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Altered coagulation and fibrinolysis have been found in the lungs of patients with ILD and adult respiratory distress syndrome and in the blood of patients with diseases and injuries that can involve the lung. None of these studies, however, examined the relationship between lung and blood coagulation abnormalities concurrently. In the present study, BAL D dimer was found in the majority of patients with sarcoidosis, but there was no correlation with plasma D dimer levels. While BAL D dimer may be marker of pulmonary disease activity, plasma D dimer may be a marker of systemic disease activity that frequently accompanies sarcoidosis and other types of ILD. Our study did not examine any association between circulating D dimer and systemic disease activity; however, activation of the blood coagulation system has been demonstrated in a variety of systemic disorders and has been shown to be a sensitive and temporally related measure of disease activity.- Blood fibrino-peptide A levels have been found to correlate with disease activity in systemic lupus erythematosus and Crohn’s disease. Circulating fibrin split products may indicate an increased risk of developing adult respiratory distress syndrome and may serve as a marker of persistent respiratory distress in this condition.
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Although we demonstrated that D dimer could be found simultaneously in the lungs and blood of some patients with sarcoidosis, there were some in whom D dimer was found in only the lungs or the blood. D dimer may have crossed from the blood into the alveolar space in patients with detectable levels in both compartments. However, BAL and plasma D dimer levels adjusted to total protein levels, and BAL D dimer levels adjusted for the loss of D dimer during BAL fluid concentration showed that patients had 70 to 500 times higher levels of D dimer per milligram of total protein in BAL fluids than in the plasma. These adjustments indicate that most, if not all, of the BAL D dimer was generated within the pulmonary compartment and could have leaked into the systemic circulation. We were surprised to find that the sarcoidosis patient with the highest BAL D dimer levels did not have detectable D dimer in the plasma. It is possible that D dimer in the lungs may not have crossed to the circulation, but this is unlikely, since very high BAL protein levels, suggesting alveolar-capillary leak, were found in this individual. Another possibility is that D dimer may have gained access to the blood but was rapidly degraded so that fibrin fragments with epitopes both to the capture and tag antibodies were absent. Finally, a particular factor in this patients plasma could have inhibited D dimer detection by EIA, but 12 serial dilutions of the plasma did not unmask any inhibitory effect in the D dimer assay.
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It is not surprising that fibrinogen or FDP were not found in the BAL fluids of healthy individuals. Fibrinogen (MW 340,000) is not among the proteins found by BAL in normal lungs. Therefore, no fibrin formation, cross-linking, or degradation should occur in the intact normal lung, though normal lung tissue and alveolar lining fluid contain other procoagulant and fibrinolytic factors including tissue factor and plasminogen. Permeability of the alveolar-capillary membrane is probably the most important factor that regulates the influx of fibrinogen into the extravascular spaces of the lung. Alveolar-capillary permeability is increased in acute and chronic pulmonary inflammation, allowing even the largest plasma proteins to enter the alveolar space. Fibrinogen leaks from the plasma into the pulmonary interstitium and alveolar spaces of patients with sarcoidosis and other ILD where fibrin turnover ensues forming various FDP. Enhanced procoagulant activity and inhibited plasminogen activator activity likely shift the balance of fibrin turnover in ILD toward fibrin formation. This may create a constant fibrin “burden” from which D dimer and other FDP can be generated continuously during active inflammation. Once the inflammation resolves, the balance of fibrin turnover may return to a profibrinolytic state, clearing deposited fibrin and FDP
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Discussion
We were able to detect activation of the coagulation system in the lungs and blood of patients with sarcoidosis. Eight of 10 sarcoidosis patients had detectable levels of BAL D dimer, while none of 18 healthy subjects had detectable BAL D dimer. Our findings are in agreement with those of Nakstad et al who measured FDP by latex agglutination in the unconcentrated BAL fluids of patients with ILD. They found FDP in the BAL fluids of 6 of 22 sarcoidosis and 10 of 29 idiopathic pulmonary fibrosis patients and no FDP in the BAL fluids of 60 healthy individuals. We found a higher prevalence of BAL fluid FDP in our sarcoidosis patients probably because we used EIA, which is more sensitive than agglutination methods for antigen detection.
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Bronchoalveolar Lavage D Dimer and Alveolitis
The eight sarcoidosis patients with BAL D dimer had significantly higher levels of BAL cells per milliliter and total protein than healthy volunteers (Table 2). The two individuals with sarcoidosis and no BAL D dimer were indistinguishable from the healthy group except that one patient had a higher than normal percentage of lymphocytes in BAL fluid. The percentage of lymphocytes was approximately twofold greater in sarcoidosis patients with measurable BAL D dimer, giving them approximately 6.5 times more lymphocytes per milliliter of BAL fluid than the two patients without BAL D dimer. Comparisons for significant differences between sarcoidosis patients with BAL D dimer and those without were not made because of the small sample sizes.
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Results
Bronchoalveolar Lavage and Plasma D Dimer
The D dimer levels in the BAL fluids and plasma of healthy volunteers and sarcoidosis patients are displayed in Figure 1. None of the 18 healthy volunteers had detectable BAL D dimer, but three had plasma D dimer at the lowest level of detection. Eight of ten patients with sarcoidosis had detectable levels of BAL D dimer, and seven had detectable plasma levels. Plasma D dimer did not correlate with BAL D dimer in these patients (r= —0.554, p = 0.10). Table 1 lists the clinical features of all ten patients with sarcoidosis.

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Autoradiography using the method of Connaghan et al was performed on BAL fluids from healthy subjects and patients with sarcoidosis as evidence that the EIA used in this study was detecting FDP containing D dimer. Autoradiography also gave a qualitative assessment of fibrinogen and the range of FDP in BAL fluids. Soluble fibrin (Desafib-X, Biopool, Umea, Sweden), D dimer (American Diagnostica, New York), and human plasma as a source of fibrinogen were used as references. Cels were cast from a 2 percent agarose suspension in 0.1 sodium dodecyl sulfate 0.05 M phosphate buffer, pH 7.00. The reference and BAL samples were boiled for 5 min in 0.1 percent sodium dodecyl sulfate and mixed with methylene blue marker dye. They were applied to a gel at a final concentration of 3 mg of protein per milliliter and electropho-resced on the cooled cell (10°C) of an LKB 2117 Multiphor apparatus for 60 min. The gels were fixed, incubated with l“I-labeled goat anti-human fibrinogen antibody (Organon Teknica Corp, West Chester, Pa), washed, and exposed to Kodak X-omat AR film.
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Preliminary experiments were performed to determine how the concentration of BAL fluids affected recovery of D dimer and whether BAL fluids inhibited D dimer measurement by EIA. Plasma was obtained from patients with disseminated intravascular coagulation, pooled, and diluted serially with normal saline solution. The undiluted and diluted plasma samples were screened for D dimer by latex agglutination (Dimertest II Latex, American Diagnostica, NY) and were found to have D dimer at a level between 3,200 and, 6,400 ng/ml of plasma as expected for patients with disseminated intravascular coagulation.

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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.
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Materials and Methods

Subjects

Blood samples and BAL fluids were obtained from 18 healthy volunteers and 10 patients with biopsy-proven sarcoidosis. Informed consent was obtained following a protocol approved by the Human Investigations Committee of the Emory University School of Medicine.
All subjects underwent standardized history and physical examination by one of two authors (G.W.S. or R.L.P.). Spirometry was performed with equipment meeting American Thoracic Society guidelines using the regression equations of Crapo and colleagues to determine the percent of the predicted normal value. Healthy volunteers were accepted for study if they had no respiratory symptoms within one month of the study, normal posteroan-terior radiographs, and pulmonary function within the normal range for age, height, and sex. Chest radiographs of sarcoidosis patients were classified as follows: type 0, no adenopathy or infiltrates; type 1, hilar adenopathy only; type 2, hilar adenopathy with parenchymal infiltrates; type 3, parenchymal infiltrates only.
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