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Reproducibility of Asbestos Body Counts in Digestions of Autopsy and Surgical Lung Tissue

Reproducibility of Asbestos Body Counts in Digestions of Autopsy and Surgical Lung Tissue

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AMERICAN JOURNAL OF INDUSTRIAL MEDICINE
Reproducibility of Asbestos Body Counts inDigestions of Autopsy and Surgical Lung Tissue
Marı´a-Isabel Velasco-Garcı´a,
BSc
,
1,2,3
Marı´a-Jesus Cruz,
PhD
,
1,2
Laura Ruano,
BSc
,
1,2
Marı´a-Angeles Montero,
PhD
,
4
Asuncio´n Freixa,
BSc
,
5
and Jaume Ferrer,
PhD
1,2
Ã
Background
Asbestos body (AB) counting by a single observer is the most commonlyused objective technique to demonstrate asbestos deposition in the lung. In this study,the accuracy and reliability of this procedure is analyzed by evaluating the degree of agreement between two experienced readers.
Methods
Lung tissue specimens from 66 individuals, most of whom had not beenexposed to asbestos, were studied: 35 were obtained in postmortem studies (upper,middle, and lower lung) and 31 were from patients who underwent surgery for lungcancer. Overall, 167 samples were analyzed. Lung tissue sections weighing 0.5 g wereobtained prospectively and processed, and the inorganic residue was analyzed by light microscopy at 400
Â
magnification by two experienced readers. Results were expressed as AB/g of dry lung tissue. Interobserver variability was analyzed using the Spearmancorrelation coefficient and agreement was evaluated by the Bland–Altman method and the kappa index.
Results
The interobserver correlation was 0.8975: 0.8029 for autopsy samples and 0.9592 for biopsy samples. Bland–Altman plots showed that most values were grouped around the 95% limits of agreement. The kappa index was 0.87 for all samples, and 0.79, 0.65, and 0.54 for upper, middle, and lower lung specimens, respectively.
Conclusions
Asbestos body counting by a single reader is a reliable method, especi-ally at low concentrations of asbestos bodies in lung tissue. Double reading may beindicated in borderline cases with asbestos body levels close to levels of 1 000 AB/g.
Am. J. Ind. Med.
ß
2011 Wiley-Liss, Inc.
 KEY WORDS: asbestos bodies; lung; light microscopy; interobserver agreement
INTRODUCTION
Asbestos is a natural fibrous silicate mineral that hasbeen widely used for industrial purposes [Col
Á
legi d’apar-elladors i Arquitectes Te`cnics de Barcelona, 1998]. Inhala-tion of asbestos fibers increases the risk of developingseveral respiratory diseases, including pleural plaques, fib-rosis, and effusion, as well as more severe diseases, suchas malignant mesothelioma, lung cancer, and asbestosis[Becklake et al., 2007]. Asbestos-related conditionsoccupy the second position in the voluntary registry of occupational diseases in Catalonia [Orriols et al., 2006].Diagnosing asbestos-related diseases may be difficult,because the clinical picture is confusing or because thereis a lack of information regarding the patient’s exposurestatus. The problem in these cases is not only clinical, but
1
ServiciodeNeumologiaHospitalUniversitarioValld’Hebron,Barcelona,Spain
2
CiberEnfermedadesRespiratorias(Ciberes),Barcelona,Spain
3
DepartamentodeMedicina,UniversidadAuto ¤nomadeBarcelona,Barcelona,Spain
4
Servicio de AnatomÕa Patolo ¤gica, Hospital Universitario Vall d’Hebron, Barcelona,Spain
5
SpanishNationalInstituteforOccupationalSafetyandHealth,Barcelona,SpainContract grant sponsor: Instituto de Salud Carlos III; Contract grant number: FIS PI07/90478;Contractgrantsponsor:Fundacio ¤CatalanadePneumologia.*Correspondence to: J. Ferrer, Jaume Ferrer Sancho Servicio de Neumologia, HospitalValld’Hebro,PasseigValld’Hebron,11908035Barcelona,Spain.E-mail:jjferrer
@
vhebron.netAccepted12April2011DOI10.1002/ajim.20966.PublishedonlineinWileyOnlineLibrary(wileyonlinelibrary.com).
ß
2011Wiley-Liss,Inc.
 
also legal, since economic compensation is frequentlyinvolved [Ferrer and Cruz, 2008]. In several countries,management of these difficult cases requires demon-stration of asbestos deposition in the lung. The finding of elevated levels of asbestos in lung tissue is definite proof of exposure and provides powerful evidence favoring thediagnosis of asbestos-related disease [Finish Institute of Occupational Health, 1997; De Vuyst et al., 1998].Asbestos body (AB) counting using light microscopyis the simplest way to determine the lung asbestos content[Kishimoto et al., 1989]. The threshold of 1 000 AB/g drytissue has been proposed as an indicator of significantasbestos lung burden. There is some agreement that abovethis threshold, there is a risk for the development of asbes-tosis [De Vuyst et al., 1998]. Nevertheless, in the case of mesothelioma, slight exposures and AB counting belowthis threshold have been reported [Finish Institute of Occupational Health, 1997]. As such, the precision of ABcounting is important, particularly in cases with AB valuesclose to the threshold. In these cases, even small errors inthe reading could imply a difference when attributing aspecific disease to asbestos.International guidelines have pointed out that samplepreparations for this purpose vary considerably and haveadvised individual laboratories to standardize their refer-ence values [De Vuyst et al., 1998]. However, little atten-tion has been focused on determining the reliability of ABreading by a single observer. AB evaluation includesdetection and counting. As in many other measurementsystems performed by humans, it depends on the personalexperience of the observer and implies a certain margin of error. Considering that the reader is an inherent source of variation in AB counting [Harper and Bartolucci, 2003], itis important to know the interobserver reproducibility of the lung AB results to determine the number of readersneeded to accurately carry out the technique.The aim of this study is to determine the reliability of the currently used method for AB counting, based onsample examination by a single observer.
MATERIALS AND METHODSStudy Population
Sixty-six individuals were included in the study, dis-tributed in two groups: 35 cadavers of persons with nomedical record of respiratory disease undergoing post-mortem examination (group A) and 31 patients with lungcancer undergoing surgery (group B) (Table I).A specific questionnaire was administered in all casesto evaluate asbestos exposure [Velasco-Gar´a et al.,2010]. The questionnaire was completed by the patients,themselves, in group B and by close relatives in group A.The study was approved by the Ethic Committee of theVall d’Hebron Hospital in Barcelona. All patients inGroup B and the relatives in Group A gave their writtenconsent.
Sample Collection Protocol
Post-mortem examinations were performed at the
 Instituto Anato´ mico-Forense de Barcelona
(ForensicAnatomy Institute of Barcelona). At group A, lung tissuespecimens 2 cm
3
in size were obtained from three regionsof the right lung: apex of the upper lobe (zone 1), apex of the lower lobe (zone 2), and base of the lower lobe(zone 3). The 105 samples obtained were fixed in formoland sent to our hospital laboratory for analysis.The surgical specimens (group B) were obtained fromthe Pathology Department of Hospital Vall d’Hebron. Thesize of the specimen was 2 cm
3
whenever possible.The region of the lung sampled depended on the sitewhere the tumor had developed: specimens came fromthe right lung in 14 patients and from the left lung in theremaining 17. Sixty-four samples were obtained in thisgroup, with more than one from some patients.All lung specimens were examined by a pathologistfrom our hospital. The presence of chronic pulmonary dis-ease was ruled out in all cases.
TABLE I.
AsbestosBodyValuesintheStudyPopulation
Nsamples Reader1AB/gdrytissue
a
Reader2AB/gdrytissue
a
k
95%CI
Total(groupsAandB) 169 43(0^32146) 42(0^30875) 0.869 0.766^0.972GroupATotal 105 21(0^1308) 19(0^1212) 0.645 0.342^0.949Zone1 35 21(0^490) 0(0^429) 0.785 0.370^1.200Zone2 35 20(0^778) 0(0^489) 0.653 0.016^1.323Zone3 35 22(0^1308) 21(0^1212) 0.535 0.033^1.038GroupBTotal 64 102(0^32146) 72(0^30875) 0.930 0.833^1.026
a
Dataexpressedasmedian(range).
2 Velasco-Garcı´a et al.
 
Preparationof Lung Samples
Two 0.5 g fragments of lung tissue that did not con-tain pleura or vessels were obtained from each specimen.One of these fragments was frozen, lyophilized, andweighed to determine the dry tissue weight, in keepingwith an international agreement to express the AB resultsin terms of grams of dry lung tissue. Once the dry weightwas known, the lyophilized sample was discarded, becausethis technique can cause changes in the concentration andsize of the fibers [De Vuyst et al., 1998].A 30 cc amount of filtered sodium hypochlorite wasadded to the non-lyophilized tissue section, and thesample was shaken for 24 h to facilitate digestion of the tissue and elimination of other organic material. Thesample was then centrifuged at 3 700 rpm for 20 min,sodium hypochlorite was eliminated, and the sample wasre-suspended in filtered distilled water. To dissolve theAB present in this liquid medium, the sample was soni-cated for 10 min using an ultrasonic water bath (UCI-50Raypa SL; 300 W, 50/60 Hz), and was then washedand re-suspended in 20 cc of filtered distilled water. Thesolution obtained was passed through a 0.45-
m
m porediameter filter (Millipore membrane filters, HAWP02500).The filter was dried overnight at 37
8
C, placed on a micro-scope slide and made transparent using acetone vapor (JSHoldings vaporizer; 240 v/110 v) for subsequent reading.In all the samples, we obtained an adequate filter loadingfor counting, and it was not necessary to dilute thesample.
AsbestosBody Counting by OpticMicroscopy
The filters were viewed with an optic microscope(CX21FS2; Olympus Life Science Europe GMBH,Hamburg, Germany) at
Â
400 magnification. All filterswere examined by two experienced readers according to aprotocol previously described by our group [Velasco-Gar-´a et al., 2010].All the samples obtained for each individual wereassessed: in group A, the values obtained for each of thethree lung zones were evaluated, and in group B, all thezones from which a sample could be obtained. In accord-ance with the criteria established by the working group of the European Respiratory Society in 1998, AB levelsexceeding 1 000 AB/g dry tissue were considered signifi-cant [De Vuyst et al., 1998].
Statistical Analysis
The data are expressed as the median and the range(r). On analysis with the Kolmogorov–Smirnov test, ABvalues did not follow a normal distribution; hence, theWilcoxon test was used to determine the differencesbetween groups. The Spearman correlation coefficient wascalculated to establish the relationships between theparameters analyzed. The statistical analysis was carriedout with GraphPad software (2002–2005).Interobserver variability in AB counts was comparedusing Bland–Altman difference plots [Bland and Altman,1986]. Logarithmic transformation of the data was thenused to correct the relationship between the ratio and theaverage. The 95% confidence interval for the differencewas used to assess the differences between the tworeaders.The kappa coefficient (
k
), corresponding to the per-centage of agreements over the total of observations,excluding agreements attributed to chance, was used todetermine the degree of interobserver variability. For thispurpose, the AB counts obtained were divided into threegroups: (1) 0–300 AB/g, (2) 301–1 000 AB/g, and (3)
>
1000 AB/g. As was reported in a previous study[Velasco-Garcı´a et al., 2010], the majority of the urbanpopulation in our setting has an AB lung content of lessthan 300 AB/g.
RESULTSStudy Population
Individuals in group A had a mean age at death of 64years (r: 35–87); 63% were men, 36% had been smokersand 24% ex-smokers. The most common cause of deathwas heart failure. Patients in group B had a mean age of 62.5 years; 87% were men, 6% were smokers and 84%ex-smokers, and all had lung cancer.
Lung Content of Asbestos
The median (range) of the values obtained by the tworeaders was 43 (0–32.146) AB/g for reader 1 and 42(0–30.875) AB/g for reader 2 (Table I). Only six individ-uals (3.6%) presented AB values higher than 1 000 AB/gof dry tissue (one from group A and five from group B).The cause of death in patient in group A with high levelsof AB was cardiovascular disease. In group B, vepatients had lung tumors, including three with adenocarci-noma and two with epidermoid carcinoma.
InterobserverVariability
Bland–Altman plots were generated for the entirepopulation analyzed (Fig. 1), for each of the groups (A andB) separately (Fig. 2), and for each of the 3 lung zones ingroup A (Fig. 3). In these plots, the x-axis is the Averagebetween the determinations of reader 1 and reader 2, andthe y-axis is the Ratio of the 2 determinations.
Reproducibility of Asbestos Body Counts 3

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