The Destructive Index and Early Lung Destruction in Smokers1- 3
D. H. EIDELMAN, H. GHEZZO,
w.
D. KIM, and M. G. COSIO
Introduction
Recently, Saetta and colleagues (1) described the destructive index (DI), in which point counting is employed to permit direct microscopic assessment of the various aspects. of lung destruction: breaks in the alveolar and alveolar duct wallsand emphysematous spaces (1). Applying this technique to the lungs of human cigarette smokers with mild airflow obstruction, they found that 01, but not Lm , was able to separate the lungs of smokers from those of nonsmokers. Speculating that the difference between 01 and Lm could be accounted for by the inclusion of septal breaks in 01, they hypothesized that septal breaks may constitute the earliest lesion of cigaretteinduced lung destruction. The purpose of this study was to investigate the nature of lung destruction in the lungs of cigarette smokers before the development of marked emphysema. Based on previous observations (1), we hypothesized that destruction of the alveolar wallsshould be evident evenin the absence of macroscopic emphysema. To test this hypothesis, we measured emphysema grade, 01, and L m in the whole lungs of nonsmokers and smokers obtained at autopsy. We separately measured the "classic emphysematous spaces" and the "breaks in the walls" of the alveoli and alveolar ducts. Further, as the emphysema seen in the lungs of smokers commonly shows a predilection for the upper lobes, we also investigated these indices in the upper and lowerlobes of these lungs. Methods Subjects Lungs were obtained from 12 lifelong nonsmokers and 16 smokers who died suddenly outside the hospital of nonrespiratory causes. Cases were selected to have no or minimal emphysema as defined by an emphysema score (ES) of 5 or less. The smoking history was obtained from next of kin. Only cases in which a definitely positive or negative 158
SUMMARY The destructive Index (DI), a measure of alveolar septal damage and emphysema, has been proposed as a sensitive Index of lung destruction that closely reflects functional abnormalities, especially 1088of el.stlc recoil. To better unde...tand the progre88lon of lung destruction In smo...., we studied the contribution of Its principal components: breaks In the .Iveolar septa (Dlb) and the presence of emphysem.tous spaces (01.), and compared them to the mean linear Intercept (L m) .nd 01 .s orlgln.lly described. To do this we employed lungs obtained at autopsy from nonsmo.....nd smo Lungs were selected by emphysema score (ES) so that .11cases were emphysema free (nons mo and seven smo.rs) or had minimal emphysema (nine smo.rs; ES 5). Of these Indices, only Dlb was significantly Increased In the lungs of smo.rs: 17.8 ± 1.2 versus 12.4 ± 1.6, P < 0.05. We also Investigated the regional dl.trlbutlon of de.tructlon by comparing results In upper and lower lobes. 01., but not Dlb, was significantly Increased In upper lobes of smoM.... These data support the notion that Incre.ses In 01 In the lungs of .mo.... that occur before Incre.... In Lm or ES reflect the presence of alveolar ..ptal brealcB and highlight the Importance of alveolar septal destruction as a precursor to the development of airspace enlargement In the lungs of cigarette smo.,.. AM REV RESPIR DIS 1991; 144:158-159
=
I
smoking history was obtained were included in the study.
Microscopic Examination Only sections that weredetermined to be free of cutting artifacts wereaccepted for use, that is, free of compression caused by cutting or tearing, for example. A total of·20 nonoverlapping fields per slidewereused for each type of measurement. If there was any structure in the field, such as an airway, septum, or blood vessel, the maximal diameter of which was larger than 0.59 mm, then that field was not used for analysis.
Preparation of Lungs All specimens were fixed with 10070 formalin by intrabronchial infusion at a constant pressure of 25 em H 2 0 for at least 24 h. Sagittal slices of lung 1 em thick were cut, and 5 to 10 randomly selected blocks of tissue (template size 2 x 2.5 em) were obtained from the subpleural slice from each lobe or lung. The randomization of tissue blocks was done according to the method described by Thurlbeck (2). This approach results in selection of samples away from areas of marked destruction from which template blocks cannot be obtained. Therefore, the tissue studied was systematically less likely to contain gross destruction than the lung as a whole. Seven to ten sections 6 IJ.m thick were stained with periodic acid-Schiff and hematoxylin-phloxine saffron (PAS-HPS) (3) for light microscopy.
(Received in original form July 3, 1990 and in revised form January 25, 1991)
Emphysema Score The midsagittal slicesof whole lungs were impregnated with barium sulfate by first soaking the lung in a tray of saturated barium nitrate, squeezing it, and placing it in a tray of saturated sodium sulfate (4). Emphysema was ranked by comparison of the whole lung slice to the grading pictures of Thurlbeck (5). The emphysema score of the lungs included in this study were0 or 5. A score of 5 represents justdetectable emphysema.
1 From the Royal Victoria Hospital, the Montreal Chest Hospital Centre, and the MeakinsChristie Laboratories, McGill University, Montreal, Quebec, Canada. :1 Supported by the Medical Research Council of Canada, the Quebec Thoracic Society, and the EL/JTC Memorial Research Fund. 3 Correspondence and requests for reprints should be addressed to D. H. Eidelman, MeakinsChristie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, Quebec H2X 2P2, Canada.
Destructive Index Measurement of DI was made by a modification of the method previously described (1). In summary, we used a light microscope with a x6.3 objective and xl0 eyepiece contain-
157
WNO DESTRUCTION IN SMOKERS
ing a graticule with 42 points (Weibel No.2; Graticules, Tonbridge, Kent, UK). Structures underlying these points were classified as normal (N), destroyed (B), or emphysematous (E). Only points falling on alveolar and/or alveolar duct spaces werecounted. Points falling on other structures, such as duct wallsand alveolar walls, were not included in the calculations. Both alveolar spaces and alveolar duct spaces were considered normal if they were surrounded by intact walls disrupted in only one place. B was counted when the wall of an alveolus was disrupted in two or more places, when there were more than two disruptions of contiguous alveoli that opened into a single duct space, or when a duct space contained two or more islands of lung parenchyma within its lumen. E was counted when a structure was lined by cuboidal epithelium but was clearly not an airway, with or without breaks in the walls, or when a classic emphysematous lesion was present. Structures lined with cuboidal epithelium were rarely encountered. A total of 3,000 points per case wereevaluated (approximately 500 points per slide). The following indices were calculated: OIb
=
B
X 100
(1)
X 100
(2)
B+B+N Ole
=
B B+B+N
01
= OIb +
Ole
(3)
TABLE
1
DATA SUMMARY Patient No.
Age
01 (0/0)
Dlb
Ole
(mm)
(0/0)
(0/0)
0 0 0 0 0 0 0 0 0
0.295 0.278 0.389 0.390 0.313 0.318 0.345 0.322 0.354 0.334 0.013
26.6 6.1 32.8 38.2 12.9 18.5 27.3 13.9 30.2 22.9 3.5
18.1 6.0 10.8 11.0 7.7 17.9 8.5 13.0 18.9 12.4 1.6
8.5 0.1 21.9 27.1 5.2 0.6 18.8 1.0 11.4 10.5 3.3
0 0 0 0 0 0 0 5 5 5 5 5 5 5 5 5
0.315 0.339 0.332 0.422 0.407 0.308 0.304 0.360 0.270 0.385 0.309 0.456 0.357 0.378 0.332 0.304 0.349 0.013
27.0 26.8 32.4 46.2 26.7 24.1 16.5 31.7 18.6 44.2 24.5 57.3 52.5 39.2 37.7 37.8 33.1 3.1
16.6 13.9 11.3 26.5 17.4 15.3 15.8 12.3 18.6 12.0 24.1 16.5 21.8 16.3 23.4 18.8 17.8 1.2
9.5 12.9 21.1 19.7 9.3 2.2 0.7 19.4 0.0 32.2 0.4 40.7 30.7 22.9 14.3 19 14.8 3.2
Emphysema Score
(y"
Lm
Nonsmokers
1 2 3 4 5 6 7 8 9
65 68 81 86 59 60 68 64
75 69.6 3.1
x SEM Smokers
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
44
n
57 82 47 60 56 45 41 64
55 69 43
45 56 52 55.8 3.0
X SEM
Definition of abbreviations: X
Where OIb and Ole are the components of 01 that correspond to alveolar septal breaks and emphysematous lesions, respectively.
= mean; SEM = standard error of the mean. TABLE
2
ANOVA TABLE, MODEL
1
Dependent Variables
Mean Linear Intercept L m Measurement of Lm was made using a microscope with a x 10 objective and x 10 eyepiece by the modified method of Thurlbeck (2). The size of the section was determined by digitizing its area, and Thurlbeck's formula for correction of Lm because of tissue shrinkage was used to obtain the final value of Lm • Data Analysis Group results are expressedas means ± SBM. A general analysis of variance (ANOVA)model was constructed to evaluate the effect of smoking and ES on the dependent variables 01, OIb, Ole, and Lm (Modell). As we were interested in possible differences between lobes in the independent variables, werepeated the ANOVA, including lobe as an additional independent factor (Model 2). In determining significance, we collapsed the ANOVA model by progressively removing terms whose p value was greater than 0.25, starting with the interaction terms. Correlation was carried out by simple linear regression. P values less than 0.05 were considered significant. All statistical tests were carried out on a microcomputer using the NCSS statistical system (NCSS, Kaysville, UT).
01
Effect Smoking (A) ES (B) AB
Dlb
Ole
7.24 (0.013) 7.98 (0.010)
3.65 (0.068)
* F and p values in parentheses. The F values obtained from the final model after elimination of factors and/or interactions with a p value greater than 0.25. This model is based on 25 independent observations (one lung per case). For Lm none of the factors or interactions were found to be significant.
Results
The age, emphysema score, Lm , and 01 data are summarized in table 1. The results of ANOVA are shown in table 2. DIb was significantly related to smoking (p = 0.013) (Figure 1) but not to ES (figure 2). In contrast, Ole appeared to be related somewhat to ES, as might be predicted, although this did not quite reach significance (p = 0.068) (figure 2). Overall 01, the sum of DIb and Ole, was significantly related to ES (p = 0.010) (figure 2). There were no significant interactions between ES and smoking for any DIb, Ole, or 01. L m, on the other hand, was not significantly related
to either ES or smoking in this population. The results of a second ANOVA including lobe as an additional independent factor are shown in table 3. All the relationships continued to hold in this model. No significant effect of lobe could be found on DIb, 01, or Lm • With regard to Ole, significant interaction was found between smoking and lobe (p = 0.041). This was accounted for by a higher value of Ole in the upper lobes of smokers (19.2 ± 4.0) compared with the lower lobes (9.9 ± 2.9, n = 16)(figure 3). This did not reach significance in nonsmokers (upper, 12.5 ± 5.8; lower, 8.3 ± 3.7;
158
EIDELMAN, GHEZZO, KIM, AND COSIO 30
A
*
20
g
20
~
w o
~
~
a: o en
o
~ 10
10
.....
O~-----
NON-SMOKERS
Fig. 1. Components of the destructive index in nonsmokersand smokers. The open bars represent nonsmokers; the hatchedbarsrepresent smokers (mean ± SEM). Olb, the component related to alveolar septal breaks, wassignificantly increased in the lungsofsmokers compared to nonsmokers (p < 0.05). Therewas no significant difference in Ole, the component related to microscopic emphysema, between smokers and nonsmokers.
n = 9). No other relationships were significant.
Correlation Between Indices 01 and Lm were significantly correlated (r = 0.761, p < 0.01); this correlation held among nonsmokers (r = 0.839, p = 0.01) andamongsmokers(r = 0.747,p
D. H. EIDELMAN, H. GHEZZO,
w.
D. KIM, and M. G. COSIO
Introduction
Recently, Saetta and colleagues (1) described the destructive index (DI), in which point counting is employed to permit direct microscopic assessment of the various aspects. of lung destruction: breaks in the alveolar and alveolar duct wallsand emphysematous spaces (1). Applying this technique to the lungs of human cigarette smokers with mild airflow obstruction, they found that 01, but not Lm , was able to separate the lungs of smokers from those of nonsmokers. Speculating that the difference between 01 and Lm could be accounted for by the inclusion of septal breaks in 01, they hypothesized that septal breaks may constitute the earliest lesion of cigaretteinduced lung destruction. The purpose of this study was to investigate the nature of lung destruction in the lungs of cigarette smokers before the development of marked emphysema. Based on previous observations (1), we hypothesized that destruction of the alveolar wallsshould be evident evenin the absence of macroscopic emphysema. To test this hypothesis, we measured emphysema grade, 01, and L m in the whole lungs of nonsmokers and smokers obtained at autopsy. We separately measured the "classic emphysematous spaces" and the "breaks in the walls" of the alveoli and alveolar ducts. Further, as the emphysema seen in the lungs of smokers commonly shows a predilection for the upper lobes, we also investigated these indices in the upper and lowerlobes of these lungs. Methods Subjects Lungs were obtained from 12 lifelong nonsmokers and 16 smokers who died suddenly outside the hospital of nonrespiratory causes. Cases were selected to have no or minimal emphysema as defined by an emphysema score (ES) of 5 or less. The smoking history was obtained from next of kin. Only cases in which a definitely positive or negative 158
SUMMARY The destructive Index (DI), a measure of alveolar septal damage and emphysema, has been proposed as a sensitive Index of lung destruction that closely reflects functional abnormalities, especially 1088of el.stlc recoil. To better unde...tand the progre88lon of lung destruction In smo...., we studied the contribution of Its principal components: breaks In the .Iveolar septa (Dlb) and the presence of emphysem.tous spaces (01.), and compared them to the mean linear Intercept (L m) .nd 01 .s orlgln.lly described. To do this we employed lungs obtained at autopsy from nonsmo.....nd smo Lungs were selected by emphysema score (ES) so that .11cases were emphysema free (nons mo and seven smo.rs) or had minimal emphysema (nine smo.rs; ES 5). Of these Indices, only Dlb was significantly Increased In the lungs of smo.rs: 17.8 ± 1.2 versus 12.4 ± 1.6, P < 0.05. We also Investigated the regional dl.trlbutlon of de.tructlon by comparing results In upper and lower lobes. 01., but not Dlb, was significantly Increased In upper lobes of smoM.... These data support the notion that Incre.ses In 01 In the lungs of .mo.... that occur before Incre.... In Lm or ES reflect the presence of alveolar ..ptal brealcB and highlight the Importance of alveolar septal destruction as a precursor to the development of airspace enlargement In the lungs of cigarette smo.,.. AM REV RESPIR DIS 1991; 144:158-159
=
I
smoking history was obtained were included in the study.
Microscopic Examination Only sections that weredetermined to be free of cutting artifacts wereaccepted for use, that is, free of compression caused by cutting or tearing, for example. A total of·20 nonoverlapping fields per slidewereused for each type of measurement. If there was any structure in the field, such as an airway, septum, or blood vessel, the maximal diameter of which was larger than 0.59 mm, then that field was not used for analysis.
Preparation of Lungs All specimens were fixed with 10070 formalin by intrabronchial infusion at a constant pressure of 25 em H 2 0 for at least 24 h. Sagittal slices of lung 1 em thick were cut, and 5 to 10 randomly selected blocks of tissue (template size 2 x 2.5 em) were obtained from the subpleural slice from each lobe or lung. The randomization of tissue blocks was done according to the method described by Thurlbeck (2). This approach results in selection of samples away from areas of marked destruction from which template blocks cannot be obtained. Therefore, the tissue studied was systematically less likely to contain gross destruction than the lung as a whole. Seven to ten sections 6 IJ.m thick were stained with periodic acid-Schiff and hematoxylin-phloxine saffron (PAS-HPS) (3) for light microscopy.
(Received in original form July 3, 1990 and in revised form January 25, 1991)
Emphysema Score The midsagittal slicesof whole lungs were impregnated with barium sulfate by first soaking the lung in a tray of saturated barium nitrate, squeezing it, and placing it in a tray of saturated sodium sulfate (4). Emphysema was ranked by comparison of the whole lung slice to the grading pictures of Thurlbeck (5). The emphysema score of the lungs included in this study were0 or 5. A score of 5 represents justdetectable emphysema.
1 From the Royal Victoria Hospital, the Montreal Chest Hospital Centre, and the MeakinsChristie Laboratories, McGill University, Montreal, Quebec, Canada. :1 Supported by the Medical Research Council of Canada, the Quebec Thoracic Society, and the EL/JTC Memorial Research Fund. 3 Correspondence and requests for reprints should be addressed to D. H. Eidelman, MeakinsChristie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, Quebec H2X 2P2, Canada.
Destructive Index Measurement of DI was made by a modification of the method previously described (1). In summary, we used a light microscope with a x6.3 objective and xl0 eyepiece contain-
157
WNO DESTRUCTION IN SMOKERS
ing a graticule with 42 points (Weibel No.2; Graticules, Tonbridge, Kent, UK). Structures underlying these points were classified as normal (N), destroyed (B), or emphysematous (E). Only points falling on alveolar and/or alveolar duct spaces werecounted. Points falling on other structures, such as duct wallsand alveolar walls, were not included in the calculations. Both alveolar spaces and alveolar duct spaces were considered normal if they were surrounded by intact walls disrupted in only one place. B was counted when the wall of an alveolus was disrupted in two or more places, when there were more than two disruptions of contiguous alveoli that opened into a single duct space, or when a duct space contained two or more islands of lung parenchyma within its lumen. E was counted when a structure was lined by cuboidal epithelium but was clearly not an airway, with or without breaks in the walls, or when a classic emphysematous lesion was present. Structures lined with cuboidal epithelium were rarely encountered. A total of 3,000 points per case wereevaluated (approximately 500 points per slide). The following indices were calculated: OIb
=
B
X 100
(1)
X 100
(2)
B+B+N Ole
=
B B+B+N
01
= OIb +
Ole
(3)
TABLE
1
DATA SUMMARY Patient No.
Age
01 (0/0)
Dlb
Ole
(mm)
(0/0)
(0/0)
0 0 0 0 0 0 0 0 0
0.295 0.278 0.389 0.390 0.313 0.318 0.345 0.322 0.354 0.334 0.013
26.6 6.1 32.8 38.2 12.9 18.5 27.3 13.9 30.2 22.9 3.5
18.1 6.0 10.8 11.0 7.7 17.9 8.5 13.0 18.9 12.4 1.6
8.5 0.1 21.9 27.1 5.2 0.6 18.8 1.0 11.4 10.5 3.3
0 0 0 0 0 0 0 5 5 5 5 5 5 5 5 5
0.315 0.339 0.332 0.422 0.407 0.308 0.304 0.360 0.270 0.385 0.309 0.456 0.357 0.378 0.332 0.304 0.349 0.013
27.0 26.8 32.4 46.2 26.7 24.1 16.5 31.7 18.6 44.2 24.5 57.3 52.5 39.2 37.7 37.8 33.1 3.1
16.6 13.9 11.3 26.5 17.4 15.3 15.8 12.3 18.6 12.0 24.1 16.5 21.8 16.3 23.4 18.8 17.8 1.2
9.5 12.9 21.1 19.7 9.3 2.2 0.7 19.4 0.0 32.2 0.4 40.7 30.7 22.9 14.3 19 14.8 3.2
Emphysema Score
(y"
Lm
Nonsmokers
1 2 3 4 5 6 7 8 9
65 68 81 86 59 60 68 64
75 69.6 3.1
x SEM Smokers
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
44
n
57 82 47 60 56 45 41 64
55 69 43
45 56 52 55.8 3.0
X SEM
Definition of abbreviations: X
Where OIb and Ole are the components of 01 that correspond to alveolar septal breaks and emphysematous lesions, respectively.
= mean; SEM = standard error of the mean. TABLE
2
ANOVA TABLE, MODEL
1
Dependent Variables
Mean Linear Intercept L m Measurement of Lm was made using a microscope with a x 10 objective and x 10 eyepiece by the modified method of Thurlbeck (2). The size of the section was determined by digitizing its area, and Thurlbeck's formula for correction of Lm because of tissue shrinkage was used to obtain the final value of Lm • Data Analysis Group results are expressedas means ± SBM. A general analysis of variance (ANOVA)model was constructed to evaluate the effect of smoking and ES on the dependent variables 01, OIb, Ole, and Lm (Modell). As we were interested in possible differences between lobes in the independent variables, werepeated the ANOVA, including lobe as an additional independent factor (Model 2). In determining significance, we collapsed the ANOVA model by progressively removing terms whose p value was greater than 0.25, starting with the interaction terms. Correlation was carried out by simple linear regression. P values less than 0.05 were considered significant. All statistical tests were carried out on a microcomputer using the NCSS statistical system (NCSS, Kaysville, UT).
01
Effect Smoking (A) ES (B) AB
Dlb
Ole
7.24 (0.013) 7.98 (0.010)
3.65 (0.068)
* F and p values in parentheses. The F values obtained from the final model after elimination of factors and/or interactions with a p value greater than 0.25. This model is based on 25 independent observations (one lung per case). For Lm none of the factors or interactions were found to be significant.
Results
The age, emphysema score, Lm , and 01 data are summarized in table 1. The results of ANOVA are shown in table 2. DIb was significantly related to smoking (p = 0.013) (Figure 1) but not to ES (figure 2). In contrast, Ole appeared to be related somewhat to ES, as might be predicted, although this did not quite reach significance (p = 0.068) (figure 2). Overall 01, the sum of DIb and Ole, was significantly related to ES (p = 0.010) (figure 2). There were no significant interactions between ES and smoking for any DIb, Ole, or 01. L m, on the other hand, was not significantly related
to either ES or smoking in this population. The results of a second ANOVA including lobe as an additional independent factor are shown in table 3. All the relationships continued to hold in this model. No significant effect of lobe could be found on DIb, 01, or Lm • With regard to Ole, significant interaction was found between smoking and lobe (p = 0.041). This was accounted for by a higher value of Ole in the upper lobes of smokers (19.2 ± 4.0) compared with the lower lobes (9.9 ± 2.9, n = 16)(figure 3). This did not reach significance in nonsmokers (upper, 12.5 ± 5.8; lower, 8.3 ± 3.7;
158
EIDELMAN, GHEZZO, KIM, AND COSIO 30
A
*
20
g
20
~
w o
~
~
a: o en
o
~ 10
10
.....
O~-----
NON-SMOKERS
Fig. 1. Components of the destructive index in nonsmokersand smokers. The open bars represent nonsmokers; the hatchedbarsrepresent smokers (mean ± SEM). Olb, the component related to alveolar septal breaks, wassignificantly increased in the lungsofsmokers compared to nonsmokers (p < 0.05). Therewas no significant difference in Ole, the component related to microscopic emphysema, between smokers and nonsmokers.
n = 9). No other relationships were significant.
Correlation Between Indices 01 and Lm were significantly correlated (r = 0.761, p < 0.01); this correlation held among nonsmokers (r = 0.839, p = 0.01) andamongsmokers(r = 0.747,p
