Skip to main content Accessibility help

Reproducibility of Immunostaining Quantification and Description of a New Digital Image Processing Procedure for Quantitative Evaluation of Immunohistochemistry in Pathology

  • Vagner Bernardo (a1), Simone Q.C. Lourenço (a1), Renato Cruz (a1), Luiz H. Monteiro-Leal (a2), Licínio E. Silva (a3), Danielle R. Camisasca (a1), Marcos Farina (a4) and Ulysses Lins (a5)...


Quantification of immunostaining is a widely used technique in pathology. Nonetheless, techniques that rely on human vision are prone to inter- and intraobserver variability, and they are tedious and time consuming. Digital image analysis (DIA), now available in a variety of platforms, improves quantification performance: however, the stability of these different DIA systems is largely unknown. Here, we describe a method to measure the reproducibility of DIA systems. In addition, we describe a new image-processing strategy for quantitative evaluation of immunostained tissue sections using DAB/hematoxylin-stained slides. This approach is based on image subtraction, using a blue low pass filter in the optical train, followed by digital contrast and brightness enhancement. Results showed that our DIA system yields stable counts, and that this method can be used to evaluate the performance of DIA systems. The new image-processing approach creates an image that aids both human visual observation and DIA systems in assessing immunostained slides, delivers a quantitative performance similar to that of bright field imaging, gives thresholds with smaller ranges, and allows the segmentation of strongly immunostained areas, all resulting in a higher probability of representing specific staining. We believe that our approach offers important advantages to immunostaining quantification in pathology.


Corresponding author

Corresponding author. E-mail:


Hide All
Bacus, S., Chin, D., Stewart, J., Zelnick, C., Mahvi, D. & Gilchrist, K. (1997). Potential use of image analysis for the evaluation of cellular predicting factors for therapeutic response in breast cancers. Anal Quant Cytol Histol 19, 316328.
Baxes, G.A. (1994). Digital Image Processing. Principles and Applications, 1st ed. New York: John Wiley & Sons.
Benali, A., Leefken, I., Eysel, U.T. & Weiler, E. (2003). A computerized image analysis system for quantitative analysis of cells in histological brain sections. J Neurosc Methods 125, 3343.
Bilous, M., Dowsett, M., Hanna, W., Isola, J., Lebeau, A., Moreno, A., Penault-Llorca, F., Rüschoff, J., Tomasic, G. & Van De Vijver, M. (2003). Current perspectives on HER2 testing: A review of national testing guidelines. Mod Pathol 16, 173182.
Bishop, P.W. (2002). An immunohistochemical vademecum. Curr Diag Pathol 8, 123127.
Bloom, K. & Harrington, D. (2004). Enhanced accuracy and reliability of HER-2/neu immunohistochemical scoring using digital microscopy. Am J Clin Pathol 121, 620630.
Brey, E.M., Lalani, Z., Johnston, C., Wong, M., Mcintire, L.V., Duke, P.J. & Patrick, C.W. Jr. (2003). Automated selection of DAB-labeled tissue for immunohistochemical quantification. J Histochem Cytochem 51, 575584.
Camisasca, D.R., Honorato, J., Bernardo, V., Silva, L.E., Fonseca, E.C., Faria, P.A.S., Dias, F.L. & Lourenço, S.Q.C. (2009). Expression of Bcl-2 family proteins and associated clinicopathologic factors predict survival outcome in patients with oral squamous cell carcinoma. Oral Oncol 45, 225233.
Caulet, S., Lesty, C., Raphael, M., Schoevaert, D., Brousset, P., Binet, J.-L., Diebold, J. & Delsol, G. (1992). Comparative quantitative study of Ki-67 antibody staining in 78 B and T cell malignant lymphoma (ML) using two image analyser systems. Pathol Res Pract 188, 490496.
Chen, W., Reiss, M. & Foran, D.J. (2004). A prototype for unsupervised analysis of tissue microarrays for cancer research and diagnostics. IEEE Trans Inf Technol Biomed 8, 8996.
De la Grandmaison, G.L., Dorandeu, A., Carton, M., Patey, A. & Durigon, M. (1999). Increase of pulmonary density of macrophages in sudden infant death syndrome. For Sci Intern 104, 179187.
Derkx, P., Nigg, A.L., Bosman, F.T., Birkenhäger-Frenkel, D.H., Houtmuller, A.B., Pols, H.A.P. & Van Leeuwen, J.P.T.M. (1998). Immunolocalization and quantification of noncollagenous bone matrix proteins in methylmethacrylate-embedded adult human bone in combination with histomorphometry. Bone 22, 367373.
Ednaggar, A.K., Lai, S., Clayman, G.L., Zhou, J.-H., Tucker, S.A., Myers, J., Luna, M.A. & Benedict, W.F. (1999). Expression of p 16, Rb, and cyclin D1 gene products in oral and laryngeal squamous carcinoma: Biological and clinical implications. Hum Pathol 30, 10131018.
Ellis, C.M., Dyson, M.J., Stephenson, T.J. & Maltby, E.L. (2005). HER2 amplification status in breast cancer: A comparison between immunohistochemical staining and fluorescence in situ hybridisation using manual and automated quantitative image analysis scoring techniques. J Clin Pathol 58, 710714.
Fritz, P., Wu, X., Tuczek, H., Multhaupt, H. & Schwarzmann, P. (1995). Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology? Pathologica 87, 300309.
Gala, J.-L., Guiot, Y., Delannoy, A., Scheiff, J.-M., Philippe, M. & Martiat, P. (1999). Use of image analysis and immunostaining of bone marrow trephine biopsy specimens to quantify residual disease in patients with B-cell chronic lymphocytic leukemia. Mod Pathol 12, 391399.
Giardina, C., Serio, G., Caniglia, D.M., Lettini, T., Ricco, R., Renzulli, G. & Pesce Delfino, V. (1994). Nuclear morphology and histological grading of oral squamous cell carcinoma (OSCC). A morphometric study. J Biol Res 70, 271279.
Goto, M., Nagatomo, Y., Hasui, K., Yamanaka, H., Murashima, S. & Sato, E. (1992). Chromacity analysis of immunostained tumor specimens. Path Res Pract 188, 433437.
Hatanaka, Y., Hashizume, K., Nitta, K., Kato, T., Itoh, I. & Tani, Y. (2003). Cytometrical image analysis for immunohistochemical hormone receptor status in breast carcinomas. Pathol Int 53, 693699.
Hendricks, J.B., Rainer, R. & Munakata, S. (1995). Computer-assisted and visual methods of assessing cellular proliferation in tissue sections from non-hodgkin's lymphoma. Anal Quant Cytol Histol 17, 383388.
Huang, X., Chen, S. & Tietz, E.I. (1996). Immunocytochemical detection of regional protein changes in rat brain sections using computer-assisted image analysis. J Histochem Cytochem 44, 981987.
Inoué, S. & Spring, K.R. (1997). Video Microscopy. The Fundamentals, 2nd ed. New York: Plenum Press.
Jacobs, J.J.L., Lehé, C., Cammans, K.D.A., Yoneda, K., Das, P.K. & Elliott, G.R. (2001). An automated method for the quantification of immunostained human Langerhans cells. J Immunol Methods 247, 7382.
Jalava, P., Kuopio, T., Juntti-Patinen, L., Kotkansalo, T., Kronqvist, P. & Collan, Y. (2006). Ki67 immunohistochemistry: A valuable marker in prognostication but with a risk of misclassification: Proliferation subgroups formed based on ki67 immunoreactivity and standardized mitotic index. Histopathol 48, 674682.
Karlsson, M.G., Davidsson, Å. & Hellquist, H.B. (1994). Quantitative computerized image analysis of immunostained lymphocites. A methodological approach. Path Res Pract 190, 799807.
Katayama, A., Bandoh, N., Kishibe, K., Takahara, M., Ogino, T., Nonaka, S. & Harabuchi, Y. (2004). Expressions of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive indicators for tumor metastases and prognosis. Clin Can Res 10, 634640.
Kennedy, J.C., El-Badawy, N., Derose, P.B. & Cohen, C. (1992). Comparison of cell proliferation in breast carcinoma using image analysis (Ki-67) and flow cytometric systems. Anal Quant Cytol Histol 14, 304311.
Kinoshita, Y., Inoue, S., Honma, Y. & Shimura, K. (1992). Diagnostic significance of nuclear DNA content and nuclear area in oral hyperplasia, dysplasia, and carcinoma. J Oral Maxillofac Surg 50, 728733.
Kirkegaard, T., Edwards, J., Tovey, S., McGlynn, L.M., Krishna, S.N., Mukherjee, R., Tam, L., Munro, A.F., Dunne, B. & Bartlett, J.M.S. (2006). Observer variation in immunohistochemical analysis of protein expression, time for a change? Histopathol 48, 787794.
Kokolakis, G., Panagis, L., Stathopoulos, E., Giannikaki, E., Tosca, A. & Krüger-Krasagakis, S. (2008). From the protein to the graph: How to quantify immunohistochemistry staining of the skin using digital imaging. J Immunol Methods 331, 140146.
Kraan, M.C., Haringman, J.J., Ahern, M.J., Breedveld, F.C., Smith, M.D. & Tak, P.P. (2000). Quantification of the cell infiltrate in synovial tissue by digital image analysis. Rheumatology 39, 4349.
Kraan, M.C., Smith, M.D., Weedon, H., Ahern, M.J., Breedveld, F.C. & Tak, P.P. (2001). Measurement of cytokine and adhesion molecule expression in synovial tissue by digital image analysis. Ann Rheum Dis 60, 296298.
Kuropkat, C., Venkatesan, T.K., Caldarelli, D.D., Panje, W.R., Hutchinson, J., Preisler, H.D., Coon, J.S. & Werner, J.A. (2002). Abnormalities of molecular regulators of proliferation and apoptosis in carcinoma of the oral cavity and oropharynx. Auris Nasus Larynx 29, 165174.
Law, A.K.W., Lam, K.Y., Lam, F.K., Wong, T.K.W., Poon, J.L.S. & Chan, F.H.Y. (2003). Image analysis system for assessment of immunohistochemically stained proliferative marker (MIB-1) in esophageal squamous cell carcinoma. Comput Methods Programs Biomed 70, 3745.
Layfield, L.J., Saria, E.A., Conlon, D.H. & Kerns, B.-J. M. (1996). Estrogen and progesterone receptor status determined by the ventana ES320 automated immunohistochemical stainer and the CAS 200 Image analyser in 236 early-stage breast carcinomas: Prognostic significance. J Surg Oncol 61, 177184.
Lehr, H.-A., Mankoff, D.A., Corwin, D., Santeusanio, G. & Gown, A.M. (1997). Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem 45, 15591565.
Lehr, H.-A., Van Der Loos, C.M., Teeling, P. & Gown, A.M. (1999). Complete chromogen separation and analysis in double immunohistochemical stains using photoshop-based image analysis. J Histochem Cytochem 47, 119125.
Leong, A.S.-Y. (2004). Quantitation in immunohistology: Fact or fiction? A discussion of variables that influence results. Appl Immunohistochem Mol Morphol 12, 17.
Mao, K.Z., Zhao, P. & Tan, P.-H. (2006). Supervised learning-based cell image segmentation for P53 immunohistochemistry. IEEE Trans Biomed Eng 53, 11531163.
Marioni, G., Blandamura, S., Giacomelli, L., Calgaro, N., Segato, P., Leo, G., Fischetto, D., Staffieri, A. & De Filippis, C. (2005). Nuclear expression of maspin is associated with a lower recurrence rate and a longer DISBEase-free interval after surgery for squamous cell carcinoma of the larynx. Histopathology 46, 576582.
Maudelonde, T., Brouillet, J.P., Roger, P., Giraudier, V., Pages, A. & Rochefort, H. (1992). Immunostaining of cathepsin D in breast cancer: Quantification by computerised image analysis and correlation with cytosolic assay. Eur J Cancer 28A, 16861691.
Molenaar, W.M., Plaat, B.E.C., Berends, E.R. & Te Meerman, G.J. (2000). Observer reliability in assessment of mitotic activity and MIB-1–determined proliferation rate in pediatric sarcomas. Ann Diagn Pathol 4, 228235.
Murray, T.J., Fowler, P.A., Abramovich, D.R., Haites, N. & Lea, R.G. (2000). Human fetal testis: Second trimester proliferative and steroidogenic capacities. J Clin Endocrinol Metab 85, 48124817.
Nagler, R.M., Kerner, H., Laufer, D., Ben-Eliezer, S., Minkov, I. & Ben-Itzhak, O. (2002). Squamous cell carcinoma of the tongue: The prevalence and prognostic roles of p53, Bcl-2, c-erbB-2 and apoptotic rate as related to clinical and pathological characteristics in a retrospective study. Cancer Lett 186, 137150.
Obenauer-Kutner, L.J., Halperin, R., Ihnat, P.M., Tully, C.P., Bordens, R.W. & Grace, M.J. (2005). Use of an automated image processing program to quantify recombinant adenovirus particles. Microsc Microanal 11, 3741.
Ornberg, R.L., Woerner, B.M. & Edwards, D.A. (1999). Analysis of stained objects in histological sections by spectral imaging and differential absorption. J Histochem Cytochem 47, 13071313.
Ostrowski, M.L., Chakraborty, S., Laucirica, R., Brown, R.W. & Greenberg, S.D. (1995). Quantitative image analysis of MIB-1 immunoreactivity. A comparison with flow cytometric assessment of proliferative activity in invasive carcinoma of the breast. Anal Quant Cytol Histol 17, 1524.
Persohn, E., Seewald, W., Bauer, J. & Schreiber, J. (2007). Cell proliferation measurement in cecum and colon of rats using scanned images and fully automated image analysis: Validation of method. Exp Toxicol Pathol 58, 411418.
Pham, N.A., Morrison, A., Schwock, J., Aviel-Ronen, S., Iakovlev, V., Tsao, M.S., Ho, J. & Hedley, D.W. (2007). Quantitative image analysis of immunohistochemical stains using a CMYK color model. Diagn Pathol 2, 8.
Ruifrok, A.C. (1997). Quantification of immunohistochemical staining by color translation and automated thresholding. Analyt Quant Cytol Histol 19, 107113.
Russ, J.C. (1990). Computer-Assisted Microscopy. The Measurement and Analysis of Images. New York: Plenum Press.
Sarker, S.K. & Patel, K.S. (1997). Mean nuclear and chromossomal DNA content of squamous cell carcinomas of the oral cavity using computerized image analysis. J Laryngol Otol 111, 141144.
Sarker, S.K. & Patel, K.S. (2001). Mean nuclear area of squamous cell carcinomas of the head and neck using image cytometry. Anal Quant Cytol Histol 23, 413417.
Schwartz, B.R., Pinkus, G., Bacus, S., Toder, M. & Weinberg, D.S. (1989). Cell proliferation in NH lymphomas. Digital image analysis of Ki-67 antibody staining. Am J Pathol 134, 327336.
Sekine, J., Uehara, M., Hideshima, K., Irie, A. & Inokuchi, T. (2003). Predictability of lymph node metastases by preoperative nuclear morphometry in squamous cell carcinoma of the tongue. Cancer Detect Prev 27, 427433.
Stute, P., Wood, C.E., Kaplan, J.R. & Cline, J.M. (2004). Cyclic changes in the mammary gland of cynomolgus macaques. Fertil Steril 82(s3), 11601170.
Taylor, C.R. & Levenson, R.M. (2006). Quantification of immunohistochemistry—Issues concerning methods, utility and semiquantitative assessment II. Histopathol 49, 411424.
Tumuluri, V., Thomas, G.A. & Fraser, I.S. (2002). Analysis of the Ki-67 antigen at the invasive tumour front of human oral squamous cell carcinoma. J Oral Pathol Med 31, 598604.
Walker, R.A. (2006). Quantification of immunohistochemistry—Issues concerning methods, utility and semiquantitative assessment I. Histopathol 49, 406410.
Walsh, C.T., Wei, Y., Wientjes, M.G. & Au, J.L.S. (2008). Quantitative image analysis of intra-tumoral bFGF level as a molecular marker of paclitaxel resistance. J Trans Med 6, 4.
Wu, C., Zhao, W., Lin, B. & Ginsberg, M.D. (2005). Semi-automated image processing system for micro- to macro-scale analysis of immunohistopathology: Application to ischemic brain tissue. Comput Methods Programs Biomed 78, 7586.
Xu, Y.H., Sattler, G.L., Edwards, H. & Pitot, H.C. (2000). Nuclear-labeling index analysis (NLIA), a software package used to perform accurate automation of cell nuclear-labeling index analysis on immunohistochemically stained rat liver samples. Comput Methods Programs Biomed 63, 5570.
Zhang, K., Prichard, J.W., Yoder, S., De, J. & Lin, F. (2007). Utility of SKP2 and MIB-1 in grading follicular lymphoma using quantitative imaging analysis. Hum Pathol 38, 878882.
Zhou, R., Hammond, E.H. & Parker, D.L. (1996). A multiple wavelenght algorithm in color image analysis and its applications in stain decomposition in microscopy images. Med Phys 23, 19771986.


Type Description Title
Supplementary materials

Bernardo Supplementary Material

 PDF (295 KB)
295 KB


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed