Stereology involves the quantitative assessment of objects in three dimensions. Examples of measurements that can be obtained include total cell count, cell density, cell size, tissue volume, and the length density and/or total length of linear structures such as nerve process or blood vessels. Current design-based stereological methods have evolved to provide results that are unbiased, efficiently obtained, and repeatable. A key stereological practice that is intended to reduce bias and aid both efficiency and repeatability has been termed systematic random sampling (SRS). According to this concept, selection of the initial tissue measurement site is randomized, but selection of ensuing measurement sites is regular. One of the most commonly used stereological tools for cell counting, especially in neuropathology, is the optical fractionator approach. This approach employs a virtual three-dimensional geometric probe called a disector that is used to count the number of objects (e.g., cells) in a well-defined, three-dimensional region (e.g., the hippocampal region of the brain). The results of disector counting are unaffected by the size, shape, or orientation of the objects. In anatomic pathology, the disector principle may be used to count cells in paired sets of thin histologic sections (physical disector) or at various focal plane depths in a subsampled series of thick histologic sections (optical disector). A disadvantage of the physical disector approach is the need to align the paired sections based on morphologic landmarks to ensure that a cell is never counted twice; however, the optical disector approach may not be feasible if thick sections cannot be prepared or if the cell type of interest cannot be reliably identified. The optical fractionator is a variation of the optical disector approach. The fractionator differs from earlier methods in that it is not necessary to determine the volume of the region in order to estimate the total cell number. This is because unlike prior methods, the entire region of interest is sampled. Advantages of using EPL for your Stereological projects: • EPL technicians have extensive expertise in the creation of thick frozen or paraffin-embedded sections for cell counting purposes • Immunohistochemical staining and evaluation can be performed using either chromogenic (diaminobenzidine, DAB) or fluorescent markers. • Stereology projects supervised by an ACVP board-certified pathologist. • EPL uses MBF Bioscience’s popular and robust StereoInvestigator software • Stereology can be conducted in accordance with Good Laboratory Practice guidelines as prescribed by the US FDA, US EPA, OECD, or other regulatory organizations.

1. Experimental Pathology Laboratories, Inc.
Stereology

2. Stereology Basics
Stereology is the science in which estimates of
three-dimensional measurements are obtained
using two-dimensional sampling.
There are several ways in which stereology differs from two-
dimensional image analysis:
– Stereological procedures may utilize glass slides
directly or digitized images
– Stereology provides a more accurate assessment of
the number of objects within a given three-
dimensional region of interest
– Special attention is given to procedures for
minimizing bias during specimen sampling and data
collection
– Stereology can be used to obtain measurements that
cannot be assessed using two dimensional image
analysis (e.g., the degree of blood vessel branching)
x
y

3. Two Dimensional Analysis vs. Stereology
2-D Image Analysis
– Counts
– Lengths
– Areas
– Color Intensity
Stereology
– Counts
– Lengths
– Surface areas
– Volumes
– Connectivity

4. When to Utilize Stereology
Many types of morphologic measurements are
amenable to stereological evaluation. Various examples
include:
– Estimates of neuronal cell counts within specified
brain nuclei
– Differential counts and volumes of ovarian
follicles
– Estimates of cell number, total volume, mean
volume and mean height of enterochromaffin
cells in the stomach
– Estimates of thyroid follicular colloid volumes
– Estimates of proximal tubule cell numbers and
tubular cell volume in the kidney
– Estimates of alveolar volumes in the lung

5. EPL Stereology Experience
• EPL has experience with modern design-
based stereological techniques
• For the past five years, EPL has worked with a
mouse model of Parkinson’s disease
• Projects utilize MBF’s Stereo Investigator
software
• Projects supervised by a toxicologic
pathologist, and performed in accordance with
GLP principles