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Validation of various adaptive threshold methods of segmentation applied to follicular lymphoma digital images stained with 3,3’-Diaminobenzidine&Haematoxylin

Anna Korzynska1, Lukasz Roszkowiak1*, Carlos Lopez2, Ramon Bosch2, Lukasz Witkowski1 and Marylene Lejeune2

Author Affiliations

1 , Nalecz Institute of Biocybernetics and Biomedical Engineering, Ks. Trojdena 4 Str., Warsaw, Poland

2 , Molecular Biology and Research Section Hospital de Tortosa Verge de la Cinta, C Esplanetes 14, Tortosa, Spain

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Diagnostic Pathology 2013, 8:48  doi:10.1186/1746-1596-8-48

Published: 25 March 2013


The comparative study of the results of various segmentation methods for the digital images of the follicular lymphoma cancer tissue section is described in this paper. The sensitivity and specificity and some other parameters of the following adaptive threshold methods of segmentation: the Niblack method, the Sauvola method, the White method, the Bernsen method, the Yasuda method and the Palumbo method, are calculated. Methods are applied to three types of images constructed by extraction of the brown colour information from the artificial images synthesized based on counterpart experimentally captured images. This paper presents usefulness of the microscopic image synthesis method in evaluation as well as comparison of the image processing results. The results of thoughtful analysis of broad range of adaptive threshold methods applied to: (1) the blue channel of RGB, (2) the brown colour extracted by deconvolution and (3) the ’brown component’ extracted from RGB allows to select some pairs: method and type of image for which this method is most efficient considering various criteria e.g. accuracy and precision in area detection or accuracy in number of objects detection and so on. The comparison shows that the White, the Bernsen and the Sauvola methods results are better than the results of the rest of the methods for all types of monochromatic images. All three methods segments the immunopositive nuclei with the mean accuracy of 0.9952, 0.9942 and 0.9944 respectively, when treated totally. However the best results are achieved for monochromatic image in which intensity shows brown colour map constructed by colour deconvolution algorithm. The specificity in the cases of the Bernsen and the White methods is 1 and sensitivities are: 0.74 for White and 0.91 for Bernsen methods while the Sauvola method achieves sensitivity value of 0.74 and the specificity value of 0.99. According to Bland-Altman plot the Sauvola method selected objects are segmented without undercutting the area for true positive objects but with extra false positive objects. The Sauvola and the Bernsen methods gives complementary results what will be exploited when the new method of virtual tissue slides segmentation be develop.

Virtual Slides

The virtual slides for this article can be found here: slide 1: webcite and slide 2: webcite.

Lymphoma; Immunohistochemistry; Nuclear quantification; Pathology; Morphometry