Background
Bronchoalveolar lavage (BAL) is a well-established tool for minimally invasive sampling of the microenvironment in the lower airways. It is performed by wedging the tip of a flexible bronchoscope within a selected bronchopulmonary segment, instilling a volume of sterile isotonic saline sufficient to reach the alveolar space, followed by suctioning of the fluid [
1,
2]. Analysis of BAL samples through biochemical, cytological, and microbiological techniques play a predominant role in the diagnosis of a wide variety of diseases in respiratory medicine. In contrast to BAL, a bronchoscopic fluid sample that has been utilized less commonly in the clinical setting is the bronchial wash, which has been defined as a non-wedged or lower volume bronchoscopic sampling where the instilled fluid does not reach the alveolar space [
2]. Specifically, the large airway bronchial wash (LABW) is obtained with the bronchoscope tip in a mainstem or lobar airway. The diagnostic value of bronchial wash and BAL has been directly compared in only a few disease states, including pulmonary tuberculosis [
3], lymphangitic carcinomatosis [
4], and peripheral lung cancer [
5]. All of these studies found that BAL had higher sensitivity.
LABW may be more advantageous than BAL as a diagnostic sample to detect pulmonary microaspiration driven by gastroesophageal reflux disease (GERD). In BAL, the presence of biochemical compounds originating from the stomach, such as pepsin and bile acids, have been shown as indicators of microaspiration in patients receiving mechanical ventilation [
6], advanced lung disease patients [
7‐
9], and lung transplant recipients [
10,
11]. Given the relative proximity of the large airways and tracheobronchial space to the gastrointestinal tract, LABW may yield biomarkers of microaspiration at more clinically useful levels than BAL.
Microaspiration biomarkers with diagnostic and prognostic value are highly sought after in clinical lung transplantation as GERD-driven microaspiration has been linked to acute rejection [
12], acute decline in pulmonary function [
13], and development of chronic lung allograft dysfunction (CLAD) [
10], presumably through inflammation and fibrosis [
14]. Although early anti-reflux surgery in lung transplant recipients may slow lung function deterioration, its related risks and complications necessitate improved patient selection [
15]. Our group recently reported that a specific bile acid, taurocholic acid (TCA), in BAL at 3 months post-transplant was associated with concurrent objective evidence of GERD, inflammation, and acute lung allograft dysfunction (ALAD) [
16]. Moreover, BAL TCA was reduced following anti-reflux surgery [
16]. Urso et al. recently showed that elevated bile acids in LABW at 3 months post-transplant were independent predictors of CLAD, death, and bacterial infections [
17]. Similarly, Nakajima et al. observed that bronchial wash from donor lungs declined for implantation in recipients due to aspiration or infection had higher bile acids compared to those from accepted donor lungs. [
18]
A direct comparison of the diagnostic and prognostic value of LABW and BAL bile acids in the context of microaspiration in lung transplant recipients would be timely and helpful in guiding future biomarker research. Leveraging the existing cohort from our previous BAL bile acid study [
16] and our institutional protocol of routinely collecting both LABW and BAL, we aimed to compare bile acid levels between sample types and their associations with relevant short- and long-term clinical outcomes. We hypothesized that LABW bile acid at 3 months post-transplant are more strongly associated with inflammation, ALAD, CLAD, and death compared to BAL bile acid.
Discussion
Our study reveals that bile acids, specifically TCA and GCA, are present at higher concentrations in LABW compared to BAL. This is consistent with the long-held theory that bile acid presence in the lungs is the result of microaspirated gastrointestinal contents [
16]. Bile acid-containing refluxate initially enters the large airways and tracheobronchial tree, which are preferentially sampled by LABW. As refluxate travels more distally to small airways and alveolar space, it mixes with airway secretions and becomes progressively diluted, leading to the lower bile acid concentrations measured in BAL. We speculate this model is applicable in health and disease states, although impaired mucociliary clearance in lung transplant recipients may have exaggerated the effect in our study population [
26].
In addition to bile acids, the vast majority of proteins are higher in LABW than in BAL. It is likely that variable dilution may play a role in our observed differences between LABW and BAL biomarker levels, since LABW involves instillation of 20 mL of isotonic saline as opposed to 50 mL for each BAL. However, the notable exception of RAGE being lower in LABW suggests that non-trivial reasons contribute, such as heterogeneous cell composition along the airways. Prior studies have shown RAGE is constitutively expressed by type 1 pneumocytes in the alveoli [
27], which may account for its higher level in BAL. In contrast, CCSP is abundantly expressed within the conducting airway epithelium [
28], explaining its higher level in LABW. In a study from another center where LABW was obtained prior to BAL (similar to our protocol), LABW recovered more epithelial cells and neutrophils, while BAL recovered more lymphocytes and alveolar macrophages [
29]. Building upon these previous observations, our study further supports the idea that LABW and BAL can provide complementary information, as highlighted in the consensus guidelines for standardization of LABW collection and processing in lung transplantation. [
2]
Similar to what we [
16] and others [
10] have reported in BAL, positive correlations exist between a majority of bile acids and inflammatory proteins in LABW. Our study examines this bile acid-inflammation link between sample types. We find that bile acids in LABW, specifically TCA and GCA, show strong positive correlations with many of the inflammatory proteins in BAL, specifically four proinflammatory cytokines and two chemokines. Conversely, TCA and GCA in BAL only correlate with two chemokines in LABW. The four BAL proinflammatory cytokines that correlate with LABW TCA and GCA (i.e. IL-1α, IL-1β, IL-6, IL-8) have all been implicated as key mediators of pulmonary inflammation and fibrosis. [
30,
31]
LABW TCA and GCA are also the only bile acids in either sample type associated with a concurrent acute decline in lung function or ALAD. In our previous study on the larger BAL-only cohort of 76 patients, TCA and GCA were shown to be associated with ALAD [
16]. After the exclusion of 15 patients who did not have matching LABW samples, BAL TCA and GCA do not demonstrate statistical significance in this present study, although both
P values closely approach the statistical significance threshold. It is unclear whether lung inflammation and ALAD in our cohort were specifically due to microaspiration or other complications after lung transplantation such as acute rejection or infection. Nonetheless, LABW TCA and GCA appear to be more useful than their BAL counterparts in aiding the diagnosis of biologically and clinically relevant microaspiration in our cohort.
Consistent with the ALAD analysis, LABW TCA and GCA are the only bile acids in either sample type which are predictive of death after adjusting for major known risk factors. When stratifying our cohort by their bile acid levels (third tertile vs. first and second), TCA is better than GCA at identifying patients at higher risk of long-term mortality. These results are similar to those in our previous study, which found multiple strong signals with TCA, including associations with GERD, ALAD, and anti-reflux surgery. This was less so the case for GCA and not at all for CA. Overall, TCA and GCA seem more predictive than CA in our study. One of the important differences between these bile acid subspecies is that TCA and GCA are conjugated, whereas CA is unconjugated. Our findings confirm a recent study by Urso et al., which observed that conjugated bile acids in BW are associated with CLAD, mortality, and bacterial infections, in addition to changes in airway lipidome and cytokines [
17]. As postulated by Urso et al., conjugated bile acids may be more deleterious to pulmonary epithelium compared to their unconjugated counterparts due to increased solubility. Our study thus provides additional evidence for the use of LABW over BAL in future mechanistic and translational research on bile acids.
Although our study focused on lung transplant recipients and microaspiration, it raises the question whether LABW sampling may be useful in general respiratory medicine. In contrast to previous studies that have compared the diagnostic yields of BAL and bronchial wash [
3‐
5], ours is the first to identify a condition where LABW may offer superior clinical utility. Other possible conditions include tracheobronchial tuberculosis [
32] or relapsing polychondritis [
33], where the large airways and tracheobronchial space are preferentially affected. Moreover, there are certain clinical situations, such as in mechanically ventilated patients with acute respiratory distress syndrome, where significant hemodynamic instability, oxygen desaturation or even cardiac arrest have been reported after full volume BAL [
34]. LABW may be considered a safer alternative to obtain an adequate sample for diagnostic purposes in these cases. We believe that future studies directly comparing between LABW and BAL in these broader clinical contexts would be helpful.
Our study’s strengths include the routine use of LABW and consistent bronchoscopy protocols at our lung transplant center. Given widely differing practices between institutions, with most centers rarely performing LABW, a multicenter study would be difficult. Another strength is our multimodal approach to analyzing samples, the completeness of follow-up, and the availability of clinical data which allowed us to evaluate relationships between biomarkers and relevant biological and clinical outcomes.
This work has several limitations which warrant discussion. Given its retrospective design, there are many potential unmeasured or unknown confounding variables which may have impacted the associations observed in our study. The study cohort was derived from a highly selected retrospective cohort of lung transplant recipients with and without GERD, approximately matched one to two. This limits the generalizability of our findings, as our study cohort may not be representative of the general lung transplant recipient population or patients with other respiratory diseases. Our sample size was limited, making it likely underpowered for multivariable survival analysis. Despite the low events and sample size in some analyses, we detected a consistent signal between LABW and BAL for TCA and GCA. However, our results should be interpreted with caution and require validation in larger studies before clinical translation.
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