Article
Cytological analysis of bronchoalveolar lavage fluid acquired by bronchoscopy in healthy ferrets: A pilot study Marjorie Bercier, Isabelle Langlois, Marilyn Dunn, Pierre Hélie, Patrick Burns, Carolyn Gara-Boivin
Abstract The objective of this study was to investigate the normal cytological evaluation of bronchoalveolar lavage (BAL) fluid in healthy adult ferrets (N = 12). These ferrets underwent bronchoscopy and BAL using sterile saline [1.5 mL/kg body weight (BW)]. Percentage of fluid recovered, total leukocyte count, differential leukocyte count, and cell count of the epithelial lining fluid (ELF) were determined. The mean percentage of lavage volume recovered from the right lung and left lung were 67.8 6 14.9% and 69.7 6 20.0%, respectively. Gender (P = 0.12) and weight (P = 0.17) did not significantly affect the mean percentage of recovered volume. The mean percentage of recovered volume (P = 0.47) and the mean leukocyte count (P = 0.17) from the right and left lung were not significantly different. Macrophages were the main leukocyte component of the lavages, followed by neutrophils, lymphocytes, and eosinophils. The mean proportion of ELF in BAL fluid was 9.3 6 3.7% v/v. Bronchoscopy is clinically useful for collecting good quality BAL samples for cytological analysis in ferrets. The leucocyte differential was established, which may help veterinarians to make better clinical decisions when treating respiratory disease. Further studies are required with a larger group in order to establish the healthy reference intervals for BAL values in ferrets.
Résumé L’objectif de la présente étude était d’examiner l’évaluation cytologique normale de fluide de lavage broncho-alvéolaire (LAB) de furets adultes en santé (N = 12). Ces furets ont subi ne bronchoscopie et un LAB avec de la saline stérile [1,5 mL/kg de poids corporel (PC)]. Le pourcentage de fluide recouvré, le nombre total de leucocytes, un comptage différentiel de leucocytes, et un compte cellulaire du liquide sécrété par l’épithélium respiratoire ont été déterminés. Les pourcentages moyens de volume de lavage recouvrés du poumon droit et du poumon gauche étaient de 67,8 6 14,9 % et 69,7 6 20,0 %, respectivement. Le sexe (P = 0,12) et le poids (P = 0,17) n’ont pas affecté significativement le pourcentage moyen du volume recouvré. Le pourcentage moyen de volume recouvré (P = 0,47) et de comptage leucocytaire moyen (P = 0,17) provenant du poumon droit et du poumon gauche n’étaient pas significativement différents. Les macrophages étaient les principaux leucocytes retrouvés dans les lavages, suivis par les neutrophiles, lymphocytes et éosinophiles. La proportion moyenne d’ELF dans le fluide de LBA était de 9,3 6 3,7 % v/v. La bronchoscopie est utile cliniquement pour prélever des échantillons de LAB de bonne qualité pour l’examen cytologique chez les furets. Le différentiel des leucocytes a été établi, ce qui pourrait aider les vétérinaires à prendre de meilleures décisions cliniques lors du traitement de maladies respiratoires. Des études supplémentaires sont requises avec un plus grand groupe afin d’établir les intervalles des valeurs de référence des LAB de furets en santé. (Traduit par Docteur Serge Messier)
Introduction Respiratory pathologies are relatively common in ferrets. Such pathologies include influenza; asthma; infectious and non-infectious pneumonias, such as endogenous lipid pneumonia; canine distemper virus; and neoplasia (1). From 2002 to 2012, 6% of all ferrets presented to our teaching hospital displayed respiratory signs. Considering that there are an estimated 10 million ferrets in North America and that they are increasing in popularity, respiratory disease may affect a large number of these animals (2). Bronchoalveolar lavage (BAL) is a diagnostic procedure used in human and veterinary medicine to evaluate the cellularity of the lower respiratory tract (3). The leukocyte differential and mor-
phology can indicate an inflammatory, infectious, degenerative, or neoplastic process. Reference values are available for rabbits, dogs, cats, and horses (4–7), but are lacking for ferrets. Considering that there is a substantial variation among species in BAL fluid cytology from healthy animals (4–7), this may also be true in ferrets and such information becomes a necessity in disease investigation. Bronchoalveolar lavage (BAL) fluid is composed of 3 distinct components: the epithelial lining fluid (ELF), the cellular component of the epithelial lining fluid, and the retrieved amount of saline, the latter of which varies greatly (8). The epithelial lining fluid is defined as the liquid lining the alveoli and the airways, which contains the total leukocyte count measured in the BAL (9,10). The concentration of cellular components in the BAL fluid depends on the concentration
Department of Clinical Sciences (Bercier, Langlois, Dunn, Burns) and Department of Pathology and Microbiology (Gara-Boivin, Hélie), Faculty of Veterinary Medicine, University of Montreal, St. Hyacinthe, Quebec J2S 2M2. Address all correspondence to Dr. Patrick Burns; telephone: 144(0)131 650 7650; fax: 144(0)131 650 7652; e-mail: [email protected] Dr. Burns’ current address is Royal (Dick) School of Veterinary Studies, Easter Bush Campus, Midlothian EH25 9RG, United Kingdom. Dr. Bercier’s present address is University of Florida, College of Veterinary Medicine, 2015 SW 16th Avenue, Gainesville, Florida 32610, USA. Received April 15, 2015. Accepted September 8, 2015. 74
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of cellular components in the ELF and the amount of dilution of the ELF in the saline retrieved, i.e., the proportion of ELF in the sample of BAL fluid (11). In light of this statement, intra-species quantitative comparison of total leukocyte count in BAL samples may not be accurate. In an attempt to standardize these measures, urea has been used as a marker of dilution in human medicine and has also been evaluated in feline, canine, and equine medicine (8–11). Theoretically, the plasmatic urea concentration is equal to the urea concentration in the ELF, since it is a small molecule that diffuses freely throughout the body (8). Using a simple dilution technique, urea can be used as a marker of dilution to standardize the apparent volume and cellular concentration of ELF in humans, horses, dogs, and cats (8–11). Despite some limitations, this technique is known to reduce error in interpreting absolute cell count in BALs. In the current literature, 1 study compared absolute cell counts of BAL fluid in ferrets acutely exposed to either ozone or filtered air (12). The BAL samples were not collected using bronchoscopy, however, which is known to yield better quality samples than a blind BAL (3). Furthermore, the experimental conditions were artificially induced and were therefore not representative of the domestic ferret population. There are no reference values available for the cytological evaluation of BAL fluid in healthy domestic ferrets. There are no published data describing the systematic bronchoscopic examination of the respiratory tract of a ferret. Finally, urea has not been used as a marker of dilution for estimating ELF in ferrets. Therefore, the objectives of this study were to investigate the cytologic leucocyte differential of bronchoalveolar lavage (BAL) in healthy ferrets using bronchoscopy, to describe the bronchoscopic technique used for sampling, and to standardize the analysis of cellular components of ELF in healthy ferrets using a urea-dilution technique.
Materials and methods Animals Twelve healthy, 6-month-old adult ferrets (Mustela putorius furo), 6 males and 6 females, were used in this study. Body weight varied from 0.64 to 1.46 kg (mean 1/2 SD: 1.04 1/2 0.08 kg). All ferrets came from a breeding farm (Marshall Bio Resources, North Rose, New York, USA). After arriving at the Faculty of Veterinary Medicine, all ferrets were allowed 7 to 9 d to acclimatize. Ferrets were housed individually in stainless steel cages, with a standard paper litter, blankets, and hiding boxes. The ferrets were fed a commercial pelleted diet (Premium Ferret Diet; Marshall Pet Products, Wolcott, New York, USA) and were provided free access to water. Physical examination, thoracic radiographs, and thoracic tomodensitometric evaluation were carried out to assess the health of all ferrets. Their general condition was assessed daily (weight, appetite, activity level, and quantity and quality of fecal output) throughout the study period. The experimental protocol was approved by the Animal Care and Use Committee of the University of Montreal.
Experimental design Ferrets were fasted for 4 to 6 h and were subsequently sedated with an intramuscular injection of midazolam (Sandoz Canada, Boucherville, Quebec), 1 mg/kg body weight (BW) and butor-
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phanol (Torbugesic; Fort Dodge Animal Health, Fort Dodge, Iowa, USA), 0.1 mg/kg BW before induction with an intramuscular injection of alfaxalone (Alfaxan; Jurox, Rutherford, New South Wales, Australia), 6 mg/kg BW. Ferrets were intubated using an uncuffed endotracheal tube (3.0- to 4.0-mm ID) (Sheridan; Teleflex Medical, Research Triangle Park, North Carolina, USA) and provided supplemental oxygen (2 L/min). A 24G venous catheter (0.7 3 19 mm; 24G 3 0.750) (BD Insyte-W; BD Infusion Therapy Systems, Sandy, Utah, USA) was placed in a cephalic vein and ferrets were maintained on a constant rate infusion of alfaxalone [3 to 15 mg/kg/h (1.36 to 6.81 mg/lb)] throughout the procedure. The bronchoscope (Storz Flexible Ureteroscope #11278; Karl Storz, Tuttlingen, Germany) was filled with 0.8 mL of a sterile solution of isotonic saline [sodium chloride (NaCl) 0.9%; Baxter, Mississauga, Ontario] and inserted into the trachea of the ferrets down to the carina. Ferrets were extubated and the endoscope was randomly advanced into the right (n = 6) or left (n = 6) bronchus until a tight fit was obtained. Sterile saline (1.5 mL/kg BW) was instilled twice in the working channel of the bronchoscope and immediately retrieved by gentle negative pressure on the syringe. The procedure was repeated on the contralateral side for a total of 2 samples for each lung. Ferrets were re-intubated to facilitate mechanical ventilation and provide supplemental oxygen between samples. Blood oxygen (SpO2) values were monitored throughout the endoscopic procedure and, if desaturation was documented (, 90), the ferret was re-intubated. Endoscopic procedures were continued when the SpO2 values were above 95%. The retrieved liquid was placed in an ethylenediamine tetra-acetic acid (EDTA) tube for cytological evaluation and urea determination. The total time for each lavage averaged 20 s and dwell time was estimated at 0 s. A multi-parameter physiologic monitor (LifeWindow LW6000; Digicare Biomedical Technology, Boyton Beach, Florida, USA) was used, which included pulse oximetry, end-tidal carbon dioxide (ETCO2), end-tidal isoflurane (ETiso%), direct arterial blood pressure, electrocardiography, and rectal temperature. All ferrets were evaluated for adverse reactions for 3 d.
Cytological evaluation Bronchoalveolar lavage (BAL) samples from the same bronchi were pooled together for every individual, for a total of 2 pooled samples per ferret. The macroscopic color and turbidity of the BAL fluid was described and the amount of lavage fluid recovered was recorded. Leukocyte and erythrocyte cells were counted in wellmixed undiluted fluid using an improved Neubauer hemocytometer (Reichert Bright-line; Hausser Scientific, Horsham, Pennsylvania, USA). Two cytospin smears, 1 of 400 mL and 1 of 200 mL, of the fluid samples were then prepared by centrifugation at 1000 3 g for 5 min (Shandon Cytospin 4 Cytocentrifuge; Thermo Scientific, Pittsburg, Pennsylvania, USA). When mucus was present, it was isolated and applied on a slide using a squash preparation technique. A Romanosky stain (Aerospray 7120 slide stainer; Wescor, Logan, Utah, USA) was applied to the cytospin smears. Microscopic evaluation was done in a blind fashion by a board-certified veterinary clinical pathologist (CGB) from the American College of Veterinary Pathologists. This consisted of an overall analysis using a 103 objective on all slides and a 300-cell leukocyte differential
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Figure 1. Bronchoalveolar (BAL) cells/microliter (mL) and differential (%) cell counts of 11 healthy domestic ferrets. Circles represent the combined samples from the left and right lung of individual ferrets. The dashed lines represent the 2.5th and 97.5th percentiles and the solid line represents the median as determined by the nonparametric bootstrap (10 000 resamplings) method. This figure and calculations are based on the recommendations of the accreditation and quality assurance of laboratory reference values (13).
count on slides from each side of the lung, using a 503 oil-immersion objective.
Urea dilution method A 1-mL arterial plasma sample (BD Microtainer; Becton, Dickinson, Franklin Lakes, New Jersey, USA) was obtained immediately before bronchoscopy to determine the plasma urea. Both the plasma and BAL urea were measured on a biochemistry analyzer (Synchron DXC 600; Beckman Coulter, Fullerton, California, USA). The urea-adjusted ELF total leukocyte count was calculated by first determining the plasma:BAL urea concentration ratio for all samples. This plasma:BAL urea ratio was then multiplied by the BAL total leukocyte count for each sample. In order to calculate the proportion of ELF within the BAL fluid (%ELF/BAL), the following formula was also used: (urea concentration in the BAL)/(plasmatic urea concentration) 3 100% (8).
Histologic examination Five ferrets were humanely euthanized with an intravascular injection of barbiturates (pentobarbital) (Euthanyl Forte; BimedaMTC Animal Health, Cambridge, Ontario), 110 mg/kg BW after anesthetic induction with isoflurane, which was delivered through a facemask. The remaining 7 ferrets were fostered by the Small Mammal Shelter of the Faculty of Veterinary Medicine, University
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of Montreal. All respiratory systems of the euthanized ferrets were grossly examined and then fixed with intratracheal instillation of 10% neutral-buffered formalin; the larynx, trachea, extrapulmonary bronchi, and whole lungs were kept whole for 24 h. Samples of trachea (3 levels), the extrapulmonary portion of the 2 main bronchi, and 6 sections of lung (cranial and caudal left lobes, cranial, and middle and caudal right lobes) were then taken for examination. Tissues were processed for histopathology, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin, eosin, phloxine (Fisher Scientific, Fair Lawn, New Jersey, USA) and saffron (HEPS) (Chaptec, Montreal, Quebec). Slides were read in a blinded fashion by a board-certified veterinary pathologist (PH) from the American College of Veterinary Pathologists.
Data analysis A t-test was used to compare the weight of males and females. A mixed-linear model was used to examine the effect of gender and weight on the percentage of volume recovered using the individual as a random factor, since each subject provided 2 measurements. The effect of sampling sequence on the percentage of volume recovered and leukocyte counts was analyzed with a mixed-linear model with side (left or right) and sequence (left-right versus right-left) as fixed factors and the individual nested within the sequence as a random factor. Statistical analyses were done using SAS Version 9.3 (Statistical Analysis System, Cary, North Carolina, USA). The level of statistical significance was set at a P-value , 0.05.
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Table I. Absolute and differential leukocyte count (leukocyte count 3 109/L) enumerated from the bronchoalveolar lavage (BAL) fluid from each ferret, as well as the mean, median, and interval range. The interval range was defined as the 2.5% to 97.5% percentiles Leukocyte count 3 109/La Absolute values Ferret ID Leukocytes Macrophages Neutrophils Eosinophils Lymphocytes 9 0.140 0.110 0.019 Rare (0) 0.010 4 0.176 0.133 0.024 0.000 0.018 10 0.240 0.180 0.032 0.001 0.026 3 0.295 0.187 0.072 Rare (0) 0.033 5 0.384 0.292 0.060 0.003 0.016 7 0.474 0.318 0.107 0.002 0.041 12 0.531 0.342 0.090 Rare (0) 0.096 1 0.534 0.311 0.175 0.002 0.044 11 0.535 0.473 0.040 Rare (0) 0.022 2 0.617 0.441 0.059 0.002 0.093 8 0.708 0.597 0.043 Rare (0) 0.068 Median 0.474 0.311 0.059 0.000 0.033 Mean 0.421 0.308 0.065 0.003 0.043 Intervalb 0.149; 0.639 0.116; 0.504 0.020; 0.0124 0.000; 0.010 0.012; 0.094 a Values are the average of the right and left counts. b 2.5th and 97.5th percentiles as determined by the nonparametric bootstrap method (10 000 resamplings) (13).
Results One male ferret had radiographic and tomodensitometric signs of bronchopneumonia. This ferret was excluded from the analysis of total and differential leukocyte counts and erythrocyte counts for the BAL, but was kept for the rest of the study. During bronchoscopy, both right and left primary bronchi were entered with ease in all ferrets and the right cranial, right middle, right accessory, and caudal bronchi were easily identified. The cranial and caudal portions of the left cranial bronchus, along with the left caudal bronchus, were easily identified in all individuals. At the level of the carina, both right and left primary bronchi appeared symmetrical in all ferrets. There was little variation in airway anatomy in the 12 ferrets. The insertion of the endoscope occluded 1 lung lobe causing a transient hypoxemia in most of the ferrets (n = 10/12). Pulse oximetry values quickly returned to normal when the endoscope was removed and mechanical ventilation was instituted. There were no other complications during the BAL procedure. All ferrets recovered well and none had adverse reactions during the 3-day observation period. On cytological evaluation, varying amounts of erythrocytes were seen in all lavage samples. Similarly, erythrocyte cell counts done using a hemocytometer showed a high count in 6 samples out of 22 (2.621 to 5.504 erythrocytes 3 109/L), a moderate count in 5 samples (1.400 to 1.662 erythrocytes 3 109/L), and a low count in 11 samples (0.048 to 0.734 erythrocytes 3 109/L). Rare numbers of mast cells were detected in 10 samples, whereas low numbers were seen in 5 samples. Rare numbers of plasma cells and multinucleated macrophages were detected in 17 and 15 samples, respectively. Single or small clusters of ciliated epithelial cells were visible in 14 samples
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and moderate clusters were seen in 8 samples. Rare to low numbers of non-nucleated and nucleated keratinized epithelial cells were detected in 13 and 17 samples, respectively, with moderate amounts seen in 5 samples. Oropharyngeal contamination was confirmed in 4 samples by the presence of Simonsiella spp. Rare extracellular bacteria (rods and coccobacilli) were seen in 13 samples, while 3 samples had moderate amounts. Rare mitotic figures were detected in 9 samples. Macrophages are the main cellular component of the lavages, followed by neutrophils, lymphocytes, and lastly, eosinophils. Results are shown in Figure 1 (13). No significant lesions were observed on macroscopic evaluation of the lungs during necropsy and bronchoscopic examination. Upon necropsy, variable, multifocal atelectasis was observed in 3 subjects, which was attributed to anesthesia prior to euthanasia. No other significant histologic lesions were seen. Average weight was significantly higher in male ferrets (1.33 6 0.08 kg) than in female ferrets (0.75 6 0.07 kg, P , 0.0001). The mean percentage of volume recovered from the right, left, and combined lungs was 68 6 15%, 70 6 20%, and 69 6 17%, respectively. Gender (P = 0.12) and weight (P = 0.17) did not significantly affect the mean percentage of volume recovered. The sampling sequence (left lung first and right lung second versus right lung first and left lung second) did not significantly affect the mean percentage of volume recovered and all individuals subjected to either sequence were homogenous for this variable (P = 0.56). The mean percentage of recovered volume from the right or left lung was not significantly different according to the sequence of sampling (no significant interaction between side and sequence: P = 0.47). The mean leukocyte count from the right or left lung was not significantly different according to the sequence of sampling (P = 0.17). The
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Table II. Derived leukocyte and differential counts of the epithelial lining fluid (ELF) of each ferret. These values are derived using the relative urea concentration in the plasma and the bronchoalveolar lavage (BAL) fluid. Urea is used in this way since it is a small, freely diffusible molecule that will quickly equilibrate between these 2 fluid compartments. The urea-adjusted ELF leukocyte count in the BAL fluid was calculated by multiplying the absolute leukocyte count by the [(plasma urea concentration)/(urea concentration in the BAL fluid)] 3 100%. The mean, median, and interval range are also described. The interval range was defined as the 2.5% to 97.5% percentiles Urea-adjusted epithelial lining fluid (ELF) leukocyte count 3 109/La Ferret ID Total cell count Macrophages Neutrophils Eosinophils Lymphocytes 1 1.421 1.104 0.209 0.004 0.103 3 2.262 1.641 0.392 0.000 0.209 10 3.021 1.916 0.732 0.000 0.343 11 3.466 2.707 0.373 0.035 0.352 5 3.741 2.851 0.582 0.108 0.159 7 5.192 3.488 1.160 0.064 0.443 4 5.209 3.739 0.509 0.093 0.706 2 5.900 5.218 0.454 0.000 0.228 12 6.308 5.284 0.422 0.000 0.603 8 6.733 4.343 1.145 0.000 1.212 9 10.161 5.923 3.337 0.145 0.843 Median 5.192 3.488 0.509 0.004 0.352 Mean 4.856 3.474 0.847 0.041 0.473 Intervalb 1.631; 7.590 1.238; 5.443 0.250; 1.704 0.000; 0.118 0.117; 0.935 a Values are the average of the right and left counts. b 2.5th and 97.5th percentiles as determined by the nonparametric bootstrap method (10 000 resamplings) (13).
mean proportion of ELF in BAL fluid was 9.3 6 3.7%. The absolute and urea-adjusted leukocyte count of BAL fluid and ELF are shown in Tables I and II (13), respectively.
Discussion In the current study, the leukocyte count and cell type of bronchoalveolar lavage (BAL) fluid acquired by bronchoscopy were determined in healthy ferrets. Bronchoscopy is easy to carry out and is clinically useful for collecting good quality BAL samples for cytological analysis in ferrets. The airway anatomy was homogenous among ferrets. The bronchoscopic technique used in this project is similar to that used in cats (5,6), rabbits (7), horses (11), and dogs (3,14). Recovery percentage of BAL fluid with this technique obtained in cats [79 6 7% (6), 59 6 24% (15)], dogs (57 6 11%) (14), rabbits (63 6 13% and 53 6 13%) (7), and humans (58 6 2%) (8) is similar to our results (69 6 17%). The success of recovery appears to be slightly better during necropsy for ferrets (81 6 8%) (12) and for dogs (78 6 6%) (10) compared to our findings. The difference appears to be relatively small, however, and our sampling technique was meant to be applied in a clinical setting, as opposed to other methods that were designed for research purposes. It is interesting to note that the variability in recovery (expressed by the standard deviation) is higher in small exotic pet species such as ferrets and rabbits (7) than in other species. We believe that this finding could be attributed to the smaller size of the patient. A small
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unrecovered volume would have a greater impact on the recovery percentage since the total volume instilled is smaller for ferrets and rabbits than in larger species. It is also interesting to note that the success of recovery is much less in horses (16) (36.7 6 1.8%) than in smaller species. This could be due to the fact that a larger pulmonary area is being lavaged in horses and a large amount of lavage fluid could be reabsorbed through pulmonary capillaries or lost into the airways, although further investigations would be required. Fixed volumes of fluid instilled were proven to yield a more variable percentage of recovery than weight-adjusted volume instilled in dogs, with the latter resulting in a more uniform ELF recovery (14). Although this could not be validated in our study, it supported our choice of a weight-adjusted volume of fluid. Alveolar macrophages were the most common types of cells in BAL fluid samples of ferrets, with relative cell counts similar to that of dogs (70 6 11%) and cats (70.6 6 9.8%) (5). Neutrophils and lymphocytes were the second and third most predominant types of cells in BAL fluid from ferrets, whereas these 2 cell types were almost equivalent in dogs (5 6 5% and 7 6 5%, respectively) (5). It is now well-known that healthy cats have larger amounts of eosinophils in their BAL fluids [16.1 6 6.8% (5), 25 6 21% (6)] than other species [2 6 4% in rabbits (7), 6 6 5% in dogs (5)] and that this is not necessarily associated with respiratory tract pathology, such as chronic obstructive pulmonary disease or inflammation (6). Unlike the cat, ferrets do not appear to have elevated relative eosinophil counts within their BAL fluid.
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The use of urea as a marker of dilution in BAL was described in humans in 1986 (8). This technique has been used in veterinary medicine for dogs (10,14), cats (9), and horses (11). Urea is known to have many advantages compared to other markers. Firstly, it is an endogenous marker, which makes sampling protocol straightforward. Secondly, it is not metabolized in the lung, which enables its use in BALs. Urea has a small molecular weight and its concentration is equal in all biologic fluids, i.e., ELF and plasma. Finally, urea is easy to measure since its concentration is very high in all fluids, so that even small variations are detectable with standard methods. In our study, the proportion of ELF in BAL fluid (9.3 6 3.8%) is higher than that observed in other species. In humans (8), horses (11), and dogs (10,14), it is estimated at , 3% (14) and , 5% in cats (9). This could be attributed to 3 possible scenarios, all of which resulted in an increase of urea concentration in the BAL fluid. When used as a marker of dilution, urea can be overestimated in BAL when dwell times are over 2 min (17) because urea can diffuse into the BAL, which would falsely increase the proportion of ELF in BAL fluid. In our study, total lavage time was approximately 20 s and dwell time was set at 0 s, making this hypothesis less likely. Secondly, lung pathology may theoretically increase the diffusion of urea into the BAL and, once again, falsely increase the proportion of ELF in BAL fluid, thus making the use of this technique questionable when disease is suspected. In our study, microscopic evaluation of the lung tissue did not demonstrate any signs of pathology. Finally, and most likely, the higher ELF/BAL% can be attributed to the species, meaning that healthy ferrets may have a higher proportion of ELF within their BAL than other species studied to date. This finding could be further investigated in a comparative study. One limitation of this study was the removal of the endotracheal tubes to facilitate the passage of the endoscope. There was the potential for hemorrhage due to the repeated insertion of the endoscope into bronchi and applied suction, which led to a moderate increase in the erythrocyte count in the BAL fluid of 6 ferrets. Considering that neutrophil concentrations are the most prominent leukocyte in peripheral blood of healthy ferrets (18), there may have been a minor contribution to the neutrophil count. Oropharyngeal contamination may have also slightly increased the neutrophil concentration. The overall effect on the leucocyte differential seems to have been minimal, however, since the results of our BAL fluid showed that macrophages were the cells with the highest percentage. The broncoscopic technique did yield bacteria in a certain number of BAL fluid samples. However, these bacteria could be easily identified as an oropharyngeal contamination due to their characteristic morphological shape (Simonsiella sp) and their extracellular presence on the surface of keratinized squamous epithelial cells (19). This study was conducted on ferrets from the same source. Differences may be identified in a larger ferret population of various origins. Future investigations are warranted to establish reference intervals with a larger population. In conclusion, this study yielded leukocyte counts and cytologic results for bronchoalveolar lavage (BAL) fluid in healthy domestic ferrets that were similar to other species. Moreover, the bronchoscopic technique described in this report is clinically relevant and can be easily carried out in practice. Considering the absence of
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information in the literature on BAL fluid in healthy ferrets, this information may help veterinarians to make clinical decisions when treating respiratory disease. Using urea as a marker of dilution, we calculated the adjusted epithelial lining fluid (ELF) cell counts for clinicians and researchers using this technique. Further studies will be needed to establish reference intervals for healthy ferrets and those with bronchopulmonary disease.
Acknowledgment This study was supported in part by the Fond en Santé des Animaux de Compagnie.
References 1. Baron HW, Rosenthal KL. Respiratory diseases. In: Quesenberry KE, Carpenter JW, eds. Ferrets, Rabbits, and Rodents — Clinical Medicine and Surgery. 3rd ed. St. Louis: Elsevier/Saunders, 2011:78–85. 2. Swiderski JK, Seim HB, 3rd, MacPhail CM, Campbell TW, Johnston MS, Monnet E. Long-term outcome of domestic ferrets treated surgically for hyperadrenocorticism: 130 cases (1995–2004). J Am Vet Med Assoc 2008;232:1338–1343. 3. Andreasen CB. Bronchoalveolar lavage. Vet Clin North Am Small Anim Pract 2003;33:69–88. 4. Zinkl J. Lower respiratory tract. In: Cowell RL, Tyler RD, eds. Diagnostic Cytology and Hematology of the Horse. 2nd ed. St. Louis: Mosby, 2002:73–86. 5. Hawkins EC, DeNicola DB, Kuehn NF. Bronchoalveolar lavage in the evaluation of pulmonary disease in the dog and cat. State of the art. J Vet Intern Med 1990;4:267–274. 6. Padrid PA, Feldman BF, Funk K, Samitz EM, Reil D, Cross CE. Cytologic, microbiologic, and biochemical analysis of bronchoalveolar lavage fluid obtained from 24 healthy cats. Am J Vet Res 1991;52:1300–1307. 7. Hawkins MG, Vernau W, Drazenovich TL, Griffey SM, Johnson LR. Results of cytologic and microbiologic analysis of bronchoalveolar lavage fluid in New Zealand White rabbits. Am J Vet Res 2008;69:572–578. 8. Rennard SI, Basset G, Lecossier D, et al. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol (1985) 1986;60:532–538. 9. Mills PC, Litster A. Using urea dilution to standardise cellular and non-cellular components of pleural and bronchoalveolar lavage (BAL) fluids in the cat. J Feline Med Surg 2006;8:105–110. 10. Mills PC, Litster AL. Using urea dilution to standardise components of pleural and bronchoalveolar lavage fluids in the dog. N Z Vet J 2005;53:423–428. 11. McGorum BC, Dixon PM, Halliwell RE, Irving P. Evaluation of urea and albumen as endogenous markers of dilution of equine bronchoalveolar lavage fluid. Res Vet Sci 1993;55:52–56. 12. Sterner-Kock A, Kock M, Braun R, Hyde DM. Ozone-induced epithelial injury in the ferret is similar to nonhuman primates. Am J Respir Crit Care Med 2000;162:1152–1156. 13. Coskun A, Ceyhan E, Inal CI, Serteser M, Unsal I. The comparison of parametric and nonparametric bootstrap methods
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for reference interval computation in small size groups. Accred Qual Assur 2013;18:51–60. 14. Melamies MA, Jarvinen AK, Seppala KM, Rita HJ, Rajamaki MM. Comparison of results for weight-adjusted and fixed-amount bronchoalveolar lavage techniques in healthy Beagles. Am J Vet Res 2011;72:694–698. 15. Ybarra WL, Johnson LR, Drazenovich TL, Johnson EG, Vernau W. Interpretation of multisegment bronchoalveolar lavage in cats (1/2001-1/2011). J Vet Intern Med 2012;26:1281–1287. 16. Jean D, Vrins A, Beauchamp G, Lavoie JP. Evaluation of variations in bronchoalveolar lavage fluid in horses with recurrent airway obstruction. Am J Vet Res 2011;72:838–842.
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17. Ward C, Effros RM, Walters EH. Assessment of epithelial lining fluid dilution during bronchoalveolar lavage. Eur Respir Rev 1999;9:32–37. 18. Lee EJ, Moore WE, Fryer HC, Minocha HC. Haematological and serum chemistry profiles of ferrets (Mustela putorius furo). Lab Anim 1982;16:133–137. 19. Burkhard MJ, Millward LM. Respiratory tract. In: Raskin R, Meyer DJ, eds. Canine and Feline Cytology: A Color Atlas and Interpretation Guide. 2nd ed. St. Louis: Saunders/Elsevier, 2010:125.
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Cytological analysis of bronchoalveolar lavage fluid acquired by bronchoscopy in healthy ferrets: A pilot study Marjorie Bercier, Isabelle Langlois, Marilyn Dunn, Pierre Hélie, Patrick Burns, Carolyn Gara-Boivin
Abstract The objective of this study was to investigate the normal cytological evaluation of bronchoalveolar lavage (BAL) fluid in healthy adult ferrets (N = 12). These ferrets underwent bronchoscopy and BAL using sterile saline [1.5 mL/kg body weight (BW)]. Percentage of fluid recovered, total leukocyte count, differential leukocyte count, and cell count of the epithelial lining fluid (ELF) were determined. The mean percentage of lavage volume recovered from the right lung and left lung were 67.8 6 14.9% and 69.7 6 20.0%, respectively. Gender (P = 0.12) and weight (P = 0.17) did not significantly affect the mean percentage of recovered volume. The mean percentage of recovered volume (P = 0.47) and the mean leukocyte count (P = 0.17) from the right and left lung were not significantly different. Macrophages were the main leukocyte component of the lavages, followed by neutrophils, lymphocytes, and eosinophils. The mean proportion of ELF in BAL fluid was 9.3 6 3.7% v/v. Bronchoscopy is clinically useful for collecting good quality BAL samples for cytological analysis in ferrets. The leucocyte differential was established, which may help veterinarians to make better clinical decisions when treating respiratory disease. Further studies are required with a larger group in order to establish the healthy reference intervals for BAL values in ferrets.
Résumé L’objectif de la présente étude était d’examiner l’évaluation cytologique normale de fluide de lavage broncho-alvéolaire (LAB) de furets adultes en santé (N = 12). Ces furets ont subi ne bronchoscopie et un LAB avec de la saline stérile [1,5 mL/kg de poids corporel (PC)]. Le pourcentage de fluide recouvré, le nombre total de leucocytes, un comptage différentiel de leucocytes, et un compte cellulaire du liquide sécrété par l’épithélium respiratoire ont été déterminés. Les pourcentages moyens de volume de lavage recouvrés du poumon droit et du poumon gauche étaient de 67,8 6 14,9 % et 69,7 6 20,0 %, respectivement. Le sexe (P = 0,12) et le poids (P = 0,17) n’ont pas affecté significativement le pourcentage moyen du volume recouvré. Le pourcentage moyen de volume recouvré (P = 0,47) et de comptage leucocytaire moyen (P = 0,17) provenant du poumon droit et du poumon gauche n’étaient pas significativement différents. Les macrophages étaient les principaux leucocytes retrouvés dans les lavages, suivis par les neutrophiles, lymphocytes et éosinophiles. La proportion moyenne d’ELF dans le fluide de LBA était de 9,3 6 3,7 % v/v. La bronchoscopie est utile cliniquement pour prélever des échantillons de LAB de bonne qualité pour l’examen cytologique chez les furets. Le différentiel des leucocytes a été établi, ce qui pourrait aider les vétérinaires à prendre de meilleures décisions cliniques lors du traitement de maladies respiratoires. Des études supplémentaires sont requises avec un plus grand groupe afin d’établir les intervalles des valeurs de référence des LAB de furets en santé. (Traduit par Docteur Serge Messier)
Introduction Respiratory pathologies are relatively common in ferrets. Such pathologies include influenza; asthma; infectious and non-infectious pneumonias, such as endogenous lipid pneumonia; canine distemper virus; and neoplasia (1). From 2002 to 2012, 6% of all ferrets presented to our teaching hospital displayed respiratory signs. Considering that there are an estimated 10 million ferrets in North America and that they are increasing in popularity, respiratory disease may affect a large number of these animals (2). Bronchoalveolar lavage (BAL) is a diagnostic procedure used in human and veterinary medicine to evaluate the cellularity of the lower respiratory tract (3). The leukocyte differential and mor-
phology can indicate an inflammatory, infectious, degenerative, or neoplastic process. Reference values are available for rabbits, dogs, cats, and horses (4–7), but are lacking for ferrets. Considering that there is a substantial variation among species in BAL fluid cytology from healthy animals (4–7), this may also be true in ferrets and such information becomes a necessity in disease investigation. Bronchoalveolar lavage (BAL) fluid is composed of 3 distinct components: the epithelial lining fluid (ELF), the cellular component of the epithelial lining fluid, and the retrieved amount of saline, the latter of which varies greatly (8). The epithelial lining fluid is defined as the liquid lining the alveoli and the airways, which contains the total leukocyte count measured in the BAL (9,10). The concentration of cellular components in the BAL fluid depends on the concentration
Department of Clinical Sciences (Bercier, Langlois, Dunn, Burns) and Department of Pathology and Microbiology (Gara-Boivin, Hélie), Faculty of Veterinary Medicine, University of Montreal, St. Hyacinthe, Quebec J2S 2M2. Address all correspondence to Dr. Patrick Burns; telephone: 144(0)131 650 7650; fax: 144(0)131 650 7652; e-mail: [email protected] Dr. Burns’ current address is Royal (Dick) School of Veterinary Studies, Easter Bush Campus, Midlothian EH25 9RG, United Kingdom. Dr. Bercier’s present address is University of Florida, College of Veterinary Medicine, 2015 SW 16th Avenue, Gainesville, Florida 32610, USA. Received April 15, 2015. Accepted September 8, 2015. 74
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of cellular components in the ELF and the amount of dilution of the ELF in the saline retrieved, i.e., the proportion of ELF in the sample of BAL fluid (11). In light of this statement, intra-species quantitative comparison of total leukocyte count in BAL samples may not be accurate. In an attempt to standardize these measures, urea has been used as a marker of dilution in human medicine and has also been evaluated in feline, canine, and equine medicine (8–11). Theoretically, the plasmatic urea concentration is equal to the urea concentration in the ELF, since it is a small molecule that diffuses freely throughout the body (8). Using a simple dilution technique, urea can be used as a marker of dilution to standardize the apparent volume and cellular concentration of ELF in humans, horses, dogs, and cats (8–11). Despite some limitations, this technique is known to reduce error in interpreting absolute cell count in BALs. In the current literature, 1 study compared absolute cell counts of BAL fluid in ferrets acutely exposed to either ozone or filtered air (12). The BAL samples were not collected using bronchoscopy, however, which is known to yield better quality samples than a blind BAL (3). Furthermore, the experimental conditions were artificially induced and were therefore not representative of the domestic ferret population. There are no reference values available for the cytological evaluation of BAL fluid in healthy domestic ferrets. There are no published data describing the systematic bronchoscopic examination of the respiratory tract of a ferret. Finally, urea has not been used as a marker of dilution for estimating ELF in ferrets. Therefore, the objectives of this study were to investigate the cytologic leucocyte differential of bronchoalveolar lavage (BAL) in healthy ferrets using bronchoscopy, to describe the bronchoscopic technique used for sampling, and to standardize the analysis of cellular components of ELF in healthy ferrets using a urea-dilution technique.
Materials and methods Animals Twelve healthy, 6-month-old adult ferrets (Mustela putorius furo), 6 males and 6 females, were used in this study. Body weight varied from 0.64 to 1.46 kg (mean 1/2 SD: 1.04 1/2 0.08 kg). All ferrets came from a breeding farm (Marshall Bio Resources, North Rose, New York, USA). After arriving at the Faculty of Veterinary Medicine, all ferrets were allowed 7 to 9 d to acclimatize. Ferrets were housed individually in stainless steel cages, with a standard paper litter, blankets, and hiding boxes. The ferrets were fed a commercial pelleted diet (Premium Ferret Diet; Marshall Pet Products, Wolcott, New York, USA) and were provided free access to water. Physical examination, thoracic radiographs, and thoracic tomodensitometric evaluation were carried out to assess the health of all ferrets. Their general condition was assessed daily (weight, appetite, activity level, and quantity and quality of fecal output) throughout the study period. The experimental protocol was approved by the Animal Care and Use Committee of the University of Montreal.
Experimental design Ferrets were fasted for 4 to 6 h and were subsequently sedated with an intramuscular injection of midazolam (Sandoz Canada, Boucherville, Quebec), 1 mg/kg body weight (BW) and butor-
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phanol (Torbugesic; Fort Dodge Animal Health, Fort Dodge, Iowa, USA), 0.1 mg/kg BW before induction with an intramuscular injection of alfaxalone (Alfaxan; Jurox, Rutherford, New South Wales, Australia), 6 mg/kg BW. Ferrets were intubated using an uncuffed endotracheal tube (3.0- to 4.0-mm ID) (Sheridan; Teleflex Medical, Research Triangle Park, North Carolina, USA) and provided supplemental oxygen (2 L/min). A 24G venous catheter (0.7 3 19 mm; 24G 3 0.750) (BD Insyte-W; BD Infusion Therapy Systems, Sandy, Utah, USA) was placed in a cephalic vein and ferrets were maintained on a constant rate infusion of alfaxalone [3 to 15 mg/kg/h (1.36 to 6.81 mg/lb)] throughout the procedure. The bronchoscope (Storz Flexible Ureteroscope #11278; Karl Storz, Tuttlingen, Germany) was filled with 0.8 mL of a sterile solution of isotonic saline [sodium chloride (NaCl) 0.9%; Baxter, Mississauga, Ontario] and inserted into the trachea of the ferrets down to the carina. Ferrets were extubated and the endoscope was randomly advanced into the right (n = 6) or left (n = 6) bronchus until a tight fit was obtained. Sterile saline (1.5 mL/kg BW) was instilled twice in the working channel of the bronchoscope and immediately retrieved by gentle negative pressure on the syringe. The procedure was repeated on the contralateral side for a total of 2 samples for each lung. Ferrets were re-intubated to facilitate mechanical ventilation and provide supplemental oxygen between samples. Blood oxygen (SpO2) values were monitored throughout the endoscopic procedure and, if desaturation was documented (, 90), the ferret was re-intubated. Endoscopic procedures were continued when the SpO2 values were above 95%. The retrieved liquid was placed in an ethylenediamine tetra-acetic acid (EDTA) tube for cytological evaluation and urea determination. The total time for each lavage averaged 20 s and dwell time was estimated at 0 s. A multi-parameter physiologic monitor (LifeWindow LW6000; Digicare Biomedical Technology, Boyton Beach, Florida, USA) was used, which included pulse oximetry, end-tidal carbon dioxide (ETCO2), end-tidal isoflurane (ETiso%), direct arterial blood pressure, electrocardiography, and rectal temperature. All ferrets were evaluated for adverse reactions for 3 d.
Cytological evaluation Bronchoalveolar lavage (BAL) samples from the same bronchi were pooled together for every individual, for a total of 2 pooled samples per ferret. The macroscopic color and turbidity of the BAL fluid was described and the amount of lavage fluid recovered was recorded. Leukocyte and erythrocyte cells were counted in wellmixed undiluted fluid using an improved Neubauer hemocytometer (Reichert Bright-line; Hausser Scientific, Horsham, Pennsylvania, USA). Two cytospin smears, 1 of 400 mL and 1 of 200 mL, of the fluid samples were then prepared by centrifugation at 1000 3 g for 5 min (Shandon Cytospin 4 Cytocentrifuge; Thermo Scientific, Pittsburg, Pennsylvania, USA). When mucus was present, it was isolated and applied on a slide using a squash preparation technique. A Romanosky stain (Aerospray 7120 slide stainer; Wescor, Logan, Utah, USA) was applied to the cytospin smears. Microscopic evaluation was done in a blind fashion by a board-certified veterinary clinical pathologist (CGB) from the American College of Veterinary Pathologists. This consisted of an overall analysis using a 103 objective on all slides and a 300-cell leukocyte differential
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Figure 1. Bronchoalveolar (BAL) cells/microliter (mL) and differential (%) cell counts of 11 healthy domestic ferrets. Circles represent the combined samples from the left and right lung of individual ferrets. The dashed lines represent the 2.5th and 97.5th percentiles and the solid line represents the median as determined by the nonparametric bootstrap (10 000 resamplings) method. This figure and calculations are based on the recommendations of the accreditation and quality assurance of laboratory reference values (13).
count on slides from each side of the lung, using a 503 oil-immersion objective.
Urea dilution method A 1-mL arterial plasma sample (BD Microtainer; Becton, Dickinson, Franklin Lakes, New Jersey, USA) was obtained immediately before bronchoscopy to determine the plasma urea. Both the plasma and BAL urea were measured on a biochemistry analyzer (Synchron DXC 600; Beckman Coulter, Fullerton, California, USA). The urea-adjusted ELF total leukocyte count was calculated by first determining the plasma:BAL urea concentration ratio for all samples. This plasma:BAL urea ratio was then multiplied by the BAL total leukocyte count for each sample. In order to calculate the proportion of ELF within the BAL fluid (%ELF/BAL), the following formula was also used: (urea concentration in the BAL)/(plasmatic urea concentration) 3 100% (8).
Histologic examination Five ferrets were humanely euthanized with an intravascular injection of barbiturates (pentobarbital) (Euthanyl Forte; BimedaMTC Animal Health, Cambridge, Ontario), 110 mg/kg BW after anesthetic induction with isoflurane, which was delivered through a facemask. The remaining 7 ferrets were fostered by the Small Mammal Shelter of the Faculty of Veterinary Medicine, University
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of Montreal. All respiratory systems of the euthanized ferrets were grossly examined and then fixed with intratracheal instillation of 10% neutral-buffered formalin; the larynx, trachea, extrapulmonary bronchi, and whole lungs were kept whole for 24 h. Samples of trachea (3 levels), the extrapulmonary portion of the 2 main bronchi, and 6 sections of lung (cranial and caudal left lobes, cranial, and middle and caudal right lobes) were then taken for examination. Tissues were processed for histopathology, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin, eosin, phloxine (Fisher Scientific, Fair Lawn, New Jersey, USA) and saffron (HEPS) (Chaptec, Montreal, Quebec). Slides were read in a blinded fashion by a board-certified veterinary pathologist (PH) from the American College of Veterinary Pathologists.
Data analysis A t-test was used to compare the weight of males and females. A mixed-linear model was used to examine the effect of gender and weight on the percentage of volume recovered using the individual as a random factor, since each subject provided 2 measurements. The effect of sampling sequence on the percentage of volume recovered and leukocyte counts was analyzed with a mixed-linear model with side (left or right) and sequence (left-right versus right-left) as fixed factors and the individual nested within the sequence as a random factor. Statistical analyses were done using SAS Version 9.3 (Statistical Analysis System, Cary, North Carolina, USA). The level of statistical significance was set at a P-value , 0.05.
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Table I. Absolute and differential leukocyte count (leukocyte count 3 109/L) enumerated from the bronchoalveolar lavage (BAL) fluid from each ferret, as well as the mean, median, and interval range. The interval range was defined as the 2.5% to 97.5% percentiles Leukocyte count 3 109/La Absolute values Ferret ID Leukocytes Macrophages Neutrophils Eosinophils Lymphocytes 9 0.140 0.110 0.019 Rare (0) 0.010 4 0.176 0.133 0.024 0.000 0.018 10 0.240 0.180 0.032 0.001 0.026 3 0.295 0.187 0.072 Rare (0) 0.033 5 0.384 0.292 0.060 0.003 0.016 7 0.474 0.318 0.107 0.002 0.041 12 0.531 0.342 0.090 Rare (0) 0.096 1 0.534 0.311 0.175 0.002 0.044 11 0.535 0.473 0.040 Rare (0) 0.022 2 0.617 0.441 0.059 0.002 0.093 8 0.708 0.597 0.043 Rare (0) 0.068 Median 0.474 0.311 0.059 0.000 0.033 Mean 0.421 0.308 0.065 0.003 0.043 Intervalb 0.149; 0.639 0.116; 0.504 0.020; 0.0124 0.000; 0.010 0.012; 0.094 a Values are the average of the right and left counts. b 2.5th and 97.5th percentiles as determined by the nonparametric bootstrap method (10 000 resamplings) (13).
Results One male ferret had radiographic and tomodensitometric signs of bronchopneumonia. This ferret was excluded from the analysis of total and differential leukocyte counts and erythrocyte counts for the BAL, but was kept for the rest of the study. During bronchoscopy, both right and left primary bronchi were entered with ease in all ferrets and the right cranial, right middle, right accessory, and caudal bronchi were easily identified. The cranial and caudal portions of the left cranial bronchus, along with the left caudal bronchus, were easily identified in all individuals. At the level of the carina, both right and left primary bronchi appeared symmetrical in all ferrets. There was little variation in airway anatomy in the 12 ferrets. The insertion of the endoscope occluded 1 lung lobe causing a transient hypoxemia in most of the ferrets (n = 10/12). Pulse oximetry values quickly returned to normal when the endoscope was removed and mechanical ventilation was instituted. There were no other complications during the BAL procedure. All ferrets recovered well and none had adverse reactions during the 3-day observation period. On cytological evaluation, varying amounts of erythrocytes were seen in all lavage samples. Similarly, erythrocyte cell counts done using a hemocytometer showed a high count in 6 samples out of 22 (2.621 to 5.504 erythrocytes 3 109/L), a moderate count in 5 samples (1.400 to 1.662 erythrocytes 3 109/L), and a low count in 11 samples (0.048 to 0.734 erythrocytes 3 109/L). Rare numbers of mast cells were detected in 10 samples, whereas low numbers were seen in 5 samples. Rare numbers of plasma cells and multinucleated macrophages were detected in 17 and 15 samples, respectively. Single or small clusters of ciliated epithelial cells were visible in 14 samples
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and moderate clusters were seen in 8 samples. Rare to low numbers of non-nucleated and nucleated keratinized epithelial cells were detected in 13 and 17 samples, respectively, with moderate amounts seen in 5 samples. Oropharyngeal contamination was confirmed in 4 samples by the presence of Simonsiella spp. Rare extracellular bacteria (rods and coccobacilli) were seen in 13 samples, while 3 samples had moderate amounts. Rare mitotic figures were detected in 9 samples. Macrophages are the main cellular component of the lavages, followed by neutrophils, lymphocytes, and lastly, eosinophils. Results are shown in Figure 1 (13). No significant lesions were observed on macroscopic evaluation of the lungs during necropsy and bronchoscopic examination. Upon necropsy, variable, multifocal atelectasis was observed in 3 subjects, which was attributed to anesthesia prior to euthanasia. No other significant histologic lesions were seen. Average weight was significantly higher in male ferrets (1.33 6 0.08 kg) than in female ferrets (0.75 6 0.07 kg, P , 0.0001). The mean percentage of volume recovered from the right, left, and combined lungs was 68 6 15%, 70 6 20%, and 69 6 17%, respectively. Gender (P = 0.12) and weight (P = 0.17) did not significantly affect the mean percentage of volume recovered. The sampling sequence (left lung first and right lung second versus right lung first and left lung second) did not significantly affect the mean percentage of volume recovered and all individuals subjected to either sequence were homogenous for this variable (P = 0.56). The mean percentage of recovered volume from the right or left lung was not significantly different according to the sequence of sampling (no significant interaction between side and sequence: P = 0.47). The mean leukocyte count from the right or left lung was not significantly different according to the sequence of sampling (P = 0.17). The
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Table II. Derived leukocyte and differential counts of the epithelial lining fluid (ELF) of each ferret. These values are derived using the relative urea concentration in the plasma and the bronchoalveolar lavage (BAL) fluid. Urea is used in this way since it is a small, freely diffusible molecule that will quickly equilibrate between these 2 fluid compartments. The urea-adjusted ELF leukocyte count in the BAL fluid was calculated by multiplying the absolute leukocyte count by the [(plasma urea concentration)/(urea concentration in the BAL fluid)] 3 100%. The mean, median, and interval range are also described. The interval range was defined as the 2.5% to 97.5% percentiles Urea-adjusted epithelial lining fluid (ELF) leukocyte count 3 109/La Ferret ID Total cell count Macrophages Neutrophils Eosinophils Lymphocytes 1 1.421 1.104 0.209 0.004 0.103 3 2.262 1.641 0.392 0.000 0.209 10 3.021 1.916 0.732 0.000 0.343 11 3.466 2.707 0.373 0.035 0.352 5 3.741 2.851 0.582 0.108 0.159 7 5.192 3.488 1.160 0.064 0.443 4 5.209 3.739 0.509 0.093 0.706 2 5.900 5.218 0.454 0.000 0.228 12 6.308 5.284 0.422 0.000 0.603 8 6.733 4.343 1.145 0.000 1.212 9 10.161 5.923 3.337 0.145 0.843 Median 5.192 3.488 0.509 0.004 0.352 Mean 4.856 3.474 0.847 0.041 0.473 Intervalb 1.631; 7.590 1.238; 5.443 0.250; 1.704 0.000; 0.118 0.117; 0.935 a Values are the average of the right and left counts. b 2.5th and 97.5th percentiles as determined by the nonparametric bootstrap method (10 000 resamplings) (13).
mean proportion of ELF in BAL fluid was 9.3 6 3.7%. The absolute and urea-adjusted leukocyte count of BAL fluid and ELF are shown in Tables I and II (13), respectively.
Discussion In the current study, the leukocyte count and cell type of bronchoalveolar lavage (BAL) fluid acquired by bronchoscopy were determined in healthy ferrets. Bronchoscopy is easy to carry out and is clinically useful for collecting good quality BAL samples for cytological analysis in ferrets. The airway anatomy was homogenous among ferrets. The bronchoscopic technique used in this project is similar to that used in cats (5,6), rabbits (7), horses (11), and dogs (3,14). Recovery percentage of BAL fluid with this technique obtained in cats [79 6 7% (6), 59 6 24% (15)], dogs (57 6 11%) (14), rabbits (63 6 13% and 53 6 13%) (7), and humans (58 6 2%) (8) is similar to our results (69 6 17%). The success of recovery appears to be slightly better during necropsy for ferrets (81 6 8%) (12) and for dogs (78 6 6%) (10) compared to our findings. The difference appears to be relatively small, however, and our sampling technique was meant to be applied in a clinical setting, as opposed to other methods that were designed for research purposes. It is interesting to note that the variability in recovery (expressed by the standard deviation) is higher in small exotic pet species such as ferrets and rabbits (7) than in other species. We believe that this finding could be attributed to the smaller size of the patient. A small
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unrecovered volume would have a greater impact on the recovery percentage since the total volume instilled is smaller for ferrets and rabbits than in larger species. It is also interesting to note that the success of recovery is much less in horses (16) (36.7 6 1.8%) than in smaller species. This could be due to the fact that a larger pulmonary area is being lavaged in horses and a large amount of lavage fluid could be reabsorbed through pulmonary capillaries or lost into the airways, although further investigations would be required. Fixed volumes of fluid instilled were proven to yield a more variable percentage of recovery than weight-adjusted volume instilled in dogs, with the latter resulting in a more uniform ELF recovery (14). Although this could not be validated in our study, it supported our choice of a weight-adjusted volume of fluid. Alveolar macrophages were the most common types of cells in BAL fluid samples of ferrets, with relative cell counts similar to that of dogs (70 6 11%) and cats (70.6 6 9.8%) (5). Neutrophils and lymphocytes were the second and third most predominant types of cells in BAL fluid from ferrets, whereas these 2 cell types were almost equivalent in dogs (5 6 5% and 7 6 5%, respectively) (5). It is now well-known that healthy cats have larger amounts of eosinophils in their BAL fluids [16.1 6 6.8% (5), 25 6 21% (6)] than other species [2 6 4% in rabbits (7), 6 6 5% in dogs (5)] and that this is not necessarily associated with respiratory tract pathology, such as chronic obstructive pulmonary disease or inflammation (6). Unlike the cat, ferrets do not appear to have elevated relative eosinophil counts within their BAL fluid.
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The use of urea as a marker of dilution in BAL was described in humans in 1986 (8). This technique has been used in veterinary medicine for dogs (10,14), cats (9), and horses (11). Urea is known to have many advantages compared to other markers. Firstly, it is an endogenous marker, which makes sampling protocol straightforward. Secondly, it is not metabolized in the lung, which enables its use in BALs. Urea has a small molecular weight and its concentration is equal in all biologic fluids, i.e., ELF and plasma. Finally, urea is easy to measure since its concentration is very high in all fluids, so that even small variations are detectable with standard methods. In our study, the proportion of ELF in BAL fluid (9.3 6 3.8%) is higher than that observed in other species. In humans (8), horses (11), and dogs (10,14), it is estimated at , 3% (14) and , 5% in cats (9). This could be attributed to 3 possible scenarios, all of which resulted in an increase of urea concentration in the BAL fluid. When used as a marker of dilution, urea can be overestimated in BAL when dwell times are over 2 min (17) because urea can diffuse into the BAL, which would falsely increase the proportion of ELF in BAL fluid. In our study, total lavage time was approximately 20 s and dwell time was set at 0 s, making this hypothesis less likely. Secondly, lung pathology may theoretically increase the diffusion of urea into the BAL and, once again, falsely increase the proportion of ELF in BAL fluid, thus making the use of this technique questionable when disease is suspected. In our study, microscopic evaluation of the lung tissue did not demonstrate any signs of pathology. Finally, and most likely, the higher ELF/BAL% can be attributed to the species, meaning that healthy ferrets may have a higher proportion of ELF within their BAL than other species studied to date. This finding could be further investigated in a comparative study. One limitation of this study was the removal of the endotracheal tubes to facilitate the passage of the endoscope. There was the potential for hemorrhage due to the repeated insertion of the endoscope into bronchi and applied suction, which led to a moderate increase in the erythrocyte count in the BAL fluid of 6 ferrets. Considering that neutrophil concentrations are the most prominent leukocyte in peripheral blood of healthy ferrets (18), there may have been a minor contribution to the neutrophil count. Oropharyngeal contamination may have also slightly increased the neutrophil concentration. The overall effect on the leucocyte differential seems to have been minimal, however, since the results of our BAL fluid showed that macrophages were the cells with the highest percentage. The broncoscopic technique did yield bacteria in a certain number of BAL fluid samples. However, these bacteria could be easily identified as an oropharyngeal contamination due to their characteristic morphological shape (Simonsiella sp) and their extracellular presence on the surface of keratinized squamous epithelial cells (19). This study was conducted on ferrets from the same source. Differences may be identified in a larger ferret population of various origins. Future investigations are warranted to establish reference intervals with a larger population. In conclusion, this study yielded leukocyte counts and cytologic results for bronchoalveolar lavage (BAL) fluid in healthy domestic ferrets that were similar to other species. Moreover, the bronchoscopic technique described in this report is clinically relevant and can be easily carried out in practice. Considering the absence of
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information in the literature on BAL fluid in healthy ferrets, this information may help veterinarians to make clinical decisions when treating respiratory disease. Using urea as a marker of dilution, we calculated the adjusted epithelial lining fluid (ELF) cell counts for clinicians and researchers using this technique. Further studies will be needed to establish reference intervals for healthy ferrets and those with bronchopulmonary disease.
Acknowledgment This study was supported in part by the Fond en Santé des Animaux de Compagnie.
References 1. Baron HW, Rosenthal KL. Respiratory diseases. In: Quesenberry KE, Carpenter JW, eds. Ferrets, Rabbits, and Rodents — Clinical Medicine and Surgery. 3rd ed. St. Louis: Elsevier/Saunders, 2011:78–85. 2. Swiderski JK, Seim HB, 3rd, MacPhail CM, Campbell TW, Johnston MS, Monnet E. Long-term outcome of domestic ferrets treated surgically for hyperadrenocorticism: 130 cases (1995–2004). J Am Vet Med Assoc 2008;232:1338–1343. 3. Andreasen CB. Bronchoalveolar lavage. Vet Clin North Am Small Anim Pract 2003;33:69–88. 4. Zinkl J. Lower respiratory tract. In: Cowell RL, Tyler RD, eds. Diagnostic Cytology and Hematology of the Horse. 2nd ed. St. Louis: Mosby, 2002:73–86. 5. Hawkins EC, DeNicola DB, Kuehn NF. Bronchoalveolar lavage in the evaluation of pulmonary disease in the dog and cat. State of the art. J Vet Intern Med 1990;4:267–274. 6. Padrid PA, Feldman BF, Funk K, Samitz EM, Reil D, Cross CE. Cytologic, microbiologic, and biochemical analysis of bronchoalveolar lavage fluid obtained from 24 healthy cats. Am J Vet Res 1991;52:1300–1307. 7. Hawkins MG, Vernau W, Drazenovich TL, Griffey SM, Johnson LR. Results of cytologic and microbiologic analysis of bronchoalveolar lavage fluid in New Zealand White rabbits. Am J Vet Res 2008;69:572–578. 8. Rennard SI, Basset G, Lecossier D, et al. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol (1985) 1986;60:532–538. 9. Mills PC, Litster A. Using urea dilution to standardise cellular and non-cellular components of pleural and bronchoalveolar lavage (BAL) fluids in the cat. J Feline Med Surg 2006;8:105–110. 10. Mills PC, Litster AL. Using urea dilution to standardise components of pleural and bronchoalveolar lavage fluids in the dog. N Z Vet J 2005;53:423–428. 11. McGorum BC, Dixon PM, Halliwell RE, Irving P. Evaluation of urea and albumen as endogenous markers of dilution of equine bronchoalveolar lavage fluid. Res Vet Sci 1993;55:52–56. 12. Sterner-Kock A, Kock M, Braun R, Hyde DM. Ozone-induced epithelial injury in the ferret is similar to nonhuman primates. Am J Respir Crit Care Med 2000;162:1152–1156. 13. Coskun A, Ceyhan E, Inal CI, Serteser M, Unsal I. The comparison of parametric and nonparametric bootstrap methods
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for reference interval computation in small size groups. Accred Qual Assur 2013;18:51–60. 14. Melamies MA, Jarvinen AK, Seppala KM, Rita HJ, Rajamaki MM. Comparison of results for weight-adjusted and fixed-amount bronchoalveolar lavage techniques in healthy Beagles. Am J Vet Res 2011;72:694–698. 15. Ybarra WL, Johnson LR, Drazenovich TL, Johnson EG, Vernau W. Interpretation of multisegment bronchoalveolar lavage in cats (1/2001-1/2011). J Vet Intern Med 2012;26:1281–1287. 16. Jean D, Vrins A, Beauchamp G, Lavoie JP. Evaluation of variations in bronchoalveolar lavage fluid in horses with recurrent airway obstruction. Am J Vet Res 2011;72:838–842.
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17. Ward C, Effros RM, Walters EH. Assessment of epithelial lining fluid dilution during bronchoalveolar lavage. Eur Respir Rev 1999;9:32–37. 18. Lee EJ, Moore WE, Fryer HC, Minocha HC. Haematological and serum chemistry profiles of ferrets (Mustela putorius furo). Lab Anim 1982;16:133–137. 19. Burkhard MJ, Millward LM. Respiratory tract. In: Raskin R, Meyer DJ, eds. Canine and Feline Cytology: A Color Atlas and Interpretation Guide. 2nd ed. St. Louis: Saunders/Elsevier, 2010:125.
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