|Year : 2015 | Volume
| Issue : 1 | Page : 17-21
|Liquid-based cytology versus conventional cytology for evaluation of cervical Pap smears: Experience from the first 1000 split samples
Vikrant Bhar Singh1, Nalini Gupta1, Raje Nijhawan1, Radhika Srinivasan1, Vanita Suri2, Arvind Rajwanshi1
1 Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Obstetrics and Gynecology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
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|Date of Web Publication||11-Feb-2015|
| Abstract|| |
Context and Aim: Screening programs using conventional cytology conventional Pap smear (CPS) have successfully reduced cervical cancer, but newer tests like liquid-based cytology (LBC) and human papillomavirus testing might enhance screening. The main aim of the present study was to assess the diagnostic accuracy of LBC versus CPS using "split samples." Materials and Methods: This was a prospective study comprising of 1000 consecutive cervical "split samples" over a period of 1 year. Split sample was obtained using cervex-brush. CPS was prepared from the brush and the brush head was suspended in the LBC vial and processed by SurePath™ LBC. Results: There were 4.3% unsatisfactory (U/S) cases in CPS and 1.7% in LBC; the main cause is insufficient cells, and excess of blood in CPS. About 25/100 (2.5%) split samples had epithelial abnormalities both in CPS and LBC (1.2%-atypical squamous cells of undetermined significance; 0.4%-low grade squamous intraepithelial lesion; 0.2%-high grade squamous intraepithelial lesion; 0.5%-squamous cell carcinoma; 0.1%-atypical glandular cells favouring neoplasia; 0.2%-adenocarcinoma). Inflammatory organisms were almost equally identified in both techniques but were better seen in LBC samples. Conclusions: LBC technique leads to significant reduction of U/S rate. LBC samples offered better clarity, uniform spread of smears, less time for screening and better handling of hemorrhagic and inflammatory samples. LBC had equivalent sensitivity and specificity to CPS.
Keywords: Cervical cancer, cervical precancer, human papillomavirus, liquid-based cytology
|How to cite this article:|
Singh VB, Gupta N, Nijhawan R, Srinivasan R, Suri V, Rajwanshi A. Liquid-based cytology versus conventional cytology for evaluation of cervical Pap smears: Experience from the first 1000 split samples. Indian J Pathol Microbiol 2015;58:17-21
|How to cite this URL:|
Singh VB, Gupta N, Nijhawan R, Srinivasan R, Suri V, Rajwanshi A. Liquid-based cytology versus conventional cytology for evaluation of cervical Pap smears: Experience from the first 1000 split samples. Indian J Pathol Microbiol [serial online] 2015 [cited 2020 Sep 21];58:17-21. Available from: http://www.ijpmonline.org/text.asp?2015/58/1/17/151157
| Introduction|| |
Liquid-based cytology (LBC) was introduced in mid-1990s as an alternative technique to process cervical samples. Since then a lot of countries in the Western world has switched from conventional Pap smear More Details (CPS) method to LBC, although amid contrasting results from various studies comparing the benefits of LBC with CPS. LBC is proposed to have many benefits over CPS such as less number of unsatisfactory (U/S) smears, , more representative transfer of cells from collecting device, evenly distributed cellular material, the choice of using residual cellular material for human papillomavirus (HPV) testing, reduced screening time and possibly higher rate of high grade squamous intraepithelial lesion (HSIL) detection. Extra slides prepared from residual LBC material has been shown to upgrade the diagnosis in 14.3% cases.  Most consistent benefit of LBC over CPS observed in various studies is reduced rate of U/S smears. ,,, Diagnostic accuracy of LBC when compared to CPS is a matter of great debate. Several studies have shown increased sensitivity of LBC over CPS, ,,, whereas others showing decreased or equal sensitivity and specificity. ,, Previous studies have also shown increased detection of glandular abnormalities in LBC preparations.  The present study was undertaken to study the differences between conventional and LBC methods in cervical Pap samples and to assess diagnostic accuracy of LBC in our setting. To the best of our knowledge, this is the first Indian study that provides information on use of LBC for cervical screening.
| Materials and Methods|| |
This was a prospective study comprising of 1000 consecutive cervical samples from women visiting the Department of Obstetrics and Gynecology over a period of 1 year. The samples taken were part of routine hospital-based screening of patients for cervical epithelial lesions. The samples were taken with cervex-brush and divided into two parts (split-sample technique). First, a CPS was prepared and was immediately alcohol-fixed. After that same brush head was detached and suspended in LBC vial containing preservative fluid, which was transferred to the cytopathology laboratory for further processing took place as per the prescribed protocol for the LBC equipment.
Cervical samples were compared for multiple parameters like morphology of various cells, U/S rates and sensitivity of two methods for detection of epithelial abnormalities as per the Bethesda system (TBS) 2001. Also wherever available, the results of cervical Pap samples were correlated with follow-up cervical biopsies/resection specimens. The study was approved by the Institutional Review Committee and an oral informed consent was taken from every patient.
Data were analyzed using the statistical package SPSS version15 for MS-Windows (SPSS Inc., Chicago, IL, USA). Pearson Chi-square test was used to analyze the data and P value was calculated wherever required. P value of 0.05 or less was considered as statistically significant.
| Results|| |
"Split samples" (CPS and LBC samples from the same patient) were reported on cytology according to TBS 2001. Break-up of "split samples" reported as per TBS 2001 is given in [Table 1].
Epithelial abnormalities detected in 1000 split Pap samples
About 25/1000 or 2.5% split Pap samples were reported to have epithelial cells abnormalities both in CPS and LBC samples. Out of a total of 25 cases, 12 cases were reported as atypical squamous cells of undetermined significance (ASCUS) (0.12% of the total); 4 as low grade squamous intraepithelial lesion (LSIL) (0.4%); 2 as HSIL (0.2%); 5 squamous cell carcinoma (SCC) (0.5%); 1 atypical glandular cells favoring neoplasia (AGC-FN) (0.1%); and 2 adenocarcinoma (0.2%) [Table 2]; [Figure 1]. The pick-up rate for clinically significant abnormalities in split-samples was similar. SCC was confirmed on follow-up biopsies in 4/5 cases. About 1/4 cases reported as LSIL had cervical intraepithelial neoplasia grade 3 (CIN3) changes on follow-up biopsy. 2/12 cases of ASCUS were reported as CIN1 on follow-up biopsies. Adenocarcinoma cases were confirmed as carcinosarcoma of the endometrium and ovarian adenocarcinoma on follow-up histology.
|Figure 1: Split sample. (a and b) High grade squamous intraepithelial lesion (a - conventional Pap smear [CPS] Pap, ×40), (b - liquid-based cytology [LBC] Pap, ×100). (c and d) Squamous cell carcinoma (c - CPS Pap, ×40), (d - LBC Pap, ×40)|
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|Table 2: Break-up of split-samples reported as epithelial cell abnormality (n = 25) with follow-up histology|
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Comparison of morphological characteristics in conventional Pap smear versus liquid-based cytology
Atypical squamous cells of undetermined significance (n = 12) and low grade squamous intraepithelial lesion (n = 4)
Low grade squamous intraepithelial lesion in LBC showed singly scattered and groups of intermediate sized squamous cells with nuclear enlargement, slight increase in nuclear: Cytoplasmic (N:C) ratio, uniformly distributed, coarsely granular nuclear chromatin and slightly irregular nuclear membranes. Koilocytosis was noted in all four cases. These changes were appreciated both in CPS and LBC samples, however nuclear details were much more clear in LBC smears and it was easier to appreciate koilocytosis in LBC smears. Small brightly orangeophillic dyskeratotic cells were conspicuous in two cases in LBC smears. Cases which fell short of LSIL but had changes more than reactive atypia were reported as ASCUS.
High grade squamous intraepithelial lesion (n = 2)
Both CPS and LBC smears showed cytological features of HSIL. There were scattered as well as groups of abnormal cells. These cells were of the size of parabasal cells and had high N:C ratio, coarse chromatin and inconspicuous nucleoli. The cell size was much smaller in LBC and nuclear chromatin was not as hyperchromatic as seen in CPS. One case showed micro-biopsies/hyperchromatic crowded cell groups in LBC sample, with loss of polarity associated with cellular abnormalities. Another feature which was seen in both cases was presence of small dyskeratotic cells/marker cells, which were conspicuous in LBC smears.
Squamous cell carcinoma (n = 5)
Five cases of SCC showed almost similar features in CPS and LBC samples. Diathesis was a characteristic feature of SCC, which was relatively difficult to pick on LBC and appeared as some fibrin-rich tangles with entrapped inflammatory cells and tumor cells. CPS samples showed excess of blood obscuring the morphology of tumor cells, which was clearer in LBC samples.
Adenocarcinoma (n = 2)
Both cases were diagnosed as adenocarcinoma, not otherwise specified, on CPS and LBC samples. One case was a metastatic ovarian papillary serous adenocarcinoma which on LBC mimicked an endocervical adenocarcinoma, as the tumor was directly sampled by cervex-brush. It was not possible to state the site of the tumor on CPS as it had tumor cell clusters in hemorrhagic background. The second case showed scattered three-dimensional clusters of tumor cells, which mimicked endometrial adenocarcinoma. The stromal component was not appreciated on either CPS or LBC. This case was confirmed as carcinosarcoma on histology.
There were 43/1000 (4.3%) U/S cases in CPS and 17 (1.7%) cases in the LBC samples with 53.5% reduction in U/S smears. This difference is statistically significant with P = 0.0006. In split samples, the main cause of unsatisfactory smears was insufficient cells-16/43 (37.2%) in CPS and 10/17 (58.8%) in LBC samples. The second major cause was low cellularity with excess blood-9/43 cases in CPS and only excess blood - 9/43 in CPS [Figure 2]. About 7/17 U/S cases in LBC were due to low cellularity and excess blood.
|Figure 2: Split sample. (a) Unsatisfactory sample due to low cellularity and blood (conventional Pap smear [CPS] Pap, ×40), (b) corresponding liquid-based cytology [LBC] satisfactory sample (LBC Pap, ×40); (c) unsatisfactory sample due to excess of inflammation (CPS Pap, ×40), (d) corresponding LBC satisfactory sample (LBC Pap, ×40)|
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Smears with altered flora (139/1000 cases) and bacterial vaginosis (BV) (121 cases) were seen more commonly in CPS as compared to LBC (altered flora (110) and BV (112) respectively). Atrophic smears (n = 43), smears showing actinomycetes-like organisms (n = 6), and smears showing leptothrix (n = 4) were equal in both groups. Trichomonas vaginalis (TV) was seen in 17 cases of CPS and 16 cases of LBC. Candida spores were picked more easily in CPS (n = 28 cases); however Candida hyphae were very conspicuously seen in LBC samples as compared to CPS (n = 12 cases) with "Shish-kebab" like appearance.
Other morphological features and differences
Although this study was not designed to evaluate screening time, it was seen that the average time needed to screen a LBC slide is 2.5-3 min as compared to CPS, which is at least 5-6 min. In LBC, benign endometrial cells are easier to identify with tight three-dimensional-groups. Their nuclei are small, kidney-bean shaped, dark with smudgy chromatin. Cytoplasm is scant to vacuolated.
| Discussion|| |
Pap smear is one of the best available screening methods for early detection of cervical precancerous lesions. LBC is an alternate technique for processing the cervical sample collected. Most Western countries have switched over from CPS to LBC, even though the sensitivity and specificity is almost similar in various comparison studies. The reason for this may be consistently reduced rates of U/S results on LBC, clarity of microscopy, improved sample processing, and small area to be screened. Furthermore, the potential for performing additional tests, including HPV testing on the residual sample, probably underpins the acceptability of LBC among gynecologists, colposcopists and pathologists. We started using LBC SurePath ® system 2 years back and this study was conducted to evaluate the performance of LBC in our setting and to study the differences in morphology of various lesions in CPS and LBC samples.
In our study, the rate of detection for epithelial cell abnormalities was similar in both CPS and LBC. There are many studies which have documented similar detection rate on both types of preparations. In a direct comparison study by Taylor et al. of 5652 cases, CPS and LBC performance and accuracy were statistically similar.  Another Japanese study with 1551 split samples, showed that the sensitivity of lesions histologically diagnosed as CIN1 or above was not significantly different between the two methods (P = 0.575-1.000) and cytologic results showed a concordance rate of 85.3% (k = 0.46) between the two methods.  Large meta-analyses by Arbyn et al. included 109 studies where positivity and/or adequacy rate was studied. In their analyses, there was no statistically difference in sensitivity and specificity between the two different methods for detection of CIN2+.  However, there are other studies in the literature indicating higher detection rates of HSIL + lesions and glandular lesions. ,
The U/S rate was reduced from 4.3% to 1.7% in LBC smears in the present study. The most common reason for U/S was low cellularity in both categories. There was no inadequate LBC sample due only to excess blood or obscuration by polymorphs/mucus or other technical artefacts. Therefore, the samples with excess blood are better handled by LBC. The reduction of U/S smears in LBC samples is consistent with many previous studies. ,, The National Institute for Clinical Excellence in UK showed lower proportions of U/S smears from 9% in conventional cytology to 1.6% in LBC.  LBC leads to almost complete elimination of most causes for U/S conventional preparation, with scant cellularity remaining as the main cause for U/S LBC.  This can also be handled by adequate visualization of the cervical os and proper sample taking.
Infectious organisms such as Candida hyphae, TV, herpes simplex virus and actinomycetes-like organisms were seen better or more easily on the LBC samples. Candidal hyphae were more easily identified in LBC as the "Shish-kebabs" of pseudohyphae skewering the squamous cells. This effect was more pronounced in the LBC. On the other hand, Candida spores were more commonly seen present in the background on the CPS, in the present study. Similar findings were observed by Fitzhugh and Heller.  Similar to our study, Takei et al. found that Trichomonas and a shift in bacterial flora were detected more often with CPS than with SurePath ® (13.4% vs. 8.3% and 38.7% vs. 30.2%, respectively). 
Therefore, the main advantage of LBC is reduction in unsatisfactory rate and availability of residual LBC sample to perform HPV DNA testing. HPV testing is of increasing importance as HPV testing is considered for incorporation into screening programs  as triaging low-grade abnormalities, co-testing with cytology and as a primary cervical cancer screening tool.
The present study compared the performance of LBC and conventional cytology in Indian setting. The detection rate of epithelial abnormalities and infections in both preparations was similar. U/S rate of CPS was 4.3% and 1.7% for LBC and this difference is statistically significant. There has been 100% conversion from CPS to LBC for cervical cancer screening in the developed world. In the Indian scenario with a low-resource setting, we need to re-consider the cost-effectiveness of LBC as compared to CPS, especially in the absence of reflex HPV testing in a majority of centers. Our study provides important context on current patterns of uptake of LBC, which is strongly dependent on a woman's age, her screening history, socioeconomic factors and ability of pay in absence of public funding. Cost-effectiveness of LBC needs to be evaluated in Indian context with benefits and harms associated with a move to LBC.
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Dr. Nalini Gupta
Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]
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