This controls for the effect of diluting the level of antibodies when adding DTT to the reaction. Hence, if the crossmatch becomes negative with the addition of phosphate-buffered saline, the results with DTT cannot be fully interpreted as the result may have become negative by diluting the antibody level. Complement-dependent cytotoxicity crossmatching was

pioneered by Terasaki and colleagues in the 1960s.3,8 It seeks to identify 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 high throughput screening pathway resulting

in lysis of the lymphocytes (see Fig. 1). 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 PR-171 purchase 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 DSAb 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 PtdIns(3,4)P2 with a desensitization protocol. 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 DSAb attached to the donor 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. In Australia this assay is not routinely used. It is also possible to have a negative crossmatch in the presence of a DSAb.