Immunophenotyping

All these CD numbers are confusing. It has nothing to do with compact discs. CD stands for clusters of differentiation.

In the 1980s laboratories began to make monoclonal antibodies against blood cells. At that time nobody had any idea how complex was the surface of the lymphocyte. It soon became clear that some antibodies reacted with some lymphocytes and some reacted with others. It was very confusing.

We had known about T and B lymphocytes since the early 1970s. B lymphocytes had immunoglobulin molecules on their surface. T lymphocytes bound tightly to sheep red blood cells. Why anybody should have mixed lymphocytes with sheep blood cells seems a mystery now, but at the time a small industry came into being mixing blood cells from various types of animals with human lymphocytes. Scientists were raiding zoos for the most exotic species. I remember that we kept an ox at a local farm to provide us with red cells for certain experiments. CLL was strange; it was certainly a B cell since it had immunoglobulin on its surface (though only about 10% as much as other B cells) but it also reacted with red blood cells, not from the sheep but from the mouse.

To solve the mystery workshops were held where people with monoclonal antibodies took them along and reacted them with various populations of white cells. Antibodies that seemed to have similar reactivity were placed in a particular "cluster of differentiation" and given a CD number. Where there were not enough different antibodies of a particular type, a provisional "workshop" number was given. Thus the antibody called B1 by the Boston workers became CD20 because there were several similar antibodies. There were several anti-B cell antibodies recognised at that time and they were given successive numbers: CD19, CD20, CD21, CD22, CD23 and CD24. In the same way the earliest anti-T cell antibodies were given the numbers CD1 through to CD8. Campath was unique; it was given the number CDw52 and only later was it allowed to drop the w.

The eighth workshop was held this year and we are now up to 239 CD numbers. Some have suggested that there may be as many as 5000 different proteins on the surface of lymphocytes so who knows where the process will end?

Immunophenotyping is the process by which a cell can be identified by listing the different antibodies that react with it. It used to be done by fluorescence microscopy. A cell was reacted with an antibody that had been labelled with a fluorescent dye. Under the microscope the dye was excited with ultra-violet light to make it glow bright green, and so label the cells that it reacted with. These days it is all done much more quickly, accurately, sensitively and quantitatively with a flow cytometer.

It is not necessary to list all the antibodies that a type of cell reacts with, only the ones that give it a distinct pattern. For CLL the pattern is CD5, CD19, CD20, CD23 positive, weakly positive for surface immunoglobulin and CD79b, and negative for FMC7.

CD5 is expressed on all mature T cells, but not normally on B cells. Its presence on CLL cells is therefore unexpected. It is also present on the cells of mantle cell lymphoma and these two conditions are frequently confused. I shall dicuss this problem in a later blog. There is also a population of normal CD5 positive B cells. In the mouse these live in the peritoneal cavity and are rather special. They are known as B1 cells. In the human CD5 positive cells are present in the follicular mantle of the lymph node and in cord blood. In tissue culture CD5 negative B cells can be induced to express CD5 after certain types of activation. The question of whether CLL is a tumor of B1 cells or whether CD5 is simply an activation marker on CLL is controversial. I will write about it on another occasion.

CD19 is a marker present on all B cells and in some circumstances is involved in signaling through the surface immunoglobulin.

CD20 is present only on B cells. On CLL cells it is more weakly expressed than on other B cells. It is now clear that FMC7 recognises part of the CD20 molecule, but this part is hidden by lipid in most CLL cells so that FMC7 is usually negative in CLL and the CD20 is weaker than normal.

CD23 is one of the receptors for IgE. It is usually undetectable in B cells except for CLL, but it is also present on monocytes and eosinophils.

CD79b is one of the immunoglobulin associated molecules. It is through this molecule that stimulation of the surgace immunoglobulin sends a signal to the inside of the cell.

Immunophenotyping has enabled us to recognise CLL cells when there are only a few present. It has therefore reduced the threshold for diagnosis of CLL from 15,000 lymphocytes to 5000. It has helped us to distinguish CLL from several imposters including splenic lymphoma with villous lymphocytes, mantle cell lymphoma and marginal zone lymphoma.