1. HLA TYPING & ORGAN TRANSPLANTATION
Neveen A. Soliman
Prof. of Clinical & Chemical Pathology & Immunology

2. Organ Transplantation
What is it?
Organs or tissues from one human being (the donor) are put into another person's body (the recipient).
Factors Effecting Transplantation?
HLA Antigens

3. Statistics on Organ Transplantation
There are more than 91,500 people on the organ transplantation waiting list.
Each day 74 people receive an organ transplantation, but 18 people on the waiting list die because a donor is not available.
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.

4. First Organ Transplantation
In 1959, Joseph Murray and his colleagues in Boston successfully transplanted a Kidney that were donated by fraternal twins and it functioned for 20 years without immunosuppression drugs.
First successful Liver transplant- In Denver on 7/23/1967
First successful Heart transplant- In Cape Town, South Africa on 1/2/68
First successful Bone Marrow transplant- Minneapolis, MN on 8/25/68

5. Why Is it Difficult?
Organ transplantation is difficult because of the HLA antigens located on the cell surface.
Human Leukocyte Antigen (HLA) also referred to as Major Histocompatibility Complex (MHC) plays a role in intercellular recognition and discrimination between self and non-self.

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

7. Types of MHC There are three classes of MHC molecules.
Class I- encodes glycoproteins expressed on the surface of nearly all nucleated cell; the major function of the class I gene is presentation of peptide antigens to cytotoxic T-cells
Class II- encodes glycoproteins expressed primarily on antigen-presenting cells, examples: macrophages, dendritic cells and B-cells, where they are present processed antigenic peptides to T helper cells.
Class III- encodes various secreted proteins that have immune function including components of the complement system; C2,C4, Factor B, &TNF, and molecules involved in inflammation.

8. Different HLA Alleles Class I- HLA A 451 alleles HLA B 782 alleles
HLA C alleles
Class II- HLA DR alleles
HLA DQ alleles
HLA DP alleles
HLA DM 11 alleles
HLA DO 21 alleles

9. Requirements for Transplant
Each transplant center has different requirements for allele matches.
For the National Marrow Donor Program:
In order to find a match doctors require that at least a minimum of 3 allele matches. HLA-A, HLA-B and HLA-DRB1.
One set of the three antigens are inherited from your mother and the other set is inherited from your father. This makes 6 antigens to match
Therefore, it is required that 4 of the 6 antigens match for cord blood donation and 5 of the 6 antigens for adult donation.

10. Requirements for Transplant
National Marrow Donor Program:

11. Chance of a Match Mother/Father: 25% chance of full match
One Sibling: 25 % chance of full match
Two Siblings: 44 % chance of full match
Three siblings: 58% chance of full match
The chance to find donors may be better for more homogenous racial groups.

12. HLA In Transplantation
There are two characteristics of the HLA genes that make them special for organ transplantation:
There high degree of polymorphism
There strong immune reactions that their products can produce in other individuals.

13. HLA Pathways
There are two pathways that can occur that cause problems in organ transplantation as a result of HLA antigens.
Direct pathway- the alloreactive responses of recipient T-cells to donor APC expressing incompatible antigens.
Indirect Pathway- allogeneic HLA antigens are taken up and processed by recipient APC and presented in context with autologous HLA molecules to recipient T-cells.

14. Problems from HLA antigens
Engraftment- immunological rejection of donor hematopoietic cells by recipient T cells that recognize incompatible HLA determinants.
Factors:
pregnancy or transfusion
HLA mismatching of the donor
Using of less intense preparative regimen before transplant
Suboptimal post-transplant immunosuppressive therapy
Depletion of T lymphocytes from marrow grafts.
Class I determinants govern graft acceptance
Class II determinants play a role in GVHD.

15. Overview
Methods: Histocompatibility test, consisting of three tests: HLA antigen typing, screening of the recipient for the anti-HLA antibodies and the lymphocyte crossmatch or compatability test.
Results: More allele mismatch more complications
Further Studies/Discussion: Outcomes of unrelated donor/recipient transplant.

16. Histocompatibility testing consists of three tests
HLA antigen typing (also called tissue typing)
Screening of the recipient for anti-HLA antibodies (also called antibody screening)
Lymphocyte cross matching (also called compatibility testing)

17. Two different methods, serological and DNA sequencing
HLA antigen typing
Two different methods, serological and DNA sequencing

18. Serological method
Lymphocytes are harvested from the blood by density gradient centrifugation
A solution of Ficoll-Hypaque is layer underneath the whole blood, and the tube is centrifuged
Red blood cells are denser and go to the bottom
mononuclear cells are less dense, and are found in the middle, just underneath the platelets
The mononuclear layer is removed, and washed. T-cells are removed usually by binding to magnetic beads coated with T-cell antibodies, and are washed away, leaving only the B-cells.

19. Serological method
This B cell enriched media is added to a microtiter plate with each well containing a different antibody to a certain HLA antigen. If a certain MHC cell is present, the antibodies will bind, forming an antigen-antibody complex.
After incubation, rabbit complement is added to each well. If an antigen-antibody complex is present, complement will be activated, and will destroy the cells with an antigen-antibody complex.
After incubation formalin is added to fix the cells and stop the complement reaction. Eosin Y is added to stain any dead cells.

20. 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.

21. DNA typing methods
Granulocytes and lymphocytes are separated from blood by lysis of the red blood cells using ammonium chloride and centrifugation.
DNA is extracted from the white cells by chloroform and ethanol and added to the wells of a microtiter tray.
Each well contains oligonucleotide primers complementary to a small segment of only one HLA allele. If the primer can attach, the HLA antigen is present on the cells.

22. DNA typing methods
DNA polymerase and oligonucleotide triphosphates are added to each well and the plate is incubated in a thermal cycler, which multiplies the sequence between the primers (same as PCR)
The DNA is removed and run on agarose gel by electrophoresis.
Since the DNA was amplified, if there is any DNA detected, HLA is present. If no DNA is seen, HLA is not present.

23. Antibody screening for anti-HLA antibodies
Purpose: to detect antibodies in the recipient’s serum that react with HLA antigens. We know what HLA type the person is, but we don’t know what antibodies they have to other HLA types

24. Antibody screening for anti-HLA antibodies
Leukocytes (neutrophils, monocytes, basophils, lymphocytes) are harvested from the blood of donors with a known HLA type and are added to a microtiter plate.
Serum from the recipient is added to each well.
After incubation, cells are washed to remove any unbound proteins
Anti-human Ab is added, incubated, and then rabbit complement is added.

25. Antibody screening for anti-HLA antibodies
If an antibody against HLA is present, it will bind to the cells, and antigen-antibody complexes will bind to the anti-human Ab, which will then activate complement.
Eosin Y is added, cells are examined under a microscope.
Pink stained cells indicates the presence of anti-HLA antibodies.
The higher the number of different HLA antibodies the lower the probability of finding a match.

26. Crossmatch test
Purpose: to detect presence of preformed antibodies in recipient that are reactive against donor tissues.

27. Crossmatch test
Peripheral blood lymphocytes from the donor are separated into B and T lymphocyte populations
T-cells are purified by magnetic beads coated with monoclonal antibodies for B-cells. The B-cells bind and are removed by magnetic force.
B-cells are purified in the same manner, but the magnetic beads are coated with monoclonal antibodies for T-cells.

28. Crossmatch test
B-cell crossmatch is performed using the same method as HLA typing
T-cell crossmatch is performed using the same method as screening test
Why do a crossmatch when screening seems sufficient?
Antibodies against low-incidence antigens are likely to be missed.
Acts as a mock transplant

29. Limitations cont…
Time between registration and identification of donor
Disease progression in patients
Age
Increase in morbidity and mortality
DNA-based matching
Reduces the odds of finding a suitable matched donor

30. Molecular Typing Methods
Sequence Specific Primers (SSP)
Sequence Specific Oligonucleotide Probe (SSOP)
Analysis of allelic polymorphism at the DNA level
Analyze Class II micropolymorphism down to a single a.a
Sequence-Based Typing (SBT).
provide the highest resolution possible
important for discovering new alleles
potential impact on transplantation.

31. Sequence Based Typing (SBT)