1. Donor Matching of Kidney Transplantation

2. 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

3. 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

4. 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

5. 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

6. 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

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

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

9. HLA Typing (Serologic Test)

10. 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

11. 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% Negative
11 ∼ 20% Doubtful negative
21 ∼ 50% Weak positive
51 ∼ 80% Positive
81 ∼ 100% Strong positive

12. 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

13. Complement-dependent Cytotoxicity NIH Assay

14. Complement-dependent Cytotoxicity NIH Assay

15. Complement-dependent Cytotoxicity NIH Assay

16. 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

17. Anti-human Globulin (Enhancement) Assay

18. Anti-human Globulin (Enhancement) Assay

19. Anti-human Globulin (Enhancement) Assay

20. 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

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

23. Detection of antidonor antibodies (5)
Flow PRA

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

26. 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

27. 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

28. 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

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

30. 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

31. 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

32. 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