The bronchoalveolar lavage has been described as a liquid biopsy, and, indeed, this is an apt description.
The bronchoalveolar lavage has been described as a liquid biopsy, and, indeed, this is an apt description. Why should the practitioner perform a BAL when pursuing a diagnosis of IAD rather than an endoscopic tracheal aspirate? First, it gives us a sample that is more representative of what is going on in the area of the small airways and alveoli, which is responsible for gas exchange. Second, it is less invasive than the tracheal aspirate, and, with practice, becomes easier to perform.
How to perform the BAL:
It is essential to have a clear picture in your mind of what your set-up and procedure will be before you proceed. Then, assemble your supplies:
· A good helper
· One 500-ml bottle OR 4X 60-ml syringes of sterile saline, warmed to approximately 90F.
· Xyalzine (0.5 – 0.8 mg/kg IV or Detomidine (0.01 – 0.02 mg/kg IV)
· Butorphenol (not necessary but VERY helpful) 0,01 – 0.02 mg/kg IV
· Aerosolized bronchodilator (optional but very useful)
· BAL tube (Bivona)
· 3-way stopcock
· 5-ml syringe
· Dilute lidocaine (10-20 mls of 0.5%)
· IV solution administration set with puffer if using bottled saline.
Adequate, even heavy sedation is critical to performing the BAL successfully. We routinely use xylazine or detomidine, and the addition of butorphenol helps to suppress the cough reflex and provides some analgesia. We find it useful to bronchodilate the horse with 5 puffs of albuterol prior to performing the lavage. It is helpful to briefly walk the owner/trainer/holder through the process before starting.
The author always warns any helpers or onlookers that it is normal and expected that the horse will experience very heavy coughing so that the owner may choose to be absent. After the horse is sedate, apply the twitch – even in heavily sedated horses, this will be useful to control the head. Clean the visible portion of the nostril to minimize contamination.
At this point, a pair of examination gloves should be donned, again, to minimize contamination. Gently insert the tube into the ventromedial meatus, and advance the tube to the level of the larynx. At this point, you will notice the horse making swallowing motions. Insert 10-20 mls of sterile dilure lidocaine to anesthetize the nasopharynx and larynx, and wait several minutes. Now, straighten the head and neck as much as possible, and pass the tube into the trachea. Horses will rarely cough until the BAL tube reaches the carina. Advance the tube slowly, stopping to rest each time the horse coughs, until the tube will no longer advance despite using gentle pressure.
At this point, fill the cuff of the tube with 8-10 mls of air. Now, rapidly infuse 120-250 mls of warmed saline, and immediately suction to retrieve the fluid. In the Lung Function Laboratory, we use a commercial pump set at -10 to – 15 cm H2O, but a large syringe can also be used to suction the fluid. Be careful not to exert too much force, as you may exceed the closing pressure of the bronchus in which you are wedged – this swill result in not being able to retrieve a sufficient amount of fluid. You should expect to retrieve between 50% and 70% of the fluid that was infused.
How to transport and process a field BAL:
Samples may be put in EDTA tubes at room temperature if they will be processed within 6 hours. They may be stored similarly for up to 24 hours if they are kept refrigerated. If you will be delayed in processing the sample, or if you will be sending the sample to a laboratory for analysis, it is very important to process the sample properly. We have particularly liked the results of adding commercial calf serum to BAL fluid in a 1:10 solution; you may also store the fluid in a 50:50 solution with ethanol. Whichever method you choose, you should try to have the sample arrive at the laboratory within 24 hours, as after 48 hours, all methods for storing BAL fluid result in some deterioration. Most laboratories will prepare the slides using the cytospin method.
It is entirely possible to process your own slides, whether you choose to examine the slides yourself, or send the slides to the appropriate laboratory. When you are first starting to perform BALs, it is probably best to send of the fluid or the slides to a cytologis who is used to examining BAL from horses. (A. Hoffman, Department of Clinical Sciences, Lung Function Laboratory, Tufts Cummings School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA 01536).
If you are preparing your own slides, you should first spin the tubes at 600 g for 10 minutes – this can be done in any tabletop centrifuge. Pour out the supernatant, - you needn't be excessively cautious, as the cells will remain firmly stuck to the side of the tube as a pellet. There will inevitably be a little bit of fluid left, as well. This is useful for resuspending the pellet with a pipette. This resuspended pellet is then used to make a slide. The traditional blood smear approach will work, but the author prefers to lightly dip a sterile swab into the fluid and then gently roll the swab across the slide in several rows. Regardless of the method for making slides it is critical to dry the slides rapidly to avoid ‘blue dot disease'.
If you stain the slides yourself, Diff Quik or any Wright-Giemsa type stain is useful for initial inspection. It is very important, however, to remember that you will not easily detect mast cells using these staining techniques, and in a large subsection of horses, excessive numbers of mast cells may be exactly the causative pathology. We routeinly stain one slide with Gram stain as well, to scan for any bacteria. If you send slides off to the laboratory, it is very important to take the time to look at the slides yourself first to make sure that they are adequate. Most cytologists would like to see at least 5 cells per high-powered field to make an accurate diagnosis.
In our laboratory, we perform both absolute cell counts as well as cell percentages; however, we primarily look at percentages, as the dilution that occurs with BAL makes it almost impossible to have an accurate cell count. We routinely use 5000 mls of saline, and expect to see 40- 60% alveolar macrophages, 40% to 60% lymphocytes, < 5% neutrophils, < 2% mast cells, and < 1% eosinophils. You will also easily detect hemosiderophages in horses with EIPH by using Diff Quik or Wright-Giemsa stains.
What does the BAL mean?
So, you've successfully performed the BAL, and you have at least 10 cells per high-powered field on your slide. How do you interpret your findings? Remember that the normal BAL has a preponderance of alveolar macrophages (~ 60%) and the remainder lymphocytes. Although up to 5% neutrophils are considered within normal limits, the normal, non-pastured horse usually has less than 1% neutrophils. There are several patterns that you will recognize in horses with non-septic inflammatory airway diseases.
The young, athletic horse with very mild signs of reduced performance – neutrophilic inflammation
This horse will have mildly elevated neutrophils (usually in the area of 10%), with scant amounts of mucus. If the horse lives in a dusty environment, you will often see particulates within the macrophages and also free within the surrounding fluid. Occasional spores may be observed free or engulfed within macrophages. If the horse is coughing, ciliated epithelial cells may be observed.
The young athletic horse with mild signs of reduced performance – mast cell inflammation
This horse has greater than 2% mast cells, and may also have increased neutrophil percentages. Otherwise, the BAL is similar to the one above. Clinically, we notice that these horses can be harder to treat successfully.
The middle-aged horse with intermittent cough +/- discharge, but without overt heaves episodes
This horse usually has markedly elevated neutrophils – up to 60%. There is often marked amounts of mucus, which may be very well-organized. Curshmans's spirals may be seen. There are frequently particulates engulfed within the macrophages and free within the fluid. If the horse is coughing, ciliated epithelial cells will often be seen.
The horse with overt heaves (recurrent airway obstruction)
This horse has markedly elevated neutrophils – often up to 90 or even 95% of the total cell count. The mucus is plentiful and well-organized. It can be difficult to determine the cell differential with confidence, as many of the cells are trapped within the copious mucus. Note well, however, that this BAL, as all the others, is NOT distinguished by bacteria. If the BAL is not done in very clean fashion, you may see bacteria and squamous cells that represent contamination from the pharynx.
Other, less usual findings
On rare occasion, horses may be found to have an eosinophilic bronchitis (normal is < 0.5% eosinophils). Horses with silicate-induced interstitial disease may have a lack of neutrophilic or mast cell inflammation; rather, they have an excess of foamy macrophages containing copious amounts of particulate matter.