Acute cellular rejection (ACR) represents one of the most common lung transplant complications affecting 29% of patients according to the most recent registry report by the International Society for Heart and Lung Transplantation.1 Although ACR is most prevalent in the first year after transplantation, its presence significantly increases the risk of subsequent development of chronic lung allograft dysfunction, most notably bronchiolitis obliterans syndrome.2 Acute cellular rejection can occur in either asymptomatic or symptomatic patients and frequently cannot be distinguished from infection. Therefore, the gold standard for diagnosis and classification of ACR has been histopathologic analysis of lung tissue obtained by transbronchial biopsy (TBB) according to the recommended guidelines.3 In most studies, the sensitivity of TBB for diagnosis of ACR ranges from 72% to 84%.4,5 In addition to inadequate tissue sampling and the risks posed by bronchoscopy and biopsy, there is a potential for high degree of interobserver variability and limited reproducibility with TBB that might lead to undertreatment or overtreatment of ACR.6
Probe-based confocal laser endomicroscopy (pCLE) is a novel bronchoscopic technique that uses a laser beam to generate autofluorescence of elastin and macrophages at the alveolar level, thereby allowing visualization of alveolar structure, blood vessels, and intra-alveolar macrophages. The fluorescent signal is recorded at the spatial resolution of 3 μm and analyzed with the assistance of computer software.7 In addition to capturing still images, this imaging technique can produce sequential video fragments that can be analyzed in real-time by a bronchoscopist. Previously, Yserbit et al8 have found in their retrospective study that pCLE demonstrated sensitivity and specificity of 96% and 83% when using the combination of 3 pCLE criteria for the diagnosis of ACR. These pCLE criteria included autofluorescent cell at the alveolar level (ACA) count, cellular autofluorescence intensity, and vascular index defined as a ratio between the thickness of autofluorescence lining in a vascular structure and its diameter. Of these 3 variables, ACA count performed best individually (sensitivity and specificity 79% each), whereas the other individual variables did not perform as well. However, as the authors have noted in their study, the clinical relevance of ACA count remains unclear as the presence of ACAs did not correlate with any other clinical variable (infection for example) and ACAs were present to some extent in 65% of patients regardless of ACR.
In this issue of Transplantation, Keller et al9 present the results of their multicenter prospective study of pCLE for diagnosis of ACR in lung transplant recipients. A total of 30 pCLEs were performed in 20 subjects, and the images obtained by pCLE were compared with the histopathologic tissue analysis obtained by TBB from the same area where pCLE was performed. The investigators, after undergoing extensive training, had initially reviewed the pCLE images individually with an unlimited amount of time and then collectively by consensus in real time. The individual and collective image analysis scores were compared. The authors have elected to study 2 pCLE variables for the diagnosis of ACR: abundance of alveolar cellularity (AAC) and perivascular cellularity (PVC). Alveolar cellularity was considered positive if several frames showed abundant cellularity in multiple alveolar structures and PVC was considered positive when at least 1 frame showed the presence of cellularity on the surface of perivascular structures. The additional analysis of the PVC criterion was performed where the number of blood vessels with PVC was recorded and correlated with the ACR grade severity as determined by TBB. The authors of this study report that PVC has demonstrated significantly better diagnostic performance for ACR and interobserver agreement compared to AAC (P < 0.01). Perivascular cellularity has exhibited significant correlation with the diagnosis of ACR by TBB (rho = 0.79, P < 0.01), whereas this has not been the case with AAC (rho = 0.20, P = 0.30). The presence of PVC has also showed high sensitivity and specificity for ACR when evaluated by the more experienced investigator. Of note, the sensitivity and specificity of pCLE have declined when performed in real time by consensus in the more experienced investigator, whereas they have improved in the less experienced investigators. Additionally, interobserver agreement has improved when performed by consensus in contrast to individual image analyses.
As previously stated, the accurate and timely ACR diagnosis remains a critical outcome in the management of lung transplant patients. New diagnostic techniques are needed to improve the performance and reproducibility of TBB. The study by Keller et al provides important novel insights and advances the field. Importantly, they show that pCLE might surpass the sensitivity and specificity of TBB in diagnosing A-grade ACR. The authors identify PVC as a sole criterion for detecting ACR through laser-generated fluorescent imaging of likely perivascular inflammatory infiltrate. Although the authors report an association between the number of blood vessels with PVC and histopathologic grading of ACR, the PVC criterion remains unable to discriminate between different grades of ACR. Additionally, pCLE is unable to determine the presence of B-grade bronchiolar inflammation due to technical limitations. As highlighted in the study, 2 remaining questions to be answered in the future include how to accurately distinguish infection from ACR (both can cause PVC) as well as how to quantify and interpret the presence of intra-alveolar macrophages.
The authors have also addressed the issue of interobserver agreement, and several important points can be concluded from this study. First, the experienced investigators have performed better when they had unlimited time compared with the real-time interpretation of pCLE images. The impact of this finding on the potential role of real-time pCLE to provide guidance for TBB will require further study. Second, the less experienced investigators have performed better after consulting with the more experienced investigators, suggesting that considerable training and experience are required to improve sensitivity and specificity for diagnosing ACR with pCLE.
In conclusion, pCLE or “optical biopsy” is a novel and possibly promising diagnostic tool for noninvasive tissue analysis. However, it remains to be determined whether this new technique has the potential to guide or replace TBB in making the ACR diagnosis. We are hopeful that future larger randomized studies will address these important questions.
1. Yusen RD, Edwards LB, Kucheryavaya AY, et al. The Registry of the International Society for Heart and Lung Transplantation: thirty-second official adult lung and heart-lung transplantation report—2015; focus theme: early graft failure. J Heart Lung Transplant
2. Martinu T, Pavlisko EN, Chen DF, et al. Acute allograft rejection: cellular and humoral processes. Clin Chest Med
3. Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant
4. Higenbottam T, Stewart S, Penketh A, et al. Transbronchial lung biopsy for the diagnosis of rejection in heart-lung transplant patients. Transplantation
5. Trulock EP, Ettinger NA, Brunt EM, et al. The role of transbronchial lung biopsy in the treatment of lung transplant recipients: an analysis of 200 consecutive procedures. Chest
6. Arcasoy SM, Berry G, Marboe CC, et al. Pathologic interpretation of transbronchial biopsy for acute rejection of lung allograft is highly variable. Am J Transplant
7. Peng M, Liang TG, Anantham D. Probe-based confocal laser endomicroscopy of the lungs. J Pulm Respir Med
8. Yserbyt J, Dooms C, Decramer M, et al. Acute lung allograft rejection: diagnostic role of probe-based confocal laser endomicroscopy of the respiratory tract. J Heart Lung Transplant
9. Keller CA, Khoor A, Arenberg DA, et al. Diagnosis of acute cellular rejection using probe-based confocal laser endomicroscopy in lung transplant recipients: a prospective, multicenter trial. Transplantation