a radiographic technique for the study lung …thorax.bmj.com/content/thoraxjnl/7/2/170.full.pdf ·...

Thorax (1952), 7, 170.

A RADIOGRAPHIC TECHNIQUE FOR THE STUDY OFLUNG PATHOLOGY

BY

GEORGE J. CUNNINGHAM AND JOHN W. MILLERFrom the Department of Pathology, St. Bartholomew's Hospital, London

(RECEIVED FOR PUBLICATION SEPTEMBER 19, 1951)

During a radiographic study of lung specimensinjected with various radio-opaque materials wewere impressed by the amount of detailed structurewhich could be identified in uninjected areas. Itwas therefore considered worth while attempting todevelop a method for the examination of uninjectedlungs by radiography, which, when used in con-junction with recognized histological methods,might provide further information on the structureof lung tissue in health and in disease.

MATERIALSTo test the method, many lung specimens obtained

surgically and post mortem and containing a widevariety of pathological lesions were radiographed. Thesespecimens had been fixed in 10% formol-saline; in themore recent ones this had been injected into the tracheaor main bronchus, but in some older specimens the lunghad been simply immersed in the fixative. The pre-paration of the lung for radiography consisted in slicingit with a sharp knife, great care being taken to ensurethat the cut surfaces were as nearly flat as possible. Ifthis was not achieved, it was difficultto fix the specimen securely in theholder. Although these slices couldoften be cut satisfactorily freehand,we found it desirable to use asimple apparatus (Fig. 1) composedof a wooden trough, the base ofwhich contained a few superimposedplates of glass. The specimen isplaced on the glass surface and heldthere firmly by hand while a sectionis cut, keeping the knife absolutelyflat. As during the sectioningprocess the knife is resting on thesides of the trough, the thickness ofthe section will depend on the depthof the trough. The latter can bevaried by changing the number ofplates of glass placed in its base.The best thickness, though varyingaccording to the pathological lesionin the lung, was usually about 0.5cm. A few hours before radiographythe slices were immersed in

methylated spirit, as we preferred to handle thespecimens in this rather than in formalin.

RADIOGRAPHIC TECHNIQUEThe tube used for the production of x rays was one

employed at the Royal College of Surgeons for the studyof bone sections (Sissons, 1950). It is similar to tubesused in crystallographic studies and emits rays at avoltage of 5 to 35 kilovolts. These rays consist of anarrow beam produced by the linear focus technique(Eastman Kodak publication, 1943), the size of the focalspot being 0.3 mm. In our initial trials a large numberof radiographs was taken at varying voltage andamperage. For reasons which will be discussed laterwhen considering the distance between film and x-raysource, most of our recent and better results wereobtained with voltages from 20 to 35 kilovolts and withcurrents of from 10 to 25 milliamperes. As the x-raybeam emitted by the apparatus was horizontal, someform of holder for the specimen was necessary. In theearlier trials small pieces of lung were used and thesecould be made to adhere to the film container by meansof their moisture, this method proving satisfactory,

FIG. 1.-Method of slicing the lung specimen.

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RADIOGRAPHIC TECHNIQUE FOR LUNG PATHOLOGY

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FIG. 2.-The holder partly'assembled with specimen in position.

especially if the exposure time was short. When largerportions of tissue were used and the exposure times wereincreased, any slight movement of the specimen-spoiledour results and forced us to employ a holder. Wewere fortunate in having the assistance of Mr. S. P.Steward, of the Physiological Department, RoyalCollege of Surgeons, and he was responsible for theholder illustrated here (Figs. 2 and 3). It has provedto be very satisfactory and our results greatly improvedwith its use. It is composed of two frames, one made ofmetal and the other of perspex. These two frames canbe approximated one to the other once the specimenhas been placed in position, and maintained there bymeans of a screw-clamp. It was thought that the perspexframe might be too fragile for the work, but it was care-fully handled and was in good condition after twomonths' continuous use. In fact its elasticity seemed tobe an advantage, as it held the specimen firmlyin position without compressing it too strongly. Eachof these frames was covered by a single layer of celluloseacetate sheeting* 3/1000 in. thick. The sheeting wasstretched tightly over the frames and fixed in positionby means of cellophane tape. The specimen was thusheld between these two layers of sheeting. Beforeplacing the slice of lung between the frames it was lightlyblotted to absorb any excess of methylated spirit.Although the cellulose acetate sheeting when moistenedby the spirit buckled to some extent, there was littledistortion left on drying and we did not have to change

Obtainable from Messrs. F. G. Kettle, 23, New Oxford Street,London, W.C. I.

FIG. 3.-The holder fully assembled and ready for radiography.

the original sheets during a trial period of two months.If such a change should become necessary it can beperformed quite easily.As this kind of radiography is usually performed in

the light, many workers have employed a light-tightcontainer in which both specimen and film are placed,the unit being assembled in a dark room. We consideredthat it would be simpler to make a light-tight holder forthe film only, as it would then be possible to arrange thespecimen in the holder in daylight. For this purpose,after trying various materials, envelopes were madefrom the type of black paper that is ordinarily used forpacking x-ray films, but after having used it for sometime we were surprised to discover that other workershad condemned its use. They found that the texture ofthe paper was visible on the radiograph and thus inter-fered with the image. These workers were using voltagesof 2 to 8 kilovolts, and this almost certainly accounts fortheir findings, as we never encountered any such diffi-culties. The film container was strengthened by a pieceof cardboard identical in size with the film and placedinside the envelope.When loading the container care had to be taken to

place the film in front of the sheet of cardboard and toavoid tearing the black paper at the top corners of theenvelope when inserting the film. The loaded filmcontainer is placed in the metal frame, thus bringing itinto direct contact with the layer of sheeting behind thespecimen. The latter is, therefore, only separated fromthe film emulsion by two thin layers of material, namelythe black paper and one layer of the cellulose acetate

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GEORGE J. CUNNINGHAM and JOHN W. MILLER

sheeting. Any space behind the film container in themetal frame is filled in with a suitable number of sheetsof cardboard (in our case three), and the back of theframe covered with a sheet of perspex. All the con-stituents of the holder were then held firmly togetherby means of three large metal clips placed as indicatedin Fig. 3.The distance of the film from the focal spot has

usually been about 12 in. (30.5 cm.) according to earlierworkers, though Barclay (1947) used 8 in. (20.3 cm.).We began by employing a distance of 20 to 24 in. (50to 60 cm.), but soon found that the cone of x rays atthat distance would not be sufficiently large to enablewhole lobes of lung to be radiographed. We aimed atobtaining a cone which would easily cover an area of41 X 61 in. (10.8 X 17.1 cm.), and this could be achievedat a distance of not less than 33 in. (83.8 cm.), though thedistance we actually used was 36 in. (91.4 cm.). Thisincrease necessitated a considerably longer exposureand required a rise in the kilovoltage from between15 and 20 to between 30 and 35. These radiographsappeared to us better defined than our previous ones.Franke's formula (Bohatyrtschuk, 1944) indicates thefactors which cause blurring of the image:

bdwhereW = the blurring, b = the distance of objectfrom film, d = the size of the focal spot, andF = the target-film distance

Thus, with an object almost in contact with the film,an increased distance from the tube is an advantageprovided that the resulting increase in exposure is nottoo great. Many of our later and better radiographs,including those illustrated in this article, were taken at35 kilovolts and 25 milliamperes. We found that underthese conditions a slice of lung 0.5 cm. thick requiredan exposure of three to four minutes.

After some initial failures with ordinary types oforthochromatic photographic emulsions we eventuallycame to use either Ilford process or line films. At thetime of writing we favour the latter, though they are atrifle slower and thus require a slightly longer exposure.We used Johnson's fine-grain contrast developer, andwere able to obtain magnifications of 25 diameterswithout loss of detail. We have, unfortunately, not beenable so far to try the special fine-grain emulsions referredto by Barclay and his colleagues in their renal studies(Trueta, Barclay, Daniel, Franklin, and Prichard, 1947).

HISTORICALAlthough hard x rays are used so much in

medicine, dentistry, and metallurgy, the uses ofsofter or Grenz rays are not so well recognized. Ofrecent years medical workers have had their atten-tion drawn to them by the researches of Barclayand his colleagues (1947, 1951) on microarterio-graphy of kidneys injected with radio-opaquematerials.

The earliest application of radiography to thestudy of the structure of soft tissues was made byGoby (1913), who studied diatoms and foraminifera.The results he obtained were remarkably good whenone considers the limited facilities at his disposal.They were not entirely satisfactory, as he employedx rays that were too hard, and the coarseness of hisphotographic emulsions prevented him from obtain-ing enlargements of more than 17 diameters. Sincethen similar studies have been made with consider-able success by other workers (Fricke, 1932; Sher-wood, 1934, 1936, 1937). Seeds as well as insectshave been studied and the distribution of radio-opaque organic spray materials on foliage deter-mined (Eastman Kodak, 1943). In 1930 Dauvilliercarried out the first successful radiograph on ahistological section of plant tissue. He employed avery thin section, used a special fine-grain emulsionphotographic plate, and claimed to have been ableto magnify his negative up to 600 diameters. Thiswork was followed by that of Lamarque (1938),who obtained beautiful radiographs of skin sectionsand called the technique histo-radiography. Hispositive prints, suitably magnified, compared veryfavourably with photomicrographs taken from thesame tissue. Since that time similar methods havebeen used for a remarkable number of differentpurposes. Forgeries in postage stamps can frequentlybe detected (Cheavin, 1948). Seemann (1943) in anexcellent article cites such uses as the detection ofgrubs and death-watch beetles in wood, the dis-tinction between natural and cultured pearls, andthe extreme ease with which natural and artificialleather can be distinguished radiographically. Healso mentions its use in the textile industry, aboutwhich Sherwood (1936) has also written. Grenzradiography has been in use in this industry for atleast 15 years, as excellent pictures showing theweave of the cloth can be obtained.

DIscUSSIONOf recent years, micro-radiography has become

increasingly employed, chiefly as a result of theresearches of Bohatyrtschuk (1944), Barclay (1947,1949, 1951), and Trueta et al. (1947). With theexception of Sissons (1950), who was studying bone,all the other workers injected the tissues with radio-opaque materials. Several organs have beenstudied, including the kidney, stomach, spleen,liver, and diaphragm (Bohatyrtschuk, 1944; Barclay,1947, 1949, 1951; Woolf, 1950; Tirman and others,1951). Barclay (1951) attempted micro-arterio-graphy of the lung, but was not satisfied with thedefinition he obtained; Tirman and others (1951)in their paper published magnified radiographs of

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injected lung specimens, but the details are noteasily recognizable. While our method closelyresembled that of Barclay (1947) and of Sissons(1950), a voltage slightly higher than that of Grenzrays was employed. Most of the workers quotedtook radiographs at a voltage below 20 kilovolts.We took our radiographs at a distance of 36 in.(91.4 cm.) to fill a half-plate field, and used a highervoltage to keep the exposure time within reasonablelimits. Had we found it necessary to use voltages of5 to 10 kilovolts, an exposure of one to two hourswould have been necessary at that distance. Thiswe felt undesirable, as it would greatly limit ournumber of exposures. A more serious objectionwas the fact that the specimen might shrink bydrying and therefore the lung slice in the holderwould be more likely to move.

In examining a number of specimens we wereimpressed by the information obtained of thegeneral distribution of the disease process. It wasobvious that a precise idea of the extent of certainlung lesions could be obtained by serially slicing thelung and making radiographs of each slice. Bythis procedure the task of the histologist in selectingsuitable areas for section would be greatly simplified.

FIG. 4-A photo-graph. a radio-graph, and ahistological sec-tion of the sameslice of tissuefrom a case ofcarcinoma ofthe lung. Thepneumonicareas can beseen extendingoutwards to thepleural surface.(About two-thirds of actualsize.)

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FIG. 5.-A simila]bronchiectatic:part of the lob

A great additional advantage was that the materialcould be sectioned histologically and yet a per-manent record kept of its macroscopic appearancesfor comparison.

In Fig. 4 a specimen from a case of bronchialcarcinoma is illustrated by a photograph and aradiograph of the specimen, together with a photo-graph of the histological section. The extent of thelesion can be well seen in the radiograph, andhistological examination proved that the shadowsextending out to the pleural surface were pneumonic,following bronchial obstruction by the neoplasm.In Fig. 5 a specimen of bronchiectasis showing basaldistribution is similarly illustrated. The smalltuberculous focus, unsuspected before operation,can be seen in all three pictures.Lamarque (1938) laid considerable emphasis on

the varying radio-densities of tissue and hoped thathisto-radiography with Grenz rays might prove a

r illustration to Fig. 4 of a portion oflung. Note the tuberculous focus in the upper)e. Actual size.

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FIG. 6.-Illustration of a portion of silicotic lung, showing nodules. Actual size.

method of analysis for different kinds of tissue.In studying the relative opacities to x rays of anumber of tissues he found that connective tissuefibrils were relatively opaque compared withordinary cellular elements. This point is wellillustrated by the portion of silicotic lung (Fig. 6).Here the detail of the silicotic nodule is so clear thatthe whorled arrangement so characteristic of colla-genous tissue can be seen if a small hand-lens isused in examining the radiograph. Again, in Fig. 7the radiograph of the portion of emphysematouslung shows the abnormal connective tissue frame-work very clearly, and the advantages comparedwith the accompanying photographs of the specimenand histological section are easily apparent. Thelung tissue would thus appear ideal for this methodof study, as its basic structure of radio-opaqueconnective tissue contrasts strongly with the alveolarspaces. Examination of the radiographs shown in thispaper emphasizes how advisable it is to inflate the

lung at the time of fixation. In Fig. 5 the lung tissuehas not been satisfactorily inflated, and this defectis most obvious in the apex of the bronchiectaticlobe. While it is likely that Lamarque's hope ofa radiographic method for analysing tissue com-position is demanding too much of the method, wehave obtained some interesting results in the radio-opacity ofa few pathological conditions. A specimenof lung from a coal-miner, kindly given to us byProfessor Gough, of Cardiff, showed the presenceof a large amount of carbon. We radiographed thisspecimen in an attempt to illustrate the precisedistribution of the pigment and found that theradio-opacity was much less than we had expected.Hence radiography in this type of lung might pro-vide information of any modification in structureof the connective tissue framework of the lungin coal-miners' pneumoconiosis. This might bevaluable, since the histological structure may becompletely obscured by dense areas of carbonpigmentation. We have been informed that such amethod has been used for revealing the pattern of apostage stamp which had been obscured by heavypostmarks of carbon-containing ink. Lamarquecut his sections very thin (4-10 p), as he was anxiousto avoid a large number of superimposed shadowswhich he would be unable to analyse. We consideredthat thick slices were essential for our purpose,and our aim was therefore in direct contrast to that



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FIG. 7.-Portion of an emphysematous ung, showing a number of bul I

of Lamarque. Like many histologists, we havealways considered that thin sections are mostunsatisfactory for giving any real idea of such anessentially three-dimensional structure as the lunglobule.The combined technique which we have illustrated

has been invaluable to use in providing a morecomplete idea of certain lung lesions. Apart fromthis the radiographic definition is sufficiently goodto enable the negatives to be easily examined bymagnification on a dissecting microscope. This factseemed to us to extend the possibilities of themethod to the examination of the more intimatepulmonary histology. It was realized, however,that radiographs of thick slices should be inter-preted with caution owing to the complexity of thepicture and the possibility of artefacts produced bythe superimposition of shadows. We consideredthat the best way to avoid these difficulties was toobtain stereoscopic radiographs, rotating the speci-men and taking two pictures, each at an angle of50 from the mid-line position. We have nowmodified a Wheatstone stereoscope to our require-ments and have obtained results which appear tobe most promising. Stereoscopic radiographs canbe considerably enlarged, and when viewed in thestereoscope they show the intimate lung structurein a most striking way. These stereoscopic viewsare similar to those obtained by examining a sliceof lung under the dissecting microscope. Theyshow, however, a greater depth of tissue, and havethe great advantage of providing a permanent

record of the macroscopic structure of the portionsof tissue to be sectioned. An added advantage isthat, if during histological examination any interest-ing feature be found, reference to the deeper struc-tures in the stereoscopic radiographs may showwhether any useful purpose would be served bycutting further sections from the block. Comparisonbetween radiograph and histological section isgreatly facilitated by their similarity in size (thesection being only slightly smaller owing to shrinkageas the radiograph is really a contact negative.This enables individual lung structures to be



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RADIOGRAPHIC TECHNIQUE FOR LUNG PATHOLOGY

measured by a simple scale during stereoscopicexamination.Our results will give some idea of the scope and

value of this method, which we believe is ideal forrecord purposes and for providing information of a

more complete character in lung specimens. Weshould at the same time like to emphasize that we

regard the detailed interpretation of these radio-graphs as an exceedingly complex matter, and one

in which much practice and further research willbe required before conclusions are too readilydrawn. At the moment we consider that its placeis to assist the pathologist in obtaining a three-dimensional view of the lesion, and that its functionis therefore ancillary to the histological method.

SUMMARY

A technique for examining slices of lung by softx rays is described. By radiography of serial slicesand subsequent histological examination an accuratepicture of the whole lesion may be obtained.The radiographs can be greatly magnified and

thus the finer details of the lung examined.Stereoscopic radiography in conjunction with

orthodox histological methods is likely to proveof great value in obtaining a better conception ofthe lung lobule in health and disease.

The radiographs provide a permanent record ofthe macroscopic appearances and are more infor-mative than ordinary photographs.

We should like to express our gratitude to ProfessorJ. W. S. Blacklock for much encouragement; toProfessor G. Hadfield for allowing us the use of thex-ray apparatus and for helpful advice; to ProfessorD. Slome for encouragement and many valuablesuggestions; to Mr. Norman K. Harrison for thephotography; to Dr. G. S. Sansom for the photo-micrography.

REFERENCES

Barclay, A. E. (1947). Brit. J. Radiol., 20, 394.- (1949). Amer. J. Roentgenol., 62, 119.

(1951). Micro-arteriography and Other Radiological TechniquesEmployed in Biological Research. Oxford.

Bohatyrtschuk, F. (1944). Acta radiol., Stockh., 25, 351.Cheavin, W. H. S. (1948). Research, 1, 208.Dauvillier, A. (1930). C.R. Acad. Sci., Paris, 190, 1287.Eastman Kodak Company X-Ray Division (1943). Radiography

of Materials. Rochester, N.Y.Fricke, H. (1932). Radiogr. and clin. Photogr., 8, No. 5, p. 12.Goby, P. (1913). C.R. Acad. Sci., Paris, 156, 686.Lamarque, P. (1938). Brit. J. Radiol., 11, 425.Seemann, H. E. (1943). In Symposium on Radiography, 1936 and

1942, p. 29. American Society for Testing Materials, Philadel-phia, Pennsylvania.

Sherwood, H. F. (1934). Radiogr. and dlin. Photogr., 10, No. 4, p. 10.(1936). J. Text. Inst., Manchr. (Trans.), 27, T162.(1937). J. biol. photgr. Ass., 5, 138.

Sissons, H. A. (1950). Brit. J. Radiol., 23, 2.Tirman, W. S., Caylor, C. E., Banker, H. W., and Caylor, T. E.

(1951). Radiology, 57, 70.Trueta, J., Barclay, A. E., Daniel, P. M., Franklin, K. J., and

Prichard, M. M. L. (1947). Studies of the Renal Circulation.Oxford.

Woolf, A. L (1950). Brit. J. Radiol., 23, 8.

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