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Journal of Histochemistry and Cytochemistry, Vol. 51, 575-584, May 2003, Copyright © 2003, The Histochemical Society, Inc.

ARTICLE

Automated Selection of DAB-labeled Tissue for Immunohistochemical Quantification

Eric M. Breya,b, Zahid Lalanic, Carol Johnstona, Mark Wongc, Larry V. McIntireb, Pauline J. Duked, and Charles W. Patrick, Jr.a
a Laboratory of Reparative Biology and Bioengineering, Department of Plastic Surgery, University of Texas M. D. Anderson Cancer Center and University of Texas Center for Biomedical Engineering, Houston, Texas
b Department of Bioengineering, Institute for Biosciences and Bioengineering, Rice University, Houston, Texas
c Department of Oral and Maxillofacial Surgery, University of Texas Health Science Center at Houston–Dental Branch, Houston, Texas
d Department of Orthodontics, University of Texas Health Science Center at Houston–Dental Branch, Houston, Texas

Correspondence to: Charles W. Patrick, Jr., Dept. of Plastic Surgery, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 443, Houston, TX 77030. E-mail: cpatrick@mdanderson.org

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland–Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels. (J Histochem Cytochem 51:575–584, 2003)

Key Words: image analysis, immunohistochemistry, growth factors, diaminobenzidene, normalized blue


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