key: cord-0856714-ueig940p
authors: Martinu, Tereza; Koutsokera, Angela; Benden, Christian; Cantu, Edward; Chambers, Daniel; Cypel, Marcelo; Edelman, Jeffrey; Emtiazjoo, Amir; Fisher, Andrew; Greenland, John R.; Hayes, Don; Hwang, David; Keller, Brian C.; Lease, Erika D.; Perch, Michael; Sato, Masaaki; Todd, Jamie L; Verleden, Stijn; der Thüsen, Jan von; Weigt, S. Samuel; Keshavjee, Shaf
title: ISHLT CONSENSUS STATEMENT FOR THE STANDARDIZATION OF BRONCHOALVEOLAR LAVAGE IN LUNG TRANSPLANTATION
date: 2020-07-15
journal: J Heart Lung Transplant
DOI: 10.1016/j.healun.2020.07.006
sha: 6c2bd00df2f58a3815fed9ccc3fb78d26984c2e8
doc_id: 856714
cord_uid: ueig940p

Bronchoalveolar lavage (BAL) is a key clinical and research tool in lung transplantation (LTx). However, BAL collection and processing are not standardized across LTx centers. This ISHLT-supported consensus document on BAL standardization aims to clarify definitions and propose common approaches to improve clinical and research practice standards. Nine areas are covered: 1) bronchoscopy procedure and BAL collection, 2) sample handling, 3) sample processing for microbiology, 4) cytology, 5) research and 6) microbiome, 7) sample inventory/tracking, 8) donor bronchoscopy and 9) pediatric considerations. This consensus document aims to harmonize clinical and research practices for BAL collection and processing in LTx. The overarching goal is to enhance standardization and multi-center collaboration within the international LTx community and enable improvement and development of new BAL-based diagnostics.

Bronchoscopic bronchoalveolar lavage (BAL) allows sampling of the small airway and alveolar compartment of the lung. In clinical lung transplantation (LTx), BAL is routinely used for monitoring the lung allograft and detecting infections. In research, BAL has been extensively used to improve our understanding of allograft dysfunction and to identify biomarkers with diagnostic and/or prognostic value for phenotyping acute rejection (AR) and chronic lung allograft dysfunction (CLAD) [1] [2] [3] [4] [5] . However, comparison of clinical and research data from different institutions, validation of findings, and ultimately clinical applicability are hindered by the high variability of BAL collection and analysis approaches. This constitutes an important barrier for collaborative projects in this setting.

General BAL standardization guidelines were published by the European Respiratory Society (ERS) in 1999, and guidelines specific to patients with interstitial lung diseases were put forth by the American Thoracic Society (ATS) in 2012 [6] [7] [8] . While these guidelines set a great precedent, BAL collection techniques still vary significantly and are often poorly described in the literature. In LTx, specific considerations about BAL collection and processing apply as, in most centers, LTx recipients undergo regularly scheduled surveillance bronchoscopies with BAL sampling, BAL is often performed in the setting of good lung function, and the overall poor outcomes after LTx create a greater mandate for research, patient enrollment and multi-center collaboration. However, the definitions and techniques used for bronchial and alveolar sampling have never been standardized across LTx centers. The objective of this consensus document, supported by the International Society for Heart and Lung Transplantation (ISHLT), is to assist in standardizing practices across LTx centers around the world by clarifying definitions and techniques, and by proposing recommendations for bronchial and alveolar sampling in LTx.

The detailed methods used for the creation of this document are presented in section A of the Supplement. Briefly, an international workgroup of 66 LTx specialists was created and divided into 9 subgroups covering 9 overarching topics. The subgroups prepared the comprehensive ISHLT BAL survey, capturing BAL collection and processing practices, and administered it to 114 LTx centers from 27 countries. The survey results (section B of the Supplement), as well as a systematic literature review (section C of the Supplement), were used for the creation of the statements. All statements were subjected to voting by all workgroup members according to the Delphi method. In the absence of a strong evidentiary base regarding best practices for the acquisition, storage, and processing of BAL fluid, the proposed statements represent consensus recommendations. In order to avoid repeatedly stating this limitation for most statements, specific grades were used to reflect the level of evidence, as well as strength of agreement within the survey, subgroups, and workforce voting (see grading system in Table 1 ). Table 1 : Grading of the statements Literature level of evidence* A: data derived from multiple RCTs or meta-analyses B1: data derived from one RCT B2: data derived from large non-randomized studies C1: data derived from small studies, retrospective studies or registries C2: expert opinion, no published data Strength of survey agreement (S) # (S1) Excellent: 81-100% (S2) Good: 61-80% (S3) Moderate: 41-60% (S4) Fair: 21-40% (S5) Poor: < 20% Strength of subgroup opinion (O) (OI) strong (OII) moderate (OIII) weak Strength of workforce agreement based on Delphi voting (V) % of workforce members who voted 8 or 9 out of 9 (i.e. high agreement) * This scale is in accordance with the grading schema proposed by the ISHLT but provides more details for grade C which was split into C1 and C2. # Based on Ref 9 Abbreviations: RCT=randomized controlled trial STATEMENTS 1. Bronchoscopy procedure and BAL collection

The ISHLT BAL survey shows considerable variability in the interpretation of BAL and bronchial wash (BW) definitions and collection techniques among centers around the world: for example, 17.2% report wedging the bronchoscope when they perform bronchial washings. Major thoracic societies have provided guidance on the performance of BAL whereas no guidelines exist regarding BW. The American Thoracic Society Clinical Practice Guideline, as well as the British Thoracic Society guideline, recommend BAL be performed with a flexible bronchoscope placed in a wedge position within a selected bronchopulmonary segment [6] [7] [8] . We agree with these prior published guidelines and propose summary statements below to solidify the definitions. The BW has not been previously defined. BAL and BW may yield different information: the BAL is considered to sample the alveoli and small airways, while the BW primarily samples larger airways. If the BW is performed in a mainstem or lobar airway, it can be referred to as large airway BW.

BAL is also distinct from what has been termed "mini-BAL", which samples airways by blind passage of a protected telescoping non-wedged lavage catheter, via the endotracheal tube in mechanically ventilated patients. It was first described in 1989 10 . Among survey respondents, only 27.6% perform "mini-BAL" in LTx recipients and of those 75 .9% indicate that the procedure is done the same as BAL but with a smaller instillation volume, indicating an important misunderstanding of the term. Therefore, we recommend avoiding the term "mini-BAL" as specified below.

-Bronchoalveolar lavage (BAL) is a method of sampling the lung allograft where sterile isotonic saline is instilled and then aspirated through a flexible bronchoscope, with the tip wedged in a segmental or subsegmental airway. The instilled volume should be sufficient to reach the alveolar space.

-Bronchial wash (BW) is a method of sampling the lung allograft where the instilled fluid does not reach the alveolar space, due either to low instillation volume or not wedging. The BW can be wedged or unwedged.

-The use of the term "mini-BAL", which usually refers to a catheter-based bronchial wash, is confusing and should be avoided.

[C2, S3-5, OI, V94%]

The indications for BAL after LTx generally fall into one of two categories: surveillance or diagnostic. Surveillance refers to a scheduled protocol bronchoscopy in the absence of any clinical suspicion of acute pathology. Diagnostic (or clinically-indicated) bronchoscopy is done for suspected pathology such as AR or infection. According to the ISHLT BAL survey, 93.4% of respondents perform BAL during surveillance bronchoscopy and 91.4% during diagnostic bronchoscopy. Of note, 34 .1% indicate that large airway BW is performed in addition to or instead of BAL during a surveillance bronchoscopy and 39% during diagnostic bronchoscopy.

Although broadly utilized, there are no prospective controlled data assessing the utility of surveillance bronchoscopy. In uncontrolled observational studies, surveillance BAL commonly identifies asymptomatic infections (pathogens found in 12-40% of cases), especially in the first 6-12 months posttransplantation [11] [12] [13] [14] and potentially even after the first year 15 . However, a small single center nonrandomized study that compared surveillance with no surveillance showed that, although more infections were diagnosed in the surveillance group, no difference was observed in freedom from CLAD or survival 16 . In the ISHLT BAL survey, surveillance bronchoscopy is utilized by 86.7% of the centers. The schedule for surveillance bronchoscopy varies, but most indicate that it is performed at 4 weeks (76.9%), 3 months (79.1%), 6 months (73.6%), and 12 months (80.2%) post-LTx.

Regarding the indication for diagnostic bronchoscopy, besides suspected infection, surveyed respondents most commonly reported performing it for suspected AR (98.1%), suspected antibodymediated rejection (AMR) (81%), and suspected CLAD (78.1%). However, apart from ruling out infection 17 , there are no strong data to show that BAL is useful to diagnose AR, AMR, or CLAD. When BAL is performed for diagnostic reasons, pathogens are identified in 39-69% of cases 11, 12, 14, 18 .

In a large cohort of LTx patients, BAL was generally well tolerated and safe while serious complications with bronchoscopy were associated with more invasive procedures like transbronchial biopsies 19 . Specific risk factors need to be considered and include oxygen requirement prior to bronchoscopy 19 , thrombocytopenia [BAL is considered safe with platelet counts >20.000 per μL 20 ], active cardiac ischemia 8 , as well as male gender, increase in body mass index after LTx, and presence of obstructive sleep apnea which increase risk of upper airway obstruction 21 . Hypoxia often presents postprocedure after the bronchoscope is withdrawn 22 . Topical lidocaine anesthesia may facilitate minimization of systemic sedation. Although lidocaine can inhibit growth of pathogens in culture, the concentrations of lidocaine measured in BAL fluid are generally well below the reported minimal inhibitory concentrations 23, 24 .

While not directly relevant to BAL standardization, in light of the current SARS-CoV-2 pandemic and concern over potential future epidemics, we would like to highlight the importance of infection control measures while performing, transporting, processing, and storing BAL. As it is an aerosol generating procedure, bronchoscopy should only be done with appropriate protection for the operators consistent with current guidelines 25, 26 and as directed by local recommendations. Any of the statements provided herein might be subject to future amendments if compliance with specific biosafety protocols is required per health and safety policies. 

As there is still no satisfactory method to determine the dilution factor during lavage, the lack of standardized technique causes great difficulty in interpreting the measurements of BAL components. Previous task force reports and guidelines provide vague guidance, but none are specific to LTx [6] [7] [8] .

The route of bronchoscopy is not specified by guidelines. In the ISHLT BAL survey, respondents reported that the oral (45.7%) or nasal (32.4%) route for bronchoscopy were preferred over laryngeal mask (11.4%) and elective intubation (10.5%). Several respondents noted that they avoid the nasal route in recipients with cystic fibrosis (CF). The middle lobe (ML) and lingula result in higher returns of instilled volumes compared to other lung regions 27 . Survey respondents confirm that the preferred choice for lavage in bilateral LTx is the ML (81%), whereas the lingula was the most common second choice (72.4%). The majority of respondents (88.6%) do not perform BAL in more than one location during the same procedure. Given the potential for iatrogenic bleeding to alter the cellular and protein components of BAL, it is recommended that BAL be performed before any biopsy or airway brushing 28 , which is consistent with the approach of most centers surveyed. Several survey respondents comment that suctioning is avoided prior to BAL and a minority indicate performing a rinse of the bronchoscope channel immediately before wedging for BAL, although prior guidelines have not addressed this technique and there are no supportive data.

Regarding the volume of normal saline to be instilled, prior guidelines have recommended between 60 to 300 mL in total: 100-300mL per ATS 7 , >100mL per ERS 6 , 60-180mL per British Thoracic Society 8 . Studies using 50 or 60-mL aliquots demonstrate that the concentration of BAL components in the first aliquot differs from subsequent aliquots [29] [30] [31] . Studies that used two 50 or 60 mL aliquots showed that the cumulative return fluid contained significant concentrations of alveolar-derived proteins, suggesting that this approach is sufficient to achieve lavage of both airways and alveoli, while a single 50-60 mL aliquot only samples airways 32, 33 . In the ISHLT BAL survey, the most common responses for volumes instilled at a time were 50 (28.6%) or 60 mL (23.8%). The mean usual total volume instilled was 100 mL (± SD 43.8, range 20-200) and the mean maximum total volume instilled was 145 mL (± SD 54.3, range 30-300).

A longer time between fluid instillation and aspiration (i.e. dwell time) results in greater diffusion of molecules from sources other than the epithelial lining fluid into the recovered lavage fluid 34 . Guidelines consistently recommend immediate aspiration (no dwell time) by low pressure suctioning to avoid airway collapse and allow maximal retrieval of instilled fluid. Most respondents of the ISHLT BAL survey (58.1%) indicate that they aspirate immediately after instillation (i.e. no dwell time). Among the 40% who preferred a dwell time, there was no consensus on the amount of time waited, ranging from 3 to 30 seconds, or 2 to 3 breaths. Slightly more respondents favor wall (vacuum) over manual suction (51.4% vs. 43.8%).

Acknowledging the discrepancies in guidelines, literature, and current approaches, we propose a LTx standard protocol based on the most common answers in the ISHLT BAL survey and the data showing that two sequential 50 mL aliquots adequately lavage both airways and alveoli 32 . 

Prior studies have shown that successive BAL return volumes retrieved from the lung have different cellular and protein contents. Rennard et al. demonstrated that the first returned aliquot was enriched for ciliated airway epithelial cells, contained higher proportions of neutrophils and fewer lymphocytes and macrophages, while being more abundant in content for some large proteins including immunoglobulins A and G 35 . Recently this has also been shown in LTx patients suggesting that the first returned BAL aliquot is more representative of the airway compartment while latter aliquots are more representative of the alveolar space 33 .

Consistent with these observations, previous BAL practice guidelines put forth by the ERS and ATS have recommended that successive return aliquots be pooled or, at minimum, the sampling method be specified 6, 36 . The results of the ISHLT BAL survey suggests that the majority of the respondents (71.4%) pool successive BAL aliquots into a single sample. Given the current literature and existing practices at LTx centers, we recommend that serial BAL aliquots obtained from the same lobe should be pooled prior to submission for clinical testing. Discarding the first obtained return aliquot was discussed: However, we deemed that this approach would be unlikely uniformly applied to all patients (given different yields of BAL) and would lead to greater discrepancy between patients and centers.

Best practices related to short-term storage of BAL samples in the bronchoscopy area or during transportation to the clinical laboratory have not been rigorously established. The results of the ISHLT BAL survey demonstrated that most of the responding centers hold BAL samples at room temperature in the bronchoscopy area and during transport to the clinical laboratory (73.3% and 79% respectively). The second most common response included holding the BAL sample on ice or placing it in a 4 o C refrigerator (21.9% and 18.1% respectively). Note is made that some highly specific studies, such as aspergillus antigen testing, may require the sample be held on ice. 

Infection after LTx is common and contributes significantly to morbidity and mortality, especially in the first year after transplant [37] [38] [39] [40] [41] . The ISHLT BAL survey found that 100% of respondents perform bacterial culture, 87.7% perform fungal culture, 86% perform acid-fast bacilli (AFB) culture, and 70.2% perform polymerase chain reaction (PCR) for viruses other than cytomegalovirus (CMV) [e.g. influenza, respiratory syncytial virus (RSV), adenovirus, etc.]. Over half of centers (59.7%) reported utilizing CF respiratory bacterial cultures, which employ specific processing and selective media to identify bacterial organisms more commonly identified in patients with CF. Fewer centers reported routinely performing CMV-specific analysis such as shell-vial assay or PCR (61.4%), Pneumocystis jiroveci (PJP) testing via either silver stain or PCR (54.4%), galactomannan (45.6%), or Nocardia species culture (28.1%). A small number of centers reported also specifically including Legionella culture. In regards to viral analysis performed, centers reported routinely testing for influenza (80.7%), RSV (75.4%), parainfluenza (73.7%), adenovirus (73.7%), rhinovirus (66.7%), human metapneumovirus (64.9%), and herpes simplex virus (HSV) (54.4%). Of centers performing viral analysis, 82.5% do so by multiplex PCR. Fewer centers (38.6%) routinely perform testing for varicella zoster virus (VZV). There is no current consensus or data regarding the appropriate microbiologic studies to perform on routine BAL collected after LTx. Infectious Disease Society of America (IDSA) guidelines support quantitative cultures of invasively obtained samples in the setting of suspected hospital-acquired pneumonia and ventilator-associated pneumonia. While quantitative culture of BAL in other settings and populations may be reasonable, the culture thresholds defining pneumonia and/or necessity to treat are not established. PCR-based detection methods are becoming increasingly available and further studies will be needed to establish their use for infection assessment in LTx patients. Furthermore, endemic infections and pandemic or local epidemic outbreaks of respiratory pathogens may warrant additional specific testing. 

No data exist in regard to the recommended laboratory processing of BAL samples in the microbiology laboratory, specifically for samples collected from LTx recipients, and most laboratories devise their own individual standard operating procedures. The IDSA and the American Society for Microbiology (ASM) published a joint document offering some guidance regarding diagnostic procedures and sample transportation, recommending that BAL fluid be placed into a sterile container that may be maintained at room temperature for up to 2 hours or in a 4°C refrigerator up to 24 hours after collection 42 . The ISHLT BAL survey found that 66.7% of centers store BAL fluid at room temperature prior to processing and 38.6% in a 4°C refrigerator. Centers reported a maximal acceptable delay of 6 hours (45.6%) or "other" (26.3%) with comments indicating acceptable delay in processing depends on the testing ordered.

The IDSA/ASM guideline does not comment on the minimum quantity needed for individual microbiologic analyses. Respondents to the ISHLT BAL survey reported a minimum quantity needed for standard post-transplant-related microbiologic analysis to be 10.9 ± 8.5 mL. The largest proportion of centers reported a minimum quantity of BAL fluid to be 10 mL (26.3%) while almost an equal number reported a minimum quantity of 5 mL (24.6%). The majority of centers do not mention BAL sample quality in their clinical reporting (63.2%) while 22.8% will comment only when BAL quality is low.

Approximately half (50.9%) of centers reported that centrifugation of BAL samples prior to further processing was not needed while 29 .8% reported that centrifugation should be performed. If centrifugation occurs, centers reported a median (range) of 10 (5-20) minutes at a speed of 1750 (250-3000) rcf/g (relative centrifugal force) or 1500 (1000-3000) rpm (revolutions per minute).

The minimum clinical information required to facilitate proper processing in the microbiology laboratory should include patient identifiers, status as a LTx recipient, relevant clinical history, and testing required, as outlined in Table 2 .

Note 

Microscopic cytology examination: Of the 105 ISHLT BAL survey participants, 61% reported they routinely request cytology with pathology review during each surveillance bronchoscopy; 22.9% request cytology evaluation for suspected infection, 13.3% for suspected rejection, 35.2% for suspected malignancy, and 7.7% stated they never request cytology evaluation for post-transplant bronchoscopies. Although commonly requested in routine lung recipient care, the value of sending BAL cytology with pathology examination as a routine study has been questioned, particularly in light of the relative cost 43, 44 . Prior studies examining the diagnostic performance of BAL cytology for infection have yielded conflicting results. Al Zaabi et al demonstrated a poor detection rate for infectious agents utilizing cytology 43 . In contrast, a study by Walts et al in 1991 showed good diagnostic capacity of cytology for non-bacterial organisms -specifically, Candida species, although this is often not a pulmonary pathogen, and Herpes simplex virus 45 . Additionally, special staining of cytology specimens may be a useful adjunct for the identification of difficult-to-culture organisms such as mucor or nocardia species or in cases of suspected PJP.

Beyond detection of infection, another common application of BAL cytology examination is in the detection of malignancy, particularly relevant in the immunocompromised LTx population. A small study by Ohori et al examining atypical epithelial cells from BAL fluid of LTx recipients compared to those from non-transplant patients with known lung carcinoma determined that the evaluation of cytologic features alone may not permit differentiation of atypical cells in non-neoplastic conditions within the lung recipient from those in malignant conditions 46 . In addition, detection of lipid-laden macrophages may indicate chronic aspiration or gastro-esophageal reflux 47 .

Cell counts: Examination of the cellular composition of the BAL fluid and the correlation of BAL cell populations with acute and chronic rejection in particular has been an intense area of research interest in the LTx community. In the ISHLT BAL survey, 71.4% of respondents reported that BAL differential cell counts are performed routinely on their posttransplant bronchoscopies. Among 54 cytology labs surveyed, 14.8% stated that they performed cell counts and/or differentials only, 18.5% perform microscopic examination only, while the majority of labs (53.7%) combine cell counts and/or differentials with microscopic examination.

The literature indicates that cytologic findings on BAL do not adequately distinguish between AR and infection 48, 49 . While the cytologic changes on BAL (an early lymphocytosis followed by a rise in neutrophils within the fluid) cannot be considered specific for acute rejection, they do raise the clinical suspicion 49, 50 . With regard to the utility of BAL cell counts to aid in the detection of CLAD, as summarized in a recent review 1 , several studies have now demonstrated a significant association between BAL neutrophilia and concurrent or future CLAD with significant neutrophil percentage cut-off identified at 16-24% 49,51-53 . A significant (>2%) eosinophilia in BAL was also associated with lower overall and CLADfree survival 54 . BAL eosinophilia may further associate with worse outcomes specifically after diagnosis of restrictive allograft syndrome 54, 55 . Although clear risk thresholds have not been established in multicenter studies, clinical examination of the BAL differential inflammatory cell count may provide useful information in the assessment of patients with loss of lung function.

There is no good evidence to support the best approach to determining BAL cell counts. Most centers mention using at least one approach for BAL cell quantification: cell count (65.7%), differential (71.4%), and/or microscopic analysis (61.0%). With respect to the inclusion of epithelial cells in the differential, the literature was not particularly revealing, as the vast majority do not provide this information (95%). Only 2 papers mention the inclusion of epithelial cells in the differential 43, 56 , which may be important in (a) assessing the representativeness of the specimen, (b) detecting potential cytopathogenic effects of viral infections and (c) uncovering signs of epithelial malignancy. 

With respect to the minimum BAL volume required for adequate cytological assessment, according to the survey respondents, 1 mL (16.7%), 5 mL (35.2%) or 10 mL (18.5%) would suffice for a standard cytologic analysis of BAL, with only a minority using more than 10 mL. Only few papers comment on this, suggesting that 15-30 mL is sufficient for a full analysis [57] [58] [59] [60] [61] .

In the relevant literature, volumes of BAL instilled or retrieved for cytologic analysis are mentioned in a minority of papers. Bollmann et al. 62 found that, while significantly larger volumes were returned with a 5x20 mL instillation protocol, cellular concentration was higher when using a 2x50 mL protocol. The latter regimen, therefore, may be preferable for cytologic diagnosis and is consistent with the recommended BAL collection outlined in section 1 of this document.

Once received by the laboratory, the majority of survey respondents (53.7%) centrifuge their samples. Among those who mention the use of centrifugation, the median (range) of time is 10 (3-20) minutes and the median (range) of speed 390 (72-616) rcf/g or 2000 (800-4400) rpm. Most papers mention the use of centrifugation, but with a wide range of conditions: median speed of 400-500g (range 40-1000g) and median duration 5-10min (range 2-20min) (see section C of the Supplement). Thirty-six papers of 140 (26%) which describe centrifuge methodology mentioned the use of the cytospin technique.

Only a minority of ISHLT BAL survey respondents employ red cell lysis (14.8%). The literature assessed does not provide significant insight in this matter, as only 7 papers comment on the use of red cell lysis, of which 6 use this technique 4,50,63-66 , and 1 does not 67 .

For morphological assessment, most laboratories use an H&E (42.6%) or H&E equivalent stain (9.3%) and/or Papanicolaou stain (40.7%), with Giemsa stains (20.4%) also being commonly employed. In addition, fungal stains are frequently used (33.3%). Of the assessed papers, the majority (69%) do not comment on the stains used, while the majority of those that do, employ a Giemsa stain in isolation (18%), or a combination of stains (e.g. Giemsa + Pap stain, with or without fungal or iron stains).

The initial step in morphological evaluation of the BAL specimen could be an assessment of the suitability of the specimen received. However, only 24.1% of laboratories surveyed comment on the quality of the BAL specimen. With regard to the literature, very few papers comment on the need for reporting the quality of BAL specimens for cytology (6.9%).

The minimum clinical information required to facilitate proper processing in the cytology laboratory is summarized in Table 2 . Based on the ISHLT BAL survey, 57.1% of centers were using BAL for research. Of the 42 centers that completed the research-specific survey section, 57.1% collected and banked BAL samples, while 42.9% collected samples for specific research projects without active biobanking. Regarding sample types, 47.6% collected raw BAL, 69% supernatant, 64.3% cell pellets, and 11.9% collect bronchial wash samples. While 61.9% of centers had no specific analyte planned at the time of sample collection, 40.5% were performing leukocyte phenotyping and microbiome analyses, 35.7% protein analyses, and 31% RNA expression studies (other endpoints listed in the Supplement S4Q11).

One major limitation in the literature is inadequate reporting on the details of the BAL collection procedure: e.g. the majority of studies do not report on instillation volume and aliquots, location of sampling, processing of the BAL (see Table 3 ).

Based on the ISHLT BAL survey, of the centers who perform BAL-based research, 65% reported not changing the BAL procedure or the total instilled volume for research. Twenty-six % of the centers increase the instilled volume when planning to use BAL for research in addition to the clinical purposes.

Among respondents who change the instilled BAL volume for research, there is a wide range of instilled BAL volume (60-200 mL, mean 137.2, SD 39.3).

There is no universally accepted protocol for the volume or the number of instilled aliquots for the optimal BAL return for research purposes. While the practice of using multiple instilled aliquots to reach a total volume of equal or greater than 100 mL has been recommended 71 and used in a number of studies 4,62,72 , uncertainty remains as to how variation in the instilled volume can affect the measurements of analytes in BAL. Indeed, decreased BAL fluid return volume has been associated with infection and rejection in LTx recipients 73 . In a study comparing two sequential 50 mL lavages, the first lavage was enriched with neutrophils, airway epithelial cells and their secreted proteins, while the second lavage had higher cell viability and alveolar surfactant protein D 33 . At a fixed total volume, the number of instilled aliquots may also affect BAL analytes, as one study observed that instilling five 20 mL aliquots resulted in higher BAL return but lower median cell count when it was compared to using two aliquots of 50 mL 62 . 

In the ISHLT survey of centers performing BAL for research, there was substantial variability in techniques: Before arrival to the research lab 35.7% of centers kept samples at room temperature, 50% placed samples on ice and 21.4% froze samples either at -20 or -80º Celsius. While three-quarters of centers processed samples within 6 hours, with some reporting significant loss in cell viability at 3 hours, 7.1% considered acceptable a delay of 24 hours before sample processing.

Filtration of BAL was also highly variable between centers. About 19% of respondents filtered raw BAL though gauze before storage or centrifugation, while many centers used no filtering, and a few used cell separation mesh filters (of varied opening size). While filtration may be particularly important for flow cytometry (to minimize clogging of the cytometer nozzle), it may not be necessary for other techniques. There is also a concern that filtration could affect results by selectively binding cells or proteins, although the evidence for this is sparse. Similarly, for centrifugation, centers used a range of speeds and times, depending on the target analyte.

A wide range of sample aliquot volumes is stored at different centers. BAL cell pellets are stored alternatively in phenol, trizol/Qiazol, RNAlater, Allprotect, DNA/RNA shield (Zymo), saline, DMSO in fetal calf serum, RPMI in fetal calf serum, or RLT buffer. At appropriate concentrations, glutaraldehyde-and formalin-containing storage buffers can inactivate viral pathogens 74 . BAL cell pellets, supernatant, and raw fluid are most commonly stored at -80 degrees Celsius, though some centers use -20 degrees and others stored in liquid nitrogen.

The recommended approach to processing and storing BAL depends on the intended analyses: 5.2.1. Generally, BAL should be kept at 4ºC and processed within 24 hours or processed within 2 hours if at room temperature (see statement 2.2). [C2, S2, OII, V97%]

[C1, S2, OI, V92%] Centrifugation forces (in G or RCF) should be reported rather than speeds (rotations per minute or RPM) since the RPM corresponds to different centrifugal forces based on the rotor size. 

Variation in BAL collection techniques can affect analyte concentrations, and there is no universally accepted method for normalization of BAL analyte concentration. Of centers collecting BAL for research, 23.8% reported not normalizing BAL analytes. The most common normalization parameter was the return volume of BAL fluid collected (16.7%), while some centers reported using total protein, albumin concentration, or plasma to BAL albumin or urea ratios. Normalization can have unpredictable effects on results.

The most frequently recommended quality metric in the survey was the time between collection and processing, though some centers recommended the percentage of epithelial cells, quantity of mucous, or protein or albumin concentrations. A quarter of centers would discard samples that have passed a threshold time (between 2 hours and 7 days) from collection to processing, 7.1% of centers would discard samples because of a high percentage of epithelial cells, and 2.4% because of high quantities of mucous. While a few studies advocate use of special techniques during bronchoscopy (e.g. double bronchoscopy, laryngeal mask airway, endotracheal tube) to attempt to minimize oropharyngeal contamination during the collection of BAL for microbiome analysis 95 , these may be impractical for use in routine clinical practice. In view of the often low microbial burdens present in BAL samples and the corresponding potential for confounding by high relative abundance of environmental contaminants 88, 96 , the subgroup consensus view was that concurrent analysis of negative control samples collected prior to bronchoscopy should be considered. Further, while personal protective equipment (e.g. gloves, gowns, masks) are routinely used during bronchoscopic procedures, similar precautions should be taken during subsequent specimen handling and processing, in order to minimize the risk of contamination by microbiota from the user. Regarding the storage of samples between collection and processing, a review of both the transplant and non-transplant BAL microbiome literature found that specimens were placed on ice or at 4°C prior to transfer in the large majority of studies, but with variability in reporting the delay between procurement and processing/analysis.

Where feasible, use of special techniques to minimize oropharyngeal contamination for microbiome analysis may be applied, and personal protective measures (e.g. gloves, masks) should be used when handling and processing specimens to minimize contamination from the user. [C2, S N/A, OI-II, V96%] 6.1.2. Where participation in microbiome studies is being considered, the most rigorous approach includes collection of control specimens for each bronchoscopy procedure, consisting of 10-20 mL of each of the following: blank fluid to be used for the BAL; blank fluid aspirated through the bronchoscope suction channel prior to insertion into the patient; oral rinse (mouth wash) sample using fluid from the same batch of fluid to be used for the BAL. [C1, S N/A, OI, V88%] 6.1.3. Where participation in microbiome studies is being considered, BAL specimens should be kept at room temperature for no more than 2 hours, and at 4°C for no more than 24 hours, prior to processing and/or freezing for longer-term storage. [C2, S1, OI-II, V91%]

There is a diversity of opinion and limited evidence regarding the need for, relative advantages or disadvantages, and techniques of fractionation of BAL prior to storage and downstream microbiome analysis. In its discussions, the subgroup recognized the range of practice, and the perceived relative merits and disadvantages of each specimen type. It was acknowledged that raw vs. fractionated specimens might be preferable in different situations, depending on the type of microbiome analysis performed, bearing in mind that BAL supernatant may be suboptimal for some types of microbiome analysis 88 . 

In review of the BAL microbiome literature, the vast majority of both transplant and nontransplant studies report storage at -80°C prior to use for microbiome analysis, with very few reporting storage of cell pellets in RNALater or other preservation agents. However, the effects of such preservation reagents on downstream microbiome analysis have not been well characterized and require further investigation.

The working group concluded that whereas 1-2 mL of BAL fluid may suffice for bacteriome analysis, studies of the mycobiome or virome may require larger volumes. 

In the literature, the guidelines on the use of BAL in interstitial lung disease 7 do not make specific recommendations for annotation or tracking of BAL samples for clinical or research purposes. Based on the ISHLT BAL survey, the majority of LTx bronchoscopists believe that samples should be de-identified and accompanied by data that includes gender, age, native lung disease, date of transplant, type of transplant, date of bronchoscopy, indication for bronchoscopy and a description of the procedures performed (Table 2) . STATEMENT: 7.1. Research BAL samples should be de-identified and accompanied by data that include gender, age, native lung disease, date of transplant, type of transplant, date of bronchoscopy, indication for bronchoscopy and a description of the procedures performed. The location of the BAL, the volume instilled and the volume retrieved should also be recorded (see Table 2 ). [C2, S2, OI, V97%]

There is no literature available regarding labeling or tracking of research BAL samples. The ISHLT BAL survey indicated that only a unique sample ID is crucial to enable database linkage, however the date and type of samples are also considered relevant information.

funding should support adequate infrastructure and dedicated personnel for BAL biobanking and data retention. Data can be retained in a secure spreadsheet, local laboratory information management system database or (particularly for multi-center studies) a secure cloud-based platform. [C2, S2, OI, V93%]

Surprisingly, the literature search showed that 26% of BAL-focused articles contained no details concerning the bronchoscopy procedure, 44% had no information about the sample collection procedure and 33% included no information about sample processing. Table 3 summarizes the frequency of detail reporting in prior BAL-focused research papers, related to the bronchoscopy procedure itself, sample collection, and subsequent sample processing. The minimal required information, as identified by the majority of the ISHLT BAL survey respondents, is indicated.

7.3 Published studies should report, at a minimum: whether the procedure was conducted according to this ISHLT Consensus Statement, timing post-transplant, route of bronchoscopy, description of other procedures accompanying the BAL, BAL location(s) , the volume instilled, the number of aliquots used, the pooling procedure, storage conditions between collection and processing, how the sample was processed in the lab (specifically regarding BAL cell pellet and supernatant separation and centrifugation conditions), details of preservation solutions and subsequent longterm storage conditions (see Table 3 ). [C2, S3, OI, V97%]

Among US regulatory agencies and organ procurement organizations, minimal donor assessment guidelines for donor lung evaluation are not uniform (AOPO policy CL4.E.5.3 and OPTN/UNOS Policy 2.11.D). Bronchoscopy in donor organ assessment can provide information that may not be readily available on chest radiographs or manual inspection 97, 98 . Fiberoptic bronchoscopy of donors may maximize organ utilization through airway clearance, anatomic assessment and identification of infectious organisms [99] [100] [101] . The ISHLT BAL survey showed that a majority (72.4%) performs bronchoscopy for culture analysis. Bronchoscopy and sample collection in donor assessment is uniformly done before procurement and varies from several days before to intraoperatively during the procurement 98, 102 [AOPO policy, OPTN/UNOS policy]. Evidence for specific timing of airway sample collection is lacking. 97, 99, 100, [103] [104] [105] [106] . Precise explanation of the sampling methodology is rarely available, making evidence-based practice recommendations problematic. The ISHLT BAL survey demonstrated significant variation in donor airway sampling methodology and technique; however, bronchoscopic aspiration of secretions (35.2%) and large airway BW (28.6%) were by far the most common practices. Additionally, 8.5% of centers perform large airway swabs, 6.7% perform a "low volume BAL" or BAL different than that done in the recipient, as compared to only 4.8% that do a standard BAL similar to what would be performed in the recipient. Complicating this observation is the limited evidence to support these practices 104, 105 and the general support for BAL by many experienced groups as the preferred sampling technique 97, 99, 100, 102, 103 . While there is evidence for "low volume BAL" in diagnosing infections 107, 108 , based on section 1 explanations, a low-volume airway sample does not necessarily reach the alveoli and should be called a BW rather than BAL. Additionally, evidence to support location or side for donor lung assessment is lacking. Based on the ISHLT BAL survey, most centers (86.7%) do not collect donor bronchial samples for research; however, 6.7% do so for specific studies and 4.8% for biobanks. In the literature, descriptions of donor lung sampling methods for research vary from "low volume BAL" to standard BAL techniques 5, 103, [109] [110] [111] [112] [113] [114] [115] [116] [117] . In general, studies in which the organ was ultimately used for transplant used less saline for sample collection 5, 103, [109] [110] [111] [112] [114] [115] [116] [117] than those that used organs for pure research 113, 118 In all studies that reported research methodology for sample collections of the donor airway in LTx, there was consistent agreement in wedged sampling from a single segment 5, 103, [109] [110] [111] [112] [113] [114] [115] [116] [117] . Location of sampling varied between studies but most commonly was from the right middle lobe 5, 103, [109] [110] [111] [112] [113] [114] [115] [116] [117] 

Evidence defining when and how to perform airway sampling in EVLP cases is limited. The majority of centers perform bronchial sampling before EVLP (17.1%) rather than during (8.6%) or after EVLP (6.7%). Within the literature, sampling before and after EVLP has been described when used to define changes to organs on EVLP 110, 111, 115 . 

Based on the ISHLT BAL survey, clinical donor assessment of airway samples most commonly includes bacterial gram stain and culture (75.2%), fungal stain and culture (59%) and AFB stain and culture (49.5%). Practices cited in the literature agree that microbiological assessment is necessary 28, 97, [99] [100] [101] 103, 104, 106, 118 , but unambiguous description of the specific clinical assessment is sometimes lacking. In general, most suggest that bacterial gram stain and culture and fungal stain and culture should be performed 97, 103, 106, 115 . A majority of transplant centers (73.3%) do not use viral assessment as part of their minimum clinical analysis. Only one manuscript 97 and 12% of transplant programs have described viral assessment as part of routine organ evaluation. Pediatric-specific considerations With no established standards in children after LTx, pediatric-specific recommendations in this document are based on the ISHLT BAL survey as well as expert opinion of the consensus panel.

With evidence that children often have silent allograft rejection or subclinical infection, especially during the first year after LTx 11 , routine surveillance bronchoscopy is widely used according to published reports in pediatric LTx recipients 119 . Of the 8 pediatric LTx centers surveyed, 100% of centers reported performing surveillance bronchoscopies with BAL. In addition, diagnostic bronchoscopy is universally performed when clinical evidence suggests a deterioration in allograft health from infection, AR, AMR, or CLAD. At a few select transplant programs where infants undergo LTx, surveillance and diagnostic bronchoscopy are performed even in the youngest post-LTx patient population 119,120 , so there are no age or size limitations.

Although contraindications to BAL were not included in an official document for pediatric airway endoscopy 121 , common complications include bronchospasm, bleeding, hypotension, hypoxemia, and tachycardia from either the procedure or sedation. Building upon these standards, our group's consensus is that contraindications for BAL collection include conditions where a risk-benefit ratio is not favorable for pediatric LTx recipients.

surveillance and diagnostic bronchoscopy for assessment of the allograft for infection and/or rejection. The potential complications associated with this procedure have to be evaluated carefully and taken into consideration. [C1, S1, OI, V96%]

The technical standards for performing bronchoscopy and BAL in children were recently developed and published by an ad hoc committee of the American Thoracic Society 121 . Expanding upon these standards, we identified relevant issues specific to the pediatric LTx population.

The longitudinal assessment is usually performed at pre-determined times during the first year post-LTx. The ISHLT BAL survey identified that surveillance bronchoscopies with BAL were universally performed by 2 weeks and at 3, 6, and 12 months post-LTx, with some variability for other time-points. Additional time-points included by 6 weeks (4/8 centers) and at 9 months (4/8) post-LTx, resulting in 6 surveillance bronchoscopies with BAL during the first post-LTx year in the majority of pediatric LTx recipients. After the first post-LTx year, surveillance bronchoscopies with BAL were performed less widely with 3/8, 2/8, and 2/8 centers reporting 18 month, 24 month, and yearly procedures, respectively.

Collection of BAL was universally performed prior to transbronchial biopsies (8/8 centers), while the majority collected BAL in one allograft (5/8) . For surveillance bronchoscopy with BAL, the preferred location was the middle lobe (8/8) and the lingula (7/8). According to the ISHLT BAL survey, all 8 pediatric centers instill 1 mL/kg to perform a BAL. Our consensus group determined the maximum volume to be that recommended for the adult population: i.e. 50 mL for children of 50 kg or greater. The ISHLT BAL survey identified that the majority of pediatric LTx experts use no dwell time before aspirating the BAL (5/8). Previous research determined manual suctioning during bronchoscopy was associated with higher percentage of BAL volume return and increased odds of performing technically acceptable procedures in children 122 . Consistent with this research and other reports 119 , the survey identified that pediatric LTx centers performing bronchoscopy with BAL in children after LTx use manual suctioning with a syringe (5/8). 

Following standard laboratory protocols for handling BAL after collection, the ISHLT BAL survey identified that routine analysis of BAL in pediatric LTx recipients included total cell count with differential counts (8/8 centers), cytology (8/8), and Oil-Red-O stain for aspiration (7/8) . A wide array of microbiologic testing is also performed for bacteria (8/8), fungi (7/8), mycobacteria (7/8), and respiratory viruses (Influenza, Parainfluenza, Adenovirus, RSV, Rhinovirus and Human metapneumovirus) (7/8). When available, polymerase chain reaction (PCR) or other molecular techniques is preferred for PJP (6/8), CMV (6/8) and respiratory viruses (7/8). Although not all pediatric LTx programs surveyed perform research using BAL from recipients (5/8), the 5 centers that do collect BAL for research split the fluid for clinical and research purposes (5/5).

In addition to following established standards for handling and transporting BAL fluid from children, specific considerations are provided to address transplant-specific issues for BAL analysis in pediatric LTx recipients: 9.3.1. BAL analysis should include total cell and differential cell counts, including eosinophils, macrophages, lymphocytes, and neutrophils [C1, S1, OI, V88%] 9.3.2. BAL analysis should include bacterial (CF respiratory culture when appropriate), fungal, and mycobacterial staining, galactomannan and cultures [C1, S1, OI, V88%] 9.3.3. BAL analysis should include PCR for common respiratory viruses. PCR for CMV and PJP should be considered when clinically appropriate [C1, S1, OI, V88%] 9.3.4. We recommend splitting of BAL for research purposes if sufficient sample is obtained to complete clinical analysis. [C2, S1, OI, V94%]

The medical literature supports the use of bronchoscopy with BAL for assessing pediatric donor lungs 123 . Donor bronchoscopy allows for inspection of the airway and clearance of secretions and the BAL or BW microbiological data can help guide antimicrobial therapy post-LTx. Although repeated bronchoscopy may be needed in some donors to manage secretions, consideration should be given to the potential risk of causing lung injury. The ISHLT BAL survey did not identify specifics about airway sampling for pediatric donors, so the pediatric experts on the ISHLT consensus panel recommended limiting fluid instillations to smaller aliquots.

9.4. Given the lack of evidence and practice variation, no airway sampling technique or volume appears superior for clinical pediatric donor lung assessment. Thus, transplant-specific considerations are based on expert opinion of the consensus panel: Donor airway samples usually involve low-volume instillations, which are unlikely to reach alveolar spaces, and should therefore be called BW samples, instead of BAL (as explained in section 1). Our recommended BW volume of instilled sterile saline for pediatric lung donors is 0.5 mL/kg up to a maximum of 20 mL per aliquot (consistent with the adult donor airway sampling recommendation). If the yield is insufficient, up to 3 aliquots can be used to allow for optimal sampling, while aiming to prevent development of new opacities on radiography or transient hypoxemia, which may interfere with donor evaluation. [C2, S1, OI, V89%]

Based on a comprehensive international survey of lung transplant centers and the best available evidence, the ISHLT BAL standardization workforce herein puts forth recommendations for BAL collection and processing in LTx. The literature review identified limited data to inform certain statements, emphasizing the need for further studies to better direct future revisions of this document. In spite of this limitation, the statements represent a consensus approach that can serve to standardize practice within the community. Members of the ISHLT BAL standardization workforce hope that this document will harmonize clinical and research practices for BAL collection and processing in LTx. The overarching goal is to enhance standardization and multi-center collaboration within the international lung transplant community and enable improvement and development of new BAL-based diagnostics. 

Bronchoalveolar lavage as a tool to predict, diagnose and understand bronchiolitis obliterans syndrome

Plasma and bronchoalveolar lavage samples in acute lung allograft rejection: the potential role of cytokines as diagnostic markers

Diagnostic value of plasma and bronchoalveolar lavage samples in acute lung allograft rejection: differential cytology

Bronchoalveolar lavage cell immunophenotyping facilitates diagnosis of lung allograft rejection

Gene set enrichment analysis identifies key innate immune pathways in primary graft dysfunction after lung transplantation

Report of ERS Task Force: guidelines for measurement of acellular components and standardization of BAL

An official American Thoracic Society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease

British Thoracic Society guideline for diagnostic flexible bronchoscopy in adults: accredited by NICE

The measurement of observer agreement for categorical data

A prospective study of protected bronchoalveolar lavage in the diagnosis of nosocomial pneumonia

Surveillance bronchoscopy in children during the first year after lung transplantation: Is it worth it?

The utility of annual surveillance bronchoscopy in heart-lung transplant recipients

The importance of bronchoscopy with transbronchial biopsy and bronchoalveolar lavage in the management of lung transplant recipients

Results of surveillance bronchoscopy after cadaveric lung transplantation: a Japanese single-institution study

Surveillance bronchoscopy in lung transplant recipients: risk versus benefit

Single-institution study evaluating the utility of surveillance bronchoscopy after lung transplantation

Bronchoscopic monitoring after lung transplantation

Bronchoscopy in the diagnosis and surveillance of respiratory infections in lung and heart-lung transplant recipients

Safety and efficacy of outpatient bronchoscopy in lung transplant recipients -a single centre analysis of 3,197 procedures

Complications of fiberoptic bronchoscopy in thrombocytopenic patients

Management of acute hypoxemia during flexible bronchoscopy with insertion of a nasopharyngeal tube in lung transplant recipients

Arterial hypoxemia induced by fiberoptic bronchoscopy

Lidocaine topical anesthesia for flexible bronchoscopy

Antibacterial properties of lidocaine

Guidance from the International Society of Heart and Lung Transplantation regarding the SARS CoV-2 pandemic

The Use of Bronchoscopy during the COVID-19 Pandemic: CHEST/AABIP Guideline and Expert Panel Report

Interlobar variation in the recovery of bronchoalveolar lavage fluid, cell populations, and angiotensin-converting enzyme in normal volunteers

Role of flexible bronchoscopy in lung transplantation

Analyses of sequential bronchoalveolar lavage samples from healthy human volunteers

Effect of changing instilled volume for bronchoalveolar lavage in patients with interstitial lung disease

Kinetic analysis of respiratory tract proteins recovered during a sequential lavage protocol

Anatomical distribution of bronchoalveolar lavage fluid as assessed by digital subtraction radiography

Sequential broncho-alveolar lavages reflect distinct pulmonary compartments: clinical and research implications in lung transplantation

Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution

Fractional processing of sequential bronchoalveolar lavage to separate bronchial and alveolar samples

Technical aspects of bronchoalveolar lavage: recommendations for a standard procedure

The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation: Thirty-fifth adult lung and heart-lung transplant report-2018; Focus theme: Multiorgan Transplantation

Host-Pathogen Interactions and Chronic Lung Allograft Dysfunction

Bacterial and fungal pneumonias after lung transplantation

Community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study

Airway pathogens during the first year after lung transplantation: a single-center experience

A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases

Cytologic examination of bronchoalveolar lavage fluid from immunosuppressed patients

The utility of cytopathology testing in lung transplant recipients

Pulmonary cytology in lung transplant recipients: recent trends in laboratory utilization

Epithelial cell atypia in bronchoalveolar lavage specimens from lung transplant recipients

Oil red O stain of alveolar macrophages is an effective screening test for gastroesophageal reflux disease in lung transplant recipients

Bronchoalveolar lavage in lung transplant patients

Bronchoalveolar lavage fluid characteristics in acute and chronic lung transplant rejection

Cytological monitoring of peripheral blood, bronchoalveolar lavage fluid, and transbronchial biopsy specimens during acute rejection and cytomegalovirus infection in lung and heart--lung allograft recipients

Upregulation of chemokines in bronchoalveolar lavage fluid as a predictive marker of post-transplant airway obliteration

Early detection of airway involvement in obliterative bronchiolitis after lung transplantation. Functional and bronchoalveolar lavage cell findings

Predictors of survival in restrictive chronic lung allograft dysfunction after lung transplantation

Elevated bronchoalveolar lavage eosinophilia correlates with poor outcome after lung transplantation

Antimicrobial peptides in lung transplant recipients with bronchiolitis obliterans syndrome

Quantitative detection of human cytomegalovirus DNA in lung transplant recipients

Cytokine gene expression in human lung transplant recipients

Analysis of bronchoalveolar lavage from human lung transplant recipients by flow cytometry

Technique and use of transbronchial biopsy in children and adolescents

The role of heat shock protein 27 in bronchiolitis obliterans syndrome after lung transplantation

Cellular analysis in bronchoalveolar lavage: inherent limitations of current standard procedure

Cumulative exposure to CD8+ granzyme Bhi T cells is associated with reduced lung function early after lung transplantation

Dynamics of human regulatory T cells in lung lavages of lung transplant recipients

Surgical correction of gastroesophageal reflux in lung transplant patients is associated with decreased effector CD8 cells in lung lavages: a case series

Predictive value of cytomegalovirus DNA detection by polymerase chain reaction in blood and bronchoalveolar lavage in lung transplant patients

Early diagnosis and effective treatment of pulmonary CMV infection after lung transplantation

Epithelial clara cell injury occurs in bronchiolitis obliterans syndrome after human lung transplantation

Resident tissue-specific mesenchymal progenitor cells contribute to fibrogenesis in human lung allografts

Pepsin, a biomarker of gastric aspiration in lung allografts: a putative association with rejection

Technical recommendations and guidelines for bronchoalveolar lavage (BAL)

Quantitative detection of HHV-6 and HHV-7 in transbronchial biopsies from lung transplant recipients

Broncho-alveolar lavage fluid recovery correlates with airway neutrophilia in lung transplant patients

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Processing of Bronchoalveolar Lavage Fluid and Matched Blood for Alveolar Macrophage and CD4+ T-cell Immunophenotyping and HIV Reservoir Assessment

Gene Expression Profiling of Bronchoalveolar Lavage Cells Preceding a Clinical Diagnosis of Chronic Lung Allograft Dysfunction

CXCR3 chemokine ligands during respiratory viral infections predict lung allograft dysfunction

The Human Respiratory Microbiome: Implications and Impact

Airway microbiota signals anabolic and catabolic remodeling in the transplanted lung

Bronchiolitis obliterans syndrome susceptibility and the pulmonary microbiome

Distal airway microbiome is associated with immunoregulatory myeloid cell responses in lung transplant recipients

Rapid adaptation drives invasion of airway donor microbiota by Pseudomonas after lung transplantation

Reemergence of Lower-Airway Microbiota in Lung Transplant Patients with Cystic Fibrosis

Microbial Communities of Conducting and Respiratory Zones of Lung-Transplanted Patients

Airway Microbiota Determines Innate Cell Inflammatory or Tissue Remodeling Profiles in Lung Transplantation

Looking Beyond Respiratory Cultures: Microbiome

Cytokine Signatures of Bacterial Pneumonia and Tracheobronchitis in Lung Transplant Recipients

Cell-associated bacteria in the human lung microbiome

Changes in the lung microbiome following lung transplantation include the emergence of two distinct Pseudomonas species with distinct clinical associations

The lung microbiome after lung transplantation

Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing

Longitudinal analysis of the lung microbiome in lung transplantation

Reestablishment of recipient-associated microbiota in the lung allograft is linked to reduced risk of bronchiolitis obliterans syndrome

Lung-enriched organisms and aberrant bacterial and fungal respiratory microbiota after lung transplant

Enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation

Spatial Variation in the Healthy Human Lung Microbiome and the Adapted Island Model of Lung Biogeography

Objective assessment of criteria for selection of donor lungs suitable for transplantation

Fiberoptic bronchoscopy in brain-dead organ donors

Strategies to optimize the use of currently available lung donors

Maximizing the utilization of donor organs offered for lung transplantation

Flexible bronchoscopy in lung transplantation

Bacterial colonization of the donor lower airways is a predictor of poor outcome in lung transplantation

The donor lung: infectious and pathologic factors affecting outcome in lung transplantation

A positive donor gram stain does not predict outcome following lung transplantation

Organ procurement for pulmonary transplantation

Torrington KG, Finelli MR. Small volume bronchoalveolar lavage used in diagnosing Pneumocystis carinii pneumonia in HIV-infected patients

The Perioperative Lung Transplant Virome: Torque Teno Viruses Are Elevated in Donor Lungs and Show Divergent Dynamics in Primary Graft Dysfunction

The effect of ex vivo lung perfusion on microbial load in human donor lungs

Profiling inflammation and tissue injury markers in perfusate and bronchoalveolar lavage fluid during human ex vivo lung perfusion

Elevated levels of interleukin-8 in donor lungs is associated with early graft failure after lung transplantation

Ex Vivo Perfusion Treatment of Infection in Human Donor Lungs

Receptor for advanced glycation end products in donor lungs is associated with primary graft dysfunction after lung transplantation

Microbial pattern of bronchoalveolar lavage in brain dead donors

Effects of exogenous surfactant instillation in clinical lung transplantation: a prospective, randomized trial

Significant role for microRNA-21 affecting toll-like receptor pathway in primary graft dysfunction after human lung transplantation

Normothermic Ex Vivo Lung Perfusion in Brain-dead Donors Reduces Inflammatory Cytokines and Toll-like Receptor Expression

Bronchoscopic procedures and lung biopsies in pediatric lung transplant recipients

Comprehensive evaluation of lung allograft function in infants after lung and heart-lung transplantation

Official American Thoracic Society technical standards: flexible airway endoscopy in children

Comparison of two aspiration techniques of bronchoalveolar lavage in children

Management of the pediatric organ donor to optimize lung donation

No specific funding was available for this project. During this project, Angela Koutsokera received grants from the Swiss National Science Foundation (grants P300PB_164733 and P3P3PB_164734/1), the University Hospital of Lausanne (Fond de Perfectionnement), the Ligue Pulmonaire Suisse (grant 2018-16) and the University of Lausanne (grant Pépinière). The funding sources for each workforce member had no role in study design, data collection, data analysis or the writing of the report.Relevant disclosures: the disclosures of the workforce participants are provided in section D of the Supplement.