Flow Cytometric Analysis of Bronchoalveolar Lavage Fluid Cells

Recommendation from the Advanced Flow Cytometry Focus Group

1. Principle

This protocol describes a procedure for the determination of cell surface markers on lymphocytes obtained from bronchoalveolar lavage fluid (BAL). Because of the potential for large numbers of macrophages and debris, a 3-color staining method utilizing CD45 gating is used to optimize recovery and purity of lymphocytes in the analysis gate. The monoclonal antibody combinations included will provide basic T cell subset information. Other monoclonal antibodies can be substituted as needed.

2. Reagents

RPMI-complete

RPMI1640

L-glutamine 2mM

Penicillin 100U/ml

Streptomycin 100ug/ml

Fetal calf serum 10%

1% paraformaldehyde

Dulbecco's PBS

FACS Lysing solution

Fluorochrome conjugated monoclonal antibodies

150 um Mesh

3. Procedure

Processing of BAL cells

1. Centrifuge BAL for 10 minutes @ 400 x g. Discard supernatant.

2. Resuspend cell pellet in RPMI-complete. Centrifuge for 10 minutes @ 400 x g. Repeat for a total of 2 washes.

3. Resuspend cells in approximately 2ml of RPMI-complete.

4. Count cells using a hemacytometer and adjust concentration to 2 - 10 x 106 /ml.

Monoclonal Antibody Staining

1. Add appropriate volume of monoclonal antibodies to 12 x 75mm tubes (see Table 1 for limited immunophenotypic panel).

2. Add 100ul of well mixed cells to each tube. Incubate for 15 minutes at room temperature in the dark.

3. Add 2ml of lysing solution (e.g. FACS lyse) and incubate for 10 minutes at room temperature in the dark.

4. Centrifuge tubes for 5 minutes @ 300 x g. Decant supernatant.

5. Add 2ml DPBS to each tube and vortex thorougly.

6. Centrifuge tube for 5 minutes @ 200 x g. Decant supernatant.

7. Resuspend cells in 0.5ml of 1% paraformaldehyde solution

Table 1:

Tube #	FITC	PE	PerCP
1	IgG1	IgG1	CD45
2	CD3	CD4	CD45
3	CD3	CD8	CD45

Flow Cytometric Analysis

1. Start up and calibrate the flow cytometer according to the laboratories standard operating procedure. Adjust compensation using the monoclonal antibody CD8 labeled with FITC, PE and third color (e.g. PerCP) as per recommendations in the extended 3- color protocol.

2. Set a region on CD45 positive / low side scatter events and collect 3000 events for each tube (Figure 2).

3. Display FL1 vs FL2 dot plots and set quadrant markers based on isotype control population (or nonstained cells if isotype control not used; Figures 3 and 4).

4. Calculations

The % CD4 positive and CD8 positive cells are determined as a percent of total Lymphocytes.

5. Controls

Laboratories must ensure the qualilty of monoclonal antibody reagents by periodic testing on a blood specimen obtained from a normal donor or on stabilized control cells.

If normal values are not obtained or the appearance of the histograms is not appropriate, a different lot of antibody should be tested. Do not use suspect lot until integrity has been confirmed.

6. Expected Values

Example histograms are presented for CD4 and CD8 phenotyping (figures 1 through 4).

7. Procedure Notes

Pre-filtering of BAL specimens through gauze prior to washing: This does not appear to be necessary in all cases and should be avoided if possible due to potential cell loss.

Depending upon cell yield, there may not be sufficient numbers of cells to stain with isotype controls. For CD4 and CD8 determinations this is not a problem. However, for dimly expressed surface markers, the isotype control should be run to guide quadrant settings. A minimum of 1 x 105 cells per tube is required for analysis.

CD45 vs SSC gating is utilized due to the presence of a large percentage of macrophages and debris in BAL specimens. If gating still appears problematic, CD14 may be used to guage lymphocyte gate purity. However, not all macrophages in BAL's express CD14.

8. Method Validation

Studies in progress

9. References

Harbeck, R.J. 1998. Immunophenotyping of Bronchoalveolar Lavage Lymphocytes. Clin. Diag. Lab. Immunol. 5:271-277.

10. Effective Date and Schedule for Review

Effective Date: August 27, 1998

Review Date: August 27, 1999

11. Authors

John L. Schmitz

Scottie Sparks

The Advanced Flow Cytometry Focus Group