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* From the Division of Pulmonary Medicine (Drs. Irani, Hofer, Russi, and Boehler) and Institute of Clinical Pathology (Dr. Gaspert), University Hospital, Zurich; Pneumology (Dr. Hess), Medical Clinic, Kantonsspital, Winterthur; and Horten Center (Dr. Bachmann), University Zurich, Zurich, Switzerland.
Correspondence to: Annette Boehler, MD, Division of Pulmonary Medicine and Lung Transplant Program, C HOER 27, University Hospital, Ramistrasse 100, CH-8091 Zurich, Switzerland; e-mail: capybara{at}compuserve.com
Abstract
Background: Endobronchial ultrasonography (EBUS) has been shown to be an adequate tool to visualize the laminar structure of the bronchial wall. The purpose of this study was to investigate the potential of EBUS to identify and quantitatively assess bronchial wall structures in lung transplant recipients.
Methods: EBUS was performed with a radial 20-MHz ultrasonic miniprobe in lung transplant recipients undergoing surveillance bronchoscopies. Sequential pictures were taken of the main bronchus (proximal of anastomosis) and proximal right intermedius bronchus or proximal left lower lobe bronchus (distal of anastomosis), respectively. From every localization, five slides were chosen. The quantitative assessment of the digitized pictures was done with the aid of image analysis software. In addition to the comparison of the different layers between patients with and without infection and rejection, respectively, the intraclass correlation coefficients (ICCs) of the different measurements were calculated.
Results: From 20 EBUS examinations performed in 10 lung transplant recipients, 200 slides were selected for quantitative assessment. A five-layer composition could be identified in all selected slides. The relative area of layer two (hypoechoic submucosal tissue) of the autologous part was significantly smaller in patients with graft rejection (p = 0.04) compared to patients without rejection, and significantly larger in patients with graft infection (p = 0.02) compared to patients without graft infection. The ICC values were calculated in 50 different slides in a subset of five consecutive patients (0.91, 0.95, 0.88, and 0.91 for layers 1, 2, 3, and 5 of the autologous and 0.70, 0.92, 0.88, and 0.84 for the allogeneic parts, respectively).
Conclusions: EBUS enables to discriminate different layers of the bronchi in humans and to measure the thickness of these layers in a reproducible fashion. Therefore, EBUS may be used to investigate and quantify inflammatory alterations of bronchial wall structures in vivo.
Key Words: acute graft rejection • endobronchial ultrasonography • lung transplant recipients • quantitative assessment
Endobronchial ultrasonography (EBUS) is successfully used for the detection of mediastinal lymph nodes and other mediastinal structures.12 Furthermore, EBUS has been shown to be a suitable tool to visualize the laminar structure of the bronchial wall.13456 The correlation between the ultrasonographic images and the real bronchial wall structures have been clarified by means of needle puncture experiments34 and by predicting the tracheobronchial wall invasion of intrathoracic malignancies with consecutive histologic confirmation.34578
After lung transplantation, the bronchial epithelium is considered a major target of alloimmunologic processes in both small9 and large101112131415 airways. Lymphocytic bronchitis and lymphocytic bronchiolitis represent acute airway rejection and can be graded according to the criteria of the International Society for Heart and Lung Transplantation (ISHLT).9 In addition to histologic findings in airway biopsies of transplanted lungs, alterations of the bronchial mucosal blood flow during graft rejection have been reported in an animal model.16 To our knowledge, it has not yet been studied whether bronchial mural structures can be quantitatively assessed by 20-MHz ultrasonic probes and whether airway alterations due to inflammatory injuries can be detected with the aid of this noninvasive technique.
The rationale of the present study was to investigate the ability of EBUS to discriminate the bronchial wall structure in lung transplant recipients undergoing routine bronchoscopy and to quantitatively assess the findings in both autologous and allogeneic parts of the large airways. The comparison of these findings with the histologic and microbiological results of the surveillance bronchoscopy was a further purpose of this study.
Materials and Methods
Consecutive bilateral lung transplant recipients who were scheduled for routine surveillance bronchoscopy were included in the study. Before BAL and transbronchial biopsies (TBBs) were performed, ipsilaterally EBUS of the proximal main bronchus (autologous tissue, proximally of the bronchial anastomosis) and the intermediate bronchus or, on the left side, proximal lower lobe bronchus (allogeneic tissue, distally of the bronchial anastomosis) was performed. To avoid local mechanical manipulation, the anastomosis itself was not investigated. Patients were excluded if they had clinical or radiologic evidence of intercurrent lung infection or a contraindication for TBB (ie, coagulopathy); moreover, single-lung transplant recipients were excluded from the study. The study was approved by the Ethics Committee of our hospital, and written informed consent from the patients was obtained.
Pulmonary Function Testing
Multiple serial pulmonary function tests (spirometry and body plethysmography; Autobox; SensorMedics; Yorba Linda, CA) were available in each patient. The latest test was performed 1 day prior to bronchoscopy. The terms baseline values and bronchiolitis obliterans syndrome were used as defined elsewhere.17
Fiberoptic Bronchoscopy With BAL and Biopsies
Bronchoscopy was performed with IV conscious sedation with midazolam (Roche; Basel, Switzerland) and hydrocodon (Knoll; Liestal, Switzerland). The upper airways were anesthetized with topical 10% lidocaine, and the lower airways were anesthetized with inhalation of 4% lidocaine and local application of 1% lidocaine. BAL of the middle lobe or lingula was performed after wedging the bronchoscope in a suitable segment or subsegment bronchus with 4 x 50 mL of 0.9% saline solution. BAL was followed by TBB. Seven to nine TBB specimens were taken from the upper and lower lobes of the same lung under fluoroscopic guidance (FB19C1 forceps; Olympus; Tokyo, Japan).
TBB Assessment: TBB samples were stained with hematoxylin-eosin and Elastin van Gieson to assess acute rejection or bronchiolitis obliterans according to standard criteria.9 Acute rejection was defined as every ISHLT grade other than A0B0. Stains for fungi and for cytomegalovirus were also performed.
BAL Cell Processing
Total cell counts were performed on the unfiltered BAL fluid using a hemocytometer (1:x21; Sysmex Corporation; Kobe, Japan). Cytocentrifuge preparations (1,800g for 5 min) were made using 50 µL of unfiltered BAL aspirate, and cell counts were performed by counting 200 cells. One portion of BAL was sent for routine microbiologic examinations. Graft infection was defined as BAL positive for any microorganism other than oral flora.
Ultrasonography and Quantitative Assessment
An ultrasonography miniprobe (UM-BS20–26R; Olympus) combined with a balloon sheath was used. Additionally, our EBUS unit consists of an Olympus EU-M30 processor and an Olympus MH-240 driving unit. Under real-time conditions, the entire EBUS examination was recorded on videotape and by means of an analog/digital converter system (MovieBox DV; Pinnacle Systems; Mountain View, CA) on a laptop computer. Further analysis of the data was performed in a second phase outside the endoscopy unit. With a step-by-step analysis (Pinnacle Studio 8, Version 8.6; Pinnacle Systems), the digitalized film was screened for at least five representative slides from each autologous and allogeneic bronchial portion. From cartilage-containing parts of the bronchial wall, those slides were selected by a blinded investigator (S.I.) that showed the most obvious and well-defined laminar structure. In order to assess as many views as possible, this procedure was applied to the complete recording available. The files were saved in tagged image file format, and the image displayed on the monitor was measured by the computer (analySIS software, Version 3.1; Soft Imaging System; Munster, Germany). As mentioned, due to its well-defined configuration, we decided to take the cartilaginous portion of the bronchus into detailed consideration. Firstly, the image size was calibrated. Secondly, the largest possible sector starting at the center of the bronchus containing cartilage was defined. Thirdly, the absolute values of the thickness of each layer and the relative value of the area of each layer were measured (Fig 1
). Due to the anatomic inconsistency of the diameter of the cartilage, the relative values of the other four layers were also calculated after exclusion of the area of the cartilage. For statistical analysis, the mean values of the five measurements were used.
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Results
Twenty EBUS examinations were performed in 10 lung transplant recipients who underwent flexible bronchoscopy for routine surveillance biopsy. A total of 200 slides were selected for further analysis. Clinical and demographic details of the lung transplant recipients are summarized in Table 1 . All patients received immunosuppressive triple therapy with cyclosporine, mycophenolate mofetil, and prednisone. All procedures were performed without complications; particularly, no EBUS-associated adverse events occurred (ie, rupture or stenosis formation). The ultrasound examination prolonged the endoscopy procedure by an average of 5.4 min (range, 4.1 to 7.3 min).
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Figures 2, 3 present the quantified layer thickness (upper half) and the relative proportions after extraction of the cartilage (lower half) in both autologous and allogeneic parts of airways. The correlation of measurements between these two tissue compartments are shown in Table 2 . A statistically significant correlation was found for layer three (inner hyperechoic marginal echo of the cartilage) between autologous and allogeneic airways.
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The results of ultrasonic measurements in cases with and without graft infection (defined as BAL containing any microorganism other than oral flora) are presented in Figure 3. A statistically significant difference between these groups was found in the relative area of layer two in the autologous part (p = 0.02) of airways.
Discussion
Several properties of EBUS make this new technique potentially useful in assessing patients after lung transplantation. EBUS is a noninvasive and safe method to assess mediastinal, peribronchial, and bronchial wall structures. Since the airways of lung transplant recipients are the key structure of alloimmunologic and nonalloimmunologic inflammatory injuries finally leading to chronic rejection, particularly the latter is a thrilling and exclusive target of EBUS. Furthermore, in contrast to invasive diagnostic tools such as lung biopsy, with EBUS any number of images from different parts of the large airways can be assessed. The dynamic property of EBUS is an additional advantage of this technique. To our knowledge, this is the first study investigating EBUS in lung transplant recipients.
Using a 20-MHz ultrasonic probe, we performed EBUS in the autologous (main bronchus) and allogeneic (intermediate bronchus on the right and proximal lower lobe bronchus on the left, respectively) parts of the large airways. At both localizations, we were able to discriminate the previously described multilayer structure of the airway wall.13456 In particular, the cartilaginous parts consistently showed a five-layer laminar composition that facilitated the quantitative assessment and individual comparison.
Using a computerized image analysis system (analySIS), we were able to quantitatively assess these layers in a consistent manner. The ICC calculations of 50 different slides confirmed reproducible measurements in both autologous and allogeneic parts of the airways. Although due to smaller diameter and irregular cartilage formation, the latter in general showed a slightly less well-defined bronchial wall pattern in the ultrasonographic examination, we found only minor differences of the ICC values between the two localizations.
Overall, only for the absolute value of layer three did we find a statistically significant correlation between the main and lower lobe/intermediate bronchi. Considering the allogeneic origin of the latter, this anatomic difference of the two compartments was not unexpected. However, we found no significant difference between the proximal and distal parts either (data not separately shown).
When we compared the measured and calculated (relative) values between examinations with and without acute rejection, we found a significant difference of the relative area of layer two (submucosa) of the autologous parts, whereas in the allogeneic parts no difference could be shown. Since the absolute values of the second layer did not differ significantly between the rejection and nonrejection group and we found a significant difference of the relative areas only in the autologous parts, this finding is difficult to interpret. One can speculate that the entire airway wall composition in the rejection group changed due to an alteration of the perfusion and/or inflammation characteristics in that group. Tanabe et al1618 showed in a dog lung transplant model that bronchial mucosal blood flow in allogeneic parts of airways was reduced in acute graft rejection. These findings may explain the difference in the sonographically assessed composition of the airway wall in patients with graft rejection. The reason for the limitation of these changes to the autologous part is speculative and may be due to altered perfusion in the allogenic parts of airways. It happened in our study population that most of the patients without rejection had infection and, hence, these findings might have been biased by other than allogeneic mechanisms as well (see below). Additionally, it should be stressed that the interrelations of the inflammatory changes of the central airways and the alterations of the lung parenchyma of lung transplant recipients are a matter of ongoing debate. In particular, the alloimmune origin of inflammatory changes seen in the central airways has not been fully clarified yet. We believe that EBUS might play a role in the future research of this topic.
In patients with graft infection, we found a significant increase of the relative area of layer two (submucosa) of the autologous part and a trend to an enlarged layer three (inner marginal echo of the cartilage) of the allogeneic parts. Particularly, the latter is an interesting finding, since cartilage involvement in lung allograft inflammation is well known in animal models19; however, due to difficult assessment in clinical routine, data concerning changes of the cartilage in human lung allografts are scarce. However, there is evidence20 for involvement of the cartilage in inflammatory injury in human lung transplant recipients as well. Therefore, EBUS has the potential to serve as a reliable, noninvasive tool to further investigate this mesenchymal cell compartment. The underlying reason for the increase of the relative area of the second layer of the autologous airways in patients with graft infection might be the result of a relative edema probably due to inflammatory alterations. Since no particular absolute value showed a significant difference between patients with and without graft infection, rather the entire airway composition than one single layer differs between these groups of patients.
This study is limited by its cross-sectional nature and the relatively small number of patients. In particular, the uneven distribution of infections between patients with and without rejection makes a quantitative comparison difficult. Since full histologic examination of the central airway walls in vivo is not possible, comparison between endonsonographic findings of the central bronchial structures and the results of TBBs were performed. The resolution of the 20-MHz ultrasonic probe is limited, which explains why the borders of the particular layers appear blurred. A 30-MHz probe providing a higher image resolution6 has been developed. We plan further investigations with this new equipment.
In summary, for the first time EBUS was performed in lung transplant recipients undergoing routine surveillance bronchoscopy. We showed a distinct laminar airway structure in both autologous (main bronchus) and allogeneic (intermediate bronchus and proximal lower lobe bronchus, respectively) portions. Furthermore, we were able to quantitatively and consistently assess the five different layers on both positions. Statistically significant differences in the airway composition were found between patients with graft rejection and graft infection. These preliminary results show the following: (1) EBUS is a safe procedure in lung transplant recipients; (2) both allogenous and allogeneic airways sonographically consist of a multilayer structure; (3) by computerizing these images, these distinct layers can be quantitatively assessed; and (4) there might be differences in airway composition between patients with graft rejection and graft infection, respectively. EBUS therefore potentially may play a role in future surveillance of lung transplant recipients.
Footnotes
Abbreviations: EBUS = endobronchial ultrasonography; ICC = intraclass correlation coefficient; ISHLT = International Society for Heart and Lung Transplantation; TBB = transbronchial biopsy
This work was presented in part at the Annual Congress of the European Respiratory Society in Glasgow, Scotland, September 4 to 8, 2004.
Dr. Boehler holds a professorship position from the Swiss National Science Foundation.
Equipment was provided by Olympus, Switzerland.
Received for publication April 28, 2005. Accepted for publication June 17, 2005.
References
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