A comparative study of 2 computer-assisted methods of quantifying brightfield microscopy images.
Immunohistochemistry continues to be a powerful tool for the detection of antigens. There are several commercially available software packages that allow image analysis; however, these can be complex, require relatively high level of computer skills, and can be expensive. We compared 2 commonly available software packages, Adobe Photoshop CS6 and ImageJ, in their ability to quantify percentage positive area after picrosirius red (PSR) staining and 3,3'-diaminobenzidine (DAB) staining. On analysis of DAB-stained B cells in the mouse spleen, with a biotinylated primary rat anti-mouse-B220 antibody, there was no significant difference on converting images from brightfield microscopy to binary images to measure black and white pixels using ImageJ compared with measuring a range of brown pixels with Photoshop (Student t test, P=0.243, correlation r=0.985). When analyzing mouse kidney allografts stained with PSR, Photoshop achieved a greater interquartile range while maintaining a lower 10th percentile value compared with analysis with ImageJ. A lower 10% percentile reflects that Photoshop analysis is better at analyzing tissues with low levels of positive pixels; particularly relevant for control tissues or negative controls, whereas after ImageJ analysis the same images would result in spuriously high levels of positivity. Furthermore comparing the 2 methods by Bland-Altman plot revealed that these 2 methodologies did not agree when measuring images with a higher percentage of positive staining and correlation was poor (r=0.804). We conclude that for computer-assisted analysis of images of DAB-stained tissue there is no difference between using Photoshop or ImageJ. However, for analysis of color images where differentiation into a binary pattern is not easy, such as with PSR, Photoshop is superior at identifying higher levels of positivity while maintaining differentiation of low levels of positive staining.
The University of Edinburgh/MRC Centre for Inflammation Research, The Queen's Medical Research Institute, Edinburgh, UK.
George H
GH
Tse
Lorna P
LP
Marson
D000818:Animals; D001402:B-Lymphocytes / Q000473:pathology*; D002478:Cells, Cultured; D006084:Graft Rejection / Q000175:diagnosis* / Q000209:etiology; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000276:immunology; D007090:Image Interpretation, Computer-Assisted; D007150:Immunohistochemistry; D016030:Kidney Transplantation; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008853:Microscopy / Q000379:methods*; D009154:Mutation / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software / Q000592:standards; D013154:Spleen / Q000473:pathology
D000949:Histocompatibility Antigens Class II