BRIEF REPORT |
Correspondence to: Richard L. Ornberg, Pharmacia Corp., 700 Chesterfield Parkway N, BB4J, St Louis, MO 63198. E-mail: Richard.L.Ornberg@.pharmacia.com
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Summary |
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Cell proliferation and apoptosis indices are important indicators for the prognosis and treatment of a variety of cancers. A method is described using differential absorption color image analysis to measure proliferation and apoptosis in tumor sections using BrdU (5' bromodeoxyuridine) incorporation and immunohistochemistry and terminal deoxytransferase nick end-labeling (TUNEL). Nuclei were labeled with streptavidinperoxidasediaminobenzidine (DAB) secondary detection. The differential absorption method uses a computer-controlled microscope equipped with a tunable filter and digital camera to take advantage of the spectral differences of stained objects of interest. Images collected at defined wavelengths are divided and scaled to form ratio images in which the hematoxylin- or DAB-stained nuclei have intensity ranges far above those of surrounding structures. Using brightness thresholding followed by selection based on nuclear size and shape parameters, binary images were formed of the BrdU/apoptotic-positive tumor and all the tumor nuclei for subsequent counting and calculations of proliferation and apoptotic indices. (J Histochem Cytochem 49:10591060, 2001)
Key Words: apoptosis, cell proliferation, BrdU, TUNEL
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Introduction |
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Proliferation and apoptosis measurements were made on human colon cancer cell tumors, either HT-29 or HCT-116 cells, grown in athymic nude mice. BrdU was administered by IP injection of bromodeoxyuridine, 0.1 mg/g body weight, in saline at 2 hr before sacrifice. Tumors were fixed in Streck STF (Omaha, NB) or 10% neutral buffered formalin and processed to paraffin according to routine procedures. BrdU immunohistochemistry was performed with reagents and protocol provided by Zymed Laboratories (San Francisco, CA). The TUNEL procedure was used (Trevigen; Gaithersburg, MD) to enzymatically attach biotin deoxy-UTP to DNA fragments in apoptotic nuclei. Both anti-BrdU and TUNEL labels were visualized by streptavidinperoxidase (DAKO; Carpinteria, CA) and either aminoethyl carbazole (AEC) (Zymed Laboratories) or diaminobenzidine (DAB) (DAKO Liquid DAB) as peroxidase substrates. Sections were counterstained with aqueous hematoxylin. BrdU- or TUNEL-positive nuclei were stained either red (AEC) or brown (DAB) and all other nuclei were stained blue. The example below illustrates the method for DAB- and hematoxylin-stained tissue.
Color image analysis by the differential absorption method takes advantage of the differences in light absorption of stained objects to isolate or segment these objects for subsequent counting and measurement. Using a light microscope equipped with a liquid crystal tunable filter, digital camera, and image analysis software as previously described (
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The validity of this method was examined by comparing proliferation indices obtained by image analysis with indices obtained by visual counting of the same image field in regions of tumor tissue having varying degrees of proliferation. As shown in Fig 2, there was a strong correlation between the two methods (correlation coefficient = 0.96) and this correlation was linear (correlation coefficient = 0.86) as expected. A systematic undercount of nuclei by the image analysis method (data not shown) produces the largest variation in the two methods, due in part to the inability to accurately determine a nuclear count when nuclei touch one another. Hence, the image analysis method required thin sections to minimize this problem.
In addition to accuracy, the differential absorption method is rapid and widely applicable. Typically, a study of 100 sections at 10 images each can be analyzed in a few hours. The operator need only select an area, focus, and initiate the program. Any marker that can be labeled with a colored reaction product can be analyzed. To date, the method has been applied to two stain systems. Multistain analysis is currently being developed. With wider use and awareness, differential absorption color image analysis can be a powerful method in modern pathology for providing easy and accurate morphometric analysis of routinely prepared tissue sections.
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Footnotes |
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Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 27, 2001.
Received for publication December 7, 2000; accepted February 16, 2001.
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Literature Cited |
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Ornberg RL, Woerner BM, Edwards DA (1999) Analysis of stained objects in histological sections by spectral imaging and differential absorption. J Histochem Cytochem 47:1-7