Introduction to Flow Cytometric AnalysisFrom $1The following is an introduction to the principles of flow cytometric analysis of hematolymphoid neoplasia. We have several normal cases posted that you can download and view using the FCS Express Reader. These cases contain a detailed explanation about each tube and what are the key features to look for on each tube. This page will just provide a general overview of the principles and thought process behind an analysis. A typical sample is usually stained with between 10-30 monoclonal antibodies or CD markers. Depending on the capabilties of the instrument (i.e. how many different wavelengths the instrument can acquire simultaneously) this will usually be divided to between 3-10 tubes per sample. 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. 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. This information is invaluable for selecting therapy and for determining prognosis and can help in the detection of relapse or of secondary leukemia. 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. 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. HLA-DR is not specific to B cells.
The antibodies are generally grouped in lineage specific groupings. A common anchor antibody, typically CD45, is present throughout all of the tubes. A typical panel looks as follows:
Above is an example panel used for peripheral blood and bone marrow samples. The antibodies and combinations were selected based on the preference of one particular laboratory. There is currently no standard recommended set of marker combinations. 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. Initial GatingCD45 is a very important anchor marker because, in conjuction with Side Scatter (or Forward Scatter), it allows one to identify most of the important populations on a single plot. By placing CD45 in each tube, one can make consistent gates for the different cell populations across all samples. a. Example peripheral blood (or bone marrow) CD45 gating dot plot.
b. Example fresh tissue CD45 gating dot plot.
Once the populations are identifed and "painted" specific colors, further analysis tools can be used as described below. Forward Scatter vs Fluorescence:Displaying 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.
Gated Dual Parameter Dot Plots:Once a population of interest is identified and painted, a more specific review of the marker expression on the selected population can be done. Dual parameter dot plots are gated population where two different markers are paired and assessed together. Mutually exclusive marking is where a population only expresses one marker and not the other. Co-expression is where a population expresses both markers. Non-expression is when the population of interest does not express either population.
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. For example, B-CLL/SLL and hairy cell leukemia (HC) are characterized by co-expression of CD5/CD19 and CD25/CD103, respectively. Fluorescence IntensityThe fluorescent staining intensity is determined by comparison of the negative control and the staining of each antibody. Negative staining is defined by fluorescent intensity similar to that of a negative control (generally in the first decade of fluorescence). The staining is positive when the fluorescence of any given marker is greater than that of the negative control (internal negative or isotype control). In general, positive staining can be categorized as dim, moderate, bright or variable. Dim staining is defined as slightly increased when compared to the negative control. Moderate staining is defined as at least one log decade brighter than the negative control. Bright staining is defined as at least two log decades brighter than the negative control. (See examples below)
In some neoplasia, fluorescent intensity may be different from that of the normal/benign counterpart. This comparison is a helpful tool in identifying and characterizing a malignancy. One example is that normal B lymphocytes express CD20 with moderate fluorescent intensity; however, in many cases of B-CLL/SLL, CD20 is very dim. (See below)
Aberrant Expression of Antigens:Benign B-cells express the following markers (e.g. CD19, CD20, CD22, CD79a) and negative for T-cell markers (CD2, CD3, CD5, CD7) and conversley, normal T-cells are positive for T cell antigens and negative for B cell markers. Other cell lineages have characteristic immunophenotypic profiles. Positive expression of a marker(s) not associated with a specific lineage, or absence of an antigen which is normally positive, is termed an aberrant antigen expression. Presence of aberrancy not only helps identify a neoplastic process, but is helpful in case followup to identify cells for minimal residual disease (MRD). Below is a table that shows aberrant antigen expression in B cell lymphomas:
Below is a table that shows aberrant expression of pan-T cell antigens in non-T cell disorders:
*****The MOST important step in flow cytometric analysis is becoming VERY FAMILIAR with the patterns and markings of NORMAL samples, so you can recognize and identify ABNORMAL. ***** Sample Normal CasesClick here for instructions on how to download the free FCS Express Reader to view and manipulate the sample cases.
Tags:
 Files (4)
Images (18) Comments (2)Viewing 2 of 2 comments: view all #1 shabs says: very good introduction to the immunophenotyping feild. Good job Posted 11:18, 15 Apr 2008 #2 KANCHANJ says: This site indeed acts as a good teaching tool to the new users of phenotyping of leukemia's Posted 18:39, 7 Sep 2008 Viewing 2 of 2 comments: view all You must login to post a comment. Page last modified 23:58, 21 Jun 2008 by Teri
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
