Donor Matching of Kidney Transplantation

History of transplantation 1954: 1st successful kidney transplantation by Dr. Murray between identical twin 1967: 1st successful liver transplantation by Dr. Starzl in child with hepatoblastoma Late of 1970s: Cyclosporine, OKT-3 (monoclonal anti-lymphocyte antibody) Late of 1980s: FK-506, MMF, polyclonal anti-lymphocyte antibody

Important factors for transplantation ABO blood group compatibility Human Leukocyte Antigen (HLA) System Hyperacute rejection caused by the presence of preformed antibodies to donor blood group ABO or HLA antigens

ABO blood groups and antigens A, B, AB and O Antigen expression on red blood cells, lymphocytes, platelets, epithelial and endothelial cells Formation of blood group antibodies occurs against those antigens not native to the host Blood type O: antibodies to both A and B Blood type AB: no antibodies to A or B antigens

HLA HLA class I (HLA-A, -B, -C) HLA class II (DR, DP, DQ) Two major considerations regarding HLA HLA antigens would be recognized as foreign to a host, unless genetically identical -> concept of matching donor and recipient HLA antigens to avoid recipient antidonor immunity HLA antigens may serve as targets for antibody responses leading to graft destruction

HLA HLA antigens HLA class I (HLA-A, -B, -C) HLA class II (DR, DP, DQ) Central role in cellular and humoral immune responses of transplant outcome HLA class I (HLA-A, -B, -C) HLA class II (DR, DP, DQ) Two major considerations regarding HLA HLA antigens would be recognized as foreign to a host, unless genetically identical -> concept of matching donor and recipient HLA antigens to avoid recipient antidonor immunity HLA antigens may serve as targets for antibody responses leading to graft destruction

Effect of HLA-A, -B, -DR Mismatching on Kidney Graft Survival

NIH-CDC assay Donor lymphocyte Rabbit complement 5% eosin Y recipient serum IgG

HLA Typing (Serologic Test)

HLA typing - 방법 각종 항혈청(eg. anti-HLA-A2 Ab)이 분주되어 있는 microplate + 환자림프구(HLA Ag) → 보체첨가 → cell lysis로 Eosin Y 등의 염색약이 침투됨-> 현미경으로 관찰

HLA-typing (serologic test) 3)현미경 판독 위상차현미경 X 100배 시야에서 판정한다 죽은 세포의 %(음성대조 well과 비교)에 의해 1+-8+의 score를 기록한다. % Dead Cells Score Interpretation 0 ∼ 10% 1 Negative 11 ∼ 20% 2 Doubtful negative 21 ∼ 50% 4 Weak positive 51 ∼ 80% 6 Positive 81 ∼ 100% 8 Strong positive

Detection of antidonor antibodies (1) Complement-dependent lymphocyte cytotoxicity (CDC) assay Mixing patient sera with target pph blood lymphocytes Mixture to allow immunoglobulin binding Bind to antibody– antigen (cell-surface antigens) complexes Determining cell viability by vital dye

Complement-dependent Cytotoxicity NIH Assay

Complement-dependent Cytotoxicity NIH Assay

Complement-dependent Cytotoxicity NIH Assay

Detection of antidonor antibodies (2) Antihuman globulin (AHG)-CDC assay Increased the sensitivity in detecting lower titer antibodies in standard CDC negative sera Complement fixing AHG (goat antihuman light chain) added before complement

Anti-human Globulin (Enhancement) Assay

Anti-human Globulin (Enhancement) Assay

Anti-human Globulin (Enhancement) Assay

Detection of antidonor antibodies (3) Flow cytometry crossmatch (FCXM) Most sensitive antibody-detecting assay Detects donor reactive antibodies independent of complement fixation Discriminate antibodies bound to T or B cells Less subjective and more standardized than the standard CDC or AHG corssmatches

Detection of antidonor antibodies (4) ELISA PRA Microtest trays containing known HLA antigens Add potential recipient serum

Detection of antidonor antibodies (5) Flow PRA

Flow Cytometry Assay NIH - CDC Negative AHG – CDC Negative Now measuring binding of IgG (absent C’)

Interpretation (1) IgG vs IgM IgG antibodies detected by sensitization or crossmatch assays: true sensitization against HLA antigens IgM antibodies: not considered typical of a true anti-HLA response

Interpretation (2) T cell vs B cell T cell (+): HLA class I antigens T cell (-) / B cell (+): HLA class II antibodies of less significance clinically / secondary to either class I or II antibodies T cell (+) / B cell (-): non-HLA antibody, as class I antigen is expressed on both T and B cells

Interpretation (3) Complement fixing vs noncomplement fixing Antibodies that must fix complement to produce a positive assay Cytotoxic Clinical importance Antibodies detected by means that are independent of complement fixation, such as flow cytometric assay Not be cytotoxic Clinical significance is unclear

Interpretation (4) Transplantation should not proceed if there is evidence of a positive crossmatch secondary to a cytotoxic IgG anti-HLA antibody

Crossmatch is Positive by CDC Add Dithiothreitol (DTT) Reduce the disulfide bonds present when the antibody is IgM CDC (+) / DTT (-) Presence of an IgM antibody only Should not preclude transplantation CDC (+) / DTT (+) IgG anti-donor antibody Absolute contraindication to transplantation

Risk assessment Current positive CDC or AHG-CDC: high risk for antibody mediated rejection (AMR) Current positive CDC: contraindication to transplantation, unless donor specific antibody (DSA) can be reduced with desensitization protocols

Current positive flow crossmatch or a remote (historic) positive CDC or AHG-CDC crossmatch: intermediated risk for AMR. Augmented immunosuppression Negative current and remote flow or AHG-CDC crossmatch: low risk for AMR. Conventional immunosuppression