Image analysis and stereology manual#
Image analysis and stereology professional#

Often these fields are chosen вЂњrandomlyвЂќ by the scientist, introducing substantial inherent user bias, and the chosen fields may not be representative of the tissue as a whole. Additional sampling bias comes into play when the microscopic fields of view are chosen. Furthermore, if the entire cerebral cortex is the region of interest, a single section through the brain will not be representative of that area as a whole.

It can be extremely difficult to section the brain at exactly the same location in every animal, which may impact the ability to compare between animals and across treatment groups. For example, the appearance of a small anatomical area such as the arcuate nucleus can change dramatically even in a single Bregma level 2. This is often not the case, particularly for certain tissues such as the brain.

One assumption made when tissues are sampled in such a way is that the entire organ or area of interest is homogeneous and that the section being analyzed is truly representative of the tissue as a whole. With sampling bias, some of the cells or objects of interest are less likely to be included in the sample than others. These sections are nonrandom tissue samples and they introduce sampling bias. With 2D methods, the most optimum tissue sections are often analyzed. In toxicology studies, the true population mean is most often unknown, and this stresses the importance of having unbiased data. This can include both type I error, in which an effect is detected that is not really present (a вЂњfalse positiveвЂќ) or type II error, in which an effect that is present is undetected (a вЂњfalse negativeвЂќ). Bias leads to a difference between the mean of the estimator and the true population mean. Furthermore, recent advances in technology have made stereology more approachable and efficient, allowing it to be used more readily in both investigational and safety assessment studies.ДЄlthough 2D morphometry can provide additional quantitative information about the tissue sections being examined, it also makes several assumptions about the tissue, all of which are sources of bias. The results are absolute estimates rather than densities or ratios. Stereology utilizes stringent sampling methods and calculations based on statistical theory and stochastic geometry that can be applied to virtually any tissue type from any species. Unlike histomorphometry, stereology does not make assumptions about the tissue and can supply accurate three-dimensional (3D) estimates of number, volume, surface area, or length.

Image analysis and stereology professional#

In turn, professional and regulatory societies are increasingly becoming wary of 2D data. Because of this, the data are inaccurate, often overestimating the object number or even showing trends that are in the opposite direction of the truth. Furthermore, histomorphometry makes several assumptions about the tissue sections, all of which are sources of bias. Counts or measurements performed on single thin 2D sections really have no correlation with what is present in a 3D structure. Although these methods can supply the pathologist with quantitative information, the resulting data only pertain to the histologic section being examined.

Image analysis and stereology manual#

With this technique, manual or automated analysis is performed on single or multiple tissue sections to obtain quantitative information, such as number of cells or other objects, linear measurements, or total area of positive staining (after the application of a cellular marker such as an immunohistochemical stain). For that reason, more sensitive techniques have been employed for these quantitative endpoints.ДЄ commonly utilized technique to obtain quantitative information from histologic tissue sections is two-dimensional (2D) morphometry. For example, a previous study found that a 33% reduction in total hippocampal neuron number could not be appreciated by pathologists, even when viewing side-by-side photomicrographs 1. However, the human eye lacks the sensitivity to detect subtle changes in cell or object quantity. Qualitative histopathology of tissue sections remains the gold standard for routine safety assessment studies, and pathologists play a unique and important role in identifying potential safety concerns.