Note ___________________________________ DIAZO REPLICATION OF THE LUNG: AN IMPROVED METHODOLOGY
Summary - - - - - - - - - - - - - - -
The methodology of diazo replication has been improved to provide replicas of 60-,um·thick lung sec· tions with high contrast and sharp delineation of airways and air spaces. The newly introduced techniques arc critical temperature control, embedment in isopentane, Bismarck brown staining, and parallel light, halogen lamp illumination. The earlier methodology for diazo replication ( 1) was presented as an adjunct procedure for the 500,utn·thick Gough-Wentworth sections (2). The main purpose was to provide replicas of emphysematous lungs that would permit reproducible, objective quantification by a variety of morphometric methods (3). The 500-,um-thick sections were adequate for emphysematous lesions == 1 mm in diameter but not suitable for delineating the fine alveolar structure of the normal lung or dilations < I mm that could nevertheless represent alveolar volume changes several hundred times normal values. In attempting to cut sections thinner than 500 ,urn. 3 major problems were encountered : (1) whole lung sectioning was difficult at the desired thickness, i.e., 60 ,um, the radius of the smallest alveolus (4); (2) thin sections that could be obtained provided insufficient contrast for reproducible diazo imaging; (3) the light source used for diazo imaging, e.g., a printing box, did not provide appropriately parallel light. This report presents an improved methodology that bypasses the obstacles mentioned and yields diazo replicas with much finer discrimination of th~ terminal airways and air spaces. The lung is fixed by continuous bronchial perfusion of 10 per cent formalin (3 per cent sodium acetate buffer) under a constant pressure of 25 em H 20, using a recirculating immersion pump. After a minimum of 48 hours of fixation, the lungs are sliced on an electric rotary slicer to provide one-inch-thick sections for alternate diazo replication and paraffin sections. The slices are washed in running water for at least 12 hours, blotted dry on paper towels, and transferred into a container filled with a gelatin mixture (I). A double layer of meshed gauze is used to cover the sections. After infiltration in a vacuum oven at 37o C for== 24 hours, the tissue in its container is placed on a block of dry ice. The tissue slice in the liquid gelatin mixture is immediately pressed to the bottom of the container until it is evenly and firmly adherent. It is preferable to use a glass container in order to visualize attachment. After
gelling at 4o C, the tissue block is freed with run· ning hot water. The block is trimmed to a rectangu· Jar shape, with a minimum of a one-half inch margin of gelatin around the tissue surfaces. A broad spatula, warmed in hot water, is used to smooth the block surfaces. A metal chuck, heated in a water bath (50° C) for several minutes and then dried, is pressed into the tissue block until the softened gelatin envelops the edges of the chuck. The preparation is immediately immersed in an ice water bath and then transferred to an isopentane-dry icc bath at a temperature of -40° C to -50° C for at least 15 min. It is important not to over-cool, because this cracks the block, or to over-warm, because multiple pockets of ice crystals will form. The temperature range can be maintained by adding small pieces of dry ice to the bath as required. A sledge microtome with increments of 2 ,urn is r·equired for sectioning at 60 ,urn, e.g., the Jung Tet· rander II. The face of the block is trimmed on a microtome, with subsequent recooling to -40° C if undue warming occurs. The sections are cut with a prechilled microtome knife, and the sections floated in a 10° C water bath, using a brush to facilitate the transfer. For staining, the gelatin is removed from the section by placing the section in a warm water bath (40° C), transferred to a 3 per cent Bismarck brown solution for 15 min, rinsed in water, and picked up on a glass slide or a 500-ml sheet of Mylar. The addition of gelatin to the rinse water (0.5 ml of 25 per cent gelatin per liter of water) ensures complete wetting of the glass or plastic slide and thus assists in picking up the section; too much gelatin, however, may result in poor adhesion of the section. A thin plastic sheet, preferably 1-ml Aclar (Allied Chemical Co., 1\'ew York, N.Y.), is used as a coverslip. A photographic contact print is obtained by placing the preparation on a sheet of K-MAX (Tcchnifax, Diazo Master Transparency; Plastic Coating Corp., Holyoke, Mass.) film, having blotted dry the apposing surface of the Aclar coverslip. The emulsion of the film faces the Aclar coverslip, and the preparation is placed in a nearly vertical position. A glass plate (one-fourth inch) provides the proper pressure for a flat photographic image. A quartz iodide spot lamp is focused perpendicularly on the preparation at a distance of 36 inches; resolution is suboptimal at shorter distances, and longer distances require excessive exposure time. The endpoint of exposure is the diappearance of the yellow color from the K-MAX master, generally 8 min. Bracketing of exposure time is recommended for each specimen, e.g., 8 min, 8 min less 15 sec, and 8 min plus 15 sec. With underexposure, the K-MAX master will not
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 114, 1976
647
648
NOTE
have a clear background; with overexposure, contrast is poor and the washing out of fine details is usually fairl y obvious. The K-MAX film is developed in a large jar containing ammonium fumes. Liquid ammonium hydroxide should not come in contact with the film. Development time is not critical; generally, a 10-min period is adequate. A number of contact prints may be made from the K-MAX master, using diazo paper of various types, or plastic sheets for transparencies. Using the K-MAX master, almost any kind of illumination is adequate for prints. A commercial printer is available, and a duplicating machine can be used. For very high volume
and low cost, offset printing can be applied. Storage of the lung tissue section in a vacuum-packed Aclar envelope, using a heat impulse sealer, permits stereomicroscopy for additional studies.
• • •
In summary, the new methodology for diazo replication now permits much finer, objective quantifications than was previously available. The very specific temperature control and the isopentane bath combine to produce an unusual embedment mixture, one that is a homogeneous white and unusually cohesive. The Bismarck brown stain appears to be
Fig. I. Diazo replica of right lung of premature infant. T h e lung was inflated through the bronchus with formalin at a pressun: of 25 em H 20 for 72 hours. This photographic print was made from a diazo replica transparency. The lung was sectioned at 50 JLm. The posterior half of the upper and lower lobes (left side) is very dark owing to poorly expanded or unexpanded lung tissue (atelectasis). Conversely, the anterior half, and the central portion of the middle lobe, appear overinflated. Arrow points to area magnified in figure 2 and asterisks indicate an identical structure. (Original magnification : X 3.5.)
NOTE
649
Fig. 2. Higher magnification of diazo replica shown in figure 1. This is a portion of the apex of the right upper lobe, at the junction of the overinflated and underinflated areas. (See arrow in figure 1.) Note the irregular inflation of terminal airways, alveolar ducts, alveolar sacs, and alveoli. The architecture of a pulmonary acinus is well shown in the center of the photo (arrow). (Original magnification: X 10.)
optimal for the factors relating to illumination and the diazo dyes. A number of other stains proved to be unsatisfactory. The parallel light provided by the halogen lamp permits much finer delineation than has been previously available, and this allows for 3X and grea.ter magnifications (figures I and 2). The diazo replicas can be analyzed by relatively inexpensive morphometric means such as point counting and linear intercepts (3), with certain advantages afforded by the 2-dimensional replica that are not available from examination of the frozen section of lung itself. In the latter respect, the 2-dimensional nature of the replica bypasses the need to make subjective decisions about measurement problems involving 3 dimensions (e.g., alveolar diameter), and the sharp contrast permits more reproducible, precise measurements. These advantages are particularly critical for large volume quantification by image analyzers and are provided by replicas either in the form of pa-
per prints or plastic transparencies. Although image analyzer studies of alveolar walls have been succcssfull y carried out with frozen sections of lung (5) , our experience indicates that replicas would be far more feasible for the a!'l alysis of both a large number of specimens and a large number of sections from each specimen. Replicas can also be used for quantifying selected cells in whole sections of human or animal lungs, e.g., by the lactate dehydrogenase method of staining type 2 pneumocytes for image analyzer studies (6) . The replicas do not, of course, provide cytologic detail, but this is readily available through the simple expedient of preserving the 60-,um section used for the diazo master in a transparent, sealed plastic (1-ml Aclar) enclosure where it can be examined by stereomicroscopy. In addition , we obtain whol e lung paraffin-embedded sectio ns from "sister" slices adjacent to the frozen section for routine histopathologic examination (7). Finally, the methodol-
650
NOTE
ogy has potential application for quantitative measurements of other organs in which appropriate contrast can be obtained, e.g., kidney tubular studies. RAY M. YuTANil RussELL P. SHERWIN Department of Pathology University of Southern California School of Medicine 2025 Zonal Ave. Los Angeles, Calif. 90033
References I. Sherwin, R. P.: Diazo replication for the pathologic study of emphysema, Am J Clin Pathol, 1968,45, l. 1 Supported by a grant from the American Lung Association, California Division.
2. Gough, J., and Wentworth, J. E.: Thin sections of entire organs mounted on page, in Recent Ad''ances in Pathology, Little, Brown and Company, Boston, 1960, p. 1180. 3. Weibel, E. R.: Morphometry of the Human Lung, Academic Press, New York, 1963. 4. Wyatt, J. P., Fischer, V. W., and Sweet, H. C.: The pathomorphology of the emphysema complex: Part l, Am Rev Respir Dis, 1964,89,533. 5. Sherwin, R. P., Margolick, J. B., and Azen, S. P.: An automated determination of ratios of type 2 pneumocytes to alveolar wall area using an image analyzer, Am Rev Respir Dis, 1973,108, 1015. 6. Margolick, J. B., Azen, S. P., and Sherwin, R. P.: An image analyzer quantitation of type 2 pneumocytes, Am Rev Respir Dis, 1973, 108, 704. 7. Gillette, L. V., and Sherwin, R. P.: A Mylar and Aclar plastic method for giant histologic sections of the lung, Am Rev Respir Dis, 1965,92,238.
Summary - - - - - - - - - - - - - - -
The methodology of diazo replication has been improved to provide replicas of 60-,um·thick lung sec· tions with high contrast and sharp delineation of airways and air spaces. The newly introduced techniques arc critical temperature control, embedment in isopentane, Bismarck brown staining, and parallel light, halogen lamp illumination. The earlier methodology for diazo replication ( 1) was presented as an adjunct procedure for the 500,utn·thick Gough-Wentworth sections (2). The main purpose was to provide replicas of emphysematous lungs that would permit reproducible, objective quantification by a variety of morphometric methods (3). The 500-,um-thick sections were adequate for emphysematous lesions == 1 mm in diameter but not suitable for delineating the fine alveolar structure of the normal lung or dilations < I mm that could nevertheless represent alveolar volume changes several hundred times normal values. In attempting to cut sections thinner than 500 ,urn. 3 major problems were encountered : (1) whole lung sectioning was difficult at the desired thickness, i.e., 60 ,um, the radius of the smallest alveolus (4); (2) thin sections that could be obtained provided insufficient contrast for reproducible diazo imaging; (3) the light source used for diazo imaging, e.g., a printing box, did not provide appropriately parallel light. This report presents an improved methodology that bypasses the obstacles mentioned and yields diazo replicas with much finer discrimination of th~ terminal airways and air spaces. The lung is fixed by continuous bronchial perfusion of 10 per cent formalin (3 per cent sodium acetate buffer) under a constant pressure of 25 em H 20, using a recirculating immersion pump. After a minimum of 48 hours of fixation, the lungs are sliced on an electric rotary slicer to provide one-inch-thick sections for alternate diazo replication and paraffin sections. The slices are washed in running water for at least 12 hours, blotted dry on paper towels, and transferred into a container filled with a gelatin mixture (I). A double layer of meshed gauze is used to cover the sections. After infiltration in a vacuum oven at 37o C for== 24 hours, the tissue in its container is placed on a block of dry ice. The tissue slice in the liquid gelatin mixture is immediately pressed to the bottom of the container until it is evenly and firmly adherent. It is preferable to use a glass container in order to visualize attachment. After
gelling at 4o C, the tissue block is freed with run· ning hot water. The block is trimmed to a rectangu· Jar shape, with a minimum of a one-half inch margin of gelatin around the tissue surfaces. A broad spatula, warmed in hot water, is used to smooth the block surfaces. A metal chuck, heated in a water bath (50° C) for several minutes and then dried, is pressed into the tissue block until the softened gelatin envelops the edges of the chuck. The preparation is immediately immersed in an ice water bath and then transferred to an isopentane-dry icc bath at a temperature of -40° C to -50° C for at least 15 min. It is important not to over-cool, because this cracks the block, or to over-warm, because multiple pockets of ice crystals will form. The temperature range can be maintained by adding small pieces of dry ice to the bath as required. A sledge microtome with increments of 2 ,urn is r·equired for sectioning at 60 ,urn, e.g., the Jung Tet· rander II. The face of the block is trimmed on a microtome, with subsequent recooling to -40° C if undue warming occurs. The sections are cut with a prechilled microtome knife, and the sections floated in a 10° C water bath, using a brush to facilitate the transfer. For staining, the gelatin is removed from the section by placing the section in a warm water bath (40° C), transferred to a 3 per cent Bismarck brown solution for 15 min, rinsed in water, and picked up on a glass slide or a 500-ml sheet of Mylar. The addition of gelatin to the rinse water (0.5 ml of 25 per cent gelatin per liter of water) ensures complete wetting of the glass or plastic slide and thus assists in picking up the section; too much gelatin, however, may result in poor adhesion of the section. A thin plastic sheet, preferably 1-ml Aclar (Allied Chemical Co., 1\'ew York, N.Y.), is used as a coverslip. A photographic contact print is obtained by placing the preparation on a sheet of K-MAX (Tcchnifax, Diazo Master Transparency; Plastic Coating Corp., Holyoke, Mass.) film, having blotted dry the apposing surface of the Aclar coverslip. The emulsion of the film faces the Aclar coverslip, and the preparation is placed in a nearly vertical position. A glass plate (one-fourth inch) provides the proper pressure for a flat photographic image. A quartz iodide spot lamp is focused perpendicularly on the preparation at a distance of 36 inches; resolution is suboptimal at shorter distances, and longer distances require excessive exposure time. The endpoint of exposure is the diappearance of the yellow color from the K-MAX master, generally 8 min. Bracketing of exposure time is recommended for each specimen, e.g., 8 min, 8 min less 15 sec, and 8 min plus 15 sec. With underexposure, the K-MAX master will not
AMERICAN REVIEW OF RESPIRATORY DISEASE, VOLUME 114, 1976
647
648
NOTE
have a clear background; with overexposure, contrast is poor and the washing out of fine details is usually fairl y obvious. The K-MAX film is developed in a large jar containing ammonium fumes. Liquid ammonium hydroxide should not come in contact with the film. Development time is not critical; generally, a 10-min period is adequate. A number of contact prints may be made from the K-MAX master, using diazo paper of various types, or plastic sheets for transparencies. Using the K-MAX master, almost any kind of illumination is adequate for prints. A commercial printer is available, and a duplicating machine can be used. For very high volume
and low cost, offset printing can be applied. Storage of the lung tissue section in a vacuum-packed Aclar envelope, using a heat impulse sealer, permits stereomicroscopy for additional studies.
• • •
In summary, the new methodology for diazo replication now permits much finer, objective quantifications than was previously available. The very specific temperature control and the isopentane bath combine to produce an unusual embedment mixture, one that is a homogeneous white and unusually cohesive. The Bismarck brown stain appears to be
Fig. I. Diazo replica of right lung of premature infant. T h e lung was inflated through the bronchus with formalin at a pressun: of 25 em H 20 for 72 hours. This photographic print was made from a diazo replica transparency. The lung was sectioned at 50 JLm. The posterior half of the upper and lower lobes (left side) is very dark owing to poorly expanded or unexpanded lung tissue (atelectasis). Conversely, the anterior half, and the central portion of the middle lobe, appear overinflated. Arrow points to area magnified in figure 2 and asterisks indicate an identical structure. (Original magnification : X 3.5.)
NOTE
649
Fig. 2. Higher magnification of diazo replica shown in figure 1. This is a portion of the apex of the right upper lobe, at the junction of the overinflated and underinflated areas. (See arrow in figure 1.) Note the irregular inflation of terminal airways, alveolar ducts, alveolar sacs, and alveoli. The architecture of a pulmonary acinus is well shown in the center of the photo (arrow). (Original magnification: X 10.)
optimal for the factors relating to illumination and the diazo dyes. A number of other stains proved to be unsatisfactory. The parallel light provided by the halogen lamp permits much finer delineation than has been previously available, and this allows for 3X and grea.ter magnifications (figures I and 2). The diazo replicas can be analyzed by relatively inexpensive morphometric means such as point counting and linear intercepts (3), with certain advantages afforded by the 2-dimensional replica that are not available from examination of the frozen section of lung itself. In the latter respect, the 2-dimensional nature of the replica bypasses the need to make subjective decisions about measurement problems involving 3 dimensions (e.g., alveolar diameter), and the sharp contrast permits more reproducible, precise measurements. These advantages are particularly critical for large volume quantification by image analyzers and are provided by replicas either in the form of pa-
per prints or plastic transparencies. Although image analyzer studies of alveolar walls have been succcssfull y carried out with frozen sections of lung (5) , our experience indicates that replicas would be far more feasible for the a!'l alysis of both a large number of specimens and a large number of sections from each specimen. Replicas can also be used for quantifying selected cells in whole sections of human or animal lungs, e.g., by the lactate dehydrogenase method of staining type 2 pneumocytes for image analyzer studies (6) . The replicas do not, of course, provide cytologic detail, but this is readily available through the simple expedient of preserving the 60-,um section used for the diazo master in a transparent, sealed plastic (1-ml Aclar) enclosure where it can be examined by stereomicroscopy. In addition , we obtain whol e lung paraffin-embedded sectio ns from "sister" slices adjacent to the frozen section for routine histopathologic examination (7). Finally, the methodol-
650
NOTE
ogy has potential application for quantitative measurements of other organs in which appropriate contrast can be obtained, e.g., kidney tubular studies. RAY M. YuTANil RussELL P. SHERWIN Department of Pathology University of Southern California School of Medicine 2025 Zonal Ave. Los Angeles, Calif. 90033
References I. Sherwin, R. P.: Diazo replication for the pathologic study of emphysema, Am J Clin Pathol, 1968,45, l. 1 Supported by a grant from the American Lung Association, California Division.
2. Gough, J., and Wentworth, J. E.: Thin sections of entire organs mounted on page, in Recent Ad''ances in Pathology, Little, Brown and Company, Boston, 1960, p. 1180. 3. Weibel, E. R.: Morphometry of the Human Lung, Academic Press, New York, 1963. 4. Wyatt, J. P., Fischer, V. W., and Sweet, H. C.: The pathomorphology of the emphysema complex: Part l, Am Rev Respir Dis, 1964,89,533. 5. Sherwin, R. P., Margolick, J. B., and Azen, S. P.: An automated determination of ratios of type 2 pneumocytes to alveolar wall area using an image analyzer, Am Rev Respir Dis, 1973,108, 1015. 6. Margolick, J. B., Azen, S. P., and Sherwin, R. P.: An image analyzer quantitation of type 2 pneumocytes, Am Rev Respir Dis, 1973, 108, 704. 7. Gillette, L. V., and Sherwin, R. P.: A Mylar and Aclar plastic method for giant histologic sections of the lung, Am Rev Respir Dis, 1965,92,238.
