Context.—Although the cytologic features of squamous cell carcinoma in ThinPrep specimens are well known, whether these features are different in cases that are easily identified than in cases that are more difficult to identify is not known.

Objective.—To determine the cytologic features of squamous cell carcinoma in ThinPrep specimens that are easy to identify versus those that are difficult.

Design.—The cytologic features of 6 cases of squamous cell carcinoma that performed poorly in the College of American Pathologists Interlaboratory Comparison Program were compared with 14 cases that performed extremely well.

Results.—After evaluation of multiple criteria, 7 different cytologic features were analyzed based on review by a consensus panel blinded to the performance of the cases. The feature that was most strongly associated with cases that performed poorly was the presence of Trichomonas vaginalis (5/6 [83%] vs 0/14; P < .001). The presence of marked nuclear pleomorphism was more common in cases that performed well (4/14 [28%] vs 0/6; P = .27), but was not significant. The number of tumor cells, the number of normal cells, and the presence of keratinization, pleomorphism, nucleoli, and diathesis were not significant. The most common misdiagnosis after Trichomonas vaginalis was reparative change.

Conclusions.—The presence of Trichomonas is characteristic of cases of squamous cell carcinoma in ThinPrep slides that are often misdiagnosed in this program. While Trichomonas is identified by participants in some of these cases, a significant percentage of participants interpreted the findings as reparative, without identifying the organism. These results emphasize the importance of distracting factors, whether identified or not, in evaluating gynecologic cytology.

It is well known that cervicovaginal cytology is not perfect and is associated with a significant error rate for both squamous cell carcinoma and its precursor lesions.1 Previous studies have shown that cases of high-grade squamous intraepithelial lesion that are missed on conventional smears may have characteristic cytologic features, including relatively few abnormal cells.2,3 Studies examining the cytologic features of cases of squamous cell carcinoma that are missed are not available.

Use of liquid-based cytology continues to increase in the United States. The cytologic features of squamous cell carcinomas in ThinPrep specimens are similar to those in conventional smears and have been well described by Clark and Dawson.4 These authors noted, however, that a diathesis was common and that the cellularity of these specimens appeared decreased. We recently discovered that a small number of ThinPrep slides with a reference diagnosis of squamous cell carcinoma in the College of American Pathologists (CAP) Interlaboratory Comparison Program in Cervicovaginal Cytology (PAP) were performing poorly. To investigate the reasons for this poor performance, we compared the cytologic features of these cases with a number of ThinPrep cases with squamous cell carcinoma that have performed extremely well.

The PAP is a glass-slide quality improvement program that is mailed quarterly. The CAP Laboratory Accreditation Program requires that all laboratories evaluating gynecologic cytology enroll in the PAP or an equivalent glass-slide program. Cytology laboratories of all types participate, the largest number (approximately 60%) being hospital laboratories. In addition, independent laboratories, federal and government laboratories, university laboratories, and others, such as those associated with a group practice or physician's office, also participate.

Participants generously contribute slides to the program. Submitted slides with a diagnosis of low-grade squamous intraepithelial lesion or higher must be confirmed by biopsy. After receipt and accessioning into the program, the slides are reviewed by at least 3 experienced cytopathologists from the CAP Cytopathology Resource Committee. Before acceptance into the program, each slide must be judged to be of good technical quality and an excellent example of the reference diagnosis. All 3 reviewers must agree on the exact target diagnosis, and this diagnosis must agree with the submitted and biopsy diagnoses prior to accepting a slide for circulation in an educational set.

The PAP consists of 5 glass slides of cervicovaginal material mailed 4 times per year. The coded answer sheets have diagnostic menus using terminology modified from the Bethesda System. Referenced slides are placed into 1 of 3 selection series: the 000 series for unsatisfactory slides; the 100 series for normal cytology, infections, and reparative conditions; and the 200 series for epithelial cell abnormalities and carcinomas.

ThinPrep cases with a reference diagnosis of squamous cell carcinoma and at least 15 individual observations by pathologists or cytotechnologists were identified. A group of cases with either poor performance or excellent performance were identified. The cases of squamous cell carcinoma that performed poorly were incorrectly assigned to the 100 series at least 10% of the time (range, 11%–50%; median, 27%). The group of cases that performed well were cases in which the participants correctly identified the cases to the correct series 100% of the time and were classified as carcinoma at least 50% of the time.

Cases were originally reviewed by 2 authors (A.A.R. and B.D.B.) to evaluate possible cytologic features that might differ between the 2 groups. The cases were then mixed together and blindly reviewed at a multiheaded microscope by the remaining authors; their interpretations are detailed in the results section. The number of abnormal cells were categorized into groups of fewer than 50, 50 to 100, 101 to 500, 501 to 1000, or more than 1000. For the purpose of analysis, the threshold that separated the greatest number of cases that performed poorly and excellently was tested statistically. The number of normal cells were categorized into groups of 500 and fewer or more than 500 cells.

The statistical significance of the various features was evaluated using a 2-tailed Fisher exact test. P < .05 was considered statistically significant.

A total of 6 cases that were frequently missed were identified and compared with a total of 14 cases that were always identified (Figures 1 and 2). The cytologic features that were evaluated are listed in the Table, along with the frequency and relative significance of these findings.

Figure 1.

Squamous cell carcinoma case that performed well in the College of American Pathologists Interlaboratory Comparison Program. Cases were characterized by marked nuclear pleomorphism (a and b), with (b) or without (a) marked keratinization (Papanicolaou, original magnifications ×100).Figure 2. Squamous cell carcinoma case that performed poorly in the College of American Pathologists Interlaboratory Comparison Program. Cases were often confused with reparative change (Papanicolaou, original magnification ×100)

Figure 1.

Squamous cell carcinoma case that performed well in the College of American Pathologists Interlaboratory Comparison Program. Cases were characterized by marked nuclear pleomorphism (a and b), with (b) or without (a) marked keratinization (Papanicolaou, original magnifications ×100).Figure 2. Squamous cell carcinoma case that performed poorly in the College of American Pathologists Interlaboratory Comparison Program. Cases were often confused with reparative change (Papanicolaou, original magnification ×100)

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Cytologic Features Evaluated in Cases of Squamous Cell Carcinoma in ThinPrep Specimens

Cytologic Features Evaluated in Cases of Squamous Cell Carcinoma in ThinPrep Specimens
Cytologic Features Evaluated in Cases of Squamous Cell Carcinoma in ThinPrep Specimens

The feature that was most strongly associated with cases that performed poorly was the presence of Trichomonas vaginalis (5/6 [83%] vs 0/14; P < .001). The presence of marked nuclear pleomorphism was more common in cases that performed well (4/14 [28%] vs 0/6, P = .27), but was not significant. The number of tumor cells, the number of normal cells, and the presence of keratinization, pleomorphism, nucleoli, and diathesis were not significant.

Interestingly, although Trichomonas was present in 5 of the 6 cases, it was a response by a participant in only 4 cases, was the most common incorrect answer in only 3 cases, and was the most common answer overall in only 2 cases. In addition, Trichomonas was noted by the review panel in only 1 case. If the 2 cases in which Trichomonas was the most common response are eliminated, the most common incorrect response was reparative change (12 responses), followed by Trichomonas vaginalis (3 responses) and negative (1 response).

Cervicovaginal cytology is not a perfect test, and it is well known and well documented to have a false-negative rate.1 Previous studies have shown that cases of high-grade squamous intraepithelial lesion that are missed on conventional smears may have characteristic cytologic features, including relatively few abnormal cells.2,3 To our knowledge, studies examining the cytologic features of cases of squamous cell carcinoma that are missed are not available.

Data acquired in the CAP PAP present an excellent opportunity for evaluating the significance of cytologic findings in a series of extremely well-characterized cases. The diagnoses are confirmed by biopsy in every case of squamous intraepithelial lesion or carcinoma, reviewed by 3 members of the committee, and most importantly are then evaluated by their performance in the program based on the interpretation of practicing cytologists. As such, the performances we document in these cases may more accurately reflect the abilities of practicing pathologists in general to interpret these types of cases, as opposed to the ability of 1 or only a few pathologists practicing in 1 or a few primarily academic institutions.

Nevertheless, there are limitations to the results that we document. Specifically, in this program one can only record a single interpretation for each case, and this may have biased the results. It is possible that once the participants identified the presence of Trichomonas vaginalis, they then did not spend additional time examining the case. Or, once Trichomonas was identified, they were more likely to interpret the cytologic changes that were present as a reactive process related to the Trichomonas rather than evaluating the significance of these findings independently. Finally, it is possible that the presence of Trichomonas altered the appearance of the carcinoma cells in the cases in which they were present. Regardless of the true reason for the decreased performance, these results reinforce the previous observations from this committee that the presence of more than 1 diagnostic entity in a case provides a “distraction factor” that can significantly reduce the ability of observers to make the other more significant diagnosis in a case.5,6 

We were surprised that cellularity was not found to be important in this study. We initially believed that one of the most significant features of cases of squamous cell carcinoma that performed poorly would be a paucity of abnormal cells. This is one of the most consistent findings in prior studies of high-grade squamous intraepithelial lesion cases that have performed poorly.2,3 We were not surprised that the overall number of abnormal cells in these cases was much higher than in prior studies. Those prior studies examined cases that were missed in the clinical laboratory. All cases in this program were initially identified in the clinical laboratory, were subsequently confirmed with biopsy, were selected by members of that clinical laboratory for submission to the program, and were reviewed by 3 members of the committee to ensure that the slides were representative of squamous cell carcinoma. As a result, it is not surprising that the overall numbers of abnormal cells in these cases were much greater than in cases in prior studies. Nevertheless, we still expected to find that cases that performed poorly would have fewer abnormal cells than those that performed well. This did not appear to be the case. Similarly, previous authors had noticed an overall decrease in cellularity in cases with squamous cell carcinoma.4 While our method of assessing this characteristic was different (counting normal cells), we were not able to confirm this observation either.

Finally, our results do suggest that a major problem with cases of squamous cell carcinoma that perform poorly is misinterpretation rather than screening error. Other than the identification of Trichomonas, the most common incorrect response was reparative change. It has previously been shown that reparative change is one of the least reproducible diagnostic categories in the program.7 Taken together, these results suggest that one should be very cautious about using the diagnosis of reparative change, and that additional educational efforts concerning the distinction between reparative change and squamous cell carcinoma in liquid-based cytology may be of value.

In conclusion, the presence of Trichomonas is characteristic of cases of squamous cell carcinoma in ThinPrep slides that are often misidentified in this program. While Trichomonas is identified by participants in some of these cases, a significant percentage of participants interpreted the findings as reparative, without identifying the organism. These findings emphasize the importance of distracting factors, whether identified or not, in evaluating gynecologic cytology.

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The authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: Andrew A. Renshaw, MD, Department of Pathology, Baptist Hospital of Miami, 8900 N Kendall Dr, Miami, FL 33176 ([email protected])