Endotracheal aspirates contain a limited number of lower respiratory tract immune cells

Marika Orlov, Eric D. Morrell, Victoria Dmyterko, Jessica A. Hamerman, Mark M. Wurfel, Carmen Mikacenic
2021 Critical Care  
Use of bronchoscopies for research during the COVID-19 pandemic has been limited due to risk of aerosol exposure and need to preserve PPE. Endotracheal aspirates (ETAs) have been used for research as they are easily obtained via a simple, non-aerosol generating procedure without need for extra PPE [1, 2] . However, there is a paucity of information regarding whether ETAs are a reasonable surrogate for bronchoalveolar lavage (BAL) to study lung specific immune responses in critically ill
more » ... tically ill patients. The purpose of this study was to compare the immune cell populations detectable in ETA versus BAL using flow cytometry to evaluate the potential utility of ETAs for research. We enrolled critically ill, non-COVID patients (n = 12) with suspected bacterial pneumonia on mechanical ventilation undergoing bronchoscopy with BAL, approved by the University of Washington Human Subjects Committee under a waiver of consent. Immediately before bronchoscopy, ETA was collected into a sputum trap via the in-line suction catheter passed through the ET tube to maximal depth. BALF was processed as described previously [3] . ETA was mixed with equal volume 0.1% DTT, incubated on ice for 15 min, and strained through a 70 µM cell strainer [4] . Cells were pelleted by centrifugation, washed in PBS, and cryopreserved. Later, cells were thawed and stained with a live/dead cell marker (eFluor780, eBiosciences), washed with PBS, and stained for 30 min with antibodies to the following extracellular markers: (eBiosciences) anti-CD45-FITC, anti-CD3-BV510, anti-CD4-BV421, anti-CD8-PE-Cy7, anti-CD14-PE, anti-CD206-PerCP-Cy5.5, and anti-CD20-APC. ETA and BALF cell proportions were measured by manual inspection of cytospins prior to cryopreservation (n = 8). Wilcoxon signed-rank test was used to compare percent populations of cells across the two groups, and Spearman's rank-order test was used to identify correlations between ETA and BALF. A majority of the patients were male (10/12, 83%), white (9/12, 75%), and average age was 54 years (range 30-72). Bronchoscopies were performed an average of 4.75 days post-intubation (range 1-11 days), and pneumonia was diagnosed by quantitative BAL culture in 50% of the samples (6/12). Manual inspection of cytospins demonstrated low percentages of neutrophils (ETA and BAL: 6% of all cells) and epithelial cells (ETA: 1% of all cells, BAL: 0%). Flow cytometric quantification of BAL showed CD206+ alveolar macrophages (36% of CD45+ cells, Table 1) and T-and B-lymphocytes (32% of CD45+ cells) to be the most abundant cell types. In contrast, the predominant cell type in ETA was CD14+ monocytes (65% of CD45+ cells). Despite differences in abundance by fluid type, we did observe moderate to high correlation in proportions for alveolar macrophage (r = 0.643, p = 0.028), CD4+ (r = 0.848, p = 0.001), CD8+ (r = 0.692, p = 0.016), and CD20+ lymphocytes (r = 0.587, p = 0.049, Fig. 1 ). Percentages of monocytes and total lymphocytes were not significantly correlated between the two samples. We show that there are immune cell subsets present in ETA in critically ill patients and that these subsets
doi:10.1186/s13054-020-03432-1 pmid:33407749 fatcat:7vvf6u3gunf47omy676uv3sbfy