Keywords frozen - imprint cytology - sensitivity
Introduction
The female reproductive system is a very significant part of a body system. It definitely
has the capability to function with nearly all others organs of body for the purpose
of reproduction.
Gynecological neoplasms are among the most common cancers in female population of
India and worldwide. There is approximately 60% of cancer burden among women which
include breast, ovary, corpus uteri, and cervical cancers. Mostly women report in
advanced stage of cancer in India. Being a developing country, there is a lack of
awareness, varying pathology of tumor, and shortage of proper screening facilities.
Ovarian and cervical cancers are the most common female genital organ cancers affecting
women worldwide and in India. According to GLOBOCAN 2020 data, total numbers of new
cases are 313,959 and deaths due to ovarian carcinoma are approximately 207,252. Total
number of new cases in corpus uteri and cervix uteri cancers are 417,367 and 604,127,
respectively.[1 ] Various new advances have been made to diagnose gynecological pathologies which
include imprint cytology and frozen sections in addition to the gold standard histopathological
techniques.
Imprint cytology is a popular technique for pathological assessment. The excised tissue
is sent fresh to the pathologist who processes it immediately. It provides rapid diagnosis
(within 20 minutes) without losing the architecture of the tissue. This procedure
can be done in less developed setting with minimal trained technicians.[2 ]
Frozen sections are used in gynecological practices mostly to help in differentiating
between benign and malignant diseases. The reason of performing this procedure is
to provide the doctors with information which enables them to perform the most apt
treatment. The diagnostic accuracy of intraoperative rapid frozen procedure in gynecological
neoplasms is assessed to be approximately 91 to 97%. This technique is performed by
embedding a fragment of tissue in Tissue-Tek OCT compound and cutting.[3 ] The present study was therefore conducted to assess the accuracy of intraoperative
diagnostic techniques and their comparison with histopathological diagnosis which
was considered as gold standard for tumor diagnosis.
Materials and Methods
The observational study was performed in the department of pathology over a period
of 12 months with a sample size of 50 samples which were included for statistical
purpose by convenient sampling. The subjects were recruited after obtaining written
informed consent and ethical clearance. Sampling methods included all the female patients
who came with complaints related to benign and malignant lesion of female genital
tract. Patients with prior chemotherapy or radiotherapy and tumors with extensive
hemorrhage or necrosis were excluded from our study. After noting all the gross features
including capsular integrity of the received specimen, it was sectioned and imprints
were made from the cyst wall and solid areas of the fresh specimen, avoiding lateral
movements, by applying a slight but firm pressure against the glass slides. A minimum
of four slides were prepared and were fixed in 90% isopropyl alcohol for 5 minutes
and stained with pap/Giemsa as well as were kept air dried. Isopropyl alcohol was
used as a fixative in our study as per our departmental standard operating procedures.
It was preferred over 95% ethyl alcohol in view of better fixation, cost effectiveness,
accessibility, and better results in staining of slides in our department. For pap
staining, the fixed smears were hydrated, dipped in Harris' alum hematoxylin, decolorized,
and differentiated using 1% acid alcohol. These differentiated smears were then put
into running tap water for bluing following which they were dehydrated and stained
with OG-6 and EA-50. Air-dried or isopropyl alcohol-fixed smears were also stained
with May–Grunwald working solution followed by Giemsa stain which was washed with
6.8 pH buffer and mounted. The smears were evaluated for cellularity, arrangement
of epithelial cells, cellular features of malignancy, necrosis, and background. Benign
category included few number of cells, arranged in sheets with no overlapping, absent
tumor diathesis, no nuclear atypia, and small nuclei with abundant cytoplasm whereas
large cell clusters with overlapping, tumor diathesis, marked nuclear atypia, and
enlarged nuclei with scant cytoplasm were included in malignant category. Any cells
having mild nuclear atypia with moderately overlapping clusters and having features
not fitting into clear benign or malignant categories on imprint cytology were labeled
as borderline category. Blinding was applied for evaluation of these criteria for
classifying tumors in various categories on imprints. Frozen sectioning (at –20°C
to –30°C on a cryostat instrument) of the same specimen was done, fixed in 10% neutral-buffered
formalin and stained in Harris' (progressive) hematoxylin followed by 1% aqueous eosin.
Simultaneous hematoxylin and eosin (H&E) sections of the main specimen were prepared
at the time of frozen reporting and compared with findings on frozen. With all the
data in hand, a correlative study of imprint cytology, frozen sectioning, and histopathology
was performed, taking histopathology as the gold standard.
Results
The study included 50 cases of suspected gynecological neoplasms ranging from 19 to
76 years of age with mean age of patients being 53.14 ± 14.17 years and median of
53.50 years. Majority of the cases were married (98%), had complaint of pain in abdomen
(70%), were multigravida (96%), and were postmenopausal (60%). Maximum number of specimens
retrieved were from ovaries (68%) followed by uterus (26%) and cervix (6%). Thirty-four
cases (68%) were labeled as malignant on radiological investigations which included
contrast-enhanced computed tomography scan and ultrasound of pelvis and abdomen.
Imprint cytology, frozen sections, and histopathology were done in all the cases which
were categorized into benign, malignant, and borderline. Thirty-eight (76%) cases
were diagnosed as malignant using histopathology whereas n = 31 (62%) and n = 26 (52%) were diagnosed as malignant using imprint cytology and frozen section,
respectively. Borderline cases of ovary which were reported as n = 1 (2%) on imprint cytology and n = 4 (8%) on frozen section were actually n = 2 (4%) cases on histopathology. Among histopathology, 30% cases belonged to serous
papillary cystadenocarcinoma of ovaries whereas among imprint cytology 24% cases were
diagnosed as endometrioid adenocarcinoma and ovarian adenocarcinoma. Similarly, 52%
cases were diagnosed as malignant on frozen section.
[Table 1 ] shows that there was a significant statistical difference between the various groups
in terms of distribution of histopathological diagnosis and frozen section diagnosis
(chi-square = 13.646, p = 0.004), hence proving their association, similarly [Table 2 ] also shows that there was a significant statistical difference between the histopathological
diagnosis and imprint diagnosis (chi-square = 27.427, p ≤ 0.001), thus proving their association.
Table 1
Association between diagnosis: frozen section and diagnosis: histopathology (n = 50)
Diagnosis: Histopathology
Diagnosis: Frozen section
Fisher's exact test
Benign
Borderline
Malignant
Total
Chi-square
p -Value
Benign
9
(45.0%)
0
(0.0%)
1
(3.8%)
10
(20.0%)
13.646
0.004
Borderline
1
(5.0%)
0
(0.0%)
1
(3.8%)
2
(4.0%)
Malignant
10
(50.0%)
4
(100.0%)
24
(92.3%)
38
(76.0%)
Total
20 (100.0%)
4 (100.0%)
26 (100.0%)
50 (100.0%)
Note: Fisher's exact test.
Table 2
Association between diagnosis: imprint and diagnosis: histopathology (n = 50)
Diagnosis: Histopathology
Diagnosis: Imprint
Fisher's exact test
Benign
Borderline
Malignant
Total
Chi-square
p -Value
Benign
9
(50.0%)
1
(100.0%)
0
(0.0%)
10
(20.0%)
27.427
< 0.001
Borderline
2
(11.1%)
0
(0.0%)
0
(0.0%)
2
(4.0%)
Malignant
7
(38.9%)
0
(0.0%)
31
(100.0%)
38
(76.0%)
Total
18 (100.0%)
1
(100.0%)
31 (100.0%)
50 (100.0%)
Note: Fisher's exact.
[Table 3 ] shows that sensitivity of imprint cytology was 77.5% which was slightly greater
than sensitivity of frozen section. The diagnostic accuracy of cases reported by intraoperative
imprint cytology was seen to be much higher, that is, 80% than frozen section which
was 76%.
Table 3
Primary diagnostic parameters (frozen section and imprint cytology impression)
Variable
Sensitivity
Specificity
PPV
NPV
Diagnostic accuracy
Impression: Imprint
77.5%
(62–89)
90.0%
(55–100)
96.9%
(84–100)
50.0%
(26–74)
80.0%
(66–90)
Impression: Frozen section
72.5%
(56–85)
90.0%
(55–100)
96.7%
(83–100)
45.0%
(23–68)
76.0%
(62–87)
Abbreviations: NPV, negative predictive value; PPV, positive predictive value.
[Fig. 1 ] shows concordant cases having similar findings in imprint cytology, frozen section,
and histopathology, whereas [Fig. 2 ] shows discordant cases as findings of imprint cytology and frozen section were not
similar on comparison with gold standard histopathology.
Fig. 1 Photomicrographs of concordant cases suggesting similar findings in imprint cytology,
frozen section, and histopathology sections. (A ) Imprint cytology showing benign sheets of cells in ovarian cyst adenoma (hematoxylin
and eosin [H&E] 40 × , 10 × ). (B ) Frozen section showing mucinous cystadenoma of ovary lined by stratified columnar
epithelium (H&E 20 × , 10 × ). (C ) Histopathological examination of mucinous cystadenoma of ovary lined by stratified
columnar epithelium with apical mucin (H&E 20 × , 10 × ). (D ) Imprint cytology showing anucleate squames in teratoma (May Grunwald-Giemsa [MGG]
20 × , 10 × ). (E ) Frozen section of mature cystic teratoma showing nest of squamous epithelium (H&E
10 × , 10 × ). (F ) Histopathological examination of mature teratoma showing hair follicle (H&E 10 × ,
10 × ).
Fig. 2 Photomicrographs of discordant cases with findings not similar when compared with
gold standard histopathology. (A ) Imprint cytology of endometrium showing adenocarcinoma with tumor cells forming
vague acini (May Grunwald-Giemsa [MGG] 40 × , 10 × ). (B ) Frozen section showing infiltrating endometrial carcinoma into the stroma (H&E 10 × ,
10 × ). (C ) Histopathology examination of endometrium showing epithelial component in mixed
Mullerian tumor (H&E 4 × , 10 × ). (D ) Histopathological examination of endometrium showing sarcomatous component in mixed
Mullerian tumor (H&E 20 × ,10 × ).
For calculating predictive values from imprint cytology, frozen section, and histopathology,
all borderline and malignant cases were grouped under malignant/borderline to calculate
predicting impression. Maximum true positive cases were diagnosed by histopathology
as it being the gold standard. True negative and false positive cases were equally
reported by both frozen and imprint cytology. False negative cases were reported by
frozen section than imprint cytology.
A statistical significance (p -value < 0.05) was observed in correlation of CA-125 with histopathology, comparison
of histopathology with imprint diagnosis, comparison of histopathology with frozen
section, and comparison of diagnosis of imprint cytology with frozen section. However,
the kappa statistical value, deciding the agreement between two methods, was lesser
in frozen versus histopathology and more in imprint versus histopathology.
Discussion
The present observational study was done in a tertiary care hospital. Therefore, cases
taken were mainly patients suffering from critical illness or suspicious female genital
tract neoplasms.
Female gynecological neoplasms have global distribution, but vary from one region
to another. Cervical, ovarian, and endometrial carcinoma are the most common type
of cancers in female genital system. Cervical cancer is the fourth most frequently
diagnosed cancer and the fourth leading cause of cancer death in women, with an estimated
604,000 new cases and 342,000 deaths worldwide in 2020. Similarly, total number of
new cases in ovarian, corpus uteri, and cervix uteri cancers are 313,959, 417,367,
and 604,127, respectively.[1 ] Dudgeon and Patrick were the first to describe the imprint smears of fresh tissues
in the rapid microscopic diagnoses of tumors.[2 ] Hence, we found from our research that intraoperative imprint cytology procedure
was better and a rapid method for diagnosis of lesions which helps in making surgeon
quick decision for surgery. In the past, many studies were done on imprint cytology
and frozen section in intraoperative diagnosis of various tumors especially ovarian
neoplasms. But we undertook this study as there was no much emphasis given on intraoperative
diagnostic role in gynecological tumors which can further help surgeons in early and
accurate diagnosis.
The total numbers of cases included in the present study were 50. The studies done
by Bokhman et al,[4 ] Lee,[5 ] and Kumar et al[6 ] included more cases since they studied the cases for a longer duration of time and
these studies were done for suspected ovarian neoplasm. The present study was done
for a limited duration of 1 year and it comprises all those suspected female genital
tract lesions whose frozen and imprint were done in our hospital.
Bokhman et al study[4 ] was one of the most comparable studies to our research as the age group in the former
was 13 to 96 years and the mean age was 54.5, whereas in comparison to our study,
the age ranged from 19 to 76 years with a mean age of 53.14 years and a median age
of 53.50 years. In one of the previous studies by Terzic et al, they have observed
immature teratoma in a 17-year-old female,[7 ] similarly we had reported one such case in a 19-year-old and findings were comparable
to the study. According Morice et al, endometrial cancers were more prevalent in patients
above 55 years of age.[8 ] We had 8 such cases of endometrioid carcinomas in which females were mostly 56 years
or above. The findings in our study were comparable with Platz and Benda which showed
that most malignant and aggressive cancers were seen with increasing age group and
TNM staging.[9 ]
Shahid et al assessed the role of intraoperative cytology in ovarian neoplasms. They
studied 50 cases, out of which 25 were labeled as benign, 24 as malignant, and 1 was
reported as inconclusive. This intraoperative cytology was compared with histopathology
which resulted in 25 malignant lesions and 25 benign lesions resulting in a sensitivity,
specificity, and diagnostic accuracy of 95.8, 96.0, and 95.8%, respectively.[10 ] In our research, we included all female genital tract lesions, comprising of 34
ovarian lesions as the most common intraoperative diagnosis. Imprint diagnosis showed
that there were 17 benign cases, 16 malignant cases, and 1 borderline case. On comparison
with histopathology, these 17 benign cases on imprint cytology, actually turned out
to be less (10 benign lesions). There were 5 cystadenomas on imprint smears which
were reported as 1 mucinous and 4 serous cystadenocarcinoma on H&E stained sections.
Two teratoma cases turned out to be immature teratoma which has a high malignant potential.
All malignant imprint smears were reported as same on histopathology and borderline
tumor was labeled as mucinous cyst adenoma. Hence, our statistics resulted in a sensitivity
of 77.5%, specificity of 90.0%, positive predictive value (PPV) of 96.9%, negative
predictive value (NPV) of 50%, and diagnostic accuracy of 80%. Our study showed discordance
among these values as we took samples from all parts of female genital tract that
came for intraoperative diagnosis whereas most studies included ovarian samples for
imprint cytology.
According to Khan et al, diagnostic accuracy was assessed in 54 patients by intraoperative
frozen section in pelvic neoplasms which also included ovarian malignancy. These frozen
sections were later compared with histopathological examination. Their frozen diagnosis
included 20 benign cases, 6 malignant cases, 1 borderline, and 3 deferred cases. On
paraffin-embedded sections, 17 cases were benign, 1 was borderline, and 2 were malignant.
The malignant and borderline cases were reported as same on histopathology. Out of
the 3 deferred cases 2 were reported as malignant and 1 as borderline. There diagnostic
accuracy was 92.6% with sensitivity of 75%, specificity of 97.6%, 90% PPV, and 93.2%
NPV.[11 ] This study was compared with our present study in which 20 cases were diagnosed
as benign lesions, 4 were diagnosed as borderline, and 26 were diagnosed as malignant
cases. Histopathology was done which resulted in diagnosing 10 benign lesions into
malignant, that is, 4 benign ovarian cysts turned out to be serous and mucinous cyst
adenocarcinomas, 5 endometrioid carcinomas were reported on frozen as endometrial
polyp, high-grade dysplasia, and complex endometrial hyperplasia, and 1 was diagnosed
as teratoma which was diagnosed as immature teratoma on histopathology. Hence, our
study statistics showed diagnostic value of frozen sections in terms of sensitivity,
specificity, NPV, and PPV as 72.5, 90.0, 45.0, and 96.7%, respectively. However, the
diagnostic accuracy of frozen sections remained low, that is, 76.0% versus 80% of
imprint cytology. There was discordance in diagnostic accuracy as number of borderline
and benign cases reported on frozen were wrong.
The study done by Negri et al emphasized the role of family history in cases of cervical
cancers[12 ] and similarly Cramer et al studied the significance of family history in ovarian
cancers and correlated with follicular phase hormone levels.[13 ] Both the studies proved their importance as there is twice the risk of cancers in
cases of positive family history. We studied such 7 cases in which 6 ovarian tumors
had a positive family history. Out of them 4 had first degree relative on maternal
side and 2 cases had second degree relative on maternal side and first degree relative
on paternal side, respectively. There was one case of cervical cancer which reported
as poorly differentiated squamous cell carcinoma and had a positive history of breast
cancer in patient's mother who had mastectomy. Thus, our findings were in concordance
with the earlier studies.
An important limitation of the present study is that lesser number of cases were included
which may have resulted in statistical bias. Difficulty faced while preparing imprints
was that yield obtained was low in cases, especially stromal tumors, and care had
to be taken not to cause capsular rupture in cystic lesions.
Conclusion
Our study concludes that the maximum cases of female genital tract neoplasms belonged
to the age group of 19 to 76 years, with 60% cases in the postmenopausal age group.
In most of the ovarian neoplasms initially complaints were abdominal distension associated
with pain. Abnormal uterine bleeding was seen in cervical and endometrial carcinomas.
In many cases of suspicious ovarian, cervical, and endometrial neoplasms, per vaginal
discharge were present along with foul smelling in some which favors a malignant outcome.
Majority of specimens taken were ovary (n = 34, 68%), as intraoperative diagnosis is more required in ovarian lesions. Serum
tumor markers CA-125 and CEA were helpful in making diagnosis in ovarian neoplasms.
Positive family histories were relevant in cases of ovarian and cervical carcinoma.
Radiology played its vital role in making diagnosis of various benign and malignant
lesions on cytology and H&E stained sections. The intraoperative imprint cytology
showed sensitivity of 77.5%, specificity of 90.0%, PPV of 96.9%, NPV of 50%, and diagnostic
accuracy of 80%. Diagnosis by intraoperative frozen section showed sensitivity, specificity,
NPV, and PPV as 72.5, 90.0, 45.0, and 96.7%, respectively. The kappa statistical value,
deciding the agreement between two methods, was lesser in frozen versus histopathology
and more in imprint versus histopathology.
