Riber-Hansen R, Vainer B, Steiniche T. Digital image analysis: a review of reproducibility, stability and basic requirements for optimal results. APMIS 2012; 120: 276-89.Digital image analysis (DIA) is increasingly implemented in histopathological research to facilitate truly quantitative measurements, decrease inter-observer variation and reduce hands-on time. Originally, efforts were made to enable DIA to reproduce manually obtained results on histological slides optimized for light microscopy and the human eye. With improved technical methods and the acknowledgement that computerized readings are different from analysis by human eye, recognition has been achieved that to really empower DIA, histological slides must be optimized for the digital 'eye', with reproducible results correlating with clinical findings. In this review, we focus on the basic expectations and requirements for DIA to gain wider use in histopathological research and diagnostics. With a reference to studies that specifically compare DIA with conventional methods, this review discusses reproducibility, application of stereology-based quantitative measurements, time consumption, optimization of histological slides, regions of interest selection and recent developments in staining and imaging techniques. Key words: Image processing; computer-assisted; pathology; immunohistochemistry; reproducibility of results.Rikke Riber-Hansen, Institute of Pathology, Aarhus University Hospital, Noerrebrogade 44, DK-8000 Aarhus C, Denmark. e-mail: rikrib@rm.dk Digital image analysis (DIA) in histopathology is a process of obtaining digitalized images by a microscope connected to a video camera and a frame-grabber or a digital camera, or by whole slide scanning to conduct computer-based analyses to extract meaningful information from the images. In histopathological research, the motivation for implementing DIA has from its invention been to optimize and standardize diagnostic and prognostic processes. The fast development in technology entailed the expectation that DIA would be able to address the intra-and inter-observer variation inherent to manual pathology, to allow truly quantitative measurements to replace semi-quantitative scores and to reduce the workload for the pathologist.For these hopes to come true, some basic requirements have to be met by the DIA process, covering all steps from removal of the tissue of interest to the final assessment. These requirements include optimization and standardization of tissue fixation, sampling and staining as well as image capturing, region of interest (ROI) selection, algorithm design and output.Generally, comparing digital studies performed over a time span of more than 60 years is complicated by the often rapid technological advancement; however, the basic expectations and requirements are unchanged. In this article, we therefore review studies focused on quality