Total Protein Assay
Total protein in unconcentrated and concentrated BAL fluids and plasma was determined using a modification of the Coomassie brilliant blue dye-binding colorimetric assay (Bio-Rad Laboratories, Richmond, Calif). One hundred sixty microliters of BAL fluid diluted with normal saline solution (in a ratio of 1:5 times for unconcentrated samples and 1:10 to 1:30 times for concentrated samples) or plasma diluted 2,500 times were added to the wells of 96-well microtiter plates (Flow Laboratories, McLean, Va). Fifty microliters of normal saline solution containing 0.0125 percent sodium dodecyl sulfate was added to decrease optical differences between different protein species.” Finally, 40 microliters of Coo-massie brilliant blue dye was added and the color allowed to develop for 15 min at room temperature. Dilutions of a bovine serum albumin and gamma globulin mixture in a ratio of65:35, respectively, were used to make standard curves for each plate. Absorbance was read at 595 nm with a Bio-Rad model 3550 microplate reader.
Bronchoalveolar Lavage and Plasma Fibrinogen and Fibrinogen Degradation Products
Bronchoalveolar lavage fluid and plasma levels of D dimer were determined simultaneously in all study subjects. The D dimer was measured by enzyme immunoassay (EIA) using the monoclonal antibody DD-3B6/22 as the capture antibody and the peroxidase-conjugated monoclonal antibody DD-42/182 as the tag antibody (Dimertest EIA kit, American Diagnostica, New York). These antibodies detect separate epitopes on FDP containing the crosslinked D dimer configuration. We used the lowest Idt concentration standard of 78 ngftnl as the detection limit of the assay. The D dimer levels in the blood are expressed as nanograms per milliliter of plasma and in the lung as nanograms per milliliter of concentrated BAL fluid.