Specimen Processing Techniques for Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration

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1 Specimen Processing Techniques for Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Jennifer W. Toth, MD, Konstantin Zubelevitskiy, MD, Jennifer A. Strow, DO, Jussuf T. Kaifi, MD, PhD, Allen R. Kunselman, MA, and Michael F. Reed, MD Division of Pulmonary, Allergy, and Critical Care, Department of Medicine, Divisions of Surgical Oncology and Cardiothoracic Surgery, Department of Surgery, Department of Public Health Sciences, and Heart and Vascular Institute, Penn State Milton S. Hershey Medical Center, Hershey, Pennsylvania Background. Endobronchial ultrasound is used for sampling thoracic pathologic processes. Histologic examination may provide added diagnostic yield to cytologic preparations owing to superior assessment of architecture and immunohistochemistry. It remains unclear whether specific specimen processing technique impacts diagnostic yield. We hypothesized that diagnostic yield using histologic analysis of core needle biopsies is higher than cytologic preparations alone. Methods. We evaluated 177 consecutive patients with mediastinal abnormalities. An interventional pulmonologist or thoracic surgeon performed endobronchial ultrasound. We compared diagnostic yields of two specimen processing techniques, fixed slides (cytology) and formalinfixed core samples (histology). Results were categorized as malignant, benign (infectious, inflammatory), normal nodal tissue, or inadequate sampling (nondiagnostic). Malignancy, a defined benign process, and normal lymph node were considered diagnostic. Results. The diagnostic yield for benign processes was higher by histologic examination (n 37) than in cytologic preparations (n 22; p ). The diagnostic yield was comparable in malignancy (p ). The combination of both techniques provided a higher overall diagnostic rate: 84% (n 148) by histology, 82% (n 146) by cytology, and 89% (n 158) using both. Using two techniques revealed discordance in 23% (n 40), demonstrating that the use of one technique alone would have resulted in missed diagnoses. Conclusions. Adding histologic analysis of tissue cores obtained by endobronchial ultrasound offers higher diagnostic accuracy than only cytologic preparation of needle aspirates. Histologic and cytologic methods offer comparable diagnostic rates for malignancy. However, diagnostic yield for benign conditions is higher using histologic examination. Together, histology and cytology provide fewer missed diagnoses than either individually. When using endobronchial ultrasound, it is ideal to routinely use both needle aspirate cytology and core biopsy histology. (Ann Thorac Surg 2013;95:976 81) 2013 by The Society of Thoracic Surgeons It is well established that investigation of mediastinal adenopathy and masses has profound implications on patient management, especially in suspected malignancy. Historically, mediastinal lymph node and mass evaluation was accomplished by several methods: imaging only (computed tomography with or without positron emission tomography), transthoracic image-guided needle biopsy, bronchoscopy with blind transbronchial lymph node aspiration (TBNA), mediastinoscopy, left anterior mediastinotomy (Chamberlain procedure), and video-assisted thoracoscopic surgery [1 5]. Each has specific risks and degrees of invasiveness, as well as effectiveness of tissue acquisition. The addition of endoscopic Accepted for publication Nov 27, Presented at the Forty-eighth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 28 Feb 1, Address correspondence to Dr Toth, Penn State Milton S. Hershey Medical Center, Division of Pulmonary, Allergy, and Critical Care Medicine, 500 University Dr, H041, Hershey, PA 17033; jtoth@ psu.edu. ultrasound provided a novel, and often safer, means to access these anatomic locations [6]. A minimally invasive technique for thoracic tissue sampling is endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). It has been particularly studied in the staging of lung cancer. Reports have also documented its application to unknown lesions and evaluation of suspected benign disease [1, 4, 7, 8]. Multiple studies have proven EBUS-TBNA to be a safe procedure [1, 9]. There are minimal nuisance complications that are common to all bronchoscopic interventions, including cough, agitation (especially with the use of conscious sedation), and minor bleeding at the biopsy sites [7]. A recent comparison between EBUS and mediastinoscopy (n 153 medically operable lung cancer patients) demonstrated no complications from EBUS- TBNA [9]. Additionally, there was no significant difference between EBUS-TBNA compared with mediastinoscopy in determining pathologic diagnoses [9]. Sensitivity and specificity for EBUS-TBNA when applied to lung cancer staging has been reported to be 79% 2013 by The Society of Thoracic Surgeons /$36.00 Published by Elsevier Inc

2 Ann Thorac Surg TOTH ET AL 2013;95: SPECIMEN PROCESSING FOR EBUS 977 to 95% and 100%, respectively, in multiple reviews and the American College of Chest Physicians Guidelines on Invasive Mediastinal Staging of Lung Cancer [2 4]. Recent investigations of EBUS-TBNA for evaluation of unknown mediastinal lesions have shown definitive benign diagnoses in 96% of patients studied [1]. In contrast, traditional ( blind ) bronchoscopic TBNA has a reported diagnostic yield of 61% [10]. Multiple techniques for specimen processing have been used in endoscopic ultrasound or EBUS-TBNA. The most common method is for each needle aspirate to be processed using one air-dried slide stained with DiffQuik (a commercial Romanowsky stain variant used to rapidly stain smears, described in Material and Methods) for on-site evaluation of sampling adequacy and a second slide fixed in 95% alcohol and stained with Papanicolaou stain for greater nuclear cytologic detail [1, 9, 11, 12]. Rapid on-site cytologic evaluation (ROSE) is highly recommended as it has been shown to increase sensitivity and specificity without increasing procedure time [9, 11 14]. Paraffin-embedded hematoxylin and eosin stained slides are often made from the centrifuged needle rinses and offer complementary information in identification of histologic patterns and enhanced ability to perform immunohistochemistry and molecular studies [12]. Finally, histologic core biopsies preserved in formalin can provide superior information to that obtained from cell blocks because they are often larger samples and architectural detail is maintained. Multidirectional processing of samples as described by Nakajima and Yasufuku [11] provides a comprehensive specimenprocessing algorithm for use in the patient with mediastinal adenopathy or mass, in whom the potential diagnoses are extensive. During this approach, tissue from needle aspirations is distributed to multiple media (including cytologic preparations, histologic core preparations, RPMI preservation, and flow cytometry) based on ROSE assessment to narrow the differential diagnoses and concentrate on the most efficient use of tissue [11]. We hypothesized that diagnostic yield using histologic analysis of core needle biopsies is higher than that from cytologic preparations alone. Material and Methods Consecutive patients referred to the Multidisciplinary Thoracic Oncology Program at Penn State Milton S. Hershey Medical Center who underwent EBUS-TBNA for mediastinal or hilar adenopathy or masses during the 12-month period between December 1, 2010, and November 30, 2011, were enrolled in this retrospective review. Institutional review board approval and waiver of the need for consent were granted (The Pennsylvania State University College of Medicine). Endobronchial Ultrasound Technique Endobronchial ultrasound-tbna was performed using a flexible ultrasound bronchoscope (convex probe EBUS, BF-UC260F-OL8, Olympus, Tokyo, Japan) with a linear transducer with a frequency of 7.5 MHz. The transducer provides a 50-degree linear continuous B-mode ultrasound image. An endoscopic image is projected at 30 degrees from the long axis of the bronchoscope, parallel to the biopsy channel. A disposable latex balloon placed over the ultrasound probe can be filled with sterile saline or water to provide a liquid interface between the sensor and the tissue [15]. An ultrasound processor (EU-C60, Olympus, Tokyo, Japan) with Doppler capability is connected to the ultrasound bronchoscope. All procedures were performed under general anesthesia using a laryngeal mask airway. Endobronchial ultrasound was performed by either an interventional pulmonologist (J.W.T.) or thoracic surgeon (M.F.R.). Specimen Acquisition and Processing Biopsy targets were based on computed tomography or positron emission tomography plus computed tomography studies. Still ultrasound images were taken at each sampling site, and measurements were made in both dimensions. The 21-gauge needle designed for the scope (NA-201SX-4021; Olympus) was used to perform all EBUS-TBNA [16]. The site was punctured and the internal stylet tapped to clear the needle of bronchial mucosa and cartilage. A 20-mL VacLoc syringe (Merit Medical Systems, Inc, South Jordan, UT) was attached to the end of the needle to provide suction. The needle was moved in and out of the target lesion at least 10 times. Suction was then released, the needle was withdrawn, and the sample was processed accordingly. Adequate sampling is optimized when there is a clear ultrasound image. Additional strategies to potentially increase adequacy include tapping the stylet several times to avoid contamination with airway mucosa, use of suction at 20 on the syringe, and needle agitation 10 to 15 times before withdrawal. Cytologic preparations were made by using the stylet to expel the sample from the needle onto a slide. If this yields minimal to no sample, air is used to push the sample out. Another slide is placed gently over the first to make a smear. One slide is air dried if ROSE is used for DiffQuik staining (Mercedes Medical, Sarasota, FL). If ROSE is not used, then both slides are sprayed with an alcohol-based fixative for later processing. A needle rinse was then obtained by expelling any remainder of the sample into Cytolyt (methanol-based buffered preservative) for cell block preparation. For histologic examination, the stylet is used to expel the sample into formalin. Sterile saline solution is then used to wash any remaining tissue or cells into the formalin. The core biopsies are then embedded in paraffin and stained with hematoxylin and eosin. When immediate results were required, one slide was air dried for DiffQuik staining and interpreted by a cytologist for adequacy of sampling. The DiffQuik staining method is a modification of a Romanowsky stain. The smear is allowed to dry and is then dipped in a fixative for 5 seconds (fast green in methanol). It is then dipped in Eosin G five times for 1 second each. This is followed by dipping the slide in thiazine dye in the exact same manner. The slide is rinsed in distilled water and allowed to dry. Immediate interpretation is then feasible. For each

3 978 TOTH ET AL Ann Thorac Surg SPECIMEN PROCESSING FOR EBUS 2013;95: Table 1. Patient Characteristics Characteristic Malignancy No Malignancy All Age (y) a Male 32 (58%) 78 (64%) 110 (62%) Total a Results are mean standard deviation. aspirate, a second slide was fixed in 95% alcohol and stained with Papanicolaou stain (Thermo Scientific, Kalamazoo, MI). Excess material was placed in Cytolyt preservative (Cytyc Corp, Marlboro, MA) and labeled as needle rinse. Cell blocks were not routinely made from needle rinses unless the cytologist determined the need for additional information not provided by cytologic slides or if the histologic samples (from core biopsies) were nondiagnostic. An additional pass from each site was performed for core biopsy. For core biopsies, tissue was expelled from the 21-gauge needle using the stylet and the needle was flushed with sterile saline solution into formalin for histologic processing, as well as additional molecular studies if indicated. The majority of sampling included three cytology slide preparations and one core biopsy for histologic analysis. Statistical Analysis The EBUS-TBNA results were classified into malignancy, a benign process, inadequate tissue sampled, and normal tissue. The result was considered diagnostic if there was demonstration of malignancy, a defined benign entity (eg, sarcoidosis), or normal tissue (eg, benign lymph node). Inadequate tissue sampling was classified as nondiagnostic. The specimen processing technique was defined as histology (core biopsy) or cytology (slides). To account for the within-subject correlation owing to multiple assessment methods per subject (eg, histology, cytology, and both), generalized linear mixed models were fit using either a multinomial distribution and a generalized logit link for nominal responses or a binomial distribution and a logit link for binary responses [17]. A logistic regression model was fit to assess the association between discordant status (yes/no) and malignancy (yes/no). Effect sizes from the generalized linear mixed models and logistic regression model were quantified using the odds ratio and 95% confidence interval. No adjustments for multiple hypothesis testing were taken into consideration, as this was an exploratory study. All hypothesis tests were two-sided, and all analyses performed using SAS software, version 9.3 (SAS Institute Inc, Cary, NC). Table 2. Diagnostic Yield of Histologic and Cytologic Analyses Yield Histology Cytology Combined Diagnostic 148 (84%) 146 (82%) 158 (89%) Nondiagnostic 29 (16%) 31 (18%) 19 (11%) Total Table 3. Histologic and Cytologic Diagnoses Diagnosis Histology Cytology Both Malignancy Small cell lung cancer Adenocarcinoma Squamous cell carcinoma Epithelioid carcinoma Poorly differentiated carcinoma Neuroendocrine tumor Melanoma Breast carcinoma Sarcoma Lymphoma Benign Inflammation Granulomatous disease Normal thyroid Inadequate sampling Normal Total Results One hundred seventy-seven consecutive patients underwent EBUS-TBNA. Patient characteristics are detailed in Table 1. There was no difference in the age or sex distribution between those diagnosed with malignancy when compared with nonmalignant conditions. One third of patients were diagnosed with malignancy. The overall diagnostic yield was 89% (Table 2). Fifty-five patients (31%) were diagnosed with malignancy (Table 3). Most were primary lung cancers (93%, n 51), and the others represented metastases or lymphoma. Benign conditions were present in 22% (n 39) of patients, with granulomatous inflammation (n 33) accounting for the majority of cases. Normal lymph node (adequate sampling proven by both histologic and cytologic techniques) was obtained in 64 patients (36%). Inadequate sampling by both techniques occurred in 11% (n 19). Diagnostic yield for each technique (histology, cytology, or both) was compared using generalized linear mixed models (Table 4). For benign diagnoses, histologic examination offered a higher diagnostic yield than cytologic analysis (p ). The diagnostic yield was also higher when both histologic and cytologic techniques were compared with cytologic preparations alone (p ). However, there was no difference in diagnostic yield between histologic and cytologic techniques for Table 4. Comparison of Diagnostic Yield Between Techniques Based on Disease Categories Category Histology versus Cytology Both versus Histology Both versus Cytology Malignancy p p p Benign p p p

4 Ann Thorac Surg TOTH ET AL 2013;95: SPECIMEN PROCESSING FOR EBUS 979 Table 5. Discordant Results Between Histologic and Cytologic Analyses Variable Agreement Discordance Malignancy 48 (87%) 7 (13%) No Malignancy 89 (73%) 33 (27%) Total 137 (77%) 40 (23%) malignant diagnoses (p ). The use of both techniques together did not increase diagnostic yield for malignancy when compared with either histologic examination alone (p ) or cytologic preparation alone (p ). Histologic and cytologic results were discordant in 23% of patients (n 40; Table 5). Discordance was lower in the malignancy group (13%; n 7). For patients with nonmalignant conditions, the discordance between the two techniques occurred in 27% (n 33). Most of the discordance can be attributed to the higher diagnostic yield of histologic examination for benign conditions (n 19), in which histologic examination obtained the diagnosis in 17. The discordance for malignant conditions (n 7) was comparable between the techniques with histologic examination identifying 3 and cytologic technique identifying 4. Of the 65 patients with normal lymphoid tissue, 13 had further tissue confirmation. Ten were concordant with the EBUS results. Of the three discordant results, two had granulomatous disease based on mediastinoscopy and one had lymphoma based on video-assisted thoracoscopic surgery. None had carcinoma. Fifty-two had no tissue confirmation. Thus, the negative predictive value of EBUS for carcinoma, as well as all malignancy, appears to be high when normal lymphoid tissue is demonstrated. Of the 52 patients without tissue confirmation after EBUS, the indication for the procedure was for benign diagnoses in 13 whereas 39 were in the setting of malignant conditions. In those with malignancy, none had subsequent imaging to suggest false-negative diagnoses. Nineteen patients had inadequate sampling or nondiagnostic results. Four of these patients had subsequent tissue sampling. Two were negative for malignancy based on surgical resection, whereas 2 were positive for malignancy based on additional bronchoscopic sampling. Of the 15 without tissue verification, 8 had subsequent imaging, none of which suggested missed malignant diagnosis. Molecular testing is currently used primarily for adenocarcinomas, particularly for advanced disease requiring chemotherapy. We identified 16 adenocarcinomas on core biopsy. Based on the consensus recommendations of a multidisciplinary thoracic tumor board, 5 cases were sent for molecular testing. Four of 5 provided adequate tissue for molecular testing, and affected treatment. Of note, immunohistochemical testing was also frequently used effectively on core biopsy specimens in settings other than primary lung adenocarcinoma. Comment Endobronchial ultrasound-tbna has revolutionized tissue acquisition for diagnosis and staging of thoracic pathologic processes, particularly mediastinal adenopathy. Here we compared two established specimen processing techniques. With 177 consecutive patients undergoing EBUS-TBNA, this represents the largest study specifically comparing histologic and cytologic specimen processing [13, 18 21]. Notably, it is one of the few studies to report 100% histologic and cytologic correlation [20]. This permitted comparison of the techniques in the same patients rather than relying on historical experience or separate control groups. The reported diagnostic yield of EBUS-TBNA is 77% to 95% [13, 18 21]. Our diagnostic yield of 89% is comparable. Some series achieve high diagnostic yields by including biopsy of pulmonary parenchymal lesions, by using endoscopic ultrasound-tbna, and by performing traditional blind TBNA with larger gauge needles. In this series, we specifically included only patients with mediastinal pathologic disease that was accessible by linear EBUS. Here we demonstrated that histologic examination provided a higher diagnostic yield than cytologic preparation for benign processes. There was no difference between cytologic and histologic techniques in the diagnosis of malignancy. In our definition, if either technique obtained a positive finding, it was considered a true positive. Thus, more cytologic specimens were incorrectly interpreted as normal lymph node. One explanation for diagnostic equivalence between the techniques when evaluating malignancy is that a limited number of cells may provide sufficient material for diagnosis, whereas diagnosis of some benign conditions (eg, sarcoidosis) requires tissue architecture, which is better obtained with larger samples. This concept is supported by the low discordance between the two techniques in malignancy, but a higher rate of discordance in nonmalignant conditions. Moreover, among benign conditions in which discordance existed, histologic examination achieved the diagnosis in 17 of 19 cases. Delattre and colleagues [22] obtained improved diagnostic yield using the strategy of replacing standard cytologic slide preparations with a liquid-based approach in a cohort of patients primarily undergoing lung cancer staging. A lower rate of inadequate sampling was obtained because of an increased yield of specimen sufficient for paraffinembedded cell blocks. In their experience, the combination of standard histologic and liquid-based cytologic processing improved overall diagnostic rates for both malignant and benign conditions. Our results suggest that routine use of both histologic and cytologic preparations is optimal. First, EBUS is frequently used when the cause of the abnormality is unknown. In this case, the higher diagnostic yield of histologic examination for benign conditions would support the routine use of both specimen processing methods. Second, even in malignancy, the discordance of only 13% resulted from 7 patients in whom 3 were diagnosed by histologic exam-

5 980 TOTH ET AL Ann Thorac Surg SPECIMEN PROCESSING FOR EBUS 2013;95: ination and 4 by cytologic preparation. Without use of both techniques, some of these diagnoses would have been missed. Third, thorough immunohistochemical analysis appears superior with larger tissue samples such as those from core biopsies. Fourth, with the increasing requirement for molecular analysis (eg, epidermal growth factor receptor mutation, EML4-ALK translocation, K-ras mutation), larger biopsies obtained by core sampling may be beneficial [12, 23], and was achieved here on 4 of 5 specimens when considered clinically indicated by a multidisciplinary tumor board. Fifth, paraffin blocks from core biopsies, consistently containing more material than the inconsistent yield of cell blocks from needle rinses, can be added to tissue banks for future molecular profiling. Certain patients with normal lymphoid tissue obtained by EBUS had subsequent tissue confirmation with no demonstration of carcinoma, but one with lymphoma, in agreement with the 91% negative predictive value of EBUS-TBNA in lung cancer reported by Yasufuku and colleagues [9]. Additionally, the usefulness of a negative result by EBUS is also supported by the absence of progression on subsequent imaging studies among the 39 patients with malignancy but no tissue verification. Thus, false-negative results in the setting of malignant indications for EBUS appear to be rare when normal lymphoid tissue is obtained. However, when inadequate sampling or nondiagnostic results occur in the setting of suspected malignancy, additional approaches to tissue diagnosis may be warranted. This study has certain limitations. Routine ROSE of cytologic preparations is recommended by many authors. Our practice has been to selectively use ROSE when it will impact clinical decision making during the procedure. For example, in a hybrid procedure in which EBUS sampling of mediastinal lymph nodes determines whether to proceed immediately with surgical resection, ROSE is routinely used. However, in a patient with low suspicion for malignancy (eg, suspected sarcoidosis), ROSE would rarely alter the immediate management of the patient. The routine use of ROSE in this study might have increased the diagnostic yield; however, it is unlikely this would alter the conclusion that use of both techniques is superior to either technique alone. Diagnostic accuracy may have been achieved because only 2 dedicated bronchoscopists, 1 an interventional pulmonologist and 1 a thoracic surgeon (both members of a high case-volume, collaborative, multidisciplinary team), performed or supervised all of the procedures. Therefore, we had consistency throughout the study in patient selection for EBUS, procedural technique, and specimen processing. Additionally, both operators were already experienced with EBUS. A perceived limitation of the study might be that tissue diagnostic confirmation by surgery was not obtained. This has been critical to proving that EBUS is effective and comparable to mediastinoscopy for lung cancer [3, 9]. Indeed, sensitivity and specificity of EBUS in lung cancer have been reported to be 79% to 95% and 100%, respectively [3, 9]. Without confirmation by tissue diagnosis using surgery, sensitivity and specificity cannot be defined. False-positive results would be unlikely. Even if false-negative results were present when using both histologic and cytologic techniques, it would not change the conclusion that both techniques used together offer improved diagnostic information compared with one technique. Notably, this study was not designed to assess sensitivity and specificity, both of which are well established. Rather, the study compared two specimen processing techniques, defining their diagnostic efficacy. In conclusion, adding histologic analysis of tissue cores obtained by EBUS offers higher diagnostic accuracy than only cytologic preparation of needle aspirates. Histologic analysis offers higher diagnostic yield than cytologic preparation for benign processes. Together, histologic and cytologic techniques provide fewer missed diagnoses than either method individually. When using EBUS, it appears optimal to routinely use both needle aspirate cytologic preparations and core biopsy histologic examination. References 1. Yasufuku K, Nakajima T, Fujiwara T, Yoshino I, Keshavjee S. Utility of endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of mediastinal masses of unknown etiology. Ann Thorac Surg 2011;91: Cameron SE, Andrade RS, Pambuccian SE. 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6 Ann Thorac Surg TOTH ET AL 2013;95: SPECIMEN PROCESSING FOR EBUS 981 DISCUSSION DR FRANK DETTERBECK (New Haven, CT): I just have a question about the categories and the normal tissue. So if you got normal lymphoid tissue, was that classified as normal? DR TOTH: It was classified as normal. DR DETTERBECK: And benign means it was specific benign diagnosis, right? DR TOTH: Specific benign diagnosis versus normal lymph node tissue, correct. DR DETTERBECK: And inadequate was if you didn t really get normal lymphoid tissue? DR TOTH: Correct. DR ANDREW CHANG (Ann Arbor, MI): Could you comment on the learning curve for this. Was there any difference in your diagnostic yield early on in your experience? Were you able to correct for that in the study? 13. Wallace WA, Monaghan HM, Salter DM, Gibbons MA, Skwarski KM. Endobronchial ultrasound-guided fineneedle aspiration and liquid-based thin-layer cytology. J Clin Pathol 2007;60: Micames CG, McCrory DC, Pavey DA, Jowell PS, Gress FG. Endoscopic ultrasound-guided fine-needle aspiration for non-small cell lung cancer staging: a systematic review and metaanalysis. Chest 2007;131: Rice DC. Endobronchial ultrasound (EBUS) biopsy of mediastinal lymph nodes. Available at sections/clinicalresources/thoracic/expert_tech-40.html. Accessed January 25, Yarmus LB, Akulian J, Lechtzin N, et al. Comparison of 21-gauge and 22-gauge aspiration needle in endobronchial ultrasound-guided transbronchial needle aspiration: results of the AQuIRE Registry. Chest 2012 Oct 15. [Epub ahead of print]. 17. Molenberghs G, Verbeke G. Models for discrete longitudinal data. New York: Springer Alsharif M, Andrade RS, Groth SS, Stelow EB, Pambuccian SE. Endobronchial ultrasound-guided transbronchial fine-needle aspiration: the University of Minnesota experience, with emphasis on usefulness, adequacy assessment, and diagnostic difficulties. Am J Clin Pathol 2008;130: Skov BG, Baandrup U, Jakobsen GK, et al. Cytopathologic diagnoses of fine-needle aspirations from endoscopic ultrasound of the mediastinum: reproducibility of the diagnoses and representativeness of aspirates from lymph nodes. Cancer 2007;111: Feller-Kopman D, Yung RC, Burroughs F, Li QK. Cytology of endobronchial ultrasound-guided transbronchial needle aspiration: a retrospective study with histology correlation. Cancer 2009;117: Jacob-Ampuero MP, Haas AR, Ciocca V, Bibbo M. Cytologic accuracy of samples obtained by endobronchial ultrasoundguided transbronchial needle aspiration at Thomas Jefferson University Hospital. Acta Cytol 2008;52: Delattre C, Fournier C, Bouchindhomme B, et al. Endoscopic ultrasound guided transbronchial fine needle aspiration: a French department of pathology s 4-year experience. J Clin Pathol 2011;64: Rusch VW. Mediastinoscopy: an obsolete procedure? J Thorac Cardiovasc Surg 2011;142: DR TOTH: We did not. We ve been doing EBUS (endobronchial ultrasound) for probably 3 years now. And this past year we have added in our thoracic surgeon to our EBUS technique. We did not look at the difference between the two. DR DETTERBECK: One other question perhaps. When you do EBUS, can you tell me a little bit about what your policy is. Do you sample a representative node in every node station? Do you go after more than one node if more than one is suspicious in a node station? Just how thorough are you? DR TOTH: It depends. I mean, it obviously depends on indication. In somebody that we re doing an EBUS for staging for a lung cancer, our practice is to start at the N3 position, examine the entire mediastinum, sample N3 and then move over to N2. So I will try to get as many as possible, with some concentration, if we have a PET (positron emission tomography) -positive, to make sure, certainly, that we re going in that direction.