Study of Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology

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1Department of Pathology, Jagadguru Shri Shivarathreswara Medical College, Jagadguru Shri Shivarathreswara University,
Mysore Karnataka, India.
2Department of OBG, JSS Medical College, JSS University, Mysore Karnataka, India.
*Corresponding author: E-mail: nandinimanoli65@gmail.com;
Chapter 5
Print ISBN: 978-93-91473-61-7, eBook ISBN: 978-93-91473-85-3
Study of Manual Liquid Based Cytology in Diagnosis
of Gynecologic Pathology
Nandini N. Manoli1*, Nandish S. Manoli2, Lopamudra Kakoti1, Akshata Kamath1
and Shweta Kulkarni1
DOI: 10.9734/bpi/nfmmr/v2/3205F
ABSTRACT
Abnormal uterine bleeding (AUB) is a commonly encountered complaint in gynecology department.
Endometrial cancer is the fourth most common malignancy in women and the most frequent
gynecological cancer in developed countries. With 5,28, 000 new cases every year, cervical cancer is
the fourth most common cancer affecting women worldwide, after breast, colorectal, and lung
cancers. Though the cytological examination has been the mainstay for early detection of cervical
cancer, and found to be useful in detection of endometrial cancer, its widespread use has not been
possible in the developing countries due to paucity of resources, man power and other facilities Its
sensitivity reduces to less than 50% when there is presence of obscuring blood, inflammation or thick
areas of overlapping epithelial cells. Manual Liquid Based Cytology (MLBC) is a cost effective
technique that enables cells to be suspended in a monolayer and thus improve detection of precursor
lesions and specimen adequacy. The residual sample can be used for other tests like Cell block and
immunocytochemistry.
Objectives: 1. Toimprove the diagnosis of gynecological cancers by a method called as Manual
Liquid Based Cytology (MLBC). 2. To study a cost effective method of studying both endometrial and
cervical cancer with help of ancillary techniques like cellblock, immunocytochemistry 3To compare the
findings between conventional pap smear (CPS) and MLBC in detection of gynecological conditions of
endometrium and cervix.
Methodology: Samples were collected using Ayres spatula by split sample technique from
transformation zone of cervix which included outpatients of gynecology dept. The women were aged
between 20-60 years, 82 cases with bleeding history were taken to study endometrial pathology, while
100 cases of white discharge per vagina were selected to study the cervix. 60 cases were later
subjected to ancillary studies like cellblock with Immunocytochemistry. Histopathological correlation
was done for cases wherever possible.
Results: The study on endometruim showed MLBC is more sensitive and specific than CPS in
diagnosis of malignant lesions. The contingency coefficient for LBC/Histopathology V/s
CPS/Histopathology was 0.572 V/s 0.556. It was observed for cervical lesion that increased detection
rate was 150% for low grade intraepithelial lesion. The Positive predictive value for diagnosing
neoplastic lesions on cell blocks was 75%, while Concordance Rate of CB/Histopathology Vs
CPS/Histopathology was 74% vs. 54%.
Conclusion: MLBC is a cost effective method for detections of cancerous lesions of endometruim
andcervix. It has been found to be useful in both Endometrial and cervical cytology in increasing the
detection of infection and neoplastic conditions of female genital tract.
Keywords: Manual liquid based cytology; endometrium; cervix.

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1. INTRODUCTION
Endometrial carcinoma (EC) is the commonest malignancy of the female genital tract in the USA and
Europe, accounting for nearly 50% of all new diagnoses of gynecological cancer [1,2,3]. The
incidence of EC increased from 13.5 to 20.3per 100,000between1993and2010intheUK, [4] and the
incidence and mortality have been reported to have increased by 1.1% and 0.3%, respectively,
annually during the same period of time in the USA [5]. The incidence of EC is also increasing in
Japan, even in younger age groups, [6] where it has become more frequent each year over a period
of 30 years, and as a proportion of all uterine carcinoma rose from 10% in 1983 to 45% in 2004 [7].
The main risk factors for ECareobesity, diabetes, estrogenuse, tamoxifen treatment polycystic ovarian
syndrome (PCOS), a history of infertility, alcohol abuse and antidepressant agents [8, 9, 10, 11].
Many of these factors are tightly linked to current lifestyles in developed countries. A small but
significant percentage (about 2-3%) of EC is attributable to Lynch syndrome, a hereditary cancer
predisposition syndrome which significantly increases the risk of colorectal, endometrial and other
cancers [1, 2] Fig. 1. With 528, 000 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 (266 000 deaths in 2012) in women worldwide [12]. 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 Fig. 2.
Fig. 1. Graph showing the incidence of endometrial carcinoma with age and ethnicity
The main focus is on the secondary prevention of cervical cancer through early detection, a focus
point of National Cancer Control Programme revised in 1985 [13]. Though the cytological examination
has been the mainstay for early detection of cervical cancer, its widespread use has not been
possible in the developing countries due to paucity of resources, man power and other facilities.
Moreover, although Conventional Pap Smears (CPS) screening leads to reduction in the rate of
invasive cancer of the uterine cervix, its sensitivity reduces to less than 50% when there is presence
of obscuring blood, inflammation or thick areas of overlapping epithelial cells [14, 15]. These problems
with the CPS, gave rise to the advanced technologies, like, Thin Prep and Sure Path commonly used
in the setup of developed countries, like UK and USA [15, 16]. Use of these technologies however are
quite resource intensive and therefore not feasible in the setup of developingcountries.

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On the other hand, Manual Liquid Based Cytology (MLBC) is a technique that enables cells to be
suspended in a monolayer and thus improves detection of precursor lesions and improvement of
specimen adequacy. MLBC has been reported to improve the effectiveness of cervical cancer
screening in a population by increasing the detection of histologically confirmed neoplastic and
preneoplastic disease while simultaneously decreasing over diagnosis of benign processes [17]. Also,
in case of MLBC, the residual sample can be used for other tests like detection of HPV, DNA and
immunocytochemistry thereby increasing the utility of MLBC [14,15].
.
Fig. 2. Worldwide incidence of cervical cancer
There are studies [15, 18] which have dealt with liquid based cytology and have found its diagnostic
accuracy comparable with conventional Pap smears. MLBC method however is specific to the
laboratory, available equipments, fixatives and polymer solutions, etc. Therefore the overall aim of our
study was to assess the utility of indigenous MLBC in comparison of CPS for low resource settings.
Specific objectives of the study were: a) To compare the morphological view of different diagnoses
according to CPS and MLBC, b) To compare the cellular and nuclear parameters according to two
methods and c) To compare the validity of the two methods in terms of sensitivity and specificity.
As MLBC can be used for ancillary studies like cell block study, immunocytochemistry and HPV
testing, one of the ancillary techniques which we studied is cell blocks prepared from residual tissue
fluids and fine-needle aspirations which can be useful adjuncts to smears for establishing a more
definitive cytopathologic diagnosis [19, 20].
Thus, our study includes MLBC in Endometrium &Cervix with an ancillary technique, cell block to
improve the diagnostic accuracy of gynecological conditions in women.
2. MATERIALS AND METHODS
The present study was undertaken to observe the presence of endometrial cells in cervical cytology
smears using conventional Pap smears and the manual method of liquid- based cervical cytology
(MLBC) in all abnormal uterine bleeding cases.
Samples were collected using the split-smear technique from 82 patients (sample size) in the age
group of 20 to 60 years attending the gynecology out-patient clinic at JSS Hospital, Mysore,
prospectively from July 2012 to July 2014 for a period of two years.
All the patients were clinically examined in detail according to the proforma and relevant radiological
findings were collected. Material collected was stained by Pap stain.
Ayre’splastic 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

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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’sspatula. The scrapings were evenly
spread onto a glass slide, and immediately fixed in ethyl-alcohol fixative (95% ethylalcohol). After
fixation, smears were stained using conventional Pap stain.
Both smears were screened and results were compared. Cyto-histologic correlation was done. The
Bethesda system 2001 was used for reporting cervical cytology for both groups.
The cases in which a D&C or hysterectomy was done, specimens were collected in 10% formalin and
allowed to fix overnight. Detailed gross examination was done and bits were given. Paraffin
embedded H and E stained sections were obtained and studied under lightmicroscopy.
Similarly another study on cervical pathology was conducted Samples were collected using the split-
smear technique from 100 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. The cases collected were patients with
clinical suspicion of cervical pathology.
The processing of the samples was done as in the study on Endometrial pathology. Both smears were
screened and 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. 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 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.
Samples 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 gynecological complaints like white discharge P/V and bleeding
P/V attending the Gynecology out-patient clinic at JSS Hospital, Mysore. In the present study KI 67
and p16 markers were done on cell block preparation.
3. RESULTS
In the present study, 82 cervical smears prepared by conventional pap and MLBC were studied, out of
which 52 cases (63.4%) were reported with the presence of endometrial cells, Among the 52 cases 38
cases were identified as benign endometrial cells and 14 cases as atypical/ malignant endometrial
cells.

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3.1 Conventional Pap Smears vs Manual LiquidBased Cytology
Among the 52 cases with endometrial cells, 46 were detected in conventional Pap smear and 38
cases in Manual Liquid Based Cytology. Out of 46 cases of endometrial cells- 35 were benign and 11
were atypical cells in conventional pap smear, while in manual liquid based cytology 28 were benign
and 10 were atypical endometrial cells out of 38 cases. (Table 1)
Table 1. Distribution of cases into benign and Atypical/malignant endometrial cells
Lesions
No.
Percentage (%)
Benign endometrial cell
28
53.9
Atypical/malignant endometrial cells
10
19.2
Absent
11
21.2
Inadequate
03
05.7
Total
52
100
A
B
C
D
Fig. 3.a. Proliferative growth occupying the entire uterine cavity b. CPS-Atypical Endometrial
Cells - Smear shows cells with high N/C ratio arranged in clusters with enlarged
hyperchromatic nuclei and fairly abundant cytoplasm. (Pap, X400) c. MLBC Atypical
Endometrial Cell - Smear shows cells with high N/C ratio arranged in clusters with enlarged
hyperchromatic nuclei and fairly abundant cytoplasm(Pap, X400) d. HPE - Endometrial
Adenocarcinoma type 1 -Section shows a tumor displaying features of endometrial
adenocarcinoma. (H & E, X10)
3.2 Morphological Distribution of Histopathological Examination
On follow up with histopathology, maximum 8 cases of leiomyoma with proliferative endometrium,
followed by 6 cases of simple hyperplasia without atypia and 4 cases each of adenomyomatous polyp
and endometrial carcinoma type 1 were diagnosed. There was only one case each of endometrial

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type 2 carcinoma and complex hyperplasia with atypia and 3 cases each of squamous cell carcinoma,
endometritis and disordered proliferative endometrium. Remaining cases were 2 proliferative
endometrium and 1 case of placental site trophoblastic reaction (Table 2) Fig. 3.
Table 2. Morphological distribution of histopathological examination of endometrial pathology
Histopathological diagnosis of endometrium
No. of cases
Percentage (%)
Proliferative phase
02
5.1
Endometritis
03
7.7
Disordered proliferative phase
03
7.7
Leiomyoma
08
20.5
Simple hyperplasia without atypia
06
15.4
Complex hyperplasia with atypia
01
2.6
Adenomyomatous polyp
04
10.2
Atrophic endometrium
03
7.7
Endometrial carcinoma type 1
04
10.2
Endometrial carcinoma type 2
01
2.6
Squamous cell carcinoma
03
7.7
Placental site reaction
01
2.6
Total
39
100
3.3 Conventional Pap Smear Findings in Correlation with Histopathology

Histopathological study was obtained in 29 out of 35 cases with benign endometrial cells and 8
out of 11 cases with atypical/malignant endometrialcells.

Among them, 21cases and 6 cases were consistent with histopathological findings of benign and
malignant endometrial pathology respectively.
3.4 Manual Liquid Based Cytology Findings in Histopathology Correlation

Histopathological study was obtained in all the 29 cases with benign endometrial cells and 8 out
of 10 cases with atypical/malignant endometrialcells.

Among them, 20cases and 6 cases were consistent with histopathological findings of benign and
malignant endometrial pathologyrespectively

MLBC is more sensitive and specific than CPS in diagnosis of malignantlesions.

The contingency coefficient by LBC/Histopathology V/s CPS/Histopathology was 0.572 V/s0.556.
Results of study of CPS vs MLBC for cervical cancer screening showed the following, Panel of
various cellular and nuclear parameters were compared between CPS and MLBC (Table 3).
Table 3. Morphological Features as Observed Through Conventional Pap Smear (CPS) and
Manual Liquid Based Cytology (MLBC) Methods
Morphological feature
CPS
MLBC
Cellularity
Unsatisfactory in 10 cases
Adequate
Cleanliness of background
Absent
Absent
Uniformity of distribution
present
Absent
Artifacts
present
Rare
Cellular overlapping
Yes (Marked)
Yes (Rare)
Architectural change
Yes
Rare
Cellular morphological
Change yes
No
Nuclear change
Not always clear
Always very clear
Cellularity was adequate in all of the MLBC cases whereas it was unsatisfactory in many CPS cases.
The background was observed to be clean in all cases of MLBC which was not the case in majority of
CPS. Uniform distribution seen in MLBC with cellular overlapping was seen more in CPS than in

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MLBC. Artifacts were present in most CPS samples. Architectural and cellular morphologic changes
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 so clear by CPS.
Diagnostic features of 100 cases according to both CPS and MLBC were divided into 10 categories.
Figs. 4 & 5
Fig. 4.a. CPS-Inflammatory smear (NILM) (PAP, X200) b. MLBC-Inflammatory smear with clear
background (PAP, X200) c. HPE-section studied shows chronic cervicitis. (H&E, X200)
Fig. 5.a. CPS-squamous cell carcinoma of cervix (PAP, X200) b. MLBC-Squamous cell
carcinoma of cervix. with clear background. (PAP, X200). c. HPE- squamous cell carcinoma of
cervical biopsy. (PAP, X200)
In the study, the comparison between conventional CPS and MLBC showed certain observations in
different diagnostic categories. Both the methods showed same number of normal smears.
Inflammatory smears diagnosis was more by CPS than by MLBC (42%). However, diagnosis of
Bacterial Vaginosis(14%) and low grade squamous intraepithelial lesion (36%) were more by MLBC
which is a significant observation. Also the number of infestations detected by MLBC method was
increased koilocyticatypia, candidiasis and leptothrix. Cytohistological correlation was done in 22
cases. Increased detection rate was 150% for low grade intraepithelial lesion. The rate of
concordance with histology was 77% for CPS and LBC, whereas, it was 86% for MLBC. The rate of
increased detection of LSIL through MLBC was 150%. (Table 4).
In addition, to compare the validity of the two methods, we estimated sensitivity and specificity of the
two considering HPE as the gold standard for the two important diagnoses, namely, LSIL and
inflammation. In the diagnosis of LSIL, MLBC was more sensitive than CPS (75% vs. 50%) with
similar specificity (100%). In case of inflammation also, MLBC was found to perform better being more
specific(92% vs. 77%) with same sensitivity(89%).
To work on the utility of MLBC on ancillary studies a 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.

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Table 4. Comparison of Classification of Cases by Conventional Pap Smear (CPS) and Manual
Liquid Based Cytology (MLBC) with that by Histo- Pathological Examination (HPE)
Categories
CPS
MLBC
HPE
Normal smear
14
15
1
Inflammatory Smear (NILM)
42
20
8
Bacterial Vaginosis
7
14
0
LSIL
14
36
8
HSIL
1
1
1
Squamous cell carcinoma
2
2
1
Adenocarcinoma
1
1
1
Menopausal
8
6
1
Unsatisfactory
9
1
0
Infestations
HPV (Koilocyticstypia)
2
3
1
Candidiasis
0
1
0
Leptothrix
0
3
0
Total
100
100
22
43 non-neoplastic lesions include chronic polypoidalendocervicitis (CPEC - 10 cases), Koilocytes (3
cases), chronic cervicitis (12 cases), normal study (10 cases), acute on chronic cervicitis (7 cases),
only hemorrhage (1case).
Neoplastic lesions included squamous cell carcinoma SCC (1 case), dysplasia (2 cases), carcinoma
in situ (4 cases)10 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. (Table 5).
Table 5. 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
Acute 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
Cases reported as No deposits were maximum when MLBC had inadequate material and were
minimum when LBC smears were not done and sediment was directly processed for cellblocks.
3.5 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. 1 case 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 3 cases of CIN were diagnosed as HSIL and LSIL.

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This shows that cellular architecture is better preserved and neoplastic lesions which are under
diagnosed on CPS and MLBC can be correctly diagnosed on cellblocks.
3.6 Comparison of Cell Block Diagnosis with Histopathology
Histopathologic 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 6)
Table 6. 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%)
3.7 Statistical Analysis
Sensitivity and specificity of conventional smears, LBC smears and cell blocks were calculated and
compared.

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 VsCPS/Histopathology is 74% vs54%.
4. DISCUSSION
Although cervical cancer was historically commoner than EC, cytological screening has progressively
decreased its prevalence.Onthecontrary,ECshowsanincreasingtrend.In developed countries, where
cervical cancer screening is performed, it is estimated that the number of women who die from EC is
almost two fold higher than the number of women who die from cervical cancer [21]. Unfortunately,
although screening strategies for cervical cancer continue to improve, screening for EC with
cytological techniques remains widely unaccepted by the medicalcommunity.
82 cases with abnormal bleeding history were sampled and smears obtained in the above mentioned
way. These 82 cases of MLBC were also prepared and compared with the corresponding
conventional pap smears. Cytology smear results for the 52 patients out of 82 cases showed
endometrial cells. Among 52 cases, histopathological correlation was obtained in 39 cases.
More recent abstracts reported endometrial cells of 1.8% and 0.5% on Pap tests in women 40 years
and older [22, 23]. Though many of these women with endometrial cells on Pap tests (61%) had their
Pap test collected in the first 14 days of the menstrual cycle [22].

However, the various studies published since 1970 shows were listed below- (Table 7)

The reported prevalence of normal endometrial cells in all smears varied between 0.1% to 1.4%
[38] and Atypical glandular cells ranging from 0.18 to 0.74% [39,40].

The increased prevalence of endometrial cells in our study may be because of confinement of
patients selection was only from bleeding history cases in which some pathology must bethere.
The presence of endometrial glandular cells was found to indicate a five times greater risk of
endometrial disease [41].
However, evaluation of prevalence of endometrial pathology might be complicated because of
selection bias and no adequate control for comparison.

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Previous studies showed Endometrial cells on Pap tests have been associated with normal
endometrial findings in 22- 97% of women, polyps in 1-41%, hyperplasias1-20%, atypical hyperplasia
in 0.6-8%, and carcinoma in 1-15% [42,43, 44].
Table 7. Pap smear studies published since 1970 in women with endometrial malignancies
(35).
Study
year
Total no.
Normal results
Abnormal results
BEC
Burk et al."[24]".
1974
154
123
31
unknown
Vuopala et al."[25]"
1977
88
9
79
abnormal
Bibbo et al."[26]"
1979
33
10
23
normal
Lozowski et al."[27]"
1986
58
23
35
abnormal
Kuebler et al."[28]"
1989
34
12
22
Unknown
Larson et al."[29]"
164
94
94
70
Normal
Zuna and Erroll et al"[30]"
1996
61
25
36
unknown
Eddy et al."[31]"
1997
112
59
53
Abnormal
Fukuda et al."[32]"
1999
99
68
31
Normal
Gu et al."[33]"
2001
76
34
42
unknown
Morimura et al."[34]"
2002
53
15
38
normal
DuBeshter et al."[35]"
2003
300
109
191
abnormal
Brown et al."36]"
2005
412
139
273
Abnormal
Thrall et al."[37]"
2007
73
41
32
abnormal
Present study
2012
52
38
14
abnormal
In our study, we had maximum of cases with leiomyoma with proliferative endometrium in 8 (20.5%)
cases, followed by endometrial hyperplasia without atypia in 6 (15.4%) cases, adenomyomatous
polyps in 4 (10.3%) cases and proliferative endometrium, disordered proliferative endometrium and
endometritis in 3 (7.7%) cases each.
There were 8 (20.5%) cases of malignancy, and 1 (2.6%) case of precursor lesion with complex
hyperplasia with atypia. So, the final diagnosis was completely benign pathology in 31 patients and
carcinoma in 8 patients.
Among all benign lesions, leiomyoma with proliferative endometrium was most common because,
leiomyoma is found to be the most common pathology associated with abnormal uterine bleeding by
causing inadequate contraction of myometrium and irregular spotting leads to shedding of
endometrium.
Recently, Obenson and coworker reported that their most common benign lesions were endometrial
polyps [45]. While in our study, adenomyomatous polyp was third most common lesion preceded by
simple hyperplasia.
Sensitivity of Pap testing for true glandular neoplasia is less than that for squamous lesions, in part
because glandular lesions arise in the endocervical canal and endometrial cavity where they may be
difficult to sample.
The low sensitivity for endometrial malignancy was improved only marginally (from 38.3% to 43.8%) by
the addition of the 2001 Bethesda System reporting category of benign endometrial cells in women
aged >40 years, but specificity of the Pap test for the diagnosis of endometrial hyperplasiaor
malignancy decreases from 99.8% to 96% [31].
Available literature suggested compared with conventional cytology, LBC may be associated with a
higher prevalence of NECs because of more consistent use of sampling instruments for LBC with
better access to this area [23].
The ThinPrepPap test has high sensitivity and specificity in detecting or suggesting the presence of
endometrial cancer. Certain cytomorphologicfeatures are helpful in distinguishing benign and malignant
endometrial lesions [32,46,47].

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Schorge et al. [33] using Thin Prep, demonstrated a sensitivity of 65.2% among 45 patients with
endometrial cancer.
In our study we also found that the MLBC method was comparable to the CPS. Though the number of
inadequate specimen for MLBC was 5 whereas 1 for conventional pap smear, the sensitivity,
specificity, negative and positive predictive value was better for MLBCs. Even on cross tabulation,
MLBC showed better histopathological correlation than conventional pap smear.
MLBC showed a slightly higher concordance coefficient (0.572) than CPS (0.556).
The advantages of MLBC are increased detection of cellular abnormalities. This is because of many
factors like adequacy of sample, type of spatula used to collect the sample, type of sampling like
whether direct to vial or split sample method. Also the polymer solution which is prepared in our
laboratory contains agarose, polyethylene glycol, alcohol and poly-L-lysine, form a thin monolayer of
cells within a clean background [15, 18].
This helps to detect nuclear changes in readable clean background MLBC method which we follow
helps to bring in a cheap method. In addition the residual specimen can be used for ancillary testing
like immunocytochemistry (IHC) on cell block and detection of HPV DNS by PCR or insitu
hybridization [15,1].
IHC markers which can be studied are p16inK4a, MIB-1, BD-ProExC and L1 [48].
To continue with the study of advantages of MLBC we took up a study on cell block and
immunocytochemistry on cell block between the periods of 2011 to2013.
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-
thromboplastin [49].
In addition, there are commercially available systems, such as the 'Shandon 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 [50].
5. USES OF CELL BLOCK ARE
It improves the diagnosis of malignant conditions of cervix due to better presentation of
cytomorphologic features, better staining characteristics of the nucleus, nucleoli and cytoplasm, clear
recognition of nuclear and cytoplasmic features [51, 52] (Table 8)
Table 8. Sensitivity and specificity of cell blocks in diagnosing neoplastic lesions of cervix
Authors
Sensitivity (%)
Specificity (%)
Gangane Net al(2007)"[53]"
86.3
100
Gupta.S et al(2007)"[54]"
87.5
100
Catteau X (2012)"[55]"
50
100
Present study
75
93
Thus, if used in adjunct of the smear it aids in providing a reliable diagnosis of cervical cancer in
majority of clinically suspected cases and thus the biopsy need can be reduced significantly in
resource poor settings [54].

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Study of Manual Liquid Based Cytology in Diagnosis of Gynecologic Pathology
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
we also found also similar advantages in our study which were observed by Wei Q et al [56] who
like us also found that cell block increased both sensitivity andspecificity.
Table 9. Table showing the different categories of cervical lesions and markers
Categories of lesions
Number of lesions(60)
P
16 Ki
67 Ecadherin
Squamous cell carcinoma
2
++
++
_
Dysplasia
21
+
+
_
Inflammatory conditions
20
_
_
+
Normal smears(NILM)
17
_
_
++
Cell block can be used to do IHC on suspicious cases which were done in our study. Stains used were
p16inK4A and KI67. It was found that both markers increased their expression as the grade of
dysplasia increased and maximum in cervical cancer. Thus helping in improving diagnostic accuracy
of cervical lesion. These were consistent with findings ofSiddham V. B et al. [57] for p16inK4a and by
Sahebali s et al [58] for KI67 (Table 9,) Fig. 6.
There are many studies done on markers and endometrium. Ghosh et al have found that there was
increased expression of Cyclin D1 and Ki-67 in patients with endometrial carcinoma relative to
proliferative endometrium and simple hyperplasia, but there was no such difference between cases of
atypical hyperplasia and endometrial carcinoma. Cyclin D1 expression had a positive correlation with
Ki-67 expression. Cyclin D1 together with Ki-67 may be a marker for endometrial carcinogenesis and
tumor cell proliferation [59].
Other marker studies on cervix have been undertaken p16 and Survivin expression can be correlated
with the clinico- pathological and prognosis of cervical cancer. p16 protein is found to be associated
with CIN grade and lymph node metastasis. Survivin protein also is the same. Both together were
positively correlated with cervical cancer (r=0.854, p,0.001) and associated with poor prognosis of
cervical cancer [60].
The advantages of liquid-based cytology include improved sensitivity and specificity since fixation
better and nuclear details are well preserved. Singh et al proved that LBC performed much better by
showing more sensitivity, specificity and accuracy than the conventional method of cytology to detect
recurrence of squamous cell carcinoma. Abnormal cells are not obscured or diluted by other epithelial
or Inflammatory cells. There is, therefore, a lowe rate of unsatisfactory cervical cytology samples [61].
In comparison to a conventional Pap smear, a liquid-based Pap smear has high specificity, and Ki-67
expression with LBC can exclude false positivity, which can substantially reduce diagnostic biopsies
and subsequent Pap smears in postmenopausal women with atypical Pap smears [62].
A B

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C D
Fig. 6.a. cell block of normal cervix. (H&E, x200) b. p16 ink4a negative on cell block of normal
cervix (x200) c. cell block of cervix-HSIL (H&E, X200) d. p16ink4a positive on cell block of HSIL
(H&E, X200).
6. CONCLUSION
Women are vulnerable group to many illnesses. Gynecological pathology with conditions related to
Endometrium and cervix are major causes of the health problems ranging from infections to neoplasia
which require early detection and treatment.
The commonly used method of conventional Pap smear (CPS) which was used as an screening
method has its limitations. It has many obscuring factors like blood and inflammatory cells which
limited the sensitivity and specificity of the test.
Liquid based cytology (LBC), a methodology was introduced in the advanced countries to overcome
these limitations by making a thin monolayer of cells with a clear background. This method but is
costly for developing countries where awareness, sources for health care are scarce. To obviate this,
a Manual Liquid based cytology method (MLBC) was introduced in our setup.
It has been found to be useful in both Endometrial and cervical cytology in increasing the detection of
infection and neoplastic conditions of female genital tract. It is also a cost effective method which can
be used to test ancillary techniques of MLBC like cell block study, immunocyto chemistry and HPV
testing.
COMPETING INTERESTS
Authors have declared that no competing interests exist.
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_________________________________________________________________________________
© Copyright (2021): Author(s). The licensee is the publisher (B P International).
DISCLAIMER
This chapter is an extended version of the article published by the same author(s) in the following journal.
Cancer Research Journal, 4(4): 58-68, 2016.