American Journal of Industrial Medicine 22:49-58 (1992)

Electron Microscopy Analysis of Mineral Fibers in Human Lung Tissue Karl Heinz Friedrichs, Dr.-lng, Michael Brockmann, MD, Margit Flscher, PhD, and Gabrlele Wick

In the present study, lung samples from 126 autopsied cases were examined to determine the content of mineral fibers using analytical transmission electron microscopy (ATEM). The cases were divided into four groups (22 lungs of persons exposed to ambient environmental pollution, 32 cases of mesothelioma, 38 cases of primary lung cancer, and 34 asbestosis cases, 13 of these with additional pleural plaques). Fibers were counted, measured, and mineralogically identified using a combination of X-ray microanalysis and electron diffraction of the non-oriented fiber. Concentration of fibrous particles (defined as particles above 1 pm in length with roughly parallel long sides and an aspect ratio of 5:l and greater) was calculated as fibers lo6@ dry lung weight. The concentration of chrysotile was found to be similar throughout the groups except for two cases in the asbestosis group with comparably high numbers of chrysotile. However, a remarkable difference for amphiboles could be observed between the groups. Asbestos bodies were mostly found in the asbestosis group. There was a rather good correlation between numbers of amphibole fibers and asbestos bodies, with an average ratio of 10:1 . For comparison purposes between occupationally exposedhon-exposed individuals, a transition was found in the concentration range of 3-lo7 asbestos fiberslg dried lung weight. o 1992 Wiley-Liss, Inc. Key words: fibrous particles, occupational asbestos exposure, amphiboles, chrysotile, asbestos bodies, mesothelioma, lung cancer, asbestasis

INTRODUCTION The diagnoses of asbestosis and mesothelioma as occupationally induced diseases may be supported by determining the asbestos fiber content in lung tissue of patients applying for compensation. For many years, studies of lung fiber burden were performed by counting asbestos bodies with the phase contrast microscope [Eitner and Otto, 19841. Currently, more quantitative information on the fiber content in lung tissue is available by use of high resolution electron microscopes fitted with energy dispersive X-ray spectrometer and facilities for electron diffraction. Until now, there is no generally accepted value of fiber concentration to distinguish between fiber burden of the “normal” population and of occupationally exposed subjects. ReMedical Institute of Environmental Hygiene, Heinrich Heine University, Diisseldorf, Federal Republic of Germany. Address reprint requests to Prof. K.H. Friedrichs, Medical Institute of Environmental Hygiene, Heinrich Heine University, Diisseldorf, Auf‘m Hennekamp 50,4000 Diisseldorf 1, Federal Republic of Germany. Accepted for publication October 22, 1991. 0 1992 Wiley-Liss, Inc.

50

Friedrichs et al.

cently, proposals were made to normalize the available analytical data [Pott et al., 19911. A further step was taken by introducing size fractions with respect to different diseases [Lippman, 19881. A wide range of methods have been used in different laboratories in analyzing lung tissue for mineral fibers [Churg, 1982; Davies et al., 1986; Rodelsperger et al., 1990; Chiappino et al., 19891. In studies on lung fiber burden it is usual to summarize similar cases in appropriate groups for comparison purposes. However, a rather crucial problem is raised by the selection into relevant groups. Classification may be performed, for example, according to likelihood of specific occupational exposure: possible-probable-unlikely [SCbastien et al., 1988; Friedrichs et al., 19891. In this study, the total number of 126 cases selected from the German mesothelioma register in Bochum covering the period from 1989-1991 has been divided into four different groups based on the medical diagnosis at autopsy, similar to a previously conducted study [Friedrichs and Molik, 19851: 22 cases exposed to ambient environmental pollution; 381 cases of primary bronchial carcinoma; 32l cases of pleural mesothelioma; and 34 cases of asbestosis, 13 of these with pleural plaques. The groups were differentiated without regard to further diseases. Tissue samples were obtained from different sites of the lung, which unfortunately could not exactly be described as to location in each case. MATERIALS AND METHODS The dried weight of each tissue sample was determined using a similar technique as described previously [Friedrichs, 19901. Briefly, this consisted of dissolving formalin-fixed lung tissue in boiling formamide for several hours and then collecting particulates on previously carbon-coated 0.4 p,m nuclepore filters. The ratio of dry weight to wet weight of tissue varied from 70/0-18%.In all preparations, the samples were neither ashed nor ultrasonicated. The dry filter was once more carbon-coated, cut into pieces, and placed on 200 mesh copper-grids. The filter substrate was dissolved afterwards with chloroform in a petri dish for several hours. First, all grids were scanned in an analytical transmission electron microscope (ATEM) with goniometer stage at low magnifications ( X 1,OOO) to check for an even particle distribution. The same magnification was later used for counting asbestos bodies in the total area of one grid per sample. Uncoated fibers were identified at magnifications ranging from X 16,000- X 31 ,OOO using a combination of morphological observation, quantitative x-ray microanalysis, and electron diffraction of the non-oriented fiber. No distinctionwas made between members of the amphibolegroup. A minimum of ten grid openings per sample (correspondingto a total area of -08mm2) was examined for asbestos and other mineral fibers which were additionally measured (length and width). The resulting analytical sensitivity was in the range of lo5 fibers/g dry weight. In this study, a fiber was defined as an elongated particle having mainly parallel long sides, a minimum length of 1 pm, and an aspect ratio of lengtwdiameter (UD) of 5:l and larger. For each sample, a blank filter was prepared to check for impurities on the surface. In addition to regular particles, low concentrations of very short elementary chrysotile fibrils were detected on empty filters. Evaluation showed that concentration of fibers, as well as their sizes, were approximately log-normally distributed and statistics could be performed by using transformed values.

'WiWwithout occupational asbestos exposure according to anamnestic data.

Electron Microscopy Analysis of Lung Tissue

51

TABLE I. Concentration of Fibers (L>1 pm, L/D >5:1; 106/pgDry Weight) in Samples From 126 Autopsies in Bochum (FRG) Mesothelioma Register Over the Period 1989-1991 Geometric Mean (and Geometric SD) ~~

Chrysotile

Amphiboles

Asbestos bodies

Other

4.5 (2.6) 5.7 (3.6) 10.7 (4.7) 7.2 (6.8)

1.9 (2.3) 3.7 (4.4) 16.6 (6.6) 106.1 (6.0)

-

6.9 (2.6) 10.0 (3.4) 26.7 (2.8) 60.1 (3.1)

~

Normal lungs Lung cancer Mesothelioma Asbestosis

1.2 (1.9) 2.8 (3.9) 11.6 (5.0)

RESULTS

Average concentrations of chrysotile, amphibole, and other fibers as well as of asbestos bodies are shown in Table I. Data are reported as geometric means (and geometric standard deviation), since this value could mostly be approximated to the corresponding type of distribution curve. Obviously, there is a similar level of 5-106 chrysotile fibers/g dry weight in groups 1 and 2, with a significant difference between 1 and 3 at the 0.1% level, while the concentration of amphiboles, asbestos bodies, and other fibers is steadily increasing from groups 1-4. It is notable that the standard deviation of mean values is high in nearly all cases (between 1.9 and 6.8). For comparison between groups, the concentration distribution seems to be more informative. Cumulative frequency distribution of chrysotile fiber concentration in the groups is shown in Figure 1. From the analysis of variance (U-test after Wilcoxon-Mann-Whitney)it can be concluded that there is no statistically significant difference for chrysotile between the groups. For amphibole fibers (shown in Fig. 2), the difference of the median is more evident (mostly at the p = 0.005 level). The cumulative frequency curve for other fibers in Figure 3 is comparable to that in Figure 2, indicating the possibility that this group of fibers may contain a number of asbestos fibers that could not definitively be identified by either analytical technique. Statistical evaluation yields similar results as obtained for amphiboles (mostly at the p = 0.01 level). Table I1 shows data (geometric mean and standard deviation) derived from analysis of variance for fiber length, diameter, and aspect ratio, respectively. Comparison of data suggests a similarity in size of chrysotile fibers in most of the groups. The most marked differences are found in the lengths of amphibole fibers (the longest uncoated amphibole fiber measured 120.5 pm) for the mesothelioma and asbestosis groups on the one side and groups 1 and 2 on the other. Consequently, the aspect ratio in the former mentioned groups is also comparably great. Other fibers were rather short but thick, thus leading to a low L/D between 11:l and 12:l. It has to be noted that the aspect ratio of all fibers found in the normal group was also low. Asbestos bodies were found only in groups 2-4. Their cores consisted mainly of amphibole asbestos (crocidolite, amosite) and only less than 5% of fibers were chrysotile. Neither marked alterations of the x-ray spectrum nor structural damage of the fiber surface could be observed. The correlation between amphibole asbestos fibers and asbestos bodies is shown in Figure 4. The ratio of fibers to asbestos bodies was calculated to be approximately 1O:l which is similar to results of other authors [Ashcroft and Heppleston, 1973; SCbastien et al., 19771. Mean values and standard

52

Friedrich et al. cumulative frequency (S-%) 100 80

-+

normal lunga

+ mrrothrllomr + lung ornoor

80

4- arbratorlr

* plrurrl plrquaa

40 20 0 .6

1.6

8

3

12

-

24

48

96

182 384

chrysotlle 10°/g dry Fig. 1. Cumulative frequency curves ( S - 8 ) of chrysotile concentration (fibers 106/g lung dry weight) in five different groups.

cumulative frequency

@-%I

+ normal lunga

+ mrrothrlloma + lung ornuor -a- rrbrrtoala ... . , . .. . .. .

.6 1.6

3

8

* plrurrl plrqura

12 24 48 98 192 384 7881638

-

amphiboles 10e/gdry Fig. 2. Cumulative frequency curves (S-%)of amphibole concentration(fibers * 106/g lung dry weight) in five different groups.

+

deviations were found to be 32 29 pm for the length and 0.27 diameter of cores. The longest asbestos body measured 180 pm.

+ 0.24 pm for the

DISCUSSION

In this investigation we determined concentrations and sizes of fibers and examined types of fibers in 126 lung tissue specimens using an ATEM. Based on the medical diagnosis at autopsy, we established four different groups, one of them classified as “normal,” without occupational exposure to asbestos for comparison purposes. There were no cases of lung cancer in this group exposed to general

Electron Microscopy Analysis of Lung Tissue

53

cumulative frequency (5-96)

-

normel lung

+ meeothdiome -l+ lung cancer

+- eebeatoale

* piourel plaque

-

other fibers 106/g dry Fig. 3. Cumulative frequency curves (S-%) of other fibers (fibers different groups.

- 106/g lung dry weight) in five

TABLE II. Size Data and Aspect Ratio of Fibers in Lung Tissue Samples From 126 Autopsies in Bochum (FRG) Mesothelioma Register 1989-1991 Geometric Mean (and Geometric SD) Fiber length (bm)

Chrysotile Normal lungs Lung cancer Mesothelioma Asbestosis

1.4 (2.1) 2.3 (2.6) 2.0 (2.3) 1.7 (1.9)

Normal lungs Mesothelioma Lung cancer Asbestosis

.05 (1.7) .06 (1.6)

.07 (1.8) .07 (1.9)

Amphiboles

Other

2.6 (2.5)

1.8 (1.6) 2.2 (1.9) 2.2 (1.9) 2.5 (2.2)

4.4 (2.7)

6.0 (2.6) 6.3 (2.8) Fiber diameter (bm) .17 (2.1) .16 (2.1) .19 (2.0) .17 (2.0)

.16 (2.0) .18 (2.6) .25 (2.4) .23 (2.5)

LID ~

Normal lungs Mesothelioma Lung cancer Asbestosis

18 (2.5) 34 (2.5) 32 (2.8) 27 (2.3)

15 (2.4) 37 (3.0) 23 (3.0) 38 (3.3)

12 (2.1) 12 (2.5) 11 (2.1) 11 (2.5)

environmental particle concentrations. In the cancer groups (2 and 3), the information about the occupational history in several cases was incomplete. The results of the present study suggest an increase of the asbestos content in occupationally exposed individuals compared with the non-exposed. However, it should be noted that the TEM results are inevitably associated with great scatter due to different reasons. Firstly, all analyzed fibers represent the state of the lung at autopsy, while nothing is known about the deposition time of fibers in the sample. Furthermore, there is no standardization as yet as to which part of the lung should be sampled. In surgical cases, the specimen is necessarily obtained from the removed

Friedrichs et al.

54

I 4 8

X

10‘

1

Fig. 4. Logarithmic scattergram of correlation between amphibole fibers and asbestos bodies (“EM evaluation).

part of the lung. Though standardization seems to be necessary since the fiber distribution in different lobes of the lung is evidently not uniform [Teschler et al., 1990; Rendall and Van Sittert, 19851, this seems to be impossible in several cases (e.g., exhumation). Standardization of medical diagnosis as a further simplification would also be helpful. Another point inducing uncertainty in interpretation of data is the lack of confident information about the patient’s occupational “asbestos” history. Information obtained from the patient himself or his relatives may sometimes be incomplete. In one case, the patient never realized a previous exposure to asbestos [Fischer et al., 19901.

Results will also depend on numerous factors that influence the analytical procedure. Important factors include all preparative steps beginning with the determination of the weight of tissue, followed by procedures of digestion, recovery, analysis, and even the reporting of results, which should be harmonized as far as possible to make results comparable between laboratories [Roggli, 1990; Davies et al., 19861. In our experience, as little preparation as possible should be applied to avoid any alterations of the original state of the sample. Relating to microscopic work, it has to be considered that the occurrence of very short fibrils of chrysotile as possible impurities of water or filters is unavoidable. Our investigations showed that it is not possible to “correct” for those artifacts since the loading (particularly of chrysotile fibers