Histologic Correlates of Gastrointestinal Ultrasound Images

Transcription

1 GASTROENTEROLOGY 1989;96: Histologic Correlates of Gastrointestinal Ultrasound Images M. B. KIMMEY, R. W. MARTIN, R. C. HAGGITT, K. Y. WANG, D. W. FRANKLIN, and F. E. SILVERSTEIN Departments of Medicine, Anesthesiology, Pathology, and Radiology, and the Center for Bioengineering, University of Washington, Seattle, Washington Endoscopic ultrasound imaging has potential for improving the diagnosis of gastrointestinal disease. However, the anatomic correlates of gastrointestinal ultrasound images have not been precisely defined. We have compared ultrasound images with the corresponding histologic sections of 81 specimens of resected and postmortem, normal and diseased gastrointestinal tissue. The five layers seen on ultrasound images of the normal gastrointestinal tract correspond to (1) superficial mucosa, (2) deep mucosa, (3) submucosa plus the acoustical interface between the submucosa and muscularis propria, (4) muscularis propria minus the acoustical interface between the submucosa and muscularis propria, and (5) serosa and subserosal fat. This interpretation takes into consideration the echoes produced by the tissue layers and the echoes produced by the interfaces between layers. Abnormal findings on ultrasound images of neoplastic and inflammatory diseases correspond to histologic tissue structure. When properly interpreted, ultrasound images of the gastrointestinal wall can provide potentially useful diagnostic information. Ultrasound can be used to image the gastrointestinal tract by both transcutaneous and endoscopic techniques, Transcutaneous techniques have been limited by poor resolution produced by attenuation from intervening structures and by echo reverberation or ring down produced by intraluminal gas (1). Endoscopic ultrasound is being used more frequently as improved equipment becomes available. Examination of the upper gastrointestinal tract and rectum with ultrasound endoscopes and rectal ultrasound probes has been advocated as a reliable method of staging neoplasia and investigating intrarectal abnormalities (2-6). Several investigators have described five layers on ultrasound images of gastrointestinal organs. These layers are parallel to the mucosal surface in the image and are distinguished by their different echo texture and intensity. Assignment of the anatomic correlates of these layers has varied. There has been controversy about whether the layers seen on ultrasound images correspond directly to the histologic layers (7-10), or whether the ultrasound appearance of layers is artificial, caused by echoes produced by the interaction of the ultrasound wavefront at interfaces between the anatomic tissue layers (11,12). We have previously described a method that allows precise comparison of an ultrasound image with the histologic section of the same area of resected tissue (13). In this paper we describe application of this method to answer the following questions about ultrasound images of the gastrointestinal tract: (a) what are the anatomic correlates of the layers seen on ultrasound; (b) can the physical principles of ultrasound explain differences between ultrasound and histologic layer thickness; and (c) do changes in the ultrasound images produced by the physical interaction of ultrasound with tissue interfere with the detection and diagnosis of inflammatory and neoplastic diseases? Materials and Methods Correlation of Ultrasound With Histology Human gastrointestinal tissue from surgical or autopsy material was obtained and imaged within 4 h of resection. Tissue specimens ~ cm 3 wide by 6 cm long were taken from the longitudinal axis of the opened gastrointestinal organ. Eighty-one specimens were studied including 72 from surgical resections and nine from autopsy material. Permission to study this tissue was obtained from the University of Washington Human Subjects Review Committee. A predominance of colorectal tissue (59 specimens) reflected the availability of surgically re by the American Gastroenterological Association /89/$ 3.50

2 434 KIMMEY ET AL. GASTROENTEROLOGY Vol. 96, No. 2, Part 1 Figure 1. The ultrasound image of a specimen of gastric antrum (top) is compared with the corresponding histologic section (bottom) to illustrate the labels used in the text. This specimen was taken from a patient with gastric outlet obstruction and has a thickened muscularis propria. The ultrasound layers are numbered sequentially U, through U 5 beginning with the mucosal surface. The submucosa (sm) is labeled H3 and the musculiuis propria (mp) is labeled H4 for the purposes of the equations given in the text. The mucosa (m) and serosa (s) are also seen on the histologic section. The horizontal bar represents 5 mm. sected tissue in our hospital. Two esophageal, 13 gastric, and seven small intestinal specimens were analyzed. Both normal and abnormal tissue were found in a number of the specimens. Normal structure was studied in two esophageal, six gastric, three small intestinal, and 26 colorectal specimens. Abnormal tissue studied included two esophageal carcinomas, five gastric carcinomas, 17 colorectal neoplasms, 20 specimens containing inflammatory bowel disease, 10 cases of other inflammatory diseases, and four other miscellaneous abnormalities. Specimens were mounted with needles into a specimen container constructed to permit accurate spatial localization of the tissue and ultrasound images (13). A prototype 8.5-MHz linear array ultrasound transducer (Advanced Technology Laboratories, Bellevue, Wash.) was held over the tissue specimen with a micropositioner to control its position and orientation. This ultrasound transducer has a lateral resolution of 0.8 mm and a scan plane or section thickness of 1.2 mm (14). The axial resolution of this transducer is 0.3 mm when measured in any of three ways: by measuring the thickness of an oil/water interface, by measuring the thickness of a water/tissue-equivalent material interface (15), and by the ability to discriminate two wires placed 0.3 mm apart in a water bath. Ultrasound images Were obtained by scanning through normal saline at room temperature placed over the tissue. After imaging fresh tissue, the saline was replaced with formalin to fix the tissue. Ultrasound imaging was repeated after tissue fixation to confirm accurate placement of the micropositioner. A double-scalpel cutter was then mounted on the micropositioner with the blades set to remove a 5-mmthick block of tissue that included the tissue imaged with ultrasound. The ends of the tissue block were marked with ink and cut to match the length of the ultrasound image, producing a block of tissue 3.2 cm long. The tissue block was embedded in paraffin and sectioned for histologic examination. A 5-/1,ffi-thick section from the center of the block was stained with hematoxylin and eosin or Masson's trichrome. This was the tissue section that was compared to the ultrasound image. The ultrasound image was photographed by a multiformat camera. Comparisons were facilitated by photographing the ultrasound image from the multiformat film and the histologic image from the histology slide so that the tissue was the same width. Assignments of the histologic c u~r e lates of the ultrasound image were made by a side-by-side comparison of these photographs. The layers seen on histology and ultrasound images and how they are labeled are illustrated in Figure 1. Layer Thickness Analysis In our early studies we observed that the third layer on the ultrasound image (U 3 in Figure 1) corresponded spatially to the submucosa (H 3 ), but appeared to be thicker on ultrasound than on histology. The fourth ultrasound layer (U 4 ) corresponded spatially to the muscularis propria (H4), but was thinner. To quantify this observation, individual layers were measured on the ultrasound images and histologic sections of three gastric and seven colonic specimens containing normal tissue. These specimens

3 February 1989 GASTROINTESTINAL ULTRASOUND INTERPRET A TION 435 were selected for their good technical quality and for having flat areas that could be measured accurately. Histologic layer thickness was measured at a magnification of X4 using an eyepiece micrometer accurate to 0.01 mm by a histopathologist. Three separate measurements of ultrasound layers were made directly from the multiformat film using a dissecting microscope (magnification, X4) and vernier calipers accurate to 0.03 mm. Ultrasound measurements were corrected for magnification on the multiformat film by dividing by a magnification factor determined by measuring the distance between a set of internal grid wires placed 5 mm apart in the agar below the tissue. Layer measurements were taken from corresponding areas of the ultrasound image and histologic section; After studying 10 specimens, it became apparent that one-to-one comparisons of ultrasound and histologic layer thickness were not possible because the total thickness of the ultrasound image was not the same as the total thickness of the histologic section, even when the ultrasound image was corrected for magnification. There are at least two potential uncertainties that might account for the discrepancy between the thickness of the ultrasound image and the thickness of the histologic section. First, artifacts in the histologic section are created by tissue shrinkage and expansion during processing. When overall specimen thickness was measured before and after histologic processing, we found that the tissue processing reduced overall thickness by 5%. There could also be small degrees of differential shrinkage of the various tissue layers. The second uncertainty in relating the overall thickness of the ultrasound image and the histologic section is that depth or thickness on the ultrasound image is really a measurement of time, not distance. Depth on the ultrasound image represents the time (t) for an ultrasound pulse emitted by a transducer to travel to and return from a reflective tissue interface rather than the actual distance in the tissue. Time is related to distance (d) by the equation d = vt, where v is the velocity of sound in the tissue being imaged. The scaling factor of an ultrasound unit assumes an average velocity of sound in tissue to be 1540 m /s. This single value is built into the imaging unit as an average value for living tissue at 37 C. However, whether this velocity is correct for gastrointestinal tissue in vitro at 20 C is unknown. Further, acoustic velocity is not the same in all tissue and it is unlikely that the different histologic layers of the gastrointestinal tract have identical acoustic velocity. These differences in overall thickness of the histologic section and the ultrasound image were corrected for by comparing ratios of layer measurements rather than absolute values (see below). Hypothesis of the Influence of Tissue Interface Echoes We hypothesized that the overall appearance of the ultrasound image should be determined by a combination of the echoes from two sources: those created at interfaces between tissue layers with different acoustic impedances and those created by the internal structure of the tissue layers. In the former case, the duration or thickness in the II. EiJ Tissue ilnd CJ Tissue Interlacc Combined ~ ~ ~ [ ~ : :;: : ' :]: EJi ::::::::: ~ a. b. c. Figure 2. The influence of the interface echo on the relative thicknesses of the echoes from the histologic layers is illustrated. The ultrasound beam passes through the tissue from the top to the bottom. Column a shows echoes produced by the internal tissue constituents. Column b illustrates the addition of the interface echo to the tissue layer echoes, highlighted to illustrate its position in the image. Column c illustrates the overall image appearance with both types of echoes. (1) If the more superficial layer is echo rich and the deeper layer echo poor, then the interface echo adds thickness to the superficial layer and subtracts thickriess from the deeper layer. (II) If the more superficial layer is echo poor and the deeper layer echo rich, then the interface echo is not distinguishable from the deeper layer and therefore does not change the relative layer thicknesses. image of an interface echo is primarily determined by the duration of the ultrasound pulse and the impulse response of the system, factors that determine the axial resolution of the system. If this hypothesis is correct, interface echoes shoulcl be visible only if they are different in amplitude from echoes produced by the internal structure of the adjacent tissue layers. The amplitude of echoes from the deeper of two adjacent tissue layers is most important to the visibility of the interface echo because the interface echo begins at a place on the image that corresponds to the tissue interface; the duration of the interface echo then extends into the area on the tissue that corresponds with the deeper tissue layer (Figure 2). If the more superficial tissue layer has echoes of the same magnitude as the interface echo, then the thickness of the interface echo will add to the thickness of the superficial layer on the ultrasound image (box l.b. in Figure 2). If the deep tissue layer has lower amplitude echoes than the interface echo, then the thickness of the interface echo will subtract from the thickness of the deep tissue layer on the ultrasound image (box l.c. in Figure 2). If on the other hand the echoes from the deeper tissue layer are of the same magnitude as the interface echo, then the echoes from the interface will combine with those of the deeper tissue layer and the thickness of the layers on the ultrasound image will be the same as those of the corresponding histology (row II in Figure 2).

4 436 KIMMEY ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 We tested this hypothesis by comparing measured thicknesses of the submucosa and muscularis propria on histology with thicknesses of the corresponding layers on ultrasound. These layers were chosen because they are thick enough to make accurate measurements and because the submucosa is echogenic and the deeper muscularis propria is echo poor. Comparisons between ultrasound and histology were made using ratios of layers to each other to reduce the uncertainties created by differences between the overall thicknesses of the ultrasound image and the histologic section. If our hypothesis is correct, the interface echo should add to the thickness of the ultrasound layer corresponding to submucosa and detract from the ultrasound layer corresponding to the muscularis propria. This calculated difference in thickness (1) should be equal to the axial resolution of the ultrasound transducer used. The above hypothesis can be restated in the form of a mathematical model using layer thickness ratios as follows: [H31H,1/[H.IH,1 = [(U 3 - I)/U,I/[(U. + DIU,), (i) where H3 represents the measured thickness of the submucosa on histology, H. represents the thickness of the muscularis propria on histology, H, represents the overall thickness of the histologic section, U 3 represents the thickness of the central or third ultrasound layer (corresponding to the submucosa), U. represents the thickness of the fourth ultrasound layer (corresponding to muscularis propria), and U t represents the overall thickness of the ultrasound image. The overall thickness measurements (H, and U t ) cancel each other so we,are left with the relationship Measurements of H 3, H., U 3, and U. from 10 specimens were entered into an equation-fitting program to solve for I. The 95% confidence interval for I was determined using the t statistic (16). The calculated I was used in Equation (3) to determine the error for each data point. Results Error = (H31H. ) - [(U 3 - I)I(U, + I)). (3) Correlation of Ultrasound With Histology Five major layers were seen on 30 of 37 ultrasound images of normal esophageal, gastric, colonic, and rectal wall. The outer layer was not seen on one esophageal and one gastric specimen that had no subserosal fat or inflammation. The second layer was not visible on one duodenal, three colonic, and one rectal specimen. These specimens had a thin mucosal layer on the corresponding histologic section. An example of the ultrasound image and corresponding histologic section of normal rectum is shown in Figure 3. The layer nearest to the mucosal surface, layer 1, is usually seen as a thin echogenic line. It closely follows the contour of the mucosal surface but is (2) much thinner than the full thickness of the mucosa. This layer is thicker in some specimens (Figure 1) than in others (Figure 3); tissue specimens with more surface irregularities appeared to have thicker first layers. The second ultrasound layer is thicker than the first and is echolucent. This layer corresponds to the remainder of the mucosa as seen on histology. Specimens with a thick mucosa were found to have a thick second layer (Figure 1). The mean thickness of the muscularis mucosae in the measured histologic specimens was 0.17 ± 0.09 mm for gastric specimens and 0.06 ± 0.03 for colorectal specimens (Table 1). The shortest distance between two echoes that can be resolved by an ultrasound transducer is determined by the axial resolution; this was measured to be 0.3 mm for the transducer used in these studies. Therefore, the second acoustic layer is too thick to be the muscularis mucosae alone in these specimens. The third or central layer is the most echogenic layer in normal gastrointestinal tissue. This layer corresponds spatially to the histologic submucosa but consistently appears to be slightly thicker on ultrasound than by histology (see Figures 1 and 3, and below). The fourth layer is echolucent and corresponds spatially to the histologic muscularis propria. A thin echogenic line is seen on the ultrasound images of specimens with a well-defined area of connective tissue between the inner circular and outer longitudinal components of the muscularis propria. This echogenic line is seen in the middle of the fourth layer in colonic specimens if the image is taken from an area corresponding to a taenia coli. The echogenic line is near the outer layer in colonic specimens if the image is taken from an area between two taenia coli (Figure 4). The fifth or outer ultrasound layer is echogenic and of variable thickness. The thickness of this layer ultrasonographically corresponds to the thickness of the histologic subserosal fat or serosal fibrous or inflammatory tissue. Influence of Tissue Interface Echoes Measurements of the histologic layers and the ultrasound layers (Table 1) of 10 normal specimens were compared in an effort to determine the influence of boundaries between the tissue layers on the ultrasound image. The thicknesses of the third and fourth ultrasound layers were compared to the thicknesses of the histologic submucosa and muscularis propria using Equation (2) (see Materials and Methods). It is emphasized that this comparison was made using ratios of the layer thicknesses listed in

5 February 1989 GASTROINTESTINAL ULTRASOUND INTERPRETATION 437 Figure 3. The ultrasound image (top) of a specimen of normal rectum is compared with the corresponding histologic section (bottom). Ultrasound layers 1 and 2 are not well seen because the mucosa (m) is thin in this specimen. Ultrasound layer 3 corresponds spatially with the submucosa (sm), but is slightly thicker. Ultrasound layer 4 corresponds to the muscularis propria (mp) but is slightly thinner. The thick subserosal fat (s) corresponds to ultrasound layer 5. The horizontal bar represents 5 mm. Table 1 because of the difference between total thicknesses of the ultrasound image and histologic section. The calculated value for the interface echo [I in Equation (2)] was 0.25 mm (95% confidence interval, mm). This value of I calculated from the layer measurements compares favorably with the measured value of the axial resolution (0.3 mm). This supports the hypothesis that the interface echo, the thickness of which is determined by the axial resolution of the ultrasound transducer, accounts for the increased thickness of layer 3 and the decreased thickness of layer 4 on ultrasound compared with the thicknesses of the submucosa and muscularis propria. Table 1. Measured Thicknesses of Ultrasound and Histologic Layers of Resected Normal Gastrointestinal Tissue Q Histologic thickness (mm) US thickness b (mm) Tissue m mscm sm mp Errore Stomach (0.02) 0.73 (0.02) 1.17 (0.18) 3.58 (0.43) Stomach (0.13) d 0.84 (0.11) 3.16 (0.16) Stomach (0.23) 1.04 (0.06) 0.82 (0.25) 2.55 (0.13) Colon (0.03) 0.32 (0.12) 2.61 (0.07) 2.37 (0.15) Colon (0.02) 0.41 (0.21) 0.94 (0.12) 1.34 (0.24) Colon (0.06) 0.26 (0.02) 1.27 (0.09) 1.65 (0.08) Colon (0.05) 0.28 (0.02) 1.17 (0.07) 1.43 (0.25) Colon (0.22) 0.22 (0.04) 0.67 (0.12) 1.22 (0.10) Rectum (0.04) 0.59 (0.12) 1.35 (0.24) 3.02 (0.38) Rectum (0.02) d 0.80 (0.14) 1.53 (0.21) m, mucosa excluding muscularis mucosae; mp, muscularis propria; mscm, muscularis mucosae; sm, submucosa; US, ultrasound. a Measurements from histology and ultrasound images are not for direct comparisons but should be used in Eql,lations (2) and (3) in Materials and Methods, where H3 and H. correspond to the thicknesses of the submucosa and muscularis propria, and U 3 and U 4 correspond to the thicknesses of ultrasound layers 3 and 4. b Mean and standard deviation of three separate measurements are listed. C The error for each data line is calculated from Equation (3) using a value of I = 0.25 mm. d Layers 1 and 2 could not be distinguished from each other.

6 438 KIMMEY ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 Figure 4. The ultrasound images (top) and corresponding histology (bottom) of a colonic specimen taken proximal to the aganglionic segment in a patient with Hirschsprung's disease is shown. The specimen is oriented in the longitudinal axis of the colon. The muscularis propria (mp) is thickened. The area of fibrous tissue between the outer longitudinal muscle and the inner circular muscle is seen as a thin echogenic line on the image (arrows). This echogenic line is closer to the center of the fourth layer when the image is made of tissue including a taenia coli (A) than when it is made of tissue between taenia (B). Histologic sections are stained with Masson's trichrome to accentuate collagen. The horizontal bar represents 5 mm. m, mucosa; s, serosa; sm, submucosa. Detection of Abnormal Tissue Structure Neoplasms were detected as a disruption in the continuity of an ultrasound layer or by diffuse layer thickening. Disruption of layer continuity was best seen in colonic neoplasms and corresponded to the area and depth of invasion seen on histology. Infiltrating gastric neoplasms produced diffuse thickening of all ultrasound layers without causing layer disruption. Mucosal neoplasms could be distinguished from extramural metastases when layer structure was carefully examined (Figure 5). The small change in ultrasound layer thicknesses produced by the interface echoes did not change the image interpretation with regard to depth of invasion of the neoplasm. A change in the echogenicity of the ultrasound layers is another sign of tissue pathology. Edema of the submucosa ~ aassociated s with thickening and a decrease in the echogenicity of the central echogenic layer (layer U 3 ) in both the colon and the ileum. Submucosal hemorrhage produced thickening of this layer without a loss of echogenicity. Neoplasms were usually of intermediate echogenicity: less echogenic than the central layer but more echogenic than the second and fourth layers. Ultrasound images of inflammatory intestinal disease also correlated with the corresponding histology. The depth of mucosal ulcers appeared the same on both the ultrasound image and the histologic section (Figure 6). Thickened layers were also recognized on the ultrasound images. For example, ultrasound images of specimens of diverticular strictures showed a very thick fourth layer corresponding to a thickened muscularis propria (Figure 7). Discussion Interpretations of ultrasound images of the gastrointestinal tract have varied widely. The presence of five ultrasound layers led early investigators to conclude that these layers corresponded directly to five histologic layers (7-10,17-20). This interpretation was argued to be too simplistic by others who tried to interpret the images based on a knowledge of the physical principles of ultrasound (11,12). Proof of either of these two hypotheses has been difficult to establish because of the limited resolution of available ultrasound instruments and because previously there has not been a method of precisely obtaining a histologic section from the same tissue plane that was imaged with ultrasound. Using careful correlation of ultrasound images with the corresponding histologic section, we propose an interpretation of ultrasound images of the gastrointestinal tract that

7 February 1989 GASTROINTESTINAL ULTRASOUND INTERPRETATION 439 accounts for the tissue structure, the ultrasound characteristics of gastrointestinal tissue, and the physics of ultrasound. This interpretation is based on studies made on resected tissue and must be extrapolated to in vivo ultrasound interpretation. However, the precise assignment of histologic correlates to in vivo images is not possible with current technology. The interpretation outlined is based on measurements of layers in normal specimens, but the appearance of images of abnormal specimens remains consistent with this interpretation. Echoes from within a tissue layer are produced when acoustical energy is reflected or back-scattered by inhomogeneities within this tissue. The key factor in the production of echoes by a tissue layer is a change in the acoustic impedance between adjacent areas within the tissue layer. Acoustic impedance is a function of the density of a tissue and its bulk Figure 6. Areas of colonic ulceration are shown (open arrows) on the ultrasound image (top) and histologic section (bottom) of a specimen from a patient with ulcerative colitis. The depth of ulceration on the image corresponds to the depth on histology. The thin line of echoes at the ulcer base could be from inflammatory cells at the bottom of the ulcer or could be echoes from the interface between the fluid bathing the tissue and the ulcer base. The horizontal bar represents 5 mm; m, mucosa; mp, muscularis propria; s, serosa; sm, submucosa. Figure 5. The ultrasound image (top) is compared with the histologic section (bottom) of a specimen of colon from a patient with an extramural metastasis (M) from ovarian carcinoma. Ultrasound layer 3, corresponding to the submucosa and ad jacent tissue interface, is compressed (arrows) but not disrupted by the metastasis. The horizontal bar represents 5 mm. modulus or stiffness. As the density of a tissue layer is relatively constant, changes in the bulk modulus within a tissue layer are the main determinant of the echogenicity of the layer. Collagen has a very high bulk modulus; tissues containing abundant collagen are echogenic (21-23). Fat within tissue is also echogenic. It is thus not surprising to find echogenic layers on ultrasound images in the same positions as the submucosa, which is dense in collagen and sometimes contains fat, and in the subserosal layer where fat is often abundant. Echoes are also created when an ultrasound beam encounters an interface between two tissue layers with different acoustic impedances (24). The thickness of this echo depends on the axial resolution of the ultrasound transducer. The vertical dimension on an ultrasound image encodes the time it takes for the ultrasound beam to encounter tissue at a certain depth and be returned to the transducer. An interface echo will begin at the vertical distance related to where the interface occurs. The remainder of the interface echo will then blend with the echoes that

8 440 KIMMEY ET AL. GASTROENTEROLOGY Vol. 96, No.2, Part 1 Figure 7. The ultrasound image (top) of a specimen of a colonic diverticular stricture shows a thickened fourth layer corresponding to the thickened muscularis propria (mp) on histology (bottom). The horizontal bar represents 5 mm; m, mucosa; s, serosa; sm, submucosa. arise from the superficial part of the deeper tissue layer. The combination of echoes produced at interfaces with the echoes created by the tissue layers themselves will determine the appearance of the final image (Figure 2). An ultrasound beam passing through the gastrointestinal wall will encounter at least six potential interfaces between tissue layers. The first interface occurs at the surface of the mucosa. In these in vitro studies the interface is between fluid bathing the tissue and the mucosal surface. This interface accounts for the first echo genic layer seen on the images. This layer varies in thickness with the smoothness of the mucosal surface. Areas with an irregular surface such as the stomach and colon can be expected to have a slightly thicker first layer because more surfaces (gastric pits and colonic crypt openings) are present to generate echoes. The remainder of the mucosa produces few echoes and forms the second ultrasound layer. The second potential tissue interface is between the mucosa and the muscularis mucosae. This interface should produce an echo if the acoustic impedances of the mucosa and muscularis mucosae are sufficiently different. The thickness of this echo is greater than the thickness of the muscularis mucosae in the specimens studied so the interface echo, if present, would be expected to combine with the echoes from the underlying submucosa. These inter- face echoes would obscure any echoes arising from the muscularis mucosae itself, so the position of the muscularis mucosae would correspond to the most superficial part of ultrasound layer 3. If the acoustic impedances of the mucosa and muscularis mucosae are not sufficiently different to produce an interface echo, then the position of the muscularis mucosae would correspond to the deepest part of ultrasound layer 2. The correlation method used in these studies is not sufficiently precise to distinguish between these two possibilities; the precise ultrasound localization of the muscularis mucosae remains unknown. It is clear from these studies, however, that the second ultrasound layer is too thick to represent the muscularis mucosae alone, as reported in earlier studies (18,20). The third tissue interface occurs between the muscularis mucosae and the submucosa. The echoes created by this interface would combine with any echoes produced by the superficial submucosa. Echoes from the muscularis mucosae-submucosa interface are not seen because the submucosa is itself echogenic. This interface blends together with echoes from the internal structure of the submucosa to form the central echo genic layer. Echoes produced at the fourth interface, between the submucosa and the muscularis propria, combine. with the echoes from the submucosa to create a central echogenic layer that is thicker than the submucosa. The interface echo also obscures the relatively fewer echoes that arise in the superficial muscularis propria. The fourth ultrasound layer is thinner than the muscularis propria by the same distance that the central echogenic layer is thicker than the submucosa. This distance is a characteristic of the ultrasound transducer used. This distance was calculated to be 0.25 mm using the layer measurements in Table 1, comparing favorably to the measured axial resolution (0.3 mm) of the ultrasound transducer used in these studies. Current ultrasound endoscopes have an axial resolution of between 0.2 and 0.5 mm (25,26). Echoes created at the fifth interface, between the muscularis propria and the subserosal tissue, blend with echoes created by any fat present in the subserosal area. If no fat is present, the interface echo alone may be all that is seen. The sixth potential interface occurs between the serosa and any surrounding tissue. Whether this interface is seen will depend on the echogenicity of the surrounding tissue. The effect of tissue layer interfaces on gastrointestinal ultrasound images should be considered when these images are interpreted. We have shown that ultrasound determination of the depth of invasion in specimens of colonic neoplasms accurately predicts

9 February 1989 GASTROINTESTINAL ULTRASOUND INTERPRETATION 441 the depth of invasion shown histologically (17). It is likely that in most clinical situations, the usefulness of endoscopic ultrasound in detecting abnormal tissue structure will not be affected by the small changes in layer thickness produced by the interface echo. It is possible that detection of small foci of malignant invasion confined to the boundary between two tissue layers could be affected by interface echoes. Areas of malignant invasion are usually hypoechoic and might be obscured by the echoes produced by the interface. The thickness of this interface echo could potentially be made very small with increased frequency and other improvements in ultrasound transducers. If histologic layer thickness measurements are used in future clinical or research studies, the third and fourth layers should be corrected by a factor that is determined by and is a characteristic of the transducer used. Ultrasound images made with transducers within the lumen of the gastrointestinal tract show great promise for improving diagnosis and directing therapy. Staging of neoplasms and identification of the nature of intramural and extramural masses should lead to more appropriate therapy. As instruments improve, more endoscopists should be able to use this technique. Correct interpretation of ultrasound images is not difficult, but requires some knowledge of applied ultrasound physics. References 1. Schoelmerich J, Diaz A, Volk BA, Spamer C, Brambs HJ, Gerok W. Clinical significance of abnormalities of the gastrointestinal tract detected by abdominal ultrasound. Dig Dis Sci 1988;33 : Gordon SJ, Rifkin MD, Goldberg BB. Endosonographic evaluation of mural abnormalities of the upper gastrointestinal tract. Gastrointest Endosc 1986;32: Rifkin MD, McGlynn ET, Marks G. Endorectal sonographic prospective staging of rectal cancer. 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Correlation of echographic visualizability of tissue with biological composition and physiological state. J Acoust Soc Am 1973;54: Goss SA, Frizzell LA, Dunn F. Dines KA. Dependence of the ultrasonic properties of biological tissue on constituent proteins. J Acoust Soc Am 1980;67: Pohlhammer J, O'Brien WD JI. Dependence of the ultrasonic scatter coefficient on collagen concentration in mammalian tissues. J Acoust Soc Am 1981;69: Hyles DL, Hedrick WR, Starchmari DE. Ultrasound physics and instrumentation. New York: Churchill Livingstone, 1985: DiMagno EP. Regan PT, Clain JE, James EM, Buxton J1. Human endoscopic ultrasonography. Gastroenterology 1983; 83: Tio TL, Tytgat GNJ. Atlas of transintestinal ultrasound. Aalsmere. the Netherlands: Mur-Kostverloren, Received April 25, Accepted September 15, Address requests for reprints to: Michael B. Kimmey, M.D., Division of Gastroenterology, Mail Stop RG-24, University of Washington, Seattle, Washington This study was supported by grant R01 OK from the National Institutes of Health and an equipment grant from Advanced Technology Laboratories, Bellevue, Washington.