5% to 94.8%) for the detection of ≥CIN2 and a specificity of 70.5% (95% CI, 68.1% to 72.7%). The analytic sensitivity (limit of detection) ranged from 150 to 2,400 copies/ml, depending on genotype. The analytic specificity, evaluated by comparing the HPV result with a combined comparator of Sanger sequencing and the Qiagen digene HC2 high-risk HPV DNA test (hc2), demonstrated overall positive agreement of 96.3% for 14 hrHPV types in women with ASC-US cytology results who were aged ≥21 years and 86.1% in women with NLIM (negative for intraepithelial neoplasia or malignancy) cytology who were aged ≥30 years. These and other performance validation studies demonstrate that the cobas HPV test is a fully automated and clinically validated robust test.

Detection of Human Papillomavirus in Anal Specimens Using the Hybrid Capture 2 Assay

The hc2 human papillomavirus DNA test (HC2) is effective when screening women for cervical dysplasia, and it might be effective in the screening for anal dysplasia. Differences between the anal and the cervical canals could affect the test performance. This prospective study (n=292) measured the HC2 signal and in agreement with a histologic endpoint of high-grade dysplasia for anal specimens collected in various ways. Sensitivities were 91%, 85%, and 62% for specimens collected in a sample transport medium and a liquid-based cytology medium processed by Gyn or Non-Gyn protocol, respectively. HC2 sensitivity and specificity to predict high-grade anal dysplasia were similar for brush or swab specimen collections, but HC2 signal was 6 times higher with the brush. Specificity and sensitivity were similar whether the sample was collected first or after a cytology sample for brush or swab, but swab specimens at the second collection had an HC2 signal (mean) 48% lower than that of the first collection, and the swab cellularity was lower. The presence of maximum stool decreased the HC2 signal in anal swab specimens. Consensus polymerase chain reaction (PCR) confirmed that the 13 human papillomavirus probe types in HC2 were optimal for performance. HC2 could potentially be further investigated for use in screening anal dysplasia. A larger prospective study is indicated.

Comparison of cobas human papillomavirus test results from primary versus secondary vials of PreservCyt specimens and evaluation of potential cross‐contamination

The preferred workflow for high-risk human papillomavirus (hrHPV) testing in the majority of laboratories involves cytology processing of samples collected in liquid-based cytology medium followed by hrHPV testing. The cobas HPV Test received approval from the US Food and Drug Administration in April 2011, and the supporting clinical trial design necessitated prealiquoting the sample used for hrHPV testing from the PreservCyt primary vial into a secondary vial that was placed on the cobas 4800 System. To validate use of the postcytology residual sample in the primary vial, the authors presented the results of cross-contamination studies and a comparison of the cobas HPV Test results from the prealiquot in the secondary vial with results obtained from the postcytology primary vial on samples processed on either the Hologic ThinPrep 2000 System (T2000) or ThinPrep 3000 System (T3000). Cross-contamination checkerboard studies with 100 samples processed on the T2000 system and 120 samples processed on the T3000 system indicated no conversion of primary vial results from positive to negative or from negative to positive. For clinical samples, approximately 1100 archived specimens from the ATHENA (Addressing the Need for Advanced HPV Diagnostics) study and 1100 combined archived and fresh primary vial specimens that had been processed on the T2000 and T3000 processors, respectively, were compared. The overall percentage agreement between the primary and secondary vials was at least 93.5%. The postcytology residual of samples collected in PreservCyt primary vials and run on the T2000 and T3000 processors provided reliable cobas HPV Test results that were comparable to results from the precytology secondary vial, without any evidence of contamination.

Performance of the cobas CT/NG Test Compared to the Aptima AC2 and Viper CTQ/GCQ Assays for Detection of Chlamydia trachomatis and Neisseria gonorrhoeae

The next-generation amplification test for Chlamydia trachomatis and Neisseria gonorrhoeae (Roche cobas 4800), a fully automated system, was compared head to head, using female samples, to Gen-Probe Aptima Combo 2 and BD ProbeTec using Viper. Endocervical swabs, female urine, and endocervical samples in liquid-based cytology medium were run on at least two of three platforms. A total of 4,316 samples were evaluated, and 281 chlamydial and 69 gonococcal infections were identified. Estimates of sensitivity and specificity were obtained relative to the patient infection standard (PIS) and using latent class analysis (LCA). Chlamydia sensitivity estimates ranged from 86.9 to 95.6% using PIS and 97.6 to 98% using LCA. Specificity was ≥ 99.6% for all sample types. Sensitivity ranged from 95.6 to 100% using PIS and 96.9 to 100% using LCA for the detection of gonococcal infections. Specificity for gonococcal infections was ≥ 99.8%. cobas 4800 performance was equivalent to the comparator assays (all P values, >0.05), and the fully automated system provides high laboratory efficiency.

Evaluation of the hybrid capture 2 assay for detecting anal high‐grade dysplasia

Hybrid Capture 2 (HC2) Human Papillomavirus (HPV) DNA Test® is FDA approved and is a proven aid in detecting HPV infections of the cervix and as an aid in diagnosing, with cytology, cervical disease. A prospective feasibility study was conducted to determine if HC2 testing has utility when screening for high-grade anal dysplasia (AIN2+). We enrolled 298 patients (45% HIV+) who had AIN2+ screening with cytology, histology and HC2 testing for two specimens: a swab into liquid-based cytology medium and either a swab or a brush collection in specimen transport medium (STM). High-resolution anoscopy was performed on all patients with biopsy of AIN2+ suspicious lesions. Cytology was benign (42%), atypical squamous cells of undetermined significance (30%), low-grade squamous intraepithelial lesion (18%), high-grade squamous intraepithelial lesion (1%), ASCUS possibly high-grade dysplasia (1.7%) and nondiagnostic (7%) and 36% had AIN2+ histology. Sensitivity and specificity for predicting AIN2+ histology for any abnormal cytology were 77 and 52%, whereas HC2 sensitivity and specificity were 91 and 40% (p = 0.005 for sensitivity), respectively. There was no significant difference in HC2 sensitivity or specificity between brush and swab or STM and residual cells from cytology. AIN2+ was found in 20% of patients with benign cytology. Only nine AIN2+ specimens were HC2-. This prospective study indicates that HC2 may be useful when screening for anal dysplasia; however, a larger study is recommended.

Novaprep ® Vial Test is a suitable liquid-based cytology medium for high risk human papillomavirus testing by Hybrid Capture 2

Background Liquid-based cytology (LBC) for cervical cancer screening presents the advantage that cytological and virological investigations can be undertaken from the same specimen. Nevertheless, the fixative may alter DNA integrity and the sample may be inadequate for HPV DNA detection. The Novaprep ® Vial Test (NVT) (Novacyt, Vélizy-Villacoublay, France) is a new device dedicated to LBC which permits an automated cell spreading over slides and an automated cell sampling for molecular analyses. Objective To determine whether the NVT was suitable for high risk (HR) HPV DNA detection with the Hybrid Capture 2 (HC2) assay (Qiagen, Courtaboeuf, France). Study design Two cervical specimens were harvested. The first sample was taken with a Rovers Cervex Brush (Therapak Corporation, Buford, USA) placed in the NVT and the second sample was taken with a DNAPAP cervical sampler placed in the Specimen Transport Medium (STM) (Qiagen). This last sample served as gold standard for HPV detection. NVT and STM samples were analyzed for HR HPV DNA with HC2 assay. Results One hundred and thirty-one samples stored in NVT and STM were analyzed. The overall HC2 positivity determined from the 99 samples classified as satisfactory for cellularity (>5000 cells/slide) was 84% whatever the collection medium was. Agreement for HPV detection between NVT and STM was 94%, with a Kappa of 0.78. Moreover, we noted that HC2 values obtained from NVT samples were correlated to those obtained from STM samples. Conclusion The Novaprep ® Vial Test adequately preserves HPV DNA and is suitable for HPV testing with HC2 if cellularity is satisfactory.

Validation of ThermoFisher's Papspin for human papillomavirus detection in cervicovaginal specimens using PCR with GP5+/GP6+ primers and the Hybrid Capture II assay

The present study aimed to validate ThermoFisher's (Thermo Fisher Scientific, Runcorn, Cheshire, UK) Papspin (PS) for human papillomavirus (HPV) testing by in-house PCR and by the Hybrid Capture II (HC2) assay and to compare the results with those obtained using Specimen Transport Medium (STM) (Digene Diagnostics, Gaithersburg, MD, USA). Forty-five patients underwent conization for known lesions ranging from atypical squamous cells of undetermined significance (ASC-US) with high-risk HPV (hr-HPV) to high-grade squamous intraepithelial lesion (H-SIL/CIN2+) or adenocarcinoma. Two negative controls were included: one patient with post-menopausal bleeding and another from whom an inflammatory cervical sample was taken without conization. Prior to conization, a gynaecologist collected two cervical samples, fixed in PS or STM, from each patient. All but four cases were tested for panHPV (GP5+/GP6+) and specific hr-HPV subtypes (HPV16, 18, 31,33) by PCR using both media and all were processed for HC2. This study demonstrates that both HPV detection techniques work with PS, showing a specificity of 78.3% for HC2 and 92.8% for PCR compared to 83.8% for HC2 and 92% for PCR using STM. The efficacy of detecting HPV in PS-preserved H-SIL/CIN2+ was very high (96% for PCR using PS and 86% for HC2 using PS), which was in the same range as for PCR using STM, and which was only slightly lower than for HC2 using STM (96% and 89%, respectively). The differences were not statistically significant. It is concluded that ThermoFisher's PS is a valid liquid-based cytology medium for cervical samples, convenient for HPV testing by PCR with GP5+/GP6+ primers and by the HC2 assay.

Classification of fixed urological cells using Raman tweezers

In this paper we report on preliminary investigations into using Raman tweezers to classify urological cell lines. This builds on earlier work within the group, whereby Raman tweezer methodologies were developed, and the application of this technique to differentiate between live prostate cancer (CaP) and bladder cells lines (PC-3 and MGH-U1 respectively) was demonstrated.In this present study we analysed chemically fixed cells using two different fixative methods; SurePath (a commercial available liquid based cytology media) and 4% v/v formalin/PBS fixatives. The study has been expanded from our previous live cell study to include the androgen sensitive CaP cell line LNCaP, primary benign prostate hyperplasia (BPH) cells as well as primary urethral cells. Raman light from the cells was collected using a 514.5 nm Ar-ion laser excitation source in back-scattering configuration mode.Principal component-linear discriminate analysis (PC-LDA) models of resulting cell spectra were generated and these were validated using a blind comparison. Sensitivities and specificities of > 72% and 90% respectively, for SurePath fixed cells, and > 93% and 98% respectively for 4% v/v formalin/PBS fixed cells was achieved. The higher prediction results for the formalin fixed cells can be attributed to a better signal-to-noise ratio for spectra obtained from these cells.Following on from this work, urological cell lines were exposed to urine for up to 12 hours to determine the effect of urine on the ability to classify these cells. Results indicate that urine has no detrimental effect on prediction results.

Recovery of human papillomavirus nucleic acids from liquid-based cytology media

Residual material from liquid-based cytology (LBC) samples, collected during cervical screening, is a valuable resource for molecular biological analysis. Because of the central role played by human papillomavirus (HPV) in the development of cervical cancer, assays are being developed to quantify HPV gene expression in LBC material. Using quantitative realtime PCR we have compared recovery of HPV DNA and RNA from two of the most widely used LBC systems (the ThinPrep system (Cytyc Corp.) and the SurePath system (TriPath Imaging, Inc.)). Recovery of RNA was unaffected by storage in ThinPrep media, however storage of cells in SurePath resulted in significantly reduced yields (between 10 4- and 10 8-fold reduction depending on extraction technique). Given the increasing prominence and importance of molecular diagnostics and prognostics this is an important finding, and must be considered in relation to choice of LBC system.

Assessment of human papillomavirus mRNA detection over time in cervical specimens collected in liquid based cytology medium

Little is known about the stability of human papillomavirus (HPV) RNA within cervical samples collected in liquid based cytology (LBC) preservation media. We addressed this by analysing patient LBC specimens for the presence of HPV RNA over a prospective time course. LBC samples in PreservCyt ® were obtained from seven women referred to colposcopy due to a cytological diagnosis of moderate or severe dyskaryosis. Aliquots were removed and subject to RNA extraction at, 6 h (base-line), 4, 7 and 14 days, post-collection. HPV mRNA was detected using the PreTect ® HPV Proofer, which detects HPV 16, 18, 31, 33 and 45 E6/E7 transcripts and human small ribonucleoprotein U1A mRNA as a sample control. HPV DNA genotyping was also performed at base-line to assess the range of types in our group. In addition to assessment of viral RNA, overall integrity of the cellular RNA extract was analysed by the RNA 6000 pico assay. Control human RNA was amplified successfully in all seven samples at each time point. Five of the seven women were HPV positive for E6/E7 viral transcripts at base-line and positivity was maintained in all five up to 14 days. Although the pattern of cellular RNA profiles generated from the samples was variable, results indicated that this extract could be amenable to gene expression profiling and that degradation did not increase as a result of storage time. It is concluded that HPV RNA in routinely collected LBC specimens in PreservCyt ® can be detected for at least 14 days from sample collection.