Presented in part at the European College of Veterinary Internal Medicine-Companion Animal Congress, Sevilla, Spain, September 2011.
Interpretation of Multisegment Bronchoalveolar Lavage in Cats (1/2001–1/2011)
Article first published online: 28 OCT 2012
Copyright © 2012 by the American College of Veterinary Internal Medicine
Journal of Veterinary Internal Medicine
Volume 26, Issue 6, pages 1281–1287, November/December 2012
Total views since publication: 34
How to Cite
Ybarra, W.L., Johnson, L.R., Drazenovich, T.L., Johnson, E.G. and Vernau, W. (2012), Interpretation of Multisegment Bronchoalveolar Lavage in Cats (1/2001–1/2011). Journal of Veterinary Internal Medicine, 26: 1281–1287. doi: 10.1111/j.1939-1676.2012.01016.x
- Issue published online: 20 NOV 2012
- Article first published online: 28 OCT 2012
- Manuscript Accepted: 5 SEP 2012
- Manuscript Revised: 20 AUG 2012
- Manuscript Received: 18 APR 2012
- Respiratory endoscopy;
- Thoracic radiology
Cytologic results from bronchoscopic BAL in cats with naturally occurring respiratory disease have not been reported, and the clinical utility of multisegment lavage has not been evaluated.
BAL cytology from 2 separate lung segments in cats will have similar cell counts, cytologic interpretation, or both.
Eighty-seven cases in 85 cats (2 examined twice) with naturally occurring lower respiratory disease.
A combined prospective/retrospective evaluation of all cats with multisegment BAL was performed. BAL fluid was evaluated for total nucleated cell counts, differential cell counts, and cytologic characteristics at each lavage site. BAL fluid was categorized as eosinophilic, neutrophilic, lymphocytic, hypercellular, or mixed. Radiographs were assessed for diffuse or focal disease.
Clinical diagnoses included inflammatory airway disease (n = 63), pneumonia (n = 15), neoplasia (n = 6), and undetermined (n = 3). Total nucleated cell counts varied between sites regardless of radiographic evidence of focal or diffuse radiographic disease. In 28/87 cases (32%), cell counts differed between lavage sites by 2.2–40 fold. BAL yielded similar cytologic interpretation of inflammation in 45/87 (52%) cases. In 8/14 cases that had BAL performed at the site of a focal radiographic infiltrate, as well as at a site of diffuse infiltrates, the same inflammatory interpretation was made at each site.
Conclusions and clinical importance
Total and differential cell counts in BAL fluid often differ between lung segments in cats with lower respiratory disease, and caution is warranted when using a single BAL cytology to define the inflammatory response in cats with spontaneously occurring lower respiratory tract disease.
University of California Davis Veterinary Medical Teaching Hospital
Bronchoalveolar lavage (BAL) is an invaluable noninvasive technique for investigating the specific cause of respiratory signs in cats. Reference ranges for BAL cell counts and cytologic distributions are available for cats,[1-3] results of nonbronchoscopic BAL have been described in cats with spontaneous lower respiratory tract disease,[4-6] and information on the safety of bronchoscopy with BAL has been reported.[7, 8] However, a comprehensive evaluation of cytologic results from endoscopic BAL of diseased cats has not been reported and specifically, the clinical utility of one segment versus multisegment lavage has not been evaluated. Inspection of pooled BAL samples from multiple lung segments improved identification of fungal organisms in dogs with fungal pneumonia, and cytologic evaluation of two-segment bronchoalveolar lavage increased detection of Pneumocystis carinii and cytomegalovirus in human patients and of bacteria in dogs with foreign body inhalation,[10-12] indicating the value of multisegment BAL in these species.
It is intuitive that recovery of infectious organisms would be enhanced by lavage of multiple lung segments, and it seems likely the magnitude and type of inflammation in BAL fluid would be similar from multiple sites in patients with diffuse inflammatory airway disease or pneumonia. However, in patients with focal respiratory disease, regional differences in BAL findings might be anticipated. This theory is supported by a study in dogs with foreign body pneumonia that reported distinct differences in cell counts, differential cellular cytology, and presence of intracellular bacteria between the site of the foreign body and an alternate site. Conversely, in a separate study of dogs with diffuse pulmonary disease identified radiographically, multisegment lavage revealed different classifications of inflammation or variable presence of infecting organisms or neoplastic cells in approximately 1/3 of cases.
Both clinical and experimental evaluation of feline lower respiratory tract disease commonly relies on interpretation of blindly obtained BAL fluid or evaluation of a single BAL sample to define disease and assess response to treatment[4-6, 14, 15] The hypothesis of this study was that BAL cytology from 2 separate lung segments in cats with radiographically diffuse lower respiratory tract disease would not differ. That is, cell counts in BAL fluid collected from separate lung segments would not vary by more than 2-fold, and cytologic interpretation in each segment would be similar, that is predominantly suppurative, eosinophilic, or lymphocytic inflammation. In contrast, we hypothesized that cats with focal radiographic infiltrates would have different cell counts or cytologic interpretation of BAL fluid obtained from separate lung segments.
Materials and Methods
Cats undergoing bronchoscopy with multiple site bronchoalveolar lavage performed between 1/2001 and 1/2011 that also had 2 or 3-view radiographic studies were included in this study. Findings on thoracic imaging were collated and reviewed by a board-certified radiologist for diffuse versus focal disease, and specific lung segments involved in focal disease were recorded. Bronchoscopy and bronchoalveolar lavage were performed as previously described. Briefly, cats were anesthetized and maintained with jet ventilation for bronchoscopy with a 2.5 mm1 or 3.8 mm2 flexible endoscope. Individual aliquot volume for BAL was 3–5 mL per site, and additional aliquots were instilled, if required, to obtain a representative sample as determined by the endoscopist. If more than 1 aliquot was instilled and aspirated at 1 site, these were combined for cytologic analysis, but distinct sites were evaluated separately. Location, volume of fluid instilled, and volume recovered were recorded for each site, and cats that had BAL performed at a site with radiographically focal disease were identified for separate analysis. A 0.5–1.0 mL aliquot of pooled BAL fluid was submitted to the UC Davis Microbiology Service for bacterial culture, including aerobic, Mycoplasma, and anaerobic assessment. Samples were plated onto 5% sheep blood agar and MacConkey's agar for isolation of aerobic organisms, pre-reduced anaerobic Brucella plates3 for anaerobic culture, and pleuro-pneumonia like organism base with thallium acetate (antifungal) and penicillin G (antibacterial) for Mycoplasma spp.4 BAL fluid from separate lung sites was submitted to the UC Davis VMTH clinical pathology laboratory for cell counts and cytologic assessment. An automated cell counter5 was used to determine the total nucleated cell count (TNCC/μL). A 500 μL aliquot of BAL fluid was placed in the well of a cytospin cup6 and cells were dispersed onto glass slides via a 5-minute spin at 113 × g. For cell counts exceeding 5,000 cells/μL, BAL fluid was diluted 1 : 10 with phosphate-buffered saline and 2 separate cytospin slide preparations, one using 500 μL of undiluted fluid and another using 500 μL of diluted fluid, were made.
Differential cell counts were performed by counting a total of 200 cells at high power (50×) examination, and cellular characteristics were evaluated by a board-certified clinical pathologist. Previously established reference intervals for feline BAL fluid were used for clinical interpretation of results,[1-3] with counts of 300–400 cells/μL containing 65–80% macrophages, and up to 7% neutrophils, 10% lymphocytes, and 20% eosinophils considered normal. BAL fluid results were considered unacceptable if <300 cells/μL were obtained or if oral contaminants such as Simonsiella bacteria or squamous cells were observed, and these samples were excluded from further analysis. BAL fluid was characterized as predominantly eosinophilic inflammation (>20% eosinophils and neutrophil% within reference limits or >50% eosinophils), predominantly neutrophilic inflammation (>7% neutrophils with eosinophil% within reference limits or >50% neutrophils), or predominantly lymphocytic (>10% lymphocytes with other cell types within normal limits). Mixed inflammation with eosinophils, neutrophils, and lymphocytes was designated when there was concurrent elevation of eosinophils >20%, neutrophils >7% and lymphocytes >10%. BAL samples with greater than 1.5 times a normal cell count (>600 cells/μL), but normal differential cell counts, were classified as hypercellular. Septic cytology was characterized by the presence of intracellular bacteria and concurrent inflammation. Karyolytic neutrophils were considered suspicious, but not diagnostic for airway sepsis. The presence of hemorrhage, epithelial hyperplasia, dysplasia, or neoplasia was recorded.
The final diagnosis of inflammatory airway disease, pneumonia, or neoplastic respiratory disease was made by the primary clinician on the basis of clinical findings, radiographic features, bronchoalveolar lavage results, and follow-up information. Medical records were reviewed by two of the authors (W.L.Y., L.R.J.) for concurrence.
Statistics: BAL TNCC and percent BAL fluid volume recovery were assessed for normality by the D'Agostino and Pearson Omnibus test.7 Results are expressed as median with range for nonparametric data and mean with standard deviation for normally distributed data. TNCC from separate lavage sites in each case was compared by the Wilcoxon matched pairs test for nonparametric data. For cases that had BAL performed at a site of radiographically focal disease, results were independently compared for differences in TNCC and cytology. The analysis was repeated for the group after removal of these samples. In addition, instances of >2-fold variability in cell counts were identified.
For each case, BAL samples obtained from separate lung segments were examined for agreement of cytologic diagnoses. Samples from individual cats were considered significantly different if a different classification of inflammation was assigned. For all analyses, significance was set at P < .05.
From January 2001 through January 2011, bronchoscopy with BAL was performed in 122 cases. Lavage was performed at a single site in 23 cases and at multiple sites in 99 cases. Twelve of these 99 cases were excluded due to a low cell count in one or more of the BAL samples, and the remaining 87 cases representing 85 cats were further examined. Two cats with inflammatory airway disease had bronchoscopic BAL performed twice during the time span of this study, 12 and 31 months after the initial evaluation. Within the cohort, there were 45 male and 42 female cats. Age ranged from 0.5 to 17.6 years (median 8.8 years). Final clinical diagnoses included inflammatory airway disease in 63 cases, pneumonia in 15, neoplasia in 6 (3 of which also had radiographic and cytologic data consistent with inflammatory airway disease), and an indeterminate diagnosis in 3 cats.
Radiographic findings were considered unremarkable in 8/87 cases. Focal infiltrates only were described in 8/87 cases as nodular (3/8), interstitial (3/8), alveolar (1/8), or bronchial (1/8). In the remaining 71 cases, diffuse infiltrates were reported including bronchial pattern (60/71), interstitial infiltrates (8/71), alveolar infiltrates (2/71), and hyperinflation (1/71). In 25 of these 71 cases, a focal radiographic pattern was superimposed on a diffuse pattern that was described as bronchial (21/25), interstitial (2/25), and alveolar (2/25) (Fig 1).
Lavage fluid was instilled in 3–5 mL aliquots (median 5 mL) for total volumes of 5–15 mL per site. There was no significant difference between volume instilled for the 1st or 2nd lavage (P = .43), and fluid volume was identical at the 2 sites in 88% of cases. Fluid volume recovery was 59 ± 24%. Total nucleated cell counts ranged from 300 to 16,100 cells/μL (median 1020 cells/μL). In 28/87 cases (32%), cell counts differed between lavage sites by 2.2–40 fold, with <2.2 fold variation in remaining cases. Pairwise comparison of low and high cell counts in separate lavage sites was possible in 86 cases and revealed significant differences between sites, with a median low count of 720/μL (300–12,250) at one site and a median high count of 1340/μL (500–16,100) at the other site, P < .0001 (Table 1). When cats with different lavage volumes at 2 separate sites were evaluated, low (700/μL) and high (1,700/μL) median cell counts remained significantly different, P = .04 (data not shown). BAL was performed at the site of focal radiographic infiltrates in 14 of 33 cats. Low and high cell counts at the 2 sites were also significantly different in this subset of cats. Statistical analysis was repeated after removal of this subset of cases from the entire group and statistical significance was retained between low and high cell counts, P < .0001. In 8 cats with radiographs that were considered unremarkable, cell counts were significantly different between sites, P = .014 (Table 1).
|Group||Low Median (range)||High Median (range)||P-Value|
|All Cases (n = 86)||720/μL(300–12,250)||1,340/μL(500–16,100)||<.0001|
|Radiographically focal disease (n = 14)||600/μL(300–5,200)||1,660/μL(600–9,200)||.001|
|Radiographically diffuse patterns (n = 64)||770/μL(300–12,250)||1,300/μL(500–16,100)||<.0001|
|Radiographically unremarkable (n = 8)||820/μL(600–1,580)||1,200/μL(800–8,100)||.014|
Cytologic assessment of BAL fluid from separate sites yielded similar cytologic interpretation of inflammation in 45/87 (52%) cases (Table 2). In cats with different lavage volumes instilled at the 2 sites, a similar percentage (43%) had the same type of inflammation reported at each site. In cases with eosinophilic inflammation at both lavage sites, the median eosinophil percent was 49% (range: 21–87%), while the median neutrophil and lymphocyte percents were 4% (range: 0–14% and 0–13%, respectively). In cases with neutrophilic inflammation at both lavage sites, the median neutrophil percent was 59% (range: 11–99%), median lymphocyte percent was 3% (range: 0–18%), and median eosinophil percent was 2% (range: 0–20%). In cases with the same type of inflammation at 2 lavage sites, cell counts varied by >2 fold between sites in 14/45 cases (31%). Inflammatory airway disease was diagnosed in 31, pneumonia in 9, neoplasia in 5 cases.
Interpretation of BAL fluid yielded different cytologic interpretations at the 2 sites in 42/87 (48%) cases (Fig 2; Table 2). In 15 cases with predominantly eosinophilic inflammation at 1 site (median eosinophils: 43%, range: 24–80%), the 2nd site was categorized as neutrophilic (92%) in 1, lymphocytic (20%) in 2, mixed eosinophilic (median eosinophils: 32%, range: 20–49%) and neutrophilic (median neutrophils: 14%, range: 11–63%) in 7, mixed lymphocytic (19%) and eosinophilic (21%) in 1, mixed neutrophilic (17%) and lymphocytic (20%) in 1, mixed eosinophilic (24–25%), neutrophilic (13–16%), and lymphocytic (13–18%) in 2, and hypercellular in 1. In 18 cases with predominantly neutrophilic inflammation at 1 site (median neutrophils: 63%, range: 13–89%), the 2nd site was mixed neutrophilic (20–52%) and eosinophilic (21–45%) in 8, mixed eosinophilic (36%) and lymphocytic (11%) in 1, mixed neutrophilic (16–58%), and lymphocytic (13–21%) in 4, mixed eosinophilic (48%), neutrophilic (26%), and lymphocytic (11%) in 1, and hypercellular in 4. In these cases with differing types of inflammation at separate lavage sites, cell counts varied by >2 fold between sites in 12/42 cases (29%). Inflammatory airway disease was diagnosed in 32, pneumonia in 6, and neoplasia in 1 case, and in 3 cases, a definitive diagnosis was not obtained.
Focal radiographic infiltrates were identified in 33 cases and BAL was collected at the site of focal radiographic change in 14 cases. In 8/14 cases, the same inflammatory interpretation was made at both sites. In 2 of the remaining 6 cases, 1 site was hypercellular while the site of focal radiographic infiltrates was neutrophilic or mixed lymphocytic and eosinophilic. In the other 4 cases, variable inflammatory responses were noted at the site of focal radiographic changes compared with the alternate site (Table 3).
|BAL Cytology: Focal Radiographic Infiltrate||BAL Cytology: Alternate Site|
|Neutrophilic and lymphocytic||Lymphocytic and eosinophilic|
|Neutrophilic and lymphocytic||Lymphocytic|
|Neutrophilic and eosinophilic||Neutrophilic|
|Lymphocytic||Lymphocytic and eosinophilic|
Of 8 cats without abnormalities on chest radiographs, cytologic evidence of inflammation was present in all. TNCC varied by >2 fold in 2/8 cases, and the inflammatory response was similar between sites in 2 cases (both neutrophilic and both hypercellular, respectively). In the remaining cats, 1 site demonstrated mixed inflammation, while the second was predominately eosinophilic in 5 and neutrophilic in 1.
In this study, 48% of cases had varying types of cellular inflammation at 2 bronchoalveolar lavage sites, despite the clinical diagnosis of diffuse lower respiratory disease and the lack of localizing radiographic changes in the majority of cats. In cases where 2 lavage sites yielded similar cytologic interpretation, cell counts varied by >2 fold in 31% of cases, suggesting perhaps that the severity of inflammation differed between sites. In contrast, in cases that had lavage performed at radiographically focal disease, interpretation of BAL inflammation was most often similar at the two sites (8/14, 57%), and cell counts varied in similar magnitude to other cases with multi-segment lavage. Therefore, contrary to our hypotheses, results of this study suggest that it is common to encounter variable total cell counts and inflammatory cell cytology in lavages performed at 2 lung sites, regardless of whether radiographic changes are focal or diffuse. These findings in conjunction with those reported in dogs indicate a complex inflammatory response in lower airway disease.
One possible explanation for results of this study would be variable technique for bronchoalveolar lavage. Lavage volume, dwell time within the bronchoalveolar segment, and percent fluid recovery could impact results, particularly of total cell count. However, because of the relatively standard size of cats examined here, similar fluid volumes were used across most cases, and similar fluid volume recovery was achieved. Additionally, in cats with different lavage volumes instilled at 2 separate sites, similar differences in cell counts and cytologic interpretation were identified as in cats with identical lavage volumes at each site. BAL produces a relatively predictable dilution of epithelial lining fluid of 1% (in humans) and 2.2–2.8% (in dogs). Sequential lavage of a single lung segment is known to alter BAL fluid cell constituents in horses, humans, and cats. Although a number of endoscopists were involved in the study, all utilized a standard instillation and aspiration technique. Therefore, it is considered unlikely that BAL technique resulted in significantly disparate cell counts or cytology at different lung segments in this study.
Another possible explanation for variable BAL findings is the use of radiographs to characterize diffuse versus focal disease, which are relatively insensitive for detection of lower respiratory tract disease. This is highlighted by the fact that 8 cats in this study were reported to have unremarkable radiographs, yet had clinical signs of respiratory disease and inflammation detected in BAL fluid. Thoracic CT provides much greater detail and would undoubtedly have provided superior definition of the presence or absence of pulmonary infiltrates. Bronchoscopic findings might have provided useful information for characterizing focal and diffuse disease; however, they were not included here because bronchoscopic findings are similar in pneumonia, inflammatory airway disease, and neoplastic diseases in cats. Bronchoscopic findings could favor collection of variable airway cytology if the endoscopist chose to perform a lavage in a visually abnormal area in comparison with a more normal-appearing airway. Bronchoscopic abnormalities might also inadvertently limit the acquisition of lavage at a focally abnormal region because it can be difficult to wedge the endoscope for lavage in a stenotic airway.
Finally, regional differences in BAL findings might reflect different pathologic processes within the lung, varied deposition of inciting factors, or focal variations in the inflammatory response within the feline lung. The most obvious explanation for different BAL findings would be a foreign body response; however, none were found here. Another possibility would be a focal pneumonia. One case examined here with variable BAL findings had a tracheo-esophageal fistula, and in 2 cases with pneumonia, airway sepsis was identified cytologically at one lavage site, but not at both, suggesting focal pneumonia. Another focal infection (ie, migrating parasitic larvae or heartworm infection) could potentially explain local airway eosinophilia. Fecal examinations and heartworm testing were inconsistently performed in cats examined here; however, no obvious cause for variable inflammation was identified for the remaining 84 cases.
Altered airway deposition of particles might lead to differing inflammatory responses. The right lung is more aligned with the trachea than is the left, as the left lobar bronchus branches at a greater angle than the right. It is possible that inhaled particulate matter preferentially enters the right lung to trigger focal inflammation, although cell count and cytology from the left and right lung did not differ in some cases examined here (data not shown). Regional disparity in the inflammatory response is an intriguing speculation. If lower airway disease in some cases examined here was triggered by inhalation of aeroallergens, it is difficult to explain why 1 lavage site would be solely eosinophilic and a 2nd site would lack eosinophils, as found in some instances here, although it could perhaps reflect regional differences in ventilation because of bronchoconstriction.
The value of obtaining total cell counts in BAL fluid and interpretation of counts has been debated, perhaps partly because processing of BAL fluid has not been standardized in human or veterinary medicine. Some endoscopists advocate straining BAL fluid through gauze to remove mucus before analysis or pelleting cells followed by resuspension for cytologic analysis. While this lessens clumping of cells in cytologic specimens, it can also result in loss of any cells adherent to or trapped in the mucus, which could impact both total and differential cell counts. Neither of these processing techniques is used by our laboratory, although it is possible that mucus in some samples could have influenced results. Hypercellular BAL samples with normal cell differentials were reported in 9 cases and without knowledge of an increased cell count, these samples would have been interpreted as within reference limits. Perhaps these hypercellular BAL samples with normal cell distribution should be considered normal; however, these cats had clinical signs of respiratory disease, diffuse radiographic infiltrates and cytologic evidence of inflammation at an alternate BAL site that confirmed lower airway disease. The potential value of BAL cell counts in assessment of lower respiratory tract disease requires further investigation.
The reference intervals used for normal BAL fluid cytology were established with single-site lavage,[1-3] and studies in naturally occurring and experimentally induced airway disease have also relied on cytologic assessment of a single lavage site to define the disease process.[1-6, 14, 15] This would appear to be reasonable because in healthy dogs, similar cytologic findings are reported for lavage of the left and right lung lobe, however, different BAL findings were reported in 37% of dogs with respiratory disease. In healthy horses, there was no significant difference in cellular composition of BAL fluid between the right and left lungs other than a significantly increased number of mast cells in the left lung. A different study found similar cytologic results for multisegment lavage in both healthy horses and horses with recurrent airway obstruction. However, similar studies have not been performed in healthy cats, and our study would suggest that assuming a uniform inflammatory response within the feline lung is not reasonable.
Feline asthma and bronchitis are commonly differentiated based on the finding of eosinophilic inflammation in the former and neutrophilic in the latter, given the lack of available pulmonary function tests or biomarkers to distinguish between the 2 disorders. It is interesting to note that horses with recurrent airway obstruction, a disorder similar to the human condition of asthma, have neutrophilic airway inflammation rather than eosinophilic inflammation. Certainly, our findings of varying types of inflammation in different lung segments of cats with inflammatory airway disease raise questions about the validity of using the inflammatory cell population within a single airway sample to define the etiology of disease or potentially to define response to treatment. Cytologic interpretation differed between lavage samples in 42/87 cases examined here, and if only a single lavage sample had been collected, a different clinical interpretation would have been made. For example, of 15 cases that had predominantly eosinophilic inflammation at 1 site, 4 lacked elevation of BAL eosinophil percentage at a 2nd site (Table 2). It cannot be determined which lavage more closely defines the clinical disease process in these cases with varying BAL cytology, and more studies are needed to refine interpretation of BAL fluid assessment in cats. This would be considered more important in a research setting because clinically, treatment of feline inflammatory airway disease (asthma or bronchitis) is based on a comprehensive assessment of the severity of clinical signs, physical examination abnormalities, and radiographic findings, in addition to BAL cytology.
This study found similar cytologic assessment in 8/14 cases that had 1 lavage performed at a site of focal radiographic infiltrates. One limitation of this study is the small number of cats that had BAL performed at a site of focal radiographic disease. Bronchoscopy is valuable because it allows both direct visualization and collection of a sample from a specific lung segment, and a prospective study of multi-segment BAL utilizing CT to define focal infiltrates would be of value. Another limitation is that referral cases examined here might not be representative of the majority of cats with lower airway disease.
This study has demonstrated that varying results are not unusual when performing multisegment bronchoalveolar lavage in cats, despite the presence of diffuse radiographic disease. Lavage of 2 lung segments may provide additional clinical information to define disease and to guide treatment. Use of both total cell counts and differential cell counts and cytologic assessment is recommended to obtain the most information on the character of disease, and caution is warranted when using BAL cytology from a single site to define the inflammatory response in cats with spontaneously occurring lower respiratory tract disease. Further study is needed to determine whether it is essential to sample a site with focal radiographic infiltrates.
Supported by the Bailey Wrigley Fund, UC Davis.
Conflict of Interest: Authors disclose no conflict of interest.
60003VB, Karl Storz Veterinary Endoscopy, Goleta CA
Olympus BF 3C160, Olympus Corporation, Melville, NY
Anaerobe Systems, Morgan Hill, CA
UC Davis Media Room, Davis, CA
Advia 120, Siemens, Deerfield, IL
Cytospin3, ThermoShandon, Pittsburgh, PA
GraphPad Prism version 5, San Diego, CA
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- 23Evaluation of variations in bronchoalveolar lavage fluid in horses with recurrent airway obstruction. Am J Vet Res 2011;72:838–842., , , .