Proposed Sheffield quantitative criteria in cervical cytology to assist the grading of squamous cell dyskaryosis, as the British Society for Clinical Cytology definitions require amendment

OBJECTIVE: In 1986, the British Society for Clinical Cytology (BSCC) published quantitative criteria to assist diagnosis in a three-tier grading system of squamous cell dyskaryosis. In dyskaryotic cells, area nuclear to cytoplasmic (NC) ratios below 50%, between 50% and 66% and over 66% were defined as equating with mild, moderate and severe grades respectively. Following the Terminology Conference in 2002, however, the BSCC recommended on their website that the three-tier model should be replaced by a new two-tier system of low- and high-grade squamous abnormalities. The latter broadly equate with the two-grade Bethesda System (TBS) for reporting squamous intraepithelial lesions. The purpose of this study was to assess the accuracy and reproducibility of the BSCC three-tier quantitative definitions, to investigate if they were applicable to liquid-based cytology (LBC) and to see how they related to the proposed new two-tier BSCC system. METHODS: Quantitative image analysis was undertaken on illustrations from the 1986 BSCC terminology publication and on microscope slides from external quality assessment and Cytology Training Centre teaching sets. RESULTS: Analysis of mean NC ratios showed that mild, moderate and severe dyskaryosis exist as statistically different populations. Overlap of NC ratio ranges, however, limits their practical application in the three-tier model, although interestingly no overlap was noted between mild and severe dyskaryosis. No grade of dyskaryosis had a mean area NC ratio over 50%, indicating that the BSCC quantitative definitions are incorrect. The mean diameter NC ratios for mild, moderate and severe dyskaryosis were found to be 40%, 49% and 66% respectively. Accordingly it is possible that those reporting cervical cytology could be interpreting the BSCC NC ratios as meaning diameter rather than area. Amalgamation of the three-tier results into the proposed two-tier model shows that the resulting mean NC area and diameter ratios identify statistically different low- and high-grade populations. The reduced degree of overlap, however, of NC ratio ranges in the two-tier model implies that NC ratios could have a useful practical role in the separation of the low- and high-grade categories. The two categories were reasonably well separated by mean area and diameter NC ratios of 25% and 50% respectively. A two-tier model combining mild with moderate rather than severe dyskaryosis was found to be a statistically valid alternative but gave rise to NC ratios that would be difficult to use in practice. Except for moderate dyskaryosis, no significant differences were identified between the mean NC ratios of either conventional and LBC preparations or LBC preparations using two different commercial methodologies (SurePath and ThinPrep). Differences, however, were noted in area measurements between SurePath and ThinPrep and this has potential implications for classifications (such as TBS) using area comparisons as their basis. In addition, it was found that the increased NC ratio, associated with higher grades of dyskaryosis is more a consequence of progressive cytoplasmic area reduction rather than nuclear area increase. The similar NC ratios of borderline nuclear changes associated with human papilloma virus and mild dyskaryosis support the BSCC proposal that these can be combined to constitute a low-grade category. This study shows that the BSCC area NC ratio criteria of grading squamous cell dyskaryosis require amendment. In addition, this study supports the new BSCC recommendation of low- and high-grade squamous cell categories. CONCLUSIONS: The study proposes Sheffield quantitative criteria to assist the grading of squamous cell abnormalities. Quantitative diameter NC ratio measurements, however, must always be accompanied by detailed assessment of qualitative morphological features and in particular those relating to nuclear chromatin. This is equally relevant to both two- and three-tier models.     

Significance of bizarre cells in cervical screening liquid‐based cytology: A prospective study of 15 cases

Objective

The aim of this study was to assess the significance of bizarre cells (cells of squamous origin with a superficial squamous cell‐type cytoplasm and characterised by multinucleation that produces bizarre nuclear shapes) in liquid‐based cytology (LBC) Papanicoaou (pap) smears with clinical and histological follow‐up correlation.

Methods

Fifteen patients, all with LBC samples containing bizarre cells, were identified in routine ThinPrep^® LBC workload. HPV testing was performed in each case using residual LBC material. Cytological‐histological correlations were reviewed.

Results

All 15 LBC samples contained bizarre cells and tested positive for high‐risk HPV types. Ten of the 15 cases were identified as atypical squamous cells ‐ cannot exclude an HSIL (ASC‐H) with secondary diagnosis of low‐grade squamous intraepithelial lesion (LSIL), while five cases were identified as high‐grade squamous intraepithelial lesion (HSIL), and a subsequent biopsy was recommended. Additionally, 13/15 cases underwent cone biopsy or hysterectomy within 1‐11 months, of which 10 showed histologically confirmed HSIL end‐points. LSIL was present in three cases. Bizarre cells were identified in the HSIL epithelium of five cone biopsies.

Conclusions

Identification of bizarre cells in LBC is straightforward and may facilitate diagnosis. The cytology of bizarre cells is associated with HSIL in cone biopsies. We recommend assigning LBC samples containing bizarre cells as ASC‐H with secondary diagnosis of LSIL.     

Nuclear grooves in normal and abnormal cervical smears

OBJECTIVE: To determine whether the frequency of nuclear grooves in intermediate squamous cells in cervical smears is related to inflammatory or neoplastic events. STUDY DESIGN: Sixty benign and 40 neoplastic, nonatrophic cervical smears, collected by conventional methods and stained by Papanicolaou stain, were selected for this study. Twenty smears of the benign cohort showed evidence of inflammation. The neoplastic cohort comprised 20 smears representative of low grade and 20 of high grade squamous intraepithelial lesion (LSIL and HSIL, respectively), with 50% in each group showing evidence of inflammation. The patients, of mixed ethnic backgrounds, were between 18 and 45 years of age. The frequency of nuclear grooves in 100 morphologically benign intermediate squamous cells were determined in each case. The results were evaluated by statistical analysis. RESULTS: The study established that the presence of inflammation had no impact on the frequency of nuclear grooves in benign intermediate squamous cells in either benign or neoplastic smears. When compared with benign smears, there was no increase in the frequency of nuclear grooves in LSIL. Smears of HSIL showed the highest frequency of nuclear grooves. The difference between HSIL and other groups was statistically significant (P < .01). CONCLUSION: The frequency of nuclear grooves in either normal or neoplastic smears is unrelated to inflammation. In smears with neoplastic changes, an increase in grooved nuclei occurred in HSIL. The mechanism and significance of this observation are unknown and warrant further study.     

Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US,ASC‐H,LSIL and HSIL)

G. Bigras, J. Wilson, L. Russell, G. Johnson, D. Morel and M. Saddik
Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US, ASC‐H, LSIL and HSIL) Objectives: Given the well‐known poor reproducibility of cervical cytology diagnosis, especially for atypical squamous cells of undetermined significance (ASC‐US) and low‐grade squamous intraepithelial lesion (LSIL), this study surveyed reproducibility in the assessment of individual cytomorphological features. Methods: One hundred and fifty cells or groups of cells, with a variety of morphological appearances, including normal cells, high‐grade squamous intraepithelial lesion (HSIL), LSIL, ASC‐US and ASC cannot exclude HSIL (ASC‐H), were precisely marked on 150 different liquid‐based cytological preparations. They were analysed by 17 observers who assessed 17 cytological features including nuclear features (chromatin texture, nuclear outline, nuclear shape, etc.), cytoplasmic features (cell shape, cytoplasmic staining, cytoplasmic clearing, etc.) and group characteristics (nuclear polarity, cellular density, etc.). A total of 43 350 data scores were collected in a database using a web‐based survey. Kendall’s W and relative entropy indexes were utilized to compute concordance indexes of respectively ordinal and nominal variables. Results: Nuclear features have significantly lower reproducibility (0.46) compared with other cytological features (0.59). The feature with least agreement is assessment of chromatin texture. A small but significant difference in concordance was found between two subsets of observers with different levels of experience. Conclusion: Most previous studies assessing reproducibility of cytological diagnoses show, at best, moderate reproducibility among observers. This study focused on agreement regarding the presence of constituent morphological features used to recognize dyskaryosis and various grades of squamous intraepithelial lesions. A map of reproducibility indexes is presented that highlights, for daily practice or teaching, the robustness of features used for cytological assessment, recognizing that diagnosis is always based on a combination of features.     

Is a liquid‐based cytology more sensitive than a conventional Pap smear?

K. Sigurdsson
Is a liquid‐based cytology more sensitive than a conventional Pap smear? Background: The comparative sensitivity of liquid‐based cytology (LBC) test and conventional Papanicolaou (Pap) smears is controversial. Material and methods: This study analyses the distribution of cytology, histology, colposcopy and large loop excision of the transformation zone among women screened in Iceland with LBC at the Cancer Detection Clinic in Reykjavik and with a conventional Pap smear outside the Detection Clinic in 2007–2011. The study material included 42 654 LBC tests from 20 439 women and 103 909 Pap smears from 61 574 women. The period 2000–2004 is used to correct for potential bias as a result of unequal distribution of the studied parameters between the study sites before the introduction of LBC. Results: The observed results indicated that women screened with an LBC sample had significantly decreased detection rates of inadequate smears, increased detection of low‐grade squamous intraepithelial lesion (LSIL)/atypical cytology and referrals to colposcopy, and an increased detection rate of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) irrespective of age. LBC increased significantly the detection rates of high‐grade squamous intraepithelial lesion or worse (HSIL+) cytology and CIN3+ histology only in women under 40 years of age. Taking into consideration the unequal prevalence of the studied parameters between the study sites in 2000–2004 indicated, however, that LBC only affected the rate of inadequate and low‐grade cytology tests under the age of 40 years. Positive predictive values for CIN2+ were not significantly different between the tests. Conclusions: The study results support the view that LBC is no more sensitive than Pap smears for the detection of HSIL+ and CIN2+ irrespective of age. LBC decreased the rate of inadequate smears, but increased the rate of low‐grade cytology under the age of 40 years and decreased the total rate of abnormal smears over the age of 40 years.     

Utility of p16(ink4a) immunocytochemistry in liquid-based cytology specimens from women treated for high-grade squamous intraepithelial lesions

OBJECTIVE: To examine whether p16(ink4a) immunocytochemical (ICC) expression detected intraepithelial disease in liquid-based cytology (LBC) specimens from women with high-grade squamous intraepithelial lesions (HSIL), whose specimen was labeled negative for intraepithelial lesion or malignany (NILM). STUDY DESIGN: Residual LBC specimens from women treated for HSIL (n = 21), whose LBC test was interpreted as NILM including marked benign inflammatory changes (BCC) were used. The control (n = 25) consisted of residual LBC specimens from women with documented HSIL. ICC for p16p(16k4a) was performed on a second ThinPrep (ThinPrep 2000, Cylyl Corporation, Boxborough, Massachusetts, U.S.A.) preparation; the percentage ofpositive cells and intensity of immunostaining were recorded. Standard LBC preparations for p16(ink4a) ICC-positive and ICC-negative control cases were reviewed. RESULTS: Twenty-four of 25 (96%) of the HSIL control group were ICC p16(ink4a) positive. In the NILM/BCC group, 2 of 21 with adequate LBC residua were ICC p16(ink4a) positive; on review both were reclassified as epithelial abnormality--1 HSIL and 1 atypical squamous cells cannot exclude HSIL. In both, subsequent colposcopic biopsy yielded HSIL. CONCLUSION: p16(ink4a) ICC positivity on NILM/BCC LBC residua from patients with HSIL may identify cases that merit cytologic review and possible reclassification. The utility of p16(ink4a) ICC in this situation requires further study.     

Measurement of subvisual changes in cervical squamous metaplastic cells for detecting abnormality.

Quantitative measures of visually normal squamous metaplastic cells exfoliated from the uterine cervix were obtained to test the hypothesis that these cells, like intermediate squamous and endocervical columnar cells, show subvisual evidence of atypia in cases of bonafide squamous intraepithelial neoplasia. The cells identified as squamous metaplastic were obtained from 14 abnormal (dysplastic) and 9 diagnostically negative cases. Although the cell populations so grouped showed no statistically significant differences in overall cell size, nuclear area or nuclear/cytoplasmic ratios, there were significant differences in nuclear and cytoplasmic densitometric features and in nuclear texture features. A combination of three features (nuclear density, texture and cytoplasmic density) permitted 76% of the cells to be categorized correctly as originating in normal versus abnormal slides. It is concluded that selected quantitative features of exfoliated metaplastic cell populations may contribute to improved diagnostic accuracy in automated screening for cervical abnormalities.     

Usefulness of AgNOR technique and CEA expression in atypical metaplastic cells from cervical smears

OBJECTIVE: To evaluate the proliferating capacity of atypical metaplastic cells in cervical smears by AgNOR technique and image analysis and investigate the probable relation with squamous intraepithelial lesions (SIL) using immunocytochemical assay for carcinoembryonic antigen (CEA). STUDY DESIGN: Eight cervical smears were stained with Papanicolaou stain for diagnosis of atypical metaplastic cells. After removal of the stain the smears were processed with a silver colloidal solution and the AgNOR area determined by image cytometry. Differences in the mean AgNOR protein area between reactive metaplastic cells and controls were tested by Student's t test. The CEA was investigated by immunocytochemical staining in smears with atypical metaplastic cells and smears from high-grade squamous intraepithelial lesions (HSIL). RESULTS: The mean AgNOR areas from atypical metaplastic cells (4.55, 6.66, 4.68, 5.30, 4.97, 6.20, 6.28, and 7.35) were significantly greater those that of intermediate metaplastic cells and cells from low-grade squamous intraepithelial lesions (LSIL) (0.77, 0.99, and 0.82, respectively). The atypical metaplastic cells showed values of mean AgNOR area intermediate between that of basal cells (3.28) and HSIL cells (7.73) or neoplastic cells (16.12). The CEA was strongly expressed by the atypical metaplastic cells. CONCLUSION: The expression of CEA in the atypical metaplastic cells underlies the probable relation to SIL. Although the organizer region areas raised high values, it would be necessary to use a greater number of cases to define whether the AgNOR area is indicative of the proliferative activity of the atypical metaplastic cells.     

Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions

D. Demirel, N. Akyürek and I. Ramzy
Diagnostic and prognostic significance of image cytometric DNA ploidy measurement in cytological samples of cervical squamous intraepithelial lesions Objective: To study the DNA ploidy pattern of uterine cervical squamous intraepithelial lesions (SILs) and its diagnostic and prognostic significance. Methods: The study included 31 cases of SIL: 11 low‐grade (LSIL) and 20 high‐grade (HSIL). Feulgen–pararosaniline staining was performed on previously Papanicolaou‐stained smears and a DNA image cytometric study was performed. An internal reference was used to calibrate the samples. Results: All 31 cases of SIL, either LSIL or HSIL, were non‐diploid. Of the 11 cases of LSIL, four were tetraploid and seven were aneuploid, whereas, of the 20 cases of HSIL, four were tetraploid and 16 were aneuploid. Stemline aneuploidy was not a significant discriminator between LSIL and HSIL (P = 0.32). Based on single‐cell analysis, HSIL cases had significantly higher DNA content than LSIL cases (P < 0.01). When a mean of 30% or more was used for the 6c‐exceeding event (6cEE) value, the sensitivity and specificity to indicate HSIL were 83% and 64%, respectively, with a positive predictive value (PPV) of 81% and negative predictive value (NPV) of 65%. All HSIL cases were cervical intraepithelial neoplasia grade 2 or worse (CIN2+) on biopsy. In addition, cases which showed recurrence had more DNA content by single‐cell analysis than those with an indolent clinical behaviour: P = 0.04 and P = 0.03 for LSIL and HSIL, respectively. Conclusions: Image cytometric DNA analysis is a useful technique for diagnostic and prognostic purposes in uterine cervical SIL when appropriate ‘c’ values are used in single‐cell analysis. We propose that a >6c DNA content of 30% is useful as a cut‐off level for predicting cases with CIN2+ in DNA image cytometry of cervical smears.     

O‐12BLAND DYSKARYOSIS: A NEW PITFALL IN THINPREP® LIQUID BASED CYTOLOGY

Liquid based cytology (LBC) has improved cell visualization and preservation in cervical cytology. There has been a reduction in inadequate rate and some data to suggest an increase in sensitivity for dyskaryosis. Training for LBC has focused on differences in distribution of abnormal cells, but in most cases the morphological appearance of the dyskaryotic cells themselves is similar to that seen in conventional cytology. We are describing a new presentation of dyskaryosis which may be a cause of false negative cytology. We have referred to this as ‘Bland dyskaryosis’ because cells appear deceptively bland on low power examination, and can be misinterpreted as metaplastic or endocervical cells. Bland dyskaryosis cells are seen in groups. The architecture of the group is very disorganized, and adjacent cells show variation in size. Cells have a high nuclear/cytoplasmic ratio and smooth nuclear membranes. Chromatin is finely granular and evenly distributed. This is an unusual presentation of high‐grade dyskaryosis and we feel that there is a learning curve in laboratories converting to liquid based cytology. The spectrum of appearances of squamous dyskaryosis needs to be delineated to allow further increases in sensitivity for dyskaryosis.     

Variables associated with squamous intraepithelial lesions among women with a cytologic diagnosis of atypical squamous cells of undetermined significance.

OBJECTIVE: To identify factors among women with atypical squamous cells of undetermined significance (ASCUS) cytologic diagnosis that are associated with either low grade squamous intraepithelial lesions (LSILs) or high grade squamous intraepithelial lesions (HSILs). STUDY DESIGN: From January 1992 to June 1995, 1,660 women with a diagnosis of ASCUS were followed until the next follow-up appointment after the ASCUS diagnosis. Logistic regression analysis with either LSIL or HSIL at follow-up examination as a dependent variable was done with the following independent variables: patient age, method of follow-up, follow-up time, type of health care coverage and household income. RESULTS: In multivariate analyses, there was a 2.7-fold increase in the odds of LSIL (OR = 2.7, 95% CI = 1.3-5.8) for young women (< or = 25 years), a 2-fold decrease in the odds of LSIL (OR = .5, 95% CI .3-.9) for long time to follow-up (> 18 weeks after ASCUS diagnosis) and an 7.8-fold increase in the odds of LSIL (OR = 7.8, 95% CI = 5.1-11.9) for follow-up by colposcopic biopsy. For the odds of HSIL, there was a 6.8-fold increase (95% CI = 3.2-14.5) for follow-up by colposcopic biopsy as compared to Pap smears and a 3-fold decrease in the odds of HSIL (OR = 3, 95% CI = .1-.7) for high-income women (> $34,857). CONCLUSION: Our findings suggest that among ASCUS patients, younger women and those with shorter follow-up time are more likely to be diagnosed with LSIL. Women with a high income showed a protective association for HSIL. Colposcopic biopsy identified more LSIL and HSIL as compared to repeat Pap smear.     

Cervical squamous intraepithelial lesions and associated cervical infections in an HIV‐positive population in Rural Mpumalanga,South Africa

P. J. Swanepoel, P. Michelow, R. Du Plessis, I. G. Proudfoot, G. A. Tarr, S. L. Bockel, C. J. Swanepoel
Cervical squamous intraepithelial lesions and associated cervical infections in an HIV‐positive population in Rural Mpumalanga, South Africa Background: The incidences of genital human papillomavirus (HPV) infection, associated squamous intraepithelial lesions and cervical squamous cell carcinoma are significantly increased in HIV‐positive women. The role of other cervicovaginal infections in the acquisition of the HPV infection, cervical carcinogenesis and genital HIV infection remains largely speculative. Methods: A retrospective study was conducted including 1087 HIV‐positive women in rural Mpumalanga province, South Africa, for the period 1 May 2009 to 31 August 2010. For each patient, the age at first presentation, cervical cytological diagnosis, subsequent follow‐up cytology and histology, and microscopically visible infections (including endemic Bilharzia) were tabulated and statistically analysed. Results: The prevalence of low‐grade squamous intraepithelial lesion (LSIL), high‐grade squamous intraepithelial lesion (HSIL), squamous cell carcinoma, atypical squamous cells of undetermined significance (ASC‐US) and atypical squamous cells, cannot exclude HSIL (ASC‐H) in the study population were 22.1%, 30.9%, 0.6%, 13.5% and 4.0%, respectively. LSIL, HSIL and squamous cell carcinoma were diagnosed, respectively, at the average ages of 35.7, 37.9 and 37.2 years. Four patients with cervical intraepithelial neoplasia grade 1 (CIN1), 32 with CIN2/CIN3 and two with cervical squamous cell carcinoma were also diagnosed with Bilharzia. Of the other infections only bacterial vaginosis had a positive statistical correlation with HPV‐induced cervical abnormalities (LSIL, HSIL or squamous cell carcinoma). Conclusion: This study confirms the high prevalence of progressive HPV‐associated cervical disease in a rural Southern African HIV‐positive population, which is at least equal to or worse than in other African HIV‐positive studies. The high incidence of Bilharzia infection in those cases that underwent cervical cone excision suggests a possible relationship with progressive HPV disease and cervical carcinogenesis. Bacterial vaginosis (perhaps in combination with Bilharzia) may compromise the normal barriers against HPV and HIV infection.     

Interobserver variability in cytologic subclassification of squamous intraepithelial lesions--the Bethesda System vs. World Health Organization classification

The aim of the study was to compare interobserver variability for The Bethesda System (TBS) and World Health Organization (WHO) classification of cervical squamous intraepithelial lesions. A total of 1,000 conventional Papanicolaou smears (156 positive and 884 negative) were examined "blindly" by three cytologists and one cytotechnician. The degree of observer agreement was expressed by kappa statistics using a program for the calculation of interobserver variation and association "Agree" (Svanholm and Jergensen, 1989). Kappa (kappa) was determined for each cytologic diagnosis within a particular classification and total for either classification. The association with and separation from other diagnoses was determined for each cytologic diagnosis in the form of conditional probability (P(j)). In WHO classification, the diagnoses of dysplasia media and dysplasia gravis showed poor reproducibility (kappa = 0.114 and kappa = 0.259, respectively), the diagnosis of dysplasia levis good reproducibility (kappa = 0.639), and the diagnosis of carcinoma in situ excellent reproducibility (kappa = 0.762). WHO classification yielded pool kappa of 0.741. In TBS classification, the diagnosis of LSIL showed good, and HSIL excellent reproducibility (kappa = 0.542 and kappa = 0.763, respectively). TBS classification yielded pool kappa of 0.699. Dysplasia media (P(j) = 0.121) and dysplasia gravis (P(j) = 0.274) were found to be morphologically poorly defined, and carcinoma in situ (P(j) = 0.777) and dysplasia levis (P(j) = 0.651) well defined diagnoses. LSIL was morphologically moderately defined (P(j) = 0.587) and HSIL well defined (P(j) = 0.789) diagnosis. Accordingly, TBS does not substantially improve diagnostic reproducibility of the cytologic diagnoses of squamous intraepithelial lesions, while providing considerably less information to the clinician than the four-grade dysplasia/CIS terminology, thus eliminating the opportunity of choosing a different procedure for the diagnosis of dysplasia media, which is of utmost importance in the population of young nulliparae.     

Comparison of manual and automated methods of liquid-based cytology. A morphologic study

OBJECTIVE: To evaluate the morphologic characteristics of gynecologic samples prepared by 3 different methods of liquid-based cytology. STUDY DESIGN: Cytologic samples from representative cases of each diagnostic category of squamous epithelial lesion, prepared by automated and manual liquid-based systems, were analyzed by 3 laboratories in the United States, Portugal and Brazil. The systems included: ThinPrep (automated, U.S. Food and Drug Administration approved; Cytyc Corp., Boxborough, Massachusetts, U.S.A.), Autocyte PREP (South American system, manual; TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.) and DNACITOLIQ (manual; Digene Brazil, S?o Paulo, Brazil). A panel of 16 morphologic parameters was evaluated: cellularity, clean background, uniform distribution, artifacts, cellular overlapping, architectural and cytoplasmic distortion, cytoplasmic vacuolization, cellular elongation, imprecise cytoplasmic borders, folded cytoplasmic borders, nuclear hyperchromasia, coarse chromatin, prominent nucleolus, irregular nuclear borders, atypical mitosis and inflammatory infiltrate. Negative, atypical squamous cells of undetermined significance, low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) cases were included. Cases without biopsies were confirmed by consensus. RESULTS: Cellularity was adequate in all samples. Clean background was observed in the vast majority of samples with all liquid-based systems. Uniform distribution was frequently found in ThinPrep and Autocyte PREP samples but not in DNACITOLIQ. Artifacts were not present in DNACITOLIQ samples, rare in ThinPrep and observed in 8 (34.7%) Autocyte PREP. Cellular overlapping was observed in all 3 system samples: 11 (31.42%) cases in ThinPrep, 16 (69.56%) in Autocyte PREP and 17 (68%) in DNACITOLIQ System. Architectural and cytoplasmic distortion were present in 3 cases of HSIL (13%) and cytoplasmic vacuolization in 2 cases of LSIL and 1 HSIL of Autocyte PREP. Cellular elongation was found in 13 (56.5%) Autocyte PREP and in 5 (20%) DNACITOLIQ samples. Inflammatory infiltrate was found in all 3 system samples but with more frequency in the Autocyte PREP (69.56%) and DNACITOLIQ System (72%). CONCLUSION: This study clearly indicated that in spite of the different methodologies, the 3 methods adequately preserved cellular structure for morphologic evaluation. The choice of method will depend on price, availability and procedures involved.     

Effect of cellularity on the sensitivity of detecting squamous lesions in liquid-based cervical cytology

OBJECTIVE: To evaluate the effect of cellularity on the sensitivity of both screening and diagnosis in a liquid-based cervical sample. STUDY DESIGN: SurePath samples (TriPath Imaging Inc., Burlington, North Carolina, U.S.A.) with known diagnoses were selected, including 18 negative, 16 low grade squamous intraepithelial lesion (LSIL) and 12 high grade squamous intraepithelial lesion (HSIL) cases. Through a serial dilution technique, samples of varying cellularity were prepared. The 275 slides were assigned random numbers and were routinely screened by 1 of 2 senior cytotechnologists, blinded to the reference diagnosis. Specimens with a screening diagnosis of atypical squamous cells of undetermined significance (ASCUS) or higher were reviewed by two pathologists, resulting in a final consensus diagnosis. Using a grid counting system, cellularity was determined for each slide. RESULTS: There was a clear demarcation in sensitivity between specimens with a cellularity of < 5,000 or > or = 5,000 squamous cells. This applied to both the sensitivity for screening and to the final consensus diagnosis. For cases with a reference diagnosis of LSIL+, at a cytotechnologist screening level of ASCUS or greater, sensitivity increased from 72.8% (< 5,000 cells) to 98.1% (> or = 5,000 cells) and for a reference diagnosis of HSIL from 85.7% to 100%, respectively. Similarly, for the consensus diagnosis, sensitivity rose from 78.5% (< 5,000 cells) to 96.6% (> or = 5,000 cells) for LSIL+ and from 82.9% to 100%, respectively, for HSIL. These differences were statistically significant (P < .001). CONCLUSION: A minimum cellularity of 5,000 squamous cells is recommended for SurePath liquid-based cervical preparations.     

Using a quality control approach to define an 'adequately cellular' liquid-based cervical cytology specimen

Objective:  To define a minimum acceptable total squamous cellularity for (ThinPrep^®) liquid-based cervical cytology (LBC) specimens using quality control techniques.
Methods:  Two hundred LBC preparations were made containing varying numbers (<200) of severely dyskaryotic squamous cells and with varying total cellularities.
Results:  Ninety-eight per cent of the LBC preparations that were missed by one or more of three cytoscreeners had fewer than 16 abnormal objects (single dyskaryotic cells or clumps of cells) and 87 dyskaryotic cells. The minimum ratio of dyskaryotic to total squamous cells that, in a preparation of 5000 squamous cells has a probability of at least 0.98 that 87 or more dyskaryotic cells will be present is 1 : 47. Twenty-three preparations diagnosed as abnormal had ratios of dyskaryotic to total squamous cells of between 1 : 2.5 and 1 : 4596. There is thus no feasible minimum acceptable squamous cellularity that will give an acceptable probability of detection of all specimen vials containing abnormal cells in the observed proportions.
Conclusions:  It is suggested that the minimum acceptable cellularity for LBC specimens is set pragmatically by the screening programme to give a feasible percentage of repeat tests.     

Image analysis of peripheral compression artefacts of ThinPrep® liquid‐based cytology preparations

J. Choi, H. S. Shim, J.‐W. Song, S. W. Chae, Y.‐N. Lee, J. E. Kim and S. H. Kim
Image analysis of peripheral compression artefacts of ThinPrep^® liquid‐based cytology preparations Objective: ThinPrep (TP), one of the Food and Drug Administration‐approved liquid‐based cytology (LBC) preparations, is widely used for gynaecological and non‐gynaecological cytology samples. A unique physical artefact caused by the compression at the periphery in TP slides has not been adequately evaluated to date. Methods: We processed four established tumour cell lines (MKN28, MKN45, KG‐1 and NB4) and mononuclear cells isolated from whole blood over Ficoll‐Plaque for TP preparations. For this part of the study, we included five normal cervical LBC preparations. We then auto‐counted and auto‐measured the area, mean grey value and Feret’s diameter in both the inner disc and peripheral rim of the preparations by image morphometry. In addition, we compared the distribution of atypical cell groups in the peripheral rim and inner disc of 132 lung aspirates, 80 thyroid aspirates, 212 cerebrospinal fluids (CSFs) and 50 gynaecological samples. Results: The areas and Feret’s diameters of the cytoplasm in the peripheral compressed rim area were statistically larger than those of cells in the inner disc. The mean grey values of cells (cytoplasm and nucleus) in the peripheral compression rim were also smaller than those in the inner disc cells, leading to decreases in nuclear and cytoplasmic chromatism. Except for the mean grey values, the differences were not significant in the cervical samples. Conclusions: Cellular morphology may be markedly distorted in the peripheral rim, regardless of cell malignancy, which may lead to the misinterpretation of cells during the screening. Accordingly, cytological diagnosis based on the findings within the peripheral rim should take this phenomenon into account. Compressed cells found in the peripheral rim should be interpreted with caution when TP slides are used for cytopathological diagnosis.     

The advantages of incorporating liquid-based cytology (TACAS™) in mass screening for cervical cancer

We incorporated liquid-based cytology (LBC) in population-based screening for cervical cancer. The usefulness of using LBC in mass screening for cervical cancer was examined. From 2009 to 2014, 157,061 individuals underwent mass screening for cervical cancer in Aomori Prefecture. From 2009 to 2011, cells were collected from 82,218 individuals and the specimens were conventionally prepared (CP). From 2012 to 2014, cells were collected from 74,843 individuals and the specimens were prepared using LBC (TACAS?). Cytology results for the 2 sets of specimens were compared and differences in cytologic features were examined. ASC-US and more severe lesions were detected at a rate of 1.13 % by CP and 1.44 % by LBC, so LBC had a 1.3-fold higher rate of detection. LBC had a 1.6-fold higher rate of LSIL detection and a 1.2-fold higher rate of HSIL detection. CP detected cancer in 20 cases at a rate of 0.024 % while LBC detected cancer in 18 cases at a rate of 0.024 %. Cytodiagnosis of the 18 cases of SCC that LBC identified revealed that 7 were SCC, 8 were HSIL, and 3 were ASC-H. Atypical cells tended to be smaller with TACAS?. LBC reduced the time needed for microscopic examination of a single specimen by 42 % in comparison to CP. LBC using TACAS? allowed the detection of slight lesions and slight changes in cells. LBC can lessen the burden on medical personnel and may lead to improved accuracy.     

Follow-up study of atypical glandular cells in gynecologic cytology using conventional Pap smears and liquid-based preparations: impact of the Bethesda System 2001

OBJECTIVE: This study evaluated the impact of the Bethesda System (TBS) 2001 in reporting of atypical glandular cells (AGC) when using conventional Pap smears (CS) and liquid-based cytology preparations (LBC). STUDY DESIGN: Follow-up information for all atypical glandular cells of undetermined significance (AGUS)/ AGC cases encountered in Queen Mary Hospital from July 2000 to June 2004 was analyzed. The difference in percentages associated with certain end points when using different reporting systems and preparation methods were compared. The age trends and time interval between cytologic diagnosis and detection of positive end points were studied. RESULTS: More than half of these cases turned out to be "negative." The majority with "negative" end points belonged to the "not otherwise specified" (NOS) groups (including atypical endometrial cells) in TBS 2001. The connotation of "favor neoplastic" carried a high positive predictive value for significant lesions. Most of the significant outcomes were discovered within the subsequent 6 months. A decreased reporting of "AGC, NOS" and an increased reporting of "atypical endocervical cells, NOS" were noted when using LBC. CONCLUSION: Subcategorization of AGC in TBS 2001 according to cellular origin and risk of malignancy, which is further enhanced by application of LBC, is useful.     

O-13ENDOMETRIAL CARCINOMA DETECTED WITH SUREPATH LIQUID BASED CERVICAL CYTOLOGY: COMPARISON WITH CONVENTIONAL CERVICAL CYTOLOGY

Liquid based cytology (LBC) has improved cell visualization and preservation in cervical cytology. There has been a reduction in inadequate rate and some data to suggest an increase in sensitivity for dyskaryosis. Training for LBC has focused on differences in distribution of abnormal cells, but in most cases the morphological appearance of the dyskaryotic cells themselves is similar to that seen in conventional cytology. We are describing a new presentation of dyskaryosis which may be a cause of false negative cytology. We have referred to this as 'Bland dyskaryosis' because cells appear deceptively bland on low power examination, and can be misinterpreted as metaplastic or endocervical cells. Bland dyskaryosis cells are seen in groups. The architecture of the group is very disorganized, and adjacent cells show variation in size. Cells have a high nuclear/cytoplasmic ratio and smooth nuclear membranes. Chromatin is finely granular and evenly distributed. This is an unusual presentation of high-grade dyskaryosis and we feel that there is a learning curve in laboratories converting to liquid based cytology. The spectrum of appearances of squamous dyskaryosis needs to be delineated to allow further increases in sensitivity for dyskaryosis.