llustration of HLA typing: Schematic diagram of cell based and solid phase (bead based) antibody screening. ( a) Two representative wells are illustrated from the panel of cells that is used. Serum is added to each well in the panel. On the top the antibody in the serum does not bind to the cells on the bottom do nor specific antibody (DSA) does bind. Bound DSA remain after wash steps, so that when complement is added, it forms the membrane attack complex, killing the cell and allowing the vital dye is taken up by and visualized. No donor specific antibody leaves live cells (i), and when DSA are present, the vital dye identifies the dead cells (ii). ( b ) Serum is added to be adscoated with purified or recombinant HLA antigen. In this case, the antibody in the serum is only specific to the bead on the bottom. Only beads with DSA already bound will bind the secondary fluorescent anti-IgG marker. Increased fluorescence defines positive beads with DSA bound to them.

Evolution of basic crossmatching techniques (figure above). ( a) In an unenhanced complement dependent cytotoxicity crossmatch (CD), when high titer DSA is bound to the cell in suffi cient density, complement is activated, the cell is killed and the vital dye is taken up identifying the dead cells. ( b) With the AHG enhancement, lower titer antibody is less dense on the cell surface and would not naturally activate complement. Adding AHG increases the overall density of complement activating antibodies on a cell that already has some DSA bound, thereby allowing complement activation with subsequent cell death as with CDC alone. ( c) In FCXM, donor-specific antibody binds the cell and a second fluorescent antibody to human IgG is used to detect even small amounts of bound antibody. When run through a flow cytometer, the DSA (which may be complement or noncomplement binding) is measured as fluorescence on the cells.

The transplanted organ is a continuous source of HLA alloantigens that can induce a rejection response at any time after transplantation. Both direct and indirect alloactivation pathways are important in the generation of donor-specific cell- mediated cytotoxicity and delayed-type hypersensi- tivity-like mechanisms of allograft rejection. Conversely, the continuous presence of small num- bers of donor APC (referred to as microchimerism) may promote and maintain allograft tolerance, rather than transplant rejection.