![]() For example, B-CLL/SL L and hairy cell leukemia (HC) are characterized by co-expression of CD5/CD19 and CD25/CD103, respectively. The diagnosis and subclassification of hematopoetic malignacies relys not only on the distinction of positive and negative expression of the analyzed marker or fluorescence intensity, but also on the identification of the coexpression of two or more markers by the same population of cells. Nonexpression - The gated lymphocytes do not express CD34 or CD117. It is said that CD3 and CD7 co-express on T Lymphocytes Mutual exclusive markers - CD19 and CD5 stain either one population on another, but never both.Ĭo-expression - there are many cells that are both CD3+ and CD7+. Non-expression is when the population of interest does not express either population. Co-expression is where a population expresses both markers. Mutually exclusive marking is where a population only expresses one marker and not the other. Dual parameter dot plots are gated population where two different markers are paired and assessed together. Once a population of interest is identified and painted, a more specific review of the marker expression on the selected population can be done. This approach generally uses less antibodies than the shotgun approach but can be more time consuming.ĭisplaying forward scatter (FS) vs Fluorecence (FL) is a useful tool that allows one to visualize all the populations in the sample and their respective size and fluorescent intensity for each marker. In the event an abnormality is detected, the sample is generally stained with a subsequent set of antibodies that are targeted towards the abnormality. Screening approach - this involves using a minimal set of antibodies (generally 10-20) for each patient. Most large labs (>70 cases per day) use this approach.Ģ. This may seem excessive and potentially expensive, as one is using more antibodies than is generally needed, but can actually be quite efficient since every sample is prepared exactly the same way and the sample rarely needs to be re-stained. The antibodies chosen generally allow you to diagnose most disorders with a minimum number of extra tubes. Shotgun approach - this approach involves using a large number of antibodies (generally 25-35) for each patient. The precise makeup of a panel is usually lab specific, and can depend on many factors. The Bethesda Consensus does provide recommended markers for each category of case types. The lab's fresh tissue panel is identical to the above, but omits tubes 6, 7, and 8. There is currently no standard recommended set of marker combinations. The antibodies and combinations were selected based on the preference of one particular laboratory. Identify T cell and plasma cell malignancyĪbove is an example panel used for peripheral blood and bone marrow samples. Idenify and characterize B cell malignancy ![]() ![]() A common anchor antibody, typically CD45, is present throughout all of the tubes. The antibodies are generally grouped in lineage specific groupings. ![]() For instance, in the diagram below, you can see that CD19 is the earliest B specific protein expressed on B cells, but that it is lost as the activated B cell becomes a plasma cell. Most of these antibodies are against surface proteins that are not only often associated with particular cell lineages, but vary in expression with maturation, and thus are referred to as differentiation antigens. This information is invaluable for selecting therapy and for determining prognosis and can help in the detection of relapse or of secondary leukemia. The use of immunologic markers to determine the lineage and degree of differentiation of a leukemia has become an essential part of the diagnostic evaluation. Combinations of antibodies allow for 1) identification of specific cell types, 2) determination of the degree of cell differentiation, and 3) recognition of abnormal cells. how many different wavelengths the instrument can acquire simultaneously) this will usually be divided to between 3-10 tubes per sample. Depending on the capabilties of the instrument (i.e. This page will just provide a general overview of the principles and thought process behind an analysis.Ī typical sample is usually stained with between 10-30 monoclonal antibodies or CD markers. These cases contain a detailed explanation about each tube and what are the key features to look for on each tube. We have several normal cases posted that you can download and view using the FCS Express Reader. The following is an introduction to the principles of flow cytometric analysis of hematolymphoid neoplasia. ![]()
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