We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.
We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in > 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.
Background
Fiberoptic bronchoscopy was developed for clinical practice; it is widely used both diagnostically and therapeutically1,2. Bronchoalveolar lavage (BAL) removes nonadherent cells and lung lining fluid from the mucosal surface; biopsy is used to sample mucosal and submucosal tissues. In research environments, BAL allows sampling of innate (lung macrophages3-5), cellular (B- and T- cells6), and humoral (immunoglobulin7) responses within the lung.
BAL is internationally accepted for research purposes8 and since 1999 the technique has been performed in >1,000 subjects in the UK and Malawi by our group. We use this technique in studies of innate, cellular, and humoral immune responses to pneumococcal antigen including experimental human pneumococcal carriage9,17, biomass smoke5, HIV and vaccination, and adjunct treatment studies into recovery from pneumonia. Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid.
In a research context BAL utilizes a different technique from that practiced by respiratory and intensive care physicians (often termed bronchial wash, washings, lavage, or BAL) who are aiming to gain diagnostic or therapeutic benefit. The research technique is designed to harvest cells and preserve viability to facilitate the growth of cells in ex vivo culture. For these reasons the research technique uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage. Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory1.
Goal
The overall goal is that the procedure should be safe and effective. Subjects should not experience any physiological disturbance and operators should consistently collect in excess of 100 ml of BAL from subjects. After the procedure, subjects should experience minimal side effects.
Safety
Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria. This protocol includes a description of the potential risks and the steps taken to mitigate them.
Contraindications to research bronchoscopy may be expressed as absolute or relative, and are included in our study protocol as exclusion and inclusion criteria. Our subjects are all screened to ensure full health.
Absolute contraindications include unstable cervical spine, unresponsive hypoxia, unstable angina, bleeding diathesis, and malignant cardiac arrhythmia.
Relative contraindications, including those conditions associated with increased complication rates, include:
General: poorly cooperative subject1, any significant general medical problem e.g. epilepsy, previous poorly tolerated bronchoscopy, known adverse reactions to lidocaine or midazolam, pregnancy, poor nutrition.
Respiratory: hypoxia [saturations (sats) on air <94%], hypercapnia, unstable asthma1, significantly impaired respiratory function1 (FEV1< 1 L), pulmonary hypertension.
Cardiovascular: uremia, within 6 weeks of myocardial infarction1, superior vena cava obstruction.
Other: immunosuppression (our group regularly performs this procedure in HIV positive subjects).
See Table 1 – Risks associated with BAL for research.
1. The Subjects are Met on Arrival by the Respiratory Research Nurse
2. The Subject is Prepared for the Procedure in the Suite by a Respiratory Research Clinician Experienced in Bronchoscopy
3. The Bronchoscope is Inserted and Positioned
4. The BAL is Performed
5. Subject Recovery
6. Cells are Isolated in the Laboratory – Perform on Melting Ice Where Possible
Good clinical results are a BAL volume of over 100 ml and a subject who has experienced at most, minor discomfort (see Table 1). This requires a relaxed and cooperative subject. The clinician must be confident, fully prepared (Materials), and understanding towards the volunteer.
Inter- and intra-subject variability in cellular yield (including differential counts) has been described. Differential cell counts from healthy subjects are typically >90% macrophages with small numbers of neutrophils (<2%) and lymphocytes (5-10%). BAL cells centrifuged onto microscope slides can be stained with a differential nuclear stain to obtain this information (Figure 1). In Figure 1, BAL cells have been fixed and stained using Hemacolor staining according to the manufacturer’s instructions. At least 300 cells should be counted to obtain reliable results for macrophages, neutrophils and lymphocytes; 500 cells for rarer cell types.
Hazard | Risk | Reduced in this protocol by: |
Effect of bronchoscopy and BAL1,14 | ||
Mild discomfort | <25%14,# | Appropriate analgesia and sedation. Confident and caring approach to the subject. |
Epistaxis | <1%# | Mild pressure for nasal intubation only. If subject discomfort or narrowed aperture – oral intubation instead. |
Endobronchial hemorrhage (hemoptysis) | <0.1%*15 | No biopsies taken. Careful control during bronchoscopy – avoiding respiratory mucosa. |
Nausea, vomiting and aspiration pneumonia | <0.02%14,# | Fasting for solid food >4 hr preprocedure1. Adequate topical anesthesia. Semirecumbent position during procedure. Careful post-procedure observation. |
Fever | 0.0114 – 1%#,16 | Relates to inflammation, and may minimized by maximal collection of BAL. Single lobe BAL only.# |
Sore nose/throat and hoarseness | <25%14,# | Adequate topical anesthesia of the nose, throat and larynx (minimising coughing). |
Infection | <0.1% in HIV negative, 1% in HIV positive14,# |
Standard bronchoscope washing procedure1. Single lobe BAL only. # Early recognition of infection: clinical examination within 1 hr and clinical contact 1-5 days after bronchoscopy. |
Chest pain/ cough | <0.2%14,# | Single lobe BAL only. Maximal BAL collection.# |
Effects of drugs: lidocaine, midazolam | ||
Arrhythmia | Very rare* | Maximum of 5 mg/kg14 lidocaine used. Warmed normal saline instilled. # Pulse oximetry (sats >90% with oxygen supplementation), cardiac monitoring throughout procedure. Benzodiazepine antagonist (flumazenil) immediately available. |
Disorientation / agitation | <0.1%†# | |
Cardio respiratory depression | Very rare* |
* Not experienced in our group in over 1,500 research procedures.
† Idiosyncratic rather than dose-dependent reaction.
# Our group’s experience in over 1,500 research procedures.
Table 1. Risks associated with BAL for research. This includes the hazard or risk and how this is reduced by using the described technique. Effects of the bronchoscopy/BAL and also the effects of any medicinal drugs used are included.
Figure 1. A Microscope Slide Showing BAL Macrophages and Occasional Neutrophils. BAL cells have been fixed in methanol and stained with Hemacolor red (eosin Y) and blue (azure B).
In order to achieve good results the following are helpful:
The authors have nothing to disclose.
Thank you to Elena Mitsi (Research assistant) and Sr. Carole Hancock (Research nurse) and theatre staff for their contribution to filming, and to David Shaw (research nurse) for his support with our research bronchoscopies. Thanks also to our volunteer Rebecca Dunphy for allowing this procedure to be filmed.
Material |
Company |
Catalogue Number |
Comments |
Fibreoptic bronchoscope, light and suction source |
unspecified |
||
Surgical gown, sterile gloves |
unspecified |
||
Sphygnomanometer |
unspecified |
For continuous patient monitoring |
|
Pulse oximeter |
unspecified |
For continuous patient monitoring |
|
3 lead ECG monitor |
unspecified |
For continuous patient monitoring |
|
Nasal oxygen delivery 2-4l/min |
unspecified |
||
Lidocaine 10% spray (Xylocaine) |
AstraZeneca |
For topical anaesthesia |
|
Lidocaine hydrochloride 2% |
unspecified |
For topical anaesthesia |
|
Lidocaine gel (Instillagel) |
Farco-Pharma, Germany |
Alternative products available from other suppliers |
|
Normal saline, sterile 200ml |
unspecified |
Warm to 30oC |
|
10ml taper-end syringe (x10) |
unspecified |
For administration of lidocaine |
|
Intravenous cannula 18G |
unspecified |
For administration of midazolam |
|
Midazolam |
unspecified |
For sedation |
|
Flumazenil |
unspecified |
Reversal of benzodiazepine sedation (emergency use only) |
|
60ml taper-end syringes (x4) |
unspecified |
For normal saline injection and BAL fluid withdrawal. May require connector to attach to the injection port of the bronchoscope |
|
Sterile gauze swab |
Vernaid, UK |
Alternative products available from other suppliers |
|
Sterile container for BAL fluid |
See text and comments |
Siliconised glass bottles reduce macrophage adherence. Alternatively, 50ml centrifuge tubes may be used (total capacity 200ml) |
|
Sigmacote |
Sigma, UK |
SL2 |
Only if siliconised glass bottles used. NB: Harmful and flammable. If used, follow precautions detailed in manufacturer’s MSDS |
Centrifuge |
unspecified |
At least 4x50ml tube capacity. Refrigeration to 4oC preferred. |
|
Shandon Cytospin centrifuge |
Thermo Scientific, UK |
For differential count only |
|
RPMI 1640 medium with L-glutamine |
Sigma, UK |
R8758 |
Alternative products available from other suppliers |
Fetal bovine serum |
Sigma, UK |
F6178 |
Alternative products available from other suppliers |
Penicillin-Streptomycin |
Sigma, UK |
P4333 |
Alternative products available from other suppliers |
Amphotericin B |
Sigma, UK |
A2942 |
Alternative products available from other suppliers |
Tissue culture plates |
Greiner Bio-One, UK |
662160 |
Alternative products available from other suppliers |
Glass microscope slides |
unspecified |
For differential count only |
|
Shandon cytofunnel |
Thermo Scientific, UK |
A78710003 |
For differential count only |
Shandon cytoclip |
Thermo Scientific, UK |
59910052 |
For differential count only |
Hemacolor staining set (fixative, red and blue reagents) |
Merck, Germany |
111661 |
Use according to manufacturer’s instructions |