- Email: [email protected]
Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
Contents lists available at ScienceDirect
Respiratory Investigation journal homepage: www.elsevier.com/locate/resinv
Original article
Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography Nobuko Hazeki , M.D.a,c, Motoko Tachihara , M.D., Ph.D.a,c,n, Ryuko Tsukamotob, Shuntaro Tokunaga , M.D.a,c, Daisuke Tamura , M.D., Ph.D.a,c, Haruko Shinke , M.D., Ph.D.a,c, Kazuyuki Kobayashi , M.D., Ph.D.a,c, Yasuhiro Sakai , M.D., Ph.D.b,c, Yoshihiro Nishimura , M.D., Ph.D.a,c a Division of Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Diagnostic Pathology Department of Pathology, Kobe University Hospital, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan c Kobe University Hospital Respiratory Center, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
art i cle i nfo
ab st rac t
Article history:
Background: The demand for adequate tissue samples for both morphological assessment
Received 18 April 2016
and molecular studies on lung cancer treatment has increased. The aim of this study was
Received in revised form
to evaluate whether cell blocks (CBs) prepared from endobronchial ultrasonography with
10 July 2016
guide sheath (EBUS-GS) rinsing following catheter aspiration provide additional informa-
Accepted 18 October 2016
tion. Methods: We produced CBs from rinse fluid obtained from washing the inside of the sheath
Keywords: Aspiration method Bronchoscopy Cell block Endobronchial ultrasonography with a guide sheath Lung cancer
with saline after conventional EBUS-GS between May 2012 and April 2013. During the first 7 months, the sheath was aspirated with 20 mL of negative pressure while moving the catheter back and forth [aspiration group (Asp)]. During the next 5 months, the sheath was not aspirated, but only rinsed out [conventional group (Con)]. Patients diagnosed with lung cancer by EBUS-GS and/or CBs were identified and evaluated. The diagnostic rate of each sampling method was compared between the two groups. The number of tumor cells was also compared between the CB and EBUS-guided transbronchial lung biopsy (EBUS-TBB) groups. Results: EBUS-GS was performed on 113 patients. Fifty-five patients were included in this study (Asp ¼30, Con ¼25). The diagnostic yield of CBs in Asp was higher than that in Con (56.7% vs 32.0%; p ¼0.06). Asp showed no significant difference in the number of tumor cells between CB and EBUS-TBB. One patient who showed negative EBUS-TBB pathological results but positive CB results was diagnosed only by immunohistological staining of CB. Conclusion: CB prepared from EBUS-GS rinsing following catheter aspiration may provide additional information. & 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.resinv.2016.10.004 2212-5345/& 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
2
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
1.
Introduction
The detection of small peripheral lesions has been facilitated by the widespread use of computed tomography. The use of endobronchial ultrasonography guide sheath (EBUS-GS) is becoming increasingly common in the diagnosis of peripheral pulmonary lesions. The current diagnostic rate of EBUS-GS is approximately 80% [1–5]. To collect a sufficient amount of tissue, several samples must be collected, increasing the burden on patients and clinicians. Diagnostically useful tumor cells may remain in the guide sheath after EBUS-GS. In our hospital, tumor cells in the guide sheath were washed out with saline and collected. These fluid samples are used in gene mutation analysis and cytological evaluation. An effective use of the sheath lavage has not been established. Cell blocks (CBs) are often prepared using fine needle aspiration specimens from several organs, such as the breast and thyroid [6–10]. CB is also helpful for diagnosing serous effusion specimens [9]. However, there have been few studies on the utility of CB in bronchoscopy [11–13]. Particularly, there have been no reports of the utility of CBs prepared from sheath lavage fluid after EBUS-GS. We hypothesized that CBs prepared from sheath lavage fluid serve as pathological samples.
Tokyo, Japan). EBUS-GS was performed using an endoscopic ultrasound system (EU-ME1; Olympus) equipped with a 20MHz mechanical radial-type probe (UM-S20-17S; Olympus) with an external diameter of 1.4 mm and a guide sheath kit (SG-200C; Olympus). The diameter of the EBUStransbronchial lung biopsy (TBB) forceps was 1.5 mm (FB-233D; Olympus). The probe with the guide sheath was inserted into the target lesion as indicated by the EBUS image through the working channel of the bronchoscope. After confirming the tumor, the probe was removed, and the sheath remained attached to the target lesion. Under guidance of the sheath, the devices [EBUS-TBB forceps, brush, and/or curette] were inserted into the lesion, and tumor specimens were collected. Forceps biopsy specimens were fixed in formalin. Cytology specimens obtained by brushing were immediately smeared onto a glass slide to prevent drying artifacts. Biopsy forceps were performed until at least three histological samples were obtained. The brushing cytology technique was conducted twice.
2.3.
After every step such as brushing, curette, and TBB, cells adhered to the devices were rinsed in the same vial with 5 mL saline and collected. The device wash was used for cytology.
2.4.
2.
Patients and methods
2.1.
Patients
This retrospective study was approved by the institutional review board of Kobe University Graduate School of Medicine (No. 1543) on February 6, 2014. Between May 2012 and April 2013, consecutive patients who underwent EBUS-GS at Kobe University Hospital were included in the study. Between May 2012 and November 2012, the distal sheath was aspirated with 20 mL of negative air pressure for 20 s while pushing back and forth [aspiration group (Asp)] after conventional EBUS-GS methods (forceps, brush, and/or curette). From December 2012 to April 2013, the sheath was not aspirated, but only rinsed [conventional group (Con)] after EBUS-GS. Patients diagnosed with lung cancer by EBUS-GS and/or CBs were enrolled in the study. Informed consent was obtained from all patients.
2.2.
EBUS-GS
CB preparation
The rinse fluid specimens were collected by washing out the guide sheath with 5 mL of saline (sheath lavage). CBs were prepared from the sheath lavage fluid. After centrifugation of the sheath lavage fluid, cellular materials were fixed with 10% buffered neutral formalin. They were then processed with alcohol, chloroform, and xylene and embedded in paraffin. One day later, the samples were cut into sections. The sections were stained with hematoxylin and eosin.
2.5.
Diagnostic yield
The diagnostic yield of each sampling method during EBUSGS was compared between the Asp and Con groups. The correlation of the positive rates between CB and EBUS-TBB was examined. Specimens containing atypical cells were defined as negative. All specimens were evaluated by an experienced cytopathologist.
2.6. Comparison of tumor cell numbers between EBUS-TBB and CB preparation
Local anesthesia of the upper airway was achieved using 2% lidocaine. Bronchoscopes with a working channel diameter of 2.0 mm were used (BF-260, BF-P260F, and BF-P240; Olympus, Abbreviations: ALK,
Device wash
anaplastic lymphoma kinase; CB,
Tumor cell clusters were scored as follows. A score of 1 was given to clusters of r 20 cells, a score of 2 was given to
cell block; EBUS-GS, endobronchial ultrasonography with guide sheath;
EBUS-TBB, endobronchial ultrasonography-guided transbronchial lung biopsy; TBB, transbronchial lung biopsy n Corresponding author at: Division of Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Fax: þ81 78 382 5661. E-mail addresses: [email protected] (N. Hazeki) [email protected] (M. Tachihara) [email protected] (R. Tsukamoto), [email protected] (S. Tokunaga) [email protected] (D. Tamura) [email protected] (H. Shinke) [email protected] (K. Kobayashi) [email protected] (Y. Sakai) [email protected] (Y. Nishimura).
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
3
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
clusters of 20–49 cells, and a score of 3 was given to clusters of Z 50 cells. We calculated the total number of tumor cells per CB preparation by multiplying the score by the number of clusters for each group. All scoring and calculations were performed by the same cytologists and pulmonologists. The numbers of tumor cells were compared between the CB and EBUS- TBB methods within the Asp and Con groups.
2.7.
Statistical analysis
Patient characteristics were analyzed by the Mann–Whitney U test or Fisher's exact test. To estimate the utility of the sheath aspiration method, differences among the two groups in the positive rates of each sampling methods were analyzed using the chi-square test. The difference in the positive rates between the CB and EBUS-TBB pathological examinations was analyzed using the Fisher's exact test. The numbers of tumor cells between the CB and EBUS-TBB methods in each group were compared and analyzed using the Wilcoxon signed rank test. All statistical analyses were performed using SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). The results are shown as the mean7standard deviation. For all statistical analyses, a p-value of o0.05 was considered to indicate a significant difference.
3.
Results
One hundred thirteen patients were examined using EBUSGS. Fifty-five patients (Asp¼ 30, Con¼ 25) diagnosed with lung cancer were extracted. Fifty-eight patients were excluded. The lesion could not be confirmed by EBUS imaging in 33 of the 58 excluded patients. Another 25 patients were excluded because of a lack of CBs (n ¼7), lack of lung cancer diagnosis (n¼ 16), and lung cancer diagnosis obtained by methods other than EBUS-GS (n ¼2) (Fig. 1). Patient characteristics are shown in Table 1. The tumor size was significantly different between the Asp and Con groups; the other characteristics did not significantly differ between groups. The diagnostic yield of CB in Asp was generally higher than that in Con [Asp, 56.7% (17/30); Con, 32.0% (8/25); p¼ 0.06]. Using other techniques such as brushing, device wash, CB, and EBUS-TBB, there were no significant differences between the two groups (Table 2). The diagnostic results of CB and EBUS-TBB are shown in Table 3. One patient [1.8% (1/55)] showed negative EBUS-TBB pathological results but positive CB results. Immunohistological staining of the CB in this case was negative for TTF-1 and positive for P63 and CK5/6. A comparison of the numbers of tumor cells between the CB and EBUS-TBB methods within each group are shown in Fig. 2. In Asp, there was no significant difference in the number of tumor cells between CB and EBUS-TBB. In contrast, in Con, the number of tumor cells in CB was significantly lower than that in EBUS-TBB (po0.01).
4.
Discussion
Our study showed that CB made from rinsing following catheter aspiration served as additional specimens for
Fig. 1 – Flow diagram for evaluation of patients. EBUS-GS: endobronchial ultrasonography with a guide sheath. Table 1 – Comparison of characteristics between the two groups. Conventional group Aspiration group Number of patients Age (years) Tumor size (long axis) (mm) Histology (Ad/Sq/LCNEC/ NSCLC/meta.)
25 74.0 (56.0–84.0) 33.5 (15.0–63.0)*
30 71.5 (59.0–83.0) 24.0 (13.0–49.0)
13/7/1/3/1
25/4/0/1/0
Ad: adenocarcinoma, Sq: squamous carcinoma, LCNEC: large cell neuroendocrine carcinoma, NSCLC: non-small cell carcinoma, Meta: metastatic lung carcinoma. Age and tumor size data are presented as median (range). Tumor size in the conventional group was significantly larger than that in the aspiration group (*po0.001).
Table 2 – Diagnostic yield of each sampling method.
Brushing Device wash CB EBUS-TBB
Conventional group
Aspiration group
p Value
19/25 22/25 8/25 19/25
22/30 23/30 17/30 27/30
N.S. N.S. 0.06 N.S.
(76.0) (88.0) (32.0) (76.0)
(73.3) (76.7) (56.7) (90.0)
CB: cell block, EBUS-TBB: endobronchial ultrasonography-guided transbronchial lung biopsy, Data are presented as number/total (%). N.S.: not significant.
Table 3 – The correlation of the positive rates between the CB and EBUS-TBB.
EBUS-TBB Negative EBUS-TBB Positive
CB Negative
CB Positive
8 22 30/55(54.6%)
1 24 25/55(45.4%)
EBUS-TBB: endobronchial ultrasonography-guided transbronchial lung biopsy, CB: cell block. *p Value for the positive rate of CB.
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
4
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
Fig. 2 – Comparison of the number of tumor cells between the two groups in pathological examination. EBUS-TBB: endobronchial ultrasonography-guided transbronchial lung biopsy, CB: cell block. **po0.01; N.S.: not significant.
pathological diagnosis. For the first time, we utilized sheath lavage liquid as pathological specimens by using catheter aspiration and the CB method. There have been few reports of the utility of the catheter aspiration technique for pulmonary lesions [14–16]. The current literature on catheter aspiration reports a high diagnostic yield for cytology. Our method using catheter aspiration to obtain tissue for histopathological analysis has not been reported previously. The number of tumor cells in CB preparations was increased by catheter aspiration. The positive rate of CB using catheter aspiration was generally higher than that of CB without using aspiration. Our study also showed that the sheath aspiration method is effective for collecting tumor cells. The purpose of this study was to assess whether CB prepared from EBUS guide sheath rinsing fluid following catheter aspiration can be used as additional pathological specimens. An effective use of sheath lavage has not been established. The fluid sample mixed with sheath lavage and device washing have been used for the molecular analysis for gene mutations in our hospital. These samples cannot be stored for future analysis because tumor cells readily swell in liquid cytological material containing saline solution. Cellular degeneration due to swelling makes it more difficult to distinguish pathological phenotypes. Cytosmears obtained by brush, curette, and biopsy forceps during EBUS-GS dry easily. Additionally, the specimens obtained by EBUS-TBB may be too small to correctly distinguish pathological structures. In our study, dry artifacts were observed in 64.7% of patients whose cytology results were negative in one or more specimens. The lost value of pathological structures was observed in 90.0% of patients whose histological diagnoses were all negative. Most were too small to be fixed after extension. Another reason was structural destruction caused by the mechanical stress of forceps. The maximum outer diameter of a guide sheath is 1.95 mm. EBUS-TBB forceps is 1.5 mm in diameter. Conventional TBB forceps is 1.9 mm in diameter. Therefore, the size of the sample obtained by EBUSGS is generally smaller than that of conventional TBB. Samples obtained using the EBUS-GS method may degrade easily. The main advantages of CBs are their ability to be used as histological material and their facilitation of additional
analyses, such as morphological assessment and molecular studies, without re-biopsy. Next-generation sequencing for lung cancer diagnosis will become important in lung cancer treatment in the near future; however, morphological assessment and immunohistochemical analysis for pathological diagnosis are essential for selecting lung cancer treatment. It is important for general clinicians to search treatable gene variations, such as epidermal growth factor receptor mutations or the anaplastic lymphoma kinase (ALK) fusion gene. Therefore, clinicians should procure large specimen samples for accurate diagnosis. CB can be stored for a long period of time for additional analyses, such as fluorescence in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction assay. The detection of ALK fusion protein was performed using biopsy specimens when both biopsy and CB specimens were both positive. In particular, CB was useful when histological specimens were negative by biopsy but positive using CB. In one patient, the EBUS-TBB pathological results were negative, but the CB result was positive. This case showed typical squamous cell carcinoma; however, ALK fusion gene analysis was not performed, as adenocarcinoma typically shows ALK fusion protein. No study patients were subjected to ALK fusion gene analysis using CB. Immunohistochemical examination of the CB preparations was very helpful for establishing a diagnosis and selecting a treatment. This case did not require re-biopsy. By increasing sample size, these cases may increase. Additionally, increasing the number of tumor cells in CBs may increase both the diagnostic rate and scope of additional analyses. In this study, we prepared CBs obtained by rinsing samples of EBUS-GS. If sheath lavage fluid is combined with other liquid cytological samples, such as the lavage fluid obtained from washing of any other devices (brush, curette, or forceps), the number of tumor cells in the CBs will increase. We conducted EBUS-TBB many times to obtain adequate specimens. Patients are exposed to the risk of bleeding and pneumothorax. CBs prepared from sheath lavage may increase the ability to conduct additional analyses in the future without conducting additional bronchoscopy. A limitation of our method is that the preparation of CBs is a time-consuming and costly process. The pathology
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
technician is burdened with a long processing time. Additionally, patient selection for preparing CBs should be further investigated. The cost-effectiveness of the CB method must also be evaluated.
[6]
4.1.
[7]
Conclusion
CB prepared from EBUS-GS rinsing following catheter aspiration can be used as a histological specimen.
[8]
Conflict of interest [9]
The authors report that no potential conflicts of interest exist with any companies whose products or services may be discussed in this article.
Acknowledgements The authors gratefully acknowledge the contributions for preparing CB preparations by the pathology technicians in Kobe University Hospital.
[10]
[11]
[12]
r e f e r e n c e s [13] [1] Herth FJ, Ernst A, Becker HD. Endobronchial ultrasoundguidedtransbronchial lung biopsy in solitary pulmonary nodules and peripheral lesions. Eur Respir J 2002;20:972–4. [2] Kurimoto N, Miyazawa T, Okimasa S, et al. Endobronchial ultrasonography using a guide sheath increases the ability to diagnose peripheral pulmonary lesions endoscopically. Chest 2004;126:959–65. [3] Paone G, Nicastri E, Lucantoni G, et al. Endobronchial ultrasound-driven biopsy in the diagnosis of peripheral lung lesions. Chest 2005;128:3551–7. [4] Dooms CA, Verbeken EK, Becker HD, et al. Endobronchial ultrasonography in bronchoscopic occult pulmonary lesions. J Thorac Oncol 2007;2:121–4. [5] Huang CT, Ho CC, Tsai YJ, et al. Factors influencing visibility and diagnostic yield of transbronchial biopsy using
[14]
[15]
[16]
5
endobronchial ultrasound in peripheral pulmonary lesions. Respirology 2009;14:859–64. Liu K, Dodge R, Glasgow BJ, et al. Fine needle aspiration: comparison of smear, cytospin, and cell block preparations in diagnostic and cost effectiveness. Diagn Cytopathol 1998;19:70–4. Istvanic S, Fischer AH, Banner BF, et al. Cell blocks of breast FNAs frequently allow diagnosis of invasion or histological classification of proliferative changes. Diagn Cytopathol 2007;35:263–9. Qiu L, Crapanzano JP, Saqi A, et al. Cell block alone as an ideal preparatory method for hemorrhagic thyroid nodule aspirates procured without onsite cytologists. Acta Cytol 2008;52:139–44. Jalal R, Aftab K, Hasan SH, et al. Diagnostic value of clot examination for malignant cells in serous effusions. Cytopathology 2009;20:231–4. Khan S, Omar T, Michelow P. Effectiveness of the cell block technique in diagnostic cytopathology. J Cytol 2012;29:177–82. Sanz-Santos J, Serra P, Andreo F, Llatjo´s M, Castella` E, Monso´ E. Contribution of cell blocks obtained through endobronchial ultrasound-guided transbronchial needle aspiration to the diagnosis of lung cancer. BMC Cancer 2012;12:34, http: //dx.doi.org/10.1186/1471-2407-12-34. Yung RC, Otell S, Illei P, et al. Improvement of cellularity on cell block preparations using the so-called tissue coagulum clot method during endobronchial ultrasound-guided transbronchial fine-needle aspiration. Cancer Cytopathol 2012;120:185–95. Alici IO, Demirci NY, Yılmaz A, et al. The combination of cytological smears and cell blocks on endobronchial ultrasound-guided transbronchial needle aspirates allows a higher diagnostic yield. Virchows Arch 2013;462:323–7. Franke KJ, Nilius G, Ru¨hle KH. Transbronchial biopsy in comparison with catheter aspiration in the diagnosis of peripheral pulmonary nodules. Pneumologie 2006;60:7–10 [in German]. Eberhardt R, Morgan RK, Ernst A, et al. Comparison of suction catheter versus forceps biopsy for sampling of solitary pulmonarynodules guided by electromagnetic navigational bronchoscopy. Respiration 2010;79:54–60. Peschke A, Wiedemann B, Ho¨ffken G, et al. Forceps biopsy and suction catheter for sampling in pulmonary nodules and infiltrates. Eur Respir J 2012;39:1432–6.
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
Contents lists available at ScienceDirect
Respiratory Investigation journal homepage: www.elsevier.com/locate/resinv
Original article
Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography Nobuko Hazeki , M.D.a,c, Motoko Tachihara , M.D., Ph.D.a,c,n, Ryuko Tsukamotob, Shuntaro Tokunaga , M.D.a,c, Daisuke Tamura , M.D., Ph.D.a,c, Haruko Shinke , M.D., Ph.D.a,c, Kazuyuki Kobayashi , M.D., Ph.D.a,c, Yasuhiro Sakai , M.D., Ph.D.b,c, Yoshihiro Nishimura , M.D., Ph.D.a,c a Division of Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Diagnostic Pathology Department of Pathology, Kobe University Hospital, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan c Kobe University Hospital Respiratory Center, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
art i cle i nfo
ab st rac t
Article history:
Background: The demand for adequate tissue samples for both morphological assessment
Received 18 April 2016
and molecular studies on lung cancer treatment has increased. The aim of this study was
Received in revised form
to evaluate whether cell blocks (CBs) prepared from endobronchial ultrasonography with
10 July 2016
guide sheath (EBUS-GS) rinsing following catheter aspiration provide additional informa-
Accepted 18 October 2016
tion. Methods: We produced CBs from rinse fluid obtained from washing the inside of the sheath
Keywords: Aspiration method Bronchoscopy Cell block Endobronchial ultrasonography with a guide sheath Lung cancer
with saline after conventional EBUS-GS between May 2012 and April 2013. During the first 7 months, the sheath was aspirated with 20 mL of negative pressure while moving the catheter back and forth [aspiration group (Asp)]. During the next 5 months, the sheath was not aspirated, but only rinsed out [conventional group (Con)]. Patients diagnosed with lung cancer by EBUS-GS and/or CBs were identified and evaluated. The diagnostic rate of each sampling method was compared between the two groups. The number of tumor cells was also compared between the CB and EBUS-guided transbronchial lung biopsy (EBUS-TBB) groups. Results: EBUS-GS was performed on 113 patients. Fifty-five patients were included in this study (Asp ¼30, Con ¼25). The diagnostic yield of CBs in Asp was higher than that in Con (56.7% vs 32.0%; p ¼0.06). Asp showed no significant difference in the number of tumor cells between CB and EBUS-TBB. One patient who showed negative EBUS-TBB pathological results but positive CB results was diagnosed only by immunohistological staining of CB. Conclusion: CB prepared from EBUS-GS rinsing following catheter aspiration may provide additional information. & 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.resinv.2016.10.004 2212-5345/& 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
2
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
1.
Introduction
The detection of small peripheral lesions has been facilitated by the widespread use of computed tomography. The use of endobronchial ultrasonography guide sheath (EBUS-GS) is becoming increasingly common in the diagnosis of peripheral pulmonary lesions. The current diagnostic rate of EBUS-GS is approximately 80% [1–5]. To collect a sufficient amount of tissue, several samples must be collected, increasing the burden on patients and clinicians. Diagnostically useful tumor cells may remain in the guide sheath after EBUS-GS. In our hospital, tumor cells in the guide sheath were washed out with saline and collected. These fluid samples are used in gene mutation analysis and cytological evaluation. An effective use of the sheath lavage has not been established. Cell blocks (CBs) are often prepared using fine needle aspiration specimens from several organs, such as the breast and thyroid [6–10]. CB is also helpful for diagnosing serous effusion specimens [9]. However, there have been few studies on the utility of CB in bronchoscopy [11–13]. Particularly, there have been no reports of the utility of CBs prepared from sheath lavage fluid after EBUS-GS. We hypothesized that CBs prepared from sheath lavage fluid serve as pathological samples.
Tokyo, Japan). EBUS-GS was performed using an endoscopic ultrasound system (EU-ME1; Olympus) equipped with a 20MHz mechanical radial-type probe (UM-S20-17S; Olympus) with an external diameter of 1.4 mm and a guide sheath kit (SG-200C; Olympus). The diameter of the EBUStransbronchial lung biopsy (TBB) forceps was 1.5 mm (FB-233D; Olympus). The probe with the guide sheath was inserted into the target lesion as indicated by the EBUS image through the working channel of the bronchoscope. After confirming the tumor, the probe was removed, and the sheath remained attached to the target lesion. Under guidance of the sheath, the devices [EBUS-TBB forceps, brush, and/or curette] were inserted into the lesion, and tumor specimens were collected. Forceps biopsy specimens were fixed in formalin. Cytology specimens obtained by brushing were immediately smeared onto a glass slide to prevent drying artifacts. Biopsy forceps were performed until at least three histological samples were obtained. The brushing cytology technique was conducted twice.
2.3.
After every step such as brushing, curette, and TBB, cells adhered to the devices were rinsed in the same vial with 5 mL saline and collected. The device wash was used for cytology.
2.4.
2.
Patients and methods
2.1.
Patients
This retrospective study was approved by the institutional review board of Kobe University Graduate School of Medicine (No. 1543) on February 6, 2014. Between May 2012 and April 2013, consecutive patients who underwent EBUS-GS at Kobe University Hospital were included in the study. Between May 2012 and November 2012, the distal sheath was aspirated with 20 mL of negative air pressure for 20 s while pushing back and forth [aspiration group (Asp)] after conventional EBUS-GS methods (forceps, brush, and/or curette). From December 2012 to April 2013, the sheath was not aspirated, but only rinsed [conventional group (Con)] after EBUS-GS. Patients diagnosed with lung cancer by EBUS-GS and/or CBs were enrolled in the study. Informed consent was obtained from all patients.
2.2.
EBUS-GS
CB preparation
The rinse fluid specimens were collected by washing out the guide sheath with 5 mL of saline (sheath lavage). CBs were prepared from the sheath lavage fluid. After centrifugation of the sheath lavage fluid, cellular materials were fixed with 10% buffered neutral formalin. They were then processed with alcohol, chloroform, and xylene and embedded in paraffin. One day later, the samples were cut into sections. The sections were stained with hematoxylin and eosin.
2.5.
Diagnostic yield
The diagnostic yield of each sampling method during EBUSGS was compared between the Asp and Con groups. The correlation of the positive rates between CB and EBUS-TBB was examined. Specimens containing atypical cells were defined as negative. All specimens were evaluated by an experienced cytopathologist.
2.6. Comparison of tumor cell numbers between EBUS-TBB and CB preparation
Local anesthesia of the upper airway was achieved using 2% lidocaine. Bronchoscopes with a working channel diameter of 2.0 mm were used (BF-260, BF-P260F, and BF-P240; Olympus, Abbreviations: ALK,
Device wash
anaplastic lymphoma kinase; CB,
Tumor cell clusters were scored as follows. A score of 1 was given to clusters of r 20 cells, a score of 2 was given to
cell block; EBUS-GS, endobronchial ultrasonography with guide sheath;
EBUS-TBB, endobronchial ultrasonography-guided transbronchial lung biopsy; TBB, transbronchial lung biopsy n Corresponding author at: Division of Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Fax: þ81 78 382 5661. E-mail addresses: [email protected] (N. Hazeki) [email protected] (M. Tachihara) [email protected] (R. Tsukamoto), [email protected] (S. Tokunaga) [email protected] (D. Tamura) [email protected] (H. Shinke) [email protected] (K. Kobayashi) [email protected] (Y. Sakai) [email protected] (Y. Nishimura).
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
3
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
clusters of 20–49 cells, and a score of 3 was given to clusters of Z 50 cells. We calculated the total number of tumor cells per CB preparation by multiplying the score by the number of clusters for each group. All scoring and calculations were performed by the same cytologists and pulmonologists. The numbers of tumor cells were compared between the CB and EBUS- TBB methods within the Asp and Con groups.
2.7.
Statistical analysis
Patient characteristics were analyzed by the Mann–Whitney U test or Fisher's exact test. To estimate the utility of the sheath aspiration method, differences among the two groups in the positive rates of each sampling methods were analyzed using the chi-square test. The difference in the positive rates between the CB and EBUS-TBB pathological examinations was analyzed using the Fisher's exact test. The numbers of tumor cells between the CB and EBUS-TBB methods in each group were compared and analyzed using the Wilcoxon signed rank test. All statistical analyses were performed using SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). The results are shown as the mean7standard deviation. For all statistical analyses, a p-value of o0.05 was considered to indicate a significant difference.
3.
Results
One hundred thirteen patients were examined using EBUSGS. Fifty-five patients (Asp¼ 30, Con¼ 25) diagnosed with lung cancer were extracted. Fifty-eight patients were excluded. The lesion could not be confirmed by EBUS imaging in 33 of the 58 excluded patients. Another 25 patients were excluded because of a lack of CBs (n ¼7), lack of lung cancer diagnosis (n¼ 16), and lung cancer diagnosis obtained by methods other than EBUS-GS (n ¼2) (Fig. 1). Patient characteristics are shown in Table 1. The tumor size was significantly different between the Asp and Con groups; the other characteristics did not significantly differ between groups. The diagnostic yield of CB in Asp was generally higher than that in Con [Asp, 56.7% (17/30); Con, 32.0% (8/25); p¼ 0.06]. Using other techniques such as brushing, device wash, CB, and EBUS-TBB, there were no significant differences between the two groups (Table 2). The diagnostic results of CB and EBUS-TBB are shown in Table 3. One patient [1.8% (1/55)] showed negative EBUS-TBB pathological results but positive CB results. Immunohistological staining of the CB in this case was negative for TTF-1 and positive for P63 and CK5/6. A comparison of the numbers of tumor cells between the CB and EBUS-TBB methods within each group are shown in Fig. 2. In Asp, there was no significant difference in the number of tumor cells between CB and EBUS-TBB. In contrast, in Con, the number of tumor cells in CB was significantly lower than that in EBUS-TBB (po0.01).
4.
Discussion
Our study showed that CB made from rinsing following catheter aspiration served as additional specimens for
Fig. 1 – Flow diagram for evaluation of patients. EBUS-GS: endobronchial ultrasonography with a guide sheath. Table 1 – Comparison of characteristics between the two groups. Conventional group Aspiration group Number of patients Age (years) Tumor size (long axis) (mm) Histology (Ad/Sq/LCNEC/ NSCLC/meta.)
25 74.0 (56.0–84.0) 33.5 (15.0–63.0)*
30 71.5 (59.0–83.0) 24.0 (13.0–49.0)
13/7/1/3/1
25/4/0/1/0
Ad: adenocarcinoma, Sq: squamous carcinoma, LCNEC: large cell neuroendocrine carcinoma, NSCLC: non-small cell carcinoma, Meta: metastatic lung carcinoma. Age and tumor size data are presented as median (range). Tumor size in the conventional group was significantly larger than that in the aspiration group (*po0.001).
Table 2 – Diagnostic yield of each sampling method.
Brushing Device wash CB EBUS-TBB
Conventional group
Aspiration group
p Value
19/25 22/25 8/25 19/25
22/30 23/30 17/30 27/30
N.S. N.S. 0.06 N.S.
(76.0) (88.0) (32.0) (76.0)
(73.3) (76.7) (56.7) (90.0)
CB: cell block, EBUS-TBB: endobronchial ultrasonography-guided transbronchial lung biopsy, Data are presented as number/total (%). N.S.: not significant.
Table 3 – The correlation of the positive rates between the CB and EBUS-TBB.
EBUS-TBB Negative EBUS-TBB Positive
CB Negative
CB Positive
8 22 30/55(54.6%)
1 24 25/55(45.4%)
EBUS-TBB: endobronchial ultrasonography-guided transbronchial lung biopsy, CB: cell block. *p Value for the positive rate of CB.
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
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respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
Fig. 2 – Comparison of the number of tumor cells between the two groups in pathological examination. EBUS-TBB: endobronchial ultrasonography-guided transbronchial lung biopsy, CB: cell block. **po0.01; N.S.: not significant.
pathological diagnosis. For the first time, we utilized sheath lavage liquid as pathological specimens by using catheter aspiration and the CB method. There have been few reports of the utility of the catheter aspiration technique for pulmonary lesions [14–16]. The current literature on catheter aspiration reports a high diagnostic yield for cytology. Our method using catheter aspiration to obtain tissue for histopathological analysis has not been reported previously. The number of tumor cells in CB preparations was increased by catheter aspiration. The positive rate of CB using catheter aspiration was generally higher than that of CB without using aspiration. Our study also showed that the sheath aspiration method is effective for collecting tumor cells. The purpose of this study was to assess whether CB prepared from EBUS guide sheath rinsing fluid following catheter aspiration can be used as additional pathological specimens. An effective use of sheath lavage has not been established. The fluid sample mixed with sheath lavage and device washing have been used for the molecular analysis for gene mutations in our hospital. These samples cannot be stored for future analysis because tumor cells readily swell in liquid cytological material containing saline solution. Cellular degeneration due to swelling makes it more difficult to distinguish pathological phenotypes. Cytosmears obtained by brush, curette, and biopsy forceps during EBUS-GS dry easily. Additionally, the specimens obtained by EBUS-TBB may be too small to correctly distinguish pathological structures. In our study, dry artifacts were observed in 64.7% of patients whose cytology results were negative in one or more specimens. The lost value of pathological structures was observed in 90.0% of patients whose histological diagnoses were all negative. Most were too small to be fixed after extension. Another reason was structural destruction caused by the mechanical stress of forceps. The maximum outer diameter of a guide sheath is 1.95 mm. EBUS-TBB forceps is 1.5 mm in diameter. Conventional TBB forceps is 1.9 mm in diameter. Therefore, the size of the sample obtained by EBUSGS is generally smaller than that of conventional TBB. Samples obtained using the EBUS-GS method may degrade easily. The main advantages of CBs are their ability to be used as histological material and their facilitation of additional
analyses, such as morphological assessment and molecular studies, without re-biopsy. Next-generation sequencing for lung cancer diagnosis will become important in lung cancer treatment in the near future; however, morphological assessment and immunohistochemical analysis for pathological diagnosis are essential for selecting lung cancer treatment. It is important for general clinicians to search treatable gene variations, such as epidermal growth factor receptor mutations or the anaplastic lymphoma kinase (ALK) fusion gene. Therefore, clinicians should procure large specimen samples for accurate diagnosis. CB can be stored for a long period of time for additional analyses, such as fluorescence in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction assay. The detection of ALK fusion protein was performed using biopsy specimens when both biopsy and CB specimens were both positive. In particular, CB was useful when histological specimens were negative by biopsy but positive using CB. In one patient, the EBUS-TBB pathological results were negative, but the CB result was positive. This case showed typical squamous cell carcinoma; however, ALK fusion gene analysis was not performed, as adenocarcinoma typically shows ALK fusion protein. No study patients were subjected to ALK fusion gene analysis using CB. Immunohistochemical examination of the CB preparations was very helpful for establishing a diagnosis and selecting a treatment. This case did not require re-biopsy. By increasing sample size, these cases may increase. Additionally, increasing the number of tumor cells in CBs may increase both the diagnostic rate and scope of additional analyses. In this study, we prepared CBs obtained by rinsing samples of EBUS-GS. If sheath lavage fluid is combined with other liquid cytological samples, such as the lavage fluid obtained from washing of any other devices (brush, curette, or forceps), the number of tumor cells in the CBs will increase. We conducted EBUS-TBB many times to obtain adequate specimens. Patients are exposed to the risk of bleeding and pneumothorax. CBs prepared from sheath lavage may increase the ability to conduct additional analyses in the future without conducting additional bronchoscopy. A limitation of our method is that the preparation of CBs is a time-consuming and costly process. The pathology
Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004
respiratory investigation ] (] ] ] ]) ] ] ] –] ] ]
technician is burdened with a long processing time. Additionally, patient selection for preparing CBs should be further investigated. The cost-effectiveness of the CB method must also be evaluated.
[6]
4.1.
[7]
Conclusion
CB prepared from EBUS-GS rinsing following catheter aspiration can be used as a histological specimen.
[8]
Conflict of interest [9]
The authors report that no potential conflicts of interest exist with any companies whose products or services may be discussed in this article.
Acknowledgements The authors gratefully acknowledge the contributions for preparing CB preparations by the pathology technicians in Kobe University Hospital.
[10]
[11]
[12]
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Please cite this article as: Hazeki N? et al. Utility of cell blocks obtained by catheter aspiration via a guide sheath during endobronchial ultrasonography. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.10.004