Effect of Preparation Technique on Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Sample Adequacy: 3 Years of Experience from a Single Center
Dilek ECE1, Gonca GEÇMEN1, Şermin KÖKTEN1, Sevda ŞENER CÖMERT2
1Department of Pathology, Dr. Lütfi Kirdar Kartal Training and Research Hospital, ISTANBUL, TURKEY
2Department of Chest Disease, Dr. Lütfi Kirdar Kartal Training and Research Hospital, ISTANBUL, TURKEY
Keywords: Adequacy, Endobronchial ultrasound, Preparation technique, Transbronchial aspiration
Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used to sample mediastinal lymph
nodes and lesions. However, the methodological details of an optimal preparation technique for aspirated material have not yet been clearly
determined. This study was an evaluation of the effect of 2 preparation techniques on the adequacy of aspirated specimens.
Material and Method: A retrospective analysis was performed of EBUS-TBNA samples obtained at the institution over a total of 36 months. Two
periods were examined. Almost all of the aspirated material was smeared onto slides and fixed with 95% alcohol in the first period of the study.
Subsequently, to improve diagnostic ability, a pair of slides was prepared from each needle pass: the first was air-dried, and the second was fixed
in 95% alcohol. The remainder of the aspirate was kept for cell block analysis.
Results: In total, 462 samples were obtained from 260 patients. The overall sampling adequacy was 74% in the first and 81.1% in the second
period (p<0.05). Approximately 14% of the specimens included a sufficient number of cells for immunohistochemical cell block evaluation in
the first period and 42% in the second period (p<0.001). Histological subtyping of non-small cell lung carcinoma was determined in 18 (56.3%)
and the primary origin of a tumor was determined based on morphological and immunohistochemical properties in 32 (84.2%) of the patients
in the first and second periods, respectively.
Conclusion: The access to a sufficient EBUS-TBNA sample and the ability to perform the appropriate preparation can impact the specific
diagnosis and treatment of patient with a single procedure.
Over the past 2 decades, endobronchial ultrasound-guided
transbronchial needle aspiration (EBUS-TBNA) has
emerged as a minimally invasive, highly accurate technique
for sampling the mediastinal lymph node (LN) (1,2). This
technique allows for both visualization of the target lesion
and the surrounding structures, and the simultaneous
collection of a cytological sample of the target lesion under
direct sonographic guidance. Since tissue diagnosis is
strongly recommended as part of patient management, and
because the qualification of the cytological material affects
the pathological conclusion, the method of acquiring and
processing the EBUS-TBNA specimen plays a critical role
in case of a patient with enlarged mediastinal LNs (3).
Recently, with the introduction of novel targeted therapies
for non-small cell lung carcinoma (NSCLC), the accurate
diagnosis and appropriate classification of subtypes have become more important in patient management (4). This
has led to increased expectations from the pathologist/
cytopathologist, for example, regarding squamous
and glandular differentiation, especially in cytological
samples. Thus, conventional cytological preparation
and cytomorphological evaluation alone do not always
provide sufficient information to determine primary or
secondary origin of adenocarcinoma, especially in cases
of poorly differentiated carcinoma. Other techniques,
such as immunohistochemistry (IHC) for subtyping, and
molecular analysis for targeted therapies, are often required
(5). Consequently, it has become apparent that the means
of acquiring and processing the cytological specimens can
help to guide optimal treatment of advanced NSCLC (6).
The success of EBUS-TBNA, as in fine-needle aspiration
(FNA) of other localizations, is directly related to the
quality of the sample and the interpretation, and thus, the
appropriate treatment of patients. The quality of the sample is dependent on obtaining and preparing specimens that
are representative of the lesion with sufficient quantity and
quality for accurate interpretation. However, such criteria
defining adequacy and the methodological details about the
preparation technique of EBUS-TBNA materials have not
yet been clearly determined (7). The daily workload, habits,
and the available facilities at each institution affect the
processing of specimens. In many centers, the handling of
EBUS-TBNA specimens generally consists of conventional
smearing and fixation with 95% alcohol and/or air-drying
and additional cell block preparation from the residual
material (8-10). In a few centers, liquid-based cytology
and/or a combination of these techniques is preferred (11).
As a result of an excessive daily workload at this center,
rapid on-site evaluation (ROSE) of FNA and EBUS-TBNA
samples is limited. Slide preparations are finalized in an
aspiration room by a clinician or other skilled personnel.
Traditionally, almost all of the aspiration material is spread
onto several slides and there is little residual material
for cell block samples. However, the introduction of
novel targeted therapies and histological subtyping of
NSCLC have increased the cell block importance. In this
retrospective study, 2 methods of examining EBUS-TBNA
material collected to evaluate mediastinal LNs and lesions
from the lung, mediastinum, and trachea were reviewed.
The purpose of the study was to compare the sampling
adequacy and cell block quality of 2 preparation techniques.
The archives of the institutions pathology and pulmonary
disease clinics were reviewed for EBUS-TBNA cases
between January 1, 2009 and January 1, 2012, a period of
36 months. The data from a total of 462 specimens of the
consecutive 260 patients were assessed. All of the procedure
notes and cytological material were retrieved.
Each EBUS-TBNA procedure was performed by a chest
physician in the bronchoscopy unit of the pulmonary
diseases clinic. A 7.5-MHz BF-UC160F convex probe
bronchoscopy (Olympus Optical Co., Tokyo, Japan;
approved by FDA) and an EU c2000 processor (Olympus
Optical Co., Tokyo, Japan) were used via the oral route.
Topical anesthesia was applied with lidocaine, and sedation
was administered using midazolam. Aspiration from a
target LN or lesion in the lung, trachea, or mediastinum
was executed using an Olympus 22-G NA-201SX-4022-C
needle (Olympus Optical Co., Tokyo, Japan; approved by
FDA). Rapid on-site evaluation was not performed during
the procedure. The number of passes was determined by
the chest physician as previously described (12).
Slide preparation was performed in a bronchoscopy suite by
a member of the EBUS-TBNA team, a staff pulmonologist,
pulmonary resident, or a nurse.
In the first period of the study, almost all of the aspirated
material was smeared onto glass slides (2-41 slides per case)
and immediately fixed with 95% alcohol. The remainder of
a pass sample was rinsed into a 95% alcohol-filled tube for
cell block preparation. All needles from the same site were
rinsed in the same alcohol tube to prepare 1 cell block per
In the second period of the study, the EBUS-TBNA team
was informed by a pathologist (DE) about alternative
processing of the aspirated material in order to improve
diagnostic ability. Depending on the quantity of aspirated
material, a pair of slides was prepared from each needle
pass in this period (1-8 slides per case). For each pair of
slides smeared, one was air-dried and the other was fixed
immediately in 95% alcohol. The remnants of aspirate from
the same localization were collected in a tube and filled
with 95% alcohol for cell block sampling.
Air-dried slides were stained using May-Grunwald-
Giemsa (MGG) stain, and 95% alcohol-fixed slides with
a Papanicolaou stain. Cell blocks were prepared using
formalin fixation and paraffin embedding techniques from
pellets obtained by centrifugation of the needle rinse fluids.
Three-μm slides obtained from cell blocks were stained with
hematoxylin and eosin. Immunohistochemical evaluation
was performed as needed.
The cytological materials were reassessed for sampling
adequacy and cytological diagnosis by a pathologist (DE)
with cytopathology experience.
Sampling adequacy was defined as acquisition of tissue
sufficient for pathological diagnosis. LN specimens that
demonstrated specific diagnoses or adequate lymphocytes
were considered adequate. An adequate amount of
lymphoid material was defined as the presence of over
40 lymphocytes visible in high power field in the most
dense cellular areas of the slides or in the presence of
clusters of anthracotic pigment-laden macrophages as
defined by Alsharif et al.(8). If no diagnosis was possible
or insufficient lymphocytes were available to verify
satisfactory LN sampling, the sample was considered
inadequate. Aspiration samples of lung, mediastinum, or
trachea lesions that demonstrated a specific diagnosis were
considered adequate. If no specific diagnosis was made,
the sample was considered inadequate, even if bronchial
epithelium or pigmented macrophages were found.
In both LN and lesion specimens, the cytological diagnosis
was malignant, atypical, benign, or non-diagnostic. The
presence of obviously malignant cells led to a classification
in the malignant category. The observation of rare cells that
were suspicious but not clearly defined as malignant was
In LN specimens, the presence of adequate lymphocytes
without evidence of a tumor was considered benign.
Benign diagnoses were categorized as metastasis-negative
or granulomatous inflammation.
Lung, mediastinum, and trachea specimens that demonstrated
a specific benign lesion, such as granulomatous inflammation,
were considered benign.
For both LN and lesion specimens, the sample was
determined to be non-diagnostic when there was inadequate
material and only blood, mucus and/or necrotic material
was seen, or when the specimen was acellular.
Cell blocks containing cells of a specific lesion (tumor or
granuloma) were assessed as adequate. These cell blocks
were examined with a high-power field, and tumor cells
and epithelioid histiocytes were enumerated. The cell count
was estimated with a scoring system of 1-4. A sample with a
small number of cells was assigned a score of 0 (<10 tumor
cells/epithelioid histiocytes), a low cell count merited a
score of 1 (approximately 10-49 tumor cells/epithelioid
histiocytes), low to moderate numbers were given a
score of 2 (approximately 50-99 tumor cells/epithelioid
histiocytes), a moderate cell count scored 3 (approximately
100-499 tumor cells/epithelioid histiocytes), and a profuse
sample was given a score of 4 (approximately >500
tumor cells/epithelioid histiocytes). A cell score of 0 was
considered inadequate. Cell blocks that demonstrated a
specific diagnosis and were given a cell score of 1 or more
were assessed as adequate for IHC evaluation.
Molecular testing at this institution is performed based on
clinician request and these data were not reviewed for the
Diagnostic yield and sampling adequacy were calculated
using the standard definitions as described by Ost et al.
(13). Diagnostic yield was defined as the frequency of
a recognized disease (e.g., NSCLC or granulomatous
diseases) on the basis of cytological findings. If, in spite
of an adequate number of lymphocytes, no diagnosis was
made, the specimen was considered non-diagnostic. These
samples were examined and classified for each patient.
Sampling adequacy was defined as cytological specimens
that demonstrated a specific diagnosis or adequate
lymphocytes. If no diagnosis was made or insufficient lymphocytes were available to verify adequate LN
sampling, the samples were considered inadequate. These
were performed on a per LN/lesion basis.
The data were summarized as the mean (±SD), and median
(range) for continuous variables and as a percentage for
categorical variables. Normal distribution was tested with
the Kolmogorov-Smirnov test. The Pearson chi-square test
was used to test categorical variables and Students t-test
for continuous variables. A p value of <0.05 was considered
significant for all tests. IBM SPSS Statistics for Windows,
Version 20.0 (IBM Inc., Armonk, NY, USA) was used to
perform the analysis.
In all, 462 EBUS-TBNA specimens obtained from 260
patients [158 males (60.8%), 102 females (39.2%)] were
included in the study. The patients age ranged from 16
to 82 years, with a median age of 55 years. From the total
of 260 patients, 177 specimens were sampled from 114
patients during the first period and 285 specimens from
146 patients were analyzed in the second period. The data
of patient and sample characteristics are summarized in
. There was no statistically significant differentiation
in the demographics of the patients from the first and
second periods of the study.
The mean number of slides per LN station/lesion sampled
was greater in the first period group than the second (12.8
vs 3.9; p<0.001).
There were a great many more cell blocks prepared
from EBUS-TBNA aspirate in the second period group
compared with the first (33.9% vs 97.9%; p<0.001). An
adequate quantity of cells for additional evaluation was
available in 14.1% and 42.1% of the samples in the first and
second period, respectively (p<0.001) (Table II).
The cytological diagnoses of the specimens are summarized
in Table III. Of the benign cytological samples, 24.9%
(44/177) in the first period and 28.1% (80/285) in the
second period revealed granulomatous inflammation
(Figure 1A-D). The overall sampling adequacy of EBUSTBNA
specimens was 78.4% on a per LN/lesion basis. In the
first group, the sampling adequacy was 74% compared with
81.1% in the second group. The difference was statistically
Click Here to Zoom
|Figure 1: Benign cytological samples. A,B) Benign adequate lymph node. A) Over 40 lymphocytes seen in high power field in the most
dense areas of the slide (MGG; x100). B) Pigmented macrophages (MGG; x1000). C,D) Granulomatous inflammation. C) Epithelioid
histiocytes (MGG; x200). D) Granuloma with necrosis in cell block (H&E; x200).
A specific diagnosis was made in 148 (56.9%) patients. The
diagnostic yields were 54.4% and 58.9% in the first and
second period, respectively. The difference was statistically
Of the total of 260 patients, 78 [30 (26.3%) and 48
(32.9%) in the first and second period, respectively] had
granulomatous inflammation, and 70 [32 (28.1%) and
38 (26%) in the first and second period, respectively]
demonstrated a malignancy (p=0.741).
Immunohistochemistry and Subtyping of Tumors
The IHC method was used for the histological subtyping of
tumors in patients with a malignancy. IHC techniques were used to evaluate cell blocks in 18.8% (6 patients) in the first
period, compared with 35.6% (21 patients) in the second
period. The histological subtyping of tumors, established
based on morphological and IHC characteristics, are
illustrated in Table IV. The extrapulmonary malignancies
determined were breast carcinoma (n=1) and renal
cell carcinoma (n=1) in the first period, and colorectal
carcinoma (n=1), breast carcinoma (n=2), and malignant
melanoma (n=1) in the second period (Figures 2A-D,3A-D).
Click Here to Zoom
|Figure 2: Cytological features of lung carcinomas. A) Squamous cell carcinoma (PAP; x400). B) Adenocarcinoma (PAP; x400). C) Small
cell carcinoma (PAP; x1000). D) Large cell neu-roendocrine carcinoma (MGG; x400).
Click Here to Zoom
|Figure 3: Cytological features of extrapulmonary metastases. A,B) Colon carcinoma. A) (PAP; x1000). B) Immunohistochemistry with
CDX2 (IHC; x100). C,D) Breast carcinoma. C) (PAP; x1000). D) Immunohistochemistry with estrogen receptor (IHC; x200).
Although, sampling adequacy is an important component
of EBUS-TBNA performance, the criteria for adequacy have
not yet been established (7). Similarly, the methodological
detail of the preparation technique is not clear. Studies have
revealed a variation in the acceptability rate of EBUS-TBNA
samples (8,9,11,14). Due to the fact that EBUS-TBNA
is a multistep process, many factors (number of needle
passes, needle size, type of sedation, aspirator experience,
aspiration localization, LN size, and the use of ROSE) have
the potential to affect the adequacy of the specimen (11-
21). Hence, factors influencing EBUS-TBNA sampling
adequacy have been the subject of many studies. Most of
these factors, including anesthesia type, needle size, the
EBUS-TBNA team, and the cytopathologist, were the same
and ROSE was not performed during our 2 study periods. Statistically, the number of needle passes, the LN/lesion
size, and the aspiration localization were not significantly
different between procedure periods. Only the preparation
technique was different in the first and the second periods
of this study. We focused on the relationship between
preparation technique and sampling adequacy and cell
block quality in this study.
The majority of EBUS-TBNA studies, as in our second
period, have used both wet-fixation with 95% alcohol and
an air-drying technique with cell block evaluation prepared
from the residual material. The adequate diagnostic
specimen rates have been reported at between 94% and
77% (8-12). Specimen adequacy was observed in 81.1%
in the second period of our study. In the first period, in
which only wet fixation with 95% alcohol was used, it was
74%. Nonetheless, in 1 study that used only wet fixation, the adequate specimen rate was reported as 92% (15). The
technique of the aspiration provides specimen adequacy.
Each pass in the study provided qualified and abundant
material, including tissue cores. A lower adequacy rate in
the first period of our study may be related to spreading
most of the material on slides, rather than using the fixation
Various slide results have been reported in EBUS-TBNA
studies. Some have described smearing slides for each
aspiration, and others per aspiration site or per case (8-
11,15). In Alsharifs study, (8) with a mean of 6 slides
prepared for each LN localization, independent of the LN
station, the specimen adequacy rate was 84.3%. Similarly,
in the second period of our study, a mean of 4 slides was
prepared for each aspiration localization, and the adequacy
rate was found to be 81.1%, independent of the aspiration site. In the first period, however, the average was 13 slides
prepared for each localization and the adequacy rate was
Although the difference in diagnostic yield was not
significant between the first and second periods of the study,
the difference in adequacy rate was statistically significant.
The cell blocks of EBUS-TBNA material from the second
period displayed substantially more cells. Smearing the
majority of the aspirated material on slides may lead to
too little material remaining for an ancillary cell block
technique. Neoplastic cells may be identified on slides, but
histological subtyping of a tumor may not be determined.
A converse habit for the preparation of slides, using just
a portion of the aspirated material, may provide both
neoplastic cells on slides and more material for an ancillary
cell block technique.
Recently it has become apparent that subtyping and
genotyping help to guide optimal treatment of advanced
NSCLC. The pathologist/cytopathologist has had to
cope with a rise in the need for accurate diagnosis and
appropriate classification of subtypes. Its well known
that adenocarcinomas and squamous cell carcinomas can
be diagnosed in most instances, especially with the aid
of cell blocks and IHC in cases of poorly differentiated
carcinomas (5). Consequently, cell block preparation from
EBUS-TBNA samples is a simple way to provide additional
information in cases of NSCLC. Sanz-Santos et al. (22)
investigated the contribution of cell blocks to the diagnosis
of lung cancer and found that 47.9% of EBUS-TBNA
specimen cell blocks from EBUS-TBNA sampling provided
additional important information. Adequate material for
diagnosis was recovered from 37.6% of the samples. Cell
block processing provided clinically significant information
for one-third of the lung cancer patients in their study.
Histological subtyping of NSCLC and the primary origin
of tumors was determined using morphological and IHC
properties in 18 (56.3%) patients in the first period and 33
(86.8%) patients in the second period of our study.
In addition to the histological subtyping of NSCLC,
molecular testing to identify key driver mutations is
required for the appropriate treatment of patients with
adenocarcinoma. These ancillary tests require an adequate
amount of tissue. Molecular tests, such as EGFR mutation,
KRAS mutation, and ALK gene rearrangement analyses can
be reliably performed on cell block material from cytological
specimens (5,22). Successful EGFR DNA sequence analysis
can be obtained from as few as 30 to 100 tumor cells (23).
In our study, cell blocks contained a minimum of 50 tumor
cells in 9 (23.1%) of 39 malignant cases in the first period
and 30 (50.9%) of 59 malignant cases in the second period.
As demonstrated in the second period of our study, leaving
a little more material aside for cell block analysis instead
of smearing the majority of the material from a EBUSTBNA
specimen on slides will provide the opportunity for
morphological diagnosis, IHC, and molecular analysis in
order to provide the appropriate treatment.
Limitations of this study include the small sample size
and the lack of molecular analysis results. However, the
reliability of the results is increased due to the single-center
approach and focus on preparation technique.
In conclusion, recent advances in therapy for NSCLC and
concurrent developments in EBUS-TBNA have allowed
for a combined use of EBUS-TBNA for tissue acquisition
and genotyping of lung carcinoma. Sufficient material and appropriate preparation, especially high-quality cell
block preparations, can affect the diagnosis, staging, and
treatment of a patient with a single procedure.
CONFLICT of INTEREST
The authors declare no conflict of interest.
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