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Introduction to Flow Cytometric Analysis

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The 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.

  ./b cell.jpg

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: 

Tube Antibody combinations Purpose
 1 CD5/CD19/CD45/CD10 Idenify and characterize B cell malignancy
 2 kappa/lambda/CD45/CD19+CD20 Identify B cell clonality
 3 FMC7/CD23/CD45/CD20 Characterize B cell malignancy
 4 CD7/CD56/CD45/CD3 Identify T cell malignancy
 5 CD8/CD38/CD45/CD4 Identify T cell and plasma cell malignancy
 6 CD15/CD117/CD45/CD34

myeloid differentiation 

 7 CD16/CD13/CD45/HLA-Dr

 myeloid differentiation

 8 CD11b/Mo2/CD45/CD64 myeloid differentiation
  9 CD45/PI DNA ploidy and S-phase fraction

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. 

The precise makeup of a panel is usually lab specific, and can depend on many factors. There are generally two approaches:

1. Shotgun approach - this approach involves using a large number of antibodies (generally 25-35) for each patient. The antibodies chosen generally allow you to diagnose most disorders with a minimum number of extra tubes. 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. Most large labs (>70 cases per day) use this approach.

2. Screening approach - this involves using a minimal set of antibodies (generally 10-20) for each patient. In the event an abnormality is detected, the sample is generally stained with a subsequent set of antibodies that are targeted towards the abnormality. This approach generally uses less antibodies than the shotgun approach but can be more time consuming.

Initial Gating

CD45 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. 

./FS SSC.jpg ./CD45 SSC gating.jpg
FS vs SSC is used initially to remove "debris" and non-cellular elements.  CD45 vs SSC on the "real cells" (non-debris) is used to identify the cellular elements based on which region they appear.

b. Example fresh tissue CD45 gating dot plot.

./FS SSC 2.jpg ./CD45 FS 2.jpg
 A FS vs SSC display is used initially to remove (gate out) "debris" and non-cellular elements.  CD45 vs FS on the "real cells" is then used to categorize the cellular elements based on which region (large or small) they appear.

 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.

./FS5.jpg ./FS19.jpg ./FS10.jpg

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.  

./Dualmutex.jpg ./Dualcoex.jpg ./DUAL 34117.jpg 
Mutual exclusive markers - CD19 and CD5 stain either one population on another, but never both. Co-expression - there are many cells that are both CD3+ and CD7+. It is said that CD3 and CD7 co-express on T Lymphocytes Nonexpression - The gated lymphocytes do not express CD34 or CD117.

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 Intensity

The 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)

./CD45 SSC NEG.jpg ./CD45 SSC MOD.jpg
./CD45 SSC DIM.jpg ./CD45 SSC BRIGHT.jpg

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)

                    

./CD20benign.jpg            ./CLL20dim.jpg
    This is an example of FS vs CD20 gated on benign lymphocytes. CD20 has moderate fluorescent intensity.       This is an example of FS vs CD20 gated on CLL lymphocytes. CD20 has dim fluorescent intensity.

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:

 CD5-negative MCL   11%
 CD23-negative B-CLL/SLL   4%
 CD10-positive HCL   12%
 CD19-positive PCD (myeloma)  2%
 CD56-positive DLBCL   <1%
 CD10-negative FL   6%

  Below is a table that shows aberrant expression of pan-T cell antigens in non-T cell disorders: 

 CD2  APL, blastic NK-Leukemia/lymphoma, mast cell disease
 CD3  Primary effusion lymphoma
 CD5  B-CLL/SLL, MCL (denovo), DLBCL, thyoma/thymic carcinoma
 CD7  AML, APL, blastic NK-Leukemia/lymphoma, monocytic leukemia, AML with NK differentiation.

*****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 Cases

Click here for instructions on how to download the free FCS Express Reader to view and manipulate the sample cases.

Case Name
(click on case name to open)
Comments  Size
Normal LN A typical (normal) sample from a lymph node 1.5 Mb
Normal PB-1 A typical (normal) sample from peripheral blood 3.75 Mb
Normal PB-2  A typical (normal) sample from peripheral blood 5 Mb
case 03  Normal sample with polyclonal B  and minimal T lymphocytosis
This case was kindly provided by the ASCP Press. It is part of Flow Cytometry in Clinical Diagnosis by John Carey, Phil McCoy and David Keren.
1527 kB  
UTMC Normal PB  Normal peripheral blood processed by UTMC for comparison with their wiki cases 4.9 MB
Tags:
FileSizeDateAttached by 
 Normal LN-1.fey
A normal sample from a lymph node
1541.92 kB19:41, 9 Apr 2008David NovoActions
 Normal PB UTMC (1).fey
Normal peripheral blood processed by UTMC.
4.88 MB00:38, 7 Apr 2009Tanya TolmachoffActions
 Normal PB-1.fey
A normal sample from peripheral blood.
3.8 MB19:41, 9 Apr 2008David NovoActions
 Normal PB-2.fey
A normal sample from peripheral blood.
4.92 MB19:42, 9 Apr 2008David NovoActions
 case_03.fey
No description
1527.05 kB23:55, 21 Jun 2008TeriActions
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Comments (5)
Viewing 5 of 5 comments: view all
very good introduction to the immunophenotyping feild. Good job
Posted 11:18, 15 Apr 2008
This site indeed acts as a good teaching tool to the new users of phenotyping of leukemia's
Posted 18:39, 7 Sep 2008
Well written and very informative, Please can i use this information in my hematology thesis with your permission and thanks a lot
Posted 19:51, 3 Feb 2009
All content on the wiki is free for non-commercial purposes. Please see http://www.clinicalflow.com/Legal/Copyright_Policy for more details.
Posted 16:53, 5 Feb 2009
good things .thank you
Posted 13:37, 12 Dec 2009
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Page last modified 22:55, 20 Nov 2009 by David Novo