Once you are aware of all the factors involved in developing the methodology, then you can analyse your samples using a variety of tools and techniques to give unbiased and accurate results. The CE within your sample should be below 15%. Of course, you’ll need to determine how much needs to be counted to get a good estimate- this is called the ‘coefficient of error’ (CE). The presence of clear structural boundaries also needs to be taken into account. The entire structure must be included in the available sections, be uniformly stained and with a tissue thickness of typically >20 µm. Fractionated SamplingĪ key component to conducting an unbiased study is to randomly choose the population to provide testing samples, also referred to as ‘fractionated sampling’. Discount those cells which fall under the lines of the top and right hand side. For example- count any cells upon which the bottom and left hand side lines of the counting frame falls. If the lines of the frame fall upon a cell, then you should establish counting criteria which you should use for all of your analysis. If cells are within the frame, then they are counted. An unbiased counting frame is widely used in many stereology programs and the counting rules are established within this frame. In particular, the ‘optical dissector’ method can be used for counting cells in thick sections of tissue.Įach cell of interest should have a unique point visible in order to be counted, however, cell shape, size, orientation or distribution should not be a factor. Using certain principles, it’s possible to make efficient and accurate estimates of the number of cells in a well defined biological structure. It is also important to avoid using any sample if it does not meet pre-established criteria. To establish a bias-free environment for stereology testing, there must be complete access to the material in question. To attain this goal, a researcher must perform unbiased analysis. When conducting any test (not just stereology) we should always be aiming for the most accurate results. For example, this can range from the percentage of connective tissue within a tumour to the size range of blood vessels within a muscle biopsy. Naturally, being able to identify and distinguish cells of interest from other cell types is of primary importance! After establishing these basics, a hypothesis can be developed and your theory tested. Methodologyīefore setting out to do stereology, the researcher needs to acquire data such as the length, surface area, and volume (which is usually done within the stereology program you’ll be using).Īdditionally, it is necessary to set certain criteria for the sample so that the most specific and accurate data can be acquired. Stereology provides quantitative data and tends to provide unbiased results, but as with all types of scientific research, biases and inaccuracies do occur, the primary reason for repeating any study. It involves many variables such as the ‘dissector principle’, ‘physical dissector, variation analyses and ‘guard volume’. Stereology can be used to estimate cell population, determine cell size, regional volume, area fraction and surface area. If that all sounds a bit too much like materials science, then for us microscopists, it’s really about the review of three-dimensional objects (mainly tissues) by making horizontal and vertical incisions. According to the International Society for Stereology, the area of scientific study encompassed by this term is that which analyzes solids.
0 kommentar(er)
