**5.1. Complement-Dependent Cytotoxicity (CDC) crossmatch**

A CDC crossmatch involves placing recipient serum (potentially containing donor-specific anti-HLA antibodies) onto donor lymphocytes (containing HLA antigens). A cytotoxic reaction (deemed 'positive') suggests the presence of preformed DSA.

CDC crossmatching was pioneered by Terasaki and colleagues in the 1960s [13, 17]. It identifies clinically significant donor specific HLA antibody mediated responses for a given recipient. Lymphocytes from the donor are isolated and separated into T and B cells. Serum from the recipient is mixed with the lymphocytes in a multi-well plate. Complement is then added (usually derived from rabbit serum). If donor-specific antibody is present and binds to donor cells, the complement cascade will be activated via the classical pathway resulting in lysis of the lymphocytes.

The read-out of the test is the percentage of dead cells relative to live cells as determined by microscopy. The result can thus be scored on the percentage of dead cells, with 0 correlating to no dead cells; scores of 2, 4 and 6 represent increasing levels of lysis. On this basis, a score of 2 is positive at a low level, consistent with approximately 20% lysis (generally taken as the cut-off for a positive result). A score of 8 represents all cells having lysed and indicates the strongest possible reaction. The use of a scoring system allows a semi-quantitative analysis of the strength of reaction. Another way to determine the strength of the reaction is to repeat the crossmatch using serial doubling dilutions of the recipient serum (often known as a 'titred crossmatch'). In this way, dilutions are usually performed to 1 in 2, 4, 8, 16, 32, 64 and so on. In the situation of a high titre of high avidity DSA it may be that many dilutions are required for the test to become negative (e.g. 1 in 128). With antibody at a low level or one with a low affinity, a single dilution may be enough to render the crossmatch result negative. This may also give an indication as to the likelihood that a negative crossmatch could be achieved with a desensitization protocol.

crossmatches to be positive. A negative B-cell crossmatch in the presence of a positive T-cell crossmatch therefore suggests a technical error. This is not unusual as B cells tend to be less

Transplantation Antigens and Histocompatibility Matching

http://dx.doi.org/10.5772/54738

383

Transplanting in the setting of a positive T-cell crossmatch, which is not due to an autoanti‐ body, is likely to generate a very poor outcome. Patel and Terasaki described the outcomes of 30 such transplants [17]. Twenty four (24) patients lost their grafts immediately to HAR while another three lost their grafts within 3 months. It is not clear why the other three patients had less severe reactions but it may relate to false positive crossmatches generated by autoanti‐ bodies given that dithiothreitol (DTT) which cleaves the multimers of IgM antibodies was not used in their assays. Other possibilities include false positive tests or lower immunogenicity

A recent study investigated whether IVIg or plasma exchange was more effective at desensi‐ tizing crossmatchpositive recipients so that they might be crossmatch-negative at the time of transplant [27]. While most patients were successfully desensitized there was a group of 10 patients who did not achieve a negative crossmatch but were still transplanted. Of this group 70% developed AMR with 50% losing their grafts. Given this data, even after reducing the antibody titre with a desensitization protocol before transplant, a persistent positive T-cell

B-cell CDC crossmatching is not as predictive of HAR as the T-cell CDC crossmatch and there has been much controversy about its role [28]. Many centres do not perform B-cell cross‐ matching for cadaveric renal transplantation because of uncertainty about the significance of a positive result. The major limitation is a rate of false positive results of up to 50% [29]. In some cases this reactivity may be due to non-HLA molecules on the surface of the B lympho‐ cytes, including the presence of Fc receptors. While a negative result is reassuring a positive result may mean a transplant is cancelled when it was safe to proceed. Another argument against the routine use of B-cell crossmatching is that antibodies to class II antigens are of less significance in generating antibody-mediated rejection. But recently it has been found that they

B-cell crossmatches are often performed as part of the immunologic assessment before live donor transplantation when there is more time to determine the significance of the result. Paired with information about the presence of DSA, determined by more specific means such as antigen-coated beads (Luminex, discussed below) the B-cell CDC cross‐ match results may be more meaningful [31]. If a B-cell crossmatch is positive and there are no detectable antibodies to class I or II antigens, the result may be falsely positive while a positive result in the presence of detectable DSA signifies that the identified DSA may be functionally relevant in that it can activate complement and were associat‐ ed with increased risk of rejection [32]. This has led to the suggestion that the B-cell

resilient than T cells and their viability can often be a concern in the assays.

crossmatch remains an absolute contraindication to transplantation.

**5.2. Positive T-cell CDC crossmatch**

of the antibodies or antigens in those cases.

**5.3. Positive B-cell CDC crossmatch**

are not so benign [30].

The basic CDC crossmatch can be enhanced by the addition of antihuman globulin (AHG). This technique increases the sensitivity of the CDC crossmatch as a result of multiple AHG molecules binding to each DSA attached to thedonor cells thereby amplifying the total number of Fc receptors available for interaction with complement component 1, which increases the likelihood of complement activation and cell lysis.

It is also possible to have a negative crossmatch in the presence of a DSA and this can happen in the following conditions:


A further consideration relates to variations in antibody levels in a given individual's serum samples, collected at different times. The most reactive serum is generally called the 'peak serum'. This may have been collected several years earlier, with the 'current serum' showing quite different reactivity. As an example, the peak serum may show a clear positive CDC crossmatch result, but as the antibody levels have fallen in subsequent sera, so too may the degree of cell lysis in the assay. This may render the CDC crossmatch negative. Nevertheless, the antibodies found in the peak sera may still be of relevance, indicating that re-exposure to the relevant antigen could initiate a memory response with the risk of early and aggressive rejection. For this reason, patients on transplant waiting lists have sera collected at frequent intervals; variations can be monitored and newly appearing HLA antibodies can be detected.

There are important differences in HLA expression between T and B cells, which influence the interpretation of the crossmatch. T cells do not constitutively express HLA class II so the result of a T-cell crossmatch generally reflects antibodies to HLA class I only. B cells on the other hand express both HLA class I and II, as well as a larger range of surface markers, icluding Fc receptors. Because of this, a positive B-cell crossmatch is more difficult to interpret than a positive T-cell cross match. It may be due to antibodies directed against HLA class I or II or both, or it may be due to antibody binding to other sites, that may or may not be clinically important. Hence, if the T- and B-cell crossmatches are positive the interpretation is that there may be either single or multiple HLA class I DSA/s or a mixture of HLA class I and II DSA. While a negative T-cell crossmatch in the setting of a positive B-cell crossmatch suggests either there may be one or more class II DSA/s but no class I antibodies or that there is a low-level DSA to a class I antigen with greater lysis of B cells relative to T cells. This is often due to the fact that B cells express higher levels of HLA class I than do T cells [26]. When class I comple‐ ment fixing HLA DSA are present at a significant level one would expect both the T and B-cell crossmatches to be positive. A negative B-cell crossmatch in the presence of a positive T-cell crossmatch therefore suggests a technical error. This is not unusual as B cells tend to be less resilient than T cells and their viability can often be a concern in the assays.
