Correlation of Anal Cytology Results From Slides Prepared by Conventional Versus Liquid-Based Techniques
Combining data from HIV-infected and -uninfected MSM, there was no unsatisfactory slide among those prepared by conventional technique, whereas 3 (1.7%) slides prepared by liquid-based technique were considered unsatisfactory (Table 2). Abnormal anal cytology from ASC-US and above was identified in 80 of 173 cases (46.2%) from conventional slides and in 56 (32.4%) from liquid-based slides. Rectal columnar epithelia were presented in 83 (48.0%) of conventional slides and 75 (43.4%) of liquid-based slides (P = 0.136). Squamous intraepithelial lesions were diagnosed in 16.9% of slides where rectal columnar cells were present and in 13.1% of slides without rectal columnar cells.
Anal cytology diagnosis using conventional and liquid-based slides agreed in 108 of 173 cases (62.4%), with a κ value of 0.49 (P < 0.001). Excluding 3 indeterminate liquid-based slides, diagnosis within 1 and 2 diagnostic categories of each other was identified in another 51 (30%) and 11 (6.5%) of slides, giving a weighted κ of 0.45, P < 0.001, and an agreement of 85.7%. When data were compared using a weighted κ between HIV-infected and -uninfected MSM, the cytology tests showed an overall lower agreement in HIV-infected participants (agreement = 82.1%, κ = 0.39, P < 0.001) than HIV-uninfected participants (agreement = 93.2%, κ = 0.58, P < 0.001).
Three cases of HSIL diagnosed from conventional slides were diagnosed as ASC-US (1), LSIL (1), and HSIL (1) by liquid-based slides. From liquid-based slides, 9 cases of HSIL diagnosed were read as ASC-US (2), LSIL (6), and HSIL (1) from conventional slides. Agreement between conventional and liquid-based slides for abnormal results from ASC-US and above was 75.1% (κ = 48.7, P < 0.001), 69.5% (κ = 40.3, P < 0.001) for HIV-infected MSM and 87.3% for HIV-uninfected MSM (κ = 65.8, P < 0.001). For abnormal results from LSIL and above, agreement between both techniques was 88.4% (κ = 46.4, P < 0.001), 85.6% for HIV-infected MSM (κ = 46.4, P < 0.001), and 94.6% for HIV-uninfected MSM (κ = 37.3, P = 0.0024). Agreement was 94.2% (κ = 14.4, P = 0.01) when results were read as HSIL and above, 92.4% for HIV-infected MSM (κ = 15.0, P = 0.03). There were no HSIL cases read from conventional cytology among HIV-uninfected MSM.
Being HIV-infected MSM was the only factor that was correlated with a higher risk for discordant anal cytology results by univariate analysis (odds ratio: 3.6, 95% CI: 1.6 to 7.8, P = 0.001). Among the sociodemographic and behavioral characteristics presented in Table 1, there was no association with discordant anal cytology results. Within the HIV-infected MSM, CD4 count, plasma HIV-RNA levels, and being on antiretroviral therapy were also not associated with discordant anal cytology results.
Detection of Histologically Confirmed AIN by Abnormal Conventional and Liquid-Based Anal Cytology Results
Of 173 participants, 96 (55.5%) had HRA and 69 (40.0%) had biopsies performed. By histology, AIN 1 was identified in 33 cases (47.8% of 69 participants who had biopsy performed) and AIN 2 and AIN 3 were identified in 20 (29.0%). The remaining (23.2%) had normal or benign histological diagnoses. Of 20 AIN 2 and AIN 3 cases, 1 AIN 2 case and 6 AIN 3 cases had HSIL diagnosis by either conventional or liquid-based slides. ASC-US or higher-grade cytological diagnoses could detect 17 AIN 2 and AIN 3 cases by conventional technique and 14 AIN 2 and AIN 3 cases by liquid-based technique (Table 4).
PPVs of Abnormal Conventional and Liquid-Based Anal Cytology for Detecting Histologically Confirmed AIN
By conventional cytology, ASC-US had 28.3% and LSIL had 39.1% PPVs in predicting histological AIN2 and AIN 3, whereas HSIL had 50% PPV in predicting histological AIN 2 and AIN 3. Using liquid-based cytology, ASC-US had 37.8% PPV, LSIL had 46.2% PPV, and HSIL had 66.7% PPV in predicting histological AIN 2 and AIN 3. The 95% CI around the PPV ratios indicated that the PPVs of liquid-based versus conventional cytology were not significantly different (Table 4).
For HIV-infected MSM, the PPVs were 29.4% for ASC-US, 38.1% for LSIL, and 50.0% for HSIL in predicting AIN 2 and AIN 3 by conventional cytology and were 40.0% for ASC-US, 41.7% for LSIL, and 62.5% for HSIL by liquid-based cytology. The PPVs were 22.2% for ASC-US and 50.0% for LSIL by conventional cytology and 28.6% for ASC-US, 100% for LSIL, and 100% for HSIL by liquid-based cytology to predict AIN 2 and AIN 3 among HIV-uninfected MSM. The 95% CI around the PPV ratios did not indicate significant differences between the 2 techniques when used either in HIV-infected or in HIV-uninfected MSM.
Our study demonstrated that anal cytology results read from slides prepared by a conventional technique had good correlation with those prepared using a liquid-based technique. In resource-limited settings, use of conventional Pap slides for cytology evaluations is more practical due to the high cost of liquid-based cytology fluid and associated equipments. The Liqui-PREP solution used in the study provides an additional advantage over other liquid-based cytology fluids in terms of cost because an automated slide preparation machine is not needed.
For cervical cytology, liquid-based cytology techniques have been shown to provide greater18–20 or at least equivalent diagnostic accuracy compared with the conventional smear.14 Studies on the use of liquid-based cytology techniques for anal cytology, however, are more limited. Use of ThinPrep was shown to yield similar diagnostic results compared with conventional smears for anal cytology, although ThinPrep provided additional benefits in reducing fecal and bacterial contamination and air-drying artifact.12,13
Previous studies have shown a wide range of unsatisfactory rates of anal cytology slides prepared by conventional techniques (17%–24%)12,13 and liquid-based techniques (7%–17%).12,13,21 Rectal columnar cells, an indicator that the rectal transformation zone was adequately sampled, were found more frequently on liquid-based cytology slides than conventional slides in several studies.12,13,22 The presence of an adequate amount of well-preserved and adequately fixed cells, however, may be as important as the presence of cells from the transformation zone when evaluating the sample quality of anal cytology slides.13 We found a very low unsatisfactory rate for both conventional (0%) and liquid-based slides (1.7%) for anal cytology, given that the presence of rectal columnar cells was not a requirement for our study.
We identified more abnormal anal cytology cases from conventional slides than from liquid-based slides, although more atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion and HSIL cases tended to be diagnosed from liquid-based slides. This is in contrast to previous findings that showed equal or higher detection rates of abnormal anal cytology with the use of liquid-based techniques because of reduction of fecal material, inflammation, bacteria, and air-drying artifact.12,13 The detection rate of SILs from ThinPrep slides in 1 study was shown to be nearly 8 times higher than conventional slides.13 This may be due to the different technique used for Liqui-PREP because the smear was made manually using a pipette, which may not result in a thin, evenly dispersed monolayer of cells seen with semiautomated liquid-based slide preparation and the amount of cells obtained on the slides may be less. ThinPrep and SurePath slides were prepared by a processor that mechanically disperses the cells, which are then drawn onto a filter by negative pressure and transferred onto a glass slide in a monolayer. In addition, the amount of cells available in our liquid-based cytology fluid may have been reduced by the antecedent preparation of the conventional anal cytology slide. However, this was not found to be an issue in a previous study.12
A systematic review did not show significant differences in sensitivity and specificity of anal cytology to detect abnormal histological diagnoses between conventional and liquid-based cytology techniques conducted across different studies.10 Although we could not comment on the absolute sensitivity and specificity in our study due to the lack of reference testing on every participant, we found the overall agreement between conventional and liquid-based cytology to be 62.4% and a κ value of 0.43, which showed moderate agreement between the 2 techniques. HIV-infected MSM had a 3.6-fold increased odds of having discordant anal cytology results. The overall agreement and agreement when abnormal results from ASC-US and above were considered were lower in HIV-infected than HIV-uninfected MSM. The PPV ratios demonstrated comparable performances of both techniques in detecting histologically confirmed AIN, both in HIV-infected and in HIV-uninfected MSM.
Conventional cytology was able to accurately read only 1 of 9 HSIL results on liquid-based cytology, whereas only 1 of 3 HSIL results on conventional cytology was correctly read by liquid-based cytology. Of 20 cases which had histologically confirmed AIN 2 and AIN 3 in our study, if referral to HRA was triggered by HSIL alone, only 1 case would have been diagnosed on conventional cytology and 6 cases on liquid-based cytology. This is in accordance with previous findings that showed that the grade of disease on anal cytology did not correspond well to the histological grade after biopsy,21,23–26 and suggested that MSM with screening anal cytology results that are abnormal at any grade should be considered for HRA.24 The high prevalence of abnormal anal cytology underlies the importance of the readiness of health systems for referral to HRA services. Setting up HRA services is challenging in both resource-rich and resource-limited settings due to the cost and the paucity of trained clinicians. Use of other tests such as HPV genotypes, E6/E7 mRNA, or abnormal cell cycle markers may be useful either as a primary screening or as a adjunctive screening method to anal cytology to detect high-grade AIN.27,28 For this purpose, liquid-based cytology provides its advantage over conventional cytology because these adjunctive tests can be conducted from the same liquid-based cytology sample.
Our study had several limitations. We had limited statistical power to study agreement of the 2 cytology techniques among subset of MSM with high-grade AIN (AIN 2 and AIN 3), the putative anal precancerous lesions, because there were only 20 AIN 2 and AIN 3 cases. We also could not make definitive conclusions on the performance characteristics of anal cytology for the detection of high-grade AIN because HRA and biopsy were not performed among the majority of MSM with normal anal cytology in this study. In addition, the sequence of anal sample collection in our study could affect the amount of cells available for liquid-based cytology. Ideally, the sequence of anal sample collection could have been reversed for a subset of slides to overcome this limitation. Last, the reading of anal cytology and anal histology slides in our study were not performed by the same cytotechnician or pathologist. The interobserver agreement for anal cytology has been reported in other studies to be moderate with a weighted κ statistic ranging from 0.72 to 0.92.29,30 All cytotechnicians and pathologists in our study have participated in quality control programs conducted during the course of the associated studies to minimize interobserver variances.
In summary, we demonstrated that anal cytology results using a conventional slide preparation technique were similar to those using a liquid-based technique, although the agreement between the 2 techniques was lower among HIV-infected MSM. With low agreement between anal cytology and histology diagnoses in general, programs, which aim to screen for high-grade AIN, would need to prepare adequate infrastructure for HRA services and treatment. The need for new biomarkers to detect high-grade AIN will become more apparent as more screening programs are established worldwide.
The study team is grateful to the individuals who volunteered to participate in this study and to staff at the Thai Red Cross AIDS Research Centre and the Faculty of Medicine, Chulalongkorn University. The content of this presentation is solely the responsibility of the authors and does not necessarily represent the official views of any of the institutions mentioned above.
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Keywords:© 2013 by Lippincott Williams & Wilkins
anal cytology; conventional; liquid based; MSM