Transbronchial Cryobiopsies in the Evaluation of Lung Allografts. Do the Benefits Outweigh the Risks?

Transcription

1 Transbronchial Cryobiopsies in the Evaluation of Lung Allografts Do the Benefits Outweigh the Risks? Anja C. Roden, MD; Ryan M. Kern, MD; Marie Christine Aubry, MD; Sarah M. Jenkins, MS; Eunhee S. Yi, MD; John P. Scott, MD; Fabien Maldonado, MD Context. Transbronchial cryobiopsy technique yields larger biopsies with enhanced quality. The benefits and safety of cryobiopsies have not been thoroughly studied in lung allografts. Objective. To compare size, quality, reproducibility of interpretation of rejection and complications of cryobiopsies with those of conventional biopsies from lung allografts. Design. All cryobiopsies (March 2014 January 2015) of lung allografts performed at Mayo Clinic, Rochester, and medical records were reviewed. For comparison, conventional biopsies from the same patient or, if unavailable, from a random patient, were selected. Two pathologists blinded to outcome reviewed all biopsies. Specimen volume, number of alveoli, small airways, and pulmonary vessels were counted and statistically compared. Results. Fifty-four biopsies (27 cryobiopsies) from 18 patients (11 men) were reviewed. A median of 3 (range, 2 5) and 10 (range, 6 12) specimens were obtained with cryobiopsies and conventional biopsies, respectively. Cryobiopsies were larger and contained more alveoli (P,.001, both) and small airways (P ¼.04). Conventional biopsies showed more fresh alveolar hemorrhage (procedural) and crush artifact/atelectasis (P,.001, both). Cryobiopsies contained more pulmonary veins and venules (P,.001). There was no significant difference between the types of biopsies with respect to the reviewers agreement on grades of rejection. Complications were more frequent in the cryobiopsy group, though the difference was not statistically significant. Conclusions. Cryobiopsies of lung allografts are larger and have less artifact. However, complications occur and should be considered. Three cryobiopsy specimens appear sufficient for histopathologic evaluation of lung allografts. (Arch Pathol Lab Med. 2016;140: ; doi: / arpa oa) Transbronchial biopsy evaluation is the gold standard to assess lung allografts for rejection and to distinguish rejection from its clinical mimickers such as aspiration, infection, abnormal drug reaction, or recurrent disease. Morphologic findings of allograft rejection might be patchy, raising the concern for sampling bias in small biopsies. In addition, bronchiolar tissue to assess for small airways rejection is not always present. Acute cellular and small airways rejection have been shown to be associated with Accepted for publication September 16, Published as an Early Online Release October 21, From the Department of Laboratory Medicine and Pathology (Drs Roden, Aubry, and Yi); the Division of Pulmonary & Critical Care Medicine (Drs Kern, Scott, and, Maldonado); and the Department of Health Sciences Research (Ms Jenkins), Mayo Clinic Rochester, Minnesota. The authors have no relevant financial interest in the products or companies described in this article. This manuscript was presented in part as an oral presentation at the 35th Annual Meeting & Scientific Sessions of the International Society for Heart and Lung Transplantation; April 17, 2015; Nice, France. Reprints: Anja C. Roden, MD, Division of Anatomic Pathology, Mayo Clinic Rochester, Hilton 11, 200 First St SW, Rochester, MN ( Roden.anja@mayo.edu). chronic airways rejection and bronchiolitis obliterans syndrome (BOS), major complications and limiting factors for lung allograft survival. 1 3 Obliterative (constrictive) bronchiolitis, the histopathologic substrate of BOS, is rarely identified on conventional biopsies, at least in part owing to the relative paucity of small airways and sampling bias. Transbronchial cryobiopsies have recently become more popular, particularly for the diagnosis of interstitial lung diseases. 4 A few reports 5 10 describe the use of cryobiopsies in immunocompromised patients, lung allografts, and endobronchial tumors. Preliminary data suggest that this technique is safe and provides larger specimens with enhanced quality. 8,10 14 Cellular structures and microscopic architecture are reported to be preserved and immunohistochemical stains can be reliably performed. 14 Although pilot studies are encouraging, a detailed histologic comparison of cryobiopsies to conventional transbronchial biopsies has not been performed in lung allografts. Herein, we compare morphometric, architectural, and histopathologic characteristics of cryobiopsies with conventional biopsies of lung allografts. In addition, we study the reproducibility of the histopathologic interpretation of rejection and compare complications between cryobiopsies and conventional biopsies. Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al 303

2 MATERIALS AND METHODS All cryobiopsies of lung allografts performed at Mayo Clinic, Rochester, Minnesota, between March 2014 and January 2015 were included in this retrospective study. The decision to perform a cryobiopsy or a conventional biopsy was at the operator s discretion, which was largely based on experience and comfort level of the performing bronchoscopist. If there was a risk for bleeding (ie, coagulopathy), conventional biopsy would have likely been preferred. Each cryobiopsy was compared to a conventional forceps biopsy of the same patient that was performed at a different date or, if not available, from a random patient. The study was approved by the Mayo Clinic Rochester Institutional Review Board (No ). Histopathologic Features Formalin-fixed, paraffin-embedded tissues were sectioned at 4 lm. At least 9 levels were stained with hematoxylin-eosin. Additional sections were stained with Grocott methenamine silver and Verhoeff-Van Gieson stains. Hematoxylin-eosin slides were independently reviewed by 2 lung transplant pathologists who were blinded to clinical information. Discrepant cases were reviewed by a third lung transplant pathologist. Cell-mediated and small airways rejection was graded according to the 2007 working formulation of lung rejection of the International Society for Heart and Lung Transplantation (ISHLT) 15 and as previously summarized. 16 In agreement studies, BX and B0 and CX and C0 were combined because of potential differences in the interpretation of the presence or absence of small airways. For the purpose of this study, we attempted to evaluate biopsies for chronic vascular rejection (ISHLT grade D1) even though chronic vascular rejection is not applicable to conventional biopsy. 15 These findings were not included in agreement studies but were reported for any case in which either or all reviewers raised the possibility of chronic vascular rejection. Antibody-mediated rejection was assessed according to the 2012 update from the Pathology Council of the ISHLT 17 and as previously summarized. 16 Any additional histopathologic findings were recorded. The number of small airways, alveoli, pulmonary arteries and veins and venules were counted in the entire biopsy specimen by 1 reviewer. The volume of tissue was determined by calculating the mean of the diameter of all biopsy pieces of 1 specimen. The mean diameter was multiplied by itself times 3 (volume of 1 biopsy piece) and multiplied by the total number of biopsy pieces. If the number of biopsy pieces was not provided, this number was identified on the slide. Biopsies were evaluated for crush artifact and atelectasis. Biopsies were also evaluated for acute hemorrhage within alveoli, which was regarded as a procedural artifact. Crush artifact/atelectasis and acute hemorrhage were scored as none, focal (,50% of alveolated lung parenchyma), or diffuse (.50% of alveolated lung parenchyma or if at least 1 reviewer felt that the feature definitely interfered with the interpretation of the biopsy) (Figure 1, A through E). Complement 4d Staining and Scoring Frozen tissue sections of all biopsies were routinely stained with C4d (clone 10-11, AbD Serotec, Biogenesis, Raleigh, North Carolina). Continuous, linear, endothelial/subendothelial staining of C4d in capillaries was considered positive as recommended by the 2007 ISHLT guidelines. 15 Scoring was semiquantitative as previously described. 16 Clinical Features Clinical information was retrieved from medical records. Routine biopsies were performed according to our institutional protocol for asymptomatic patients at 1, 3, and 6 months after transplant and later at the discretion of the transplant clinician; indication biopsies were performed in the setting of signs or symptoms of graft compromise and as follow-up of a previous biopsy that had findings of rejection. Bronchiolitis obliterans syndrome was calculated as FEV 1 (forced expiratory volume in the first second of expiration) less than 80% in at least 2 consecutive lung function tests of the patient s maximum FEV 1 post transplant. 18 Cryobiopsies Bronchoscopic cryobiopsies were performed in the operating room by interventional pulmonologists under moderate to deep conscious sedation by using a combination of midazolam and fentanyl with or without propofol. Anesthesia support was available for all procedures even though patients did not require ventilation. Patients were fiberoptically intubated with a size 8, cuffed endotracheal tube with an Arndt bronchial blocker (Cook, Bloomington, Indiana) positioned deflated in the mainstem on the side chosen for biopsies as previously described. 19 All anticoagulants were discontinued before the procedure as recently recommended. 20 The bronchoscope (Olympus XT180, Olympus America Inc, Center Valley, Pennsylvania) was then advanced to the segment chosen for biopsies, typically the anterior and lateral segments of the lower lobes. Only 1 side was biopsied per patient. Once wedged in position, fluoroscopy was used to track the Erbe 1.9-mm cryoprobe (Erbe Elektromedizin GmBH, Tübingen, Germany), which was advanced all the way to the pleura, then retrieved by approximately 2 cm. A single patient underwent biopsy with a 2.4-mm probe and therefore was excluded from the study. A freeze time of 3 to 5 seconds was then applied before the bronchoscope and the cryoprobe were removed en bloc. The tip of the cryoprobe was then submerged in saline at room temperature to thaw the specimen, which was then transferred to formalin. If any blood became visible in the endotracheal tube during that time, we considered possible severe bleeding, and the bronchial blocker was inflated to conceal the bleeding segment of the lung until the scope could be reinserted. In general, 3 cryobiopsy samples per patient were obtained and fixed in formalin. Two additional conventional forceps biopsy samples were taken, 1 piece was placed in Zeus media to eventually be frozen for immunofluorescence studies and 1 was fixed in Trump media. Complications were retrieved from medical records. Mild bleeding was defined as bleeding easily controlled with suction only, while moderate bleeding was defined by the need to inflate the endobronchial blocker or with or without the need for additional interventions (such as ice-cold saline). Severe bleeding was defined as any life-threatening bleeding requiring transfusion or escalation of care (hospitalization or admission to the intensive care unit, need for surgery). Statistical Analysis The biopsy characteristics were compared between cryobiopsies and conventional biopsies by using linear regression (continuous measures) or logistic regression (binary measures) with generalized estimating equations to account for repeated biopsies among the unique patients included in this analysis. The rejection grade reported from the 2 reviewers was compared within each biopsy, and it was noted whether a biopsy was in agreement or not. The percentage of agreement was then compared by using the same generalized estimating equation method described above. In each model, the single predictor variable was biopsy type. P,.05 was considered significant. All analyses were performed with SAS version 9 (Cary, North Carolina). RESULTS Patient Demographics Fifty-four allograft biopsies, including 27 cryobiopsies (50%) and 27 conventional biopsies (50%), from 18 patients were reviewed. Demographics and clinical characteristics of the entire study population, and separated by type of biopsy, are summarized in Tables 1 and 2, respectively. The distribution of number of biopsies per patient is shown in Table Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al

3 Figure 1. Scoring of procedure-related acute hemorrhage (A through C) and crush artifact/atelectasis (A, D, E). Hemorrhage was scored as none (A, no acute hemorrhage), focal (B,,50% of alveolated lung parenchyma and reviewers did not feel that it interfered with the interpretation of the biopsy), and diffuse (C,.50% of alveolated lung parenchyma and/or interfered with interpretation of biopsy). Crush artifact/atelectasis was scored as none (A), focal (D, arrow,,50% of alveolated lung parenchyma and reviewers did not feel that it interfered with the interpretation of the biopsy), and diffuse (E,.50% of alveolated lung parenchyma and/or interfered with interpretation of biopsy) (hematoxylin-eosin, original magnification 340 [A through E]). Cryobiopsies Are Larger and Contain More Alveoli and Small Airways Than Conventional Biopsies The volume of the cryobiopsies was significantly larger and the number of alveoli was significantly greater when compared to conventional biopsies (P,.001, both) (Figure 2, A and B; Figure 3, A and B; Table 2). The number of small airways was greater in cryobiopsies than conventional biopsies (P ¼.04) (Table 2). The number of pulmonary veins and venules was significantly greater in cryobiopsies than in conventional biopsies (P,.001) (Table 2). The number of biopsies with possible chronic vascular rejection was similar between cryobiopsies and conventional biopsies (P ¼.31) (Table 4). Cryobiopsies Show No Diffuse Crush Artifact/Atelectasis and Only Rarely Diffuse Hemorrhage Conventional biopsies showed more focal and diffuse acute hemorrhage and crush artifact/atelectasis than cryobiopsies (P,.001) (Table 2). Although a few cryobiopsies revealed focal hemorrhage and/or crush artifact/atelectasis, only 2 cryobiopsies displayed diffuse acute hemorrhage. However, even though the hemorrhage was diffuse, the alveoli were well expanded and the reviewers did not feel that the acute hemorrhage hampered the histopathologic evaluation (Figure 4, A through D). The number of biopsies with additional histopathologic findings was similar between cryobiopsies and conventional biopsies. Some biopsies had multiple additional histopathologic findings as summarized in Table 2. Reproducibility of Rejection Is Similar for Cryobiopsies and Conventional Biopsies Morphologic findings of rejection are summarized in Table 4. There was no significant difference in the reviewers agreement on ISHLT grades A, B, and C rejection between the 2 biopsy types (P ¼.51, P¼.79, P¼.55, respectively). However, both reviewers felt that cryobiopsies were much easier to interpret mainly owing to the large size of the biopsy, well-expanded alveoli, and the overall lack of crush artifact/atelectasis. While 9 cryobiopsies and 7 conventional biopsies were from patients who had BOS at the time of biopsy, none of these biopsies were morphologically recognized as ISHLT grade C1 rejection by any reviewer. Among 27 cryobiopsies, reviewer A identified 3 cases and reviewer B identified 2 cases with C1 rejection (2 of these cases were in agreement). In conventional biopsies, reviewer A found 1 and reviewer B found 3 cases with C1 rejection (1 of these cases was in agreement). None of these cases had clinical evidence of BOS at time of biopsy. None of the biopsies showed morphologic findings to suggest antibody-mediated rejection. C4d was negative in all cases. Cryobiopsies in Lung Allografts May Be Associated With Serious Complications More complications were observed in association with cryobiopsies than with conventional biopsies, although the difference was not significant (P ¼.25) (Table 2). One patient required intubation and mechanical ventilation post bronchoscopy with conventional biopsy for 6 days owing to Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al 305

4 Table 1. Summary of Demographics and Clinical Characteristics of the Entire Study Population and Biopsies Characteristic Number Biopsies, No. (%) Total No. of biopsies 54 Cryobiopsies 27/54 (50.0) Conventional forceps biopsies 27/54 (50.0) No. of specimens per biopsy, median (range) Cryobiopsies 3 (2 5) Conventional forceps biopsies 10 (6 12) Cryobiopsies controlled with a 19/27 (70.4) conventional biopsy from the same patient, performed at a different date, No. (%) Cryobiopsies controlled with a conventional biopsy from another patient, No. (%) 8/27 (29.6) No. of unique patients 18 Sex, No. (%) Male 11/18 (61.1) Female 7/18 (38.9) Age of patient at time of earliest biopsy 48.4 ( ) included in study, median (range), y Time between transplantation and biopsy, 276 ( ) median (range), d Indication for transplantation, No. (%) Usual interstitial pneumonia 8/18 (44.4) Pulmonary veno-occlusive disease 3/18 (16.7) Pulmonary arterial hypertension 3/18 (16.7) Cystic fibrosis 2/18 (11.1) Emphysema 1/18 (5.5) Pulmonary Langerhans cell histiocytosis 1/18 (5.5) hypercarbic and hypoxic respiratory failure and tachycardia. One patient had a delayed pneumothorax after cryobiopsy; however, an endobronchial mass composed of fibrosis and granulation tissue was bronchoscopically removed during the same procedure. Additionally, a patient had a delayed hemothorax after cryobiopsy. That patient had undergone a transplant 3 years prior because of pulmonary fibrosis in the setting of scleroderma. The patient also had a history of deep vein thrombosis 2 years ago for which he underwent treatment with warfarin. At time of biopsy the international normalized ratio was 1.1. The biopsy was unremarkable except for a fibrinous thrombus in a small vessel. Three days following biopsy a large unilateral hemothorax was diagnosed; an intercostal arteriogram was negative for acute bleeding from intercostal or internal mammary artery. A chest tube evacuated 600 ml of blood, and multiple blood transfusions were given. Because of continuous loculated pleural fluid, thoracoscopic decortication was performed 3 days later, during which a large amount of old blood was identified. Ten days after biopsy the patient had acute deep vein thrombosis of the leg; therefore, anticoagulation was continued. He was released from the hospital 15 days after biopsy. COMMENT We show that cryobiopsies of lung allografts provide significant larger tissue volumes and higher numbers of alveoli and small airways for histopathologic review. Moreover, cryobiopsies have significantly less crush artifact/atelectasis and fresh hemorrhage than conventional biopsies. Overall, we feel that the histopathologic assessment of cryobiopsies of lung allografts is easier than the interpretation of conventional biopsies owing to the high number of generally well-expanded alveolar spaces with only occasional alveolar collapse and/or procedural hemorrhage. To our knowledge, our study is the most detailed histopathologic comparison of cryobiopsies and conventional biopsies in lung allografts. Similar to our results, other authors have shown a 2- to 5- fold higher mean diameter 6,13,21 or 3- to 4-fold higher mean specimen areas 7,8,12,21 for cryobiopsies than for conventional biopsies in the setting of interstitial lung disease, lung cancer, immunocompromised hosts, and lung allografts. Although the volume of tissue recovered with cryobiopsy is approximately 4-fold of that recovered with conventional biopsies in our study, the number of alveoli, small airways, and veins and venules is only approximately 2-fold higher in cryobiopsies. This discrepancy is likely due to the 3- dimensional nature of the volume of the tissue in contrast to the 2-dimensional tissue section that was used to count alveoli, small airways, and vessels. However, the higher number of alveoli observed in cryobiopsies is only beneficial if the alveoli are well expanded. In our study, two-thirds (19 of 27, 70.4%) of cryobiopsies have no crush artifact/ atelectasis in contrast to less than 4% (1 of 27, 3.7%) of conventional biopsies. Similarly, two-thirds (19 of 27, 70.4%) of cryobiopsies have no acute hemorrhage, while only less than 4% (1 of 27, 3.7%) of conventional biopsies are free of acute hemorrhage. In contrast, more than half (16 of 27, 59.3%) of conventional biopsies have diffuse crush artifact/atelectasis, while none of the cryobiopsies show diffuse crush artifact/atelectasis. Similar to our results, Yarmus et al 7 showed a significantly higher percentage of open alveoli in cryobiopsies than in conventional biopsies in a study of 21 lung allograft biopsies, using a 1.9-mm probe. Other authors 6,8,12,22 did not identify any crush artifact or compressed alveoli in the cryobiopsy specimens. Although none of our cryobiopsies demonstrated diffuse crush artifact/atelectasis, these features are focally present in approximately one-third (8 of 27, 29.6%) of our cryobiopsies. The presence of some crush artifact/atelectasis in cryobiopsies is not surprising, as alveoli might collapse after thawing of the specimen and might not be entirely reexpanded in formalin. Only a few studies compare findings of artifacts between cryobiopsies and conventional biopsies. Similar to our study in which we found that all but 1 of the conventional biopsies had crush artifact/atelectasis, Griff et al 12 show that all conventional biopsies (n ¼ 10) have parenchymal damage due to compression. While we identify alveolated lung parenchyma in all cryobiopsies and conventional biopsies, in a morphometric study by Griff et al, 12 only 73% (11 of 15) of cryobiopsies and 56% (10 of 18) of conventional biopsies had alveolated lung parenchyma, a difference that was not statistically significant. In our study no biopsy lacks alveolated tissue completely, but some biopsy pieces show only sparse alveolated parenchyma and are predominantly composed of large airways. Given that the specimen size of the cryobiopsies is significantly larger, we hypothesized that cryobiopsies would contain more small airways. Indeed, in our study cryobiopsies have a significantly higher number of small airways than conventional biopsies and, although not 306 Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al

5 Table 2. Summary of Clinical Characteristics of Patients and Histopathologic Findings Based Upon Type of Allograft Transbronchial Biopsy Performed Cryobiopsies (n ¼ 27) Conventional Biopsies (n ¼ 27) P Value Time between transplantation and biopsy, median (range), d 314 ( ) 184 ( ).02 No. (%).58 Protocol biopsies 10 (37.0) 8 (29.6) Indication biopsies 17 (63.0) 19 (70.4) Volume of tissue per biopsy, a median (range), cm ( ) 0.13 ( ),.001 No. of alveoli, median (range) 5228 ( ) 2469 ( ),.001 No. of small airways per biopsy, median (range) 2 (0 6) 1 (0 4).04 Biopsies without small airways, No. (%) b 3 (11.1) 7 (25.9).28 Biopsy specimens from patients with BOS at time of biopsy, No. (%) 9 (33.3) 7 (25.9).31 No. of biopsies with ISHLT grade C1 from patients with BOS at 0 0 time of biopsy No. of pulmonary arteries, median (range) c 2 (0 7) 2 (0 7).83 No. of pulmonary veins, median, (range) c 9 (3 16) 5.5 (0 12),.001 Biopsies with acute (likely procedure-related) hemorrhage, No. (%) None 19 (70.4) 1 (3.7) Reference Focal 6 (22.2) 14 (51.9),.001 d Diffuse 2 (7.4) 12 (44.4),.001 e Biopsies with crush artifact/atelectasis, No. (%) None 19 (70.4) 1 (3.7) Reference Focal 8 (29.6) 10 (37.0),.001 d Diffuse 0 16 (59.3)... f Biopsies without or with diffuse crush artifact/atelectasis and/or f hemorrhage, g No. (%) No artifact or hemorrhage 12 (85.7) 0 Diffuse artifact and/or hemorrhage 2 (14.3) 23 (100.0) Biopsies with additional findings, No. (%) 14 (51.9) 11 (40.7).61 Large airway inflammation 5 1 Focal organizing pneumonia 1 2 Granuloma 1 0 Thrombi 2 3 Calcifications 2 0 Focal pulmonary alveolar proteinosis 2 0 Focal fibrosis 2 0 Acute lung injury 0 3 Intimal proliferation of artery versus obliterative bronchiolitis h 0 1 Focal lymphocytic infiltrate, not sufficient for ACR 0 1 Procedural complications, No. (%) 7 (25.9) 4 (14.8).25 Mild bleeding 5 1 Moderate bleeding 0 1 Severe bleeding 0 0 Delayed hemothorax 1 0 Pneumothorax 0 1 Delayed pneumothorax 1 0 Intubation post bronchoscopy 0 1 Abbreviations: ACR, acute cellular rejection; BOS, bronchiolitis obliterans syndrome; ISHLT, International Society for Heart and Lung Transplantation. a Information available on 25 cryobiopsies and all conventional biopsies. b As defined by reviewer A. c Data available from 26 cryobiopsies and 24 conventional forceps biopsies. Verhoeff-Van Gieson stain failed in the other biopsies. d Focal versus none. e Diffuse versus none. f Cannot be calculated with generalized estimating equation methodology because of sparse data (zero cell count). g Includes only biopsies with none on both or diffuse on one or the other. Excludes biopsies with focal. h In a biopsy with extensive crush artifact. statistically significant, fewer cryobiopsies lack small airways. In contrast to our findings, in a study by Fruchter et al, 6 all 40 cryobiopsies of lung allografts contained terminal bronchioles. A larger 2.4-mm cryoprobe was used in that study, which may explain these differences. Since cryobiopsies provide larger and architecturally better preserved specimens with a higher number of alveoli and small airways, we hypothesized that cryobiopsies would improve the reproducibility of the interpretation of rejection. However, in our study the agreement for ISHLT grade A, B, Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al 307

6 Table 3. Distribution of Number of Allograft Transbronchial Biopsies per Patient No. of Biopsies/Patient (Total No. of Biopsies ¼ 54) No. of Patients (Total No. of Patients ¼ 18) Percentage of Total No. of Patients and C rejection between 2 lung transplant pathologists is not superior for cryobiopsies than for conventional biopsies. This might be, at least in part, due to the relatively low variability of the data, with most biopsies showing no rejection. This result might also reflect the fact that both reviewers are from the same institution and frequently share difficult cases, which could lead to a better agreement overall. Although in our study the agreement between 2 pathologists for acute cellular rejection and small airways rejection is actually slightly lower in the cryobiopsy group than the conventional biopsy group (74% versus 81% for grade A rejection, 89% versus 93% for grade B rejection, respectively), this difference is not significant. Moreover, the agreements are similar to those of a recent study of interobserver variability between 2 pathologists in grading transbronchial lung transplant biopsies that found 74% agreement for grade A and 89% agreement for grade B. 23 Similar to our study, Bhorade et al 23 also found disagreements between grade A0 and A1, A1 and A2, and A0 and A2 (higher grades of rejection are not reported in detail). Chronic vascular rejection is characterized by fibrointimal thickening of arteries and veins. 15 According to the current guidelines for histopathologic assessment of rejection, 15 chronic vascular rejection is not applicable to conventional forceps biopsies most likely because these biopsies generally contain only a few vessels, which are often crushed, and the distribution of vascular changes is generally patchy. The larger size might make cryobiopsies more suitable for the evaluation of chronic vascular rejection. Indeed, our study shows that cryobiopsies contain significantly more pulmonary veins and venules than conventional biopsies; however, larger studies need to evaluate the usefulness of cryobiopsies for evaluation of chronic vascular rejection. According to the ISHLT guidelines at least five pieces of well-expanded alveolated lung parenchyma are required for an assessment of acute rejection. In our study we show that with a median of 3 cryobiopsy pieces we obtain a number of alveoli that is twice as high as the number of alveoli obtained with a median of 10 pieces by conventional forceps biopsies. The agreement between pathologist reviewers is as good for cryobiopsies as for conventional forceps biopsies. However, additional studies are needed to evaluate the accuracy of diagnosis of rejection, which might be better in cryobiopsies. Such studies require that cryobiopsies and forceps biopsies be obtained during the same procedure. There are some fundamental technical differences between cryobiopsies and conventional biopsies. For instance, cryobiopsy uses cold to freeze the tissue to the probe. Conceivably, given the large biopsy size, a higher rate and severity of complications could be expected. We observe a higher number of complications in the cryobiopsy group, and while the difference with conventional biopsies is not significant, our limited sample size prevents firm conclusions. Mild bleeding is the most common complication in cryobiopsies, which is likely due to the rather large specimen size, and the impossibility to keep the bronchoscope wedged after biopsy (as both the bronchoscope and the cryoprobe need to be removed en bloc). With cryobiopsy, a delayed hemothorax occurred in 1 patient, likely due to a combination of parenchymal hemorrhage and visceral pleural tear. Likewise, a delayed pneumothorax was observed in another patient after cryobiopsy. In contrast to our study, Fruchter et al 6 showed fewer complications with cryobiopsies than forceps biopsies; however, that difference was not statistically significant. In that study significant bleeding was apparent in only 2.5% (1 of 40) of cryobiopsies in contrast to 7.5% (3 of 40) of forceps biopsies. While no Figure 2. Boxplots of volume (A) and number of alveoli (B) of cryobiopsies and conventional biopsies. The horizontal line in the middle of a shaded box represents the median; the lower and upper ends of a shaded box are the 25th and 75th percentiles (Q1 and Q3); and individual points represent observations that fall beyond a distance equal to (interquartile range) beyond Q1 or Q Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al

7 Figure 3. Cryobiopsies are significantly larger and have more alveoli than conventional biopsies. (A) This cryobiopsy had a volume of 1.08 cm 3 and contained 3183 alveoli. (B) The conventional biopsy had a volume of cm 3 and contained 1615 alveoli (hematoxylin-eosin, whole slide images). pneumothorax was identified in cryobiopsies, 2.5% (1 of 40) of patients with forceps biopsies had a pneumothorax. Aktas et al, 13 who performed biopsies for the diagnosis of lung cancer, noted no significant differences in bleeding in cryobiopsies and conventional biopsies. Casoni et al, 22 using a 2.4-mm probe to perform a biopsy for 69 patients with interstitial lung disease, observed prolonged bleeding only in 1 case; however, pneumothorax occurred in 28% (n ¼ 19) of the study population; 14 patients required chest tube drainage. One of the patients had a procedure-related massive pneumothorax and died 7 days after biopsy owing to acute exacerbation of idiopathic pulmonary fibrosis. Our experience, however limited, suggests that serious complications may occur acutely in the setting of transbronchial cryobiopsies. However, the higher number of complications in the cryobiopsy group is unlikely related to the longer time between transplant and biopsy for these patients. The time between transplant and biopsy was overall shorter for the patients with conventional biopsy owing to our study design that matched cryobiopsy with a conventional forceps biopsy from the same patient whenever possible. Most patients who underwent cryobiopsy had an earlier but not a later conventional biopsy. The small sample size precludes definitive comparison with conventional biopsies, but the trend toward higher number of complications with cryobiopsies is in our opinion sufficiently concerning to Table 4. Distribution of 54 Allograft Biopsies According to International Society for Heart and Lung Transplantation (ISHLT) Grade of Rejection Cryobiopsy, No. (%) (n ¼ 27) Conventional Biopsy, No. (%) (n ¼ 27) ISHLT Grade of Rejection Reviewer A Reviewer B Both Reviewers Agreed Reviewer A Reviewer B Both Reviewers Agreed A0 12 (44.4) 17 (63.0) 20 (74.1) 19 (70.4) 19 (70.4) 22 (81.5) A1 4 (14.8) 3 (11.1) 2 (7.4) 2 (7.4) A2 8 (29.6) 5 (18.5) 4 (14.8) 3 (11.1) A3 3 (11.1) 2 (7.4) 2 (7.4) 1 (3.7) A4 0 (0.0) 0 (0.0) 0 (0.0) 2 (7.4) B0/BX 25 (92.6) 22 (81.5) 24 (88.9) 22 (81.5) 22 (81.5) 25 (92.6) B1 2 (7.4) 5 (18.5) 5 (18.5) 5 (18.5) B2 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) C0/CX 24 (88.9) 25 (92.6) 26 (96.3) 26 (96.3) 24 (88.9) 25 (92.6) C1 3 (11.1) 2 (7.4) 1 (3.7) 3 (11.1) D0 22 (81.5) N/A 21 (77.8) N/A D1 5 (18.5) 6 (22.2) Abbreviation: N/A, not applicable. Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al 309

8 Figure 4. Low-power view shows diffuse hemorrhage in a cryobiopsy (A) and a conventional biopsy (B). Because of the well-expanded alveoli in the cryobiopsy, the diffuse hemorrhage does not appear to considerably interfere with the interpretation of the biopsy (C). Together with the focal crush artifact and the small size of the specimen, the diffuse hemorrhage in the conventional biopsy hampers the histopathologic interpretation (D) (hematoxylin-eosin, original magnifications 340 [A and B] and 3200 [C and D]). recommend cryobiopsies only for selected patients, or as part of a research protocol. It should be emphasized here that long-term complications of cryobiopsies in fragile patient populations undergoing frequent surveillance bronchoscopies are unknown at this time. Clearly, additional long-term data are needed. Although the available data are not sufficient to conclude which lung transplant patients should undergo cryobiopsy rather than conventional forceps biopsy, cryobiopsies might be considered (1) for patients for whom clinical suspicion of rejection persists after negative conventional forceps biopsy results or (2) when the morphologic diagnosis is equivocal, and (3) perhaps when there is concern for obliterative bronchiolitis. Even though in our study there is no increase in the morphologic diagnosis of obliterative bronchiolitis for patients who clinically have BOS, the number of small airways is significantly higher in the cryobiopsy specimens. In general, cost should not influence the decision between cryobiopsy and conventional biopsy, since there are no added costs to the cryobiopsy technique except if an endobronchial blocker is used as in our institution. Usually, anesthesia is the same (conscious sedation) without any need for mechanical ventilation regardless of type of biopsy. The cryoprobe is reusable. Our study has several limitations. (1) Although the pathologists are blinded to the type of biopsy, a larger specimen size together with well-expanded alveoli is suggestive of cryobiopsy. (2) Patients are not randomly assigned to cryobiopsy or conventional biopsy; the decision is at the discretion of the operator. (3) We do not perform a direct comparison of conventional biopsy and cryobiopsy during the same procedure, which would allow for determination of the role of sample size in the diagnostic accuracy of rejection, because of the potentially increased risk of complications; the fact that such complications, especially if they occur late, are not attributable to a certain procedure; and the fact that an ongoing large prospective study is specifically addressing this question (ClinicalTrials.- gov identifier: NCT ). (4) The long-term morphologic complications of repeated cryobiopsies in lung transplant recipients are unclear. In conclusion, cryobiopsies are of higher volume and contain more alveoli and small airways than conventional biopsies in lung allografts. Cryobiopsies appear to be more easily interpretable owing to their larger size and reduced crush artifact/atelectasis and procedural hemorrhage. Three cryobiopsy pieces might be sufficient for the evaluation of allograft rejection. An increased number of 310 Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al

9 pulmonary veins might allow for better assessment of chronic vascular rejection but larger studies are necessary. Cryobiopsies of lung allografts appear to be as safe as conventional biopsies. However, the absence of statistical evidence of increased complications in cryobiopsies does not necessarily imply that cryobiopsies are safe. There are currently no large series on cryobiopsies in transplant patients, and as such case reports of complications, even rare, should lead us to err on the side of caution. The occurrence of any significant complication should be carefully considered pending larger studies. Furthermore, longer follow-up is necessary to evaluate for potential late morphologic complications such as increased areas of fibrosis. References 1. Verleden SE, Ruttens D, Vandermeulen E, et al. Bronchiolitis obliterans syndrome and restrictive allograft syndrome: do risk factors differ? Transplantation. 2013;95(9): Davis WA, Finlen Copeland CA, Todd JL, Snyder LD, Martissa JA, Palmer SM. Spirometrically significant acute rejection increases the risk for BOS and death after lung transplantation. Am J Transplant. 2012;12(3): Glanville AR, Aboyoun CL, Havryk A, Plit M, Rainer S, Malouf MA. Severity of lymphocytic bronchiolitis predicts long-term outcome after lung transplantation. Am J Respir Crit Care Med. 2008;177(9): Gorenstein A, Neel HB III, Sanderson DR. Transbronchoscopic cryosurgery of respiratory structures: experimental and clinical studies. Ann Otol Rhinol Laryngol. 1976;85(5, pt 1): Kropski JA, Pritchett JM, Mason WR, et al. Bronchoscopic cryobiopsy for the diagnosis of diffuse parenchymal lung disease. PloS One. 2013;8(11):e Fruchter O, Fridel L, Rosengarten D, Raviv Y, Rosanov V, Kramer MR. Transbronchial cryo-biopsy in lung transplantation patients: first report. Respirology. 2013;18(4): Yarmus L, Akulian J, Gilbert C, et al. Cryoprobe transbronchial lung biopsy in patients after lung transplantation: a pilot safety study. Chest. 2013;143(3): Babiak A, Hetzel J, Krishna G, et al. Transbronchial cryobiopsy: a new tool for lung biopsies. Respiration. 2009;78(2): Fruchter O, Fridel L, Rosengarten D, Rahman NA, Kramer MR. Transbronchial cryobiopsy in immunocompromised patients with pulmonary infiltrates: a pilot study. Lung. 2013;191(6): Hetzel J, Eberhardt R, Herth FJ, et al. Cryobiopsy increases the diagnostic yield of endobronchial biopsy: a multicentre trial. Eur Respir J. 2012;39(3): Franke KJ, Theegarten D, Hann von Weyhern C, et al. Prospective controlled animal study on biopsy sampling with new flexible cryoprobes versus forceps: evaluation of biopsy size, histological quality and bleeding risk. Respiration. 2010;80(2): Griff S, Ammenwerth W, Schonfeld N, et al. Morphometrical analysis of transbronchial cryobiopsies. Diagn Pathol. 2011;6: Aktas Z, Gunay E, Hoca NT, et al. Endobronchial cryobiopsy or forceps biopsy for lung cancer diagnosis. Ann Thorac Med. 2010;5(4): Hetzel J, Hetzel M, Hasel C, Moeller P, Babiak A. Old meets modern: the use of traditional cryoprobes in the age of molecular biology. Respiration. 2008; 76(2): 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. 2007;26(12): Roden AC, Maleszewski JJ, Yi ES, et al. Reproducibility of Complement 4d deposition by immunofluorescence and immunohistochemistry in lung allograft biopsies. J Heart Lung Transplant. 2014;33(12): Berry G, Burke M, Andersen C, et al. Pathology of pulmonary antibodymediated rejection: 2012 update from the Pathology Council of the ISHLT. J Heart Lung Transplant. 2013(1);32: Meyer KC, Raghu G, Verleden GM, et al. An international ISHLT/ATS/ERS clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome. Eur Respir J. 2014;44(6): Hohberger LA, DePew ZS, Utz JP, Edell ES, Maldonado F. Utilizing an endobronchial blocker and a flexible bronchoscope for transbronchial cryobiopsies in diffuse parenchymal lung disease. Respiration. 2014;88(6): Baron TH, Kamath PS, McBane RD. Management of antithrombotic therapy in patients undergoing invasive procedures. N Engl J Med. 2013;368(22): Pajares V, Puzo C, Castillo D, et al. Diagnostic yield of transbronchial cryobiopsy in interstitial lung disease: a randomized trial. Respirology. 2014; 19(6): Casoni GL, Tomassetti S, Cavazza A, et al. Transbronchial lung cryobiopsy in the diagnosis of fibrotic interstitial lung diseases. PloS One. 2014;9(2):e Bhorade SM, Husain AN, Liao C, et al. Interobserver variability in grading transbronchial lung biopsy specimens after lung transplantation. Chest. 2013; 143(6): Arch Pathol Lab Med Vol 140, April 2016 Cryobiopsies for Lung Allografts Roden et al 311