Berylliosis

DURING THE PAST FEW YEARS, there has been increased interest in noninvasive procedures for the retrieval of cells and soluble material from the lungs. Induced sputum analysis is a particularly promising approach because it provides specific information about both the cellular and molecular aspects of airways inflammation. Sputum induction was developed initially for the investigation of lung cancer and respiratory infections. (1,2) It was also used for the diagnosis of Pneumocystis carinii in patients infected with the human immunodeficiency virus. (3) The method was modified recently for application in asthmatics, and it focused on several markers of inflammation. (4) In several studies, investigators have demonstrated the utility of sputum cell analysis in the investigation of the pathogenesis, pathophysiology, and treatment of both asthma (5,6) and occupational asthma. (7) Peleman et al. (8) found that the determination of cellular composition was also useful in the management of chronic airflow limitation.

In the current study, we describe the use of the induced sputum procedure in the evaluation of 3 patients in whom occupational lung disease was suspected.

Method

Spirometry. Spirometry was performed with a Masterlab spirometer (Masterlab E. Jaeger [Wurzburg, Germany]). The measurements were conducted in accordance with standard protocols and guidelines posited by the American Thoracic Society. (9) We chose the best of 3 consecutive measurements to represent the spirometry results.

Sputum induction. Sputum induction was performed with an aerosol of hypertonic saline generated by a DeVilbiss Aerosonic Ultrasonic Nebulizer 5000D/ 5000I (DeVilbiss Health Care Corp. [Somerset, Pennsylvania]); we used a slight modification of the method used by Pin et al. (10) In summary, subjects inhaled nebulized 3.5% saline for up to 20 min. Ten min following the initiation of nebulization, and every 5 min thereafter, each subject was asked to rinse his or her mouth with water, after which he or she was encouraged to cough and expectorate sputum into a sterile plastic container. The nebulization was terminated after a period of 20 rain, or sooner if the sputum sample was of sufficiently good quality.

Sputum examination. We used the method of Popov et al., (11) with some modifications, (12) to examine the sputum. The sputum was processed within 2 hr of collection. The sputum was poured onto a Petri dish; all portions that contained few or nonsquamous epithelial cells were selected and placed in an Eppendorf tube, and the plug's weight was recorded.

Dithiothreitol (DTT [Sputalysin, Calbiochem Corp. {San Diego, California}]) was freshly prepared in accordance with the manufacturer's instructions. The added volume, which was twice the recorded weight of the plugs, was mixed mechanically in a shaking water bath at 37 [degrees]C for 15 min, thus ensuring complete homogenization. To stop the effect of DTT, we diluted the suspension further with phosphate-buffered saline solution to a volume equal to the sputum plus DTT. The cell suspension was filtered through 52-[micro]m nylon gauze (BNSH Thompson [Scarborough, Ontario, Canada]), and the total cell count was measured with a hemocytometer (Neubauer chamber [Bright Line Sigma {St. Louis, Missouril}]). The filtered cell suspension was diluted with RPMI 1640, supplemented with 10% fetal calf serum (Biological Industries [Beit Haemek, Israel]), thus achieving a concentration of [10.sup.3]/[micro]l. Cytocentrifuge slides (Shandon Southern Instruments [Sewickley, Pennsylvania]) were stained by Giemsa, and 200 nonsquamous cells were counted. We expressed the results as a percentage of the total nonsquamous cell count.

Mineral particles examination. We used 1 ml of sputum to analyze mineral particles by electron microscopy, as was described by Brody in 1995. (13) Briefly, the same volume of formalin (i.e., 1 ml) was added, and the samples were kept refrigerated at 4 [degrees]C until the time of examination. The organic material was dispersed by a 14% formamide solution, treated in an ultrasonic bath for 30 sec, and filtered onto a 0.4-[micro]m carbon-coated Nuclepore filter (Millipore Filter Corp. [Bedford, Massachusetts]).

We analyzed the chemical, morphological, and size distribution for individual particles. The size distribution and chemical composition of selected specimens were investigated by x-ray analysis with a JEOL 840 scanning electron microscope (SEM) (London, U.K.) equipped with a Link 10,000 energy-dispersive system (EDS) (Link Oxford Analytical Instruments [Oxford, U.K.]). The spectrometer of the EDS system separates elements according to energy rather than wavelength. Quantitative analysis was performed with the ZAF 4 program, and 500 particles larger than 0.4 [micro]m in diameter were analyzed for their elements and size distribution. A petrographic microscope was used to identify the minerals. (13) For the identification of silica, fresh suspensions of induced sputum were examined by light microscopy (Olympus BH-2 [Olympus {Hamburg, Germany}]) with phase contrast and a polarizing attachment for the detection of birefringent particles, as has been described elsewhere. (14)

Case Reports

Patient 1

A 46-yr-old female nonsmoker was referred to our clinic. She had progressive cough and dyspnea on exertion. Five years earlier, she had undergone a clinical evaluation that included computerized tomography (CT) of the chest and a lung biopsy. The chest CT had shown diffuse interstitial fibrosis, peripheral emphysematous bullae, and signs of pulmonary hypertension (i.e., dilatation of the pulmonary arteries). The lung biopsy had shown nonspecific interstitial pneumonia, with some areas of unusual interstitial pneumonia in the 1st stages. The patient was treated with corticosteroids, which provided brief relief from her symptoms. The exacerbation of dyspnea led her to again seek medical care. On this occasion, she also exhibited hypoxemia with minimal exertion, and she was referred to our clinic. Her medical history was unremarkable for serious illness or lung disease.

The patient's occupational history revealed that she had been employed full time as a teacher for 24 yr, and she had used various blackboard chalks daily. There were no other exposures to potentially hazardous materials.

Pulmonary function tests revealed combined restrictive and obstructive patterns. Saturation at rest was 87%, and it decreased to 80% after 10 steps were climbed. This patient has since undergone lung transplantation; the histology of the resected tissue was compatible with unusual interstitial pneumonia.

A polarizing light microscopic study of induced sputum cells showed the presence of polarizing particles. Chemical analysis by SEM-EDS and petrographic microscopy was performed, and the most abundant particles present (1-8 [micro]m) were calcium sulfate (CaS[O.sub.4]), silica (Si[O.sub.2]), and silicates (SiCaFe and AlSi) (Fig. 1). A similar analysis, performed on the lung tissue resected during her transplantation, revealed a similar composition.

[FIGURE 1 OMITTED]

Patient 2

A 27-yr-old female nonsmoker who had no serious illnesses in her medical history, and who had been employed as a dental technician for 3 yr, was referred to the emergency room because of shortness of breath, weakness, nausea, vomiting, diarrhea, and a weight loss of 12 kg. Chest x-rays revealed an increased interstitial pattern with hilar lymphadenopathy. A lung scan showed diffuse pulmonary emboli in both lungs. Echocardiography revealed pulmonary hypertension (i.e., 90 mm Hg), accompanied by severe tricuspid valve insufficiency and enlargement of the right atrium and ventricle. Her pulmonary function tests showed a severe restrictive pattern with a severe decrease in diffusing capacity for carbon monoxide (D[L.sub.CO]/alveolar ventilation [[V.sub.A]]: 46% predicted) and increased hypoxia in the blood gases (partial pressure of oxygen [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]: 67 mm Hg). The final diagnosis of sarcoidosis was made by open-lung biopsy, which revealed abundant noncaseating granulomas. Particles of hard metals (i.e., titanium and iron) and Si[O.sub.2] were found in the tissue.

Given this individual's occupational history, she was referred to our clinic for induced sputum analysis and for the beryllium lymphocyte transformation test (BeLTT), to rule out a possible misdiagnosis of silicosis/berylliosis. Chemical analysis of the sputum by SEMEDS and petrographic microscopy showed abundant particles (i.e., 1.6-2.5 [micro]m) of clay minerals [i.e., AISiFe, AISiCa, and [Al.sub.2][Si.sub.4][(OH).sub.2]], Si[O.sub.2], and barite (BaS[O.sub.4]) (Fig. 2). The BeLTT showed an increased stimulation at [10.sup.-4] M and [10.sup.-5] M of beryllium sulfate (BeS[O.sub.4]).

[FIGURE 2 OMITTED]

Patient 3

A 73-yr-old male nonsmoker was referred to our laboratory for sputum analysis after he had been followed at another hospital for 3 yr for progressive shortness of breath. He had immigrated to Israel in 1947, and had been employed as a clerk in a municipal office throughout his working years in Israel. However, during World War II, the patient had been a forced laborer under Nazi captivity in the mining industry in Silesia, where he worked for 12 hr every day for 7 consecutive months. He described the work environment as being "heavy with dust," and he also indicated that the miners had not been provided with any protective equipment.

A routine chest x-ray done in 1988 showed increased pulmonary markings. Given the progressive dyspnea and cough that occurred following minimal exertion, he underwent a clinical evaluation in 1996. The chest CT revealed diffuse interstitial fibrosis. Pulmonary function testing showed a restrictive pattern, and there was a moderate decrease in diffusion capacity. Additional deterioration was observed in diffusion capacity (56% predicted) in 1998.

Chemical analysis of his sputum by SEM-EDS and petrographic microscopy showed abundant particles (1-4.5 [micro]m) of clay minerals, quartz, aluminum, and asbestos fibers. A representative spectrum that identifies the asbestos is shown in Figure 3.

[FIGURE 3 OMITTED]

Discussion

The technical availability of the fiber optic bronchoscope, and the development of bronchoalveolar lavage (BAL), have facilitated the sampling of inflammatory response in patients with interstitial lung diseases. BAL is useful in diagnosing specific conditions, staging the extent of diseases, and, in some cases, predicting response to treatment. (15,16) Although this technique is essentially a noninvasive procedure when applied under proper selection guidelines, and there is minimal risk to the patient, frequently there are problems with patient compliance. Furthermore, the methodology is impractical for repeated sampling and screening, and BAL cannot be used in patients who suffer from other diseases that contraindicate bronchoscopy.

In contrast, induced sputum testing is much easier to conduct and is totally noninvasive, compared with bronchoscopy and BAL. Induced sputum provides an opportunity for the investigator to perform frequent evaluations in order to follow a dynamic course of pulmonary inflammatory disease and to monitor the effect of treatment.

Previously, few studies had been conducted in which induced sputum was evaluated in subjects who were suspected of experiencing occupational exposures: eosinophil counts were performed in the induced sputum of asthmatic isocyanates-sensitized subjects, (17) and the frequency of bronchial dysplasia was investigated in the sputum of previously exposed miners. (18) Some investigators have studied the relevance of asbestos bodies in spontaneous sputum production (19-21); in several recent studies, samples collected by sputum induction and bronchoscopy in healthy subjects and in patients with asthma and chronic bronchitis have been compared. (22,23) However, it was only during the past year that the 1st studies comparing induced sputum vs. BAL in interstitial lung diseases appeared in publication. Our group recently reported that induced sputum may reveal the status of hazardous dust exposure (silica and hard metals) as effectively as does BAL. (12) We found that BAL and induced sputum specimens retrieved during the evaluation of silica and hard-metal workers yielded similar quantitative and qualitative results, and that a similar size distribution of particles was present in the samples and chemical analysis of the particles. In another study, we described a fatal case of accelerated silicosis with a component of mixed-dust pneumoconiosis in a young hard-metal grinder. (24) In that study, we also reported that polarizing light microscopic analysis of BAL and induced sputum cells showed similar polarizing particles, which are characteristic for silica. (13,14)

In the present case-study report, we have demonstrated how induced sputum can assist in the evaluation and diagnosis of suspected occupational lung diseases. We used the plug-selection method of Popov et al., (11) which minimizes contamination by saliva compared with the use of whole-sputum preparations. (6) We could observe the particles in the macrophages, which are the main phagocytes in the lung (Fig. 4). The cell preparations in all patients contained at least 20%-35% macrophages (Table 1).

[FIGURE 4 OMITTED]

In the 1st case report, a polarizing light microscopic study of induced sputum cells revealed polarizing particles characteristic of silica. (13,14) Chemical analysis by SEM-EDS and petrographic microscopy revealed that the most abundant particles in the induced sputum were those of CaS[O.sub.4], Si[O.sub.2], and SiCaFe and AlSi silicates. This case was similar to 3 other cases of idiopathic interstitial pneumonia with bullae in schoolteachers, reported by Ohtsuka et al., (25) who provided evidence of the deposition of chalk in the lungs of these teachers and its contribution to the development of a heretofore unrecognized occupational lung disease. In contrast to the study by Ohtsuka et al., in which evaluation was based on postmortem material, the evaluation in our case was based on induced sputum analysis alone.

The 2nd case was a young dental technician who was initially misdiagnosed via open-lung biopsy as having sarcoidosis. Her history included a relatively short period of exposure to a mixture of silica, hard metal, and beryllium. The results of this case emphasized the vital importance of an occupational history anamnesis of patients suspected of having sarcoidosis. Moreover, it demonstrated that the use of combined noninvasive techniques, such as induced sputum and BeLTT, can be substituted for invasive ones, and can be the 1st investigative choice in the diagnosis of silicosis by induced sputum, and berylliosis by BeLTT.

The 3rd case was a clerk who had a relatively short-term but high-level exposure to asbestos as a miner during World War II. Later, after immigrating to Israel, he was employed for the remainder of his working life as an office clerk. He developed progressive diffuse interstitial fibrosis 40 yr following exposure. The clinical work-up excluded other possible diagnoses that could have accounted for both the clinical and CT findings. A suspected diagnosis of asbestosis resulting from his work as a miner was confirmed by sputum analysis, which revealed asbestos fibers. Previous reports (19-21) have indicated that only in high burdens of lung asbestos fibers can such fibers be observed in sputum. Our 3rd case illustrated the usefulness of sputum analysis for the diagnosis of occupational lung diseases--even when there had been remote exposure.

With respect to the safety of inducing sputum, investigators have found this process to be safe for use in patients with asthma (26) of varying severities, (27,28) and in individuals with chronic obstructive pulmonary disease. (29) The sputum-induction procedure produces a minimal fall in forced expiratory volume in 1 sec (which can be inhibited by pretreatment with salbutamol (27)), as well as a very minimal reduction in arterial oxygen saturation [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. (27) Despite the fact that patients with interstitial lung diseases generally lack evidence of obstructive reversible disease, we include the administration of [[beta].sub.2]-agonists in our protocol whenever induced sputum testing is performed outside the hospital setting, in the event that any unexpected adverse effects result from the use of hypertonic saline.

In conclusion, the data reported herein, as well as in our previous studies, (12,24) indicate that the induced sputum technique--a safe and simple procedure--can serve as a useful tool in the evaluation of patients with suspected occupational lung diseases.

The authors thank Esther Eshkol for her editorial assistance in the preparation of this study.

This study was supported by a grant from the Committee for Research and Prevention in Occupational Safety and Health, Ministry of Labor and Social Affairs, and the Manof Foundation of the National Insurance Institute, Israel.

Submitted for publication August 6, 2001; accepted for publication November 26, 2001.

Requests for reprints should be sent to Elizabeth Fireman, Ph.D., Institute of Pulmonary and Allergic Diseases, Tel-Aviv Sourasky Medical Center, 6 Weizman Street, Tel-Aviv 64239, Israel.

E-mail: fireman@tasmc.health.gov.il

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YEHUDA LERMAN National Institute of Occupational and Environmental Health Ra'anana, Israel and The Sackler Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel

YEHUDA SCHWARZ Institute of Pulmonary and Allergic Diseases Tel-Aviv Sourasky Medical Center Tel-Aviv, Israel and The Sackler Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel

GABRIELA KAUFMAN National Institute of Occupational and Environmental Health Ra'anana, Israel

ELIEZER GANOR Department of Geophysics and Planetary Sciences The Sackler Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel

ELIZABETH FIREMAN Institute of Pulmonary and Allergic Diseases Tel-Aviv Sourasky Medical Center Tel-Aviv, Israel and The Sackler Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel

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