Analytical Procedure

DOE-SPEC-1142-2001

7.3.8. Lymphocytes ________%;

Alveolar macrophages ________%

Eosinophils ________ %;

Neutrophils _________%;

Basophils __________%;

RBC/WBC __________

7.3.9. BROWNISH INTRACELLULAR MATERIAL?: (YES/NO) (indicates

smoker)

7.3.10. NUCLEAR DEBRIS? (YES/NO) (indicates problems with lavage procedure)

7.4.

ANALYTICAL PROCEDURE. After the lavage sample has been received in the

BeLPT laboratory.

7.4.1. Note volume of lavage on summary sheet. Give a brief description of the

fluid (cloudy, bloody, etc.).

7.4.2. Mix fluid and remove 3 - 5 ml of lavage for CBC and differential.

7.4.3. Transfer lavage to 50 ml centrifuge tube(s), and spin for 5 minutes at 400 x g.

Wash the cells three times to be certain all surfactant proteins are removed. A

calcium and magnesium-free balanced salt solution such as Hanks or

Dulbeccos should be used for the washings.

7.4.4. After washing, re-suspend the cell pellet in the complete medium to a

concentration of 1.0 x 106 mononuclear cells/ml. Dispense 0.1 ml of cell

suspension into wells of the plate. This provides 1 x 105 cells/well. Higher cell

concentrations may produce lower counts (diminished stimulation) in the

BAL-BeLPT.

Note: It may be difficult to obtain sufficient cells to achieve the required final

cell concentration. Accordingly, the assay should be performed on the original

lavage cell preparation and a Ficoll-Hypaque separation is done only if there

are sufficient cells. The cell recovery in the gradient is considerably less than

100%. Laboratory experience in cell recovery should be the guide. In patients

with end-stage lung disease there are often increased numbers of neutrophils

which can be removed by Ficoll-Hypaque gradient centrifugation (if

PMN>20%).

7.4.5. The recommended plate arrangement requires 6 x 106 mononuclear cells in

6.0 ml medium. When sufficient cells are available, the BeLPT on the lavage

sample should be set up and harvested as described for the peripheral blood

BeLPT (see section 6.2).