EQUINE VETERINARY EDUCATION. Equine uet. Educ. (1999) 11 (6) 330-336. Tutorial Article. Bronchoalveolar lavage technique and cytological diagnosis.
EQUINE VETERINARY EDUCATION Equine uet. Educ. (1999) 11 (6) 330-336
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Tutorial Article Bronchoalveolar lavage technique and cytological diagnosis of small airway inflammatory disease A. M. HOFFMAN
Department of Clinical Sciences, l h f t s University School of Veterinary Medicine, 200 Westboro Rd, North Grafton, Massachusetts 01536, USA.
Introduction
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Bronchoalveolar lavage (BAL) is a method to recover a sample of cells that occupy the lining of the airways and alveoli (Viel 1980; Hoffman et al. 1997~).Cytology of BAL provides a microscopic ‘window’on the status of tissues adjacent to airspaces, that exude cells into airspaces. For instance, it is a sensitive diagnostic test for small airway inflammatory disease (SAID)or the more advanced form, chronic obstructive pulmonary disease (COPD). Despite clear evidence that BAL cytology can be used to diagnosis SAID, it is under-utilised as a diagnostic tool. Preliminary diagnosis of SAID can be made, based on history (signs of exercise intolerance, cough), endoscopic findings (mucus visible in the airways), radiographic findings (lung interstitial o r bronchointerstitial patterns) and results of lung functions testing (see p 322). However, BAL evaluation provides the specific cell types affecting each horse which ultimately impacts on treatment decisions. Furthermore, BAL assessment permits one to confirm that the lung is a source of poor performance (i.e. due to SAID or EIPH), especially when coupled with lung function testing. At the same time, BAL allows one to evaluate multiple problems in the respiratory tract, such as upper and lower airway disease, or SAID and exercise-induced pulmonary haemorrhage (EIPH), or diffuse infection (e.g. septic bronchitis) and SAID. The technique of performing BAL and cytology in SAID are discussed in this paper. Case examples that demonstrate how lung function tests and BAL complement each other in the outpatient diagnosis of SAID are provided at the end of this article.
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Technique of BAL The method for BAL was devised originally for horses by Viel (1980),using an endoscope and, later, a nonendoscopic technique was described (Fogarty 1990). The technique was reviewed recently (Hoffman and Viel 1997). The procedure can be performed in the field assuming one can assure excellent restraint of the horse. There are several aspects of restraint that are important. First, the horse should not move its head about, therefore shifting the position of the BAL tube or endoscope employed for BAL.
Second, coughing should be reduced to a minimum during the procedure. Third, a short acting sedative is appropriate since the procedure takes less than 10 min to complete.
To achieve these goals, one might choose to administer i.v. xylazine (0.8 mgkg bwt) or detomidine (0.01 mgkg), with or without i.v. butorphanol (0.02-0.04 mgkg bwt) to obtain adequate sedation. Xylazine and detomidine have bronchodilatory properties that may decrease coughing and bronchospasm as well. A twitch may also be necessary to avoid head tossing and to facilitate passing the instrument into the nasal cavity or trachea. Although we do not place the horse in stocks of any kind for BAL a t our clinic, this has been a preference elsewhere. To reduce the likelihood of cough, a bronchodilator is administered, prior to BAL. A rapidly acting bronchodilator such as albuterol aerosol (450 pg), ipratropium bromide aerosol or i.v. aminophylline (1 mgkg bwt. qs 500 ml 0.9% NaC1) is used. To provide further restraint and minimise coughing and discomfort, a solution of lidocaine without epinephrine (150 ml, 0.5%)is warmed (37°C)and prepared in large syringes (60 ml) for instillation into the airways during the passage of the tube (see below). A commercial tube (BAL tubell, or >2 m endoscope can be used for BAL. An endoscope is unnecessary unless one wishes to visualise other structures. A tube is our preference, as it reduces the need to clean the endoscope, and has an inflation cuff behind the tip, which reduces spillage of the instilled saline. A small amount of lubricant is smeared on the tip. The BAL tube is passed through the ventral meatus into the pharynx. Pre-measuring the tube t o a position just rostra1 to the glottis ( 3 0 4 0 cm from the opening of the nares) can help to estimate position. A small amount of the lignocaine solution is then instilled blindly onto the rima glottis. Waiting 1 or 2 min a t this point allows the lidocaine to take affect. Next, stretch the head of the horse into a horizontal position relative t o the ground and advance the tube into the trachea, as if intubating the horse for anaesthesia. A complete lack of resistance, absence of a swallowing response and aspiration of air confirm that the tube is properly positioned in the trachea, rather than the oesophagus. Once the tube is in the trachea, the position of the head can be returned to natural angle.
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Figs la-e):Bronchoalveolar cytology (1000 x mag, WrightGiemsa stain). (a) An alveolar macrophage (AM)and a darkly granulated must cell (MC)are shown in a sample from a normal horse. (b) From a horse with exerciseinduced pulmonary haemorrhage, a typical haemosiderin-laden macrophage (haemosiderophage, HSP) and lymphocyte (L).(c) In a horse with marked small airway inflammatory disease is shown neutrophils (PMN),mast cells (MC)and a n alveolar macrophage (AM).(d) A n eosinophil (EOS) and alveolar macrophage in a normal horse. (e) Fibrillar mucus containing predominantly neutrophils was recovered from a horse with chronic obstructive pulmonary disease (100 x mag, Wright-Giema stain).
A normal horse coughs several times, but a horse with small airway disease may cough repetitively, if it has not been pretreated with a bronchodilator. Additional boluses (20-30 ml) of lidocaine can be instilled a t this point. Advance the tube a t a moderate pace, until there is gentle resistance. One can also sense the rhythmic rostral-caudal movement of the lung and diaphragm if the tube is properly wedged into an airway. Next, the cuff of the BAL tube is inflated with
air (10 ml) and the tube secured in position by pressing the tube against the nasal septum. Make sure the head stays in a comfortable, but relatively fixed position, to avoid movement of the tube. A solution administration set is connected (e.g. via dispenser pin) to a rigid plastic bottle of warmed (approximately 37°C)saline (500 ml), and attach the distal end of the solution set is attached to the Bivona tube via a 3-way stopcock. An insumation bulb is attached via a short length of tubing to the plastic bottle. One aliquot of 250 ml(1/2 of bottle) is instilled under the pressure of the insufflation bulb. Gentle suction, using a metered pump
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Specimen handling and cytological diagnosis of SAID
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The 2 aliquots are pooled and a well-mixed sample placed into large tubes (5-10 ml) containing EDTA for centrifugation (600 x g, 5 min). It is important to centrifuge the samples to obtain an adequate number of cells to examine microscopically. In many horses, the sample requires cytocentrifugation for concentration of cells onto a slide. It is also paramount to air dry the
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The method of lavaging the airways with an unwedged tube is actually a bronchial lavage. The fluid recovered is typically dilute, cells are scarce, and reference ranges for cell differentials of BAL samples can not be used for interpretation.
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Fig 2: Age is compared to the percentage of cells (neutrophils, must cells or eosinophils) counted in BAL fluid samples. The technique used is described in detail in the text. Briefly, sterile saline was infused in 2 x 260 aliquots, recovered sequentially and pooled for analysis. Five hundred cells were enumerated. Fmm these graphs one can see that neutrophils tend to increase with age of the horse, and other inflammatory cells are found in younger horses. In part, this relates to the influx of neutrophils in BAL fluid from horses with COPD, resulting in a 'dilution'of the other cell types.
(10-15 cm HzO) or series of syringes can be used to remove the fluid. Next, the second aliquot (250 ml) is instilled, and similarly recovered. Volumes recovered for the first aliquot (50-150 ml) are lower than for the second aliquot (150-300 ml), with an expected total volume recovered around 250 ml.
smears as quickly as possible to preserve cell quality, using a fan if necessary.Air-dried smears can be stained with Diff-Quik, Wright-Giemsa, MayGruenwald, or similar stains. Prussian blue stain might be used if one desires staining of haemosiderin. Toluidine blue stain can be employed to better visualise mast cells. Smears are examined a t high power (900-1000 x) and S O 0 cells are examined (Fig 1). A number of studies have reported 'normal' values for BAL cell differentials in their sample population (Viel 1980; Derksen et al. 1985, 1989; Fogarty 1990; Fogarty and Buckley 1991; Vrins et al. 1991). In general, BAL was a repeatable method. Perhaps the most important technical factor that influenced 'normal' values reported in the literature, were differences in the volume of fluid instilled for lavage. If smaller volumes (e.g. 300 ml) were instilled, the volume recovered is lower and the cell differential was richer in mast cells and neutrophils observed (Sweeney et al. 1992). There were only minor differences detected in lung cytology of the left and right lung (Sweeney et al. 1992, 1994). Using these techniques, a normal cell differential for the equine BAL in our clinic includes 40-70% macrophages, 30-60% lymphocytes, 6 mg/ml), demonstrating airway hyper-reactivity. Bronchoalveolar lavage cytology: Alveolar macrophages 58%, lymphocytes 32%, mast cells 6% (elevated), and neutrophils 4%; a few scattered epithelial cells, goblet cells, and fine mucus strands; no bacteria seen.
Deatment A mast cell blocker was selected; disodium cromoglycte aerosol was given 3 x day and albuterol aerosol (450 pg) was given at least 30 min before exercise, for the first 2 weeks of treatment. Lung function tests were repeated after 30 days with medications withdrawn for 24 h. Baseline RRS (cmH2ONs) at 1Hz = 0.55; 2 Hz = 0.48; 3 Hz = 0.46, demonstrating normal RRS and negligible frequency dependence. F,,, (resonant frequency) was within normal limits (2.0 Hz).
Histamine challenge: PClOO RRS = 8.4 mg/ml (normal >6 mg/ml), demonstrating normal airway reactivity. Long-term control: Disodium cromoglycate or Nedocromil sodium.
Case 3
History
Lung function tests: Baseline RRS (cm H20Ns) a t 1 Hz = 0.88, 2 Hz = 0.67, 3 Hz = 0.65, demonstrating a n elevated baseline RRS and frequency dependence. Fre, (resonant frequency) was also elevated (2.8 Hz). Histamine challenge: PC~OORRS= 2.4 mg/ml (normal >6 mg/ml), demonstrating marked airway hyper-reactivity. Bronchoalveolar lavage cytology: Alveolar macrophages 44%, lymphocytes 32%, mast cells 4% and neutrophils 15%; fine mucus strands. No bacteria seen. Interpretation: increased mast cells and neutrophils, excess mucus; evidence of SAID.
Deatment Treatment was given with a Fluticasone aerosol.
Clinical response: coughing at the beginning of the ride, still observed after 30 days. The horse had much more energy and is back to full work. Lung function tests after 30 days: Baseline RRS (cmH2ONs) at 1 Hz = 0.56; 2 Hz = 0.55; 3 Hz = 0.61, demonstrating a normal RRS, and no frequency dependence. F, (resonant frequency) dropped to within normal limits (2.2 Hz). Histamine challenge: PClOO RRS = 4.4 mg/ml (normal >6 mg/ml), demonstrating airway hyper-reactivity, but to a lesser extent. Long-term control plan: Fluticasone aerosol, 1720 pg once per day. Lung function tests after 90 days: Baseline RRS (cmH2ONs) at 1 Hz = 0.33; 2 Hz = 0.35; 3 Hz = 0.34; demonstrating a further reduction in RRS and fres (1.8 Hz), and frequency dependence of Rm was absent. At this time, the horse was clinically normal, and competing at the expected level.
A 12-year-old Dutch Warmblood gelding used for dressage presented with a history of exercise intolerance, lethargy, lack of impulsion and cough; prior history of seasonal cough. The temperature, pulse, respiratory rate and auscultation and percussion of the thorax were within normal limits. After rebreathing into a plastic bag, the horse coughed productively a few times.
Long-term management: This horse was prescribed Fluticasone, at 860-1720 pg per day, indefinitely. “he drug was withdrawn at appropriate periods before competitions.
Previous management
A 22-year-old Quarter Horse mare presented with seasonal cough and exercise intolerance; this summer, the horse shows signs of laboured breathing (‘heaves’) which is controlled temporarily with dexamethasone injections. This mare had laminitis and the use of systemic corticosteroids was a serious concern of the owner.
Antihistamines, occasional use of oral dexamethasone. Environmental management consisted of removing hay from the diet and maximising turnout. There was no improvement in 2 months.
Case 4
History
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Previous management The cough was controlled with clenbuterol syrup (1.6 pgkg ) given on a n ‘as needed’ basis and intermittent treatment with dexamethasone (40 mg i.v. a s needed); the mare was fed haylage and housed out of doors a t all times.
Results Radiographs: Moderate bronchointerstitial pattern and hyperinflation of lung. Lung function tests: Baseline RRS (cmH2ONs) a t 1 Hz = 2.4, 2 Hz = 0.88, 3 Hz = 0.89, demonstrating a high baseline Rm, and marked frequency dependence. F,,, was markedly elevated (6.8 Hz). Bronchodilator challenge: Albuterol (450 pg) was administered and the following lung function data was obtained: RRS (cmH~O/l/s)at 1 Hz = 0.96,2 Hz = 0.76,3 Hz = 0.66; fres decreased to 3.4 Hz. The absolute values for RRS, frequency dependence and f,,, decreased significantly, indicating t h a t albuterol or similar beta-2 adrenoceptor agonists would effectively control bronchospasm. Bronchoalveolar lavage cytology: Alveolar macrophages 12%, lymphocytes 16%, mast cells 2%, and neutrophils 70%; mucus, goblet cells observed; no bacteria seen.
Deatment
Manufacturer’s address ‘Bivona Medical Technologies, Gary Illinois, USA
References Derksen, F.J., Scott, J.S., Miller, D.C., Slocombe, R.F. and Robinson, N.E. (1985) Bronchoalveolar lavage in ponies with recurrent airway obstruction (Heaves). Am. Rev. Resp. Dis. 132,1066-1073. Derksen, F.J., Brown, C.M., Sonea, I., Darien, B.J. and Robinson, N.E. (1989) Comparison of transtracheal aspirate and bronchoalveolar lavage cytology in 50 horses with chronic lung diesease. Equine vet. J . 21,23-26. Fogarty, U. (1990) Evaluation of a bronchoalveolar lavage technique. Equine vet. J . 22,174-176. Fogarty, U. and Buckley, T. (1991) Bronchoalveolar lavage findings in horses with exercise intolerance. Equine vet. J . 23, 434-437. Hare, J.E. and Viel, L. (1998) Pulmonary eosinophilia associated with increased airway responsiveness in young racing horses. J . vet. 2nt. Med. 12, 163-170. Hoffman, A. and Viel, L. (1997) Techniques for sampling the respiratory tract of horses. Vet. Clin. N . Am.: Equine Pract. 13, 463-475. Hoffman, A. and Mazan, M.R. (1999) Programme of lung function testing horses suspected with small airway disease. Equine vet. Educ. 11,322-328. Hoffman, A,, Mazan, M.R. and Manjerovic, N. (1998a)Association between airway reactivity and bronchoalveolar lavage cytology in horses with exercise intolerance. Am. J . vet. Res. 59, 176-181.
Prednisone (0.8 mgkg 1 week, then 0.6 mgkg 1 week, then 0.4 mgkg for 2 weeks); starting on the third week of treatment, fluticasone aerosol (1720 pg twice per day) was started; salmeterol aerosol was used to control cough and bronchospasm for the first 2 weeks, then discontinued.
Hoffman, A.M., Lilly, C.M., Umkauf, L. and Ellenberg, S. (1998b3 Elevated Leukotriene C4 in BAL fluid from horses with cough and exercise intolerance. Proceedings of the World Airways Symposium. p 25.
Lung function tests after 30 days: Baseline RRS (cmHzO/l/s) a t 1 Hz = 1.24; 2 Hz = 0.88; 3 Hz = 0.65; demonstrating frequency dependence. F,,, (resonant frequency) was improved (3.6 Hz).
Rush-Moore, B., Krakowka, S., Cummins, J.M. and Robertson, J.T. (1994) Efficacy of oral interferon-alpha for treatment of inflammatory airway disease in standardbred racehorses. Proceedings of the 23th Veteterinary Respiratory Symposium. p 19.
Bronchodilator challenge: Albuterol (450 pg) was administered and the following lung function data was obtained: RRS (cmH20/l/sjat 1Hz = 0.66,2 Hz = 0.70,3 Hz = 0.65. This is considered a n significant bronchodilator response, and might indicate that there is room for ongoing improvement in baseline lung function.
Sweeney, C.R., Rossier, Y., Ziemer, E.L. and Lindborg, S.R. (1994) Effect of prior lavage on bronchoalveolar lavage fluid cell population of lavaged and unlavaged lung segments in horses. Am. J . uet. Res. 55, 1501-1504.
Long term control: Fluticasone aerosol, 1720 pg b.i.d. Lung function tests in 90 days: Baseline RRS (cmH2ONs) at 1 Hz = 1.0, 2 Hz = 0.80, 3 Hz = 0.66, demonstrating frequency dependence. f,,, (resonant frequency) was improved (3.0 Hz). Overall there was very slight improvement.
Mair, T.S., Stokes, C.R. and Bourne, F.J. (1987) Cellular content of secretions obtained by lavage from different levels of the equine respiratory tract. Equine vet. J . 19, 458-462.
Sweeney, C.R., Rossier, Y., Ziemer, E.L. and Lindborg, S. (1992) Effects of lung site and fluid volume on results of bronchoalveolar lavage fluid analysis in horses. Am. J . vet. Res. 53,1376-1379. Viel, L. (1980) Structural-Functional Correlations of the Lung in the Light Horse. MS Thesis, University of Guelph, Canada. Vrins, A,, Doucet, M. and Nunez-Ochoa, L. (1991)A retrospective study of bronchoalveolar lavage cytology in horses with clinical findings of small airway disease. J . vet. med. A. 38, 472-479.
