Type 3 Procollagen Peptide in Bronchoalveolar Lavage Fluid: Methods

18 Nov

Study Population

Eighty four patients (37 women, 47 men) with sarcoidosis verified by biopsy, mean age 33.4 (range 20 to 64) years, underwent bronchoalveolar lavage. Sixty-one were newly presenting at the time of lavage and the median duration of disease from diagnosis in the remaining patients was 33 (range 6 to 126) months. None of the patients was receiving cortocosteroid therapy at the time of investigation. Twenty were current smokers. Four healthy volunteers and seven hospitalized patients without lung disease were included in the study as a control group, (one woman, ten men; mean age, 30.3 years). Pulmonary function tests, performed before lavage, indicated normal lung (unction in the control subjects. All subjects gave their informed consent for lavage.

Bronchoalveolar Lavage
Before bronchoscopy, patients were given intramuscular atropine (0.6 mg) and pethidine (50 mg) and the upper respiratory tract was anesthetized with lignocaine. A fiberoptic bronchoscope was securely wedged in a subsegmental bronchus in the right middle lobe, 180 ml of sterile 0.9 percent saline solution at 37°C infused in three 60 ml aliquots, and gentle suction applied after each infusion. The volume of the aspirated fluid was recorded and the fluid strained through sterile surgical gauze to remove mucus. The fluid was then centrifuged at 400 g for 15 minutes and the supernatant stored at – 20°C for subsequent analysis.

Analysis of Lavage Fluid
Cells removed from the lavage fluid were resuspended in Hanks’ balanced salt solution to a concentration of 2 x 10 cells/ml. Total T-lymphocytes and T-lymphocyte subsets were analyzed by fluorescent microscopy using murine monoclonal antibodies as previously described.® Before analysis, lavage fluids were centrifuged at 1,000 g for 15 minutes. Phenylmethylsulphonylfluoride (PMSF, final concentration 0.1 mM) was added to prevent protease digestion and the fluid concentrated (x20) by ultrafiltration in membrane cones. To minimize protein loss on concentration, the fluids were first concentrated by a factor of 50 to 100 and the cone membranes washed three times with aliquots of the filtrate. These were then combined to give final x 20 concentrates. Lavage fluid N terminal type 3 procollagen peptide was analyzed by radioimmunoassay. Studies by other workers have established that this assay system is specific for the aminoterminal sequence of type 3 procollagen and the antibody employed does not cross react with type 1 collagen, type 1 procollagen, 7s collagen, laminin or fibronectin. In our laboratory, the detection limit of the assay was found to be 0.2 ng per ml of concentrated fluid. Lavage fluids were also analyzed for fibronectin, angiotensin converting enzyme (ACE), and protein.