Proficiency testing in cytology laboratories in Ontario, Canada: a decade of experience. III. A precision study of consistency and reproducibility in cytology reporting

The Ontario Laboratory Proficiency Testing Program's testing model and the results of early surveys of cytology laboratories have been previously described. To test consistency and accuracy, the same slides from one survey were recirculated to the same laboratory in the next survey. There was no statistical difference in either the accuracy or consistency in reporting by the various categories of personnel. Analysis of the data by category of diagnosis showed the reporting of "no abnormal cells" and "benign atypia" to be slightly more accurate and "moderate dysplasia" and "severe dysplasia" to be slightly less accurate than the reporting in other categories. Consistency in reporting appeared to be slightly less accurate in severe dysplasia than in the other categories. The analysis, which includes the results from all licensed cytology laboratories in Ontario, provides evidence that, in the reporting of cervical cytologic material, it is feasible to differentiate among the various categories within the spectrum of squamous epithelial abnormalities with a general level of accuracy and consistency that, although satisfactory, is not yet ideal.     

Prediction of cervical neoplasia diagnosis groups. Discriminant analysis on digitized cell images

The purpose of this study was to develop discriminant analysis models for predicting cervical dysplasia/neoplasia case diagnoses using cytometric features derived from the digital image analysis of cell monolayers. The data base consisted of 925 cells from 27 cases diagnosed either as moderate dysplasia (n = 10), severe dysplasia (n = 5), carcinoma in situ (n = 8) or invasive carcinoma (n = 4) on both tissue biopsy and monolayer preparations. Cell features examined were cell diameter, nuclear diameter, nuclear mean optical density (OD), nuclear integrated OD (IOD), nuclear OD standard deviation, normalized IOD, nuclear texture and nuclear-cytoplasmic ratio. Features derived from cells visually classified as moderate dysplasia correctly predicted the case diagnosis of moderate dysplasia versus more severe disease for 85% of the cells. Prediction models using summary measures (mean and variance) derived from all visually classified abnormal cells within each case correctly separated all cases into their respective diagnostic categories. These findings suggest that dysplastic cells in a cytologic sample have features that collectively reflect the tissue diagnosis, regardless of the visual differences among the cells. Such information has potential use for diagnosis and possibly for prognosis.     

Karyometric image analysis for intraepithelial and invasive cervical lesions

OBJECTIVE: To derive an objective, numeric measure for the progression of intraepithelial and invasive squamous cell cervical lesions. STUDY DESIGN: Thin-layer cervical cytology preparations from colposcopically confirmed normal cervix, low grade squamous intraepithelial lesions, high grade squamous intraepithelial lesions and carcinoma were identified from a cross-sectional study. Fifty-nine cases representing 4 diagnostic categories were selected, and 2,375 nuclei from epithelial cells representative of the diagnostic category were randomly selected for imaging and measurement from these cases. Additionally, 1,378 visually normal appearing intermediate cells from low and high grade squamous intraepithelial lesions, as well as from carcinoma cases, were identified for analysis. The nuclei were quantitatively characterized, and discriminant analyses were performed to derive a progression curve from normal cytology to carcinoma. RESULTS: The lesion signatures show a clear increase in nuclear abnormality with increasing progression. A progression curve was derived based on mean discriminant function scores for each diagnostic category and on the mean nuclear abnormality values for the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. CONCLUSION: A numeric assessment of lesion progression for cervical precancerous and cancerous lesions based on karyometric measurements is possible and may provide an objective, precise characterization of each lesion as well as a basis for improved performance in automated cytology-based cervical cancer screening.     

Canonical analysis of cells in normal and abnormal cervical smears

Over 4,000 cells from 105 normal and 96 abnormal uterine cervical scrapes were prepared according to the UCLA monolayer procedure, stained by a routine Papanicolaou method and visually classified by two cytopathologists and a technologist into seven classes: parabasal, metaplastic, mild dysplasia, moderate dysplasia, severe dysplasia, carcinoma in situ and invasive carcinoma. Canonical analysis was used to correlate effects-coded class membership variables with 23 cell features derived from digital image analysis. In general, nuclear texture measures derived from linear combinations of run-length correlations along with features derived from a Markov transitional probability matrix provided the best predictors of cell class. After cells were divided into benign (moderate dysplasia or less) and malignant (severe dysplasia or worse) groups, discriminant analysis correctly classified 84% of the benign cells and 91% of the malignant cells.     

Evaluation of contextual analysis for computer classification of cervical smears

A procedure for automated analysis of cervical smears has been implemented in an image cytometry system. Smears are described exclusively in terms of global and contextual information extracted by pattern-recognition algorithms and represented by a vector of proportions of cellular object types. Linear discriminant functions, based on a Fisher criterion, are derived to classify smears with a cross-section of diagnoses into two broad categories, normal and abnormal. Results obtained from 83 smears indicate 78% correct classification. In contrast to most automated systems, good classification results were obtained in normal smears with benign changes caused by inflammation and with postmenopausal atrophia and in abnormals with mild dysplasia. These findings suggest that contextual analysis may be sensitive to subtle changes in cellular morphology and to progressive patterns of dysplasia. When used with standard isolated cell analysis, contextual analysis may provide additional complementary information for automated cervical prescreening.     

Diagnostic significance of "severe dysplasia" in sputum cytology

The diagnostic significance of a cytologic diagnosis of "severe dysplasia" on sputum samples was assessed. In a group of 46 patients with diagnoses of severe dysplasia, follow-up showed no malignancy of the lung in 25 patients (54%) and a malignant process in 21 patients (46%). These groups were compared to 52 patients with correct negative and 202 patients with correct positive sputum diagnoses. Of the patient characteristics investigated, age, previous sputum production, vital capacity and low forced expiratory volume were not significantly related to a sputum cytodiagnosis of severe dysplasia. In contrast, severe dyspnea showed a significantly higher frequency in patients with a sputum cytodiagnosis of severe dysplasia, but without an underlying malignant lung process. Follow-up disclosed a malignant tumor in 10 of 13 patients with disease; the presence of severely dysplastic cells in sputum specimens from such patients should be considered a warning signal for an underlying malignant lung process. Since severe dysplasia should be considered a premalignant epithelial lesion, patients with sputum cytodiagnoses of severe dysplasia should undergo bronchoscopy, with multiple bronchial brushings of all areas showing suspicious mucosal changes, together with segmental bronchial washings. In case a malignant process cannot be located, sputum examinations should be repeated at three-month intervals.     

Retrospective evaluation of gynecologic cytodiagnosis. II. Interlaboratory reproducibility as shown in rescreening large consecutive samples of reported cases

Two laboratories exchanged and rescreened a large sample of cases with cervicovaginal smears they had consecutively accessioned to examine the reproducibility of gynecologic cytodiagnosis under optimum conditions. At least a "working agreement" (diagnoses within +/- 1 category on a ten-category scale) was achieved in diagnoses of normal, benign reaction and squamous abnormality (from minimal dysplasia though invasive cancer) in 18,859 cases (96.8%), of endometrial abnormality in 21 cases (42%) and of "unsatisfactory" in 99 cases (20.7%). Larger differences occurred in greater than or equal to 30% of cases except in the categories of "normal" and "benign reaction," reaching a maximum of 82% for moderate dysplasia. Reexamining 382 cases decreased disagreement by category to the 20% to 65% range only in the five categories of dysplasia plus carcinoma in situ. Agreement was not predicated on the presence of endocervical cells or squamous metaplasia; the basis for "unsatisfactory" calls was not uniform. Comparison of the laboratories' diagnoses with referee diagnoses or, on 178 cases, with tissue diagnoses also demonstrated differences in diagnostic criteria.     

Proficiency testing in cytology laboratories in Ontario, Canada: a decade of experience. II. Results of initial surveys

The results of the initial surveys in the cytology proficiency testing of the medical laboratories in the Province of Ontario, Canada, showed a high correlation between the opinions of the testing committee and the participants in the categories of "no abnormal cells," "metaplasia" and forms of "benign atypia." The proportion of times that slides were tested in the categories of dysplasia and malignancy in the surveys increased from 38% by the end of survey 3 to 46% by the end of survey 5. A progressive improvement in the diagnostic accuracy was demonstrated in the categories of malignancy and severe dysplasia while results were more variable in the categories of moderate and mild dysplasia. Several educational activities were initiated following survey 3, including development and circulation of demonstration sets of marked glass slides for repeated circulation to participants as well as copies of a slide/tape presentation describing the program and specific case material.     

Marker features for malignancy in ectocervical cells

Marker features for malignancy have recently been observed in ectocervical cells, even in cells that are visually normal in appearance. This study assessed the statistical significance of these marker features using a mixed-model nested-design analysis of variance (ANOVA). Features in blue intermediate cells from patients with normal cytology, moderate dysplasia, and severe dysplasia/carcinomain situ, nonkeratinizing cells from patients with moderate dysplasia, severe dysplasia/carcinomain situ, and invasive cancer, and dysplastic cells from areas of metaplasia from patients with moderate dysplasia, severe dysplasia/carcinomain situ, and invasive cancer were tested. ANOVA clearly demonstrated that the marker features differentiate between cells of the same cell type originating from patients in different diagnostic categories. In every instance, the differences owing to the diagnostic category were statistically significantly greater than those caused by patient-to-patient variability. Although the discriminating marker features in the intermediate cells were almost exclusively spectral features reflecting staining differences, morphometric features were also marker features in the dysplastic cells.     

The use of an automated image cytometer for screening and quantitative assessment of cervical lesions in the British Columbia Cervical Smear Screening Programme

The use of an automated image cytometer for screeing and quantitative assessment of cervical lesions in the British Columbia Cervical Smear Screening Programme
The development of an automated device to screen cervical cytology slides for the detection of pre-invasive lesions of the cervix has been the goal of many individuals for over 30 years. The increasing sophistication of the technology of automation and increasingly powerful computer technology have enabled a number of these systems to reach the stage at which they have become a practical reality. The Department of Cancer Imaging at the British Columbia Cancer Agency has developed such a device over the past few years. This study reports the preliminary results of a trial to determine the reliability of the device for the screening and quantitative assessment of cervical cells. A training set of over 1000 cervical slides was used to train the image cytometer. A test set of 1030 slides was screened by the image cytometer and in the Cytology Screening Laboratory. At the 50% sample split the sensitivity of the image cytometer was 95% for severe dysplasia and 90% for moderate dysplasia, compared with a sensitivity of 90% for both of these lesions using conventional screening. A combination of nuclear texture features was found which can be used for the quantitative assessment of both abnormal cells and apparently normal intermediate cells.     

Marker features for malignancy in ectocervical cells. Statistical evaluation

Marker features for malignancy have recently been observed in ectocervical cells, even in cells that are visually normal in appearance. This study assessed the statistical significance of these marker features using a mixed-model nested-design analysis of variance (ANOVA). Features in blue intermediate cells from patients with normal cytology, moderate dysplasia, and severe dysplasia/carcinoma in situ, nonkeratinizing cells from patients with moderate dysplasia, severe dysplasia/carcinoma in situ, and invasive cancer, and dysplastic cells from areas of metaplasia from patients with moderate dysplasia, severe dysplasia/carcinoma in situ, and invasive cancer were tested. ANOVA clearly demonstrated that the marker features differentiate between cells of the same cell type originating from patients in different diagnostic categories. In every instance, the differences owing to the diagnostic category were statistically significantly greater than those caused by patient-to-patient variability. Although the discriminating marker features in the intermediate cells were almost exclusively spectral features reflecting staining differences, morphometric features were also marker features in the dysplastic cells.     

AgNOR counts in cervical smears under normal and other cytopathologic conditions

OBJECTIVE: To investigate the diagnostic value of AgNOR counts in cervical smears in the process of cervical carcinogenesis and in discriminating the different grades of squamous intraepithelial lesion (SIL). STUDY DESIGN: Silver nitrate staining for AgNOR counts was performed in 50 cervical smears of cytologically diagnosed normal, inflammatory, low grade SIL (LSIL) (mild dysplasia), high grade SIL (HSIL) (moderate and severe dysplasia) and squamous cell carcinoma. The smears were derived from the ongoing routine outpatient cytology screening at Queen Mary's Hospital, Lucknow, India. RESULTS: In normal and inflammatory smears, the number of AgNOR dots varied from 1 to 2, in mild dysplasia from 2 to 4, in moderate dysplasia from 4 to 6 and in severe dysplasia from 6 to 8. Frank cervical carcinoma cases revealed 8-10 dots. Thus, a progressive increase in AgNOR counts was observed when the severity of pathologic lesions increased. Statistical analysis revealed a significant difference in AgNOR counts between normal and inflammatory smears, but it was highly significant between inflammatory and LSIL cases, between LSIL and HSIL, and between severe dysplasia and frank malignancy. CONCLUSION: This study underscored the diagnostic importance of AgNOR counts, especially in discriminating between LSIL and HSIL of the cervix. Another study is under way to assess the potentiality of AgNOR counts as tumor markers in cervical carcinogenesis.     

A Quality Control System Involving Peer Review of Abnormal Cervical Smears

An internal quality control system which is used in the centralized cytology laboratory of a population-based cervical cancer screening programme in Florence is described. It includes a peer review procedure. Abnormal cervical smears are circulated among all the cytologists and a consensus on the final diagnosis is reached. This daily procedure is designed to evaluate the performance of each cytologist and of the laboratory as a whole but can also be considered a valuable training opportunity. During an 18-month period 1197 smears were reviewed by 15 readers using a reporting form with six main categories of reporting (from ‘regative’ to ‘invasive carcinoma’), plus an ‘unsatisfactory’ category. Overall the concordance between the 15 cytologists, assessed using the kappa statistic (range 0.46–0.71; median 0.60), was good. the level of agreement increased when a weighted kappa statistic (range 0.55–0.78; median 0.68) was used. Kappa values were also calculated for specific categories and suggested an increasing concordance with increasing severity of the lesions, the categories of ‘severe dysplasia’ and ‘invasive carcinoma’ showing the highest agreement. the poor results for the ‘moderate dysplasia’ confirmed the need for combining this group with the ‘severe dysplasia’, as proposed in the Bethesda system.     

Cytologic phenomena accompanying uterine cervix electrocoagulation

In the study of cellular and tissue response to electrocoagulation of the uterine cervix, two cytologic phenomena accompanying the delayed healing process were described: the "contact-developed lucid cell" and the "regression field," which were limited to smear samples exhibiting the transitory appearance of abnormal cells after electrocoagulation. While "contact-developed lucid cells," which were firmly attached to abnormal target cell nuclei in a "cell-in-a-cell" pattern, had a variable effect upon subsequent smear scores, the "regression field," which is similar to that described by others during immunologic rejection of kidney transplants, was consistently followed by a shift of the smear score from the dysplasia range to the normal range. We suggest that abnormal cells differentiated during the healing process as well as those present both before and after treatment are subject to an immune rejection induced by uterine cervical electrocoagulation.     

Histopathologic criterion of dysplasia of the uterine cervix and its biological nature.

A histopathologic diagnostic standard for abnormal epithelium of the uterine cervix, based on its biological behavior, has been proposed, and dysplasia and carcinoma in situ have been classified according to this standard. Benign dysplasia by this classification may be considered as a benign lesion, and only the lesion which satisfies the standard for atypical dysplasia should be considered as dysplasia, having a significance as a precursor to cervical cancer. This would be clinically convenient.     

Immunocytochemical staining of cervical smears for the diagnosis of cervical intraepithelial neoplasia

A total of 233 cervical smears were stained by immunocytochemical methods for epithelial membrane antigen (EMA); the findings were compared with those from Papanicolaou-stained smears from the same women. Squamous epithelial cells from normal cervices did not stain, but cells shed from cervices with cervical intraepithelial neoplasia (CIN) did express the EMA marker. Metaplastic cells from normal and abnormal cervices also frequently stained. The results confirm that this marker detects cervical intraepithelial neoplasia in vitro, but its potential use in an automated screening program may be limited by the staining of the metaplastic cells.     

Intermediate cell markers for malignancy. Consistency of expression

Studies of samples from a larger patient population confirmed the consistency of expression of the previously reported markers for malignancy in normal-appearing intermediate cervical cells in samples from patients with abnormal cytology (moderate dysplasia and severe dysplasia/carcinoma in situ). Based on samples of only 30 cells per case, a false-negative rate of 10% to 30% was estimated. The expression of the marker features thus provides a clear indication of uterine abnormal cytology; the lack of expression, however, does not entirely rule out the possibility of uterine abnormalities. The use of larger sample sizes and better staining protocols could further enhance the usefulness of marker feature studies in the prescreening for cancer.     

Atypical squamous cells of undetermined significance: audit and the impact of potential litigation. Retrospective review of 682 cases

For quality assurance purposes, the frequency of 'abnormal' cytological diagnoses of the non-systematic National Cervical Cancer Screening Programme (NCCSP) was evaluated. In 1999, an unexpected high number of Class (Cl) III cases (i.e. atypical squamous cells of undetermined significance) was reported. The cytological and histological results were reviewed in order to detect a possible cause for this threefold increase. The abnormal Papanicolaou (PAP) smears examined by conventional methods from 1 January 1990 to 31 December 2002 were analysed. The smears of 682 cases diagnosed in 1999 with a Cl III category were reviewed in 2000 and correlated with the available histological diagnoses provided by the Central Department of Pathology. Of the 682 Cl III cases, 176 cases (26.1%) had no follow-up, 314 cases (46.0%) had repeat cytology and 192 cases (28.2%) an histological correlate corresponding to 90 (46.9%) benign lesions, 78 (40.6%) squamous intraepithelial lesions, two (1%) invasive cervical cancers (one squamous and one glandular). Twenty-two Cl III cases (11.5%) were histologically within normal limits. Retrospective smear review confirmed 330 Cl III diagnoses (48.3%), 127 cases (18.6%) were recategorized as Cl IIIG (i.e. atypical glandular cells of undetermined significance), 22 cases (3.2%) as Cl IIID (i.e. mild to moderate dysplasia) and six cases (0.9%) as Cl IVa (i.e. severe dysplasia and/or carcinoma in situ). A total of 197 original Cl III cases had to be reclassified in the Cl II category (28.9%), only two cases showing mild and moderate dysplasia on histology. Thus, 195 cases (28.6%) comprised cytological overdiagnoses. The Cl III category being, by definition, a delicate and often subjective diagnosis, all external influences such as pressure of litigation should be avoided to reduce cytological overdiagnoses as a result of an unnecessary 'fear-factor'.     

Expert consultation for cervical carcinoma smears. Reliability of selected-field videomicroscopy

OBJECTIVE: To evaluate the diagnostic accuracy of videomicroscopy image selection for expert consultation in cervical cytology. STUDY DESIGN: One hundred diagnostically difficult cervical cytologic smears were selected and rescreened by a general pathologist who chose, from each slide, four or five fields featuring abnormal cells. Video images were digitized and stored on a 512 x 512-pixel matrix using an image acquisition and transmission system. Five experts each reviewed 20 of the 100 cases, and a sixth reviewed all 100 cases. Diagnoses based on selected digitized images were compared to those based on conventional examination of whole slides. RESULTS: Intraobserver agreement was fair to excellent for all six experts (kappa value: 0.47-0.81); it was complete or acceptable in 68.4-85% of cases. Compared to the reference diagnosis, interobserver agreement was not significantly different whether cases were examined by screening the entire slide or by videomicroscopy of selected fields. The marked discordance in four cases concerned very small cells the significance of which was misinterpreted on videomicroscopy because of poor image quality due to lack of focus setting. CONCLUSION: This exploratory study showed that selection of videomicroscopy images seems as reliable as conventional examination of slides for expert consultation on diagnostically difficult cervical cytologic smear cases.     

Prognosis of moderate dysplasia. Predictive value of selected markers in routinely prepared cervical smears

Digital image analysis was used to extract features from 1,123 abnormal cells in 23 routinely prepared, Papanicolaou-stained cervical smears. All slides examined had a cytologic diagnosis of moderate dysplasia. Seven slides came from patients who eventually progressed to either a severe dysplasia or carcinoma in situ; the other 16 slides came from patients whose dysplasias regressed without evidence of more serious disease. Linear discriminant analysis correctly classified approximately 73% of the cells from the regression group and 66% of the cells from the progression group. Cell features contributing to the majority of variance in the model were the mean optical density of the nucleus, an autocorrelation measure, the mean optical density of the cytoplasm and the nuclear-cytoplasmic ratio. At the patient level, 13 (81.2%) of the 16 slides from the regression group and 6 (85.7%) of the 7 slides from the progression group were correctly classified. These figures yield a sensitivity of 66.7%, a specificity of 92.9%, a predictive value of a progression prognosis of 85.9% and a predictive value of a regression prognosis of 81.2%. The overall efficiency of the model was 82.6%. These preliminary results should encourage further studies for the identification of markers to indicate which patients are at high risk for progression of their cervical dysplasias.