Enhancing recovery of endocervical component on gynecologic cytology specimens processed by thin-layer technology

OBJECTIVE: To address the causes and report the corrective measures required for resolving the initial problem of high rates of cervical vaginal cytology specimens reported as having no endocervical component on SurePath (AutoCyte, Inc., Burlington, North Carolina, U.S.A.) liquid-based, thin-layer technology at an academic center cytology laboratory. STUDY DESIGN: Analysis of 511 cases lacking endocervical/transformation zone component out of 9,221 SurePath thin-layer gynecologic specimens processed in a one-year period. The study encompassed a review of sample collection techniques by physicians and nurses, specimen processing, cytologic features of endocervical/squamous metaplastic cells processed by the SurePath method and statistical analysis of endocervical cell recovery rates after implementation of corrective measures. RESULTS: Absence of endocervical/transformation zone component varied from an initial 18% in the first month to an average of 5.3% after corrective actions were implemented. Current rates of SurePath thin-layer specimens having no endocervical component are lower than those for conventional smears. CONCLUSION: Since SurePath was only recently introduced to the market, there are no previously published data addressing how to optimize the recovery of endocervical component on liquid-based, thin-layer specimens processed by this methodology.     

Decrease in numbers of glandular cell groups in post-LLETZ liquid-based cytology preparations

Objective:  Large loop excision of the transformation zone (LLETZ) has become standard of care in the management of cervical squamous neoplasia and with cone biopsy glandular intraepithelial neoplasia. Controversy remains about the long-term effects of this traumatic procedure. The aim of this study was to count and compare the number of endocervical glandular cell groups in pre- and post-LLETZ cervical preparations using liquid-based cytology to establish a cyto-morphological correlate of destruction of the transformation zone.
Methods:  The cytology/histology correlation audit records of the Cytopathology Department of St Luke's Hospital in 2003 and early 2004 were used to select patients with a cytological diagnosis of high grade dyskaryosis followed by LLETZ. Only those cases with post-LLETZ cytological follow-up were selected. Cases using conventional smears were excluded. One hundred and twenty slides (60 pairs of slides) in total were retrieved. The cases underwent review and all groups of >3 glandular cells in each slide were counted by AM while blinded as to whether smears were pre- or post-LLETZ. Medians were compared using a Mann–Whitney U -test.
Results:  The median number of groups of endocervical glandular cells of the pre-treatment group was 5.5 and of the post-treatment group was 2.0. There were significantly fewer endocervical glandular cell groups in the post-LLETZ population ( P  = 0.03).
Conclusions:  The number of endocervical glandular groups in cervical cytological preparations decreases significantly following LLETZ procedure. This suggests that cytological follow-up may not be as useful in glandular neoplasia cases. Few or absent glandular cell groups in post-LLETZ preparations may have implications for adequacy assessment.     

Flow cytometry of human gynecologic specimens using log chromomycin A3 fluorescence and log 90 degrees light scatter.

Flow cytometry and electronic cell sorting are being investigated to screen gynecologic specimens for cervical neoplasia. Cellular DNA content is quantitated by Chromomycin A3 fluorescence and cell size is quantitated by 90 degrees light scatter; the logarithms of the measured intensities are used to produce a two parameter histogram. To determine the cell types responsible for signals in various histogram regions, systematic electronic cell sorting is performed. The sorted fractions are sedimented into microscope slides and stained by the Papanicolaou technique. The cells in each fraction are identified by conventional cytomorphologic criteria. Morphologic analysis of sorted cells reveals histogram regions corresponding to specific cell types. One very important region contains the highest concentration of signals from abnormal cells and is therefore the best region to analyze for specimen abnormality. However, because a significant number of signals in this region are from normal cells, specimens cannot be diagnosed by their analysis. Another important histogram region is composed primarily of signals from endocervical columnar and metaplastic cells. The presence of such cells is a good criterion for specimen adequacy, therefore analysis of signals in this region is essential to assess specimen adequacy for automatic screening.     

Comparison of two preparation methods for endocervical evaluation

OBJECTIVE: To assess the validity of SurePath liquid-based preparation method for examination of endocervical brush specimens as a substitute for conventionally prepared cytology methods for evaluating the endocervical canal during colposcopic examination and biopsy. STUDY DESIGN: Paired SurePath liquid-based test slides and conventional smears were obtained using an endocervical brush in a split sample protocol before biopsy at the time of colposcopy. The level of agreement between cytologic results obtained was assessed. Accuracy and operating characteristics were evaluated compared to histologic follow-up. RESULTS: Agreement between cytology results for the methods was excellent. The overall kappa was 0.924 (p = 0.0000). There was exact agreement on interpretation between the methods in 283 of 299 cases (94.6%). Cytohistologic follow-up results correlation were: SurePath liquid-based Pap test results and conventional smear results agreed with histology results in 47.8% and 49.2% of cases, respectively. Allowing for a discrepancy within 1 level of severity of cytologic grade, agreements were 76.6% and 77.2%, respectively. CONCLUSION: This study demonstrates that the SurePath method is equivalent to conventional endocervical brush cytology preparation and performs well for detection of cervical intraepithelial lesions and cancer. SurePath is acceptable for endocervical evaluation as a substitute for endocervical curettage at colposcopic biopsy.     

Comparison of conventional Papanicolaou smear and SurePath® liquid-based cytology in the Copenhagen population screening programme for cervical cancer

OBJECTIVE: To compare diagnostic performance of conventional Papanicolaou smear with SurePath liquid-based cytology in a population screening programme. METHODS: A retrospective comparison was performed on data from two 18-month periods of the screening programme for cervical cancer in the municipality of Copenhagen with conventional Papanicolaou technique (n = 82,116) and liquid-based cytology (n = 84,414). RESULTS: After the conversion to liquid-based cytology the percentage of unsatisfactory samples decreased from 2.3% to 0.3% (P < 0.001), whereas the number of normal cervical samples lacking an endocervical component increased from 8.5% to 8.9% (P < 0.005). The percentage of samples with atypical cells and cells suspicious for malignancy increased from 3% to 4.2% (P < 0.001) and from 1.9% to 2.4% (P < 0.001), respectively. The subsequent histological follow-up showed normal findings decreased from 70.5% to 68.9% and from 28.0% to 26.1%, respectively. However, in relation to the entire screening populations, there was an increase of normal findings from 2.12% to 2.89% after primary atypical diagnosis and from 0.53% to 0.62% after diagnosis of suspicious cells after conversion to the liquid-based technique. CONCLUSIONS: This study showed the number of unsatisfactory samples to be significantly reduced with the liquid-based technique. The data suggest that there is an increased detection rate of cervical precancerous lesions with liquid-based cytology, but the number of false positive tests is still high. The specificity of the two tests seems similar, but this cannot be ascertained exactly, because of the fact that follow-up of negative cases is unavailable.     

Does liquid-based technology really improve detection of cervical neoplasia? A prospective, randomized trial comparing the ThinPrep Pap Test with the conventional Pap Test, including follow-up of HSIL cases

OBJECTIVE: To compare the sensitivity, specificity and specimen adequacy of the ThinPrep Pap Test (TP) with the conventional Pap Test (CV) in a low-risk population with subsequent follow-up of HSIL cases. STUDY DESIGN: A prospective, randomized, controlled design was chosen to compare the TP with CV. Cytologic diagnosis and specimen adequacy were evaluated and compared with histology data in high grade squamous intraepithelial lesion (HSIL) cases. Fifteen gynecologists in private practice, all trained in colposcopy, participated in the trial. Cytologic diagnosis, specimen adequacy and follow-up of the cytologic HSIL cases were compared in the two groups. In total, 1,999 patients were included, 997 in the TP group and 1,002 in the CV group. Randomization assignments were designated on cytology case report forms, which were placed in sealed envelopes. Each envelope had a sequential randomization number on the outside to allow tracking and authentication of randomization assignments. RESULTS: Comparison of results between CVs and TPs revealed no statistically significant differences in all diagnostic categories, ranging from "within normal limits" to HSIL. Specimen adequacy, however, was superior with CVs (P < .001). The cytologic diagnosis of HSIL correlated with the histologic diagnosis in 91% of the TP group and 100% of the CV group. CONCLUSION: Because there was no statistically significant difference in sensitivity and specificity of the two techniques, improved detection of cervical abnormalities and better specimen adequacy might not be a consequence of utilizing liquid-based preparations but of a better sampling technique. Removing mucus and cellular debris from the cervical surface with a cellulose swab before sampling cells with a proper sampling device results in the same sensitivity and specimen adequacy and is much less expensive than the liquid-based technique.     

The Value of Gross Visual Assessment of Specimen Adequacy for Liquid-Based Cytology During Ultrasound-Guided,Fine-Needle Aspiration of Thyroid Nodules

Objective: Major problems of fine-needle aspiration (FNA) of thyroid nodules arise due to nondiagnostic results caused by inadequately obtained FNA specimens. The purpose of this study was to evaluate the value of visual assessment of liquid-based cytology specimens during FNA of thyroid nodules for predicting sampling adequacy.Methods: For 3 months, visual assessment of FNA specimens was used for 534 consecutive nodules in 534 patients. The FNA specimens were visually graded immediately following aspiration for each nodule, and the visual grades were classified into 2 categories: inadequate (<6 cell groups) and adequate (>6 cell groups). The cytology results were classified as diagnostic or nondiagnostic based on the Bethesda system. We compared the ultrasound features and FNA characteristics between the diagnostic and nondiagnostic results. Multiple logistic regression analysis was used to determine factors independently predictive of nondiagnostic results. We also evaluated the interobserver agreement regarding the visual assessment.Results: Visual assessment was feasible in all patients, and the nondiagnostic rate was 11.6% (62 of 534). Nondiagnostic results were more frequent in the inadequate visual assessment group (38.1%) than in the adequate visual assessment group (10.5%) (P = .001). Independent predictive factors for nondiagnostic results were inadequate visual assessment (odds ratio, 5.18), >50% vascularity (odds ratio, 3.98), and macrocalcification (odds ratio, 3.60). Interobserver agreement for the prediction of visual assessment was good (κ value, 0.767; P<.001).Conclusion: Immediate visual assessment of a specimen during FNA of a thyroid nodule is a feasible method for predicting sampling adequacy.Abbreviations: FNA = fine-needle aspiration LBC = liquid-based cytology US = ultrasound     

The role of cervical cytology and colposcopy in detecting cervical glandular neoplasia

Objective:  To determine the role of cervical cytology and colposcopy in the management of endocervical neoplasia.
Setting:  Colposcopy unit and cytology laboratory in a teaching hospital.
Sample:  Group 1 included 184 smears showing endocervical glandular neoplasia from 129 patients and group 2 included 101 patients with histology showing endocervical abnormalities in a 6-year period (1993–1998). Follow-up of 6–11 years to 2004 was available.
Methods:  Group 1 were identified from the cytology computer records. Group 2 were identified from histology records on the cytology database and a record of histology cases kept for audit purposes. The clinical records were examined retrospectively.
Results:  The positive predictive value (PPV) of abnormal endocervical cells in smears was 81.1% for significant glandular/squamous [cervical glandular intraepithelial neoplasia (CGIN)/cervical intraepithelial neoplasia grade2 (CIN2 or worse)] lesions. The PPV of colposcopy was 93.5% for significant glandular/squamous lesions of the cervix. The postcolposcopy probability of a significant lesion when colposcopy was normal was 87.5%. The sensitivity of colposcopy in detecting endocervical lesions was 9.8%. The sensitivity of cervical smears in detecting a significant endocervical abnormality (CGIN or worse) was 66.3%. The false negative rate for cytology of endocervical glandular lesions was 4.0%.
Conclusions:  Endocervical glandular neoplasia detected on cytology is predictive of significant cervical pathology even when colposcopy is normal, which supports excisional biopsy in the primary assessment of these smears. The high concomitant squamous abnormality rate justifies the use of colposcopy to direct biopsies from the ectocervix. Cervical cytology is the only current screening method for cervical glandular abnormalities but sensitivity is poor.     

Rapid detection of HSV from cytologic specimens collected into ThinPrep fixative

OBJECTIVE: Herpes simplex virus (HSV) infection is associated with substantial morbidity and mortality in neonates. A diagnosis of HSV on cervical cytologic studies could lead to a cesarean section, with an increase in the risk of maternal morbidity. The identification of viral lesions in sexually active women has medical and social implications. There have been reports of false positive diagnoses of HSV in patients with altered endocervical cells and with cervical intraepithelial neoplasia 3. We evaluated a polymerase chain reaction (PCR)-based assay to detect HSV-1 and HSV-2 in routinely collected cervical cytology specimens in ThinPrep fixative (Cytyc Corp., Marlborough, Massachussets, U.S.A.). STUDY DESIGN: DNA was extracted from five cases that demonstrated cytologic changes suggestive of an HSV infection. PCR amplification with subsequent gel electrophoresis was performed to detect the presence of HSV. RESULTS: HSV DNA was detected in three of five cases that were cytologically diagnosed as suspicious or strongly suspicious for HSV infection. CONCLUSION: The combination of the ThinPrep liquid-based method for cervical cytology with PCR allows prompt confirmation of the diagnosis of HSV without sacrificing the diagnostic morphology on the slide.     

Liquid-based cytology findings of glassy cell carcinoma of the cervix. Report of a case with histologic correlation and molecular analysis

BACKGROUND: Glassy cell carcinoma is a rare form of poorly differentiated carcinoma of the cervix with no obvious squamous or glandular differentiation. Its liquid-based cytology findings have not been described before. CASE: A 46-year-old Filipina presented with vaginal bleeding due to a bulky cervical tumor. The liquid-based cytology preparation was of moderate cellularity and contained small clusters of polygonal to elongated tumor cells admixed with amphophilic, granular, necrotic debris. The malignant cells possessed round to oval nuclei; a thin nuclear membrane; finely dispersed chromatin; prominent, solitary nucleoli; abundant, cyanophilic cytoplasm; and discrete cell borders. Occasional tumor cells showed phagocytosis of polymorphs. The background contained a mixed population of inflammatory cells. Eosinophils, though present, were not readily identified in the cytologic specimen. There was no evidence of dyskeratosis, cytoplasmic vacuolation or koilocytosis. Histologic and ultrastructural examination of the tumor biopsy showed classic features of glassy cell carcinoma. Molecular analysis using polymerase chain reaction and restriction fragment length polymorphism revealed the presence of human papillomavirus (HPV) DNA in the liquid-based cytology sample. The HPV genotype, however, did not belong to any of the commonly encountered prototypes. CONCLUSION: Glassy cell carcinoma of the cervix may show distinct, though subtle, cytomorphologic features in liquid-based preparations. The findings, however, are slightly different from those in conventional cervical smears. Awareness of this rare entity is important, as glassy cell carcinoma is often associated with more aggressive clinical behavior.     

High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage

Objective:  With moves to introduce human papillomavirus (HPV) triage at sentinel sites in England, it is essential that optimal storage and transport conditions are determined for efficient HPV detection using residual liquid-based cytology specimens.
Methods:  Two cytology laboratories with comparable workloads sent residual cervical cytology specimens collected in BD Surepath™ Preservative Fluid to the Specialist Virology Centre for HPV testing. Storage and transport of specimens was at ambient (site A) or refrigerated (site R) temperatures. The effect of temperature on the ability to detect high-risk human papillomavirus (HR-HPV) using Digene Hybrid Capture^® 2 High-Risk HPV DNA Test (hc2) and Roche AMPLICOR^® HPV Test (AMPLICOR) was assessed. All specimens with discordant results were tested using Roche Linear Array HPV Genotyping test.
Results:  A total of 796 residual cytology specimens, with cytology ranging from normal to severe dyskaryosis, were provided (399 from site A and 397 from site R). Ambient storage and transit of cervical specimens in SurePath medium did not appear to affect significantly the suitability of the specimen for HPV testing, as measured by the concordance of the HR-HPV screening assays for ambient versus refrigerated specimens and by the proportion of specimens which tested invalid.
Conclusion:  Residual cytology specimens in SurePath medium, stored and transported at ambient temperature, appear suitable for HR-HPV detection by AMPLICOR beyond the manufacturer's recommended time and potentially up to four weeks.     

A comparison between the Accu-Pap device and the extended-tip wooden Ayre spatula for cervical cytology sampling

The wooden Ayre spatula with an extended endocervical tip was compared with both designs of the plastic Accu-Pap sampler in two series of 100 consecutive patients to compare their adequacy in obtaining samples for cervical cytology. There was no significant advantage noted in the smears taken by either spatula. In terms of the sequence of smears, the second smear generally contained more endocervical cells and less often showed an absence of diagnostic cells.     

Influence of benign cellular changes in diagnosis of cervical cancer using IR microspectroscopy

IR spectroscopy and principal components analysis (PCA) of endocervical cells and smears diagnosed with benign cellular changes were investigated to determine the influence of these potential confounding variables in the diagnosis of cervical cancer. Spectral differences in all cell and diagnostic types investigated were found in the phosphodiester and carbohydrate regions. However, the spectral differences in other bands were not distinct enough to allow differentiation between groups. The PCA was successfully used to obtain a separation of normal ectocervical smears from normal endocervical cells and smears diagnosed with inflammation, Candida albicans, and bacterial vaginosis. A separation with a slight overlap of abnormal ectocervical smears from normal endocervical cells, inflammation, and bacterial vaginosis was obtained with PCA. Candida was not separated from abnormal ectocervical smears with any success.     

Diverse glandular pathologies coexist with high-grade squamous intraepithelial lesion in cyto-histological review of atypical glandular cells on ThinPrep specimens

Objective: To identify in cytology, high‐grade squamous intraepithelial lesions with endocervical glandular extension in cases previously diagnosed as atypical glandular cells (AGC), analyse possible reasons for the diagnostic pitfall and document the frequency of glandular pathology coexisting with high‐grade cervical intraepithelial lesion in histology. Methods: Thirty‐nine ThinPrep® cervical smear (Pap) tests reported as AGC of undetermined significance and showing high‐grade lesions on histology [cervical intraepithelial neoplasia (CIN) 2 or 3, endometrial or extrauterine adenocarcinoma] were reviewed retrospectively to identify the cases of high‐grade squamous intraepithelial lesion with endocervical glandular extension, using the Bethesda 2001 system. Cyto‐histological correlation was performed. Results: A high frequency of diverse glandular pathologies coexisted with high‐grade cervical intraepithelial lesions on histology. This included endocervical glandular extension in 63%, benign glandular pathology in 33% and pre‐neoplastic or malignant glandular pathology (endocervical glandular dysplasia, adenocarcinoma in situ and metastatic breast carcinoma) in 17% cases. On cytology, the sensitivity was 40%, specificity was 80% and positive predictive value was 86% for endocervical gland extension in high‐grade squamous intraepithelial lesions. Conclusions: Special efforts to recognize endocervical glandular extension in high‐grade squamous intraepithelial lesions and glandular neoplasia coexisting with squamous intraepithelial lesions from the heterogeneous category of AGC can contribute to increasing the diagnostic accuracy. The identification of endocervical glandular extension on cervical cytology would alert the gynaecologist to perform a thorough assessment of the endocervix during colposcopy. This could also help to decide on the need to perform deeper conization rather than loop electrosurgical excision procedure to ensure negative margins when colposcopic biopsy shows CIN 2 or 3.     

Cytomegalovirus Infection of the Cervix Detected By Cytology and Histology: A Report of Five Cases

We present four cases of cytomegalovirus (CMV) infection detected by cervical cytology of asymptomatic young women. One patient who had undergone bone marrow transplantation was immunosuppressed, but no factors predisposing to CMV infection were identified in the other three patients. A cervical biopsy specimen from a fifth patient, who was also asymptomatic, demonstrated the locus of CMV infection to be in endocervical gland cells. Immunocytochemical and polymerase chain reaction studies on this biopsy specimen confirmed that the histological changes were caused by CMV. the finding of CMV-infected cells in cervical cytological or biopsy specimens is a rarity. Our observations in asymptomatic, non-immunocompromised women suggest their presence is an incidental finding, unlikely to have any clinical significance.     

A single ThinPrep® slide may not be representative in all head and neck fine needle aspirate specimens

Objectives:  Ideally, head and neck aspiration should be performed by trained aspirators within the setting of a one-stop clinic, where smeared material is available for immediate assessment. However, this may not always be possible for practical reasons and the use of liquid-based techniques in head and neck cytology is increasing. Although liquid-based cytology has been extensively validated for use in gynaecological cytology, no studies have investigated whether or not a single ThinPrep ^® slide is representative for head and neck aspirate specimens. We performed a prospective audit of head and neck fine needle aspiration specimens processed by the ThinPrep ^® method to investigate whether a single ThinPrep ^® slide was representative.
Methods:  A prospective audit of 115 consecutive head and neck aspirates was carried out. A single ThinPrep ^® slide was prepared and a diagnosis recorded. The remainder of the specimen was then spun down and prepared as a cell block. The ThinPrep ^® and cell block diagnoses were compared.
Results:  In 36 cases (31%), the cell block provided additional information that contributed to the diagnosis. In 14 (12%), the cell block was regarded as essential to the diagnosis.
Conclusions:  A single ThinPrep^® slide may not provide representative diagnostic material in all head and neck aspirates. This should be taken into consideration when contemplating the use of liquid-based methods for non-gynaecological cytology.     

Comparison of spatula and nonspatula methods for cervical sampling

A comparison between nonspatula (cotton swab and Cytobrush) cervical sampling methods and spatula (wooden Ayre spatula and plastic extended-tip Szalay Cyto-Spatula) sampling methods was made in 109 cases. Based on the presence of endocervical cells, there were statistically significant qualitative differences between the non-spatula methods as well as between the spatula methods, but not between the Cytobrush and Cyto-Spatula smears or the cotton swab and Ayre spatula smears. In all kinds of inflammatory lesions, the spatula samples were more accurate and diagnostic than the nonspatula ones. In all cases of cervical intraepithelial neoplasia and in most cases of squamous metaplasia, the Cyto-Spatula sample was the most accurate. It is concluded that the Szalay Cyto-Spatula method is superior to the other cervical sampling methods because it provides well-preserved cells from both the endocervix and the ectocervix in one smear. The Cytobrush should be used in conjunction with spatula sampling (combination method) for effective sampling of the cervix. The Cytobrush alone is effective mainly for endocervical sampling while the Ayre spatula alone is effective mainly for ectocervical sampling; the cotton swab is ineffective for both endocervical and ectocervical sampling.     

Cytologic features of ciliated adenocarcinoma of the cervix: a case report

BACKGROUND: Ciliation is a normal finding in the endometrium, fallopian tubes and cervix. Because cilia are characteristically lost when malignant tumors arise at these sites, the detection of cilia on light microscopy is frequently used to support a benign diagnosis. Ciliated carcinomas of müllerian duct origin, however, do occur, albeit rarely, and can pose a potential diagnostic difficulty in cytologic specimens. CASE: A woman with a histologically confirmed ciliated adenocarcinoma of the cervix had prior liquid-based cervical cytology showing atypical, ciliated glandular cells that initially raised the diagnostic consideration of tubal metaplasia. A concurrent biopsy, however, revealed focally ciliated adenocarcinoma of the cervix. CONCLUSION: Awareness of the ciliated variant of adenocarcinoma of the cervix is important to avoid overreliance on ciliation as a definitive feature of benignity in cervical cytologic specimens.     

Impact of liquid-based gynecologic cytology on an HIV-positive population

OBJECTIVE: To examine the imprint of liquid-based technologies for cervicovaginal cytology on HIV-positive women, who are at high risk for cervical intraepithelial neoplasia. STUDY DESIGN: We performed a retrospective search of the cytopathology files of Johns Hopkins Hospital for the cervicovaginal cytology of HIV-positive women to examine the effect of liquid-based technology on this population. RESULTS: Significant intraepithelial lesions (SILs) (low grade SIL or greater) were identified in 24% of the conventional smears and 23% of the liquid-based cytology. Atypical squamous cells of undetermined significance (ASCUS)/atypical glandular cells of undetermined significance was diagnosed in 15% of the conventional smears and 9% of the liquid-based preparations (P = .02). In patients with ASCUS diagnoses and tissue follow-up within 7 months, significant SILs were identified in 29% with conventional smears and in 65% with liquid-based cytology. CONCLUSION: There was no statistically significant difference in the rate of SILs between conventional smears and liquid-based cervicovaginal preparations in HIV-positive women. The diagnosis of ASCUS on liquid-based cytology may have an increased likelihood of representing a significant SIL in comparison to conventional smears. For the high-risk, HIV-positive population, immediate colposcopy and biopsy may be warranted following ASCUS diagnoses on liquid-based cytology.