Comparison of Cervical Cell Morphology Using Two Different Cytology Techniques for Early Detection of Pre-Cancerous Lesions

Cervical cancer is one of the foremost public health concerns after the breast cancer. Eighty-three percent of the global cervical cancer occurs in developing countries where its mortality rate is approximately 80% of 470,000 patients (Parkin et al., 2002). Its involvement in overall cancer burden is substantial in all societies whether developed or developing nations. Cervical cancer causes 270,000 deaths globally per year, major portion of these mortalities belongs to underdeveloped countries (Ferlay et al., 2010). Cervical cancer screening has reduced cervical cancer and its mortality rate up to certain extent (Hicks et al., 2006). The major contributing factors responsible for cervical cancers are sexual activities, lack of education and infectious etiology including Herpes simplex virus, Human Papilloma virus, and HIV (Chih-Ming et al., 2004). Cervical cancer can be prevented by primary as well as secondary measures. Secondary prevention through cytological examination has been the mainstay for early detection of cervical cancer (Nandini et al., 2012). Cervical


Introduction
changes can be perceived much before its progress into invasive carcinoma and this is the basis of cytological screening (Kavatkar et al., 2008). Cytological examination could be very helpful for timely diagnosis of cervical cancer, but this preventive measure is not commonly practiced in developing countries due to scarcity of resources, technical personals and other facilities (Nandini et al., 2012). The reason behind low screening settings in developing countries is little or absolutely no conception of precautionary measures, economic hindrance, social and cultural issues and of course lack of knowledge (Blanks et al., 2007). In Pakistan, the burden of disease is increased, cervical cancer is the 4th common cancer in Pakistani women with an age standardized incidence rate (ASIR) of 6.5 per 100,000 (Sardar et al., 2008).
Number of studies revealed that despite of the higher socio-economic status of South Asian females , the Pap screening is very uncommon. Therefore, the major reason is not low economic resource but lack of awareness (Gupta et al., 2002;Chaudhry et al., 2003;Sutton et al., 2004). Awareness of the population about cervical cancer screening could lead to encouraging steps towards implementation of screening setup in Pakistan (Sardar et al., 2008).
The Pap smear test can diagnose cervical cancer during precancerous conditions therefore it is mostly used as screening tool in developed nations where this test reduce incidences as well as mortality associated with cervical cancer (Nygard et al., 2002;Hewitt et al., 2004;Canfell et al., 2006). Cervical screening by using conventional Pap method rivets the microscopic examination of cell samples taken from the ecto-and endocervix, directly on glass slides and subsequent staining by using the Pap procedure (King et al., 1992).
Timely cervical screening by using Pap method has reduced approximately 50-70% death rate associated with invasive cervical carcinoma, but its sensitivity is affected significantly in the presence of blood cells, mucous and areas of overlapping epithelial cells during specimen collection (Sherwani et al., 2007;Kavatkar et al., 2008). Most parts of slide when prepared for regular Pap smear contain cell debris, inflammatory cells and sheets of epithelial cells that can interfere with correct diagnosis.
Secondly, delay in fixation of smear after taking sample may leads to deformation of cellular morphology. Cells in fact relocating on slide do not correspond to the total number of cells collected, many of them left on the device Xian (2011). Interpretation of results needs technical expert that are hardly available in developing countries. The problems associated with the conventional Pap smear lead to the developments of advanced technologies such as Thin Prep and Sure Path most commonly used in developed countries like UK and USA (Kavatkar et al., 2008;Deshou et al., 2009). Residual sample after LBC can be further utilized in the detection of HPV (Sherwani et al., 2007;Kavatkar et al., 2008). However the techniques, tools, and supplies used for liquid-based methods are costly for resource limited settings (Judy et al., 2006).
Alternately, manual liquid based cytology for cervical screening could be the most appropriate choice (Johnson et al., 2000). The universal stain for cervical cytological screening is Papanicolaou stain. It gives a polychromatic, transparent staining reaction with crisp nuclear and cytological features (Bibbo, 1991). However it utilizes a considerable amount of ethyl alcohol and it is time consuming (Sato et al., 2004). Ethyl alcohol can be replaced by acetic acid with certain modification in conventional pap staining procedure that could be economical and may give satisfactory results in lesser time (Biswas et al., 2008).

Materials and Methods
The current study was conducted on 80 patients from Get Well medical clinic, G-10 Islamabad, Pakistan. Samples were collected with the help of vaginal speculum and cervical broom with detachable head that takes samples from endo as well as ectocervix at a time. The extended central bristles were inserted into cervical canal and short bristles on sides were positioned to take samples from ectocervix. Broom was completely rotated at 360° for five times. Cells collected were transferred on two differently labeled microscopic slide by paint-stroke like motion with one side of brush first and then from other side of brush on same slide. Second slide was also prepared in same manner from same patient. Slides were then fixed immediately with fixative. As study was based on splitsample method, the head of the broom was detached and placed in the pre sterilized and labeled bottle containing preservative prepared in lab of IBMS, KMU, Peshawar. The preservative contained Sodium chloride, sodium citrate, 10% formalin and alcohol (Kavatkar et al., 2008).
Specimen were collected in three different places i.e. two on microscopic slides and one in preservative solution form single patient. Samples were then brought to lab in IBMS for further processing. Specimen were subjected to three different methods named manual liquid based cytology, conventional Pap staining and acetic acid Pap staining.

Manual liquid based cytology
Two slides were prepared from samples carried in preservative from clinic by following manual liquid based cytology procedure. One was stained with standard Papanicolaou stain and other was stained with acetic acid Pap staining. MLBC slides were prepared by the procedure stated in two different studies (Nandini et al., 2012;Kavatkar et al., 2008).
After MLBC we had total four slides from each patient. Two slides obtained directly from clinic and two slides with polymeric film prepared after manual liquid based cytology. One slide from scrape smear and one slide of polymeric film were subjected to standard Papanicolaou staining, while second scrape smear slide from clinic and other polymeric slide after MLBC were subjected to modified acetic acid Pap staining (Biswas et al., 2008).
The samples were blind examined by the supervisor and one other histo-pathologist and interpreted individually. For evaluation of both techniques i.e. conventional Pap smear and manual liquid based cytology, the  Table 1 on the basis of criteria defined in study conducted by (Dwivedi et al., 2012).
Staining quality of four different techniques named standard Pap staining, MLBC Pap staining, acetic acid Papanicolaou and MLBC acetic acid pap staining were assessed by nuclear and cytoplasmic staining quality with scoring from 0 to 3 given in Table 2. (Alves et al., 2004;Biswas et al., 2008).

Quality index
After individual evaluation of screening techniques on the basis of morphological and diagnostic parameter and subsequent assessment of staining techniques through nuclear and cytoplasmic staining, overall evaluation of best techniques among smear preparation and staining was done by means of quality index originally devised by Chan et al., 1988. The maximum score possibly attained by one case, after keeping in view for all of the six morphological parameters could be 14 and two staining parameters , could be 4. One the basis of single case the highest attainable score with one technique was calculated by multiplying the number of subjects in study with maximum possible score for each evaluation. A "Quality Index" was then found by the ratio of obtained score by maximum score: Quality index=actual score obtained/maximum score possible.

Diagnostic parameters
Total 80 cases were selected for screening study and while performing MLBC, membrane remained intact in 66 cases and was disrupted in rest of 14 cases. Sixty six cases with intact membrane were categorized as satisfactory and unsatisfactory on the basis of Bethesda system (Solomon et al., 2001). While doing MLBC fifty cases were satisfactory and out of remaining, fourteen contained more than 75% inflammatory infiltrate and blood however, two cases are lack of transformation zone. Duplicate slides of same 66 patients from gynecology clinic were subjected to CPS cytology, out of which forty-seven cases were satisfactory and remaining nineteen were unsatisfactory.
In the whole forty-seven cases that were regarded as satisfactory on the basis of both MLBC and CPS were sorted only for normal smear and inflammatory smear for comparison of two techniques. Eighteen cases were reported with inflammatory smear and twenty nine cases with normal smear when performed with MLBC, whereas twenty two cases were seen with inflammatory smear and 25 were diagnosed as normal smear with CPS shown in Table 3.

Morphological parameters Cellularity
Adequacy: in current study normal conventional Pap Smear (CPS) contains fewer well preserved superficial squamous cells and intermediate squamous cells with normal morphology however; in Manual Liquid Based Cytology (MLBC) stained by both acetic acid as well as    School of Pathology and ≥8000 for CPS. All samples smears that were selected contained enough cell count to satisfy Bethesda system. Most of the samples contains the cells from 4 to 6 cells in field with 405 objective and 22 eye piece, which means they contains more than 5000 cells Figure 1.    Pap staining, there is increased cellularity for both types of well-preserved cell that improves the chances of adequacy for smear selection in Bethesda scrutiny revealed by Figure 1. However in case of Inflammatory smear CPS is not free from obscuring blood and inflammatory cells whereas MLBC gives more clear picture. When slides of MLBC smear follows acetic acid staining, they give results with even more clear background as shown in Figure 2. Cellular count: recommended cellular count for sample selection is ≥5000 for MLBC according to American DOI:http://dx.doi.org/10.7314/APJCP.2014.15.2.975 Comparison of Cerevical Cytology Findings Using a Modified Staining Technique scoring index of staining quality shown in Table 4.

Statistical analysis
Morphological parameters like uniformity of distribution and folded cytoplasmic borders have P value greater than 0.005 (p>0.005) showing no significant difference between two techniques. i.e. CPS and MLBC with P value 0.471 and 0.108 respectively by using ANOVA. However, p<0.005 for both cellular overlapping and cleanliness of background showing highly significant difference between two techniques as shown in Table 7. Average scores of all four parameters are shown in Figure  8.
Statistics involved in comparison of both nuclear and cytoplasmic staining with all four staining techniques is highly significant with p<0.005 for cytoplasmic staining and p<0.005 for nuclear staining by using ANOVA. The average score is shown in Figure 9.

Discussion
Number of studies were performed for comparison of Conventional Pap smear and Manual liquid based cytology by using standard Pap staining in subsequent steps of staining. However, in current study both techniques were compared by using standard Pap staining method and modified acetic acid Pap staining individually on the basis of different morphological parameters, nuclear staining and cytoplasmic staining. Despite of extensive use of CPS for cervical screening this method has certain shortcomings (Deshou et al., 2009). Maksem et al., suggested the new method in 2001 that could possibly reduce these problems, this new method involves the use of polymeric membrane and named as Manual liquid based cytology. (Maksem et al., 2001). In this method membrane is formed from polymeric solution containing Carbowax and, agrose (Maksem et al., 2006) in which cells are suspended. Polymeric film is then applied on microscopic glass slide (Maksem et al., 2001).
In current study we have compared the clarity and overall beneficial aspects of MLBC technique and  When compare three systems MLBC stained with acetic acid give best results in view of three parameters uniformity of distribution, clear background and artifacts as shown in Figure 3, however, MLBC followed by Pap staining give intermediate results as revealed by Figure 4.

Cellular overlapping and folded cytoplasmic boarders
MLBC shows little to moderate cellular overlapping having score 2 shown in Figure 4. When compared with CPS that gives cellular overlapping with score 1 in more than 80% of cases, Figure 5 However, there is no significant difference in cytoplasmic border folding in both methods. Quality index for both screening technique is given in Table 4. However, individual scores of each morphological parameter is given in Tables 5 and 6.

Staining parameters
Staining of four methodologies was compared on the basis of scoring for two parameters i.e. nuclear and cytoplasmic staining. Acetic acid papanicolaou staining technique gives maximum scores i.e. 45 good category, 18 satisfactory and 3 unsatisfactory defined in material and methods for nuclear staining as shown in Figure  6. however, scores were 47 excellent, 14 optimum, and 5 poor for cytoplasmic staining given in Figure 7. Remaining three staining techniques are categorized as modified acetic staining of MLBC slides, MLBC stained by Pap stain and CPS in descending order based on the conventional Pap smear test in terms of scoring that could lead us to a better method for diagnosis. In present study different factors like cost effectiveness, time economy and better preservation of marked nuclear as well as cytoplasmic features of modified acetic acid pap staining are amalgamated with advantageous features of MLBC techniques which was then compared with simple Pap smear test, acetic acid papanicolaou staining and conventional Pap staining to map out for the most acceptable technique which could be better for cervical screening with respect to time economy, cost, clarity of features and of course correct diagnosis.
In our study 27% cases were unsatisfactory according to CPS and 24% cases were found unsatisfactory with MLBC due to scant cell, presence of blood and mucous. The percentage of unsatisfactory smears is much lower in studies like in Bergeron et al.,reported (0.14%), Garbar et al., 2005 found (0.9%). The only possible reason behind high percentage in our study could be the use of expensive automated methods in those studies, however in current study manual method was used for membrane preparation.
In LBC, the sample is first placed in fixative and then proceed for further processing instead of making slides directly as in CPS, thereby cellular structure are more well preserved and reduced drying artifacts as cells are immediately fixed Brud (2003). According to one study LBC enhance the specimen adequacy, decrease the count of unsatisfactory smears and improves screening results (Celik et al., 2008). Liquid based cytology also improves adequacy by increase in cellularity (Nandini et al., 2012). Similarly, in current study cellular structure are more well preserved in MLBC slides as compare to CPS, therefore MLBC gave more clear picture of cellularity which is beneficial for correct diagnosis. In MLBC there is marked decrease in artifacts, contaminating mucus and blood. Cells are evenly distributed on slides and centrifugation in this method offers a proper mixing (Afaf et al., 2012). Likewise in present study the average scoring of morphological parameters like cellular overlapping, clarity of background and artifacts is significantly different in both techniques. Manual liquid based cytology gives more clear results with clear background, less artifacts and lesser degree of cellular overlapping when compared with conventional Pap smear. However folded cytoplasmic borders are observed equally in slides prepared by both techniques and no significant difference is observed between these two techniques for folded cytoplasmic borders statistically. Cells are evenly distributed in slides prepared by MLBC as compared to Pap smear test in current study.
Visual inspection with acetic acid (VIA) has been extensively used for cervical cytology in economically poor countries, which encourages the use of acetic acid in staining of Pap smear slides in this study. In modified acetic acid papanicolaou, staining 1% acetic acid is used instead of ethyl alcohol in various steps. Acetic acid is used as dehydrating agent and it is economical and easily available (Dighe et al., 2006). In this study, all four techniques named acetic acid papanicolaou staining, conventional Pap staining, MLBC acetic acid staining and MLBC Pap staining are compared on the basis of two staining parameters i.e. nuclear staining and cytoplasmic staining. All four staining methods gave maximum number of excellent scoring followed by optimal and then poor score in cytoplasmic staining. But the result of average score of cytoplasmic staining was significantly different statistically due to number of cases in excellent, optimum and poor category. Excellent staining revealed maximum transparent cytoplasm, poor might be due to lesser penetration in thick part of cytoplasm (Biswas et al., 2008) In techniques which use membrane method, overall the result was clear as compare to acetic acid papanicolaou staining and CPS because membrane method removed unnecessary waste material like blood, mucous and debris and as cellular overlapping is also very few in this method, stain was evenly distributed in cytoplasm and features are more prominent. As for as nuclear staining is concerned acetic acid papanicolaou staining gave sharp nuclear features and crisp nuclear chromatin in maximum number of cases, however there were moderate number of satisfactory cases which are acceptable for diagnosis and only few cases were unsatisfactory but overall average score is maximum for modified acetic acid papanicolaou staining. In satisfactory score, chromatin is mildly hazy with little diffused nuclear features. MLBC acetic acid gave highest results in good category as in acetic acid papanicolaou staining but few in number followed by satisfactory and unsatisfactory category in descending order, rest of two staining methods MLBC Pap and CPS gave overall acceptable score for correct diagnosis.
By using two different methods for screening, some morphological features attained maximum score by using one techniques, while others got equal score by both techniques. To decide the overall favorable screening technique, quality index was calculated that was based on the ration of individual score to the maximum attainable score by considering all five morphological parameters. The quality index of MLBC is higher with value of 0.65 as compare to CPS having value 0.56, which means MLBC is more superior techniques for screening as compare to CPS. However, for quality index for staining quality of cytoplasm and nucleus, modified acetic acid staining method is very favorable with index of 0.82 followed by MLBC with modified acetic acid pap staining having index value 0.77. Conventional Pap staining is acceptable with index score of 0.59.
In conclusion, it is concluded from present study that MLBC methodology could be used for cervical screening in low resources setting instead of liquid based cytology and the slides prepared by MLBC can be stained with modified acetic acid pap staining, this can compensate the time and the cost additionally required by MLBC instead of conventional pap smear test.