Liquid-based Cytology Samples Research Articles

Human Papillomavirus Distribution among Women in Western Shandong Province, East China using Reverse Blot Hybridization Assay

Cervical cancer is the third most common cancer in women worldwide and there is a significant association between human papillomavirus (HPV) infection and cervical cancer. Certain HPV groups, labeled high-risk (HR) HPV groups, are strongly associated with malignancies of the human cervix. HPV prevalence and genotype distribution were analyzed using the REBA HPV-ID ® (YD Diagnostics, Yongin, Korea) assay based on the reverse blot hybridization assay (REBA) with a total of 324 liquid-based cytology samples from women in Western Shandong Province, East China and results were compared with cytological diagnosis. Most of the HPV genotypes that were detected in high-grade cervical lesions were HR-HPV genotypes such as HPV 16, 18, 33, 53, and 58. The prevalence of these HR-HPV genotypes increased in high-grade cervical lesions. However, from low- to high-grade cervical lesions, the ability to detect LR-HPV genotypes decreased. Additionally, in general, the single HPV genotype infection rate increases in proportion to the severity of the lesion. The study findings suggest that a currently available preventive vaccine against HPV 16 and 18 may have limited effectiveness for prevention of all HPV infection in this province. Finally, based on these findings, these data could guide national or regional vaccination programs in the Western Shandong Province of East China to substantially reduce the burden of cervical lesions.

Prevalence of type-specific oncogenic human papillomavirus infection assessed by HPV E6/E7 mRNA among women with high-grade cervical lesions

Human papillomavirus (HPV) infection is a major cause of premalignant dysplasia and cervical cancer. There are no data on the prevalence of genotype-specific HPV infection assessed by HPV E6/E7 mRNA in women representative of the Korean population across a broad age range. A total of 630 women aged 17-90 years were enrolled in this study. ThinPrep liquid-based cytology samples were evaluated using the CervicGen HPV RT-qDx assay, which detects 16 high-risk (HR) HPV genotypes (set 1: HPV 16, 31, 33, 35, 52, and 58; set 2: HPV 18, 39, 45, 51, 59, and 68; and set 3: HPV 53, 56, 66, and 69). The overall prevalence of HPV infection was 33.2% (n=209), and oncogenic high-risk HPV was detected in 75.9% (n=107) of 141 women with high-grade cervical lesions. HPV 16 was the most common HPV genotype among women with high-grade cervical lesions and histologically confirmed cervical intraepithelial neoplasia grade 2 and above (CIN2+) in the Republic of Korea (41.6%). Among women aged over 30 years, 182/329 (55%) had invasive cervical cancer and 135 (74%) of these were infected with oncogenic HR-HPV types (in particular 25% with HPV 16). Among patients diagnosed with CIN2+, the positivity rate of HR-HPV was the highest in women aged 40-49 years. These results suggest that the determination of specific HPV genotypes is very important for evaluating the potential impact of preventive measures, including the use of prophylactic vaccines, on reducing the burden of cervical cancer.

Trends in Cervical Cytology Screening and Reporting Practices: Results From the College of American Pathologists 2011 PAP Education Supplemental Questionnaire.

The College of American Pathologists periodically surveys laboratories to determine changes in cytopathology practices. We report the results of a 2011 gynecologic cytology survey. To provide a cross-sectional survey of gynecologic cytology practices in 2010. In 2011, a survey was sent to 1604 laboratories participating in the College of American Pathologists gynecologic cytology interlaboratory comparison education program and proficiency testing programs requesting data from 2010 on the following topics: terminology/reporting, cytotechnologist workload, quality assurance, reagents, and ancillary testing. Six hundred and twenty-five laboratories (39%) replied to the survey. The nonstandard use of "low-grade squamous intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion" is used by most laboratories to report the presence of low-grade squamous intraepithelial lesion with possibility of high-grade squamous intraepithelial lesion. Most laboratories also report the presence or absence of cells from the transformation zone. Most respondents do not limit cytotechnologist screening workload during the work shift. Only about one-third of laboratories (188 of 582; 32%) use image-assisted screening devices. Rapid prescreening as a quality assurance measure is used by only 3.5% (21 of 594) of the laboratories. When used for screening, most laboratories use the imager for retrospective review of slides to detect human locator and interpretive errors. Most laboratories receive both liquid-based cytology samples (mainly ThinPrep, Hologic, Marlborough, Massachusetts) and conventional Papanicolaou tests. Expiration dates of liquid-based cytology test vials are not usually recorded. The field of gynecologic cytology is evolving rapidly. These survey results offer a snapshot of national gynecologic cytology practices in 2010.

Retrospective Rescreening of Negative Cervical Cytology Samples Preceding Histologically Proven CIN2-3 and Squamous Cell Carcinoma: An Educational Opportunity to Understand and Prevent Laboratory Errors

Objectives: We aimed to analyze the false-negative (FN) liquid-based cytology diagnoses from the 5 years preceding all the 2013 histologically proven cervical intraepithelial neoplasia (CIN)2-3 and squamous cell carcinoma (SCC) and to propose corrective actions. Study Design: This was a retrospective, blinded rescreening (‘5-year look-back') of liquid-based cytology samples with negative categorizations, which occurred before histologically proven CIN2-3 and SCC. Results: The FN rate was 7.8% (21/256 samples preceding CIN2-3 and 0/13 samples preceding SCC). Slides confirmed as ‘negative', ‘interpretation error' and ‘screening error', respectively, were 3.3% (9/269), 2.6% (7/269) and 1.9% (5/269). In 9/12 cases, error was associated with small atypical cells. In 7/12 cases, these diagnostic cells were less than 5/10 HPF. Inflammation and prominent reactive changes were present in 5/12 cases. Five patients had a positive clinical history. In 2 cases, there were multiple-cell-layer artifacts. Dense groups of small blue atypical cells were missed in 2 other cases. Dotting was imprecise in 6/7 samples. Conclusion: Considering the above results, we specifically reoriented our continuous education activities, focusing rapid rescreening on scanty, isolated, small, atypical cells and dense cell groups. Prior to final diagnosis, pathologists should systematically review the entire surface of the dotted slides, with special attention being devoted to slides with multiple cell layers and tridimensional groups.

Human papillomavirus genotypes distribution by cervical cytologic status among women attending the General Hospital of Loandjili, Pointe-Noire, Southwest Congo (Brazzaville).

HPV infection is associated with cervical cancer, one of the major public health problems in developing countries. In the Republic of Congo, despite of the high age-standardized incidence rate estimated at 25.2 per 100,000 women, molecular epidemiology data on HPV infections are very limited. We investigated HPV genotypes distribution in cervical smears among patients attending the General Hospital of Loandjili, Southwest Congo. A cross-sectional hospital-based study was conducted on 321 women. Liquid-based cytology samples were collected for cytological diagnosis and HPV detection. Nested-PCR was performed using MY09/MY11 and GP5+/GP6+ primers with genotyping by direct sequencing. Type-specific PCR for HPV-6, -11, -16, -18, -31 and -33 was also used to assess multiple infections. Out of 321 women examined, 189 (58.8%) had normal cytology, 16 (5.0%) had ASCUS and 116 (36.1%) had cytological abnormalities. HPV-DNA was detected in 22 (11.6%), 6 (37.5%), and 104 (89.6%) normal cytology, ASCUS and cytological abnormalities respectively. HPV16 was the most prevalent genotype regardless of cytological status followed by HPV70 in women without lesions and HPV33 among those with lesions. HR-HPV prevalence varied significantly according to the cervical cytology (P = 0.000). Among women without lesions, two peaks of HPV infections were observed in age group less than 30 years (60.0%) and in age group 50-59 years (7.1%). Age, age of first sex, multiple sexual partners and pregnancies were the risk factors for HPV infection in women without lesions. Our findings could be used as evidence data base for future epidemiological monitoring in this region.

Cobas 4800 HPV detection in the cervical, vaginal and urine samples of women with high-grade CIN before and after treatment

AimsTo assess the performance of a clinically validated human papillomavirus (HPV) test (the Cobas 4800 HPV test) in urine and self-taken vaginal specimens within a colposcopy population and to assess...

Human papillomavirus (HPV) types 16 and 18 in liquid-based cervical cytology samples.

Human papillomavirus (HPV) DNA testing is replacing cervical cytology as a primary cervical cancer screening tool. The aim of this study was to determine the frequency of occurrence of HPV types 16 and 18 in liquid-based cytology (LBC) cervical samples in our set-up. This study comprised of 302 LBC cervical samples. HPV 16 and HPV 18 were detected by polymerase chain reaction (PCR), and the results were compared between normal (n = 155), inflammatory (n = 99), squamous (n = 37) and glandular abnormalities (n = 11). Of our patient cohort, 73.8 % was ≤40 years old. We found HPV 16 DNA in 91/302 (30.1 %) cases and HPV 18 DNA in 21/302 (6.95 %). HPV types 16 and 18 were detected in 25.8 and 4.5 % cytologically normal samples, respectively. HPV 16 was positive in 29.3 % of inflammatory samples. Squamous cervical abnormalities were more often HPV positive (HPV 16 in 48.6 %; HPV 18 in 29.7 %) than glandular abnormalities (36.4 and 18.2 %, respectively). We found high-risk HPV DNA in more than one third of the tested women. A good number of these HPV-positive cases were negative in cervical cytology.

Human papillomavirus type 16 L1/L2 DNA methylation shows weak association with cervical disease grade in young women

BackgroundPersistent infection with human papillomavirus (HPV) type 16 causes the majority of cervical cancers. Genital HPV infection is very common, but neoplastic progression is uncommon. There is an urgent need for biomarkers associated with cervical neoplasia, to enable triage of women who test positive for HPV. ObjectivesTo assess the ability of quantitative measurement of HPV16 DNA methylation to separate samples of different cytological and histological grades from young women, among whom rates of HPV infection are high. Study designDNA methylation was quantified by pyrosequencing of bisulphite converted DNA from liquid based cytology samples from 234 women (mean age 20.6 years) who tested positive for HPV16 and showed varying degrees of neoplasia. Methylation was assessed at CpGs in the HPV E2 and L1/L2 regions. ResultsThe performance of methylation-based classifiers was assessed by ROC curve analyses. The best combination of CpGs (5600 and 5609) achieved AUCs of 0.656 (95% CI=0.520–0.792) for separation of cytologically normal and severely dyskaryotic samples, and 0.639 (95% CI=0.547–0.731) for separation of samples with or without high-grade neoplasia (CIN2+/−). ConclusionsThe data are consistent with HPV L1/L2 methylation being a marker of the duration of infection in a specific host. Assessment of HPV DNA methylation is hence a promising biomarker to triage HPV-positive cytology samples, but may have limited utility in young women. Future studies assessing the likely utility of HPV DNA methylation as a potential triage biomarker must take account of women’s age.

Improving Laboratory Efficiency by Automation of Preanalytic Processing of ThinPrep Specimens for Real-Time PCR High-Risk HPV Testing.

Cervical specimens collected in liquid-based cytology (LBC) media are the most common sample type used for high-risk human papillomavirus (HPV) testing. Since preanalytic steps such as vortexing and decapping vials, liquid transfer to a sample input tube with matching unique identifier, and recapping the original vials are required for processing LBC samples prior to running the Abbott RealTime High Risk HPV assay (Abbott, Wiesbaden, Germany), a full manual execution can be complicated, especially in high-throughput diagnostic contexts. Here, a custom-configured worktable setup for the Tecan Freedom EVO (Tecan, Männedorf, Switzerland) designed to automate and control preanalytic steps for ThinPrep (Hologic, Marlborough, MA) samples was used to evaluate the impact of automated versus manual preanalytics. Archival results for manual processing of 226 samples were compared with those obtained with the Tecan protocol, observing a very good overall concordance for final assay interpretation (95.6%). High overall agreement (100%) resulted also from retesting 99 samples by both the preanalytical protocols. High reproducibility was observed analyzing 23 randomly selected samples by automated preprocessing in triplicate. Hence, the new configuration of the Tecan platform translates the manual steps required to process ThinPrep specimens into automated operations, controls sample identification, and allows for saving hands-on time, while maintaining assay reproducibility and ensuring reliability of results, making it suitable for screening settings.

Vaginal Intraepithelial Neoplasia Detected with Cervical Liquid-Based Cytology: Old Concerns or New Facilities?

Background: The detection of vaginal intraepithelial neoplasia (VAIN) in cervical samples is not a common finding. Therefore, we aimed to report VAINs detected in liquid-based cytology (LBC) from women examined at Hospital das Clinicas of Faculty of Medicine, Sao Paulo State University. Materials and Methods: We evaluated LBC samples from women referred to gynecology examination for different reasons (previous abnormal PapTest, follow up of treated cervical lesion, ecc) and women examined for regular screening proposals, and compared with biopsy diagnoses, including the controversial diagnoses of vaginal intraepithelial neoplasia (VAIN). Results: From 1866 patients, 1423 (76.3%) cases were negative and 443 (23.7%) were positive for any cellular alteration. Age of patients ranged from 12 to 86 years. We detected 25 histologically confirmed VAIN (1.3% p = 0.0002 by Fisher’s exact test IC 95% 0.0090 - 0.0198) and 1.1% VAIN (p = 0.0031 by Fisher’s exact test IC 95% 0.0077 - 0.0179). Conclusion: The identification of VAIN in routine is feasible; the professionals involved with cytological examination should be aware of these lesions in Pap test samples.

Long-term risk of cervical intraepithelial neoplasia grade 3 or worse according to high-risk human papillomavirus genotype and semi-quantitative viral load among 33,288 women with normal cervical cytology.

In this prospective cohort study, we estimated the long-term risk of cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) by high-risk human papillomavirus (hrHPV) genotype and semi-quantitative viral load at baseline among 33,288 women aged 14-90 years with normal baseline cytology. During 2002-2005, residual liquid-based cervical cytology samples were collected from women screened for cervical cancer in Copenhagen, Denmark. Samples were HPV-tested with Hybrid Capture 2 (HC2) and genotyped with INNO-LiPA. Semi-quantitative viral load was measured by HC2 relative light units in women with single hrHPV infections. The cohort was followed in a nationwide pathology register for up to 11.5 years. In women aged ≥30 years at baseline, the 8-year absolute risk for CIN3+ following baseline detection of HPV16 was 21.8% (95% confidence interval [CI]: 18.0-25.6%). The corresponding risks for HPV18, HPV31, HPV33, and other hrHPV types, respectively, were 12.8% (95% CI: 7.6-18.0%), 11.3% (95% CI: 7.7-14.9%), 12.9% (95% CI: 7.0-18.8%) and 3.9% (95% CI: 2.7-5.2%). Similar absolute risk estimates were observed in women aged <30 years. Higher HPV16-viral load was associated with increased risk of CIN3+ (hazard ratio = 1.34, 95% CI: 1.10-1.64, per 10-fold increase in viral load). A similar trend, although statistically nonsignificant, was found for viral load of HPV18. The 8-year absolute risk of CIN3+ in women with HPV16-viral load ≥100.0 pg/ml was 30.2% (95% CI: 21.9-38.6%). Our results support that hrHPV genotyping during cervical cancer screening may help identify women at highest risk of CIN3+.

A population-based observational study comparing Cervista and Hybrid Capture 2 methods: improved relative specificity of the Cervista assay by increasing its cut-off.

BackgroundHigh-risk human papillomavirus (HR HPV) testing has been shown to be a valuable tool in cervical cancer screening for the detection of cervical pre-cancer and cancer.MethodsWe report a purely observational study evaluating HR HPV prevalences in residual liquid-based cytology (LBC) samples using both the Cervista™ HPV HR Test and the Digene Hybrid Capture 2 High-Risk HPV DNA Test (HC2) in a sample of 1,741 women aged ≥30 years of a German routine screening population of 13,372 women. Test characteristics were calculated and a novel method for measuring test performances was applied by calculating ratios of sensitivity or specificity.ResultsThe overall agreement of both tests for detection of HR HPV was excellent (κ = 0.8). Relative sensitivities for the detection of histologically confirmed severe cervical intraepithelial dysplasia (CIN3+) were similar for both HPV-tests, which was confirmed by the ratio analysis. However, discrepancy analysis between the Cervista HPV HR test and HC2 revealed a high false positive rate of the Cervista HPV HR test in the cytology normal category.ConclusionsPerformance of the Cervista HPV test in cervical specimens with abnormal cytology is comparable to HC2 as both tests were highly sensitive and specific for the detection of high grade cervical disease. We also demonstrate evidence that modification of the cut-off values drastically reduces the false positive rate in the cytology normal category without affecting the detection of CIN3+, which ultimately improved specificity of the Cervista HPV HR assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0674-1) contains supplementary material, which is available to authorized users.

High feasibility of liquid-based cytological samples for detection of EGFR mutations in Chinese patients with NSCLC.

Activating mutations of epidermal growth factor receptor (EGFR) could predict response to tyrosine kinase inhibitor (TKI) treatment in patients with non-small cell lung cancer (NSCLC). However, the detection of EGFR mutation is frequently challenging in clinical practice for the lack of tumor tissue. The aim of this study was to investigate the feasibility of performing EGFR mutation testing on various types of liquid-based cytology (LBC) samples. A total of 434 liquid-based cytology samples were collected from March 2010 and November 2013. Among them, 101 with diagnosis of lung adenocarcinoma had paired surgically resected specimens. The ADx Amplification Refractory Mutation System (ADx-ARMS) was used to determine EGFR mutation status both in LBC and resected samples. All liquid-based cytology samples were adequate for EGFR mutation analysis. The mutation rate was 50.5% in the 434 NSCLC patients with LBC samples and the incidence rates of EGFR mutation were consistent among different specimens. We also detected EGFR positives in 52.5% (53/101) patients with paired histologic specimens. The concordance rate of EGFR mutation between LBC samples and paired histologic specimens was 92.1%. Our results suggest that liquid-based cytology samples are highly reliable for EGFR mutation testing in patients with NSCLC.

Preparation of Compact Agarose Cell Blocks from the Residues of Liquid-Based Cytology Samples

BackgroundInevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA).MethodsThe cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining.ResultsThe SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases.ConclusionsThis agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

EGFR mutational genotyping of liquid based cytology samples obtained via fine needle aspiration (FNA) at endobronchial ultrasound of non-small cell lung cancer (NSCLC)

ObjectivesEpidermal growth factor receptor (EGFR) gene mutation status should be determined in all patients with advanced, non-squamous non-small cell lung carcinoma (NSCLC) to guide targeted therapy with EGFR tyrosine kinase inhibitors. EGFR mutations are commonly tested by Sanger sequencing or allele specific polymerase chain reaction (ASPCR) on formalin-fixed paraffin-embedded (FFPE) samples including cell blocks (CB) that may fail due to absence of tumor cells. The cell pellet from cytology specimens obtained at the time of endobronchial guided ultrasound fine needle aspiration (EBUS FNA) (EBUS-TBNA, transbronchial needle aspiration) represents an alternative resource for additional tissue. Here we demonstrate the utility of using the FNA cell pellet versus for the detection of EGFR mutations in NSCLC. Materials and methodsFor internal validation, 39 cytology samples from patients with NSCLC referred for EGFR testing were analyzed using the EGFR rotor-gene Q (RGQ) PCR assay (Qiagen).Thereafter, a consecutive series of 228 EBUS FNA samples were tested. ResultsThe ASPCR assay demonstrated acceptable intra-assay, inter-assay and inter-lot reproducibility, sensitivity, and specificity. For the consecutive series, only 6/228 (2.6%) failed analysis (5 due to insufficient DNA yield). Of 228 EBUS FNA cell pellets tested 32 (14.0%) demonstrated clinically relevant mutations. Results and conclusionASPCR can reliably detect EGFR gene mutations in FNA preparations from patients with NSCLC obtained at EBUS.

Amplification of 3q26 and 5p15 regions in cervical intraepithelial neoplasia.

To analyze different amplification patterns of 3q26 and 5p15 regions in low-grade and high-grade cervical intraepithelial neoplasia. Experimental research. Department of Obstetrics and Gynecology at a medical faculty in Slovakia. A group of 83 patients referred for colposcopic examination. Amplification of 3q26 and 5p15 regions was analyzed on the 100 most atypical cells from a cervical cytology slide by fluorescent in situ hybridization using a multicolor hybridization probe. Chi-squared and Man-Whitney U-tests were used for statistical analysis. Liquid-based cytology samples and biopsy samples obtained during colposcopic examination correlated with high-risk human papillomavirus status and with amplification patterns of selected regions analyzed by fluorescent in situ hybridization. The number of cells with 3q26 and 5p15 gain rises with the severity of the lesion p < 0.01. The sensitivity of 3q26 amplification for CIN2+ lesions was 72.1% (95% confidence interval 56.3-84.7) and specificity was 90.0% (95% confidence interval 76.3-97.1). The sensitivity of 5p15 amplification for CIN2+ lesions was 69.8% (95% confidence interval 53.9-82.8) and specificity was 85.0% (95% confidence interval 70.2-94.3). Evaluation of telomerase components can help in differential diagnosis of low-grade and high-grade cervical lesions and in individualized management of these patients.

Human papillomavirus vaccination and cervical cytology in young minority women.

Continued surveillance of human papillomavirus (HPV) vaccination is necessary to identify clinical benefits, particularly given the low rate of vaccine uptake and completion and vaccination of many young women after sexual debut. We studied the effect of catch-up HPV vaccination on cervical cytology and HPV infection in sexually active, low-income and minority young women. We conducted a cross-sectional study of 235 women aged 21 to 30 years undergoing routine cervical cytology testing. Demographic and behavioral characteristics were self-reported. HPV vaccination status was determined by self-report and verified with electronic medical records. Liquid-based cytology samples were tested for HPV genotypes. Adjusted prevalence ratios between HPV vaccination and (1) abnormal cervical cytology result and (2) HPV genotype were estimated. At the time of the study, 96 women (41%) had received at least 1 HPV vaccination, 54 of whom had completed the series; 97% of women were vaccinated after sexual debut. Twenty-four (10%) women had an abnormal cervical cytology result. The prevalence of abnormal cytology was 65% lower in women who received at least 1 HPV vaccination versus unvaccinated women (adjusted prevalence ratio, 0.35; 95% confidence interval, 0.14-0.89). Among 232 women with genotype results, 46 (20%; 95% confidence interval, 15%-26%) had HPV infection detected. HPV types 16, 18, 45, 53, and 66 were most prevalent. The prevalence of abnormal cytology was lower in vaccinated versus unvaccinated women, despite receipt of vaccination after sexual debut. Continued assessment of HPV vaccine effectiveness before and after sexual debut on HPV infection and cervical dysplasia is needed.

Expression of HPV‐related biomarkers and grade of cervical intraepithelial lesion at treatment

New human papillomavirus (HPV)-related biomarkers may allow better identification of clinically significant lesions that warrant excision and, conversely, identification of the false positive cases that have been overreported by traditional techniques. The aim of this study was to investigate how the expression of several HPV-related biomarkers correlates to the severity of the lesion at treatment. Prospective observational study. University Hospital (2009-2011). All women submitted for excisional treatment. A liquid-based cytology sample was obtained before treatment and was tested for HPV typing, mRNA E6 & E7 with NASBA or flow cytometry and p16. All women had histological diagnosis in the form of excisional cone (gold standard). Correlation of HPV biomarker positivity rates to the grade of the lesion at treatment histology. Two hundred women were recruited: 23 were found to have negative histology (11.5%), 79 (39.5%) CIN1, 50 (25.0%) CIN2 and 48 (24.0%) CIN3. All biomarkers (HPV DNA typing, HR HPV, single HPV 16/18, mRNA E6 & E7 expression and p16) revealed an increased linear positivity rate with increasing severity and grade of the lesion (chi-squared test for trend p < 0.05). This was stronger for HPV (all and high-risk) followed by mRNA with NASBA, flow cytometry, HPV 16/18 and ultimately p16 immunostaining. The linear correlation between various HPV-related biomarkers and the grade of the lesion suggests that these biomarkers may prove to be useful in the prediction of CIN grade and, as a result, the need for treatment.

Histology Verification Demonstrates That Biospectroscopy Analysis of Cervical Cytology Identifies Underlying Disease More Accurately than Conventional Screening: Removing the Confounder of Discordance

BackgroundSubjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation.MethodsWithin a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn’s test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease.ResultsConventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation.ConclusionsHistology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool.

Liquid-based trans-epithelial flexible brush cytology of high-grade laryngeal mucosal lesions

Laryngeal cytology, which was popular in the 1950s and 60s, has lost its appeal due to difficulties in obtaining adequate specimens and frequent false negative results which are often associated with smears of suboptimal quality. Flexible laryngoscope and brushes, and liquid-based cytology (LBC) allow for improved collection and quality of laryngeal mucosal samples. In LBC the cells are optimally fixed and significantly retrieved. The samples are virtually devoid of air drying artefact and obscuring elements such as blood, mucus, necrotic debris and inflammation. The diagnostic accuracies of liquid-based transepithelial flexible brush cytological (LBTEFBC) samples and flexible biopsies (FB) were compared with those of biopsy under general anaesthesia (BUA) in 49 and 46 patients respectively. Cytology specimens were graded and scored using a recently proposed oral cytological grading and scoring system. When compared with BUA, the cytology showed 97% sensitivity and 100% specificity, and FB yielded 77.1% sensitivity and 100% specificity. Reactive/mildly dysplastic lesions can be discriminated from high-grade dysplasias/invasive squamous cell carcinomas with 100% sensitivity and specificity. LBTFBC is a simple, cost-effective, relatively atraumatic office-based procedure, and with the recently proposed classification, it can be accurate in the detection of high-grade laryngeal lesions.