Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

The overall aim of this procedure is to perform bronchoalveolar lavage in a safe and effective manner. This is accomplished firstly by ensuring the safety, cooperation and comfort of the subjects which were selected. Using clear inclusion and exclusion criteria.

Pre-procedural checks and observations are also performed. Then once in the bronchoscopy room, after all equipment is checked, the subject is given local anesthetic and offered conscious sedation. Next, after intubation of the vocal cords, the bronchoscope is advanced to a good position within the RML.

This ensures a good BAL fluid volume yield. Finally, the BAL fluid is obtained using gentle handheld suction and is then transported without delay on ice for immediate laboratory processing. Ultimately, this technique harvest cells and preserves viability to facilitate the growth of cells in ex vivo culture.

The main advantage of this technique over existing methods practiced by respiratory and intensive care physicians, often term bronchial wash washings lavage, or bronchi alveolar lavage or bowel who are aiming to gain diagnostic or therapeutic benefit is that our technique uses gentle handheld suction of instilled fluid. This is designed to maximize bowel volume returned and apply minimal she force to the ciliated epithelia in order to preserve the structure and function of cells within the bowel fluid. The bronchoalveolar lavage procedure is performed as a day case with appropriate pre and post procedure checks as per standard hospital policy, including ensuring that the written consent form is correctly signed and dated.

Upon arrival, the subject is met by the respiratory research nurse who then performs all necessary pre-procedure checks, including ensuring that the subject has been fasting for a minimum of four hours for solid food and two hours for liquids. If the subject meets all of the required criteria, they then change into a hospital gown and pre-procedure observations, including blood pressure, heart rate, and oxygen saturation measurements are taken. A small gauge cannula is inserted ideally into the left hand.

This will remain in C two until the end of the post-procedure recovery period. The subject is then transferred to the bronchoscopy suite where an experienced respiratory research bronchoscopist is present. Monitoring equipment is then attached to the subject.

This includes a three lead ECG, A, fig o manometer, and a pulse oximeter. Anesthetic support is not needed. Nasal cannula are applied to all volunteers before the bronchoscope is inserted, oxygen is delivered if saturations drop below 96%at up to four liters per minute.

At this point, the operator performs pre-procedure checks on the bronchoscope. This includes ensuring effective suction by aspirating sterile normal saline. Once the operator is sure that the bronchoscope is performing optimally, topical anesthesia is administered to the nasal passages and the oral mucosa of the subject by the application of instill agel and Xylocaine respectively.

If sedation is appropriate and has been requested by the subject, an appropriate dose of midazolam is administered intravenously. As with all procedures involving anesthesia, an appropriate reversal agent should be immediately available. In addition, ensure that resuscitation equipment is also on hand.

The subject is intubated via the nose. If this is not possible, then the subject may be intubated via the mouth as long as an appropriate mouth card is used at the larynx. Topical analgesia is administered using four to six milliliters of 4%lidocaine once the vocal cords are passed.

Further, mucosal analgesia is administered using two milliliter aliquots of 2%lidocaine at the carina at the division of the right lower lobe and right middle lobe or RML and at the RML entrance. Finally, the bronchoscope reaches the right middle lobe and ideally is positioned in the medial segment in a secure distal position. Perform the wink test using the suction button to ensure that the bronchoscope is not positioned in too distal a position which may cause airway collapse.

Four 60 milliliter syringes are prefilled with a 60 milliliter volume, two 50 milliliter volumes, and a 40 milliliter volume of warmed normal sterile saline. When the subject and bronchoscopist are the first syringe of saline is instilled by the bronchoscopy assistant. Whilst the bronchoscopist maintains the position in the RML, then the assistant performs gentle hand suction using the same port and syringe.

The procedure is then repeated a further three times using each of the syringes in succession with a maximal volume of 200 milliliters used in this specific technique, the retrieved BAL fluid is expelled gently into plastic containers that may be pres siliconized already held on melting ice. Pres ization inhibits cell attachment and maximizes cell return. The BAL fluid appears hazy against the light with surface soap bubbles that are formed by proteins and surfactants.

Finally, the bronchoscope is slowly, fully withdrawn. The subject proceeds to award for monitored recovery and the BAL fluid is transported without delay on ice. For immediate processing, allow the subject to recover for up to four hours in the care of a respiratory research nurse.

During this time, allow the subject to rest but not eat or drink until at least 60 minutes after the procedure. Once their ability to swallow is assessed as safe, this is to reduce the risk of aspiration. Post-procedural observations are monitored and recorded.

A clinical examination occurs prior to discharge. The subject is also briefed about the common side effects and given a contact number in case significant side effects occur. A follow-up appointment with the subject is performed one to five days post procedure by a doctor or nurse.

This may be either over the telephone or in person. Once in the research laboratory, perform all manipulations on wet ice. Where possible.

First, record the volume of bronchoalveolar lavage fluid. Then filter the fluid through a double layer of sterile gauze swab to remove mucus plugs and collect the filtrate into sterile 50 milliliters. Centrifuge tubes pellet the cells by centrifugation at 500 G at four degrees Celsius for five to 10 minutes.

Remove the super natin and wash with 50 milliliters of cold normal saline. Repeat the centrifugation. Once more, resuspend the cells in an appropriate volume of culture medium.

Usually two milliliters will give a good cell density for counting. Then perform cell counting using a hemo, cytometer and trian blue according to standard procedures, adjust the density of the cell suspension to one times 10 to the six cells per milliliter by adding an appropriate volume of media. Then transfer one milliliter of the cell suspension into each well of a 24.

Well plate incubate the cells at 37 degrees Celsius for three hours to allow macrophage adherence for lymphocyte work. After three hours of incubation, the culture medium is gently pipetted up and down three times and collected. This medium contains lymphocytes and non adherent macrophages and may be used to assess lymphocyte function.

It may also be used in further purification steps for macrophage work. After three hours of incubation, carefully aspirate the media and replace with fresh, warm medium without antibiotics, the macrophages are adherent to the tissue culture plate here. BAL cells have been centrifuged onto a microscope slide using a thermo shandin cyto centrifuge and stained with a differential nuclear stain to reveal the cell types.

Most of the cells are macrophages such as the cell labeled M, but the occasional neutrophil labeled N and lymphocyte labeled L are seen. After watching this video, you should have a good understanding of how to safely and effectively perform bronchoscopy and bowel for research purposes using this technique importantly, including good anesthesi of the base of the tongue and the posterior oropharynx, checking the bronchoscope for effective suction and the absence of air leaks pre procedure and choosing the most appropriate right middle lobe. Sub segmental branch operators should consistently collect in excess of a hundred mils of bowel from subjects who experience minimal side effects.