Bilateral bronchoalveolar lavage cytology profiles in a warmblood horse population during a 1‐year period
INTRODUCTION The bronchoalveolar lavage (BAL) cytology profile is highly influenced by the stabling environment as well as the endoscopic and BAL cytology techniques, , , , , , , and asymptomatic stabled horses often have BAL neutrophil proportions above 5%. , Bronchoalveolar lavage entails the sampling of pulmonary epithelial lining fluid from the terminal bronchioles and alveoli. More than 40 years after its introduction, no international standardized method for performing BAL in horses, including BAL volume, lung site, and BAL cytology reference intervals based on scientific studies has reached global agreement. Nevertheless, an endoscopic examination and BAL cytology is considered an invaluable diagnostic technique for the diagnosis of asthma in horses and for further differentiation between mild‐moderate asthma and the severe form of asthma. , The 2 BAL reference intervals utilized for mild‐moderate asthma in horses are published in the ACVIM consensus statement, the “restrictive” with a differential cell count of; neutrophils ≥5%, eosinophils ≥1%, and mast cells ≥2%, and the “alternative” with a differential cell count of; >10% neutrophils, >5% eosinophils and >5% mast cells. Traditionally, the collection of BAL fluid is performed either by blindly placing a BAL tube or as part of an endoscopic examination, collecting the BAL fluid through the working channel of the endoscope, often aiming for the right lung. An explanation for choosing the right lung has been a more straight position of the right main stem bronchus, thus favoring sampling of the right lung. , There are differences between BAL sampling of the right or left lung site both with regard to the asthma diagnosis, , , , but also with regard to cellular components. , , There are chronic histological changes in the lung tissue of healthy horses with >5% neutrophils in BAL cytology, emphasizing the clinical relevance of low grade airway inflammation (AI). The objectives of the present study were to determine whether the lung site sampled would influence the horse's BAL cytology profile, and if a pooled BAL sample would be superior with regard to accuracy of BAL cytology and AI in a cohort of healthy and subclinical asthma warmblood horses. We hypothesized, that (1) a pooled BAL cytology sample from both lungs would be more accurate to BAL cytology from either right or left lung with regard to BAL cytology and AI. Thus, the pooled BAL sample would equalize any cytological differences between the separate lungs and consequently be representative of the individual horse's general lung condition. (2) BAL cytology and AI in the right and left lung, would be consistent in a 1‐year period. Airway inflammation was defined by applying the asthma reference intervals to the BAL differential cell count results of this cohort of asymptomatic horses.   MATERIALS AND METHODS 2.1 Study design and horses included This study was conducted as a cross‐sectional study with follow up, a sample size of 67 BAL cytology samples (right, left, pooled) was estimated based on 80% power (1‐β), a significance level (α) of .05 (3 dependent groups, SigmaPlot, 15.0), based on a limit of detection of 2.5% and a SD of 8. All included horses were located at the same facility in 4 identical stables with same management strategies, were bedded on wood shavings and fed hay or haylage, were ridden exercised 5 days a week and turned out on pasture for 4 to 9 hours a day everyday all year round. The horses were warmblood geldings, except 1 Friesian in year 2021 and 2 in year 2022. The horses had a mean age of 13.6 (±5.4) years for the 59 horses included in 2021, a mean age of 13.6 (±5.6) years for the 70 horses included in year 2022 (Study part 1), and a mean age of 13.6 (±5.6) in year 2021 for the 53 horses included in both years (Study part 2). Inclusion criteria were that the horses should be clinically sound, evaluated by clinical examination with focus on the respiratory system before the endoscopic procedures. The airway sampling was part of a yearly health examination, performed in 2 days each year in July. The Ethical Committee of University of Copenhagen Large Animal Teaching Hospital granted the ethical approval (permit no. 2020‐013) for the endoscopic procedures on the horses. 2.2 Endoscopic examination including bronchoalveolar lavage All horses were sedated with a standard sedation of 0.6 mL detomidine (Domosedan 10 mg/mL, Orion Pharma, Denmark) before the procedure, except the Friesians that were more sensitive to sedation and only received half the dose. The horses were placed in a stock and the nostrils were cleaned with soaked gauzes before the endoscopy. The endoscope (3000 mm length, 10.4 mm sheath diameter, Karl Storz 60 130 PKS G28‐300, 78532 Tuttlingen, Germany) was inserted via the ventral meatus to the nasopharynx and further down the trachea. The endoscope was first introduced to the right stem bronchus until the endoscope wedged into a bronchus located in the caudo‐dorsal lung field. A total volume of 250 mL sterile isotonic saline (0.9% Braun, Germany) was infused and immediately manually aspirated. The endoscope was flushed with 20 mL of sterile saline and 5 mL of air to before being pulled back to the carina and the procedure was then repeated in the left lung. From each separate lung site, the total aspirated volume was noted and 8 mL of the sample were transferred into an EDTA tube for cytology. In 2022, further 10 mL from each lung were pooled and 8 mL was transferred to an EDTA tube for cytology. Between horses, the endoscope and biopsy channel were cleaned using sterile saline and chlorhexidine (0.2%, Glostrup Pharmacy, Denmark). 2.3 Cytological analysis Cytospin smears were prepared within 30 minutes by transferring 200 μL of each BAL fluid EDTA sample to the funnel of a cytospin and centrifuged at 93×g for 8 minutes (StatSpin Cytofuge 12, USA). The cytospin samples were then fixed in methanol (ChemSolute, Th. Geyer, Denmark) and stained using May‐Grünwald‐Giemsa (Merck, Germany). Each cytospin slide was examined using a bright‐field microscope at 630× magnification. The examination included a differential cell count based on 500 cells, including alveolar macrophages, lymphocytes, neutrophils, eosinophils and mast cells, distributed over 5 equally distributed areas in the slide. 2.4 Case definition The included horses did not have any abnormalities detected on a clinical examination and an anamnesis of coughing or poor performance was not available. Based on this limited information, the BAL cytological results of each sample categorized the sample as control or with AI based on reference intervals published in the ACVIM consensus statement. The 2 sets of reference intervals for equine asthma that can be applied to BAL cytology samples are as follows: (1) neutrophils ≥5%, eosinophils ≥1%, and mast cells ≥2%, referred to as restrictive for this study and (2) >10% neutrophils, >5% eosinophils and >5% mast cells, referred to as alternative for this study. A final AI diagnosis for the individual horse was applied if 1 or both lungs had values above the BAL cytology reference intervals. 2.5 Statistical analysis Data normality was checked using the Shapiro‐Wilk test and as data were not normally distributed, nonparametric tests for associations between continuous outcome variables (BAL volume aspirated in percentage of volume aspirated, BAL mast cells, BAL neutrophils and BAL eosinophils for respectively the left, right and pooled BAL [only for 2022] samples as well as between the different sampling years 2021 and 2022) were applied. Data are expressed as medians with interquartile ranges. A mixed effect model with horse as random effect was applied to explore the difference and effect size (ES) in BAL variables between year 2021 and 2022. A Wilcoxon's signed rank test and ES was applied to explore difference in BAL variables between left and right lung side for year 2021. A Friedman's test and Kendall's (W) effect measure was applied to explore difference between BAL variables (left, right, pooled) for year 2022. Spearman's rank correlation (rho) was used to evaluate the correlation in BAL cytology for the left vs right lung. Finally, the accuracy of the different lung site to detect AI were evaluated against each of the alternative and restrictive reference intervals. Measures of accuracy were evaluated by sensitivity (Se), specificity (Sp), area under the receiver operating characteristic curve (AUC), and a McNemar test. All analyses were assessed at significance level of <.05.   Study design and horses included This study was conducted as a cross‐sectional study with follow up, a sample size of 67 BAL cytology samples (right, left, pooled) was estimated based on 80% power (1‐β), a significance level (α) of .05 (3 dependent groups, SigmaPlot, 15.0), based on a limit of detection of 2.5% and a SD of 8. All included horses were located at the same facility in 4 identical stables with same management strategies, were bedded on wood shavings and fed hay or haylage, were ridden exercised 5 days a week and turned out on pasture for 4 to 9 hours a day everyday all year round. The horses were warmblood geldings, except 1 Friesian in year 2021 and 2 in year 2022. The horses had a mean age of 13.6 (±5.4) years for the 59 horses included in 2021, a mean age of 13.6 (±5.6) years for the 70 horses included in year 2022 (Study part 1), and a mean age of 13.6 (±5.6) in year 2021 for the 53 horses included in both years (Study part 2). Inclusion criteria were that the horses should be clinically sound, evaluated by clinical examination with focus on the respiratory system before the endoscopic procedures. The airway sampling was part of a yearly health examination, performed in 2 days each year in July. The Ethical Committee of University of Copenhagen Large Animal Teaching Hospital granted the ethical approval (permit no. 2020‐013) for the endoscopic procedures on the horses.   Endoscopic examination including bronchoalveolar lavage All horses were sedated with a standard sedation of 0.6 mL detomidine (Domosedan 10 mg/mL, Orion Pharma, Denmark) before the procedure, except the Friesians that were more sensitive to sedation and only received half the dose. The horses were placed in a stock and the nostrils were cleaned with soaked gauzes before the endoscopy. The endoscope (3000 mm length, 10.4 mm sheath diameter, Karl Storz 60 130 PKS G28‐300, 78532 Tuttlingen, Germany) was inserted via the ventral meatus to the nasopharynx and further down the trachea. The endoscope was first introduced to the right stem bronchus until the endoscope wedged into a bronchus located in the caudo‐dorsal lung field. A total volume of 250 mL sterile isotonic saline (0.9% Braun, Germany) was infused and immediately manually aspirated. The endoscope was flushed with 20 mL of sterile saline and 5 mL of air to before being pulled back to the carina and the procedure was then repeated in the left lung. From each separate lung site, the total aspirated volume was noted and 8 mL of the sample were transferred into an EDTA tube for cytology. In 2022, further 10 mL from each lung were pooled and 8 mL was transferred to an EDTA tube for cytology. Between horses, the endoscope and biopsy channel were cleaned using sterile saline and chlorhexidine (0.2%, Glostrup Pharmacy, Denmark).   Cytological analysis Cytospin smears were prepared within 30 minutes by transferring 200 μL of each BAL fluid EDTA sample to the funnel of a cytospin and centrifuged at 93×g for 8 minutes (StatSpin Cytofuge 12, USA). The cytospin samples were then fixed in methanol (ChemSolute, Th. Geyer, Denmark) and stained using May‐Grünwald‐Giemsa (Merck, Germany). Each cytospin slide was examined using a bright‐field microscope at 630× magnification. The examination included a differential cell count based on 500 cells, including alveolar macrophages, lymphocytes, neutrophils, eosinophils and mast cells, distributed over 5 equally distributed areas in the slide.   Case definition The included horses did not have any abnormalities detected on a clinical examination and an anamnesis of coughing or poor performance was not available. Based on this limited information, the BAL cytological results of each sample categorized the sample as control or with AI based on reference intervals published in the ACVIM consensus statement. The 2 sets of reference intervals for equine asthma that can be applied to BAL cytology samples are as follows: (1) neutrophils ≥5%, eosinophils ≥1%, and mast cells ≥2%, referred to as restrictive for this study and (2) >10% neutrophils, >5% eosinophils and >5% mast cells, referred to as alternative for this study. A final AI diagnosis for the individual horse was applied if 1 or both lungs had values above the BAL cytology reference intervals.   Statistical analysis Data normality was checked using the Shapiro‐Wilk test and as data were not normally distributed, nonparametric tests for associations between continuous outcome variables (BAL volume aspirated in percentage of volume aspirated, BAL mast cells, BAL neutrophils and BAL eosinophils for respectively the left, right and pooled BAL [only for 2022] samples as well as between the different sampling years 2021 and 2022) were applied. Data are expressed as medians with interquartile ranges. A mixed effect model with horse as random effect was applied to explore the difference and effect size (ES) in BAL variables between year 2021 and 2022. A Wilcoxon's signed rank test and ES was applied to explore difference in BAL variables between left and right lung side for year 2021. A Friedman's test and Kendall's (W) effect measure was applied to explore difference between BAL variables (left, right, pooled) for year 2022. Spearman's rank correlation (rho) was used to evaluate the correlation in BAL cytology for the left vs right lung. Finally, the accuracy of the different lung site to detect AI were evaluated against each of the alternative and restrictive reference intervals. Measures of accuracy were evaluated by sensitivity (Se), specificity (Sp), area under the receiver operating characteristic curve (AUC), and a McNemar test. All analyses were assessed at significance level of <.05.   RESULTS 3.1 Study cohort In 2021, 59 of 67 eligible horses were included, 3 horses had only unilateral BAL performed because of coughing, and inability to secure the endoscope and 5 because of insufficient number of cells at the cytological evaluation. In 2022, 70 horses out of 74 eligible horses were enrolled, of which 4 horses were excluded due coughing and inability to secure the endoscope. For Study part 2; 53 horses had repeated BAL samples for both years and were included in the longitudinal study. The total aspirated BAL fluid volume was 97 mL ± 30 mL (40% ± 13%) from the right lung and 107 mL ± 40 mL (44% ± 17%) from the left lung, and no significant difference was found in volume aspirated between lungs ( P = .08) (BAL volume aspirated, data only available for 2022). 3.2 Bronchoalveolar lavage cell count and correlations for right, left, and pooled samples within years In 2021, a weak correlation between lungs for the BAL mast cell count was found, and the median BAL mast cell count was significantly higher in the left lung relative to the right lung ( P < .001, ES 0.61). No significant difference in median BAL neutrophil cell count ( P = .43, ES 0.20) and a moderate correlation was found between right and left lung. No significant difference in median BAL eosinophil cell count ( P = .59, ES 0.12) and a strong correlation was found between right and left lung (Tables and ). In 2022, the median BAL mast cell count was again significantly higher in the left lung compared to both the right lung ( P < .001) and the pooled BAL samples ( P < .001). Further, no significant difference was found between the right lung and pooled sample ( P = .98), an overall high level of agreement was found (W = 3.51). A moderate correlation was found for BAL mast cell count between right and left BAL samples. For BAL neutrophils, no significant difference in median cell count and a strong correlation was found between the right and left BAL samples. For BAL eosinophils, no significant difference in cell count was found between the right, left or pooled BAL samples, but the correlation for BAL eosinophil cell count between right and left BAL samples were only negligible (Tables and ). 3.3 Categorization of AI , control and final AI diagnosis based on bronchoalveolar lavage cytology from right, left, and pooled samples In year 2021, 17% (10/59) and 66% (39/59) of the included horses had AI based on BAL cytology from the right lung, while 27% (16/59) and 83% (49/59) horses had AI based on BAL cytology from the left lung when alternative and restrictive reference intervals were applied, respectively. According to final AI diagnosis, 32% (19/59) and 92% (54/59) horses had AI when alternative and restrictive reference intervals were applied, meaning that a BAL cytology result for 1 or more cell types were above the reference intervals in either the right, the left lung or both lung sites. In year 2022 26% (18/70) and 81% (57/70) of the included horses had AI based on BAL cytology from the right lung, while 30% (21/70) and 87% (61/70) of the horses had AI based on BAL cytology from the left lung and further 20% (14/70) and 80% (56/70) of the horses had AI based on the pooled BAL cytology results. According to final AI diagnosis, 36% (25/70) and 91% (64/70) of the horses had AI for alternative and restrictive BAL reference intervals, respectively (Table ). 3.4 Accuracy of AI in right, left, and pooled bronchoalveolar lavage samples For year 2022 applying the alternative reference intervals for the final AI diagnosis as gold standard; BAL from the left lung had the highest sensitivity of 84%, a specificity of 100%, an AUC of 0.92 and was not significant different (McNemar, exact 0.13). BAL from right lung and the pooled BAL sample both had a lower sensitivity and a significant difference was found when compared to final AI diagnosis (Table ). 3.5 Bronchoalveolar lavage cell count and correlations for right, left, and pooled samples between years Repeated BAL cytology samples for 53 horses from right and left lung in a 1‐year period, showed a higher BAL mast cell count in 2022 compared to year 2021 for both right and left lung (median 1.1 [0.7‐1.5] vs 2.3 [1.6‐3.1]; P < .001, median 1.6 [0.6‐3‐4] vs 3.1 [2‐3.8]; P = .02, overall ES 0.49). The BAL eosinophils cell count from both lungs were higher in 2021 compared to 2022 (left 0.2 [0.1‐1.4] vs 0 [0‐0.1], right 0.3 [0.1‐10.2] vs 0 [0‐0.1] P < .001, overall ES 0.30). A significant increase in neutrophils was found for the right lung but not for left lung (median 3.6 [2.4‐5.2] vs 3.6 [1.5‐7.8]; P = .02, median 3.2 [1.9‐6] vs 3.8 [1.4‐7.7]; P = .78, overall ES 0.35) between year 2021 and 2022. Eight horses had AI repeatedly in 2021 and 2022 according to the alternative BAL reference intervals. Between 2021 and 2022, the localization of AI changed from bilateral to unilateral in 13% (1/8) of horses, and likewise 38% (3/8) of horses changed from being unilateral to bilateral affected. Applying the restrictive BAL reference intervals, 41 horses had AI repeatedly in both years, where 7% (3/41) of these changed localization of AI from bilateral to unilateral and 32% (13/41) changed from unilateral to bilateral AI during this period. In 2% (1/41) of horses, the location of AI switched from affecting 1 lung to the other.   Study cohort In 2021, 59 of 67 eligible horses were included, 3 horses had only unilateral BAL performed because of coughing, and inability to secure the endoscope and 5 because of insufficient number of cells at the cytological evaluation. In 2022, 70 horses out of 74 eligible horses were enrolled, of which 4 horses were excluded due coughing and inability to secure the endoscope. For Study part 2; 53 horses had repeated BAL samples for both years and were included in the longitudinal study. The total aspirated BAL fluid volume was 97 mL ± 30 mL (40% ± 13%) from the right lung and 107 mL ± 40 mL (44% ± 17%) from the left lung, and no significant difference was found in volume aspirated between lungs ( P = .08) (BAL volume aspirated, data only available for 2022).   Bronchoalveolar lavage cell count and correlations for right, left, and pooled samples within years In 2021, a weak correlation between lungs for the BAL mast cell count was found, and the median BAL mast cell count was significantly higher in the left lung relative to the right lung ( P < .001, ES 0.61). No significant difference in median BAL neutrophil cell count ( P = .43, ES 0.20) and a moderate correlation was found between right and left lung. No significant difference in median BAL eosinophil cell count ( P = .59, ES 0.12) and a strong correlation was found between right and left lung (Tables and ). In 2022, the median BAL mast cell count was again significantly higher in the left lung compared to both the right lung ( P < .001) and the pooled BAL samples ( P < .001). Further, no significant difference was found between the right lung and pooled sample ( P = .98), an overall high level of agreement was found (W = 3.51). A moderate correlation was found for BAL mast cell count between right and left BAL samples. For BAL neutrophils, no significant difference in median cell count and a strong correlation was found between the right and left BAL samples. For BAL eosinophils, no significant difference in cell count was found between the right, left or pooled BAL samples, but the correlation for BAL eosinophil cell count between right and left BAL samples were only negligible (Tables and ).   Categorization of AI , control and final AI diagnosis based on bronchoalveolar lavage cytology from right, left, and pooled samples In year 2021, 17% (10/59) and 66% (39/59) of the included horses had AI based on BAL cytology from the right lung, while 27% (16/59) and 83% (49/59) horses had AI based on BAL cytology from the left lung when alternative and restrictive reference intervals were applied, respectively. According to final AI diagnosis, 32% (19/59) and 92% (54/59) horses had AI when alternative and restrictive reference intervals were applied, meaning that a BAL cytology result for 1 or more cell types were above the reference intervals in either the right, the left lung or both lung sites. In year 2022 26% (18/70) and 81% (57/70) of the included horses had AI based on BAL cytology from the right lung, while 30% (21/70) and 87% (61/70) of the horses had AI based on BAL cytology from the left lung and further 20% (14/70) and 80% (56/70) of the horses had AI based on the pooled BAL cytology results. According to final AI diagnosis, 36% (25/70) and 91% (64/70) of the horses had AI for alternative and restrictive BAL reference intervals, respectively (Table ).   Accuracy of AI in right, left, and pooled bronchoalveolar lavage samples For year 2022 applying the alternative reference intervals for the final AI diagnosis as gold standard; BAL from the left lung had the highest sensitivity of 84%, a specificity of 100%, an AUC of 0.92 and was not significant different (McNemar, exact 0.13). BAL from right lung and the pooled BAL sample both had a lower sensitivity and a significant difference was found when compared to final AI diagnosis (Table ).   Bronchoalveolar lavage cell count and correlations for right, left, and pooled samples between years Repeated BAL cytology samples for 53 horses from right and left lung in a 1‐year period, showed a higher BAL mast cell count in 2022 compared to year 2021 for both right and left lung (median 1.1 [0.7‐1.5] vs 2.3 [1.6‐3.1]; P < .001, median 1.6 [0.6‐3‐4] vs 3.1 [2‐3.8]; P = .02, overall ES 0.49). The BAL eosinophils cell count from both lungs were higher in 2021 compared to 2022 (left 0.2 [0.1‐1.4] vs 0 [0‐0.1], right 0.3 [0.1‐10.2] vs 0 [0‐0.1] P < .001, overall ES 0.30). A significant increase in neutrophils was found for the right lung but not for left lung (median 3.6 [2.4‐5.2] vs 3.6 [1.5‐7.8]; P = .02, median 3.2 [1.9‐6] vs 3.8 [1.4‐7.7]; P = .78, overall ES 0.35) between year 2021 and 2022. Eight horses had AI repeatedly in 2021 and 2022 according to the alternative BAL reference intervals. Between 2021 and 2022, the localization of AI changed from bilateral to unilateral in 13% (1/8) of horses, and likewise 38% (3/8) of horses changed from being unilateral to bilateral affected. Applying the restrictive BAL reference intervals, 41 horses had AI repeatedly in both years, where 7% (3/41) of these changed localization of AI from bilateral to unilateral and 32% (13/41) changed from unilateral to bilateral AI during this period. In 2% (1/41) of horses, the location of AI switched from affecting 1 lung to the other.   DISCUSSION This prospective study evaluates BAL cytology from both right and left lung in a large cohort of stabled warmblood horses over a 1‐year period. The cohort included, was a homogenous group of horses with regard to stabling and management, all stabled in well‐ventilated stables, on low dust bedding and mostly fed haylage, environment recommended for asthmatic horses. , , , Results of this study from both year 2021 and 2022 found that the BAL cytology from the left lung had the highest accuracy compared to the final diagnosis of the individual horse regardless of test applied. When applying the alternative reference intervals to the pooled BAL samples, the authors found the lowest accuracy compared to the final AI diagnosis. A final AI diagnosis was applied if 1 of the lungs had BAL cytology differential cell count above the reference intervals published in the ACVIM consensus statement. The prevalence of horses presented with only unilateral localized AI was 14% to 16% in both years. Of these horses, the pooled sample was only able to correctly classify AI in 3 out of 15 and 3 out of 12 cases applying the restrictive and alternative reference intervals. In contrast, a study by Hermange et al found the pooled BAL samples to be representative of the BAL samples from right and left lung site in a diseased group of horses at a referral center. As this group only includes diseased horses, as well as including another methodical application of a lower reference interval for the pooled BAL samples the 2 studies are difficult to compare. Comparison of the BAL cytology results between right and left lung revealed no difference for macrophages, lymphocytes, neutrophils and eosinophils and a strong correlation for BAL neutrophils between lungs for both years, but an only weak to moderate correlation for BAL eosinophils, counting for both year 2021 and 2022. This is in agreement with most of the current literature investigating cytological differences between right and left lung, , , , but contrary to a large French study, including clinically healthy racing Trotters, as Depecker et al found significant higher BAL neutrophil percentages in the right lung compared to the left. Results from both years in this study, showed higher BAL mast cell counts in the left lung in comparison to the right and pooled BAL cytology sample, a result that had been reported, but conflicting results has also been published. The anatomical design of the trachea and main bronchi, creating a straight position of the right main stem bronchus and thus favoring sampling of the right lung , it could be speculated to cause a larger amount of environmental dust to be inhaled into the right lung lobe, but this has yet to be proven. This theory of the increased amount of dust in the right lung lobe, further conflicts with the findings of a higher BAL mast cell count in the left long lobe compare to the right. The variation in agreement of BAL cytology cell count between studies could also be explained by volume infused for BAL, slide variation, staining method and technique, number of cells counted as well as experience of enumerators. , , Most variation will be minimized or even eliminated by applying a standardized BAL cytology protocol, including BAL procedure and subsequently laboratory methods. Even though the accuracy between lungs found in this study seems promising, large variation in especially BAL neutrophils was found for individual horses, for example, a horse had a BAL neutrophilic cell count of right, left and pooled 31%, 8% and 28%, respectively. The effect size found in this study show that the horse factors account for the largest variation, which has also previously been reported. As neutrophilic mild‐moderate asthma is the most common asthma subtype in the literature, , , a reliable BAL count of this cell types is important. The longitudinal study including 53 horses with repeated BAL samples showed cytological variation between BAL samples collected in year 2021 and 2022, as the mast cell count decreased, and eosinophil cell count increased. The differences in these 2 cell populations might play a role in the number of horses in which the localization of AI changed from bilateral to unilateral, or vice versa, over the 1‐year study period. In only a single horse, AI switched from affecting the left lung to the right lung, according to restrictive reference intervals. Longitudinal studies on BAL cytology changes have previously been evaluated; a study evaluated BAL cytology in a 3 3 month period, with no difference. Studies evaluating BAL cytology differences in 6 to 18 month periods, approximating summer and winter, found a seasonal variation in BAL neutrophils, , although only statistically significant in the shorter study. It could be hypothesized, that the longitudinal cytological differences seen in this study, could be explained by difference in exposure to dust particles, weather conditions or subclinical viral infections, as the endoscopy and BAL sampling was done by the same person (JF) and the cytological method was consistent between years. Further, the overall stabling and management factors were kept constant over the 1‐year period. The variation in BAL cytology seen with lung and time in this study might have an impact if an asthma diagnosis is attempted with only a 1‐sided BAL. The consequence of performing only a 1‐sided BAL is a risk of underdiagnosing asthma, which will lead to a lack of a proper treatment strategy as well as management improvement, and as an extreme consequence, an increased risk of developing severe equine asthma. The longitudinal variation in AI, along with the significant difference in BAL mast cell count between lungs, further supports the suggestion of evaluating both individual lungs when a diagnosis of equine asthma is suspected. The volume of BAL fluid retrieved, in the 2022 cohort, was not significantly different between the lungs. Results from previous studies are ambiguous, as 1 study showed, like our study, no difference between lungs and no correlation with disease, while another study showed a lower aspirated volume in severe asthma horses. Limitations of the present study include the lack of diseased horses, as the horses included performed to the owner's expectations and were clinically healthy, and thereby we gained no knowledge about the behavior of the BAL cytology in more diseased individuals. A diagnosis of asthma requires an anamnesis, and clinical signs in addition to BAL cytology, whereas the included horses had only BAL cytological evidence of AI. In the racehorse population BAL cytology and tracheal mucus score above reference intervals has been associated with a decreased likelihood of winning, , justifying the application of BAL cytology without clinical sign in this specific horses population. Evidence how AI, without clinical signs, affect warmblood horse population is sparse. , Only a May‐Grünwald‐Giemsa staining was included for the BAL cytology, preferably a toluidine blue could have been added for improved mast cell reliability. , In conclusion, the location of AI in the individual horse varied with time, leading to some horses changing from unilateral to bilateral AI, or vice versa. Additional results from this study indicated that a pooled BAL sample was the least accurate sample compared to BAL samples obtained from either right or left lung, further; the left lung had the highest accuracy in relation to a final diagnosis. The accuracy, including both AUC and a McNemar test indicated a more correct classification of AI utilizing BAL from the left lung. Compiling to the restrictive reference intervals, one must further have in mind the high risk of a false negative result (4%‐25%) when evaluating the BAL sample from only 1 lung. Based on the overall results of this and previous studies, , , , , the authors recommend cytological evaluation of both individual lungs for the evaluation of especially mild‐moderate asthma. The mild‐moderate asthma diagnosis in particular, can be present without coughing and thereby a mild‐moderate asthmatic horse presenting for poor performance can be wrongly cleared by a unilateral BAL. A 2‐sided BAL inherently comes with some disadvantages, as preferably a 3 m endoscope should be available for the procedure, alternatively a short 1.5 m endoscope for guidance of an otherwise blindly placed BAL tube could be used. This comes with an increased cost for the owner.   Authors declare no conflict of interest.   Authors declare no off‐label use of antimicrobials.   Approved by the University of Copenhagen, Large Animal Teaching Hospital Ethical Committee (permit number 2020‐013).   Authors declare human ethics approval was not needed for this study.   