Extracellular traps are evident in Romanowsky‐stained smears of bronchoalveolar lavage from children with non‐cystic fibrosis bronchiectasis

Abstract Background and Objective The importance of extracellular traps (ETs) in chronic respiratory conditions is increasingly recognized but their role in paediatric bronchiectasis is poorly understood. The specialized techniques currently required to study ETs preclude routine clinical use. A simple and cost‐effective ETs detection method is needed to support diagnostic applications. We aimed to determine whether ETs could be detected using light microscopy‐based assessment of Romanowsky‐stained bronchoalveolar lavage (BAL) slides from children with bronchiectasis, and whether the ETs cellular origin could be determined. Methods Archived Romanowsky‐stained BAL slides from a cross‐sectional study of children with bronchiectasis were examined for ETs using light microscopy. The cellular origin of individual ETs was determined based on morphology and physical contact with surrounding cell(s). Results ETs were observed in 78.7% (70/89) of BAL slides with neutrophil (NETs), macrophage (METs), eosinophil (EETs) and lymphocyte (LETs) ETs observed in 32.6%, 51.7%, 4.5% and 9%, respectively. ETs of indeterminate cellular origin were present in 59.6% of slides. Identifiable and indeterminate ETs were co‐detected in 43.8% of slides. Conclusion BAL from children with bronchiectasis commonly contains multiple ET types that are detectable using Romanowsky‐stained slides. While specialist techniques remain necessary to determining the cellular origin of all ETs, screening of Romanowsky‐stained slides presents a cost‐effective method that is well‐suited to diagnostic settings. Our findings support further research to determine whether ETs can be used to define respiratory endotypes and to understand whether ETs‐specific therapies may be required to resolve airway inflammation among children with bronchiectasis.


INTRODUCTION
[5] Amy S. Bleakley, Steven Kho, Robyn L Marsh and Gabriela Minigo contributed equally to this study.
There is increasing interest in defining bronchiectasis endotypes to direct clinical management. 6Neutrophil extracellular traps (NETs) are a potentially important endotype marker that could be targeted with existing therapies, including macrolide antibiotics [7][8][9] and inhaled dornase alfa. 10NETs are string-like structures comprised of extracellular DNA, chromatin and antimicrobial peptides that protrude from neutrophils. 11,12NETs can protect against infection by trapping and killing extracellular pathogens; however, they may also promote inflammation and tissue damage if not cleared effectively. 13,14In adults with bronchiectasis or chronic obstructive pulmonary disease (COPD), NETs are associated with lung function decline, 15 disease severity, 8,16 exacerbation risk 16 and treatment responses. 8,15NETs have been observed in bronchoalveolar lavage (BAL) from children with cystic fibrosis, 10 protracted bacterial bronchitis (PBB), 17 bronchiectasis 17,18 and unspecified causes of chronic cough related to neutrophilic inflammation 10 ; however, we are unaware of any studies that have determined NETs prevalence among paediatric populations.
Other immune cells can also release extracellular traps (ETs).Macrophages, the most abundant airway immune cell, can form extracellular traps (METs) 19,20 and these structures have been detected in BAL from children with cystic fibrosis or other causes of chronic cough. 10Eosinophil extracellular traps (EETs) have been observed in respiratory specimens from asthma 21 and COPD 22 patients, but their role in disease processes and treatment responses remains poorly understood.There is some evidence that lymphocytes may also produce ETs (LETs) 23 ; however, we are unaware of any studies that have reported LETs in respiratory specimens.The presence and clinical significance of METs, EETs and LETs in paediatric bronchiectasis are unknown.
ETs testing is typically done in research laboratories using specialist immunoassays, immunofluorescent staining, confocal microscopy or functional assays. 10,21,22,24Such techniques are often unavailable in diagnostic laboratories where BAL testing is limited to differential cell counts performed using Romanowsky-stains (e.g., Wright or Giemsa stains) and bacterial culture tests.In malaria, Giemsa-stained slides have been validated for detecting NETs in blood smears. 25It is not yet known whether this method could be extended to BAL or other respiratory specimens that may be complicated by mucus, cellular aggregates, biofilms and upper airway secretions. 17Availability of a simple ETs detection method that is well-suited to diagnostic contexts is expected to substantially advance the field by enabling characterization of novel endotype descriptions that are currently absent in paediatric bronchiectasis.
In this study, we aimed to determine: (i) whether ETs could be detected in Romanowsky-stained BAL slides from children with bronchiectasis; and (ii) whether the ETs' cellular origin could be identified.The secondary aims were to determine ETs prevalence among children with bronchiectasis and to assess whether the presence of ETs in BAL was associated with clinical parameters, BAL inflammatory markers or signs of lower airway infection.

Study design
A cross-sectional analysis was performed using archived Romanowsky-stained BAL slides from 100 children with bronchiectasis or chronic suppurative lung disease (CSLD) who had been prospectively recruited to an observational study of chronic wet cough between August 2010 and October 2013.A sample size of 100 children was arbitrarily chosen a priori, as there were no prior data to inform an ETs prevalence estimate.The date range was selected to include specimens that had previously undergone bacterial culture and cytokine analyses. 26A second analysis to assess whether ETs may have been induced as an artefact of sample processing was performed using: (i) peripheral blood from a healthy adult volunteer and (ii) BAL prospectively collected from children recruited to the same observational study between June and August 2021.For all BAL analyses, children were recruited via the paediatric respiratory clinic at Royal Darwin Hospital, Darwin, Australia with written informed consent from their parent/carer, as described previously. 26

Diagnostic criteria
Bronchiectasis was diagnosed by a paediatric respiratory physician based on chest high-resolution computed tomography (cHRCT), as described previously. 1 CSLD was diagnosed where children had a wet/productive cough for >4 weeks, cHRCT was not done or did not fulfil the criteria of bronchiectasis, and the cough resolved following >2 weeks of antibiotics. 1 Bronchiectasis severity was scored using a modified-Bhalla scale, as described previously. 27

BAL collection and processing
Bronchoscopy with BAL collection was performed in accordance with the European Respiratory Society

SUMMARY AT A GLANCE
The burden of paediatric bronchiectasis is an ongoing global challenge.Extracellular traps (ETs) are a potentially important marker for defining paediatric bronchiectasis endotypes.We demonstrate Romanowsky-stained BAL can be used as a simple and cost-effective ETs detection method.Using this approach, we determined that ETs were present in BAL slides from 79% of 89 children with non-cystic fibrosis bronchiectasis.Multiple ET types were observed, with ETs derived from macrophages (METs) and neutrophils (NETs) the most commonly detected types.guidelines, 28 as described previously. 29Briefly, BAL was collected as two separate lavages and maintained on ice for up to 3 h prior to processing.The first lavage (Lavage-1) was used for bacterial culture and the second (Lavage-2) for leukocyte counts and cytokine measures (IL-6, IL-8, IL-1β and IFN-γ), as detailed in Appendix S1 in the Supporting Information.Slides for differential counts were prepared by applying unfiltered Lavage-2 containing approximately 5 Â 10 4 leukocytes onto silane-coated microscope slides (Sigma-Aldrich, St. Louis, USA) using a cytofunnel™ (ThermoFisher Scientific, Waltham, USA) with centrifugation at 80g for 8 min in a Cytospin 4 (ThermoFisher Scientific).Slides were fixed with Quick Dip Fixative (Fronine, Riverstone, Australia) for 3 min, and air dried before staining with Quick Dip stain (Fronine).This stain is a modified-Romanowsky stain that achieves equivalent leukocyte staining as Giemsa and Wright stains.Leukocyte percentages were calculated from a minimum 300 cells.BAL neutrophilia was defined as a neutrophil percentage >15.

ETs detection
ETs detection was performed according to a previously validated method for Giemsa-stained peripheral blood smears. 25riefly, the BAL cytoslides were examined by a senior microscopist (S.K.) under high power magnification (400Â) on an Olympus CX31 light microscope (Tokyo, Japan).Areas the size of one field of view around the edge of the Cytospin area were excluded.BALs were scored positive for ETs where extracellular string-like structures with similar coloured staining to the nuclei of surrounding cells (dark blue or purple) were present, with or without contact with nucleated cell(s).Where possible, the cellular origin of each ET was estimated microscopically based on the morphology, size and physical contact with surrounding cell(s), as well as the presence and type of associated granules.ETs observed attached to readily identifiable cells were categorized as either NETs, METs, EETs or LETs (Figure 1).A final category of 'indeterminate ETs' was assigned where the cellular origin could not be determined.Where necessary, magnification was increased to 1000Â with oil immersion to assess the cellular origin of individual ETs.ETs percentages were calculated from a minimum of 300 leukocytes.To assess ETs quantification across operators, the number of ETs in 15% of slides was reviewed by a second microscopist (A.B.) who was blinded to primary data.The absolute ET count per mL of BAL was estimated by referencing the total leukocyte counts (as previously determined using a haemocytometer). 26

Testing for artefactual ETs induction
Additional testing was performed to examine whether ETs were induced during specimen processing.This was done in two stages: (i) using a fresh peripheral blood mononuclear cell (PBMC) suspension to test centrifugation effects; and (ii) using prospectively collected BAL to test temperature and processing time effects.For the PBMC experiment, fresh cells were collected by Ficoll ® separation 26 of peripheral blood from a healthy volunteer.Purified PBMCs were applied to slides using methods identical to those used for BAL.For the BAL experiments, Lavage-2 was split into two equal aliquots immediately after collection.One aliquot was stored on ice and the other at room temperature during transportation to the laboratory (<15 min).Slides from both aliquots were prepared at time 0 and 3 h after arrival at the laboratory, as described above.

Statistical analysis
Continuous variables are presented as medians with inter-quartile ranges (IQR) with groups compared using the Wilcoxon rank sum test.Categorical variables are presented as frequencies and percentages with groups compared using the χ 2 test.Correlations were determined using Spearman correlation.A p-value <0.05 was considered statistically significant.Concordance was determined using Lin's concordance coefficient and Pearson correlation.Statistical analyses were performed using STATA 16 (Version 15; StataCorp, USA).Hierarchical group average cluster analysis with heat map generation was performed using R (Version 4.0.2;Austria) with the BiocManager, 30 Heatplus, 31 gplots, 32 and RColorBrewer packages. 33

Cohort characteristics
BAL slides from all 100 children were obtained from the archive; however, slides from 11 children were excluded from further analysis due to low sample and/or staining quality.Characteristics of the 89 children included in the analysis are presented in Table 1.Most of the children (84.3%) were aged <4 years (median 2.3 years, IQR 1.6-3.1 years).Eighty-two children (92.1%) had radiographic evidence of bronchiectasis and 7 (8%) had a clinical diagnosis of CSLD.Given the small number of children with CSLD, data from these children were combined with those of the bronchiectasis group in all subsequent analyses.Bronchiectasis severity ranged up to a modified-Bhalla score of 20 (median 8, IQR 6-12).BAL neutrophil percentage ranged from 0% to 99.9% (median 10.7%, IQR 4.3%-30.7%),with airway

Assessing ETs cellular origins
A total of 504 ETs were observed across the 89 slides.Of these, the cellular origin (neutrophil, macrophage, eosinophil or lymphocyte) of 65.1% (328/504) could be determined (Figure 1).The remaining 34.9% (176/504) were categorized as indeterminate ETs due to the ET either being: (i) in contact with multiple nucleated cells; (ii) detached from the original cell or (iii) obscured by squamous cells or amorphous debris (Figure 2).Among the 70 ETs-positive slides, the cellular origin of all ETs in the smear could be determined for 17/70 (24.3%)BALs.In the remaining slides, identifiable and indeterminate ETs were co-detected in 39/70 (55.7%), whereas only indeterminate ETs were observed in 14/70 (20%; Figure 3).

ETs originated from multiple lineages
NETs (173/504, 34.3%) and METs (141/504, 28%) were the most common identifiable ETs, observed in 32.6% and 51.7% of BALs, respectively (Table 1, Figure 1A-D).BAL from 13% of children were positive for METs only, and a smaller proportion (3%) only contained NETs (Figure 3).EETs were observed in slides from 4 (4.5%)children (Table 1; Figure 1E,F), all of whom had elevated BAL The origin of some extracellular traps (ETs) could not be determined from the Romanowsky-stained BAL slides (Indeterminate ETs).Representative 400Â projection images of Romanowsky-stained BAL slides showing examples of indeterminate ETs.These ETs were often observed in BAL slides with dense cellularity, resulting in ETs being in contact with multiple potential nucleated cells of origin (A).In some cases, ETs were detached from nucleated cell of origin (B).Unsatisfactory BAL smear, showing clumps of ETs with origin obscured by amorphous debris (C).Scale bar: 20 μm.

ETs not induced during processing
Given the high proportion of ETs-positive BALs and the high number of indeterminate ETs, we next sought to determine whether ET formation may have been triggered during BAL processing.This was done by undertaking quality control experiments using PBMCs from a healthy adult volunteer and prospectively collected BAL specimens from children.ETs were not present in the PBMC cytoslides, indicating that Cytospin processing alone did not induce ETs.In prospective testing of fresh BAL specimens, BAL stored on ice for up to 3 h did not alter the number of ETs observed, consistent with a lack of artefactual ET formation (Table S1 in the Supporting Information).

Relationships between ETs and clinical markers
We next undertook exploratory analyses to examine whether the presence of ETs was associated with demographic variables (age, bronchiectasis severity, gender and antibiotic use), BAL cytokine markers and culture of respiratory pathogens.Due to the high number of BALs containing indeterminate ETs, these analyses examined relationships between detection of any ETs (including indeterminate ETs) and the clinical variables.There was an association between gender and detection of ETs (p = 0.048), with males more likely to have ETs present (40/70, 57%) than females (13/19; 42.9%).No association was observed with age, bronchiectasis severity, antibiotic use or cytokine measures (Table 2).H. influenzae was cultured more often from ETs-positive (11/66, 16.7%) than ETs-negative (1/19, 5.3%) BAL; however, this difference was not statistically significant (Table 2).

DISCUSSION
Use of Romanowsky-stained BAL to detect ETs presents a simple and cost-effective method that is well-suited to diagnostic applications and to informing specialist techniques for confirming ET cellular origins.To our knowledge, this is the first study to demonstrate direct ETs detection from Romanowsky-stained BAL and the first to examine ETs prevalence among children with bronchiectasis.Among this cohort of 89 children, ETs were detected in 79% of BALs and were derived from multiple leukocyte lineages (predominantly METs and NETs).Absolute ET counts were positively correlated with BAL total cell counts, absolute neutrophil and macrophage counts, IL-8 and IL-6.
F I G U R E 3 Multiple ET types were frequently co-detected in BAL.This cluster analysis with heat map shows the relative proportions of ET types (columns) in BAL from each child (rows).The red and beige colours in the heatmap represent relative high and low proportions of ET types, respectively.
Our results are consistent with earlier studies describing NETs in sputum and BAL from patients with a range of respiratory conditions, 16,21,34 including adults with bronchiectasis 8 and children with cystic fibrosis or other causes of chronic cough. 10Results from our experiments with PBMCs and fresh BAL indicate that the high number of ETs-positive specimens in our study is unlikely to be a specimen processing artefact.This interpretation is supported by the absence of ETs in 21% of the slides examined.Instead, our results support the findings of earlier studies suggesting ETs formation is an important component of the innate response to lower airway infection.Positive correlations between ETs absolute counts and markers of neutrophilic inflammation are consistent with the hypothesis that ETs contribute to airway inflammation among children with bronchiectasis; however, these observations require confirmation with leukocyte-specific ETs tests.
We did not observe an association between ETs and age, bronchiectasis severity, antibiotic use or culture of bacterial pathogens.We did detect a statistically significant association between ETs and gender; however, replication of this finding is required as a mechanistic explanation of this result is unclear.Further testing is also warranted to confirm the observed absence of an association between ETs detection and culture of respiratory pathogens, especially given higher recovery of H. influenzae from ETs-positive compared to ETs-negative BALs.Collectively, these findings should be interpreted cautiously as our study was not primarily designed to assess such relationships and few specimens were ETs-negative.
An important observation from our use of Romanowskystained slides is that multiple leukocyte lineages in BAL can generate ETs, with METs and NETs the most commonly T A B L E 2 ETs detection was not significantly associated with clinical parameters, BAL inflammatory markers or culture of a respiratory pathogen in univariate tests.The role of METs in chronic airway diseases is poorly understood; however, in vitro studies have demonstrated MET formation can be triggered by exposure to respiratory pathogens 10 and neutrophil elastase. 35Positive correlations between absolute numbers of both METs and NETs with BAL IL-8 and IL-6 suggest a generalized ETosis response related to the underlying drivers of neutrophilic inflammation.Our observation is in accordance with earlier work by King et al. 10 demonstrating the presence of both NETs and METs in BAL was associated with neutrophilic inflammation among children with cystic fibrosis and other causes of chronic cough.Further studies are needed to determine whether co-detection of NETs and METs is associated with increased inflammation.

ETs-positive
The presence of EETs in BAL was an interesting finding builds upon earlier reports demonstrating a sub-population of children with bronchiectasis have lower airway eosinophilia. 29EETs have previously been described in asthma, 36 eosinophilic pneumonia 37 and chronic rhinosinusitis 38 ; however, we are unaware of any studies describing EETs among patients with suppurative lung diseases.The presence of eosinophilic granules aided identification of EETs from Romanowsky-stained smears; however, follow-up studies using EETs-specific testing methods will be necessary to understand the role of these structures in bronchiectasis.It remains to be determined whether the presence of EETs in BAL indicates a distinct bronchiectasis endotype requiring targeted therapy.
The observation of LETs was an unexpected finding.LETs have been considered in acne 39 and autoimmune diseases 40 but we are unaware of any reports describing LETs involvement in respiratory conditions.There is some evidence from in vitro studies that LETs may be generated in response to infectious stimuli, 41 whereas other reports suggest LET-associated inflammation may disrupt wound healing and promote tissue damage. 39Follow-up testing with lymphocyte-specific staining methods is needed to confirm the presence of LETs in BAL and sputum and to elucidate the lymphocyte cell types that may be generating these ETs. 40trengths of our study include direct ETs detection using a simple and cost-effective method that could be used to screen specimens prior to application of specialist leukocyte-specific methods.Use of Romanowsky-stained slides is well-suited to implementation in diagnostic laboratories where Wright and Giemsa-staining of BAL is performed routinely.Direct ETs detection also enabled the first determination of ETs prevalence among children with bronchiectasis.
Limitations of the study include our use of archived slides.While this supported identification of most ETs, the high proportion of indeterminate ETs restricted analyses of relationships between specific ET types and clinical markers.Specialist staining techniques remain essential to characterize ETs cellular origins and to elucidate relationships between specific ET types and different bronchiectasis endotypes.The study's cross-sectional design prevented assessment of whether ETs contribute to bronchiectasis disease progression.The study sample size (arbitrarily selected due to a lack of prior data about ETs prevalence) was not powered to assess relationships between specific ET types and the clinical and inflammatory markers.Nevertheless, our results are broadly consistent with earlier reports and provide novel data that informs future research directions.As with other paediatric respiratory studies, 10 we did not include a healthy control cohort, as BAL was only collected where children underwent bronchoscopy for clinicallyindicated reasons.Our study population also has a high burden of early-onset CSLD, and therefore it is unclear whether our findings will be generalisable to other paediatric bronchiectasis populations.Further ETs research among paediatric populations with milder suppurative lung diseases, including PBB (an important antecedent of bronchiectasis), 42 should be conducted to understand the role of ETs across the CSLD disease spectrum.Larger studies are also warranted to determine associations with frequency of exacerbation and lung function and to investigate the impact of antibiotics on ETs production.
In conclusion, multiple ET types are commonly present in BAL from children with bronchiectasis.The presence of ETs can be assessed using Romanowsky-stained BAL slides, providing a simple and cost-effective screening method that is well-suited to implementation in diagnostic laboratories.While this approach facilitates identification of most ETs in BAL, leukocyte-specific methods remain essential to confirming cellular origins of all ETs.Our results build upon earlier work demonstrating that ETs are a common feature of respiratory conditions characterized by persistent or recurrent lower airway infection.Further research is needed to determine whether different ET types, either alone or in combination, have protective and/or pathogenic roles in paediatric bronchiectasis, and whether ETstargeting treatments may be required to resolve persistent airway inflammation.
8,15,16ETs are increasingly recognized to have clinical importance in chronic airway conditions, including associations with lung function, disease severity, exacerbation risk and treatment responses among adults with COPD or bronchiectasis.8,15,16It is not yet known whether such relationships are generalisable to children with bronchiectasis.
Note: Bold indicates statistically significant differences.Abbreviations: BAL, bronchoalveolar lavage; ETs, extracellular traps (any type); IFN, interferon; IL, interleukin.aAntibioticuse data were available for 87/89 children.There was one missing datapoint from ETs-positive BALs (n = 69) and one from ETs-negative BALs (n = 18).bIL-8and IFN-ɣ data were available for 62/89 children.IL-1β and IL-6 were available from 60/89 and 61/89 children, respectively.Missing IL-1B and IL-6 datapoints were all from ETs-negative BALs.cBAL culture data were available for 85/89 children.Univariate tests were restricted to S. pneumoniae and H. influenzae only, as too few samples (<5) were positive for other species.T A B L E 3 Spearman's correlation between absolute count of any extracellular traps (ET) counts and age, bronchiectasis severity score, BAL cellular characteristics and cytokine levels.identified