Thorax 1992;47:144-149

Microscopic and macroscopic measurements ofemphysema: relation to carbon monoxide gas

transfer

A McLean, PM Warren, M Gillooly, W MacNee, D Lamb

AbstractBackground Studies of the relationbetween the severity of structuralchange in emphysema and physiologicalabnormality have been based onmacroscopic assessments, which havenot been truly quantitative or sensitiveenough to detect early changes. With ahighly reproducible method for measur-ing emphysema using histological sec-tions and a semiautomatic imageanalysis system, this quantitative assess-ment of emphysema was compared witha semiquantitative macroscopic assess-ment of emphysema and measurementsof carbon monoxide gas transfer.Methods Microscopic and macroscopicmeasurements of emphysema on 44thoracotomy specimens were compared;only two were from non-smokers. Air-space wall surface area per unit volumewas measured microscopically with anautomatic image analyser and expressedas both the mean airspace wall surfacearea per unit volume and the mean valueof the five fields with the lowest values.Macroscopic emphysema was measureddirectly on a tracing of the midsagittalslice using a digitising tablet attached toa microcomputer and expressed as apercentage of the total area of lung. Incases with centriacinar emphysema thenumber of discrete lesions was counted.Results The area of macroscopicemphysema ranged from 0 to 78% of thetotal area of lung examined, but mostpatients had less than 1% involvement sothat the distribution was highly skewed.Both mean airspace wall surface areaper unit volume and the mean of fivefields with the lowest airspace wallsurface area per unit volume werenormally distributed, with mean air-space areas ranging from 8-8 to 25 4mm2/mm' (mean 18-1 mm2lmm'). Inlobes with centriacinar emphysema thenumber of discrete lesions correlatedwith airspace wall surface area per unitvolume and with preoperative carbonmonoxide transfer factor (TLCO) perunit lung volume. However, othermeasurements of macroscopic emphy-sema did not correlate with loss ofalveolar wall surface area, and there wasconsiderable overlap between subjectswith no or minimal macroscopic

emphysema and those with more severedisease. TLCO correlated with both meanairspace wall surface area per unitvolume and the mean of five fields withthe lowest airspace wall surface area perunit volume but not with the severity ofmacroscopic emphysema.Conclusion If emphysema is to bequantified it must be measured micro-scopically; macroscopic measurementsdo not, in general, reflect the microscopicloss of airspace wall.

Reid defined emphysema as "a condition ofthe lung characterised by an increase beyondnormal in the size of airspaces distal to theterminal bronchiolus."' Other definitions differin whether the terms dilatation, destruction,or fibrosis are included.2' Althoughemphysema affects airflow, lung compliance,and gas transfer, its precise contribution tochronic obstructive airways disease is poorlyunderstood,' and in anatomical terms it isdifficult to measure accurately as normal air-space size has yet to be established.

Centriacinar and panacinar emphysema arethe two most common forms of the disease.They differ in their distribution within theacinus and to a lesser extent within the lung.6Measurements and analysis of the two formsof emphysema have often been carried outseparately,7'2 but they are now commonlyregarded as part of a spectrum."2-19

Several qualitative methods have beendevised to assess the severity of emphysemamacroscopically.2202' After comparing severaltechniques Thurlbeck et al recommended thepanel grading system because of itsreproducibility, speed, and ease of use.'022This is a semiquantitative and subjectivemethod in which whole lungs slices are gradedfrom 0 to 100 against a panel of standards.However, standards do not represent a truearithmetic scale, the method does not measurethe percentage of lung affected and cen-triacinar and panacinar emphysema are notscored separately. Alternatively, the role ofmacroscopic emphysema as a proportion oflung volume and airspace surface area can bequantified by counting points2' and by meanlinear intercept techniques,24 or, as in ourstudy, by measuring individual fields with ahigh degree of accuracy with computer basedimage analysers.

Department ofPathologyA McLeanM GilloolyD LambDepartment ofRespiratory MedicineP M WarrenW MacNeeMedical Building,University ofEdinburgh,Edinburgh EH8 9AGReprint requests to:Dr Lamb

144

Microscopic and macroscopic measurements of emphysema: relationship to carbon monoxide gas transfer

Macroscopic assessment of emphysema ispotentially insensitive to quite considerablelosses of surface area. The average alveolardiameter is about 0-25 mm25 whereas thethreshold value for detecting an emphy-sematous airspace macroscopically is 1 mm.23This represents a 64 fold increase in airspacevolume and, more importantly in terms ofsurface area available for gas exchange, a 75%reduction in surface area per unit volume (24mm'/mm' as opposed to 6 mm2/mm3).

In this study we used an image analyser tomeasure airspace wall surface area per unitlung volume-the surface area to volumerelation of airspaces within the lung. Resultsobtained from this analysis were comparedwith an objective assessment of macroscopicemphysema. Macroscopic and microscopicmeasurements were compared with pre-operative measurements of single breathcarbon monoxide gas transfer.

Materials and methodsForty four lobes were obtained at thoracotomyfrom patients aged 46 to 74 years. All but twowere smokers. In 29 cases the specimens wereobtained by a lobectomy with a peripheraltumour confined to the segment of origin. Theremaining 15 specimens were obtained atpneumonectomy: only the lobes free of tumourwere used. Immediately after resection thelobes were infused with formalin intrabron-chially at an applied pressure of 25 cm H2Ountil fully distended. Once fixed the lungs werecut into 1 cm parasagittal slices.

MACROSCOPIC ASSESSMENTThe mid-sagittal lung slices were immersedin water and examined with a hand lens.Emphysematous areas were traced on to a clearpolythene sheet placed over the slice, and theircombined areas were measured on a digitisingtablet linked to a microcomputer. Difficulty inoutlining small centriacinar lesions-whichnever accounted for more than 1% or 2% ofthetotal area-was overcome by measuring thediameter of each lesion with Vernier callipersand calculating their area assuming a circularconfiguration. Their summed cross sectionalarea was then added to the digitised figure.Macroscopic emphysema was expressed as thepercentage area of the mid-sagittal sliceoccupied by airspaces of at least 1 mm indiameter. The number of centriacinar lesionswas also recorded. To facilitate further analysisspecimens were divided into four groups-namely, those with no emphysema, those withpure centriacinar emphysema, those with pure

Table I Summary statistics ofpatients and details of lobes studied

Variable Mean Range SD n

Age (years) 61 46-74 6-6 44AWUV (mm2/mm3)Mean 18-12 8-78-25 40 3-31 44LF5 12-61 1-88-21-26 4 50 44

Macroscopic emphysema (% area) 8 0-79 19-3 44

AWUV = airspace wall surface area per unit volume.LF5 = mean of five lowest AWUV fields.

panacinar emphysema, and those with bothtypes of emphysema.

MICROSCOPIC ASSESSMENTSix blocks (2 cm by 2 cm) were taken formicroscopic assessment from each of the firsttwo lateral subpleural slices with a transparentgrid marked out in 2 cm squares and computergenerated random numbers. Blocks wereembedded in glycol methacrylate to givenegligible processing artefact,26 and sectionswere cut at 3 um and stained with haematoxylinand eosin.

Airspace wall surface area per unit volumewas measured with an IBAS2 image analyser(Kontron, Watford), a Bosch TYK 9A tele-vision camera with Chalnicon tube, and anOrtholux II light microscope with stabilisedvoltage illumination. Fields were chosen byrandomly selecting one of the 12 stained sec-tions and then locating a field from within thatsection with numbered coordinates from anEngland Finder (Graticules, Tonbridge, Kent)and computer generated random numbers.Fields containing structures such as bronchi,bronchioles, or accompanying vessels wererejected. The images of airspace walls wereenhanced, and objects such as inflammatorycells were removed by applying a size filter or byinteractive editing. Objects within the imagesuch as small vessels whose inner aspects werenot included in the calculation of airspaceperimeter were filled interactively. Total air-space wall perimeter per unit area was thenmeasured as mm per mm2 and airspace wallsurface area per unit volume (AWUV) cal-culated by using the formula:AWUV = Airspace wall perimeter per unit

area x 4/X27A minimum of 20 fields in each case were

analysed to obtain a stable running mean. Themean was deemed to be stable if it did not varyby more than 3% for five consecutive fieldswhile showing no consistent upwards or down-wards trend. To obtain a figure representing aportion of the lung with the least number ofalveoli the mean for the lowest five airspace wallsurface area per unit volume fields (LF5AWUV) was calculated for each case.

Reproducibility was assessed by selecting sixsections from the sample pool and measuringairspace wall surface area per unit volume in asingle random field from each section. TheEngland Finder coordinates ofthese fields werestored, and each field was relocated andmeasured repeatedly over 10 days withoutreference to previous results. The coefficient ofvariation (CV) was calculated for the resultsfrom each section by using the formula:

CV = standard deviation/meanThe coefficients of variation for all six sectionswere less than 1 %.

CARBON MONOXIDE GAS TRANSFERSingle breath carbon monoxide transfer factorwas measured preoperatively in 36 of the 44patients by Ogilvie's technique28 to obtain thetransfer factor (TLco) and transfer factor perunit lung volume (Kco).29

145

McLean, Warren, Gillooly, MacNee, Lamb

ANALYSISAs the extent of macroscopic emphysema wasnot normally distributed within the sample thenon-parametric Spearman correlation co-efficient was used to relate macroscopic tomicroscopic measures of emphysema and torelate both to TLco and Kco measurementswith the statistical package for the socialsciences.

ResultsSummary statistics for all variables are given intable 1. Airspace surface area per unit volume(AWUV), expressed as both the mean valueand the mean of the five lowest fields (LF5AWUV), approximated to a normal distribu-tion whereas macroscopic emphysema whenexpressed as a percentage of total lung areawas not normally distributed (figure 1). Inapproximately half the lobes the area ofmacroscopic emphysema was 1% or less.Fifteen of the 44 lobes showed no macroscopicemphysema and 17 had pure centriacinaremphysema, six pure panacinar emphysema,five a mixture of centriacinar and panacinaremphysema, and one paraseptal emphysemaalone.

RELATION OF AWUV TO MACROSCOPICMEASUREMENT OF EMPHYSEMA AND TO TLCO ANDKco

AWUV did not correlate with body height andboth mean and LF5 AWUV correlated poorlywith the severity of macroscopic emphysema(figure 2). The overlap in AWUV values be-tween cases with up to 1% and more than 1%macroscopic emphysema was extensive. Caseswith up to 1% macroscopic emphysemahad mean AWUV values ranging from 15-5 to25-4 m2/mm3, while in those with moresevere emphysema the values ranged from 8-8to 22-6 mm2/mm3. For LF5 AWUV theequivalent values were 9-9 to 21-3 mm2/mm3and 1-9 to 18-7 mm2/mm3 respectively. Onlytwo cases (figure 2)-both with extensivemacroscopic emphysema-lay outside therange ofmeanAWUV values seen in lobes withup to 3% macroscopic emphysema. Mean andLF5AWUV values showed a highly significantlinear relation with both TLco and Kco (figure3) irrespective of the type of macroscopicemphysema present (table 2). The strongestcorrelations were between Kco and AWUVsince both are expressed per unit lung volume

Figure I Frequencyhistogram of area ofmacroscopic emphysemaexpressed as percentage oftotal lung area in mid-sagittal slice.

30 -

>. 20-0

cLD

0)LL 10

(table 2). The severity of macroscopicemphysema correlated poorly with Kco (figure4) and there was considerable overlap in TLCOand Kco values between cases with up to 1%and more than 1% macroscopic emphysema(4-56 to 10-4 and 2-72 to 9-12 mmol/min/kParespectively for TLco and 1.05 to 1 88 and 041to 1-62 mmol/min/kPa/l respectively for Kco).

CENTRIACINAR EMPHYSEMA ONLYIn all ofthe 17 lobes showing pure centriacinaremphysema less than 5% of the mid-sagittalslice was affected. Mean andLF5AWUV in thisgroup correlated significantly with both TLCOand Kco (table 2), the strongest correlationbeing between LF5 AWUV and Kco(r = 0-91, p < 0.001). The area of macro-scopic centriacinar emphysema (as a percen-tage oflung area) showed little correlation withAWUV, expressed as either mean or LF5values, or with.TLco or Kco. However, thenumber of discrete centriacinar lesions (mean16-35 (SE 3 6), range 2-58) correlated withboth mean (r = 0-60, p < 0-005) and LF5AWUV (r = 0 73, p < 0-001) (figure 5). Thenumber of centriacinar lesions also correlatedwith Kco (figure 6) but not TLCO.

PANACINAR ONLY AND MIXED EMPHYSEMAIn the six lobes with panacinar emphysemaonly the distribution of AWUV was notuniform. In some cases it approximated to anormal distribution while in others it was

highly skewed. Five lobes showed a mixtureof macroscopic panacinar and centriacinaremphysema. As panacinar emphysema was thepredominant form of macroscopic emphysemaby far, these five cases were analysed with thesix cases of panacinar emphysema above. Thepercentage area of macroscopic emphysemabore little relation to either mean or LF5AWUV. For example, when AWUV rangedfrom 14 to 16 mm2/mm3 the severity of macro-scopic emphysema ranged from 3% to 51% byarea (figure 2).

30 -

'E 25-E

E 20-E

¢ 15-

E 10-

o No macroscopico * CAE only

8 o PAE only00 * CAEandPAE0

.

.

oE

a0

_ . I I I. I . I . I . I . I . I . * . .-

-10 0 10 20 30 40 50 60 70 80 90 100Area of macroscopic emphysema

11.1|0 1 2 3 4 5 6 7 8 9 10 30 50 70 90

20 40 60 80 100

Area of macroscopic emphysema(% total lung area)

(% total lung area)Figure 2 Mean airspace wall surface area per unitvolume plotted against the area of macroscopicemphysema expressed as percentage of total lung area.The scatter ofpoints shows that the relation between thesevariables is poor. CAE = centriacinar emphysema;PAE = panacinaremphysema; AWUV = airspacewall surface area per unit volume.

n-

b I. . . . . . .

L| . II N I

Li

146

Microscopic and macroscopic measurements of emphysema: relationship to carbon monoxide gas transfer

Table 2 Correlation coefficients (r) for relation between AWUV and TLCO and Kco

All cases Centriacinar emphysema only Any panacinar emphysema(n = 36) (n = 17) (n = 11)

AWUV TLCO Kco TLCO Kco TLCO Kco

Mean fr 0-61 0-66 0-81 077 054 073\ p value <0 001 <0 001 0 001 0-003 0-082 0-02

LF5 f r 073 0-84 0-84 091 076 091l p value <0 001 <0 001 0 001 <0 001 0-015 0 001

AWUV = airspace wall surface area per unit volume.LF5 = mean of lowest five AWUV fields.

Figure 3 Relationbetween single breathtransfer coefficient (Kco)and LF5 AWUV. Thesetwo variables are expressedin comparable units(r = 0-84, p < 0 001).LF5 = mean of the fivelowestfields. Otherabbreviations as infigure 2.

2 1 -

19 0

0 00 0 *0

00

0

80.

0

o No macroscopic* CAE onlyo PAE only* CAE and PAE

0 5 10 15 20 25

LF5 AWUV (mm2/mm3)30

- 1-7 -

a-

C: 1 3-E

Eo~0

UE0-9 -

0-1

00

o 000

00

0

0 10 20 30 40Number of centriacinar lesions

Figure 6 Relation between the number of centriacinarlesions and single breath transfer coefficient (Kco)(r =-089,p < 0001).

Figure 4 Relationbetween single breath Kcoand area ofmacroscopicemphysema expressed aspercentage of total lungarea. Abbreviations as infigure 2.

2 1 -

1 7-

-~ 1 3-7L

.' 1-3 -

E 09-

0C-05-

0

io

so3*o

0

0

-

o No macroscopic* CAE onlyo PAE only* PAE and CAE

..

.

.0

0-1 1-10 0 10 20 30 40 50 60 70 80

Area of macroscopic emphysema(% total lung area)

Figure 5 Relationbetween number ofcentriacinar lesions andLF5AWUV (r =-0 73,p < 0001).Abbreviations as in figures2 and 3.

20

E

EE>

U-

15'

10'

5.-

0

o00 0

0

0 0

0 0 CP

0

0

0

0 0

10 20 30 40 50 6'Number of centriacinar lesions

Mean AWUV was correlated significantlywith Kco and LF5AWUV with both TLCO andKco (table 2). The severity of macroscopicemphysema was poorly associated with bothTLCO and Kco.

DiscussionIn this study airspace wall surface area per unitvolume was measured by an image analysistechnique. The linear intercept technique23permits a more rapid assessment of airspacesurface area, but image analysis has theadvantage of giving highly reproducibleindividual field values, permitting the con-

struction of histograms of airspace surface area

for individual cases and comparison of thedistribution of the airspace surface area withinlobes.Both the linear intercept and airspace wall

surface area per unit volume are based on

absolute measurement of airspace size and are

therefore directly related and to a certain extentinterchangeable.0 In this study airspace wallsurface area per unit volume was independentof patient height, thus substantiating previousfindings that the linear intercept3" and alveolardensity32 do not vary with height. Althoughtotal alveolar number and surface area are

related to height,32 the almost threefold range ofairspace wall surface area per unit volumevalues in this study cannot be explained on thisbasis and must be due, at least in part, to loss ofairspace wall.The measurement of airspace wall surface

area per unit volume provides a continuous

2 1

17~a-

-: 1 3-

0

E 0.90

g05

.*

U

0

0-150

i,i

147

-

McLean, Warren, Gillooly, MacNee, Lamb

assessment of airspace size from normal togross macroscopic abnormality. Macroscopicassessment, on the other hand, is discontinuousas it is based on an arbitrary threshold airspacesize of 1 mm in diameter. The airspace wallsurface area per unit volume can be remarkablylow in lungs with minimal amounts of emphy-sema macroscopically, and there is consider-able overlap between such cases and those withmuch more extensive disease. Thurlbeck alsofound an overlap in linear intercept valuesbetween macroscopically emphysematous andnon-emphysematous lung.33 Duguid et alfound that macroscopic lesions were associatedwith an almost 50% reduction in airspacesurface area per unit volume.0 However, therewere only four normal patients in their study,patients were not matched for age, and manyof the abnormal cases were of patients withcoal workers' pneumoconiosis as well asemphysema. Thus assessment of macroscopicemphysema does not necessarily parallelunderlying airspace destruction. In our studythis was true for the sample as a whole and alsofor the subgroups with mixed, panacinar, orpure centriacinar disease. In particular,although ranging as high as 79% by area, theseverity of panacinar disease correlated poorlywith the airspace wall surface area per unitvolume.There were some exceptions to the generally

poor relation between macroscopic emphy-sema and airspace destruction. In patientswith macroscopic centriacinar emphysema thenumber of discrete lesions counted macro-scopically correlated with both mean airspacewall surface area per unit volume and the meanof five fields with the lowest values of surfacearea (although the area of macroscopicemphysema did not). These relations wereimpressive as macroscopic centriacinaremphysema never affected more than 5% ofthemid-sagittal slice. This relation between thenumber of centriacinar lesions and airspacewall surface area per unit volume suggests thatcentriacinar lesions develop on a background ofgeneral parenchymal loss. In support of thisthere was a strong correlation between thenumber of centriacinar lesions and Kco inpatients with centriacinar emphysema. Theseobservations may explain why Hayhurst et alfound that centriacinar lesions were associatedwith a reduction in lung density when com-pared with lungs showing no macroscopicemphysema.34 It must be remembered,however, that centriacinar emphysema has todate always been measured macroscopically asit is not yet possible to quantify centriacinaremphysema microscopically. Relations bet-ween microscopic and macroscopic cen-triacinar emphysema remain to be established.The results of this study suggest that

emphysema can be measured accurately onlyby microscopic measurements. Decrease inlung density and carbon monoxide gas transferdo not relate to the severity of macroscopicemphysema but correlate strongly with air-space wall surface area per unit volume.' Moreimportantly the relations between airspace wallsurface area per unit volume, lung density, and

carbon monoxide gas transfer are constantirrespective of the presence, type, or severity ofmacroscopic emphysema. Some authors havesuggested that the relations between carbonmonoxide gas transfer and macroscopicemphysema assessed by panel grading andpoint counting is linear."1802935 However, whenthe data are available it is obvious that theserelations are not truly linear.2935 In particular,subjects with no macroscopic emphysema canhave gas transfer function which does not differfrom that of subjects with severe macroscopicemphysema.The fields with the largest airspace size of

those measured are represented by the mean offive fields with the lowest airspace wall surfacearea per unit volume. This may represent areasof acquired emphysema but in many cases thelarger airspaces measured are the alveolarducts. The stronger correlation between themean value of the lowest five fields and Kcocompared with mean airspace wall surface areaper unit volume and Kco may reflect theimportance of the proximal acinar air:'aces inthis single breath technique for measunag gastransfer.Our results indicate that macroscopic

measurements ofemphysema do not reflect theloss of airspace wall surface area per unit lungvolume accurately. Considerable microscopicloss of surface area may precede the presence ofmacroscopic emphysema, so the incidence andseverity of smoking related parenchymaldamage must be higher than that reported byauthors using macroscopic criteria alone.Moreover, since centriacinar emphysemaseems to develop on a background of generalloss of airspace wall this may explain theapparent relation between centriacinar lesionsand Kco.

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