Cross Matching - Professor Richard Kirk 2023

Professor Richard Kirk
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Cross Match Techniques & cPRA
At the time of transplant donor cells and recipient plasma are sent to the HLA laboratory for compatibility testing using the CDC and Flow techniques (see below). Mulley and Kanellis have written an excellent review on this subject in Nephrology 2011;16:125-133.

Complement Dependent Cytotoxicity
Complement Dependent Cytotoxicity (CDC) is the so called "wet crossmatch" and performed in all transplants. When the donor organ arrives, recipient serum and donor cells (usually splenic) are sent to the HLA lab for the CDC crossmatch. Donor cells and recipient serum are mixed together and complement added. If there are HLA antibodies of sufficient strength in the recipient serum against the donor cells there is donor cell death. As T cells and B cells both express Class I HLA antigens if Class I antigens are present in the serum against the donor both T & B cells will lyse. If only Class II antibodies are present then only B cell lysis will occur (as T cells do not express Class II antigens). This CDC test is reported within 24 hours of the transplant and fortunately is usually negative for both T & B donor cell lysis. If it is positive, then the virtual cross match should be reviewed and additional testing undertaken as non IgG and autoantibodies may cause cell death in vitro but are considered not to cause in vivo issues.

Flow Crossmatch
This is more sensitive than the CDC crossmatch and is commonly used in renal transplant as nephrologists usually have the alternative of dialysis for their patients and can therefore have a low tolerance towards accepting organs for candidates with donor specific antibodies. The donor cells and recipient serum are added together, and then washed. Antihuman IgG antibodies tagged with a fluorescent dye are then added. If there are donor specific IgG antibodies (DSA) present in the serum then they will be attached to the donor cells, the anti human IgG antibodies tagged with a fluorescent dye will target these DSAs and be visible when light is applied. Again T & B cell reactions can be identified separately.
Virtual Cross Match
The virtual crossmatch technique for detecting HLA antibodies relies on HLA molecules coated onto beads fixed to columns. Serum (transplant candidate or recipient) is applied to the columns. If HLA antibodies are present with the same specificity as the HLA molecules on the column, they bind to the beats and can be detected using anti HLA antibodies, marked with a fluorescent dye. The degree of fluorescent (MFI or mean fluorescent index) allows identification of the class and specificity of the HLA antibody and also semi quantification of the amount. This is the principle of the single antigen test - often called the Luminex test. Lachmann et al have written a good review in Transfus Med Hemother 2013;40:182–189
Fig 2. Adapted from Transfus Med Hemother 2013;40:182–189
Panel Reactive Antibody
The Panel Reactive Antibody (PRA) test was originally undertaken using a panel of donor cells to which transplant candidate serum was applied with complement added (the complement dependent cytotoxic (CDC) method). The proportion of donor cells lysed gave the %PRA number. As this was a CDC test it not only took into account the donor HLA but also the antibody strength - weak antibodies in the serum would be incapable of causing cell lysis.

Calculated PRA (cPRA) and the Virtual Crossmatch
More recently knowledge of the HLA genotypes in the donor population allows the likelihood of a potential donor reacting to recipient HLA antibodies to be calculated (calculated PRA or cPRA). In the example above with HLA-A2 antibodies, the cPRA would be 50%. When there are multiple HLA antibodies in the transplant candidate's serum the cPRA may approach 100% meaning the chance of a compatible donor is virtually zero. It should be noted, however, that this theoretical calculation of the cPRA does not take into account the antibody strength, or the antibody class. Class I antibodies are 10 times more potent than Class II. Thus, whilst the cPRA% might be high, depending on the HLA strength and class, no reaction might occur with a particular donor. This is why it is necessary to know not only the breadth of sensitization (i.e. the cPRA%) but also the strength and class of each antibody as assessed by MFI. Furthermore whilst the information from single antigen bead testing is invaluable, it is important to recognize that it is an in vitro test and may not predict the in vivo effect of these antibodies as that depends on other factors - not least HLA expression on the donor cell surface. These complexities mean it is essential to work with a HLA specialist to assess and balance the risk to the patient of not proceeding vs proceeding with a particular donor.
*Note: Anti-CD20 depleting cytotoxic agents (Rituzimab) may cause a positive B cell CDC, flow or virtual cross-match in the absence of HLA or other non-HLA antibodies even months after application.
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