Comparison of bronchoscopic and nonbronchoscopic bronchoalveolar lavage in healthy cats

Kimberly S. Hooi Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Alice M. Defarges Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Andrea L. Sanchez Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Stephanie G. Nykamp Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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J. Scott Weese Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Anthony C. G. Abrams-Ogg Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Dorothee Bienzle Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Abstract

OBJECTIVE To compare bronchoalveolar lavage (BAL) accomplished by use of a bronchoscopic (B-BAL) and a nonbronchoscopic (NB-BAL) technique in healthy cats.

ANIMALS 12 healthy cats.

PROCEDURES Two BALs were performed in a randomized order 2 weeks apart in each cat. Cats were anesthetized, and a 2.9-mm fiberoptic bronchoscope (B-BAL) or 8F red rubber catheter (NB-BAL) was wedged in a bronchus. Two 5-mL aliquots of saline (0.9% NaCl) solution were infused into the left and right caudal lung fields and aspirated manually with a 20-mL syringe. Proportion of BAL fluid (BALF) retrieved, depth of wedging, and anesthetic complications were recorded. Total nucleated cell count, differential cell count, and semiquantitative scores of cytologic slide quality were determined for all BALF samples. Results were compared with ANOVAs and Wilcoxon signed rank tests.

RESULTS Proportion of retrieved BALF and depth of wedging were significantly greater for B-BAL than NB-BAL. Differential cell counts and cytologic slide quality did not differ significantly between techniques. Complications included transient hemoglobin desaturation (24/24 [100%] BALs) and prolonged anesthetic recovery time (4/24 [17%] BALs). Anesthetic recovery scores did not differ significantly between techniques.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that NB-BAL was noninferior to B-BAL with regard to ease of performance, anesthetic variables, and cytologic slide quality for cats without clinical respiratory tract disease.

Abstract

OBJECTIVE To compare bronchoalveolar lavage (BAL) accomplished by use of a bronchoscopic (B-BAL) and a nonbronchoscopic (NB-BAL) technique in healthy cats.

ANIMALS 12 healthy cats.

PROCEDURES Two BALs were performed in a randomized order 2 weeks apart in each cat. Cats were anesthetized, and a 2.9-mm fiberoptic bronchoscope (B-BAL) or 8F red rubber catheter (NB-BAL) was wedged in a bronchus. Two 5-mL aliquots of saline (0.9% NaCl) solution were infused into the left and right caudal lung fields and aspirated manually with a 20-mL syringe. Proportion of BAL fluid (BALF) retrieved, depth of wedging, and anesthetic complications were recorded. Total nucleated cell count, differential cell count, and semiquantitative scores of cytologic slide quality were determined for all BALF samples. Results were compared with ANOVAs and Wilcoxon signed rank tests.

RESULTS Proportion of retrieved BALF and depth of wedging were significantly greater for B-BAL than NB-BAL. Differential cell counts and cytologic slide quality did not differ significantly between techniques. Complications included transient hemoglobin desaturation (24/24 [100%] BALs) and prolonged anesthetic recovery time (4/24 [17%] BALs). Anesthetic recovery scores did not differ significantly between techniques.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that NB-BAL was noninferior to B-BAL with regard to ease of performance, anesthetic variables, and cytologic slide quality for cats without clinical respiratory tract disease.

Bronchoalveolar lavage is a minimally invasive technique used to obtain luminal cells from terminal bronchi, bronchioles, and alveoli for investigating pulmonary disease.1 In human, canine, and equine medicine, certain aspects of BAL techniques can affect the quality of BALF samples and therefore the diagnostic and clinical use of those samples.2,3

Two common techniques for performing BAL in cats have been described.1,4–6 One involves a sterile catheter that is blindly wedged into a terminal bronchus (NB-BAL),4,5 and the other technique consists of a flexible bronchoscope wedged into a bronchus in the lung lobe of interest (B-BAL).1,6 Nonbronchoscopic BAL can be performed without a bronchoscope, which may be useful when such equipment is unavailable. However, blindly wedging the catheter in NB-BAL precludes visual examination of the airways and restricts the ability of clinicians to collect samples from multiple lung lobes or focal lesions, which may reduce the diagnostic value of the procedure for cats with focal small airway or pulmonary parenchymal disease.7 In contrast, B-BAL allows for examination of the airways, including visual examination of airway collapse, determination of the amount of mucus and erythema, and retrieval of foreign bodies.8 Samples may be collected from multiple lung lobes or focal lesions during B-BAL; however, depending on the diameter of the bronchoscope, extubation may be required, which can cause respiratory compromise and anesthetic complications.1,6,9 The purpose of the study reported here was to compare the quality of BALF samples obtained by use of NB-BAL and B-BAL techniques in healthy cats. We hypothesized that NB-BAL would be noninferior to B-BAL for procedural variables, anesthetic complications, and cytologic quality of samples.

Materials and Methods

Animals

Twelve healthy domestic shorthair cats were included in the study. Sample size calculation was based on data that indicated BALF retrieval from healthy cats of 56%5 and 79%1 for NB-BAL and B-BAL, respectively. Therefore, it was estimated that 12 cats undergoing both techniques would be required to detect a difference of 10% in BALF retrieval between B-BAL and NB-BAL (power, 98%; α, < 0.05). Data regarding differences in quality of cytologic preparations related to different BAL protocols were unavailable.

The 12 cats were deemed healthy on the basis of results of a physical examination, CBC, serum biochemical analysis, fecal flotation, Baermann test, and ELISA to detect antibodies against FIV and FeLV antigen; measurement of total thyroxine concentration; and evaluation of 3-view thoracic radiographs by a board-certified veterinary radiologist (SGN). Cats had regularly received vaccinations and anthelmintic preventatives, and they did not have signs of respiratory tract disease during the 6 months preceding enrollment. Cats were cared for in a facility accredited by the Canadian Council on Animal Care. The study protocol was approved by the University of Guelph Animal Care Committee.

Study design

A randomized crossover study was conducted. Each cat underwent both B-BAL and NB-BAL; there was a 2-week interval between procedures. The order of the techniques and side on which the initial BAL was performed were determined by use of a random number table.

Anesthesia

Beginning approximately 28 hours before induction of anesthesia, each cat received 4 doses of terbutaline sulfatea (0.01 mg/kg, SC, q 8 h); the last injection was administered 2 to 4 hours before induction of anesthesia. Cats were sedated by an IM injection of dexmedetomidineb (5 μg/kg) and butorphanol tartratec (0.3 mg/kg). Anesthesia was induced by IV administration of propofold titrated to effect; anesthesia was maintained with a constant rate infusion of propofol that provided an adequate plane of anesthesia. To facilitate intubation, lidocainee was topically applied on the larynx; each cat then was intubated with a sterile endotracheal tube.f All cats received IV fluids, and supplemental oxygen was provided via a 3.5F sterile feeding tubeg inserted through the endotracheal tube. Cats were monitored during anesthesia by means of continuous ECG, pulse oximetry, and physical assessments. Adequate Spo2 was defined as > 95%. Procedures were terminated when Spo2 was < 90% for > 10 minutes or < 85% for > 5 minutes or a cat was bradycardic (heart rate < 80 beats/min) and unresponsive to standard interventions. The anesthesiologist scored the quality of anesthetic recovery after each BAL was completed (Appendix 1). The scoring system involved the duration of oxygen supplementation required after extubation, development of pneumothorax, requirement for ventilatory assistance, and potential procedure-related fatality, which are the most commonly reported complications related to BAL.6

BAL

An esophageal marking catheterh was inserted before each BAL procedure to measure the wedging depth of the inserted catheter and bronchoscope. For the NB-BAL technique, cats were placed in lateral recumbency, and the dependent lung was lavaged first. To lavage the opposite lung lobe, cats were repositioned in the alternate lateral recumbency. An 8F soft red rubber catheteri with a 0.035 hydrophilic guidewirej was wedged into a terminal bronchus. Location of placement was confirmed with fluoroscopyk; fluoroscopic images were acquired in a dorsoventral projection. For the B-BAL technique, cats were placed in sternal recumbency. A 2.9-mm flexible fiber-optic endoscopel was advanced through the endotracheal tube into the lung lobe of interest and wedged into a terminal bronchus; location of the endoscope was confirmed with fluoroscopy.

The right and left caudal lung lobes were lavaged during both techniques. Two 5-mL aliquots of sterile saline (0.9% NaCl) solution, each of which was followed by 2 mL of air, were infused and then were sequentially retrieved via gentle pulsatile manual aspiration with a 20-mL syringem attached to a conical plastic adaptern connected to the end of the feeding tube (NB-BAL) or to the working channel of the endoscope (B-BAL). Dwell time (time from infusion of saline solution to the first attempted aspiration) was < 20 seconds during each collection. Aspiration was continued until fluid was no longer recovered. The bronchoscope was cleaned and sterilized with a standard cold sterilization method2 between successive uses.

Amount of fluid retrieved, proportion of fluid retrieved, lowest Spo2, depth of wedging, and anesthetic recovery score were recorded. Fluoroscopic still images obtained during BAL were used to calculate depth of wedging by measuring the length of the inserted bronchoscope (B-BAL) or guidewire (NB-BAL) from the cranial aspect of the second rib to the point of wedging. Measurements were performed on images captured at peak inspiration.

BALF analysis

The BALF samples were immediately placed in sterile tubes on ice; they were delivered within 60 minutes after collection to a laboratoryo for processing. Each BALF cytology sample was labeled with a unique identification code. Cytologic evaluation was performed on each sample collected from each lung lobe. A separate small portion of each sample was pooled from both lung lobes of each cat and submitted for aerobic bacterial culture, Mycoplasma culture, and Mycoplasma PCR assay.p Total nucleated cell counts were determined by electrical impedance with an automated cell counter.q A 200-μL aliquot of each sample was cytocentrifugedr (180 × g for 6 minutes) and used to prepare 2 slides. Two additional slides were prepared from fluid centrifuged at 500 × g for 5 minutes. Slides were stained with Wright stain, and differential cell counts of a minimum of 500 nucleated cells of all slides were performed at 400 × magnification by a board-certified veterinary pathologist (DB) who was unaware of the source of each sample. For each slide, 7 variables associated with sample and slide quality were scored2,10 (Appendix 2). Mean scores ≥ 2 for cellularity and cell preservation were required for a BALF sample to be considered of adequate diagnostic quality. Samples were considered excellent when mean cellularity and cell preservation scores were ≥ 3. Inflammation in BALF was defined as a total nucleated cell count > 0.5 × 109 cells/L or ≥ 17% eosinophils, ≥ 7% neutrophils, or ≥ 5% lymphocytes, as reported in another study.10

CT

Seven days after the second BAL procedure, noncontrast CTs of the thorax of each cat was performed to further investigate potential causes of abnormal BALF cytologic findings. Cats were sedated by an IM injection of dexmedetomidine (5 μg/kg) and butorphanol tartrate (0.3 mg/kg) and placed in sternal recumbency in a transparent positioning device as described elsewhere.11 During CT, cats received supplemental oxygen (fraction of inspired O2, 40%) via tubing that delivered oxygen into the positioning device. The CT protocol was as follows: pitch, 1.375; kV, 120; mA, 140; slice thickness, 0.625 mm; increment, 0.625; gantry rotation speed, 1 second; and a detail algorithm. Motion artifacts during CT scanning were noted, and CT was repeated when motion artifact was considered to interfere with interpretation.

Dirofilaria immitis immunoassay, NT-proBNP assay, and assessment of coagulation variables

After CT was completed, a jugular blood sample (5 mL) was obtained from each cat and used for coagulation tests (prothrombin and activated partial thromboplastin times), a D immitis lateral-flow immunoassay,t and an NT-proBNP assay.u Blood collected for detection of antibodies against D immitis was allowed to sit undisturbed at room temperature (21°C) for 20 minutes to clot; it then was centrifuged to separate the serum. Serum was harvested and stored frozen (−80°C) until used for testing.

Statistical analysis

Data distributions were assessed for normality with Shapiro-Wilk tests. Nonparametric data were logit transformed, and pairwise comparisons between BAL techniques were made with the Wilcoxon signed rank test. A multivariate ANOVA was used to compare the effect of BAL technique and lung lobe of sample collection. Analyses were performed with a statistical software program.v Values were considered significant at P < 0.05.

Results

Animals

The 12 cats enrolled in the study consisted of 7 castrated males and 5 spayed females. Mean ± SD age was 6.2 ± 0.87 years (range, 4.5 to 6.9 years), and mean body weight was 5.3 ± 1.2 kg (range, 3.1 to 7.0 kg). All cats had unremarkable findings for physical examination and results of a CBC, serum biochemical analysis, fecal flotation, and ELISA for FIV and FeLV; measurement of total thyroxine concentration; and thoracic radiography.

Bronchoscopy and BAL

Bronchoscopic evaluation did not detect gross changes in any of the cats. Duration of BAL did not differ significantly (P = 0.35) between B-BAL (median, 28 minutes; range, 12 to 50 minutes) and NB-BAL (median, 28 minutes; range, 21 to 47 minutes; Table 1). A significantly (P = 0.01) greater number of attempts was required to catheterize the lung lobe of interest when performing NB-BAL (median, 1.5 attempts; range, 1 to 6 attempts) than when performing B-BAL (median, 1 attempt; range, 1 to 2 attempts). For NB-BAL, successful catheterization of the lung lobe was achieved on the first attempt in only 6 of 12 catheterizations. Median total volume of infusate was 1.89 mL/kg (range, 1.43 to 3.18 mL/kg). Depth of wedging was significantly (P = 0.01) greater for B-BAL (median, 90.25 mm; range, 72.80 to 122.00 mm) than for NB-BAL (median, 81.60 mm; range, 67.20 to 92.90 mm). The proportion of BALF retrieved was significantly (P = 0.01) greater for B-BAL (median, 70%; range, 10% to 100%) than for NB-BAL (median, 55%; range, 20% to 80%).

Table 1—

Values for procedure variables and results of cytologic analysis for NB-BAL and B-BAL in 12 cats.

FactorVariableNB-BALB-BALDifference*P value†
BALDuration (min)28 (21–48)28 (12–50)00.35
 Lowest Spo2 (%)78.0 (54–87)82.5 (58–92)–4.50.46
 Infusate retrieved (%)55 (20–80)70 (10–100)–150.01
 Length of catheter at wedging (mm)81.6 (67.2–92.9)90.3 (72.8–22.0)–8.650.01
 No. of attempts to wedge catheter in bronchus1.5 (1–6)1 (1–2)0.50.01
 TNCC (×109 cells/L)1.00 (0.37–2.80)1.04 (0.31–3.80)–0.040.80
Cytologic score‡Cellularity3 (1–4)3 (1–4)00.72
 Cell preservation4 (0–4)4 (2–4)01.00
 RBCs0.5 (0–3)0 (0–3)0.50.20
 Epithelial cells1 (1–3)1 (0–4)00.58
 Bacteria0 (0–1)0 (0–0)00.50
 Hemosiderophages3 (2–4)3 (1–4)00.71
 Mucus1 (0–4)2 (0–4)–10.42
Cell typeMacrophages (%)68.5 (24–90)70.0 (41–91)–1.50.85
 Lymphocytes (%)5.0 (1–17)5.5 (1–23)–0.50.85
 Eosinophils (%)13.0 (0–73)10.0 (0–28)30.20
 Neutrophils (%)8.5 (0–36)9.5 (2–36)–10.27
 Mast cells (%)0 (0–1)0 (0–0)01.00

Values reported are median (range).

Represents median value of NB-BAL minus median value of B-BAL.

Values were considered significant at P < 0.05.

Each variable for this factor was scored by use of criteria described elsewhere2,12 on a scale of 0 to 4, except for RBCs, which was scored on a scale of 0 to 3. TNCC = Total nucleated cell count.

See Appendix 2 for scoring description.

Impact of BAL on anesthetic recovery

Anesthetic recovery scores did not differ significantly (P = 1.00) between B-BAL (median, 1; range, 1 to 2) and NB-BAL (median, 1; range, 1 to 2). Anesthetic recovery score for 1 cat was 2 for both techniques, and anesthetic recovery scores for 2 other cats was 2 for one technique but not for the other technique. For these 3 cats, a prolonged period of oxygen supplementation was required after extubation to maintain Spo2 > 95%. For the remaining BAL procedures, all cats recovered without complications and did not require oxygen supplementation for > 10 minutes after extubation to maintain Spo2 > 95%. The lowest Spo2 recorded during the BAL procedures did not differ significantly (P = 0.46) between B-BAL (median, 83%; range 58% to 92%) and NB-BAL (median, 78%; range, 54% to 87%).

BALF analysis

Total or differential cell counts for macrophages, neutrophils, eosinophils, lymphocytes, and mast cells did not differ significantly between BAL techniques (Table 1). Cytologic quality of slide preparations also was not significantly different between the 2 techniques. Median cellularity score for samples obtained by both techniques was 3 (range, 1 to 4). One sample obtained by use of B-BAL and 3 samples obtained by use of NB-BAL had a cellularity score of 1. Median cell preservation score did not differ significantly for samples obtained by use of B-BAL (4; range, 2 to 4) and NB-BAL (4; range, 0 to 4). One sample obtained by use of NB-BAL yielded poor cell preservation. There were no significant differences in median scores for RBCs, epithelial cells, or mucus between B-BAL and NB-BAL.

Dark gray-blue cytoplasmic granules were detected in macrophages of BALF samples from all cats. Examination of specimens stained with Prussian blue stain indicated that these granules contained iron, which confirmed that these macrophages were hemosiderophages. A moderate to marked number of hemosiderophages was present in all samples, and the median score did not differ significantly between BAL techniques (Table 1).

Results of differential cell counts did not differ significantly between the 2 techniques. On the basis of a total nucleated cell count > 0.5 × 109 cells/L or an increased proportion of eosinophils or neutrophils, results for 12 BAL samples were consistent with findings for cats with asthma,10,12 and results for all 24 BAL samples were suggestive of bronchitis.10,12

Aerobic bacterial culture and Mycoplasma PCR assay results were negative for all BALF samples. Mycoplasma culture yielded 4 positive results. Mycoplasma felis and Ureaplasma spp were recovered in samples from 1 cat obtained by use of B-BAL and NB-BAL, respectively, and in samples from another cat obtained by use of NB-BAL and B-BAL, respectively. Three cats with positive results for culture of Mycoplasma spp or Ureaplasma spp had eosinophilic inflammation evident on cytologic examination, and 1 cat with positive results for culture of Ureaplasma spp had neutrophilic inflammation evident on cytologic examination.

CT findings

Despite the lack of clinical or radiographic signs of respiratory tract disease, CT changes were identified in 9 of 12 cats. Four cats had ground-glass opacities in the right cranial lung lobe (n = 1), right cranial and caudal lung lobes (2), and right caudal lung lobe (1); 2 cats had focal soft tissue opacities in the left cranial lung lobe; 2 cats had ground-glass opacities and soft tissue opacities in the right and left cranial lung lobes; and 1 cat had a diffuse reticular pattern affecting all lung lobes. Four cats had lesions in the right caudal lung lobe. Of these cats, 3 also had CT lesions in another lung field where BAL had not been performed (cranial lung lobe). Eight cats had evidence of thickened bronchi on thoracic CT that had not been evident on thoracic radiographs. Seven of these 8 cats had BALF with higher eosinophil (n = 3), neutrophil (2), or eosinophil and neutrophil (2) counts in BALF samples obtained with both techniques.

D immitis immunoassay, NT-proBNP assay, and assessment of coagulation variables

All cats had negative results for antibodies against D immitis, and coagulation times were within reference intervals. One cat had a high NT-proBNP concentration (175 pmol/L). Echocardiography of that cat revealed hypertrophic cardiomyopathy. The NT-proBNP concentration for all other cats (median, 61 pmol/L; range, 24 to 85 pmol/L) was within the reference interval (< 100 pmol/L).

Discussion

In the study reported here, both B-BAL and NB-BAL were relatively easily performed and yielded samples with no significant differences in quality. Both techniques allowed for collection of samples from specific lung lobes, which is useful in cats by use of small airway or pulmonary parenchymal disease because cytologic findings can differ among lung lobes.7 Samples were obtained from only the caudal lung fields, but it would be of interest to compare the feasibility of collection of samples from other lung fields with these techniques. Use of fluoroscopy to guide the catheter for NB-BAL could be useful when a small-diameter bronchoscope is not available.

Maintenance of an intubated airway throughout the entire BAL procedure in each cat likely contributed to the low complication rate. Laryngospasm from repeated intubation and insertion of the bronchoscope during bronchoscopy was avoided,13 and the risk of bronchospasm was reduced by premedication with bronchodilating agents.9 Therefore, NB-BAL may constitute a safe procedure in intubated cats when bronchoscopy is not available. A limitation of NB-BAL is the inability to visually examine the airway mucosa, but the appearance of the mucosa did not contribute important information for cats with respiratory tract disease in another study.8

To allow for retrieval of BALF from the same lung lobes and comparison of BALF cytologic findings between the 2 techniques, fluoroscopy was used to determine catheter location and to guide sample collection for NB-BAL. Therefore, the NB-BAL technique described was a modification of the blind NB-BAL technique. For the blind NB-BAL technique, the catheter is wedged in the distal portion of a bronchus where lavage is initiated, and the location of sample collection is unknown. Compared with results for the blind NB-BAL technique, use of fluoroscopy to confirm the location of sample collection for NB-BAL would have increased the procedural time and number of attempts required to wedge the catheter in the desired location.

The proportion of BALF retrieved ranged from 55% to 80%, which is consistent with other reports1,4–6 that involved highly variable infusion volumes. A significantly greater proportion of BALF was retrieved with B-BAL than with NB-BAL, which likely was attributable to differences in instrumentation, depth of wedging, and body position.

Outer diameter of the bronchoscope and catheter was similar for B-BAL and NB-BAL; however, rigidity, internal diameter, and length of tube differed and could have affected the pressure required to aspirate and retrieve fluid. To minimize pressure differences between instruments, a mechanical suction unit could have been used to provide consistent continuous aspiration of BALF. Use of such an aspiration technique for cats has not been evaluated, but improved retrieval of BALF from dogs by use of this technique has been reported.2,3,14

Because the bronchoscopic technique allowed for direct visual examination of the airways, it was not surprising that it resulted in a greater wedging depth. Visual examination was not possible during NB-BAL; therefore, incomplete wedging of the catheter may have led to leakage of infusate into other lung lobes and less fluid retrieval, compared with fluid retrieval for B-BAL.

Differences in body position also may have contributed to differences in the depth of wedging and percentage of fluid retrieved, as described elsewhere.15 Although samples were collected from the caudal lung lobes for both techniques, differences in body position may have influenced the portion of the lung field that was infused and the location where the infused fluid pooled.

Surprisingly, positioning the cats in lateral recumbency did not result in the NB-BAL catheter readily entering the dependent lung lobe, as was suggested in another report.4 Rather, the catheter was not in the correct location on the first attempt for half of the NB-BAL procedures. Fluoroscopy was useful for visual evaluation of the location and the passage of the catheter. If fluoroscopy were to be used to guide sample collection for NB-BAL, it would likely be feasible to perform this technique on cats positioned in sternal recumbency, which would be beneficial for visual evaluation of both the left and right lung lobes with minimal superimposition and should facilitate access to and sample collection from all lung lobes.

Discrepant results for Mycoplasma culture and PCR assay were found for 4 BALF samples. It has been suggested in some studies,16,17 but not all studies, that PCR assay is more sensitive than culture. In the present study, samples that had positive results for Mycoplasma culture yielded only low numbers of Mycoplasma spp or Ureaplasma spp, and the Mycoplasma PCR assay had a higher limit of detection than did Mycoplasma culture. Therefore, it was feasible that a PCR assay may yield negative results, despite positive results for culture. Discordant Mycoplasma culture results between the 2 BAL techniques could have arisen from transient nonpathogenic colonization of the small airways or pulmonary parenchyma with Mycoplasma spp or Ureaplasma spp or failure of growth after transient storage in saline solution.18,19 Ureaplasma spp were cultured from 1 NB-BAL sample with neutrophilic inflammation. That same cat had neutrophilic inflammation in the sample collected by use of B-BAL, but Ureaplasma spp were not identified. Considering the absence of clinical or radiographic signs suggestive of Ureaplasma infection, it was most likely that a positive culture result reflected nonpathogenic colonization of the small airways or pulmonary parenchyma. In cats with clinical signs of small airway or pulmonary parenchymal disease, results of Mycoplasma culture should be considered in conjunction with findings of thoracic imaging and BALF cytologic examination to determine the potential clinical relevance.19

In the study reported here, 43 of 48 samples had cellularity and cell preservation scores ≥ 2, which were considered to be of adequate diagnostic quality.20,21 Five samples had low cellularity scores (3 NB-BAL and 1 B-BAL) or excessive cell lysis (1 NB-BAL). Potential causes of low cell yield on glass slides despite adequate cell counts include excessive mucus content of samples that prevents cell deposition on slides during cytocentrifugation, inclusion of a large number of epithelial cells in the BALF, inadequate mixing of samples, and processing errors.22–26 Of the 4 samples with low cellularity scores, 2 were from BALs with limited fluid retrieval (approx 30%), and 2 were from BALs with adequate fluid retrieval (50% to 80%).21,27 For those samples, there was no evidence of admixture of epithelial cells or excessive mucus content, and the nucleated cell count was within the reference interval. Therefore, it was most likely that samples were not adequately mixed to provide a homogeneous cell distribution prior to cytocentrifugation. Cell lysis can result from excessive suction applied during BALF retrieval, excessive centrifugal force during cytocentrifugation, or exposure to hypotonic fluids. The cause of the limited quality of the 5 slides was not investigated.

Hemosiderophages were present in all samples. Hemosiderophages are not considered a typical finding in BALF of clinically normal cats, but they are common in cats with conditions such as asthma, neoplasia, heartworm disease, coagulopathies, and cardiac disease.15,28 Considering that hemosiderophages were present in the first BAL samples, it was unlikely that they were the result of hemorrhage during the procedure because of the short procedure time and the time required for acute hemorrhage to manifest as hemosiderophages. Heartworm disease, coagulopathies, cardiac disease, and bronchopneumonia were ruled out on the basis of negative antibody test results, coagulation times within reference intervals, NT-proBNP concentrations within the reference interval, and negative results for bacterial culture, respectively. One cat had hypertrophic cardiomyopathy as indicated by echocardiographic findings and a high NT-proBNP concentration, but there was no evidence of congestive heart failure. Therefore, the BALF cytologic findings for 10 cats of the study were consistent with asthma or chronic bronchitis, despite the fact the cats were clinically normal at the time of enrollment. The CT examination revealed that thick bronchial walls were more common in cats with eosinophilic than neutrophilic inflammation, which is also consistent with asthma.29–32 The proportion of eosinophils in BALF obtained from clinically normal cats was < 7%33,34 and < 17 %.1,33 Such discrepancies may result from differences in antigenic exposure, animal housing density, air quality, and other factors associated with group housing. For the purposes of the present study, we used reference limits listed in another study.12 Pulmonary changes were identified with thoracic CT, but not radiography, in all cats, except for 3, at study entry, which is consistent with reports7,15 of the relative insensitivity of thoracic radiography for the diagnosis of structural changes in cats with disease of the small airways or pulmonary parenchyma. Ideally, thoracic CT would have been performed on all cats before the start of the study, but because we did not anticipate that cats would have asthma, radiography was considered sufficient to rule out major respiratory tract disease. Lesions identified by use of CT were considered to indicate true lung disease because the lung lobes that were not lavaged also were affected. Furthermore, considering there was evidence of eosinophilic or neutrophilic (or both) inflammation, it was likely that most cats had subclinical asthma or bronchitis (or both). Histologic or cytologic examinations might have further characterized the lesions,7,15 but they were not performed.

The study had several limitations. The 2 BAL techniques were evaluated in research cats deemed to be healthy on the basis of results of physical examination, screening hematologic evaluation, and thoracic radiography. The low anesthetic and procedural complication rates for both BAL techniques may have been attributable, in part, to the health status of the subjects, and these rates might be higher in cats with respiratory compromise. Cytologic findings for BALF were abnormal in most cats. Although cats were found to have subclinical asthma or bronchitis (or both), these conditions were considered unlikely to have impacted our comparison of the results for the BAL techniques and might even be more relevant for patients requiring BAL for diagnostic investigation. Furthermore, the crossover design should have limited the impacts of any procedure-associated effects. Cats in research colonies are housed in environments that differ from those for client-owned cats, and research cats may be exposed to a greater density of inhaled allergens, which would account for subclinical asthma. Another limitation was the relatively small sample size, which may have resulted in type II errors.

In the present study, NB-BAL was not inferior to B-BAL regarding ease of performance, patient anesthetic stability, and quality of cytologic preparations for cats with no clinical signs of respiratory tract disease. Although B-BAL allowed for greater depth of wedging, visual examination of the bronchial mucosa, and greater fluid retrieval, there were no significant differences in cytologic quality or differential cell counts between techniques. Future studies may incorporate fluoroscopy during NB-BAL to guide placement of catheters to reach specific lung lobes or focal lung lesions.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Hooi to the Department of Clinical Studies at the University of Guelph as partial fulfillment of the requirements for a Doctor of Veterinary Science Degree.

Supported by the Pet Trust Foundation at the University of Guelph.

Presented in abstract form at the 2016 American College of Veterinary Internal Medicine Forum, Denver, June 2016.

The authors thank Drs. Lynne O'Sullivan and Shari Raheb for assistance with cardiac evaluation of cats and Dr. Alex zur Linden for assistance with CT imaging of cats.

ABBREVIATIONS

BAL

Bronchoalveolar lavage

BALF

Bronchoalveolar lavage fluid

B-BAL

Bronchoscopic bronchoalveolar lavage

NB-BAL

Nonbronchoscopic bronchoalveolar lavage

NT-proBNP

N-terminal pro B–type natriuretic protein

Spo2

Oxygen saturation measured by pulse oximetry

Footnotes

a.

Chiron Compounding Pharmacy, Guelph, ON, Canada.

b.

Dexdomitor, Zoetis Canada Inc, Kirkland, QC, Canada.

c.

Torbugesic, Zoetis Canada Inc, Kirkland, QC, Canada.

d.

Fresenius Kabi Canada Ltd, Richmond Hill, ON, Canada.

e.

Lidodan Endotracheal, Odan Laboratories, Point-Claire, QC, Canada.

f.

Sheridan/CF endotracheal tube, Teleflex Medical Canada Inc, Markham, ON, Canada.

g.

Kangaroo polyvinyl chloride feeding tube with radioopaque line, Covidien, Saint Laurent, QC, Canada.

h.

Sizing catheter, Infiniti Medical, Menlo Park, Calif.

i.

Rusch soft rubber bladder catheter, Teleflex Medical Canada Inc, Markham, ON, Canada.

j.

Weasel Wire, Infiniti Medical, Menlo Park, Calif.

k.

BV Endura Mobile C-arm, Philips Healthcare, Markham, ON, Canada.

l.

Flex X, Karl Storz Endoscopy Ltd, Mississauga, ON, Canada.

m.

Monoject 20-mL syringe with Leur-lock tip, Covidien, Mansfield, Mass.

n.

Plastic multipurpose tubing adapter, Cook Inc, Bloomington, Ind.

o.

Animal Health Laboratory, University of Guelph, Guelph, ON, Canada.

p.

Universal mycoplasma detection kit (ATCC 30–1012K), ATCC, Manassas, Va.

q.

Z2 Coulter counter, Beckman Coulter, Mississauga, ON, Canada.

r.

Shandon Cytospin 4, Thermo Fisher Scientific Inc, Waltham, Mass.

s.

GE Bright Speed, General Electric Healthcare, Milwaukee, Wis.

t.

Heska Solo Step FH, Heska, Barrie, ON, Canada.

u.

Cardiopet proBNP test–feline, IDEXX Laboratories Inc, Markham, ON, Canada.

v.

SAS, version 9.3, SAS Institute Inc, Cary, NC.

References

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Appendix 1

Description of scoring system used to assess anesthetic recovery of cats after BAL.

ScoreDescription
1Very calm, smooth, and no excitement; no additional sedation required; and no oxygen supplementation required > 10 min after extubation to maintain Spo2 ≥ 95%.
2Mild excitement; no additional sedation required; and oxygen supplementation needed for 10 to 30 min after extubation to maintain Spo2 ≥ 95%.
3Severe excitement; additional sedation required; and oxygen supplementation needed for 30 to 60 min after extubation to maintain Spo2 ≥ 95%.
4Airway complication requiring reintubation; additional sedation required; cardiac arrest followed by successful resuscitation or oxygen supplementation needed for > 60 min after extubation to maintain Spo2 ≥ 95%; and cat transferred to intensive care unit.
5Cardiac arrest without successful resuscitation (death or euthanasia).

Appendix 2

Criteria for microscopic assessment of cytocentrifuge preparations of BALF obtained from 12 cats by use of B-BAL and NB-BAL.

VariableScoreDefinition
Cellularity0< 10
(No. of leukocytes/slide)110–100
 2101–200
 3201–500
 4≥ 501
Cell preservation0< 10
(% of well-preserved cells/slide)110–25
 226–50
 351–80
 4≥ 81
Epithelial cells (No. of cells/slide)0Absent
 1< 50
 251–100
 3101–200
 4≥ 201
RBCs (percentage/slide)0≤ 1
 12–3
 24–5
 3≥ 6
Bacteria (No. of organisms/slide)0Absent
 1< 5
 26–10
 311–20
 4≥ 21
Hemosiderophages (No. of cells/slide)0Absent
 1< 5
 26–10
 311–20
 4≥ 21
Mucus (No. of strands/slide)0Absent
 1< 3
 24–6
 37–10
 4≥ 11

Scoring system was created by use of criteria described elsewhere2,10 (Adapted from Woods KS, Defarges AM, Abrams-Ogg AC, et al. Comparison between manual aspiration via polyethylene tubing and aspiration via a suction pump with a suction trap connection for performing bronchoalveolar lavage in healthy dogs. Am J Vet Res 2013;74:523–529. Reprinted with permission).

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