recent concepts in cytology for early detection of ... · preparation of cell blocks [4-6].however...

1Cervical Cancer: Recent Research and Review Studies | www.smgebooks.comCopyright Manoli N.This book chapter is open access distributed under the Creative Commons Attribution 4.0 International License, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited.

Gr upSMRecent Concepts in Cytology for Early

Detection of Cervical Cancer

ABSTRACTWith 528000 new cases every year, cervical cancer is the fourth most common cancer

affecting women worldwide, after breast, colorectal, and lung cancers; it is most notable in the lower-resource countries of sub-Saharan Africa. It is also the fourth most common c Liquid-based cytology has recently become an alternative to conventional pap smears in the detection of intraepithelial lesions as well as in invasive carcinoma of the uterine cervix. Several reports have discussed its benefits to cytologic diagnosis. In most of these reports, a significant rise in sensitivity was achieved with liquid-based procedures, without major losses in specificity. a use of cancer death (266000 deaths in 2012) in women worldwide.

LBC reduces the proportion of inadequate smears, caused by obscuring factors like blood and inflammation. It leads to an increase in the number of cellular abnormalities detected because it forms a mono-layered suspension of cells with minimal over-lapping. LBC reduces the screening time, resulting in greater efficiency of the pathologist who can handle a larger number of preparations. The single most important advantage is the opportunity to use the residual sample for additional testing, including molecular studies for the presence of infectious agents, such as HPV-DNA and preparation of cell blocks.

Nandini Manoli1, Nandish Manoli2, Akshata kamath1 and Shweta kulkarni1

1Department of Pathology, JSS Medical College, a Constituent of JSS University, India2 Department of Obstetrics and Gynecology, JSS Medical College, a Constituent of JSS Univer-sity, India

*Corresponding author: Nandini Manoli, Department of Pathology, JSS Medical College, a Constituent of JSS University, India, Tel: 9448978276 Email: [email protected], [email protected]

Published Date: January 26, 2016


We have made an attempt to study manual liquid based cytology(MLBC) and compared it with conventional pap smear (150cases) with study on an ancillary technique of cell block with immunocytochemistry(IHC) (60 cases). The study finds MLBC as a comparable method to adapt to in resource poor settings like ours and ancillary techniques like cell block can be applied with IHC markers studied were p16inka4a, and ki67.which showed good responses.

Keywords: Manual Liquid Based Cytology, Cervix, cytology, Ancillary techniques, Cell block, Immunocytochemistry

INTRODUCTIONWith 528000 new cases every year, cervical cancer is the fourth most common cancer affecting

women worldwide, after breast, colorectal, and lung cancers; it is most notable in the lower-resource countries of sub-Saharan Africa. It is also the fourth most common cause of cancer death (266000 deaths in 2012) in women worldwide. Almost 70% of the global burden falls in areas with lower levels of development, and more than one fifth of all new cases are diagnosed in India [1].

It is the most common form of cancer among Indian women and accounts for an estimated 1,00,000 cases developing annually in the country [2,3]. (Figure 1)

Figure 1: Globscan world incidence of cervical cancer 2012.


The conventional Papanicolaou test remains a main tool for cervical cancer screening in the general population in developing countries in spite of unrealistic expectations about its performance, which entails false negative results even in optimal screening programs [4,5].

Liquid-based cytology has recently become an alternative to conventional pap smears in the detection of intraepithelial lesions as well as in invasive carcinoma of the uterine cervix. Several reports have discussed its benefits to cytologic diagnosis. In most of these reports, a significant rise in sensitivity was achieved with liquid-based procedures, without major losses in specificity. Some studies have shown more accurate diagnosis of glandular lesions [4,5].

There are numerous advantages of LBC over CPS. LBC reduces the proportion of inadequate smears, caused by obscuring factors like blood and inflammation. It leads to an increase in the number of cellular abnormalities detected because it forms a mono-layered suspension of cells with minimal over-lapping. LBC reduces the screening time, resulting in greater efficiency of the pathologist who can handle a larger number of preparations [6].

The single most important advantage is the opportunity to use the residual sample for additional testing, including molecular studies for the presence of infectious agents, such as HPV-DNA and preparation of cell blocks [4-6]. However the technology, equipment and supplies for these liquid-based sample preparation methods are costly. Trained individuals and periodic maintenance of the equipment are required. Unfortunately, the cost of most commercially available LBC systems is prohibitively expensive for resource-limited settings [4,5].

Many workers like Alves et al. [4], Lee et al. [5], Maksem JA et al. [7] and Kavatkar AN et al. [8]

have worked on an improvised manual method for preparation of liquid based cytology smears for cervical cancer screening and found it comparable with CPS.

The advent of fluid based collection of gynaecologic cytologic specimens has provided the possibility of cell block preparation and monolayer cytologic specimens from these specimens [9].

Cell blocks prepared from residual tissue fluids and fine-needle aspirations can be useful adjuncts to smears for establishing a more definitive cytopathologic diagnosis and they can be a valuable diagnostic adjunct in cytological preparation in selected fluid based cervico vaginal specimens [9,10].

Cell blocks increase specificity and/or reduce false-positive results in the same manner as do cell blocks within non gynaecologic cytology [11,12].

Prospective studies on cytological material are hampered by insufficient amounts of diagnostic specimens from the same patient. Cell block processing methods of cytological material may solve this problem [13].

Here we present a similar study where in the cell blocks are prepared by adding equal amount of cell block fixative to the deposit got during manual liquid based cytology (MLBC) procedure. The diagnostic aid and accuracy of cell blocks is studied and evaluated.


LBC is currently recommended for cervical cancer screening [14] with a major advantage of allowing ancillary techniques such as those used in immunocytochemistry and molecular biology [15-21] cell blocks made from cell or tissue remnants of LBC can also be used for immunocytochemistry or imunohistochemistry of specific biomarkers for accurate diagnosis of malignancy [20-23].

The use of biomarkers has demonstrated the ability to overcome the issues with both false positive and false negative results, leading to an improved positive predictive value of cervical screening results.

Numerous protein bio-markers for the detection of cervical disease have been identified. Many of these proteins are involved in cell cycle regulation, signal transduction, DNA replication and cellular proliferation.

Biomarkers currently under investigation for use in cervical cancer screening that appear to improve the detection of women at greatest risk for developing cervical cancer, include Ki-67, P16INK4A, BD pro ex c and HPVL1. These biomarkers are reported to have a role in the triage of indeterminate cytology cases, discrimination of true high grade cervical dysplasia from mimics in histology and may serve as predictive markers to identify lesions most likely to progress to high grade cervical disease and cancer [24].

METHODOLOGYSamples were collected using the split-smear technique from 150 patients (sample size) in the

age group of 20 to 80 years attending the gynaecology out-patient clinic at JSS Hospital, Mysore. All the patients were clinically examined in detail according to the proforma and details of other relevant laboratory investigations were collected only if necessary. Material collected was stained by Pap stain.

Ayre’s spatula was used to scrape the cervix. It was immediately put into a vial containing the fixative solution. The fixative contained 0.5 gm of sodium chloride, 0.5 gm of sodium citrate, 50 ml of 10% formalin and 50 ml of isopropyl alcohol in 100ml. The sample was collected and mixed with equal parts of fixative. It was centrifuged at 2000 rpm for 5 minutes. The supernatant was decanted and excess fixative was blotted.

1-2 ml of polymer solution (containing 2 gm of agarose, 10 ml of polyethylene glycol, 2 ml of poly-L-lysine and 88 ml of distilled water) was added to the deposit. It was again centrifuged at 2000 rpm for 5 minutes. The deposit was pipetted in a circular motion on to a glass slide. The slides were placed on a metal tray and dried in an hot air oven at 50 degrees (Indirect heat fixation) and further fixed by dipping in 95% alcohol for 5 minutes. Stained with the conventional Pap stain.

Simultaneously, scrape smears were also collected using Ayre’s spatula. The scrapings were evenly spread onto a glass slide, and immediately fixed in ethyl-alcohol fixative (95% ethyl alcohol). After fixation, smears were stained using conventional Pap stain.


Both smears were screened and the results were compared. Cyto-histologic correlation was done in those cases in which a colposcopic biopsy was also done. The Bethesda system 2001 was used for reporting cervical cytology for both groups.

Whenever possible, ancillary techniques were applied for preparations of cell blocks and HPV- DNA testing from the residual cytocentrifuged sample.

The cases in which a colposcopic biopsy or hysterectomy was done, specimens were collected in 10% formalin and allowed to fix overnight. The detailed gross examination was done and bits were given. Paraffin embedded H and E stained sections were obtained and studied under light microscopy.

To extend our work on ancillary techniques we took the study further , to work on cell block as a ancillary technique with IHC on cell blocks whenever necessary. The cell blocks studied were lesser than the liquid based cytology cases.

The study was undertaken to prepare the cell blocks from samples of manual liquid based cytology (MLBC) and to compare it with conventional pap smears and liquid based smears and correlate with histopathology wherever possible.

Source of dataSamples of MLBC of which cell block preparation was made were collected from about 60

patients in the age group of 20 to 70 years with gynaecological complaints like white discharge P/V and bleeding P/V attending the gynaecology out-patient clinic at JSS Hospital, Mysore.

All the patients were clinically examined in detail according to the proforma and details of other relevant laboratory investigations were collected only if necessary.

Sampling procedure1. Samples were collected by a plastic Ayre’s spatula from the transformation zone of the cervix

by 360○ rotation around the cervix.

2. The Sample was then transferred on a clean glass slide and fixed by a spray fixative and after fixation they are stained by Papanicolaou (pap) stain.

3. Another rotation is taken and this sample is transferred to the liquid fixative.

The cell block is prepared from the liquid fixative deposit by adding equal amount of cell block fixative (10% formalin and 95% alcohol), got during MLBC procedure.

The cell block obtained is routinely processed as a histopathological tissue and stained with routine hematoxylin and eosin stain.The conventional smears, MLBC smears and cell blocks were screened and compared.

In the present study ki 67 and p16 markers were done on cell block preparations.


Table 1: Age-wise distribution of cases.

Age Group Number of cases Percentage (%)

11-20 04 2.7

21-30 27 18.0

31-40 48 32.0

41-50 46 30.6

51-60 15 10.0

61-70 05 3.3

71-80 01 0.6

81-90 01 0.6

99 03 2.0

Total 150 100

RESULTSThe present study was undertaken to prepare cervical cytology smears using the manual

method of liquid-based cervical cytology (MLBC) and compare it with the conventional Pap smears, observe morphological features and correlate with histopathological findings in suspicious cases of malignancy to determine its diagnostic accuracy.

During the period of this study from July 2009 to July 2011, a total of 1652 Conventional Pap smears were obtained, comparative study for 150 cases of Conventional Pap smear with Manual liquid-based cytology were prepared, accounting for 11%. Among 150 cases, histopathological correlation was obtained in 44 cases.

AgeAge group of patients ranged from 20 years to 80 years with the youngest patient aged 19

years and oldest 88 years. Mean age was 41.76 years. Majority of the patients were in the age group of 31-40 years. (Table 1)

Sample adequacySatisfactory cell samples were obtained in 147 cases out of the 150 cases.

Cytology In the present study 150 cervical smears prepared by MLBC were studied, out of which 88

cases (58.6%) were reported as non-neoplastic lesions and 62 cases (41.4%) were reported as neoplastic lesions. (Table 2)

Table 2: Distribution of cases into non-neoplastic and neoplastic.

Lesions No. Percentage (%)

Non neoplastic 88 58.6

Neoplastic 62 41.3

Total 150 100


Out of the 150 cases, the most common non-neoplastic lesion was inflammatory smear (31 cases), followed by normal study (22 cases), bacterial vaginosis (15 cases), menopausal/atrophic smear (7 cases), HPV and leptothrix infestation (3 cases each), candidal infestation and Trichomonas vaginalis (2 cases each). (Figure 2-10)

Fig 2 a) b) c)Figure 2: (a) MLBC (Pap, 40 X)- NILM- Smear is satisfactory and shows superficial and intermediate

squamous cells of normal morphology. Background is clear. (b) CPS (Pap, 10 X)-NILM- Smear is satisfactory and shows superficial and intermediate squamous cells of normal morphology with overlapping of cells.

Background shows numerous RBCs and mucus. (c) HPE coorelation (5x, H and E).

Fig 3 a) b cFigure 3: (a) MLBC (Pap, 40 X)- Inflammatory Smear- Smear is satisfactory and shows superficial and

intermediate squamous cells of normal morphology with quite a few inflammatory cells in the background. Cluster of squamous metaplastic cells noted. (b) CPS (Pap, 10 X) - Inflammatory Smear- Smear shows

superficial and intermediate squamous cells. Morphology is not clear due to obscuring factors like blood and large number of inflammatory cells in the background.

Fig 4 a) b)Figure 4: a) MLBC (Pap, 40 X)-Bacterial Vaginosis- Smear shows superficial and intermediate squamous

cells. Clue cells noted. Background shows a few inflammatory cells. b) CPS (Pap, 40 X) - Bacterial Vaginosis- Smear shows superficial and intermediate squamous cells with overlapping of cells. Clue cells noted.

Background shows abundant acute inflammatory cells.


Fig 5 a) b)

Figure 5: (a) MLBC (Pap, 40 X) - Atrophic smear- Smear shows predominantly intermediate squamous cells. (b) CPS (Pap, 10 X) - Atrophic smear- Smear shows a cluster of predominantly intermediate

squamous cells with a few superficial squamous cells with overlapping of cells.

Figure 6: (a) MLBC (Pap, 40 X)- HPV- Smear shows good number of koilocytes with perinuclear halos. (b) CPS (Pap, 40 X) - HPV- Smear sho ws good number of koilocytes with perinuclear halos, Numerous

superficial and intermediate squamous cells noted. Background shows abundant acute inflammatory cells. (c) Cell Block (H and E, 40 X) - HPV- Section shows superficial squamous cells with many of them showing

koilocytic changes. A few inflammatory cells seen amidst these cells. (d) HPE, (H and E,10X) showing koilocytic change in cervix.

Fig 6 a) b)

c) d)


Fig 7 a) b)Figure7: (a) MLBC (Pap, 40 X) - Candidal Infestation- Smear shows candidal spores and hyphae. (b) CPS

(Pap, 10 X)-Candidal infestation- The smear was obscured by clusters of overlapping cells, large number of inflammatory cells and RBCs.

Fig 8 a) b)Figure 8: (a) MLBC (Pap, 40 X) - Leptothrix infestation- Long curving filamentous organisms,

morphologically consistent with Leptothrix seen. A few superficial and intermediate squamous cells noted. (b) CPS (Pap, 10 X) –Inflammatory Smear- Smear was obscured by overlapping numerous superficial and

intermediate squamous cells.

Fig 9 a) b)Figure 9: (a) MLBC (Pap, 40 X) - Trichomonas Vaginalis-Smear shows round to oval cyanophilic organisms

with eccentrically located vesicular nucleus. Neutrophils are seen attached to squamous cells. (b) CPS (Pap, 10 X) - Trichomonas Vaginalis-Smear shows numerous round to oval cyanophilic organisms with

eccentrically located vesicular nucleus. Few superficial and intermediate squamous cells seen. Background shows numerous acute inflammatory cells.


The most common neoplastic lesion was LSIL accounting for 41 cases, followed by squamous cell carcinoma (8 cases), adenocarcinoma, HSIL and atypical glandular cells (4 cases each) and 1 case as high-grade carcinoma. (Figure 11-14). 3 cases were diagnosed as unsatisfactory. (Table 3)

Fig 10 a) b) c)Figure 10: (a) MLBC (Pap, 40X) Herpes virus infestation- Smear shows superficial and intermediate squamous cells. Occasional cells show multinucleation. Background shows a few inflammatory cells.

(b) CPS (Pap, 40X) Herpes virus infestation- Smear shows superficial and intermediate squamous cells. Multinucleated giant cell noted. (c) HPE of cervix showing Bowen”s disease(H&E,20X).

Fig 11 a) b) c)Figure 11: (a) MLBC (Pap, 40 X)-LSIL- Smear shows superficial and intermediate squamous cells with

many of them showing nucleomegaly and hyperchromasia. The nucleus occupies less than half the area of the cell. (b) CPS (Pap, 10 X) - LSIL- Corresponding CPS showing over -lapping of dysplastic cells with

background showing abundant inflammatory cells (c) HPE (H&E, 40 X) - LSIL- Section shows crowding of cells in the basal third of the epithelium. Nuclei are enlarged, hyperchromatic and show loss of polarity.

Koilocytes are seen.

Fig 12 a) b)


c) D)Figure 12: (a) MLBC (Pap, 40X)-Squamous cell carcinoma (Keratinizing) - Smear shows pleomorphic cells with high N/C ratio, arranged in singles and small clusters. The cells have dense orangeophilic cytoplasm, large round to oval vesicular nucleus with coarse granular chromatin and irregular nuclear membranes.

Tadpole cells seen and ‘clinging diathesis’ noted. (b) CPS (Pap, 40 X) -Squamous cell carcinoma (Keratinizing) - Smear shows pleomorphic cells with high N/C ratio, in singles and small clusters. The cells have dense orangeophilic cytoplasm, large round to oval vesicular nucleus with coarse granular chromatin

and irregular nuclear membranes. Tadpole cells seen. Tumor diathesis noted in the background. (c) Cell Block (H & E, 40 X) - Squamous cell carcinoma-Section shows a tumor displaying features of squamous cell carcinoma. (d) HPE (H & E, 10 X) - Squamous cell carcinoma- Section shows a tumor displaying features of

squamous cell carcinoma.

Fig 13 a) b cFigure 13: (a) MLBC (Pap, 40 X) - Endocervical Adenocarcinoma- Smear shows pleomorphic cells with

high N/C ratio arranged in clusters and acinar pattern. The cells have large scanty cytoplasm, large round to oval vesicular nuclei. Background shows a few inflammatory cells. (b) CPS (Pap, 10 X) - Endocervical Adenocarcinoma- Smear shows pleomorphic cells with high N/C ratio arranged in clusters and acinar pattern. The cells have large scanty cytoplasm, large round to oval vesicular nuclei. Background shows

numerous squamous cells and abundant inflammatory cells. (c) HPE (H & E, 10 X) - Endocervical Adenocarcinoma-Section shows a tumor displaying features of endocervical adenocarcinoma.


Fig 14 a) b cFigure 14: (a) MLBC (Pap, 40 X) - HSIL - Smear shows superficial and intermediate squamous cells with

many of them showing nucleomegaly, hyperchromasia and nuclear membrane irregularities. The nucleus occupies more than two- third area of the cell. (b) CPS (Pap, 10 X)- HSIL- Smear shows superficial and

intermediate squamous cells with few clusters of dysplastic cells obscured by cellular overlapping , acute inflammatory cells and RBCs. (c) HPE (H&E, 45 X) - HSIL- Section shows crowding of cells in the basal two-third of the epithelium. Nuclei are enlarged, hyperchromatic and show loss of polarity. Few mitotic figures

noted.

Table 3: Morphological distribution of cases diagnosed on MLBC.

SL NO. MLBC Diagnosis Number of cases Percentage (%)

1 Inflammatory 31 20.7

2 Normal 22 14.7

3 Bacterial Vaginosis 15 10.0

4 Menopausal/Atrophic 7 4.7

5 HPV 3 2.0

6 Leptothrix 3 2.0

7 Candida 2 1.3

8 Trichomonas Vaginalis 2 1.3

9 LSIL 41 27.3

10 SCC 8 5.3

11 Adenocarcinoma 4 2.7

12 HSIL 4 2.7

13 Atypical glandular cell 4 2.7

14 High-grade carcinoma 1 0.7

15 Unsatisfactory 3 2.0

Total 150 100


The 150 cases were compared with the conventional Pap smears obtained simultaneously. On the conventional Pap smears, the most common non-neoplastic lesion was inflammatory smear (55 cases), followed by normal study (24 cases), menopausal/atrophic smear (13 cases), bacterial vaginosis (9 cases), HPV and trichomonas vaginalis (2 cases each). No cases of candidal and leptothrix infestation were identified.

The most common neoplastic lesion was LSIL accounting for 16 cases, followed by squamous cell carcinoma (6 cases), HSIL (4 cases), adenocarcinoma (3 cases), atypical glandular cells and high-grade carcinoma (1 case each). 14 cases where diagnosed as unsatisfactory. (Table 4)

Table 4: Morphological distribution of cases diagnosed on CPS.

SL NO. CPS Diagnosis No. of cases Percentage (%)

1 Inflammatory 55 36.7

2 Normal 24 16.0

3 Menopausal/Atrophic 13 8.7

4 Bacterial Vaginosis 9 6.0

5 HPV 2 1.3

6 Trichomonas vaginalis 2 1.3

7 Candida 0 0

8 Leptothrix 0 0

9 LSIL 16 10.7

10 SCC 6 4.0

11 HSIL 4 2.7

12 Adenocarcinoma 3 2.0

13 Atypical glandular cell 1 0.7

14 High-grade carcinoma 1 0.7

15 Unsatisfactory 14 9.3

Total 150 100

Cellularity was adequate in most of the MLBC cases while the number of unsatisfactory smears were more in CPS cases. Clean background was observed in all cases of MLBC which was seen in majority of CPS. Uniform distribution was seen in MLBC but cellular overlapping was seen more in CPS than in MLBC. Artifacts were present in most CPS samples. Architectural and cellular morphologic change were present in most of CPS samples. Inflammatory infiltrate were prominently present in CPS but decreased in MLBC cases. Nuclear changes were very clear by MLBC, but not very clear by CPS. (Table 5, 6)


Table 5: Comparison of 8 morphological parameters between the 150 MLBC and CPS cases.Morphological Features MLBC CPS

Cellularity Unsatisfactory in 3 cases Unsatisfactory in 14 cases

Clean Background Present Absent and obscuring factors noted

Uniform Distribution Present Absent

Artifacts Rare, if present due to heat fixation Present usually, drying artifacts

Cellular overlapping Rare, monolayer in most of the cases Marked

Architectural changes Rare Present

Cellular morphologic change Absent Present

Nuclear changes Clearly seen Usually obscured

Table 6: Comparison of diagnosis on MLBC V/s CPS.SL NO. Categories MLBC cases CPS

1 Inflammatory 31 24

2 Normal 22 55

3 Menopausal/Atrophic 7 13

4 Bacterial Vaginosis 15 9

5 HPV 3 2

6 Trichomonas vaginalis 2 2

7 Candida 2 0

8 Leptothrix 3 0

9 LSIL 41 16

10 SCC 8 6

11 HSIL 4 4

12 Adenocarcinoma 4 3

13 Atypical glandular cell 4 1

14 High-grade carcinoma 1 1

15 Unsatisfactory 3 14

Total 150 150

Histopathological Correlation was obtained in 44 cases out of the 150 cases. MLBC diagnosis correlated with 34 cases (77.27%) and 10 cases (22.73%) did not correlate with the MLBC diagnosis. (Table 7)

Table 7: Cytohistopathological correlation of MLBC.

SL NO. Histopathological Correlation No. of cases Percentage (%)

1 Yes 34 77.27

2 No 10 22.72

Total 44 100


Statistical analysisMLBC is more sensitive than CPS in diagnosing LSIL, but CPS is more specific than MLBC in

diagnosing LSIL. CPS is more sensitive than MLBC in diagnosing inflammatory smears, but MLBC is more specific than CPS in diagnosing inflammatory smears. (Table 8, 9)

Table 8: Sensitivity and Specificity of MLBC and CPS in diagnosis of LSIL.Method Sensitivity Specificity

MLBC 100 77.7

CPS 62.5 100

Table 9: Sensitivity and Specificity of MLBC and CPS in diagnosis of Inflammatory smears.Method Sensitivity Specificity

MLBC 77.7 100

CPS 100 62.5

Concordance Rate of MLBC/Histopathology Vs CPS/Histopathology for LSIL is 81.8% Vs 45.4%. Increased Detection Rate for LSIL is 157 %.

The further study was done between 2011 to 2013. Of 60 cases in our study, 10 cases had no deposit and cell block diagnosis could not be offered. This inadequate material may be due to sampling errors. Of the rest 50 cases with adequate material 7 were neoplastic and 43 were non- neoplastic. (Table 10)

Table 10: Distribution of cases into non-neoplastic and neoplastic.Lesions No. Percentage (%)

Non neoplastic 43 86

Neoplastic 7 14

Total 50 100

43 non-neoplastic lesions include chronic polypoid endocervicitis( CPEC- 10 cases), Koilocytes ( 3 cases) , chronic cervicitis (12 cases), normal study ( 10 cases) ,acute on chronic cervicitis (7 cases), only hemmorhage (1case).

Neoplastic lesions included squamous cell carcinoma SCC (1 case), dysplasia ( 2 cases) ,carcinoma in situ (4 cases). (Table1)

Adequacy of samples10 cases of the cell blocks had no deposits. Of these only 3 cases were reported as satisfactory

on conventional smears and 7 cases were unsatisfactory on conventional smears and had inadequte samples for MLBC smear. This shows that adequte sampling procedure plays a major role for the better results by cell blocks.

Cases reported as No deposits were maximum when MLBC had inadequte material and were minimum when LBC smears were not done and sediment was directly processed for cell blocks.


Comparison of neoplastic lesions diagnosed on cell blocks with CPS and MLBC

7 cases of cell blocks were diagnosed as neoplastic including carcinoma in situ. They included squamous cell carcinoma SCC (1 case), dysplasia (2 cases), carcinoma in situ CIN (4 cases).

SCC was diagnosed as HSIL in CPS and LBC. 1case of dysplasia was diagnosed as inflammatory on CPS and MLBC and other case of dysplasia was diagnosed as HSIL on CPS and MLBC. 1 case of CIN was unsatisfactory on CPS and 3cases of CIN were diagnosed as HSIL and LSIL. (Table 11,12)

Table 11: Morphological distribution of cases diagnosed on cell block.SI.NO Cell block diagnosis No of cases percentage(%)

1 SCC 1 1.7

2 CIN 3 1 1.7

3 CIN 2 2 3.3

4 CIN 1 1 1.7

5 Dysplasia 2 3.3

6 Chronic cervicitis(CC) 12 20

7 CPEC 10 16.7

10 Normal study 10 16.7

8 Acut on chronic cervicitis (ACC) 7 11.7

9 Koilocytes 3 5

10 Only hemorrhage 1 1.7

11 No deposit 10 16.7

12 Total 60 100

Table 12: Comparison of neoplastic lesions diagnosed on cell bocks with CPS and MLBC.SL NO CPS diagnosis LBC diagnosis CB diagnosis

1 HSIL HSIL SCC

2 LSIL HSIL CIN3

3 LSIL - CIN2

4 LSIL - CIN2

5 Unsatisfactory LSIL CIN1

6 Inflammatory Inflammatory Dyspalsia

7 HSIL HSIL Dysplasia

Total 7 5 7

This shows that cellular architecture is better preserved and neoplastic lesions which are under diagnosed on CPS and MLBC can be correctly diagnosed on cell blocks. (Table 8)

Comparison of cell block diagnosis with histopathologyHistopathologic correlation was obtained in 19 cases, of these 4 cases were neoplastic and 15

cases were non neoplastic. Of 19 cases, cell block diagnosis correlated with histopathology in 14 cases. (Table 13)


Table 13: Histopathological correlation of cell blocks and CPS.

SL NO. Histopathological Correlation Cell blocks CPS

1 Yes 14 (74%) 10 (53%)

2 No 5 (26%) 9 (47%)

3 Total 19 (100%) 19 (100%)

Statistical analysis

Sensitivity and specificity of conventional smears, LBC smears and cell blocks were calculated and compared. (Table 14)

Table 14: Sensitivity and Specificity of cell blocks, MLBC and CPS in diagnosis of neoplastic lesions.Method Sensitivity (%) Specificity (%)

Cell block 75 93

LBC 66 84

CPS 50 71

• Positive predictive value for diagnosing neoplastic lesions on cell blocks: 75%

• Efficacy of cell blocks in the diagnosis of neoplastic lesions: 89%

• Concordance Rate of CB/Histopathology Vs CPS/Histopathology is 74% vs 5

DISCUSSIONThe conventional Papanicolaou smear (CPS) has been the mainstay of screening for cervical

cancer and its precursor lesions for approximately 50 years without major changes in the techniques related to preparation and interpretation. Despite its success as a preventive screening tool for cervical cancer, CPS has its limitations [25,26].

False negatives in CPS maybe related to inadequate sampling, inadequate transfer of sample onto glass slide or deficiencies in microscopic assessment of the slide [26]. A false-negative Papanicolaou smear result is defined as “the failure to demonstrate abnormality by Papanicolaou examination in a woman who has disease”[27].

As with most screening tests, the Pap smear suffers from imperfect sensitivity and specificity. The sensitivity of the Papanicolaou smear , which ranges from 50% to 90% is reflected by the false-negative rate [27].

Although a clinician may have excellent collection and sampling technique, only approximately 20% of the cells collected are smeared on the glass slide in CPS[26,27,28]. To overcome these problems, a new slide preparation method was introduced called the liquid-based cytology (LBC) [29].

The sample is collected in a similar way to the Pap smear from the cervix, the spatula is rinsed directly into the preservative fluid. The sample is centrifuged and treated with a polymer solution. The sediment is deposited onto a glass slide as a thin layer of cells [8,30].


In our study, we have improvised a new method called as the Manual Method of Liquid-based Cytology (MLBC) [4-8]. In this method, the cervix was scraped using wooden and plastic spatulas. Split sample technique was used where the cells were smeared onto a glass slide for CPS and remaining cells were rinsed into a liquid fixative. Plastic spatulas seem to yield good amount of material, increasing the adequacy of smears [31,32].

The sample was further centrifuged with a polymer solution, manually vortex-mixed and the sediment was obtained. The cells were seen uniformly dispersed by a membrane, onto the glass slide [4-8]. Further it was subjected to heat-fixation and stained with the Papanicolaou stain.

150 cases were sampled and smears obtained in this way. These 150 cases of MLBC were compared with the corresponding conventional pap smears.

In cases in which a colposcopic biopsy or hysterectomy was done, histopathological correlation was also obtained. We got correlation for 44 cases out of the 150. In our study, we found that the MLBC method was comparable to the CPS.

Specimen adequacy was significantly increased as also observed by Lee KR et al. [33,34] Bernstein SJ et al. [27] Bishop JW et al. [35] and Hutchinson et al. [34-36],

whereas the number of “satisfactory but limited by” (SBLB) specimens was not reduced. (Table 15)

Table 15: Comparison of Sample Adequacy on MLBC and CPS.

Authors LBC (%) CPS (%)

Sherwani et al. [ 37] 83.1 31.9

Betash N et al. [38] 94.7 92.1

Present study 98 86.6

Increased detection of cellular abnormalities by liquid based method depends on many factors like adequacy of sample, type of spatula used to collect sample and type of sampling whether direct to vial or split-sample method [39].

Comparison of unsatisfactory rates has varied with some investigators reporting increased rates for liquid-based methods, [4,8,40,41] with some investigators reporting decreased [5,42,35]

and some no significant change [42,39].

In our study, 3 (2%) cases were unsatisfactory on MLBC compared to 14 (9.3%) cases on CPS agreeing with workers like Sherwani RK et al. [37] Deshou H et al. [43] Bishop JW et al. [35] and Rimiene J et al. [44] and who have found decrease in number of unsatisfactory samples.

Liquid-based cytology has recently become an alternative to conventional Pap smears in detection of intraepithelial lesions as well as in invasive carcinoma of the uterine cervix. Several reports have discussed its benefits to cytologic diagnosis. In most of these reports a significant rise in sensitivity was achieved with liquid based procedures without major losses in specificity [45].


In a study by Papillo et al. [46,47] they described an increase in the number of biopsy-documented cases of SIL, particularly LSIL, with a concomitant reduction in debatable diagnosis.

An important study from Costa Rica by Hutchinson et al. [36,46] documented that the ThinPrep method had heightened the sensitivity but significantly lowered the specificity when compared with CPS. These features were also emphasized by Koss [24,44] and their findings were comparable with our study.

The sensitivity of CPS to LBC as found by various authors like Abulafi O et al. [45] are 68% by conventional and 78% by ThinPrep. Specificity rates were 79% by conventional and 86% by ThinPrep as observed by Davey E et al. [48] who had a sensitivity of CPS about 70%-80% and about 85%-95% for LBC.

Our study shows the sensitivity and specificity of MLBC and CPS for LSIL group. The findings were sensitivity of MLBC vs CPS was 100% vs 62.5% whereas specificity was 77.7% vs 100%. Thus, MLBC is more sensitive for the diagnosis of LSIL compared to CPS but, CPS is more specific. This agrees with the findings of Bernstein SJ et al. [27] and Alves VAF et al. [5] (Table 16)Table 16: Comparison of rates of sensitivity and specificity of different studies and present study.

Authors Sensitivity Specificity

LBC CPS LBC CPS

Bolick DR et al.[49] 95.2 85 58 36

Lee et al. [5] 71.4 82.1 57.1 89.7

Davey E et al.[48] 85-95 70-80 86 79

Sherwani et al.[37] 97.6 53.7 50 50

Betash N et al.[38] 83 66 98 86

Deshou H et al.[43] 95.4 78.9 - -

Rimiene et al.[50] 78.1 68.7 91.8 93.8

Present Study 100 62.5 77.7 100

Note: In our study, sensitivity and specificity was detected for LSIL as highest number of cases were found in this category which is comparable to that of Deshou et al. [43], Sherwani et al. [37] and Bolick et al. [49] who have found increased detection rates for LSIL.

Klinkhamer et al. [46,50] reviewed and analysed available literature on Autocyte Prep, SurePath and Thin-Prep systems and concluded that the Thin-Prep system had a higher sensitivity for detection of squamous precursor lesions. Similar conclusions were found by Negri et al. [46,51].

There are 3 possible explanations for improved sensitivity:

1. LBC improves sample collection by markedly increasing number of cells that leave collection device into vial.

2. LBC reduces obscuring elements and thus allows for detection of abnormal cells that could have been otherwise hidden on CPS.


3. Better preservation of abnormal cells on LBC slide allows for more definitive categorization of abnormal cells as intraepithelial lesions rather than classifying them in equivocal ASCUS category [52].

In our study, we found an increased detection rate of 157 % for LSIL. Studies in developed countries using split sample method by Autocyte PREP have shown detection rates ranging from decreased detection rates of 10% [39] to increased detection rates of 117% reported by Wilbur DC et al. [53] and 67% by Bishop JW et al. [35]. Austin MR et al. [54], showed an overall increased detection rate of 16%. But most studies including ours show an overall increased detection rate of epithelial cell abnormalities by the LBC method.

Many workers like Vassilakos et al. [55]. Hutchinson et al. [36] Marino and Fremont Smith [40]

have found better biopsy correlation with HSIL diagnosis when compared to CPS.

There is a significant difference in concordance rate between CPS and LBC (45.4% Vs. 81.8%). Thus LBC showed a significantly higher histological versus cytological concordance rate, as also observed by Deshou H et al. [43]

Bishop JW et al. [35] observed a false-negative rate of 10.5% using the AutoCyte PREP compared to CPS with a false-negative rate of 21.5%. Our study was comparable with this study.

MLBC also plays a major role in the increased detection of infectious agents over CPS. This is due to removal of obscuring factors like blood, mucus and inflammatory cells. In our study, we found 25 cases (16.6%) of infectious diseases by MLBC comprising of 15 cases of bacterial vaginosis, 3 cases each of HPV changes and leptothrix infestation and 2 cases each of candidal infestation and trichomonas infestation. CPS detected only 13 cases (8.6%). Our study was comparable with authors like Papillo et al. [34,47].

A higher percentage of conventional cases with endocervical component has been observed in various studies and has been attributed to the split-sample collection protocols and use of the residual sample for the thin-layer preparations [33,56,57].

Direct-vial studies which allow the entire cervical sample to be rinsed into the liquid-preservative fluid, allow an equal percentage of thin-layer slides to have the endocervical component when compared to CPS. [58,59] Therefore, Direct-vial sampling is the intended use of new, liquid-based cell preparation methods and can reasonably be expected to show improved performance as compared to split sample trials [31,35].

In our study, split-sample technique was used. Therefore, no increase in rates of endocervical component were noted on MLBC.

Selvaggi and Guidos [60] noted that the adequacy of liquid preparations not unlike that of CPS, depended greatly on the collection instruments and skills of the provider.

The advantages of liquid-based cytology [4-6,27,61] are mainly that by the liquid based


method, the specimen is collected in a preservative solution and allows for long term storage of the liquid sample. The polymer solution used contains agarose, polyethylene glycol, alcohol and poly-L-lysine which help to a form a thin monolayer of cells within a clean background [4-8].

Thus by giving a clear background and removal of contaminating mucus and blood, liquid based cytology improves the quality of screening of slides [5,7,8,43,55] and also it reduces reading time [62,63].

The sample enrichment which can be possible in a liquid preservation media helps in harvesting a more representative sample. In the thin-layer preparation, cells function as part of the enrichment process by reducing unreadable, thick areas where cells of interest can hide [57].

Although LBC is recommended for cervical cancer screening, it involves the use of automated devices which are high cost per test. [4,7,8,30,43]

MLBC which we followed is an inexpensive, cost-effective method of LBC which we have adapted and are comparing it with conventional pap smears (CPS) for its adequacy and utility.

The other advantages of MLBC method is that the residual specimens can be used for ancillary testing like immunocytochemistry by cell block preparations [9,46] and detection of HPV-DNA by PCR or In-situ DNA Hybridization [5,30,42,41,64,65].

Limitations of the study were that split sample method which we followed to compare between CPS and LBC method depends on proper collection and smearing method which will enhance the effectiveness of LBC [54].

We found that cases with endocervical component were noted more in CPS than LBC which has been attributed to the split sample collection protocol. This can be overcome by direct sampling method [35,47].

Developed countries have automated computer assisted systems for reading of slides like the PAPNET system and Focal point analyser which increases the detection rate by reducing the screening time but is a costly method for routine screening in our country [48,66].

Cell Blocks from LBC specimens were found to aid in diagnosis of 20% of specimens in ThinPrep preparations and were critical to the diagnosis in 5% of cases-cell blocks have the ability to reduce both the false positive and false negative rates of the lab test [32,67].

To continue with the study of advantages of MLBC we took up a study on cell block and immunocytochemistry on cell block between the period of 2011 to 2013.

Cell blocks can be prepared from all types of cytological specimens, except preparations with low cellularity such as cerebrospinal fluids. There are several techniques to produce cell blocks, such as cytocentrifugation, either with direct formalin fixation or fixation after addition of plasma-tromboplastin [68].


In addition, there are commercially available systems, such as the ‘Shandon Cytoblock Cell block Preparations System’, which offers a standardized technique with a high reproducibility. Cell blocks perform in a highly reproducible way when stained with most antibodies, except for some used in the work- up of lymphoid lesions. One distinct advantage of cell blocks is that many slides can be prepared for extensive panels of immunostains. In addition, the quality control of cell block staining is identical to that of histopathology. The morphology of cell blocks is identical to that seen in histological specimens and therefore familiar to most pathologists. The technique, which is available in most laboratories, is relatively time-consuming and the cost is comparable to that of the cytospin technique [69].

Laboratories that only process cell blocks will have the advantage of using controls similar to those used in histopathology. Blocks with several tissue cores with various antigens expressed seem to be best practice. Sections from such blocks can either be placed on each patient’s slide or on one slide, which is tested in parallel with patient material [69].

Several of the steps in immunocytochemistry (ICC) are similar to those in IHC especially if automated staining is used, and will accordingly be well controlled and standardized laboratories with such automated platforms. Moreover if the cytological material used for ICC is in the form of cell blocks he quality control will be identical to that of IHC [69].

It is note worthy that irrespective of the type of in-house control slides used a high standard of immunocytoquality of immunocytochemical staining could be obtained on cytology samples prepared and fixed in different ways. The quality of immunocytochemical staining on in house control slides, as determined by the final UK NEQAS ICC score, was on average good regardless of the slide preparation and fixation technique; the only exceptions were cell block preparations, which achieved significantly better scores. Cell blocks closely resemble Formalin-Fixed Paraffin-Embedded sections and can be stained using methods already established in general immunohistochemical laboratories, which probably explains their providing the best quality of imunocytochemical reactions [70].

Adequate sampling and processing of cell blocks

In our study out of 60 cases, 10 cases had no deposits on cell blocks due to low cellularity. These findings were consistent with study by Wei Q et al. [71] in which 6 cases, Nitin Gangane et al. [72] in which 17 cases were excluded due to low cellularity and Richard et al. [9] who found that cell blocks can be technically productive only if there were large cell groups on ThinPrep.

Similar findings by George M V et al. [73], who observed that, Liquid based cervicovaginal specimens are relatively less cellular than their non-gynaecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve.

This problem of inadequate cellularity can be evaluated by using following features [74].

http://www.jove.com/author/George+M._Varsegi


1. Concentration and alignment of singly scattered cells in a narrow plane adjacent and parallel to the cutting surface of the cell block.

2. Inclusion of a beacon-like dark AV-marker as a signpost

Few of our cases we entirely processed the material of MLBC without splitting the specimen, which yielded better results. These findings are consistent with studies by Gangane N et al. [72] and Richardson K et al. [7].

Diagnostic aid and accuracy of cell block

In present study 14% of cases were diagnosed as malignant and 86% of cases were benign which is consistent with study by Gangane N et al. [72].

We found that cell block showed an increase in both sensitivity and specificity in the diagnosis of neoplastic conditions of cervix, which is consistent with study by Wei Q et al. [71] Catteau X et al. [75], Gupta S et al. [76] and Rofagha KS et al. [18] (Table 17) The increased sensitivity of cell blocks in the diagnosis of malignant conditions of cervix may be due to better preservation of cytomorphologic features, better staining characteristics of the nucleus, nucleoli, and cytoplasm ,clear recognition of nuclear and cytoplasmic features [10,76].

Table 17: sensitivity and specificity of cell blocks in diagnosing neoplastic lesions of cervix.

Authors Sensitivity (%) Specificity (%)

Gangane Net al.[73] 86.3 100

Gupta S et al.[78] 87.5 100

Catteau X[77] 50 100

Present study 75 93

When used as an adjunct to the smear, CBs aid in providing a reliable diagnosis of cervical cancer in the majority of the clinically suspected cases and thus the biopsy load can be reduced significantly in resource-poor settings [76].

In the present study, one case which was diagnosed as HSIL turned out to be SCC in cell blocks and a case of inflammatory smear in pap was reported as dysplasia in cell blocks. These discrepancies between cytology and cell block diagnosis were also observed in the studies by by Wei Q et al. [71], Luis A et al. [77], Richard K et al. [9] and Rofagha SK et al. [12].

The sensitivity and specificity of pap smear remains low even in the hands of experts [78,79,80], inter-observer variability still is significant and an irreducible false-negative rate inherent in the process [12,80].

Despite faithful yearly examinations, approximately 50% of the women who develop carcinoma of the cervix will have no abnormal cells present on their retrospectively reviewed smear and the remainder of patients will have cells present that were not detected due to screening or interpretive errors [12].


It was believed that in most of the specimens, the discrepancy between findings in cytology samples and findings in cell blocks was attributable to the lack of diagnostic cells in the residual material from which the cell blocks were prepared [81].

The morphological details including preservation of the architectural pattern like cell balls, papillae and three dimensional clusters, excellent nuclear and cytoplasmic details, and individual cell characteristics can also be obtained with the CB method. The tissue fragments can easily be interpreted in a biopsy-like fashion [76,82]. Some consider cervical biopsy to be the gold standard for diagnosing cervical lesions, but others have suggested that cytologic analysis is the more accurate technique [81,83].

Since a large majority of lesions identified were LSILs, a repeat Pap smear in these cases represented a more cost-effective strategy than cell block preparation, although a cell block preparation may be useful in selected cases [84,85].

Smears with large clusters or true tissue fragments of abnormal cells are better visualized using the cell block technique [12].

Therefore the use of cell blocks as an adjunct to the diagnosis of challenging cytologic cases has the potential to reduce the rate of both false-positive and false-negative results with the Pap test [77].

Cyto-histo correlationIn our study, cell blocks agreed with histopathology in 74% of cases as compared to 64% in

case of pap smears. Similar findings were found in a study, in which the tissue, cytology, and cell block findings were concordant in 15 cases (28%). Tissue agreed more closely with the cell block than with the cytology in eight cases, and more closely with the ThinPrep cytology [12].

Ancillary techniques on cell blocksIn our study, immunohistochemistry was done in suspicious cases. Stains used were p16INK4A

and ki 67(MIB1) p16 was positive in CIN and SCC. p16 expression increased as the grade of the tumor increased SCC >CIN3 >CIN2 >CIN1.

These findings were consistent with studies by Yoshida T [85] and Mood NI et al. [86]. P16 has excellent inter- and intraobserver reproducibility [74] and several studies have demonstrated the effectiveness of p16 INK4a for improving the cytological detection of HSIL in either LBC or cell block preparations [87].

In our study significantly higher number of ki positive cells were found in HSIL, with full thickness staining and in cases with normal study, staining was confined to basal one third .These findings were consistent with studies by, S Sahebali et al. [88] Zeng Z et al. [89] and Anne E et al. [90]. p16INK4a and Ki-67 staining can be useful in resolving diagnostic problems, such as the differentiation of squamous metaplasia or atrophy from squamous epithelial lesions of the cervix particularly when attempting to distinguish low-grade SIL or HPV effect from reactive changes [80,91-93].


Pap test alone has diagnostic limitations and Immunocytochemical analyses been used as supportive tools in the diagnosis of cervical lesions and the CB provides ease of rapid evaluation and appropriate material for the advanced [80,81].

Pit falls of cell blocks in gynaecologic cytology

1. It is possible that the limitations are due to fragmentation due in part to the centrifugation steps used in the preparation of cell blocks [71].

2. The major argument in the use of cell block in cervical cytology has been increased turn over time [77].

3. The lack of sufficient material in 25% of specimens are the main limitations of cell block analysis [80].

4. The added cost of performing immunohistochemical studies and the lack of sufficient material in 25% of specimens are the main limitations of cell block analysis [80].

Recent concepts and biomarkers in cell block in cervical cancer

The use of immunological techniques in cytology has expanded rapidly over the last 20 years. Akin to the situation in histopathology it has led to an increased diagnostic accuracy. In addition, tumour cell characteristics that determine sensitivity to targeted therapies can also be defined on cytological material [67].

Most laboratories process an array of material such as fine needle aspirates (FNAs), cell suspensions and various types of exfoliative collections, Proper specimen processing is of utmost importance for optimal ICC the most commonly used process are direct smears. cytospin centrifugation, cell blocks and liquid- based cytology (LBC) preparation [67].

We are now expected to perform on cytological specimens, many ancillary tests that are traditionally performed on histological material. ICC has become an important ancillary method not only for diagnosis but also to assess prognostic and predictive factors on cytological material [94,95].

It is well known that slide preparation and fixation technique has a great impact on the quality of ICC reactions. However, equally good ICC result can be achieved in cytological samples fixed in 95% alcohol, buffered formalin, methanol or ethanol- based fixatives [95-99]. Brief fixation in any of the above fixatives in adequate of most cytological preparation.

The need for automation in IHC was raised many years ago [101]. Automated ICC offers the opportunity to reach new levels of quality, reproducibility and consistency while reducing labour and reagent costs. In addition, the controlled environment of automated ICC may increase the speed and timeliness of results. Automation contributes decisively to the standardization of ICC-techniques and improves the confidence of the pathologist in the ICC reaction. So, the fact that the majority of laboratories are using automated ICC on cytological samples is good news and a decisive step towards standardization.


A reasonable differential diagnosis is usually based on the cytomorphology, clinical information and the probability of certain disease occurring according to the age group of the patient and the anatomical location sampled. Another important factor is the availability of markers for the entity within the differential diagnosis perhaps the most essential factor in determining the clinical value of ICC is the experience of the cytopathologist. This has a major impact on several steps, from the selection of the markers to the evaluation of results and rendering of a diagnosis. Because most diagnostic problem in cytology can be narrowed down to a few possibilities, the choice of antibodies can also be restricted to two or three. This approach requires the input of the cytopathologist and necessitates active participation in the formulation of a working diagnosis. Considering the limited amount of tissue in cytological specimens, large pre-determined panels of antibodies should be avoided and a judicious choice of antibodies based on morphological differential diagnosis, combined with the pertinent clinical information should be encouraged. This last issue is crucial when dealing with cytological samples [102].

Reduction of this variability by using automation, appropriate controls and customized dilution of antibodies according to different samples is likely to improve the quality of ICC and will lead to standardization for the technique. However, it is equally or even more important to standardize criteria for high-quality ICC reactions and to encourage all laboratories performing this technique to use proper quality control and quality assurance measures [102].

p16ink4a immunomarker

The protein p16ink4a, derived from the host p16ink4a blocks the activity of the cyclin dependent kinases CDK4/6, resulting in greater binding of pRB to the transcription factor E2F, thus down –regulating progression through the G1-S Transition checkpoint of the cell cycle. Unbound E2F also acts in a negative feedback loop with Prb .When HR-HPV infects the host cell, the viral oncoprotein E7 binds a and inactivates pRb, thereby releasing E2F, which promotes cell cycle progression, a molecular switch that is usually activated by CD K4/6.Thus the p16ink4a induced feedback loop is lost, and p16ink4a is over expressed. As a result, the protein accumulates in the nucleus and cytoplasm of affected cells and can be detected by immunocytochemistry, usually with an brown reaction in cytological samples, p16ink4a is one of the most promising and most studied. several studies have tested it either LBC or cell block preparations and majority have demonstrated the effectiveness of p16ink4a for improving the cytological detection of HSIL [106].

Limitations in the interpretation of the p16ink4a stain include:

a. the lack of standardized criteria in cytology for the evaluation of staining intensity and nuclear versus cytoplasmic staining

b. the absence of consensus for threshold values of positivity

c. the presence of sporadic p16ink4a immunoreactivity in normal , atrophic, and metaplastic squamous cells as well as endocervical cells , inflammatory cells, and bacteria [103].


MIB-1

Ki-67 is an antigen that identifies proliferating cells and is expressed in all phases of the cell cycle (G1,S,G2/M). MIB-1 is a monoclonal antibody that detects this antigen in the nuclei of fixed cells or tissues embedded in paraffin . HPV infection activates host cell cycle progression with increased cell cycle kinetics , and this phenomenon is reflected in an increased MIB-1 staining index.with MIB -1 immunostaining , positive cells typically show dark brown staining limited to the nucleus (Figure 10). MIB-1 tends to stain mainly basal and para-basal cells , but intermediate and superficial cells can also be stained by this antibody.MIB-1 complements p16ink4a in the analysis of histological sections by differentiating SIL from atrophic epithelium with atypia [104].

BD-ProExC

MCM 2 functions during DNA replication by loading the pre-replication complex onto DNA and by unwinding DNA through helicase activity to permit DNA synthesis. TOP2A is responsible for the enzymatic unlinking of DNA strands during replication, these two proteins play a significant role in the regulation of DNA replication during S-phase. They are both over-expressed when the S-phase cycle induction is aberrant. a cocktail of two monoclonal antibodies that targets the expression of these two proteins was tested for the first time for the detection of cervical HSIL in cytological samples in 2006 [105].

BD-ProExC staining like MIB-1, is exclusively nuclear, and its histological distribution in cervical intraepithelial neoplasia is very similar to that of MIB-1 (Figure 11). In cytological specimens again like MIB-1, ProEx C occasionally stains normal glandular cells and tubal metaplasia [106].

Also, ProExC and MIB-1 stain random nuclei of para-basal cells in atrophic epithelium. This immunoreactivity of atrophic cell nuclei is a pitfall in the cytological distinction between HSIL and atrophy [107].

L1

L1 is primarily the name of the major capsid protein of HPVs. LI is also the name of an antibody against a protein of the HPV 16 capsid that is expressed in the early productive phase of the viral life cycle and is progressively lost during cervical carcinogenesis [108].

Therefore this antibody has an inverse pattern of distribution along the spectrum of evolution of SIL when compared to other biomarkers.studies using LBC samples report the highest li staining in LSIL cases, which reflects productive HPV infection [107].

Li negative cases tend to progress to hsil, while li-positive cases do not. Furthermore, the combination of li and p16ink4a antibodies in LBC samples and cell blocks has been proposed for prognostic prediction of LSIL [109-111].

Markers tested in combinations

Several studies have tested more than one biomarker on the same sample. The complementary role of P16INKPA and MIB-1 or LBC and cell block preparations which in the combination of complementary to LBC, improving diagnostic accuracy for HSIL and squamous cell carcinoma.


Fig 15 a) b)

c) d)

Figure 15: (a) Normal Ectocervix-cell block(H&E,40x). (b) p16 ink4a Negative in Normal Cervi x-Cell Block. (c) high grade intraepithelial lesion (HSIL)-cell block(H&E,40X). (d) p16ink4a positive cells seen in

HSIL- cell block.

Fig 16 a) b) c)

Figure 16: (a) Normal Ectocervix-cell block(H&E,40x). (b) high grade intraepithelial lesion (HSIL)-cell block(H&E,40X). (c) ki-67positive in HSIL-Cell Block showing ascending positive index.


CONCLUSIONManual liquid based cytology is a good method for early detection of cervical cancer. It does

help to use ancillary techniques in the early detection of cervical cancer, they are cell block, IHC and HPV testing. Our study has worked on cell block and IHC which proved to be useful in the diagnosis of various conditions of the cervix.

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https://www.researchgate.net/publication/46108767_Immunocytochemical_expression_of_p16INK4a_and_HPV_L1_capsid_proteins_as_predictive_markers_of_the_cervical_lesions_progression_risk

https://www.researchgate.net/publication/46108767_Immunocytochemical_expression_of_p16INK4a_and_HPV_L1_capsid_proteins_as_predictive_markers_of_the_cervical_lesions_progression_risk

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