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* From the Institute of Pulmonary and Allergic Diseases (Drs. Fireman, Greif, Schwarz, and Man), the Ministry of the Environment (Dr. Ganor), and the Occupational Health and Rehabilitation Institute, Ra'anana (Drs. Ribak and Lerman), the Tel-Aviv Sourasky Medical Center and the Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel.
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We compared BAL with IS analysis of 5 workers exposed to asbestos and 14 exposed to silica and hard metals. Pulmonary function tests and BAL were performed by conventional methods. IS induction was performed after a 20-min inhalation of 3.5% saline solution with an ultrasonic nebulizer. Giemsa-stained cytopreparations were differentially counted. T-lymphocyte subsets were analyzed by flow-activated cell sorter, and messenger RNA (mRNA) was transcribed by reverse transcriptase-polymerase chain reaction. Mineralogic particles were analyzed by scanning electron microscopy and polarizing light microscopy and quantified by an analyzer.
Results: The percentage of neutrophils was significantly lower in BAL fluid than in IS specimens, whereas no differences were found in the percentage of lymphocytes and subsets profile. Asbestos fibers were found in BAL but not in IS samples from workers exposed to asbestos. Polarizing particles were found in both samples. Similar mineral elements were found in qualitative analysis by scanning electron microscopy. Quantitative studies showed similar size distribution with a small shift toward larger particles in sputum; mRNA showed the same cytokine profile.
Conclusions: A comparison of BAL and IS specimens in the evaluation of the study population yielded similar quantitative and qualitative results. Further research is needed to evaluate the hypothesis that IS, being a noninvasive technique, may be useful in monitoring exposed workers.
Key Words: BAL • induced sputum • occupational lung diseases
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several studies have illustrated the usefulness of sputum cell analysis to investigate the pathogenesis, pathophysiology, and treatment of asthma.4 5 Other studies compared samples collected by sputum induction and bronchoscopy in healthy subjects with those from patients with asthma and chronic bronchitis.6 7 A few studies that evaluated induced sputum (IS) in subjects suspected of occupational exposures were conducted: eosinophil counts were performed in IS of asthmatic isocyanates-sensitized subjects,8 and the frequency of bronchial dysplasia was investigated in sputum of retired miners.9 Researchers have studied the relevance of asbestos bodies in spontaneous sputum production.10 11 However, the use of IS in the assessment of exposure to hazardous dust and the evaluation of patients with pneumoconiosis in comparison with that performed by BAL has not yet been reported (to our knowledge).
In the present article, we used IS methodology as a biological method—as opposed to clinical and environmental techniques—to assess workers exposed to hazardous dust. The main objective of this study was to compare the cell counts, type of inflammation, and mineralogic analysis between samples obtained by BAL and IS in hazardous dust workers with suspected pneumoconiosis.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Group 1 (Table 1 ) included five patients exposed to asbestos. Patients 1 and 2 worked in a large electric power plant and were retired at the time of study. They had worked there for 23 to 43 years before their referral for evaluation. Asbestos had been widely used in this power plant to provide thermal insulation of the heat generated by turbines and steam pipes. Two cases of malignant mesothelioma in a clerk and insulation worker had been reported in this plant.12
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Patient 5 had been working as a fitter for the last 26 years in a military setting. His job included the cutting and sawing of asbestos sheets used for walls, a type of work known to produce high airborne levels of asbestos.14
Group 2 (Table 1) comprised 14 patients from different plants. They were exposed mainly to silica dust or to a mixture of silica and hard metals. Patients 1 to 3 worked for 36 years in the production of clay for ceramic products and manufacturing: the process involved sifting sand, mixing it with water, and packaging. Patient 4 had been a tile layer for 40 years, a job that involved cutting ceramic tiles. These patients had retired 1 to 2 years before admission to our clinic. Patient 5 had been working in the cutting process of producing natural and artificial marble stone and had also been exposed to adhesive materials for the last 16 years. Patient 6 had been employed as a hard-metal grinder for a period of 13 years. He worked alone full time in a small shop, sharpening industrial saws using six grindstones that were being operated simultaneously.
Patients 7 through 14 worked in different sections in a large foundry. They constructed patterns for forming and assembling molds that required the melting and refining of the metal, pouring the metal into the mold, and finally removing all adherent sand and superfluous metal from the finished casting. The molds in the foundry were made of silica sand bound with clay. These patients had worked in the foundry for 12 to 34 years before they were sent for evaluation. All of them were actively employed.
Environmental Measurements
Environmental data are available for 9 of 19 subjects included
in the present study. The measurements obtained in the other work
places we considered for inclusion were sporadic and unreliable.
Early dust counts performed in the power plant (patients 1 and 2; Table 1 ) in the 1960s indicated high pollution levels at the storage and handling stages of asbestos. In 1972, the asbestos fibers count was introduced, and 70 workstations were monitored continuously using personal samples. Levels reported during the late 1970s were still high, reaching 40 fibers/mL in polishing and grinding tasks and 7 fibers/mL near the disk saw. It is noteworthy that several stations were reported to have been closed after these environmental data were obtained.13
Patients 7 to 14 (Table 1) worked in the same factory. Environmental measurements performed in 1998 in the foundry showed total crystalline silica levels that reached 3.99 mg/m3 and 12.15 mg/m3 in cave casting and shake-out processes, respectively (threshold limit value-time weighted average for total crystalline silica regulated in Israel was 0.3 mg/m3; the iron oxide level was 18 mg/m3 [threshold limit value-time weighted average = 5 mg/m3]).
Protocol
The study involved three visits to our department. Occupational
and medical history, physical examination, chest radiograph evaluation
(by three independent certified readers), and pulmonary function
tests were performed at the first visit. During the second visit (at
least 1 week after the first visit), the subjects underwent
bronchoscopy and BAL after obtaining informed consent to bronchoscopy.
Sputum induction was carried out on the third visit, which took place 2
weeks after the second one.
BAL
BAL was performed using a flexible fiberoptic video bronchoscope
(Pentax; Japan). Subjects were premedicated with meperidine, 50
mg, and atropine, 0.5 mg, and the airways were anesthetized by
inhalation of 4% lidocaine. Boluses of 50 mL 0.9% saline solution
previously warmed to 37°C, up to a total volume of 150 to 200 mL were
instilled with the bronchoscope wedged into a subsegmental bronchus of
the middle lobe or lingula. The cells were recovered by gentle
aspiration. The percentage of BAL fluid and the average total cells
recovered were 58.2 ± 8.2% and
11 ± 5 x 106 cell/mL, respectively.
Preparation of Bronchoalveolar Cells
The recovered fluid was collected into specimen traps, filtered
through sterile gauze, and centrifuged at 400g for 15 min at
4°C. The pellet was washed three times with cold phosphate-buffered
saline solution (Biological Industries; Beit Haemek, Israel), and the
cells were adjusted to a final concentration of
106 cells/mL in RPMI 1640 medium supplemented
with 2% fetal calf serum (Biological Industries). One hundred
microliters of this suspension was loaded into cuvettes of a
cytocentrifuge (Cytospin; Shandon, Southern Products Ltd; Runcorn,
Cheshire, UK). The slides were spun at 1,000 rpm for 5 min and the
fluid blotters were removed. After the slides were dried in air, a
differential cell count was performed on Giemsa-stained (Merck;
Darmstadt, Germany) preparations by counting a minimum of 500 cells.
Spirometry
The spirometry was performed by a spirometer (Masterlab; E.
Jaeger; Wurzburg, Germany). The measurement was performed using
standard protocols according to American Thoracic Society
guidelines.15
The best of three consecutive measurements
was chosen.
Sputum Induction
Sputum induction was performed with an aerosol of hypertonic
saline solution generated by a nebulizer (DeVilbiss Aerosonic
Ultrasonic Nebulizer 5000D/5000I; DeVilbiss-Health Care Corp; Somerset,
PA) with an output of 0.5 mL/min and particle size of < 5 µm
aerodynamic mass median diameter using a method slightly modified from
Pin et al.16
Briefly, subjects inhaled nebulized 3.5%
saline solution for 
20 min by ultrasonic nebulizer through a
mouthpiece without a valve or nose clip. Ten minutes after the start of
nebulization and every 5 min thereafter, subjects were asked to rinse
their mouths with normal saline solution to minimize contamination with
saliva. The patient was encouraged to cough and expectorate sputum into
a sterile plastic container. The nebulization was stopped after 20 min
or earlier if the sputum sample was of sufficiently good quality.
Sputum Examination
The method of sputum examination described by Popov et
al17
was used with some modifications.16
Sputum was processed as soon as possible within 2 h. It was poured
onto a Petri dish and all portions with little or nonsquamous
epithelial cells considered to originate from the lower respiratory
tract were selected under an inverted microscope and were placed in an
Eppendorf tube, whereupon the weight was recorded.
Dithiothreitol (Sputalysin; Calbiochem Corp; San Diego, CA) was freshly prepared in a dilution of 1:10 with distilled water according to the manufacturer's instructions. The volume added was twice the recorded weight of the plugs and was mixed mechanically with the sputum by aspiration in and out of a pipette about 20 times to ensure mixing. The sample was then placed in a shaking water bath at 37°C for 15 min to ensure complete homogenization. To stop the effect of dithiothreitol, the suspension was further diluted with phosphate-buffered saline solution to a volume equal to the sputum plus dithiothreitol. The cell suspension was filtered through a 52-µm nylon gauze (BNSH Thompson; Scarborough, Ontario, Canada) to remove debris and mucus, and the volume of the filtrate was recorded. The total cell count was measured using a hemocytometer (Neubauer chamber). The filtered cell suspension was diluted with RPMI 1640 medium supplemented with fetal calf serum to achieve a concentration of 103/µL. One drop was placed in each cup already in place in the cytocentrifuge (Shandon III Cytocentrifuge; Shandon Southern Instruments; Sewickley, PA), and cytospin slides were prepared by centrifuging at 1,000 rpm for 5 min. Separate Cytospin slides were stained by Giemsa. The cell counts were performed by scanning the cytospin preparations, starting at the top left corner in an undulating manner from top to bottom while moving across the slide using high-power (x500) magnification. Two hundred nonsquamous cells were counted, and the results were expressed as a percentage of the total nonsquamous count.
Mineral Particles Examination
Five-milliliter samples of naive BAL fluid and 1-mL samples of
sputum were used to analyze mineral particles by scanning electron
microscopy. The same volume of formalin was added, and the samples were
refrigerated at 4°C until examination. The organic material of the
naive fluid was dispersed by adding five times the volume of the 14%
formamide solution. The samples were then treated in an ultrasonic bath
for 30 s and were filtered onto a 0.4-µm carbon-coated filter
(Nuclepore Filter; Millipore Filter Corp; Bedford, MA). All reagents,
including the distilled water, were prefiltered through a 0.1-µm
membrane (Nuclepore).
The chemical composition of selected specimens was investigated counting 500 particles (< 0.4 µm diameter) by radiographic analysis using a scanning electron microscope (model 840; JEOL; London, UK) equipped with a 10,000 energy-dispersive system (Link; Oxford Analytical Instruments; Oxford, UK). The spectrometer of the energy-dispersive system separates the elements according to energy rather than wavelength. In addition, a petrographic microscope was used to identify minerals. Moreover, fresh suspensions of BAL fluid were examined with a light microscope (model BH-2; Olympus; Hamburg, Germany) equipped with phase contrast and a polarizing attachment for detecting the presence of birefringent particles to identify silica as described elsewhere.18
Quantitative Analysis
Particle size analysis and dynamic shape characterization were
performed using a laser technique based on the time of transition
theory19
using an analyzer (CIS-100 Analyzer; Galai
Production Ltd; Migdal Haemek, Israel). By this method, an exact
quantitative analysis was obtained according to particle size. The
range diameter of the particles measured was 0.5 to 10 µm. Two drops
of a suspension of BAL fluid or sputum cells (106
cells/mL) were introduced into a quartz cuvette containing stirred
phosphate-buffered saline solution. An HeNe laser beam crossed the
particle suspension, and the signal was registered by a photodiode
placed directly behind the cuvette.
Evaluation of Phenotype of BAL and Sputum Cells
Flow cytometric analysis was performed on a dual flow-activated
cell sorter (FACS 440) equipped with an Ar+ and Kr laser
(Becton-Dickinson; Mountain View, CA). Data were collected and
analyzed using appropriate computer programs (Consort VAX, Disp4, and
Disp2D; Becton-Dickinson). The information was collected on a
logarithmic scale. The selection of lymphocyte population was based on
side scatter and expression of CD 45. Lymphocytic subsets were
identified by monoclonal antibody (Becton-Dickinson) as follows: CD3,
total T cells; CD4, helper T cells; and CD8, suppressor-cytotoxic T
cells. Monoclonal antibodies were directly conjugated to either
phycoerythrin or fluorescein isothiocyanate. Cells were incubated for
10 min with proper preparation (Epics Coulter Q-Prep) and were
read either immediately or after 24 h.
Messenger RNA Preparation and Reverse Transcriptase-Polymerase
Chain Reaction Performance
RNA was extracted by adding 100 µL 4.0 mol/L
guanidinium thiocyanate (Sigma; St. Louis, MO) to
105 BAL and IS cells. The mixture was overlaid
onto 100 µL 5.7 CsCl (Sigma) and was centrifuged overnight at 35,000
rpm (Kontron Electronik GmBH; Zurich, Switzerland) at 15°C.
The RNA was reversed transcribed by 3 µL 200 U/mL MMLV virus reverse
transcriptase according to the manufacturer's protocol (Gibco
Laboratories; Gaithersburg, MD) for 1 h at 42°C.
Complementary DNA products were amplified according to the
manufacturer's protocol (GeneAmp-Clontech Lab Inc; Palo Alto, CA) with
0.25 µL Taq polymerase (5 U/mL; Promega Biotec; Madison,
WI) using a thermal cycler (MiniCycler PTC-150; MJ Research Inc;
Watertown, MA). Five-micromolar primers (Clontech) of
glyceraldehyde-3-phosphate dehydrogenase messenger RNA (mRNA)
served as internal control concurrently with interleukin (IL)-1
,
IL-1ß, IL-6, transforming growth factor (TGF)-ß, interferon
(IFN)-
, IL-2, IL-4, and IL-5 (Clontech). Eight microliters of the
reverse transcription-polymerase chain reaction product was
fractionated by electrophoresis in agarose, stained with ethidium
bromide, and matched with predicted size 174/HaeII digest
(Pharmacia; Uppsala, Sweden).
Statistical Analysis
The data were expressed as mean ± SD. Comparison of
differential cell counts and flow cytometric data between BAL and IS
was made using the nonparametric Wilcoxon test. A p value < 0.05 was
considered to indicate significant statistical differences.
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differential cells counts and T-cell subsets of all patients according to the type of exposure (as described in the study population) are listed in Table 2 . The percentage of macrophages in BAL fluid was significantly higher than that in sputum for both groups, whereas the percentage of neutrophils in BAL fluid was significantly lower compared with that in sputum. There were no significant differences in the levels of lymphocytes and the profile of T-cell subsets (group 1: percent lymphocytes and CD4/CD8 ratio, 11.8 ± 7.6% and 3.9 ± 1.7 in BAL fluid vs 11.3 ± 2.8% and 4.0 ± 1.2 in sputum [p = 0.893 and p = 0.723, respectively]; group 2: percent lymphocytes and CD4/CD8 ratio, 9.5 ± 7.8% and 1.4 ± 0.7 in BAL fluid vs 6.4 ± 5.5% and 1.9 ± 1.0 in sputum [p = 0.638 and p = 0.128, respectively]).
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, IL-1, IL-1ß, TGF-ß, IL-6, IL-5,
IL-2, and IL-4 (Table 4
) were performed on cells from BAL fluid and IS obtained from two
workers in group 1 (1 and 2 in Table 3
) and from two workers in group 2
(5 and 7 in Table 3 ). There were no differences in the profiles of the
mRNA recovered by both methods.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BAL, which has been used in recent years for the diagnosis of interstitial lung diseases, is an invasive procedure that requires special expertise and willingness on the part of the subjects to undergo the procedure. It is impractical for repeated sampling and for screening. In contrast, IS is simpler, less invasive, and less expensive than BAL.
Our first objective in the current study was to compare directly the cellular, immunologic, and microchemical constituents of the IS and BAL samples obtained by bronchoscopy in workers with suspected pneumoconiosis, undertaking this to investigate a noninvasive method both for screening and studying exposed populations. Similar comparative studies have been done for normal subjects6 and patients with asthma and chronic bronchitis,6 7 but to the best of our knowledge, this is the first report of a comparison of the two methods among workers exposed to hazardous dust. No attempts were made to study normal control subjects because the aim of our work was to compare the yield of the two methods in an exposed population.
We found that IS samples contained a significantly higher percentage of neutrophils and a lower percentage of macrophages compared with the samples recovered by BAL. These findings are similar to those found for normal subjects and asthmatics,6 7 and they suggest that the portion of IS derived from the lungs arises predominantly from neutrophil-rich secretions20 mainly recovered from large airways, whereas the BAL technique samples the distal alveolar space, which consists mainly of macrophages.21 In contrast, we found that the percentage lymphocytes and lymphocyte subtypes are similar in IS and BAL fluid. Our results agree with previous studies22 in showing a predominant CD4+ phenotype with a CD4/CD8 ratio higher than normal values in the BAL of asbestos-exposed workers. In workers with silica exposure, our findings reflected those reported by others researchers23 that there is an increase in the CD8+ T cells with fewer CD4+ T cells compared with the values of the asbestos-exposed workers.
A similarity in the pattern of the mRNA profile of cytokines shown by us in the IS and BAL fluid of the two representative patients of each group may indicate the involvement of the same inflammatory cells as was previously shown in BAL fluid during long-term occupational exposure to asbestos, coal, or silica.24
All specimens recovered by BAL and IS were analyzed by light microscopy and analytical scanning electron microscopy to identify asbestos fibers and minerals. In three of the asbestos-exposed workers, fibers were found in the BAL fluid but not in the IS. These results are similar to previous reports10 that indicated that only in lungs with high asbestos burdens can these fibers be seen in the sputum. Other studies showed that negative results from a single sputum sample have little reliability and that several samples should be obtained from each asbestos worker.11
The specimens from workers with hard-metal disease and silica exposure were also examined by polarizing light microscopy. This method was shown to be a reliable, simpler, and more rapid way in detecting silica compared with scanning electron microscopy.18 The mineral particles were seen in macrophages and epithelial cells. The observation that transbronchial epithelial cells and alveolar epithelial cells can internalize inhaled25 or instilled26 mineral particles was previously made by others, but little is known about adverse effects in those cells and in underlying interstitial tissues in relation to those exposures.27 Macrophages serve as the major defensive phagocytic cell that rapidly removes particles to avoid tissue damage. The samples of sputum tested here were derived from workers exposed for long periods. The presence of particles in epithelial cells is a marker of the overload phenomenon in which neither mucociliary clearance nor alveolar macrophages form a perfect defense.27 28 29
To the best of our knowledge, this study is the first to present qualitative and quantitative analyses of chemical particles by an IS technique. Our results suggest that the technique may be used to verify the type of exposure and number of particles. The qualitative analysis of chemical particles among silica and hard-metal workers showed very similar patterns when recovered by both IS and BAL. The quantitative analysis was done by the CIS-100 Analyzer, which allows a rapid analysis using minute quantities of biological material. In contrast the analysis by scanning electron microscopy is more expensive and time-consuming. The results showed that approximately 70% of particles internalized by macrophages or epithelial cells that are present in IS as well as in BAL samples were smaller than 2.5 µm (<PM 2.5). The US Environmental Protection Agency is currently proposing regulations that will target particles with aerodynamic diameters < 2.5 µm. IS measurements may also enable investigation of the remaining ± 30% of the particles, especially those with diameters of 4 to 5 µm, which are deposited in the lower lung fields. These particles represent the largest fraction of the weight of the dust that enters the lower lung fields.
We wish to emphasize that the present work was performed under optimal clinical laboratory conditions. The use of the present method for large-scale screening of workers would necessitate the performance of the sputum induction at the work place, because transporting these workers to a laboratory can cause unacceptable inconvenience in the work schedule. It follows, then, that the induction should be performed on site but by skilled technicians because insufficient expectorated material can yield false-negative results. Moreover, the samples must be transported to the laboratory within at least 2 h from the time the sputum was induced.
In conclusion, our findings indicate that the IS technique, recognized as being a safe30 and simple procedure, may possibly be a tool in the evaluation of workers with suspected silicosis or hard-metal disease. Further research is needed to evaluate the hypothesis that the quantitative and qualitative analysis of particles recovered by IS as shown in this study can serve as a biological monitoring method in the periodic health examinations of healthy workers exposed to hazardous dusts. Biological monitoring of workers exposed to toxic agents has gained increasing acceptance as a means of accurately determining exposure to toxic materials.31 Exposure to > 100 different chemicals such as metals (eg, lead, cadmium) and solvents (eg, toluene, trichloroethane) can be estimated in an individual by measuring the chemical or its metabolite in blood, urine, or exhaled air. However, measurements of hazardous dust have been based mainly on occupational history and environmental monitoring. The use of IS may add a new dimension to the traditional occupational parameters of past history and environmental measurements in investigating hazardous dust exposures.
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Acknowledgements |
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Footnotes |
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Abbreviations: IFN = interferon; IL = interleukin; IS = induced sputum; mRNA = messenger RNA; PM = particulate matter; TGF = transforming growth factor
Received for publication April 21, 1998. Accepted for publication January 4, 1999.
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Leader of the Working Group:, I.D. Pavord, Members of the Working Group:, P.J. Sterk, F.E. Hargreave, J.C. Kips, M.D. Inman, R. Louis, M.M.M. Pizzichini, E.H. Bel, et al. Clinical applications of assessment of airway inflammation using induced sputum Eur. Respir. J., July 1, 2002; 20(37_suppl): 40S - 43s. [Full Text] [PDF]
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