The Contribution of Additional Sampling in Cholecystectomy Materials: A Multicenter Prospective Study
Samir ABDULLAZADE1, Fahire Göknur AKARCA2, Güldal ESENDAĞLI2, Nesrin TURHAN3, Esra ERDEN4, Berna SAVAŞ4, Fatma MARKOÇ5, Deniz TUNÇEL6, Banu Özgüven YILMAZ6, Burcu SAKA7, Sevinç Hallaç KESER8, Selma Şengiz ERHAN9, Zühal GÜCİN10, Özgül SAĞOL11, Anıl Aysal AĞALAR11, Sevinç ÇELİK12 , Hatice ÖZER13 , İpek ERBARUT SEVEN14 , Çiğdem Ataizi ÇELİKEL14, Özgür EKİNCİ2, Hatice Reyhan EĞİLMEZ13, Serdar BALCI15, Gülen AKYOL2
1Department of Pathology, İzmir Tepecik Education and Research Hospital, İZMİR, TURKEY
2Department of Medical Pathology, Gazi University, Faculty of Medicine, ANKARA, TURKEY
3Department of Pathology, Turkey Yüksek İhtisas Education and Research Hospital, ANKARA, TURKEY
4Department of Medical Pathology, Ankara University, Faculty of Medicine, ANKARA, TURKEY
5Department of Pathology, Dr. Abdurrahman Yurtaslan Ankara Oncology Education and Research Hospital, ANKARA, TURKEY
6Şişli Etfal Education and Research Hospital, İSTANBUL, TURKEY
7Department of Medical Pathology, Medipol University, Faculty of Medicine, İSTANBUL, TURKEY
8Department of Pathology, Lütfi Kırdar Education and Research Hospital, İSTANBUL, TURKEY
9Okmeydanı Education and Research Hospital, İSTANBUL, TURKEY
10>Department of Medical Pathology, Bezmialem Vakıf University, Faculty of Medicine, İSTANBUL, TURKEY
11Dokuz Eylül University, Faculty of Medicine, İZMİR, TURKEY
12Bozok University, Faculty of Medicine, YOZGAT, TURKEY
13Cumhuriyet University, Faculty of Medicine, SİVAS, TURKEY
14Marmara University, Faculty of Medicine, İSTANBUL, TURKEY
15Pathologist, ANKARA, TURKEY
Keywords: Gallbladder, Cholecystectomy, Dysplasia, Cancer, Macroscopy
Cholecystectomy materials are frequently encountered in routine practice. The aim of this study was to determine the true frequency
of gallbladder lesions, the diagnostic consistency, and standardization of reports after macroscopic sampling and microscopic evaluation based
on previously defined criteria.
Material and Method:14 institutions participated in the study within the Hepato-Pancreato-Biliary Pathology Study Group. Routinely examined
cholecystectomies within the last year were included in the study in these institutions. Additional sampling was performed according to the
indications and criteria. The number of blocks and samples taken in the first macroscopic examination and the number of blocks and samples
taken in the additional sampling were determined and the rate of diagnostic contribution of the additional examination was determined.
Results: A total of 5,244 cholecystectomy materials from 14 institutions were included in the study. Additional sampling was found to be necessary
in 576 cases (10.98%) from all institutions. In the first macroscopic sampling, the mean of the numbers of samples was approximately 4 and the
number of blocks was 2. The mean of the numbers of additional samples and blocks was approximately 8 and 4, respectively. The diagnosis was
changed in 144 of the 576 new sampled cases while the remaining 432 stayed unaltered.
Conclusion: In this study, it was observed that new sampling after the first microscopic examination of cholecystectomy materials contributed to
the diagnosis. It was also shown that the necessity of having standard criteria for macroscopic and microscopic examination plays an important
role in making the correct diagnosis.
Cholecystectomies are frequently encountered in the
pathologists daily routine and are usually performed for
various benign etiologies. The common opinion is that all
cholecystectomy materials should undergo pathological
examination (1). Furthermore, there are a few studies discussing the pathological examination of the entire
cholecystectomy material (2). Gallbladder carcinomas are
difficult to detect clinically and radiologically in the early
stages and 75% of malignant cases are not resectable at the
time of diagnosis (3). There are various methods for the
macroscopic examination of cholecystectomy materials (4-8) and all these methods are important in detecting
incidental gallbladder cancer. The Hepato-Pancreato-
Biliary (HPB) Pathology Study Group has also conducted
a multicenter retrospective study to assess gallbladder
lesions and establish common macroscopy and microscopy
protocols in our country (9).
There are still some problems in the microscopic approach
to the epithelial lesions of the gallbladder. The lesions
to be reported, and the indications and criteria for
additional sampling are topics of discussion. Although
cholecystectomy material is frequently encountered
in routine pathology practice, gallbladder epithelial
anomalies and neoplasms are uncommon (10, 11).
Gallbladder epithelial lesions include metaplastic lesions
(antral/pyloric metaplasia, intestinal metaplasia), benign
epithelial neoplasms (adenomas/adenomyomas), biliary
intraepithelial neoplasia (BilIN) (dysplasia/carcinoma
in situ), and invasive carcinomas (11). In the differential
diagnosis of these lesions, especially the ones that are not
macroscopically evident, there are inconsistencies among
the observers in approaching these lesions in addition to
the problems experienced due to the nature of the lesions.
Protocols that should be followed in the sampling and
microscopic evaluation of the gallbladder remain important
and controversial since high-grade dysplasia and even
invasive carcinomas cannot be diagnosed macroscopically
in general (12).
A total of 23 pathologists from 14 institutions (8 University
Hospitals and 6 Training and Research Hospitals)
participated in this prospective study within the HPB
Pathology Study Group. There is no expert consultation by a single qualified expert for dysplastic lesions. This study
included cholecystectomy materials routinely analyzed in
these institutions within the last year. We used a form for
patient consent. In the first macroscopic examination of
the material in accordance with the decided method, the
surgical margin of the ductus cysticus was sampled in a
way that the sectional side could be seen completely and
was sampled completely by removing a full slice from the
fundus to the ductus cysticus (Figure 1
) (9). All polyps,
if present, in the material (including cholesterol polyps)
were sampled. Microscopic examinations were continued
by examining the new samples in order to find out if any
neoplastic polyp with focal epithelial atypia (including
denuded epithelium), intestinal metaplasia, or high- or lowgrade
dysplasia were detected. In this method, the criteria
defined for the reanalysis of macroscopic examination and
for additional sampling (AS) were as follows (12):
1) If pyloric-gland metaplasia or mucinous pyloric gland
nodules smaller than 3 mm is detected, there is no need
2) If a pyloric gland lesion larger than 3 mm is detected,
the material and the container are reevaluated in terms
of preinvasive papillary lesions. (No need for AS if the
pathology is not observed.)
3) If intestinal metaplasia is detected, 2 cassettes of AS are
taken as 2 to 3 samples per cassette.
4) If focal epithelial atypia is detected, 2 cassettes of AS are
taken in the same way.
5) If severe atypia is found in addition to the denuded
epithelium, 4 cassettes are taken in a similar way.
6) If high-grade dysplasia is detected, at least 12 cassettes
are taken in the same way.
7) If a neoplastic polyp with dysplasia of any size is detected,
the lesion is sampled completely and, in addition, five
cassettes are obtained from the surrounding mucosa.
8) If invasive carcinoma is detected, 7-12 cassettes are taken
to show the depth of the lesion and the relationship with
the hepatic bed.
In accordance with these methods, the number of blocks
and samples taken in the first macroscopic examination of
the materials and after AS, as well as the number of new
sampled cholecystectomy materials (cases), were reported.
The rate of change in histopathological diagnosis following
additional examinations of new sampled cholecystectomy
materials was reported.
A total of 5,244 cholecystectomy materials from 14
institutions were included in the study. AS was found to
be necessary in 576 cases (10.98%) from all institutions.
Males made up 189 patients while the remaining 387 were
female. The average age was 54.3 years. The number of
specimens in the first macroscopic sample ranged from 2
to 28 (mean 4.34) and the number of blocks ranged from
1 to 28 (mean 2.01). Among additional sampled cases, the
number of additional samples varied between 2 and 51 (mean 8.04) and the number of additional blocks between 1
and 29 (mean 4). Of the 576 new sampled cases, 432 had no
change in the diagnosis, while the diagnosis was changed
in 144 cases. Adenocarcinoma was found in 10 cases, highgrade
dysplasia in 7, low-grade dysplasia in 40, reactive/
regenerative atypia in 4, neoplastic polyp in 3 (biliary
adenoma-tubular, tubulopapillary, villous), and intestinal
metaplasia in 38 (Figure 2
Click Here to Zoom
|Figure 3: A) Adenocarcinoma (H&E; x40). B) High grade dysplasia (H&E; x200). C) Intestinal metaplasia (H&E; x100). D) Low grade
dysplasia (H&E; x100).
Epithelial lesions of gallbladder are common in routine
practice since cholecystectomy is a frequently performed
surgery (10,13). However, neoplasms and epithelial
anomalies of the gallbladder are rare. Since even highgrade
dysplasia and invasive carcinomas cannot be
macroscopically detected in general (12), it is important to
develop an easy-to-use, effective and up-to-date protocol
for sampling as well as for microscopic evaluation of the
It has been reported that the clinical course is excellent in
high-grade dysplasia cases in which invasive carcinoma was
detected after AS and also in the in situ carcinoma/highgrade
dysplasia cases in which the invasion was excluded
after examining the entire material (14,15). In terms of treatment, one of the most important points to keep in
mind is that cholecystectomy alone is sufficient in Tis and
T1 tumors (16,17), whereas the surgical resection should be
extended in cases where deeper invasion is detected (18,19).
Among other pathologies of the gallbladder, pyloric
metaplasia is the most common type. Instead of this term,
pseudopyloric, antral or mucosal gland hyperplasia is
also used. Polyps with pyloric metaplasia, if larger than
1 cm, are categorized as neoplastic (or adenoma) in the
presence of dysplasia in the metaplastic area. When chief
and parietal cells are seen, heterotopia is the most likely
diagnosis instead of metaplasia (10). Intestinal metaplasia,
which is characterized by the presence of goblet cells, is less
commonly seen than pyloric metaplasia.
It is however known that intestinal metaplasia is related
to carcinoma, in a similar way as in the stomach (10). Examples of other metaplasia types include squamous,
neuroendocrine, and pancreatic acinar cell metaplasia
(10). AS is strongly suggested in cases where intestinal
metaplasia is detected due to the carcinoma relationship
described above. Considering the continuity of the
gallbladder with bile ducts extending into the liver, the
importance of pathology in the cholecystectomy material
can be understood. Therefore, identification of all lesions
(including hyperplasia and metaplasia) in cholecystectomy
material in pathology reports is important both in terms of
the database and clinical follow-up of the patients.
In our study, the rate of adenocarcinoma was 1.7% and
this rate is compatible with the literature. The rate of
incidental carcinoma in the literature ranges from 0.25%
to 2% (20-22). In the case of adenocarcinoma found in a
cholecystectomy material, the treatment plan also changes.
Extension of the surgical resection area, lymphadenectomy, or chemo-radiotherapy can be applied to these patients. In
this respect, it is important that the parameters required for
treatment planning are included in the pathology report.
The required parameters can be listed as the type of the
procedure, histological type of the tumor, the location
and size of the tumor, whether lymph nodes contain
metastases or not, and the depth of invasion. In this regard,
the macroscopy guide of the HPB Pathology Study Group
including gallbladder carcinomas, has been updated and
published on the website (23). The latest protocol, also
recommended by the College of American Pathologists,
emphasizes that the pathology report should include
the localization, size, grade, spread, surgical margins of
the tumor as well as lympho-vascular and perineural
invasion, regional lymph nodes, grade, and additional
histopathological findings such as dysplasia/adenoma,
cholelithiasis, chronic and acute cholecystitis, intestinal
metaplasia, diffuse calcification (porcelain gallbladder),
and primary sclerosing cholangitis in the liver bed (24).
There are various methods in the literature for macroscopic
examination and sampling of gallbladder resection materials
(4-8). The issue of how many new samples should be taken in
a case of dysplasia is still controversial (25, 26). In addition,
the criteria for discrimination between low-grade dysplasia
and reactive atypia as well as true diagnosis of such lesions
have not been clearly identified (27). Although the rate of
dysplasia was reported to be 3.3% (11), this rate varies in
the literature. In another study on this subject, this rate was
reported to be 0.4-33.8% (28). It has also been reported
that the gallbladder should be examined completely when
dysplasia was observed in the first examination, while there
is a study indicating that 4 samples may be sufficient in such
cases (25). The authors of this study indicated that the low
rate of dysplasia in their series (<0.5%) is due to the lower
risk for dysplasia of their patient population than other
studies or insufficient sampling / low number of samples
(i.e. 1 cassette with 2 samples). In a study of Adsay et al. (12),
which was conducted in 2013, it was stated that the risk in
the population should be taken into account when looking
for a neoplastic lesion as the neoplastic lesions cannot
always be detected easily, especially during the macroscopic
examination. They also emphasized the important
knowledge about the presence of various epithelial lesions
in association with neoplastic lesions that can be seen in
gallbladder. In the recent consensus meeting held in 2015,
it was concluded that at least 3 samples should be obtained
from routine cholecystectomy materials including the
ductus cysticus margin in the geographic regions where
the incidence of gallbladder cancer is high, while materials
bearing dysplasia or cancer should be fully examined (29).
There are several studies conducted on the frequency of
epithelial anomalies and sampling methods in our country
(4,30). Argon et al. (4) recommend that a longitudinal
sample starting from the neck of the gallbladder to the
fundus should be obtained and placed into the cassette
as a rolled-up (Swiss roll) figure. Higher rates of pyloric
metaplasia, intestinal metaplasia, low-grade dysplasia,
and invasive carcinoma were reported with this method
compared to the method where the fundus and the body
were examined separately. Bolat et al. (30) reported
increased rates of metaplasia, dysplasia, epithelial
hyperplasia and inflammation by increasing the number
of samples obtained from cholecystectomy material.
Several methods were suggested in the literature about how
macroscopic sampling should be done and the methods to
be followed when epithelial atypia is detected (10,13,31).
In our study, the mean number of blocks was 4 in AS cases.
In the study of Wrenn et al. (20), which includes the cost
analysis, the rate of important pathological lesions detected
by histopathological examination of cholecystectomy
materials was found to be low. Nonetheless, it is necessary to
continue to perform histopathological examination in the
light of the cost analysis. The authors assert that evaluation
of risk factors, intra-operative findings and on-table
evaluation of the materials may be an alternative approach
(20). In an activity-based cost study conducted in Turkey,
the laboratory cost of pathology materials was compared to
prices indicated in the Healthcare Regulation Report (32).
According to this study, cholecystectomy materials have
one of the lowest costs among pathology materials. Given
the prevalence and low cost in addition to the importance
of early detection of malignancies, it can be assumed that
AS is not a significant burden for an institution. However,
there is no large-scale study on this topic in our country.
The limitations of our study can be listed as the lack of a
standard way for creating the database (i.e. mismatched
terminology of the lesions among the institutes or lesions
found to be too unimportant to mention in the pathology
reports), lack of expert consultation by a single qualified
expert for dysplastic lesions; and the lack of demographic,
socioeconomic, pre- and postoperative follow-up data and
cost-effectiveness analysis of AS during the pathological
examination of cholecystectomy specimens.
In conclusion, it was found that obtaining new samples
from the gallbladder after the first microscopic examination
of material contributed to the diagnosis. In addition, the
importance of specifying and using standard criteria for
macroscopic and microscopic examination was emphasized
in the current macroscopy guide of the HPB Pathology Study Group (23). In our study, it was concluded that all
pathological findings observed in the cholecystectomy
material must be specified in the pathology report.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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