Microscopy and Microanalysis

Biological Applications

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

Vagner Bernardoa1 c1, Simone Q.C. Lourençoa1, Renato Cruza1, Luiz H. Monteiro-Leala2, Licínio E. Silvaa3, Danielle R. Camisascaa1, Marcos Farinaa4 and Ulysses Linsa5

a1 Universidade Federal Fluminense, Faculdade de Medicina, Programa de Pós-graduação em Patologia, Rua Marquês do Paraná, 303 - 4o andar – sala 1, Hospital Universitário Antônio Pedro - Centro, 24033-900, Niterói, RJ, Brazil

a2 Universidade do Estado do Rio de Janeiro, Departamento de Histologia e Embriologia, Laboratório de Microscopia e Processamento de Imagens, Av. Prof. Manoel de Abreu, 48 - 3o andar - Maracanã, 20550-170, Rio de Janeiro, RJ, Brazil

a3 Universidade Federal Fluminense, Instituto de Matemática, Departamento de Estatística, Rua Mário Santos Braga s/n - 7o andar Campus do Valonguinho - Centro 24020-140, Niterói, RJ, Brazil

a4 Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Biomineralização. CCS, Bloco F, Sala F2-027, 21941-590, Rio de Janeiro, RJ, Brazil

a5 Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco I, Instituto de Microbiologia Professor Paulo de Góes, Av Carlos Chagas Filho, 373 Cidade Universitária, 21941-902, Rio de Janeiro, RJ, Brazil


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.

(Received June 26 2008)

(Accepted April 29 2009)


c1 Corresponding author. E-mail: vagnerbernardorj@gmail.com


In memoriam: This article is dedicated to Renato Cruz and Luiz Henrique Monteiro Leal, brilliant researchers who passed away during the preparation of this manuscript. Their memory lives on.