Comparison between Bronchial and Alveolar Samples of Bronchoalveolar Lavage Fluid in Asthma

Comparison between Bronchial and Alveolar Samples of Bronchoalveolar Lavage Fluid in Asthma* Thierry Van Vyve, M.D.;t Pascal Chanez, M.D.; Jean-Yves Wcoste, ~1.D.; Jean Bousquet, M.D., Ph.D.; Fran~ois-Bernard Jtlichel, M.D., F.C.C.P.; and Philippe Godard, M.D., F.C.C.P.

Background: Cell content of BALF may vary according to the segment of the lung washed. It was proposed to separate BALF into several aliquots, the first sample being more related to bronchi. The present study compared bronchial and alveolar samples by fractionating aliquots of BALF in normal and asthmatic subjects. Methods: One hundred asthmatic subjects (mean ± SEM: 37± 1.5 yr in age) were compared with 31 normal subjects (mean±SEM: 32±2.2 yr in age). None of the subjects was a smoker and none was taking drugs that might interfere with the results. The severity of asthma was defined by the clinical score of Aas examining the chronic severity of asthma and ranging from 1 to 5 (range: 1 to 4; mean ± SEM: 2.2 ± 0.1) and FEV. (range: 45 to 130 percent; mean ± SEM: 82± 1.8 percent of predicted values). Bronchoscopy was done in a standardized manner. A first aliquot of 50 ml of saline was injected and the BALF recovered was stored (bronchial sample). Then, four aliquots of 50 ml of saline each were instilled and the BALF recovered was pooled (alveolar sample). After centrifugation, total and differential cell counts (May Grunwald-Giemsa) were carried out on

bronchial and alveolar samples. Results: The alveolar sample contained significantly more cells per milliliter of BALF than the bronchial sample in normal (p<0.OO77, Wilcoxon test) and in asthmatic subjects (p=O.OOOI, Wilcoxon test). Both in normal and asthmatic subjects, bronchial samples contained significantly more neutrophils and epithelial cells and fewer macrophages and lymphocytes than alveolar samples. In asthmatic subjects, the bronchial sample contained a significantly greater percentage of eosinophils than the alveolar sample. Eosinophils were significantly increased in asthmatic subjects for both the bronchial and alveolar samples. Bronchial and alveolar eosinophilia both were correlated with the Aas score (r=O.25, p=O.024 and r=O.38, p=O.OOO6, respectively, by Spearman Rank test). Conclusions: This study shows in a large number of subjects that the cell content of bronchial and more distal segments of the lung is not comparable, indicating that studies should not give pooled data in asthmatic subjects. Moreover, it confirms the presence of BALF eosinophilia in asthmatic (Chest 1992; 102:356-61) subjects.

Since the initial report hy Reynolds and Newhall, I BAL has been used widely to sample the lower respiratory tract and represents an important clinical2 and research tool. This technique makes it possible to investigate cells and mediators recruited into the airway lumen. 3 •4 Quantitative measures are easy but BAL examines an ill-defined segment of the lung including small and large airways as well as alveoli. 4 Since BAL analysis is widely performed for the study of bronchial diseases, attempts have been made to obtain bronchial samples. By means ofinflated balloons and catheters it is possible to obtain a true bronchial sample but this technique is difficult, only applicable to large airways and the volume of fluid recovered is relatively small. 5 .6 It was also proposed to separate BALF into several aliquots, the first sample being more related to bronchP·7-16 and the others to the more distal parts of the lung including bronchioles and

alveoli. These studies were substantiated by Kelly et al 17 who, by digital subtraction radiography, estimated that the first aliquot introduced during lavage stayed close to the bronchoscope and probably sampled only the proximal airways, whereas the other aliquots were spread more widely in the airways. Although it is likely that the first sample examines the proximal airways, differences may be observed behveen normal subjects and patients \\'ith chronic obstructive lung diseases. Among the technical factors that might interfere with the cell recovery of the lavage, the volume of the injected aliquots 9 and the technique of reaspiration could be potentially important. In 1981, the use of fiberoptic bronchoscopy and BAL was extended to asthma. 1H-2o Many studies \\'ere performed and improved considerably our understanding of asthma. The major difference between asthmatic and normal subjects in the BALF cell content was the increased number of eosinophils. 7,9,15.16.1H-3.1 Ho\\'ever, differences beh\'een bronchial and more distal samples are not completely clear in asthmatic subjects and further studies are needed. Bronchial and alveolar samples obtained by BAL \\'ere compared in a large group of asthnlatic subjects

*From Service des Maladies Respiratoires, CII U, ~Iontpellier, France. tPresently at Cliniques Universitaires Saint-Luc, Brussels, Belgium. This work \\'as supported by a grant from Ie Fonds Special des Maladies Respiratoires: 89 MRD4. Manuscript received June 10; revision accepted November 8.

356

Comparison of BALF in Asthma (Van Vyve et al)

with variable severity and normal nonsmoking subjects in an attempt to differentiate cell content of both samples and to examine whether differences exist between these groups of subjects. MATERIALS AND METHODS

Patient Population We studied 100 asthmatic patients who were 18 to 71 years of a~e (mean ± SEM, 37 ± 1.5 yr). Asthma was defined according to the criteria of the American Thoracic Society14 and all patients had a reversible airway obstruction characterized by a 15 percent increase in FEV. after the inhalation of 200 f.Lg of albuterol. None of the patients was a current smoker and none had smoked \\1thin the previous two years. Patients were excluded from the study if they had taken systemic corticosteroids in any form during the previous two months, if they had inhaled corticosteroids, nedocromil sodium, cromolyn sodium, during the previous month, if they had taken ketotifen during the previous week or if they had taken theophylline during the 48 h before the test. Treatment with ~2-agonists was withheld only for 8 h. We also studied 31 normal nonsmoking subjects (age 18 to 59 years; mean ± SEM, 32 ± 2.2 yr) as a control group. Their pulmonary function was in the normal range. They had no allergic diseases and had never had asthma. The study was approved bv the ethics committee of the universitv and the patients gave infor~ed consent. .

Asthma Score and Pulmonary Function The clinical severity of asthma was assessed according to the scoring system of Aas. ~ Very mild forms received a score of 1, and incapacitating disease requiring medications received a score of 5. The grading was based on events that took place over a one-year period and considers both the symptoms (the number and duration of asthma episodes, total duration of symptoms, and presence or absence ofsymptom-free intervals between attacks) and the requirement for medications. It does not take into account the patient's pulmonary function. A score of 5 would be given to patients who are receivin~ an antiinflammatory drug. To evaluate the chronic severity of asthma, we used the Aas score because we have previously shown that it was correlated \\1th FEV., 7,36 the reversibility of airway obstruction after the inhalation of albuterol, 36 the endoscopic score, 37 the activation of alveolar macrophages JO and the level of eosinophil cationic protein in BALF as well as the BAL eosinophilia. 7 Pulmonary function was assessed just before bronchoscopy by means of a How-volume loop performed with a Pneumoscreen (E. Jaeger Laboratories, \Vtirzburg, Germany), and normal values \\'ere defined according to the standards of Knudson et al. J.lI

Etiologic Investigations of Asthma All patients were studied in the same manner. Allergy was defined by the presence ofcutaneous sensitivity to allergens and an elevated total serum IgE. Sensitivity to a1ler~ens, including a battery of extracts of common food allergens and aeroallergens h)und in the Montpellier area,39 was evaluated by skin-prick tests. Total serum IgE levels (Phadebas paper radioimmunosorbent test and Phadiatop; Pharmacia Diagnostics, Uppsala, Sweden) were determined. Senlm specific IgE was measured by the Phadebas radioallergosorbent test (Pharmacia Diagnostics) in patients with positive skin-prick tests. All patients had sinus and chest radiographs.

Fiberoptic Bronchoscopy Fiberoptic bronchoscopy was performed as previously described. 20 BrieRy, after premedication with 0.5 mg atropine and 5 mg diazepam and local anesthesia with 2 percent lidocaine applied

to the upper respiratory tract, a BFTR Olynlpus fiberoptic bronchoscope was inserted into the trachea and the airwavs \\'ere systematically examined. The BAL was carried out in on~ of the subsegmental bronchi of the middle lobe using the injection of a first aliquot of 50 Inl of saline at room temperature followed by fi>ur aliquots of 50 ml each, reaspirated by gentle syringe suction. The first aliquot considered to he a bronchial sample was collected and processed separately from the subsequent a1iquots which were pooled and considered to be the alveolar sample. Immediately after lava~e, mucus was removed from the Huid hy filtration through gauze. During bronchoscop}~ oxygen and epinephrine were readily available, and the patient had an intravenous infusion to provide venous access. Nebulization with 1 mg of salbutamol was performed after the procedure if bronchospasm was noted. The procedure was carried out in patients \\,ho had various sta~es of asthma and in some patients who had severe asthma.

Examination of BAL Cells After recovery, bronchial and alveolar samples were strained through a monolayer of surgical ~uze to remove mucus. After mixing, an aliquot of 5 ml of each sample was used to obtain a total cell count usin~ a hemacytometer. Cells were examined on the two specimens by an investi~ator who was unaware of the subject group. Cell differential counts were done after cytocentrifugation (Cytospin, Shandon, UK) and staining by May Griinwald Giemsa by counting 200 cells on each slide. Macrophages, lymphocytes, eosinophils, neutrophils and epithelial c..-ells (includin~ ciliated and goblet cells) were enumerated and results were ~ven both in percentages and numbers of cells per milliliter of Huid recovererd to correct for variable re<--overy.

Statistical Analysis Statistical analyses were made by two-tailed nonparametric tests. Correlations between Aas score, FEV. and BAL differential cell counts in bronchial and alveolar samples were done by the Spearman rank test. RESULTS

Characteristics of the Patients and Tolerance of Bronchoalveolar Lavage Characteristics of the patients are shown in Table 1. Twenty-three patients had an Aas score of 1 (mild asthma), 38 had a score of 2 (moderate asthma), 31 a score of 3 (moderately severe asthma) and 8 a score of 4 (severe asthma). No patient had a score of 5, since this implies an anti-inflammatory treatment, and the patients received no medications except inhaled ~2­ agonists. The FEV. ranged from 45 to 130 percent of predicted values (mean±SEM, 82.0±1.8 percent). Fifty-six patients were allergic. Tolerance of BAL was excellent as there was no severe attack during endoscopy, and only two mild exacerbations were noticed but they did not require the cessation of the procedure Table 1- Characteristics of ABthmatic Patients Aas Score 1 2 3 4

No.

Age, yr Mean±SEM

23 38 31 8

33± 1.2 35± 1.5 38 ± 1.4 48 ± 1.2

Sex M

F

FEV.,% Mean±SEM

Allergy

12 26 10 5

11 12 21 3

89.6± 1.5 79.3±2.5 70.4±2.6 53.8± 1.2

14 20 16 6

~

CHEST I 102 I 2 I AUGUSl: 1992

357

Table 2-Comparison between Bronchial and Alveolar Samples in Asthmatic and Control Subjects· Asthmatic Patients

BALF recovery No. of cells Macrophages Lymphocytes Eosinophils Neutrophils Epithelial cells Macrophages Lymphocytes Eosinophils Neutrophils Epithelial cells

%

1,000 cells/ml % % % % %

1,000 cells/ml 1,000 cells/ml 1,000 cellslml 1,000 cellslml 1,000 cells/ml

Control Subjects

Bronchial Sample

Alveolar Sample

Bronchial Sample

Alveolar Sample

26.4±0.lt 101.1 ± 9.69t 68.4±2.41t 5.0±0.59t 5.5± 1.13t§ 8.4± 1.51t 11.3± l.38t 66.5±6.28t 5.3±0.8t 5.5± 1.6311

47.9± 1.4511 153.5±9.3 82.0± 1.26 10.1 ±0.84 2.9±0.6§ 2.7±0.48 2.4±0.45 124.0±7.4 16.6± 1.92 4.7± 1.0711 4.6± 1.13 2.6±0.46

26.9±0.4t 103.8 ± 12.3t 77.1 ±2.59t 6.7± LOt 0.4±0.1 4.9± LIt 8.6± 1.9t 79.8± 10.0t 8.3±2.8t 0.5±O.2 6.0± 1.51l 7.6± 1.9*

6O.0± 1.2 146.3± 12.6 86.1 ± 1.7 10.5± 1.4 0.3±O.1 1.7±O.5 1.8±O.8 125.5± 11.2 15.4±2.9 0.3±O.2 2.5±0.8 2.5± 1.4

11.2±3.~

9.9± 1.9t

*Results are expressed in mean ± SEM. tSignincant difference for p=O.OOOI, Wilcoxon W test, between bronchial and alveolar samples. tSignincant difference for p
and resolved after nebulization with 1 mg of salbutamol.

Recovery of Bronchial and Alveolar lLlvage Fluid The percentage of fluid recovered from the bronchial and alveolar samples in asthmatic and normal subjects is shown in Table 2. The percentage of recovery of the bronchial sample was significantly smaller than that of the alveolar sample in both asthmatic and normal subjects (Wilcoxon W test, p=O.OOOl [Table 2]). The percentage of recovery of both the bronchial and the alveolar samples was not significantly different in allergic and nonallergic asthmatic subjects (Mann-Whitney U test, p>O.05). In normal subjects, the percentage of alveolar recovery was significantly greater than in asthmatic subjects (Mann-Whitney U test, p=O.OOl [Table 2]).

Cellular Content of Bronchial and Alveolar Samples of Asthmatic Subjects The number of cells per volume of recovered BALF and the differential cell counts in asthmatic subjects are shown in Table 2. The bronchial samples contained significantly fewer total cells per milliliter than the alveolar samples. The percentages of neutrophils, epithelial cells and eosinophils were significantly increased in the bronchial samples. On the other hand, the percentages of macrophages and lymphocytes were significantly reduced in the bronchial samples. When differential cell counts were expressed in absolute numbers ofcells per milliliter ofBALF, significant differences were found between the two samples for neutrophils, epithelial cells, macrophages and lymphocytes but not for eosinophils. In the present study, 358

squamous cells were not reported because they were observed only occasionally. Total cell numbers and differential cell counts were not significantly different in allergic and nonallergic asthmatic subjects (MannWhitney U test, p>O.05).

Cellular Content of Bronchial and Alveolar Samples of Normal Subjects The number ofcells per volume ofrecovererd BALF and the differential cell counts in normal subjects are shown in Table 2. The bronchial samples contained significantly fewer total cells per milliliter than the alveolar samples. The percentages of neutrophils and epithelial cells were significantly increased in the bronchial samples. On the other hand, the percentage of macrophages and lymphocytes were significantly reduced in the bronchial samples. When differential cell counts were expressed in absolute numbers of cells per milliliter of BALF, we found significant differences between the two samples for neutrophils, epithelial cells, macrophages and lymphocytes. There was no significant difference between mean eosinophil percentages or absolute numbers in both samples. In the present study, squamous cells were not reported because they were observed only occasionally.

Comparison between Asthmatic and Normal Subjects The comparison of results in both samples between asthmatic and normal subjects was performed for all parameters (Table 2). Eosinophils were significantly increased in asthmatic subjects for the bronchial and alveolar samples. No other cell population was found to differ significantly between the two groups. In asthmatic subjects, BALF neutrophilia ranged from 0 Comparison of BALF in Asthma (Van Vyve et al)

o control ~II 3~I Aas

30

~

,E

25

II 4---.J

~

Q) (,)

c c c

-

20

score

15

~ ~

:i:

Q.

10

0

c::

"(j)

~

5 0 bronchial sample

alveolar sample

1. Absolute numbers of eosinophils in control suhjects and asthmatic patients. Results expressed in Inean ± SE~1. FIGURE

to 79 percent in the bronchial saolple and froln 0 to 21 percent in the alveolar sample whereas in norlnal subjects it ranged in bronchial and alveolar samples, respectively, from 0 to 21 percent and 0 to 11 percent. Ho\\rever, there was no significant difference between both groups.

Correlations between the Severity of Astll1na and Cell Counts Correlations between the severity of asthola assessed either by FEV I or the clinical score of Aas and the different parameters \vere studied. The Aas score was significantly correlated with FEV I (r = 0.47, p = 0.0001, Spearman rank test). In the bronchial sample, the percentage or absolute number of eosinophils \\ras significantly correlated with the Aas score (r= 0.25, p = 0.024 and r= 0.28, p = 0.01, respectively, Spearman rank test). In the alveolar saolple, the percentage or absolute number of eosinophils \vas significantly correlated with the Aas score (r = 0.38, p = 0.0006 and r = 0.31, p = 0.0044, respectively, Spearman rank test). About 20 percent of norlnal subjects and from 50 to 83 percent of asthmatic subjects according to the Aas score had eosinophils in the BALF. Patients with nlild asthola (Aas score of 1) had a significantly greater percentage or nunlber (Fig 1) of eosinophils than normal subjects (p<0.OO9 and p<0.OO5, respectively, Mann-Whitney U test). DISCUSSION

The study presented herein shows that the total and differential cell counts of bronchial and alveolar samples differ both in asthmatic and in normal subjects. In both groups, bronchial samples contained significantly more neutrophils and epithelial cells than alveolar samples. In asthmatic subjects only, an increased percentage of eosinophils was found in the bronchial sample. The only significant difference

behveen asthmatic and normal subjects was the increased percentage and number of eosinophils in both bronchial and alveolar samples of asthmatic subjects. Current recommendations specify bronchoscopy only in mild asthmatic subjects,40·41 but we have had extensive experience in fiberoptic bronchoscopy in asthmatic subjects of variable severity7.19.20 and more than 1,000 such patients have had bronchoscopy over the past years in our clinic without any serious complications. The safety of this study was therefore discussed in detail in advance and it was approved by the ethics cOlnmittee of the university. Previous studies attempted to differentiate the cell content of bronchial and alveolar samples. The best technique is to perform a true bronchial lavage in an isolated airway segment using a double-balloon catheter. 5 Eighteen normal subjects and 14 asthmatic subjects were studied and it was observed that the bronchial sample contained a significantly greater number of neutrophils and epithelial cells than unfractionated BALF.5 However, since this technique cannot be performed on a large scale, other investigators proposed to fractionate the lavage, the first aliquot being more related to a bronchial sample. In Iuany studies, sequential constant volumes of fluid \vere injected,H,Il,14 and differential cell counts were compared in the different aliquots recovered. Martin et alii and Yasuoka et al 14 showed that neutrophils \\'ere increased in the first sample, whereas Davis et alH did not observe any difference. Two studies fractionated BALF by separating a first aliquot of 20 Inl frool the next four aliquots of the same volume in three different sites. Thompson et al 12 examined 28 patients with chronic bronchitis, 15 asymptomatic smokers and 25 nonsmoking normal subjects and found an increased number of neutrophils in the bronchial sample. Rennard et al 13 studied 18 nonsmoking normal subjects and 109 patients suffering from various diseases and confirmed the increased number of neutrophils and epithelial cells in the bronchial sample. In another study, Adelroth et al 16 fractionated BALF by separating a first aliquot of 20 ml from the next two aliquots of 60 ml each in 22 asthmatic subjects and 12 normal subjects and showed in both groups an increased number of neutrophils and epithelial cells in the bronchial sample. In a previous study from our group exalnining 43 asthmatic subjects, we found a nonsignificant increase of neutrophils and a significant increase of epithelial cells in the bronchial sample." The present study confirms previous data showing an increase of neutrophils and epithelial cells in the bronchial sample of normal subjects. Moreover, in a large group of asthmatic subjects, the same result is noticed. It is therefore likely that the first aliquot is more related to a bronchial sampling since results are close to the double-balloon technique. 5 Kelly et al,17 CHEST I 102 I 2 I AUGUSt 1992

359

by digital subtraction radiography, estimated that the first aliquot introduced during lavage stayed close to the bronchoscope and probably sampled only the proximal airways, whereas the other aliquots were spread more widely in the airways. Moreover, this hypothesis seems to be correct in both normal subjects and patients with chronic obstructive lung diseases, eg, asthmatic subjects as seen in the present study and in others7,lS,16,21 and those with chronic bronchitis. 11,12 These studies indicate that the bronchial sample should be separately analyzed from the subsequent aliquots more related to the distal parts of the airways and the lung. Eosinophilic inflammation is known to be a hallmark of bronchial asthma and several studies had reported a significant eosinophilia in BALF of asthmatic patients,7,9,15,16,18-33 but bronchial and more distal samples were only differentiated in four studies7,15,16,21 which all reported that eosinophils were increased in both samples. In the present study, a large group of 100 asthmatic subjects was studied and we confirmed the increased percentage or number of eosinophils in both bronchial and alveolar samples in comparison with normal subjects. The correlation between the clinical severity of asthma and BALF eosinophils was already reported. 7 The present study confirmed in a larger population the correlation between BALF eosinophilia and the Aas score but the significance was greater for the alveolar than for the bronchial sample. It may be surprising that alveolar" eosinophils correlate better with the clinical severity of asthma than the ubronchial" ones. The Aas score is a chronic score of asthma and does not take into account the recent events that may be of paramount importance for the BALF cell counts. On the other hand, the so-called alveolar sample is likely to sample both alveoli and bronchioles and the obstruction of chronic asthma may be more prominent in the small airways as suggested by ventilation perfusion scintigraphy.42 The present study showed a nonsignificant increase of epithelial cells in both bronchial and alveolar samples of asthmatics. Desquamation of epithelium has been described in asthmatic patients and recent studies reported a significant increase of epithelial cells in BAL of asthmatic subjects. 16,21,22,43 The lack of a significant difference between normal and asthmatic patients in our study is likely to be related to the method of BALF sampling. We always used a gentle suction with a syringe to avoid peripheral blood contamination, whereas other investigators have used a vacuum pump suction making it possible to sample more epithelial cells that are less viable (Campbell, unpublished data) and therefore more easily shed in asthmatic subjects. A significant increase of neutrophils in BALF content was documented during the late asthmatic reacU

360

tion occurring after allergen challenge,22,24-27,31,32 whereas in chronic asthma it was never reported in previous studies. 18-21 ,23,29,3o,33 In the present study, we did not observe a significant increase of neutrophils in both the bronchial and alveolar samples of asthmatic subjects. Allergen challenge represents an acute inflammatory response where neutrophils often are present,44-47 whereas in the chronic inflammation of asthma neutrophils are likely to be absent. As in other studies,18-21,23,29,30,33 lymphocyte and macrophage numbers were not significantly different in asthmatic and normal subjects. This study, using a simple method of fractionated lavage, confirms that total and differential cell counts of bronchial and alveolar samples are not comparable in asthmatic subjects and further BAL studies in asthmatic subjects should process separately bronchial and alveolar samples. Bronchial and alveolar eosinophilia are the only difference observed between both groups of subjects, thus confirming the importance of this cell in asthma. ACKNOWLEDGMENT: The writers thank Dr. F. Paganin and J. Taib for their kind help and Dr. A. Campbell for reviewing the manuscript. REFERENCES

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