|Year : 2008 | Volume
| Issue : 2 | Page : 190-194
|Study of a manual method of liquid-based cervical cytology
Anita N Kavatkar1, CA Nagwanshi1, SM Dabak2
1 Department of Pathology, BJ Medical College, Pune, India
2 Department of Gynecology and Obstetrics, BJ Medical College, Pune, India
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| Abstract|| |
We report a study of a manual liquid-based cytology (MLBC) method. Slides are prepared by using a polymer solution and allowing it to dry, forming a membrane. The aims of the study were to prepare cervical cytology smears using the manual method, observe morphology, compare with direct scrape smears and correlate with histopathology wherever possible. Out of 105 cases, the membrane was intact, indicating a good MLBC preparation, in 97 cases. Simultaneous conventional smears were taken in 81 patients. There was an 88.8% agreement in the diagnoses of general category in both groups. The diagnosis of negative for intraepithelial lesion or malignancy (NILM) in both groups was made in 70 cases. The MLBC preparation was unsatisfactory in two cases which showed high grade squamous intraepithelial lesion and low grade squamous intraepithelial lesion, respectively, on the conventional smear. One MLBC smear diagnosed as atypical squamous cells of undetermined significance was reported as NILM on conventional smear. Cytohistologic correlation was done in nine cases, all of which showed cervicitis on histopathology. The MLBC method was found to be comparable to the conventional scrape smear. Further study of this method as a cost-effective alternative to the mechanized methods would be worthwhile.
Keywords: Cervical cytology, low-resource settings, cervical cancer, early diagnosis, manual liquid-based cytology
|How to cite this article:|
Kavatkar AN, Nagwanshi C A, Dabak S M. Study of a manual method of liquid-based cervical cytology. Indian J Pathol Microbiol 2008;51:190-4
| Introduction|| |
Cervical cancer is the second most important common cancer among women worldwide. According to World Health Organization (WHO, 2003), 470,000 new cases are diagnosed each year and 80% of deaths occur in developing countries.  Invasive cervical cancer is the end result of a long pathological process that begins with precursor lesions called cervical dysplasia or squamous intraepithelial lesions (SIL).  Early changes in the cervix, specifically cervical intraepithelial neoplasia (CIN), can be detected years before invasive carcinoma develops and this is the basis for the effectiveness of cytological screening. Cervical cancer screening is routinely done using scrape smears from the cervix, which are then stained by the Papanicolaou stain.
Liquid-based cytology is a technique that enables cells to be suspended in a monolayer and thus better morphological assessment is possible. It includes the preparation and evaluation of cells collected in a liquid fixative. It is being introduced in developed countries to improve the sensitivity of the Pap test. During recent years, it has also been used for nongynecologic cytology, e.g. in breast cytology. Two technologies - Thin Prep (Cytyc Corp.) and SurePap (Tripath imaging, Inc.) have been more widely used.
The advantages of liquid-based cytology include improved sensitivity and specificity since fixation is better and nuclear details are well preserved. Abnormal cells are not obscured or diluted by other epithelial or inflammatory cells. There is, therefore, a lower rate of unsatisfactory cervical cytology samples. The residual cell suspension can be used to make further cytological preparations or used for other tests like detection of human papilloma virus (HPV) DNA. Other ancillary techniques like immunocytochemistry can also be performed on the residual sample.
The more widely used technologies for liquid-based cytology require expensive equipment. We report a study of a manual membrane-based method using simple equipment - a vortexer and laboratory centrifuge. Slides are prepared by using a polymer solution and allowing it to dry, forming a membrane.
The aims and objectives of the study were to prepare cervical cytology smears using the manual method of liquid-based cervical cytology, to observe morphological features, compare results of direct smear examination with manual liquid-based smears and correlate with histopathological findings in those cases in which a biopsy was also done.
| Materials And Methods|| |
Samples for the manual membrane-based method were collected from patients attending the gynecology out patient clinic at Sassoon General Hospital, Pune. Simultaneous cervical scrape smears were also taken. Sassoon hospital is a government run hospital with facilities for tertiary care. The study was approved by the Institutional Ethical Committee.
A brush-like device, Cervex-brush (Rovers medical devices), was used to scrape the cervix. The manufacturer's instructions, viz. insertion of long bristles into endocervical canal, short bristles against the ectocervix and five full 360º rotations in clockwise direction only, were followed. The brush head was then detached and immediately put into a vial containing the fixative solution. A commercially manufactured fixative, (SurePath preservative fluid), was used in half the cases and another fixative, prepared in the laboratory, was used for the remaining cases. The fixative prepared in our laboratory contained water, sodium chloride, sodium citrate, 10% formalin and alcohol. Further processing was done as follows after a minimum duration of 24 h:
- Collection of sample.
- Specimen is vortex mixed.
- Centrifuge at 800 g for 10 min.
- Decant fixative and blot excess fixative.
- Add 1-2 ml of polymer solution to tube.
- Vortex mix.
- Apply 3-6 drops of suspension to glass slide.
- Allow to dry.
- Stain with conventional Pap stain.
Scrape smears were collected using Ayre's spatula. Smears were made in the conventional way and immediately fixed in ether-alcohol fixative. After fixation, smears were stained using conventional Pap stain.
Both smears were screened independently by two observers and results were compared. Cyto-histologic correlation was attempted in those cases in which a biopsy was also done. The Bethesda system for reporting cervical cytology was used in both groups.
| Results|| |
Manual method of liquid-based cervical cytology was done in 105 cases. Smears were categorized as satisfactory or unsatisfactory and cellularity was judged subjectively, based on the Bethesda system.  The membrane-based preparation was satisfactory in 92 cases (Transformation zone present in 81) and unsatisfactory in 13 cases (scanty cellularity in 10 and blood in 3).
Out of the 105 cases, the membrane was intact, indicating a good manual liquid-based cytology (MLBC) preparation, in 97 cases. However, it was totally peeled off in eight cases and these were considered unsatisfactory for evaluation. In five cases, the specimen was unsatisfactory due to blood or scanty cellularity despite a good MLBC preparation. During the preparation of the slides, the fixative was decanted. Both the fixatives used gave satisfactory results. The morphology of cells was satisfactory for evaluation and cells were seen mostly uniformly spread without overlap [Figure 1]. In eight cases, there were clumps of cells seen along with uniformly distributed cells; however, these did not completely obscure the morphology. Polymorphs were seen separately from the squamous cells and did not obscure the morphology in any case.
Out of the 105 cases, conventional smears were taken in 81 patients. These were also categorized as satisfactory or unsatisfactory using the criteria given by the Bethesda system [Table 1].
The general category of negative for intraepithelial lesion or malignancy (NILM) was reported in both groups in 70 cases [Table 2]. In two cases, the MLBC preparation was unsatisfactory and the diagnosis on the conventional smear was high grade squamous intraepithelial lesion (HSIL) and low grade squamous intraepithelial lesion (LSIL), respectively. Four cases of candida, eight cases of bacterial vaginosis and three cases of trichomonas infection were identified on the MLBC preparation only. One case of candida and one case of trichomonas infection were identified on the conventional smear only [Table 3].
One MLBC smear was reported as atypical squamous cells of undetermined significance (ASCUS) [Figure 2] and was reported as NILM on conventional smear [Table 2].
Cytohistologic correlation was done in nine cases. Three patients underwent hysterectomy, cervical stump was excised in two cases, polypectomy was done in one case and biopsies of the cervix were taken in three cases, out of which one was a loop electrosurgical excision procedure (LEEP) biopsy. All cases showed cervicitis on histopathological examination. Squamous metaplasia was reported on histopathology and both conventional and MLBC cytology slides in one case. Koilocytic change was reported in four cases on histopathological examination, but was not reported on either the conventional or the MLBC cytology preparation.
| Discussion|| |
In the manual membrane-based method, 'membrane' refers to the formation of a membrane from a suspension of cells in solution. Cells are suspended in a polymer solution which is then applied to the slide. The polymer solution contains agarose, polyethylene glycol, alcohol and poly-L-lysine.  Drying of the polymer solution forms a uniform dispersion of those cells which are then sealed into a partly insoluble membrane. The membrane holds the cells onto the glass slide. Soluble components are extracted in the first stage of staining.  Cells trapped in the meshwork of the polymer are homogenously distributed within the resulting membrane with the help of a wetting agent. It is the membrane, not the cells, that adheres to the glass slide.  The fixative is decanted from the centrifuged cell button. In our study, the morphology was satisfactory with both fixatives. The MLBC method can be used with a number of fixatives. 
In our study, the MLBC method was found to be comparable to the conventional scrape smear. There was an (72/81) 88.8% agreement in the diagnoses of general category. A good overall agreement between the MLBC method and original cytology diagnosis on SurePath preparations has been reported.  In another study, 3 HSILs and 14 LSILs more were detected in the MLBC slides as compared to the original diagnoses on ThinPrep preparations.  In the present study, two HSILs on conventional smear were diagnosed as unsatisfactory on MLBC. One case of ASCUS was diagnosed on MLBC and was reported as NILM on the conventional smear.
An increased risk of cervical cancer is associated with having multiple sexual partners and with early age at initiation of sexual activity. Human papilloma virus (HPV) is recognized as the primary causal factor in the development of cervical carcinoma.  Screening for carcinoma cervix has an advantage because the natural history of the disease allows it to be detected at the preinvasive stage of dysplasia or carcinoma in situ .  Mortality from cervical cancer has decreased by more than 70% since cervical cancer screening became widely available in the United States.  A single cervical cytology test is associated with a significant false negative rate. The sensitivity of a conventional Pap smear More Details is estimated to be 70-80% and about 85-95% for liquid-based cytology tests. 
In liquid-based methods, the specimen is collected in a preservative solution rather than being directly spread on the slide. Cellular structure is better preserved because the cells are immediately fixed. The process prevents drying artefacts and removes most contaminating mucus, red blood cells bacteria and yeast.  Two methods approved by the Food and Drug Administration (USA) are widely used - the ThinPrep (TP), (Cytyc Corporation, Marborough, MA) and SurePath (SP), (Tripath Imaging Inc., Burlington, NC) (previously known as Autocyte Prep.). TP was approved for cervicovaginal cytology in 1996 and SP in 1999. Both have also been used for nongynecological cytology.  Liquid-based methods add to the cost of a standard Pap smear. However, several studies have reported that liquid-based methods improved the quality of screening through improved specimen adequacy, reduced rate of unsatisfactory smears and increased detection of epithelial abnormalities. ,
The ThinPrep method has been widely reported to improve specimen adequacy and show fewer unsatisfactory results. ,,,, It has been shown to have a higher diagnostic accuracy with a 74% agreement between diagnoses of SIL by TP and biopsy.  A higher number of HSIL and LSIL were detected by ThinPrep. ,,,, The number of false negative results were fewer.  Significantly lower error rates and more accuracy than conventional Pap was found. , In one metaanalysis of 47 articles, the sensitivity of TP was found to be 76% and specificity 86%, as compared to a sensitivity of 68% and specificity of 79% for conventional smears, relative to histology.  No significant difference in false positive rates between conventional and liquid-based methods was found. 
The SurePath method has also been reported to have a lower proportion of unsatisfactory slides. , The cell enrichment process included in SurePath ensures that more amounts of potentially obscuring blood is handled, than the filtration method of the ThinPrep system.  The sensitivity to detect CIN was increased, at the cost of specificity. 
A major advantage of liquid-based collection is that residual specimens can be used for ancillary testing like immunocytochemistry, detection of HPV DNA and studies of DNA methylation. Ongoing research includes immunocytochemical studies of expression of the cell cycle regulator p16 and studies of methylated genes using polymerase chain reaction (PCR). ,, Recently, automated screening devices, viz. FocalPoint and PAPNET, have also been used for screening. , Another alternative inexpensive test, the PapSpin, that uses a conventional centrifuge to deposit the cells from the liquid onto a glass slide, has been reported. 
A large number of women with borderline atypical cytology findings require additional management to determine the presence of a significant lesion follow-up, repeat cytology, or colposcopic examination. Human papilloma virus DNA testing has emerged as another testing modality for cervical cancer and precursor lesions. HPV testing can be used as an adjunct to atypical cytology results, as a primary screening test, as a follow-up test after colposcopy or treatment and as a quality assurance measure.  Further studies are required for HPV testing on residual samples from the MLBC method, using appropriate fixatives.
Visual inspection with acetic acid (VIA) has been widely investigated as an alternative to cytology for the control of cervical cancer in low resource settings  (WHO). Tests for HPV DNA have shown efficacy in the triage of equivocal diagnosis (ASCUS in the Bethesda system). All tests, however, have to be applied within an organized setting, preferably as a component of a National Cancer Control Program  (WHO).
In conclusion, the MLBC method was found to be comparable to the conventional scrape smear. It has potential benefits viz. better morphology, preservation of specimen for ancillary studies and opportunities for research. Hence in view of the advantages of liquid-based cytology, it would be worthwhile to further study this method as a cost-effective alternative to the mechanized methods.
| Acknowledgments|| |
The authors would like to thank Indian Council of Medical Research, New Delhi, for sponsoring a Fellowship of one of the authors. They would also like to thank Johns Hopkins Medical Institutions, Division of Cytology, Baltimore, USA for technical support for the Fellowship.
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Anita N Kavatkar
P 301, Pinnac Memories Co-op Society Phase II, Sr. No 23/3+4, Near Rahul Nagar, Kothrud, Pune - 411 038
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]
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