A patient is being assessed for transplant. He has a panel-reactive antibody (PRA) of 30% by the complement-dependent cytotoxicity (CDC) technique but 0% for Class I and II antigens by flow cytometry (Luminex).
How can you explain the difference between these tests?
Panel reactive antibody refers to the percentage of a panel of antigens representative of the diversity in a given population to which a recipient has antibodies. The goal of the test is to identify anti-HLA antibodies, which can cause hyperacute or acute antibody-mediated rejection. There are different techniques by which these antibodies can be detected.
The complement-dependent cytotoxicity (CDC) assay uses lymphocytes from a variety of individuals. The recipient’s serum and complement is mixed together with the lymphocytes. If the recipient’s serum contains antibodies against the lymphocytes of a particular sample, then those lymphocytes will be killed. For example, if the cells in 20 out of 50 samples are killed, then the PRA is 40%. Advantages of the CDC test are its simplicity, and its ability to detect antibodies that activate complement, which would likely be injurious to graft. Disadvantages include its lower sensitivity to detect antibodies; lack of specificity to identify which anti-HLA antibodies are present; and inability to distinguish between HLA and non-HLA (non-pathogenic) antibodies, IgG and IgM (non-pathogenic) antibodies, or between class I and II anti-HLA antibodies.
The other method to detect antibody is by flow cytometry. Recipient serum is mixed with beads coated with HLA antigens. Anti-HLA antibodies will bind only to beads that contain the antigen to which they have specificity. A fluorescent-conjugated anti-human globulin is then added and binds to the anti-HLA antibodies bound to the beads. The beads are then passed through a Coulter counter, which measures the degree of fluorescence as a measure of the amount of antibody present. This technique is more sensitive than the CDC test; does not require complement activation; will not give false-positive results with non-HLA or IgM antibodies; can distinguish between class I and II anti-HLA antibodies; and can allow for very precise identification of specific antibodies.
In this case, the cause of the 30% PRA by CDC is either non-HLA antibodies or anti-HLA IgM antibodies, neither of which would be detected by flow cytometry, and neither of which are believed to be of clinical significance.
