|Year : 2021 | Volume
| Issue : 3 | Page : 120-126
|Role of Endobronchial ultrasound guided transbronchial needle aspiration with cellblocks in diagnosis and subtyping of intrathoracic lesions: Two year experience from a tertiary care center
Vikrant Verma1, Ajmal Khan2, Ram Nawal Rao1, Alok Nath2, Zia Hashim2
1 Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Pulmonary Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
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|Date of Submission||23-Mar-2021|
|Date of Decision||20-Jun-2021|
|Date of Acceptance||24-Jun-2021|
|Date of Web Publication||24-Aug-2021|
| Abstract|| |
Background: Endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) allows precise real-time sampling of intrathoracic lesions and is a minimally invasive, safe, and cost effective technique with high diagnostic yield. The aim of the current study is to evaluate utility of EBUS-TBNA cytology with cellblocks (CBs) in the diagnosis and subtyping of the intrathoracic lesions. Materials and Methods: It was a prospective study conducted from October 2015 to October 2017. We received 233 cases of intrathoracic lesions for EBUS-TBNA, of which CB was made in 217 cases. A flexible endobronchial ultrasound scope was used to sample the lesions. Results: There were 89 females and 128 males amongst the total 217 patients (age range: 14–85 years, mean age: 48.35 years). The smears from EBUS-TBNA contained adequate material in 137/144 [95.1%] non-neoplastic cases and 34/36 [94.4%] of the neoplastic cases, whereas the CBs provided adequate material in 94/144 [65.3%] non-neoplastic cases and 37/40 [92.5%] neoplastic cases. The CB helped subtyping the malignancy in 19 cases and of these immunohistochemistry (IHC) was done on the CB in 15 cases. The biopsy was non-diagnostic in 17/36 cases of granulomatous pathology and 4/18 neoplastic cases diagnosed with EBUS-TBNA.3. Conclusions: EBUS-TBNA provides rapid diagnosis through cytology and the material recovered in the same setting for the CB preparation can be used for immunohistochemical analysis and it may at times provide the diagnosis in cases where the smears are non-diagnostic.
Keywords: Cellblocks, endobronchial ultrasound-guided transbronchial needle aspiration, intrathoracic, lung lesions, mediastinal
|How to cite this article:|
Verma V, Khan A, Rao RN, Nath A, Hashim Z. Role of Endobronchial ultrasound guided transbronchial needle aspiration with cellblocks in diagnosis and subtyping of intrathoracic lesions: Two year experience from a tertiary care center. J Cytol 2021;38:120-6
|How to cite this URL:|
Verma V, Khan A, Rao RN, Nath A, Hashim Z. Role of Endobronchial ultrasound guided transbronchial needle aspiration with cellblocks in diagnosis and subtyping of intrathoracic lesions: Two year experience from a tertiary care center. J Cytol [serial online] 2021 [cited 2022 May 26];38:120-6. Available from: https://www.jcytol.org/text.asp?2021/38/3/120/324485
| Introduction|| |
Endobronchial ultrasound guided transbronchial needle aspiration (EBUS-TBNA) is the modality of choice for rapid diagnosis and subtyping of intrathoracic lesions. It allows for assessment of lung masses and also their spread to mediastinal nodes. Furthermore, it yields adequate material for the diagnosis of the non-neoplastic lesions like tuberculosis which is present with nonspecific clinico-radiological findings.
Cellblock (CB) material obtained with EBUS-TBNA increases the diagnostic yield. It provides good architectural details similar to histological specimens. The aim of the current study was to evaluate utility of EBUS-TBNA cytology with CBs in the diagnosis and subtyping of the intrathoracic lesions.
| Materials and Methods|| |
This was a prospective study that included 233 cases of intrathoracic lesions who underwent EBUS-TBNA from October 2015 to October 2017. Both air-dried and alcohol fixed smears were made. A total of 217 cases were studied after excluding the hemorrhagic and inadequate samples (16 cases). There were 89 females and 128 males amongst the total 217 patients. The age range was 14 to 85 years and the mean age was 48.35 years. The mean age of those who presented with a non-neoplastic lesion was 45.6 years and the age range was 16 to 78 years. Of the neoplastic cases, there were 27 males and 13 females, the mean age was 57.1 years, and the age range was 14 to 80 years. Analysis of CBs from EBUS guided TBNA in all the cases of intrathoracic lesions was done in our study. This study was approved by Institutional Ethics Committee (IEC Code No. 2016-32-MD-EXP). No further invasive procedure as mediastinoscopy, video-assisted thoracoscopic surgery (VATS), or thoracotomy was done in this study in any case. The smears were considered as representative of lymph node sampling if the number of lymphocytes was more than 40 in the most cellular areas with most lymphocytes, under 40× magnification or if there was presence of clusters of anthracotic pigment-laden macrophages.
A written consent was obtained from each patient after explanation of the risks and benefits involved before performing the procedure of EBUS-TBNA in all cases.
Lymph nodes or the lung lesions identified on contrast enhanced computed tomography (CECT) scan of the thorax were used as a guide for EBUS-TBNA. The procedure was performed in a bronchoscopy suite with prior informed consent duly signed by the patient or their relatives.
The procedure was performed under conscious sedation using intermittent intravenous midazolam injection. All patients were given nebulized anesthesia (1%–2% lignocaine) locally before the procedure to achieve anesthesia in the oropharynx and the vocal cords. A flexible endobronchial ultrasound scope (BF-UCT180-F, Olympus, Japan) was passed through the oral route. After initial visualization of the tracheo-bronchial tree, ultrasonic evaluation of the lesion was done to characterize the lesion for its size, character, and vascularity. Once the target lesion was identified, the EBUS-TBNA needle (NA-201SX-4021, Olympus, Japan) was inserted through the working channel of the scope and the lesion was punctured under real-time US guidance with stylet of the needle in place. Once adequate length of the needle was inside the node, stylet of the needle was jabbed 2–3 times to and fro to remove any bronchial tissue inside the needle. After removal of stylet, a constant negative pressure of 25–30 mm of H2O was applied and the needle was jabbed 10–15 times in the nodes under vision and the acquired sample was collected on the slide and Eppendorf tube. Minimum of three passes were taken from each node, including a separate pass to acquire the sample for CB preparation.
Both air-dried and wet ethanol-fixed smears were made (5 to 8 smears per patient) from the aspirated material in each case for May-Grünwald-Giemsa (MGG), hematoxylin-eosin (H and E), and Papanicolaou stain.
The CBs were prepared through fix sedimentation method and Shandon Cytoblock method. In the fixed sedimentation method, the acquired FNA material was poured in Eppendorf tube containing 10% buffered formalin. This was then centrifuged at 1000 rpm for 10 minutes and supernatant was discarded. Sediment was then removed and put in to a filter paper, placed in a cassette, and processed in an automated tissue processor like routine histopathology specimens.
In the Shandon Cytoblock Cell Block method, to the acquired FNA sample, double amount of REAGENT 2 (blue colored fluid, containing formaldehyde) was added to the specimen pellet and mixed. The Cytoblock cassette was fitted into the Cytoclip. Cytofunnel disposable chamber was placed over the prepared Cytoblock and FNA sample was poured in the Cytofunnel. The assembled Cytoclip was placed into the Cytospin rotor and set for 5 minutes at 1500 rpm. The Cytofunnel assemblies were then removed and one drop of Reagent 1 (calcium chloride) was placed on top of the cell Button and then the Cytoblock cassette was closed. Cassettes were processed in a standard automated tissue processor.
Various special stains applied on smears and CBs were Ziehl-Neelsen (ZN) staining, perioidic acid schiff (PAS), perioidic acid schiff after diastase (PAD), and gram stain and reticulin stain on the CBs. Immunohistochemical markers applied on CBs as required in included Pan-Cytokeratin (Pan-CK), TTF1, CK7, CK5/6, p63, Synaptophysin, Chromogranin, neuron-specific enolase (NSE), CD56, Vimentin, Desmin, smooth muscle actin (SMA), S100, Myogenin, prostate specific antigen (PSA), ER, PR, Her2neu, CK19, CD99, leukocyte common antigen (LCA), CD3, CD20, TdT, and Ki-67.
The patient's clinical records were retrieved from the hospital information system and patient record files from department of pulmonary medicine. For all patients, clinical data including age, sex, presenting symptoms, clinical examination findings, radiological findings, cytohistological findings, and biochemical parameters (if any) were obtained. The IBM SPSS Statistics 21 software was used to analyze the data.
| Results|| |
In this prospective study, EBUS-guided TBNA was done from the mediastinal mass in 197 cases and from the lung mass in 20 cases. The most common presenting complaint of the patients was cough (147 cases) followed by breathlessness (90 cases) and fever (87 cases). The other less common complaints were chest pain (33 cases), loss of appetite (20 cases), loss of weight (18 cases), joint pain (6 cases), hoarseness of voice (6 cases), haemoptysis (5 cases), weakness (5 cases), and dysphagia (3 cases), in the decreasing order of frequency. Of the total 217 cases, the number of neoplastic cases was 40, whereas the non-neoplastic cases comprised of 144 cases. In 33 cases, the smear, as well as the CB, was inadequate for evaluation, and a large proportion of these cases were from the early period when the EBUS-TBNA was a relatively new technique in the institute; the diagnostic yield improved over the time.
There were 184 adequate aspirates, of which 139 aspirates were diagnostic of a pathology and 45 cases showed reactive hyperplasia. Hence, the diagnostic yield of EBUS-TBNA was 64.1% (139/217) while the overall yield was 84.8% (184/217). The smears from EBUS-TBNA contained adequate material in 137/144 [95.1%] of non-neoplastic cases and 34/36 [94.4%] of neoplastic cases. In four neoplastic cases, only CB was prepared and no smear was prepared. The CBs provided adequate material in 94/144 [65.3%] of non-neoplastic cases. The diagnostic rate of the CBs was high in the neoplastic cases and contained diagnostic material in 37/40 [92.5%] of neoplastic cases.
The detailed categorization of the non-neoplastic cases is provided in [Table 1]. The cytology smears were diagnostic in more number of cases as compared to the CBs; however, CBs provided the diagnosis in seven granulomatous cases where the smears were non diagnostic, thereby increasing the diagnostic yield. There were 16 cases of mycobacteriologically proven tuberculosis in this study. Diagnosis of tuberculosis was made on the basis of positive ZN staining (11/16; 68.8%), culture (11/13; 84.6%), or genexpert (7/8; 87.5%).
Among the non-neoplastic cases, the biopsy from the lung mass or the mediastinal mass were available in 36 cases of granulomatous pathology, 13 cases of reactive lymphoid hyperplasia, 2 cases with caseation necrosis without granuloma, and 1 case with a diagnosis of suppurative pathology with EBUS-TBNA. Of the 36 cases of granulomatous pathology in which the biopsy was available, 19 cases showed granulomatous pathology, whereas 17 cases did not reveal any definite pathology on biopsy. Hence, EBUS-TBNA is indispensible in the workup of the cases with suspected diagnosis of tuberculosis or sarcoidosis. Interstitial lung disease was diagnosed on lung biopsy in three of the cases where the lymph node aspirates revealed reactive lymphoid hyperplasia. EBUS-TBNA plays a vital role in such cases of interstitial lung diseases with associated reactive mediastinal lymphadenopathy, since it helps to rule out any other pathology. One case with diagnosis of reactive lymphoid hyperplasia on EBUS-TBNA revealed small cell lung carcinoma on biopsy, and one case with only necrosis on EBUS-TBNA revealed squamous cell carcinoma on the biopsy of the lung mass. Hence, the importance of the biopsy in cases with high suspicion of malignancy and negative results on EBUS-TBNA must be considered. Furthermore, granulomas were noted on lung biopsy in one case of reactive lymphoid hyperplasia on EBUS-TBNA and one case with a diagnosis of suppurative pathology on EBUS-TBNA.
The list of individual neoplastic cases is provided in [Table 2]. The cases where smears revealed malignant cells but the subtyping of malignancy was not possible were also labeled as diagnostic of malignancy. The diagnostic rate of CB was very high in the neoplastic cases, i.e. 92.5% (37 of 40 cases). The utility of CB preparation in the neoplastic cases is highlighted in [Table 3]. The CBs add to the diagnostic yield of EBUS-TBNA and also aid in the classification of neoplastic lesion through immunohistochemistry (IHC). IHC was done on CB in 15 cases in this study. Furthermore, it was found that in several cases the biopsy from the primary mass was non-diagnostic, whereas EBUS-TBNA with the CB allowed for the diagnosis of malignancy [Table 3].
|Table 3: Significance of cellblock from EBUS-TBNA of neoplastic intra-thoracic lesions (n=40)|
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The representative images of both non-neoplastic and neoplastic cases along with corresponding special stains and IHC are shown in [Figure 1],[Figure 2], [Figure 3].
|Figure 1: Tuberculous lymphadenitis: (a) EBUS-TBNA of subcarinal LN, (b and c). Cytology smear (MGG 400x) and CB (H and E 400x) show granuloma, (d) Positive ZN staining (1000x); Granulomatous pathology (likely Sarcoidosis): (e) EBUS-TBNA of 4R LN, (f) Smear (MGG 400x) and (g) CB (H and E 200x) show granuloma, (h) Reticulin rich granuloma suggestive of sarcoidosis (400x); Lymphoblastic lymphoma: (i and j) Smear (MGG 200x) and CB (H and E 200x) show small to medium sized atypical lymphoid cells, (k) Positive staining for CD3 and negative CD20 (inset), (l) Strong positive TdT (IHC 200x), Ki-67 is ~ 70% (inset)|
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|Figure 2: Adenocarcinoma: (a) Smear shows atypical cells (MGG 400x) with cytoplasmic PAD staining (400x) (inset), (b) CB shows acini of tumour cells (H and E 200x), (c and d) Positive CK7 and TTF1 (IHC 200x); Squamous cell carcinoma: (e) Smear (MGG 400x) and CB (H and E 400x) show atypical polygonal epithelial cells (MGG 400x), (g and h) Positive CK5/6 (IHC 200x) and p63 (IHC 400x); Small cell carcinoma: (i and j) Smear (MGG 100x) and CB (H and E 200x) show round to ovoid tumour cells with nuclear moulding, (k) Positive Synaptophysin and Chromogranin (inset) (IHC 200x), (l) Ki-67 ~70% (IHC 200x)|
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|Figure 3: Primitive Neuroectodermal Tumour: (a) Smear (MGG 200x) and (b) CB (H and E 200x) show small round tumour cells, (c) Positive CD99 (IHC 200x), (d) Vimentin and NSE (inset) (IHC 200x); Metastatic papillary thyroid carcinoma: (e and f) Smears show papillae of cells (MGG 200x) with occasional intranuclear inclusions (MGG 400x), (g) CB shows follicles of tumour cells (H and E 200x), (h) Positive CK19 (IHC 200x); Metastatic prostatic carcinoma: (i) Smear (MGG 200x) and CB (H and E 200x) show tumour cells in closely packed acini, (k and l) Positive Pan CK (IHC 200x) and PSA (IHC 400x)|
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| Discussion|| |
EBUS-TBNA is a minimally invasive technique that has evolved as the procedure of choice for evaluation of intrathoracic masses including neoplastic as well as non-neoplastic pathologies. It is a novel technique which combines the endoscopic visualization with high-frequency ultrasound imaging. Hence, it allows precise sampling of lesions adjacent to the tracheobronchial tree, including mediastinal and hilar lymph nodes. Earlier conventional TBNA (cTBNA) was used as a non-invasive modality for sampling of the mediastinal lesions. However, the yield of EBUS-TBNA is much higher as compared to cTBNA for both benign and neoplastic cases. In a study of over 100 cases of mediastinal lymphadenopathy and or mass lesions, positive predictive value of EBUS-TBNA for malignancy was nearly 100%. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of EBUS-TBNA, EUS-FNA, and combined EBUS-TBNA and EUS-FNA evaluated in a study were noted as 81.8%, 100%, 100%, 81.8% and 90%; 81.8%, 100%, 100%, 75%, and 88.2%; and 90.9%, 100%, 100%, 90.0%, and 95.0%, respectively. EBUS-TBNA can also aid in the diagnosis of extrathoracic malignancies with intrathoracic metastasis. In our study, sampling via EBUS-TBNA revealed four cases of extrathoracic malignancy with mediastinal lymph node metastasis, including ductal carcinoma breast (2 cases), papillary thyroid carcinoma (PTC) (1 case), and prostatic adenocarcinoma (1 case).
Similar to the previous studies, the smears from EBUS-TBNA contained adequate material for evaluation of majority of both non-neoplastic (137/144 [95.1%]) and neoplastic cases (34/36 [94.4%]) and it provided a high overall yield in our study [Table 4]. The diagnostic yield was 64.1% (139/217).
Combining CBs with the conventional smears increases the diagnostic yield for both non-neoplastic and neoplastic cases. The addition of CBs to EBUS-TBNA smears increased the yield from 80.6% (175/217) to 84.8 (184/217) % in this study, by providing diagnosis in nine cases (seven granulomatous pathology and two neoplastic cases) where the smear was non-diagnostic [Table 5].
|Table 5: Contribution of cellblock to the yield of EBUS-TBNA (benign or malignant cases)|
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In a similar study, the contribution of CBs to the diagnostic yield of EBUS-TBNA for cases of malignancy and sarcoidosis was assessed. When cytological examination of smears was not diagnostic, but CB found granulomatous inflammation or malignancy, it was defined as “contribution to diagnosis by CB.” When cytological examination of smears was reported as only malignancy, but CB or immunohistochemical staining (IHCS) reported the subtype of cancer, it was defined as “contribution to subclassification by CB” [Table 6].
|Table 6: Contribution of cellblock in diagnosis of neoplastic and granulomatous cases, and for the subtyping of the neoplastic cases|
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The CBs from EBUS-TBNA have high diagnostic rates for neoplastic cases and may at times have even better diagnostic yield than conventional smears. In this study, there were two neoplastic cases for which the smear was non-diagnostic and the diagnosis was provided only by the block. Furthermore, the CBs play a vital role in the subtyping of the malignancies since the immunohistochemical studies can be performed with a greater ease on the CBs as compared to the conventional smears. There were 19 cases in this study where the smears showed malignant cells, but the subtyping of malignancy was not possible and the CB helped subtyping these neoplastic lesions. IHC was applied in 15 of these 19 cases for subtyping.
| Conclusions|| |
Through this study, we conclude that EBUS-TBNA plays a vital role in the evaluation of neoplastic as well as non-neoplastic intrathoracic lesions as it provides rapid diagnosis through cytology and material can also be recovered for the CB preparation in the same setting. The CB material can be processed like histopathological samples after embedding them in formalin blocks and it may at times provide the diagnosis in cases where the smears are non-diagnostic. Furthermore, the CBs can be used for immunohistochemical analysis with the same ease as the biopsy specimens, thereby providing the exact subtyping in the neoplastic cases. Hence, making CB preparation indispensable for proper workup in EBUS guided TBNA of the intrathoracic lesions.
We express our deepest gratitude to our family members and the staff of Department of Pathology and Pulmonary medicine for their persistent co-operation.
Institutional Ethics Committee (IEC) Code No.: 2016-32- MD-EXP.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Ram Nawal Rao
Professor, Department of Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow - 226 014, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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