I. Preoperative preparation
Same as fiberoptic bronchoscopy (fibronectomy) preoperative preparation, BAL is routinely done after fibronectomy and before biopsy. 2% lidocaine is used as local anesthetic.
BAL operation technique
1.Selection of lavage site: For diffuse interstitial lung disease, choose the right middle lobe (B1 or B5) or the left lingual segment of the lung, and for limited lung lesions, perform BAL in the corresponding bronchopulmonary segment.
The recovered liquid was immediately filtered through a double layer of sterile gauze to remove the mucus, and the total amount was recorded; it was put into a silicone plastic bottle or a silicone coated sterilized glass container (to reduce cell adhesion), placed in a thermos containing ice, and sent to the laboratory immediately. The mucus was immediately sent to the laboratory for examination.
Bronchoalveolar lavage fluid (BALF) laboratory tests
I. BALF total cell count and sorting count detection
1. The above recovered lavage fluid was packed into plastic centrifuge tubes and centrifuged at 1,200r/min for 10min at 4 ℃, and the supernatant (stock solution or 10-fold concentrate) was stored at 70 ℃ and used for the detection of soluble components.
2. The cellular components precipitated by centrifugation were washed twice with Hank’s solution (without Ca2+ and Mg2+) under the same conditions, each time for 5 min. 3-5 ml of Hank’s solution was added after the supernatant was discarded to make cell suspension. The lavage solution can also be applied to reduce cell loss.
3. Count the total number of cells in BALF on a modified Neubauer counter, usually expressed as 1 × 109/L. If the cell count is too high, the cell count should be increased. If the cell count is too high, the cell count is adjusted to 5 × 109/L by diluting with Hank’s solution, and the tube is immersed in crushed ice.
4. Cell sorting: Using a cell centrifugal smear device, add 100 μl of spare cell suspension (cell concentration of 5 × 109/ L) and centrifuge at 1 200 r/min for 10 min at 4 ℃ to spread a certain number of BALF cells directly on the slide by centrifugation. The slides were immediately dried with cold air, fixed in anhydrous ethanol for 30 min and then stained, usually with Wright or HE staining.
5.Count 200 cells under 40 times light microscope for cell sorting.
Detection of T lymphocyte subpopulation in BALF
1, Using indirect immunofluorescence method, the above obtained BALF cell components were made into cell suspension with 3~5ml of 10% calf serum RPMI 1640 culture medium.
2.Pour the cell suspension into a flat dish and incubate it for 2h in a 5% CO2 incubator at 37 ℃ to remove the alveolar macrophages.
3, Remove the cell suspension, then rinse with Hank’s solution and centrifuge once, discard the supernatant and leave 20-100μl. After the wall treatment, the alveolar macrophages were significantly reduced and the lymphocytes were relatively increased in the cell suspension.
4, the wall-treated cell suspension was divided into three small conical centrifuge tubes, 20-30μl per tube, and 20-40μl of monoclonal antibodies CD3, CD4 and CD8 were added to the specimen with a microsparger, and mixed in a refrigerator at 4 ℃ for 1~2h.
5, remove the specimen, first rinse with Hank’s solution and centrifuge 2 times at 12 000 r/min for 20s, then add 20-40μl of each goat anti-mouse fluorescent antibody and place in a refrigerator at 4 ℃ for 30min.
6. Remove the specimen and rinse it twice by centrifugation with Hank’s solution at the same speed and time, discard the supernatant and leave 20μl to mix the cells thoroughly, take 1 drop on the slide and coverslip. Count 200 lymphocytes under the fluorescence microscope and calculate the positive rate of the cells labeled with fluorescence.
A few notes:
1. The tip diameter of the fibrinoscope used for bronchoalveolar lavage should be about 5.5-6.0 mm, and it should be properly wedged into the bronchial orifice of the segment or sub-segment to prevent the mixing of airway secretions and spillage of lavage fluid and to ensure the recovery of BALF.
2.The cough reflex must be adequately suppressed during the lavage process, otherwise it is easy to cause mucosal damage to the bronchial wall and cause the mixing of lavage fluid, which also affects the recovery volume.
3, a qualified BALF specimen should be: BALF without atmospheric secretions mixed in; recovery rate > 40%, surviving cells accounted for more than 95%; red blood cells < 10% (except for trauma / bleeding factors), epithelial cells < 3% ~ 5%; smear cell morphology intact, no deformation, uniform distribution.
4. The dilution of alveolar lining fluid varies due to many factors affecting the detection of soluble components in BALF, such as perfusion volume and recovery volume, alveolar epithelial permeability, etc. Although internal or external markers are used for the standardization of BALF soluble components, there are still differences in the test results, and their clinical value is limited, so this draft does not regulate the detection method of BALF liquid components.
5.This specification is not suitable for the operation of bronchial lavage (BL) and whole lung lavage techniques with small amount of liquid.
6.The normal reference values of BALF cytology test in healthy non-smokers:
(1) Total cell fraction: total cell count (0. 9-0. 26) × 109/L, including alveolar macrophages 0. 93 ± 0. 03, lymphocytes 0. 07 ± 0. 01, neutrophils and eosinophils are < 0. 01.
(2) T lymphocyte subsets: total T cells (CD3+) 0. 7, T helper cells (CD4+) 0. 5, T suppressor cells (CD8+) 0.3, CD4+/CD8+ ratio 1. 5 to 1. 8.