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Effect of Bronchial Lavage Volume on Cellular and Protein Recovery
Effect of Bronchial Lavage Volume on cellular and Protein Recovery· Stephen Lam, M.D., F.C.C.P.;]ean C. Leriche, M.B., Ch.B.; Karin Kijek, R. T.; and Don PhiUips, R. T.
To investigate the optimal lavage technique to study the airway response in patients with asthma, differential volume lavage was carried out in 22 normal subjects and 18 patients with red cedar asthma. Ten mI of ftuid was instilled into a main-stem bronchus followed by 5, 10, 20, 50 and 100 mI into different segmental bronchi. Small volume lavage «20 m1) in a main stem bronchus or a segmental bronchus recovered more epithelial cells and neutrophils while a lavage volume of 20-100 mI in a segmental bronchus recovered more lymphocytes, alveolar macrophages, albumin, IgG and alpha-entitrypsfn, In patients with
asthma, a higher porportion of epithelial cells was observed in the 20 ml segmental bronchus lavage 24 to 48 hours after bronchial challenge with plicatic acid, the chemical compound responsible for red cedar asthma. There was an increase in eosinophils and IgG with all volume lavages, but the changes were most marked in the 10 ml main bronchus lavage and the 10 and 20 mI segmental bronchus lavage. The results show the importance of using a systematic lavage protocol to characterize the cellular and protein changes in the airways of patients with asthma. A single volume lavage in one site may not encompass all the changes observed.
A,though there are numerous reports on the use of bronchoalveolar lavage to study various lung diseases,':" the optimal lavage technique to study airway diseases had not been examined. The technique of sampling bronchial fluid varies from direct bronchoscopic aspiratiorr'" to saline lavage at different sites. 4-6.8-~ The volume of fluid used for lavage ranges from 1 ml to 300 ml in these studies. Even within a single study, the volume of lavage fluid infused may vary from one patient to anothenv':" The site of lavage was also different. The tip of the bronchoscope was in the trachea, 5. 7 carina," a main bronchus.t?" or a
based on the clinical history and confirmed by bronchial challenge with plicatic acid (the chemical compound responsible for red cedar asthma). The clinical features of red cedar asthma had been reported in detail previously. 13
subsegmental bronchus. 8.11.~
To what extent these
variations influence the composition of the lavage fluid is not known. The purpose ofthis study is to determine the effect of the lavage volume on the cellular and protein recovery in normal subjects and patients with bronchial asthma. MATERIALS AND METHODS
Subjects Forty subjects were studied. This includes 22 normal healthy individuals and 18 patients with occupational asthma due to exposure to Western red cedar. The mean age of the normal subjects was 25 years (range 18 to 44 years). The mean age of the patients with asthma was 33 years (range 19 to 53 years). All subjects were nonsmokers. The normal subjects had no respiratory symptom or recent respiratory illness within the past three years. They also hadnormal results of spirometry. For the red cedar patients, the diagnosis was made ·From the Respiratory Division, Vancouver General Hospital, University of British Columbia and the Cancer Control Agency of British Columbia, Vancouver, Canada. Supported by the Medical Research Council of Canada, Grant No. MA-1703. Manuscript received March 15; revision accepted June 18. Reprint requests: Dr: Lam, UBC Respiratory Division, Vancouver General Hospital, Main Floor, 2775 Heather Street, Vancouver, Be, Canada V5Z 3]5
868
Methods Inhalation provocation tests were performed with plicatic acid and the method of inhalation provocation was described in detail previously." The test solutions were delivered by a Bennett twin nebulizer and the average output from the nebulizer at an oxygen Row rate of 5 Umin was 0.25 mllmin. The particle size of the aerosol has a mass median diameter of 3.1 ....m. The patients were asked to breathe the nebulized aerosol at tidal breathing. On a control day, phosphate-buffered saline solution, the diluent for plicatic acid, was inhaled for 0.5, 1.0, 1.5, and 2.0 min at 100min intervals. The FEV. was measured with a 13.5 L Collins water spirometer before the inhalations, 10 min after each inhalation, and at 10 min intervals for the first hour after the final inhalation. The FEV. was then measured at hourly intervals for the next eight to ten hr and again at 24 hr after challenge. On the second day, plicatic acid (5 mg/ml in phosphatebuffered saline solution, pH 7.4) was inhaled in a similar manner at 10 min intervals until a total of 5 min inhalation has been given. The volume of plicatic acid nebulized was determined by measuring the amount left in the nebulizer. A reaction was defined as a response in which there was a decrease in the FEV. greater than 20 percent of the baseline value. All patients developed a late asthmatic reaction either alone or as part of a dual asthmatic reaction. Late asthmatic reaction usually began several hours after challenge; was maximal in four-six hours and lasted up to 24 hours or more. Dual asthmatic reaction had two components, an immediate reaction with spontaneous recovery within one-two hours followed by a late reaction. The amount of plicatic acid required to provoke an asthmatic reaction varied between 0.5 to 7 mg. Fiberoptic bronchoscopic examination was performed following premedication with 0.8 mg atropine 1M and local anesthesia with 2 percent lidocaine (Xylocaine). The asthmatic patients received salbutamol 200 ....g by inhalation 15 minutes before bronchoscopic examination. Sterile isotonic saline solution warmed to body temperature was used for lavage. In patients with red cedar asthma, bronchial lavage was carried out 24 to 48 hours following bronchial challenge with plicatic acid when their forced expiratory volume in 1 s was greater than 1.5 L. Bronchial Lavage Volume Effect (Lam et III)
Table I-Effect ofVolume on CeURecovery in Normal Subjects·
No. samples Fluid recovery (%) Cells per ml X 10- 4 Viability (%) Epithelial (%) Neutrophil (%) Macrophage (%) Lymphocyte (%) Eosinophil (%) Degenerated (%)
Main Bronchus 10ml
5 ml
10 ml
Segmental Bronchus 20ml
50 ml
100 ml
12 33±15 22.9±20.0 55±16 21±12 28±12 41±13 3±2 0 7±4
6 35±21 11.3±10.1 54±11 22±19 22±19 48±18 4±2 0 4±2
18 38±8 11.3±6.9 56±13 21±10 22±10 47±13 3±2 0 7±5
22 41±8 12.7±8.4 67±11 16±9 10±9* 63± 14* 5±4 0 5±5
7 47±9 11.3±4.0 70±14 14±8 5±2 65±10 13±8t 0 4±3
17 5O±7 14.4±5.0 78±13 5±5t 2±2 78±11 12±7 0 2±5
*Alldata shown as mean ± SO. tSignificantly different from the preceding volume; p
nostics).The lower limits for the assayswere: total protein 100....g/ml, albumin 5 g/ml, IgG 5 ....glml, llS-IgA 1 ....g/ml and alpha-antitrypsin 3 glml. All proteins were quantitated in unconcentrated fluids. The study was approved by the Ethics Committee on Human Experimentation of the University of British Columbia. Statistical Analysis
Since a maximum of four sites was used for lavage in each subject, the number of samples in each lavage volume is not similar. Comparison within the same group is made using the unpaired t-test. The results from the asthmatic subjects are compared with those from the same lavage volume in normal subjects by the unpaired t-test. RESULTS
The effect of the lavage volume on cellular and protein recovery in the 22 normal subjects is shown in Tables 1 and 2. The percentage of fluid recovered was less with smaller volume lavages, especially in the main bronchus. The total cell count per ml of aspirated fluid was slightly higher in the main bronchus, but was essentially similar in the other volume lavages. Viability of the cells recovered was higher with the large volume lavages (Table 1). The proportion of bronchial epithelial cells and neutrophils was lower when the lavage volume was greater than 20 ml. The reverse was observed with alveolar macrophages and lymphocytes where a lower proportion was found with a lavage volume of less than 20 ml (Table 1). The level of albumin and IgG increased with volume. They were significantly higher in the 100 mllavage compared with the
Table 2-Effect ofLavage Volume on Protein Content in Normal Subjects-
Protein Albumin
IgG
IgA
AAT
Main Bronchus 10 ml
5 ml
10 ml
Segmental Bronchus 20ml
SO ml
100 ml
287±138 29±19 3±6 26±21 <3
105±79 39±22 5±9 33±21 <3
125±86 26±15 7±9 22±16 <3
123±55 35±25 12±7 24±15 <7±1
141±58 43±18 12±11 13±4 8±2
142±91 78±49* 2O± ur 25±15 9±2
*Recovery of proteins expressed as ....glml of unconcentrated lavage fluid ± SO. tSignmcantly different from the preceding volume; p
AAT=alpha-antitrypsin.
CHEST I 88 I 6 I DECEMBER, 1985
857
Table 3-Effect ofVolume Oft CeU Becooery in Patienta with Asthma·
No. samples Fluid recovery (II» Cells per ml X 10- 4 Viability (II» Epithelial (II» Neutrophil (%) Macrophage (%) Lymphocyte (II» Eosinophil (If,) Degenerated (If,)
Main Bronchus 10ml
5 ml
10 ml
Segmental Bronchus 20 ml
50ml
100 ml
8 4O±21 22.7±24.6 59±13 29±19 14±9 14±12§ 2±1 27± 18§ 14±9t
4 36±30 14.6±20.7 52±4 36±30 16±23 18± 13t 2±1 21± 12; 7±5
12 35±14 10.3±7.4 55±10 27±16 15±11 21± 10§ 2±1 25±13§ 9±4
18 33± lOt 17.8±23.3 61±17 25±13t 10±6 33± 14§ 4±2 14±9§ 14± 11+
4 27± 15t 7.3±4.0 70±20 15±6 6±3 46±16 15±17 9±9+ 10±3+
13 45±11 19.9± 11.9 82±12 6±4 3±2 6O±23 16±14 4±5+ 11 ± 16+
.A1ldata shown as mean ± SO.
tSignificantly different from normal subjects in 'Iable 1; p
smaller volume lavages. IgA concentration did not vary signi6cantly with volume. Alpha-antitrypsin in low concentrations was found in approximately 70 percent of the 100 mllavage samples and 25 percent of the 20 mllavage samples. It was undetectable in the smaller volume lavages. Albumin, IgG, IgA and alpha-antitrypsin account for over 90 percent of the total protein measured with the 100 mllavage, but only 20 percent of the total protein with the main bronchus lavage (Table 2). The same lavage protocol was applied to the patients with asthma. The results were compared with that of the normal subjects. The percentage of 8uid recovered from the asthmatic patients was significantly lower with the 20 and 50 mllavages compared to the normal individuals. The total cell count per ml of fluid recovered was not significantly different in all volume lavages between the two groups (Table 3). However, the proportion of epithelial cells in small volume lavages was higher in the asthmatic patients. The results reached statistical significance for the 20 ml lavage. The proportion of eosinophils was Significantly higher in all the lavages from asthmatic patients, especially in the 10 ml main bronchus lavage and the 10 or 20 ml segmental bronchus lavage. The proportion of alveolar macrophages was significantly lower when a volume of20 ml or less was used for bronchial lavage in the asthmatic patients (Table 3). There was no signifi-
cant difference in the proportion of neutrophils or lymphocytes in all volume lavages between the two groups. The albumin content was significantly higher in the asthmatic patients when a volume of 20 ml or less was used for lavage. There was an increase in IgG content in all volume lavage in the asthmatic patients, but it was most marked in the 10 ml main bronchus lavage and the 10 or 20 ml segmental bronchus lavage (Table 4). DISCUSSION
U sing a systematic lavage protocol, we studied the effect of the site of lavage and the volume of lavage fluid instilled on the composition of 8uid recovered by aspiration. We postulate that lavage with smaller volurnes, especially in a main bronchus, would sample preferentially bronchial 8uid while larger volumes would sample fluid lining the small airways and alveoli. Bronchial fluid should contain predominantly bronchial epithelial cells, while alveolar fluid should contain predominantly alveolar macrophages. A fluid volume of greater or less than 20 ml instilled into a segmentallsubsegmental bronchus was found to be the dividing point in the present study. A fluid volume of less than 20 ml recovered more epithelial cells while a fluid volume of greater than 20 ml recovered an increasing proportion of alveolar macrophages. Neu-
Table 4-Protein Content in Lavage Fluid from Potienta with Asthma·
Protein Albumin
IgG
19A
Main Bronchus 10 ml
5 ml
10 ml
Segmental Bronchus 20ml
50 ml
100 ml
703 ± 488 275±207+ 5O± 16§ 33±12
162±73 73±41 16±7* 54±4t
240± 191 9O±63§ 28± 171 31±30
281±291 125± 189 26± 13§ 28±18
175±66 72±50 28± 17+ 17±12
268±222 106±40 36± 16+ 26±22
·Recovery of proteins expressed as JLglml of unconcentrated lavage 8uid mean ± SD. tSignificantly greater than that from normal subjects in Table 2; p
trophils are more numerous with smaller volume lavages, possibly reflecting the normal response in the more central airways to inhalation of various dusts and fumes in the environment. Lymphocytes, albumin, IgG and alpha-antitrypsin were present in higher proportion or concentration in the large volume lavages, probably reflecting the site of origin or accumulation of these cells and protein molecules in the peripheral airways and alveoli. A similar increase in albumin with volume was also observed by Rennard and Crystal" using sequential 20-ml lavages. An increase in protein content with volume was not found by others, but the size of the first lavage aliquot was 60 ml or greater and repeated washing was carried out in the same bronchopulmonary segment. 15.16 To further test the hypothesis that small volume lavage samples, preferentially bronchial fluid, we applied the same lavage protocol to a group of patients with red cedar asthma following induced bronchoconstriction. Evidence of airway inflammation with exudation of proteinaceous fluid high in albumin and IgG content, eosinophils and sloughing of bronchial epithelial cells were observed mainly in the 10 ml main stem bronchus lavage and the 10 and 20 ml segmental bronchus lavage. Some of the changes would have been missed had lavage been carried out with a fluid volume of greater than 20 ml. The 5 ml lavage was found not to be useful because it gave highly variable results. This is probably related to the large dead space of the suction channel of the bronchoscope relative to the volume of fluid instilled. The site of lavage is also potentially important. The increase in total protein, albumin and IgG in the asthmatic patients were most marked with the main stem bronchus lavage compared to segmental bronchus lavage. However; the results were more variable probably due to contamination with lidocaine, minor aspiration of oropharyngeal secretions, and mixing with secretions lower down in the respiratory tract with coughing and the normal mucociliary escalator mechanism. Despite these considerations, when investigating the changes associated with an asthmatic reaction other than cellular and protein changes, as for example, release of various chemical mediators, it might be important to lavage the main stem bronchus in addition to a segmental bronchus. Our study underlines the importance of
standardizing the site oflavage, as well as the volume of fluid instilled in the use of bronchial lavage to study airway diseases such as bronchial asthma. REFERENCES
1 Reynolds HY, Newball HH. Analysis of proteins and respiratory cells obtained from human lungs by bronchial lavage. J Lab Clin Med 1974; 84:559-73 2 Hunninghake GW, Gadek JE, Kawanami 0, Ferrans NJ, Crystal RG. Inflammatory and immune processes in human lung in health and in disease; evaluation ofbronchoalveolar lavage. Am J Patho11979; 97:149-206 3 Chernick WS, Barbero GJ. Composition of tracheobronchial secretions in cystic fibrosis of the pancreas and bronchiectasis. Pediatrics 1959; 29:739-45 4 Deuschl H, Johansson SGO. Immunoglobulins in tracheobronchialsecretion with special references to IgE. Clin Exp Immunol 1974; 16:401-12 5 Falk GA, Okinaka AJ, Siskind GW Immunoglobulins in the bronchial washings of patients with chronic obstruction pulmonary disease. Am Rev Respir Dis 1972; 105:14-21 6 Mandel MA, Dvorak KJ, Worman L~ Decosse JJ. Immunoglobulin content in the bronchial washings of patients with benign and malignant pulmonary disease. N Engl J Med 1976; 295: 694-98 7 Wiggins J, Hill SL, Stockley RA. Lung secretion sol-phase proteins: composition of sputum with secretions obtained by direct sampling. Thorax 1983; 38:102-07 8 Merrill ww, Naegel G~ Reynolds H~ Reaginic antibody in the lung lining fluid. J Lab Clin Med 1980; 96:494-500 9 Crimi E, Scordamaglia A, Crimi ~ Zupo S, Barocci S. Total and specific IgE in serum, bronchial lavage and bronchoalveolar lavage of asthmatic patients. Allergy 1983; 38:553-59 10 Keimowitz RI. Immunoglobulins in normal human tracheobronchial washings. J Lab Clio Med 1964; 63:54-9 11 TomiokaM, Ida S, Shindoh Y, Ishihara 1: TakishimaL Mast cells in bronchoalveolar lumen of patients with bronchial asthma. Am Rev Respir Dis 1984; 129:1000-05 12 Rankin JA, Snyder PE, Schachter EN, Mathay RA. Bronchoalveolar lavage. Its safety in subjects with mild asthma. Chest 1984; 85:723-28
13 Chan-Yeung M, Lam S, Koerner S. Clinical features and natural history of occupational asthma due to Western Red Cedar (Thuja plicata). Am J Med 1982; 72:411-15 14 Rennard SI, Crystal RG. Fibronectin in human bronchopulmonary lavage fluid. J Clin Invest 1981; 69:113-22 15 Merrill M, O'Hearn E, Rankin J, Naegel G, Mathay RA, Reynolds H~ Kinetic analysis of respiratory tract proteins recovered during a sequential lavage protocol. Am Rev Respir Dis 1982; 126:617-20 16 Davis GS t Giancola MS, Costanza MC, Low RB. Analysis of sequential bronchoalveolar lavage samples from healthy human volunteers. Am Rev Respir Dis 1982; 126:611-16
CHEST I 88 I 6 I DECEMBER, 1985
858
To investigate the optimal lavage technique to study the airway response in patients with asthma, differential volume lavage was carried out in 22 normal subjects and 18 patients with red cedar asthma. Ten mI of ftuid was instilled into a main-stem bronchus followed by 5, 10, 20, 50 and 100 mI into different segmental bronchi. Small volume lavage «20 m1) in a main stem bronchus or a segmental bronchus recovered more epithelial cells and neutrophils while a lavage volume of 20-100 mI in a segmental bronchus recovered more lymphocytes, alveolar macrophages, albumin, IgG and alpha-entitrypsfn, In patients with
asthma, a higher porportion of epithelial cells was observed in the 20 ml segmental bronchus lavage 24 to 48 hours after bronchial challenge with plicatic acid, the chemical compound responsible for red cedar asthma. There was an increase in eosinophils and IgG with all volume lavages, but the changes were most marked in the 10 ml main bronchus lavage and the 10 and 20 mI segmental bronchus lavage. The results show the importance of using a systematic lavage protocol to characterize the cellular and protein changes in the airways of patients with asthma. A single volume lavage in one site may not encompass all the changes observed.
A,though there are numerous reports on the use of bronchoalveolar lavage to study various lung diseases,':" the optimal lavage technique to study airway diseases had not been examined. The technique of sampling bronchial fluid varies from direct bronchoscopic aspiratiorr'" to saline lavage at different sites. 4-6.8-~ The volume of fluid used for lavage ranges from 1 ml to 300 ml in these studies. Even within a single study, the volume of lavage fluid infused may vary from one patient to anothenv':" The site of lavage was also different. The tip of the bronchoscope was in the trachea, 5. 7 carina," a main bronchus.t?" or a
based on the clinical history and confirmed by bronchial challenge with plicatic acid (the chemical compound responsible for red cedar asthma). The clinical features of red cedar asthma had been reported in detail previously. 13
subsegmental bronchus. 8.11.~
To what extent these
variations influence the composition of the lavage fluid is not known. The purpose ofthis study is to determine the effect of the lavage volume on the cellular and protein recovery in normal subjects and patients with bronchial asthma. MATERIALS AND METHODS
Subjects Forty subjects were studied. This includes 22 normal healthy individuals and 18 patients with occupational asthma due to exposure to Western red cedar. The mean age of the normal subjects was 25 years (range 18 to 44 years). The mean age of the patients with asthma was 33 years (range 19 to 53 years). All subjects were nonsmokers. The normal subjects had no respiratory symptom or recent respiratory illness within the past three years. They also hadnormal results of spirometry. For the red cedar patients, the diagnosis was made ·From the Respiratory Division, Vancouver General Hospital, University of British Columbia and the Cancer Control Agency of British Columbia, Vancouver, Canada. Supported by the Medical Research Council of Canada, Grant No. MA-1703. Manuscript received March 15; revision accepted June 18. Reprint requests: Dr: Lam, UBC Respiratory Division, Vancouver General Hospital, Main Floor, 2775 Heather Street, Vancouver, Be, Canada V5Z 3]5
868
Methods Inhalation provocation tests were performed with plicatic acid and the method of inhalation provocation was described in detail previously." The test solutions were delivered by a Bennett twin nebulizer and the average output from the nebulizer at an oxygen Row rate of 5 Umin was 0.25 mllmin. The particle size of the aerosol has a mass median diameter of 3.1 ....m. The patients were asked to breathe the nebulized aerosol at tidal breathing. On a control day, phosphate-buffered saline solution, the diluent for plicatic acid, was inhaled for 0.5, 1.0, 1.5, and 2.0 min at 100min intervals. The FEV. was measured with a 13.5 L Collins water spirometer before the inhalations, 10 min after each inhalation, and at 10 min intervals for the first hour after the final inhalation. The FEV. was then measured at hourly intervals for the next eight to ten hr and again at 24 hr after challenge. On the second day, plicatic acid (5 mg/ml in phosphatebuffered saline solution, pH 7.4) was inhaled in a similar manner at 10 min intervals until a total of 5 min inhalation has been given. The volume of plicatic acid nebulized was determined by measuring the amount left in the nebulizer. A reaction was defined as a response in which there was a decrease in the FEV. greater than 20 percent of the baseline value. All patients developed a late asthmatic reaction either alone or as part of a dual asthmatic reaction. Late asthmatic reaction usually began several hours after challenge; was maximal in four-six hours and lasted up to 24 hours or more. Dual asthmatic reaction had two components, an immediate reaction with spontaneous recovery within one-two hours followed by a late reaction. The amount of plicatic acid required to provoke an asthmatic reaction varied between 0.5 to 7 mg. Fiberoptic bronchoscopic examination was performed following premedication with 0.8 mg atropine 1M and local anesthesia with 2 percent lidocaine (Xylocaine). The asthmatic patients received salbutamol 200 ....g by inhalation 15 minutes before bronchoscopic examination. Sterile isotonic saline solution warmed to body temperature was used for lavage. In patients with red cedar asthma, bronchial lavage was carried out 24 to 48 hours following bronchial challenge with plicatic acid when their forced expiratory volume in 1 s was greater than 1.5 L. Bronchial Lavage Volume Effect (Lam et III)
Table I-Effect ofVolume on CeURecovery in Normal Subjects·
No. samples Fluid recovery (%) Cells per ml X 10- 4 Viability (%) Epithelial (%) Neutrophil (%) Macrophage (%) Lymphocyte (%) Eosinophil (%) Degenerated (%)
Main Bronchus 10ml
5 ml
10 ml
Segmental Bronchus 20ml
50 ml
100 ml
12 33±15 22.9±20.0 55±16 21±12 28±12 41±13 3±2 0 7±4
6 35±21 11.3±10.1 54±11 22±19 22±19 48±18 4±2 0 4±2
18 38±8 11.3±6.9 56±13 21±10 22±10 47±13 3±2 0 7±5
22 41±8 12.7±8.4 67±11 16±9 10±9* 63± 14* 5±4 0 5±5
7 47±9 11.3±4.0 70±14 14±8 5±2 65±10 13±8t 0 4±3
17 5O±7 14.4±5.0 78±13 5±5t 2±2 78±11 12±7 0 2±5
*Alldata shown as mean ± SO. tSignificantly different from the preceding volume; p
nostics).The lower limits for the assayswere: total protein 100....g/ml, albumin 5 g/ml, IgG 5 ....glml, llS-IgA 1 ....g/ml and alpha-antitrypsin 3 glml. All proteins were quantitated in unconcentrated fluids. The study was approved by the Ethics Committee on Human Experimentation of the University of British Columbia. Statistical Analysis
Since a maximum of four sites was used for lavage in each subject, the number of samples in each lavage volume is not similar. Comparison within the same group is made using the unpaired t-test. The results from the asthmatic subjects are compared with those from the same lavage volume in normal subjects by the unpaired t-test. RESULTS
The effect of the lavage volume on cellular and protein recovery in the 22 normal subjects is shown in Tables 1 and 2. The percentage of fluid recovered was less with smaller volume lavages, especially in the main bronchus. The total cell count per ml of aspirated fluid was slightly higher in the main bronchus, but was essentially similar in the other volume lavages. Viability of the cells recovered was higher with the large volume lavages (Table 1). The proportion of bronchial epithelial cells and neutrophils was lower when the lavage volume was greater than 20 ml. The reverse was observed with alveolar macrophages and lymphocytes where a lower proportion was found with a lavage volume of less than 20 ml (Table 1). The level of albumin and IgG increased with volume. They were significantly higher in the 100 mllavage compared with the
Table 2-Effect ofLavage Volume on Protein Content in Normal Subjects-
Protein Albumin
IgG
IgA
AAT
Main Bronchus 10 ml
5 ml
10 ml
Segmental Bronchus 20ml
SO ml
100 ml
287±138 29±19 3±6 26±21 <3
105±79 39±22 5±9 33±21 <3
125±86 26±15 7±9 22±16 <3
123±55 35±25 12±7 24±15 <7±1
141±58 43±18 12±11 13±4 8±2
142±91 78±49* 2O± ur 25±15 9±2
*Recovery of proteins expressed as ....glml of unconcentrated lavage fluid ± SO. tSignmcantly different from the preceding volume; p
AAT=alpha-antitrypsin.
CHEST I 88 I 6 I DECEMBER, 1985
857
Table 3-Effect ofVolume Oft CeU Becooery in Patienta with Asthma·
No. samples Fluid recovery (II» Cells per ml X 10- 4 Viability (II» Epithelial (II» Neutrophil (%) Macrophage (%) Lymphocyte (II» Eosinophil (If,) Degenerated (If,)
Main Bronchus 10ml
5 ml
10 ml
Segmental Bronchus 20 ml
50ml
100 ml
8 4O±21 22.7±24.6 59±13 29±19 14±9 14±12§ 2±1 27± 18§ 14±9t
4 36±30 14.6±20.7 52±4 36±30 16±23 18± 13t 2±1 21± 12; 7±5
12 35±14 10.3±7.4 55±10 27±16 15±11 21± 10§ 2±1 25±13§ 9±4
18 33± lOt 17.8±23.3 61±17 25±13t 10±6 33± 14§ 4±2 14±9§ 14± 11+
4 27± 15t 7.3±4.0 70±20 15±6 6±3 46±16 15±17 9±9+ 10±3+
13 45±11 19.9± 11.9 82±12 6±4 3±2 6O±23 16±14 4±5+ 11 ± 16+
.A1ldata shown as mean ± SO.
tSignificantly different from normal subjects in 'Iable 1; p
smaller volume lavages. IgA concentration did not vary signi6cantly with volume. Alpha-antitrypsin in low concentrations was found in approximately 70 percent of the 100 mllavage samples and 25 percent of the 20 mllavage samples. It was undetectable in the smaller volume lavages. Albumin, IgG, IgA and alpha-antitrypsin account for over 90 percent of the total protein measured with the 100 mllavage, but only 20 percent of the total protein with the main bronchus lavage (Table 2). The same lavage protocol was applied to the patients with asthma. The results were compared with that of the normal subjects. The percentage of 8uid recovered from the asthmatic patients was significantly lower with the 20 and 50 mllavages compared to the normal individuals. The total cell count per ml of fluid recovered was not significantly different in all volume lavages between the two groups (Table 3). However, the proportion of epithelial cells in small volume lavages was higher in the asthmatic patients. The results reached statistical significance for the 20 ml lavage. The proportion of eosinophils was Significantly higher in all the lavages from asthmatic patients, especially in the 10 ml main bronchus lavage and the 10 or 20 ml segmental bronchus lavage. The proportion of alveolar macrophages was significantly lower when a volume of20 ml or less was used for bronchial lavage in the asthmatic patients (Table 3). There was no signifi-
cant difference in the proportion of neutrophils or lymphocytes in all volume lavages between the two groups. The albumin content was significantly higher in the asthmatic patients when a volume of 20 ml or less was used for lavage. There was an increase in IgG content in all volume lavage in the asthmatic patients, but it was most marked in the 10 ml main bronchus lavage and the 10 or 20 ml segmental bronchus lavage (Table 4). DISCUSSION
U sing a systematic lavage protocol, we studied the effect of the site of lavage and the volume of lavage fluid instilled on the composition of 8uid recovered by aspiration. We postulate that lavage with smaller volurnes, especially in a main bronchus, would sample preferentially bronchial 8uid while larger volumes would sample fluid lining the small airways and alveoli. Bronchial fluid should contain predominantly bronchial epithelial cells, while alveolar fluid should contain predominantly alveolar macrophages. A fluid volume of greater or less than 20 ml instilled into a segmentallsubsegmental bronchus was found to be the dividing point in the present study. A fluid volume of less than 20 ml recovered more epithelial cells while a fluid volume of greater than 20 ml recovered an increasing proportion of alveolar macrophages. Neu-
Table 4-Protein Content in Lavage Fluid from Potienta with Asthma·
Protein Albumin
IgG
19A
Main Bronchus 10 ml
5 ml
10 ml
Segmental Bronchus 20ml
50 ml
100 ml
703 ± 488 275±207+ 5O± 16§ 33±12
162±73 73±41 16±7* 54±4t
240± 191 9O±63§ 28± 171 31±30
281±291 125± 189 26± 13§ 28±18
175±66 72±50 28± 17+ 17±12
268±222 106±40 36± 16+ 26±22
·Recovery of proteins expressed as JLglml of unconcentrated lavage 8uid mean ± SD. tSignificantly greater than that from normal subjects in Table 2; p
trophils are more numerous with smaller volume lavages, possibly reflecting the normal response in the more central airways to inhalation of various dusts and fumes in the environment. Lymphocytes, albumin, IgG and alpha-antitrypsin were present in higher proportion or concentration in the large volume lavages, probably reflecting the site of origin or accumulation of these cells and protein molecules in the peripheral airways and alveoli. A similar increase in albumin with volume was also observed by Rennard and Crystal" using sequential 20-ml lavages. An increase in protein content with volume was not found by others, but the size of the first lavage aliquot was 60 ml or greater and repeated washing was carried out in the same bronchopulmonary segment. 15.16 To further test the hypothesis that small volume lavage samples, preferentially bronchial fluid, we applied the same lavage protocol to a group of patients with red cedar asthma following induced bronchoconstriction. Evidence of airway inflammation with exudation of proteinaceous fluid high in albumin and IgG content, eosinophils and sloughing of bronchial epithelial cells were observed mainly in the 10 ml main stem bronchus lavage and the 10 and 20 ml segmental bronchus lavage. Some of the changes would have been missed had lavage been carried out with a fluid volume of greater than 20 ml. The 5 ml lavage was found not to be useful because it gave highly variable results. This is probably related to the large dead space of the suction channel of the bronchoscope relative to the volume of fluid instilled. The site of lavage is also potentially important. The increase in total protein, albumin and IgG in the asthmatic patients were most marked with the main stem bronchus lavage compared to segmental bronchus lavage. However; the results were more variable probably due to contamination with lidocaine, minor aspiration of oropharyngeal secretions, and mixing with secretions lower down in the respiratory tract with coughing and the normal mucociliary escalator mechanism. Despite these considerations, when investigating the changes associated with an asthmatic reaction other than cellular and protein changes, as for example, release of various chemical mediators, it might be important to lavage the main stem bronchus in addition to a segmental bronchus. Our study underlines the importance of
standardizing the site oflavage, as well as the volume of fluid instilled in the use of bronchial lavage to study airway diseases such as bronchial asthma. REFERENCES
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