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dates. This occurs when a recipient is sensitized as a consequence of receiving multiple blood transfusions, receiving a previous kidney transplant, or from pregnancy. Transplantation of a kidney into a recipient that is sensitized against donor Class I HLA antigens is at high risk to develop hyperacute antibody-mediated rejection. All transplant candidates are screened to determine the degree of humoral sensitization to HLA antigens. This is accomplished by testing the patient's serum against a reference panel of lymphocytes that express the spectrum of HLA phenotypes. This methodology is referred to as panel reactivity and is quantified as the percentage of the panel to which the patient has developed antibody. This measurement is expressed as percent panel reactivity (PRA). For each patient this value varies between 0-100% and may change over time. Patients on the cadaveric kidney waiting list have PRA determinations on a regular basis. Information is kept that defines the peak percentage PRA and current percent PRA. Patients highly sensitized will have a very high PRA level that can remain elevated for years. The implications are that the waiting time will be very long before a patient receives a kidney to which he/she is nonreactive. Organ allocation point systems take into consideration the PRA level for maximizing organs to those individuals with high PRA that are found to have a negative crossmatch with a particular donor.

d. Crossmatching. Crossmatching is an in vitro assay method that determines whether a potential transplant recipient has preformed anti-HLA Class I antibody against those of the kidney donor. This immunologic test is conducted prior to transplantation. A negative crossmatch must be obtained prior to considering accepting a kidney for transplantation. The patient's stored or fresh serum is reacted against donor T-cells from cadaveric lymph nodes, or cadaver/living donor peripheral blood. The relevant antibodies are cytotoxic IgG anti-HLA Class I. Occasionally, a recipient will have IgM activity to HLA antigen. This can be determined by a crossmatch assay in which the recipient's serum is treated with dithiothreitol (DTT) to denature the IgM and eliminate the IgM response. Platelet absorption and determination of autoantibody are other useful techniques to carefully characterize and interpret the results of a positive crossmatch. Anti-HLA Class II antibody is less important and is detected utilizing donor B cells.

The technique of crossmatching is referred to as a microlymphocytotoxicity test. It was developed in the late 60's by Terasaki and has been one of the most important advances in kidney transplantation to prevent hyperacute rejection.6 There are several methodologies. The standard test is referred to as the NIH method. Purified donor lymphocytes are incubated in recipient serum in the presence of complement. Complement reacts with bound anti-HLA antibody to kill the cell. Viability staining is performed to determine if a reaction has occurred. To increase the sensitivity of the standard crossmatch, anti-IgG globulin may be added to enhance binding of complement. Flow cytometry crossmatching is even more sensitive and has gained in popularity making it the standard procedure for most transplant programs. Binding of recipient antibody to donor T-cells is determined by detection of fluorescein-labeled monoclonal mouse anti-human antibody reactive to the anti-HLA antibody. The degree of reactivity is quantified by channel

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