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Preface

Preface. 1953, in Paris , a little carpenter fell down from upstairs, which broke his spleen and kidney. In the surgery, the doctor found that the patient had only one kidney. When removing the kidney, the patient got uremia.

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Preface

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  1. Preface 1953, in Paris , a little carpenter fell down from upstairs, which broke his spleen and kidney. In the surgery, the doctor found that the patient had only one kidney. When removing the kidney, the patient got uremia. To save his life, his mother decided to donate one of her kidneys to her love son. The first kidney transplantation was done in a hospital of Paris. As everyone expected, the surgery was very successful. And the carpenter recovered quickly. But at last the little carpenter died of putrescence of kidney. This tragedy also encouraged scientists to find out the fact of transplant rejection.

  2. Statistics on Organ Transplantation There are more than 91,500 people on the organ transplantation waiting list Total • Each day 74 people receive an organ transplantation, but 18 people on the waiting list die because a donor is not available Died • There are 55,000 people waiting for a kidney, 17,000 waiting for a liver and 3,000 waiting for either a heart or liver transplant. Organ

  3. It is unlikely that extended family members will match you. However, your parents and/or children may also be tested to confirm your HLA typing and to make sure no possible donors are overlooked. About 70% of patients who need a transplant do not have a suitable donor in their family. 70%

  4. Now we all know the accident is due to the rejection of immune cells to the new tissue. And most of the responses of immune system have MHC/HLA restriction and proper HLA typing is the key to the successful transplantation.

  5. Why is HLA so important? How is HLA typing carried out?

  6. HLA Typing in Transplantation

  7. What is HLA? • Human Leukocyte Antigens (HLA) are proteins found on the surface of most cells in the body, but are found in greatest number on white blood cells. • And is also referred to as Major Histocompatibility Complex (MHC),which plays a role in intercellular recognition and discrimination between self and non-self.

  8. The HLA complex is a collection of genes arrayed within a long continuous stretch of DNA on chromosome 6. Each HLA type is associated with a different class of MHC molecule.

  9. HLA Inheritance Mother Father A 1 2 A 9 10 3 4 B 11 12 B 5 6 C 13 14 C 7 8 DR 15 16 DR Child 1 Child 2 Child 3 Child 4 A 1 9 A 1 10 A 2 9 2 10 A B 3 11 B 3 12 B 4 11 B 4 12 C 5 13 C 5 14 C 6 13 C 6 14 DR 7 15 DR 7 16 DR 8 15 DR 8 16

  10. Problems from HLA antigens A close match between your HLA markers and your donor's can reduce the risk that your immune cells will attack your donor's cells or that your donor's immune cells will attack your body after the transplantation, which is also called host-versus-graft disease(HVGD) or graft-versus-host disease (GVHD) .

  11. In the early time, people carry out the transplantation surgery without HLA typing and then the rejection occurs very often. Just as the case before. The photo here shows necrotic kidneys. Immune rejection

  12. HLA typing basics A well-matched donor is important to the success of your transplant. You inherit half of your HLA markers from your mother and half from your father, so each brother and sister who has the same parents as you has a 25% chance of matching you.

  13. HLA typing requirements Genes of classical HLA include A,B,C,DR,DQ and DP. In transplantation, we mainly match the three pairs of HLA-A,HLA-B and HLA-DR genes.

  14. Figure . HLA Matching of Patient and Donor On the left side of the Figure, the donor matches the patient at all 6 HLA markers. On the right side, one of the donor's A markers does not match the patient's.

  15. Can the surgery on the right side be carried out ? Many studies show that this kind of surgery could be carried out because the matching of HLA-DR is more important than the matching of HLA-A and HLA-B.

  16. Kidney graft survival and HLA typing

  17. Role of HLA typing Improves the chances for a successful transplant 1 2 Promotes engraftment. Reduces the risk of a post-transplant complication. 3

  18. How to do HLA typing? Ever since people knew the importance of HLA typing, there have been many HLA typing methods. We’d like to introduce three of them.

  19. PCR technique Mixed lymphocyte reaction Serological typing

  20. Serological method Add known HLA antiserum to tested peripheral blood lymphocytes. After reacting, add complement. Then mixed with dyes, the stained cells are dead ones. As we all know, the complement system can be activated by the Ag-Ab complex, then the stained lymphocytes has the antigens corresponding to the known anti-serum .

  21. Serological method • Cells are examined under a phase contrast microscope, and cells that are pink are positive. • If 60% or more of the cells are stained they are considered positive for the HLA antigen.

  22. Mixed lymphocyte reaction

  23. Principle as the figure shows. Stimulated cells through processing no longer proliferate. Response cells which has not been treated, can identify the HLA antigen of foreign stimulated cells and then are in proliferation. It must synthesize DNA in the process of cell proliferation and thymidine (TdR) is an essential material for DNA synthesis, so measurement using isotope labeled thymidine H can more accurately reflect the extent of cell proliferation.

  24. Mixed lymphocyte reaction

  25. PCR Technique *PCR-SSP(Polymerase Chain Reaction-Sequence-Specific Primers ) *PCR-SSO(Polymerase Chain Reaction-Single Strand Origin) *PCR-RFLP ( restriction fragment length polymorphism) *PCR-Sequencing *PCR-SSCP(PCR-single strand conformational polymorphism)

  26. PCR-SSP Design a set of allele-specific primers (sequence specific primer, SSP), using PCR technology to get HLA type specific proliferative products. It can be directly analyzed by electrophoresis band to decide HLA type. Thus greatly simplifies the experimental steps .

  27. PCR-SSCP SSCP(PCR-single strand conformational polymorphism )is the most commonly used method of typing according to the configuration . Amplify the aimed product , denature it to form a single chain. Different sequences( or even only one different nucleotide ) can form different spatial structures, thereby we can get the result because of different electrophoresis rate.

  28. Other Applications of HLA Typing Paternity testing

  29. Criminal investigation

  30. Reporter:辛颖慧 Material collecting:黄海燕杨晓科 李继川 PPT maker:周松涛

  31. Thank You!

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