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Immunophenotyping Lymphomas

IMMUNOPHENOTYPING refers to the technique of identifying molecules that are associated with lymphoma cells and that help to characterize them. The molecules are analyzable because in most cases they are expressed on the outer cell surface membrane. The molecules are characterized by using special antibodies that bind to them specifically . In this context these molecules are called "antigens," and the specific part of the molecule to which the antibody attaches is called the "epitope".

Cell surface antigens

The antibodies themselves have been manipulated so that they bear artificial reporter moieties. The technique is called "immunophenotyping" for 2 reasons: 1) It is dependent on the activity of antibodies, which are immunological substances 2) It is used chiefly to identify lymphoid and hematopoietic cells, which are part of the immune system.

How It Works:

  1. A solution of antibodies (called serum) is poured over the lymphoma cells, which may be in liquid suspension or may be in a very thin section of tissue on a slide.
  2. If the lymphoma cells bear the molecules (antigens) for which the antibodies are specific, the antibodies bind to the cells.
  3. The cells are washed to prevent non-specific binding; the specific antibodies remain bound.
  4. The cells with the bound antibodies are manipulated so that the reporter moiety attached to the antibodies declares its presence. This can be done several ways:
    • Immunofluorescence: The reporter molecule may fluoresce when exposed to light of the appropriate frequency. If the specimen is a tissue section, it can be examined under a fluoresent microscope, and cells with bound antibodies will glow in the dark (like Halloween).
    • Flow cytometry: Flow cytometer
      If the specimen is cells suspended in liquid, the cells can be run through a flow cytometer. This will expose cells in a stream of liquid one at a time to a beam of laser light. If they bear the right bound antibodies, they will fluoresce and the cytometer will record the signal.
    • Immunohistochemistry: Sometimes cells in a tissue section are identified with antibodies that have attached enzymes (usually horse radish peroxidase or alkaline phosphatase). When the tissue section is exposed to an appropriate chromogenic substrate, the enzyme on the bound antibodies will cause the substrate to change color and precipitate on the cells.

How the Antibodies Are Made:
  • Animals (mice, rabbits, goats) are immunized with the antigen for which a corresponding antibody is desired. Serum from the immunized animals is collected and purified. The antibodies thus procured are called "polyclonal antibodies". Because they are naturally made, they consist of many different kinds of antibodies with varying degrees of specificity for the antigen.
  • Alternatively, through the miracles of modern science (invent anything this insanely great, and your mailbox will contain a Nobel Prize), individual B-cells that secrete the desired antibody can be isolated from immunized animals. The selected cell is then fused with a neoplastic plasma cell that also has a natural propensity to make antibodies. This fused hybrid, called a "hybridoma" cell, secretes only one kind of antibody, without the variation seen in the first method above. This hybridoma cell is propagated to produce quantities of serum rich in a "monoclonal antibody".

What Good Is Immunophenotyping:Immunoglobulin

Immunophenotyping is used clinically chiefly to diagnose and categorize lymphomas and leukemias. Immunophenotyping is helpful because in many instances different kinds of benign and malignant lymphoid cells resemble each other in routinely stained tissue sections and smears. For example the small cells of small lymphocytic lymphoma are dead-ringers for benign small lymphocytes. (Can you tell them apart?) When lymphoma is suspected, the technique helps to distinguish between malignant and benign lymphoid proliferations, between B- and T-cell processes, and between sub-categories of B- and T-cell lymphomas (see this technique illustrated in a normal node).

  • It is especially useful in the case of B-cell lymphomas that express surface immunoglobulin (and most of them do). Immunoglobulin molecules contain a light chain and a heavy chain. In a random, benign collection of lymphoid cells, the kappa light chains are present on roughly 2/3rds of the cells and lambda light chains on 1/3rd. If you applied an antibody to kappa light chains, it would mark 2/3rds of the cells; an antibody to lambda would mark 1/3rd. On the other hand, a malignant collection of lymphoid cells is monoclonal, at least at first approximation. By definition, then, all the cells will bear identical surface immunoglobulin molecules with either kappa or lambda light chains, but not a mixture. Thus an antibody to a specific kind of light chain (kappa or lambda) will mark either all or none of the cells.
    Light chain staining

    The image above shows immunoperoxidase light chain staining, where each of 2 frames represents the same tissue stained with antibodies to different light chains. All the cells stained by lambda antibody are positive (deep orange).
    Light chains flow

    Another illustration shows the equivalent study done by flow cytometry. The cloud of positive events in the lambda frame has been shifted significantly left (positively) compared with the kappa frame.
  • Antibodies can also distinguish between B- and T-cells because these cells bear distinct kinds of surface molecules.
  • Furthermore, because specific subtypes of lymphomas are at least partly distinguished by their surface molecules, antibodies to these molecules can help tell them apart.

Cluster Designation Numbers:

Researchers in many scattered laboratories painstakingly identified many lymphoid surface antigens and developed antibodies to them. Every so often, in world congresses, it would become apparent that antibodies originating from different labs and bearing different names were marking the same antigen/molecule. At that point, the antigen would be assigned a cluster designation, or CD number, meaning that a known cluster of antibodies were binding to this known antigen. Any of these antibodies might be referred to by one of several idiosyncratic laboratory names or by the CD number of the antigen. For example, antibodies that recognize CD20, a characteristic B-cell molecule, might be called alternatively L26, B1, or, customarily, CD20. This is very confusing and is one reason why hematopathologists keep lots of aspirin in their desks and think their salaries should be huge. Nonetheless, if you study lymphomas, you will have to know at least the basics of this scheme. Here are the basics:

  • All lymphoid cells (well, almost all): Lymphoid cells are reactive for CD45 (leukocyte common antigen, or LCA). Immature cells and plasma cells in particular, however, may be negative.
  • B-cells: Almost all of these are reactive for CD19, CD20 and CD22. Certain low-grade B-cell lymphomas are reactive for two markers otherwise usually found on T-cells: CD5 and CD43. Follicular center cell lymphomas (as well as very different fish, lymphoblastic lymphomas and Burkitt lymphomas) are frequently CD10(+).
  • T-cells: Pan T-cell markers (present on almost all T-cells) include CD2, CD3, CD5, and CD7. Most T-cells mark with either CD4 (helper cells) or CD8 (suppressor cells or cytotoxic cells). Immature thymus T-cells may not conform to these generalities.
  • Natural-killer cells: These tough guys are frequently associated with CD16, CD56, or CD57.
There is a nice table of CD antibodies at Washington State University.

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