Discussion

The procedure of tracheal washing (TW) and sample analysis is an important tool in the assessment and management of lower airway disease in the horse. The presence of pulmonary disease frequently produces secretions that can be collected from the trachea. Methods of collection are either by transtracheal puncture or using a flexible fibreoptic endoscope. The technique of tracheobronchial aspiration by a transtracheal approach is described by Beech (1981). There are a number of potential untoward sequelae with transtracheal aspiration, which makes it a technique of limited application. The clinical advantages of endoscopic collection of tracheobronchial secretions outweigh those of the transtracheal approach. It is a well-tolerated, simple, quick, relatively noninvasive procedure that allows visual inspection of the airways and control of the collection procedure. Whilst it is possible to obtain direct aspirates of neat secretions it is preferable that a saline wash technique is employed. A saline wash allows collection of a sample of sufficient volume and cellular content when there is little or no mucopus present and facilitates aspiration when viscous mucopus is present. By using a fixed volume of saline a degree of standardisation of the procedure is achieved. The technique of TW employed by the author is a variation to that described by Greet (1982) and Dixon (1995).

Equipment required for TW:
- Clean endoscope: at least 120 cm working length, but preferably 130 - 140 cm.
- Light source.
- Sterile plastic catheter: to fit down biopsy channel and of sufficient length to be able to have syringe attached, for washing and aspiration, whilst protruding from distal end of endoscope.
- Sterile saline or lactated Ringer's solution. Syringe: 20 - 50 ml. Sterile plastic collection pot of at least 20 ml volume.
- Twitch.
- Two experienced helpers.

Procedure:
- Exercise the horse prior to sampling (provided clinical assessment of the horse deems this appropriate) as this encourages secretions from airways of lungs into trachea.
- TW should be carried out within 2 h of exercise.
- Do not allow access to food once exercised and 'tie up'.
- Apply nose twitch - this restrains horse and stimulates abduction of the arytenoid cartilages, leading to easier passage of endoscope from nasopharynx into tracheal lumen.
- Sedation is only required if horse is particularly fractious.
- Keep head steady in an elevated position.
- Insert sterile catheter into biopsy channel of a clean endoscope until approximately 2 - 3 cm from distal end.
- Pass the endoscope via nares and ventral meatus for distance of approximately 35 cm.
- Allow second handler to fix scope at nares with one hand, allowing their free hand to advance endoscope under instruction.
- Visualise nasopharynx for discharges, lymphoid hyperplasia or other abnormalities.
- Advance endoscope through rima glottis into lumen of rostral trachea.
- Observe nature and amount of discharges within tracheal lumen whilst continuing to advance to approximately 90 cm from the nares.
- Advance catheter so that it is readily observed protruding from distal end of endoscope.
- Lavage the walls of the tracheal lumen with 20 ml sterile saline, (saline will flow distally). A pool of saline mixed with secretions should readily be visualised at the dependant part of the trachea near the thoracic inlet.
- Retract the catheter back into the endoscope and advance endoscope to within 1 - 2 cm of the pool of secretions.
- Advance the catheter once more and aspirate the pool of fluid.
- After collection of fluid, advance the endoscope to assess distal trachea and carina region.
- Transfer the collected tracheal aspirate from the syringe to a sterile container.
- Submit the sample to laboratory for cytology and bacteriology.

Whitwell and Greet (1984) found in their study of 233 TW from 191 horses that the cells and debris washed from the walls and lumen of the trachea were nonhomogenous with some derived from the trachea and some from the more distal airways and alveoli so analysis of TW should be representative of lower airway disease status.
The amount of mucopus observed within the trachea can be graded to reflect whether it is absent or present in scanty, moderate or profuse amounts. Whitwell and Greet (1994) defined disease by finding an increased amount of visible mucopus during endoscopy, an increased proportion of neutrophils and nucleated cell count over 1000 cells/ml in the wash. This author does not quantify cell counts in the assessment of the presence of disease, but relies on visual assessment of the airways, presence and amount of mucopus, cytology, particularly the proportion of inflammatory cells, and quantitative bacteriology in assessing disease, though he does take into account semi-quantitive subjective assessments of cell numbers. As commented by Lane (1984), attempts to quantify the cell counts are questionable since it is influenced by the technique used, volume of dilution with saline and volume of discharge present.
Some concerns arise with endoscopic collection of TW samples as to the value of bacteriology, with the potential contamination of samples with bacterial and cellular components of the upper respiratory tract. It is important that the procedure is carried out rapidly and that the endoscope is smoothly passed from the upper respiratory tract to avoid contamination into the trachea. Frequently samples produce no bacterial growth indicating that upper respiratory tract contamination can be avoided. It is important that quantitative bacteriology is carried out, as high numbers (>103 colony forming units per ml) of particular bacterial species are likely to be significant.