objectives
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Objectives. Overview of HLA genes and their function Importance of HLA in solid organ transplantation Overview of HLA typing and histocompatibility testing in solid organ transplantation. HLA testing in solid organ transplantation. Dr. Robert Liwski, MD, PhD, FRCPC - PowerPoint PPT PresentationTRANSCRIPT
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Objectives
• Overview of HLA genes and their function
• Importance of HLA in solid organ transplantation
• Overview of HLA typing and histocompatibility testing in solid organ transplantation
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HLA testing in solid organ transplantation
Dr. Robert Liwski, MD, PhD, FRCPCMedical Director, HLA Typing LaboratoryDivision of HematopathologyDepartment of Pathology and Laboratory MedicineDalhousie [email protected]
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Halifax HLA Laboratory
• HLA testing for patients from all Atlantic provinces
• Solid organ transplantation– Kidney (+/- pancreas)– Liver– heart
• Hematopoietic stem cell (bone marrow) transplantation– HLA identical siblings– HLA matched unrelated donors
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HLA testing..... is similar to pre-transfusion testing.
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HLA testing..... is similar to pre-transfusion testing.
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HLA testing..... is similar to pre-transfusion testing.
• ABO, D antigen typing and transfuse ABO, D matched blood
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HLA testing..... is similar to pre-transfusion testing.
• ABO, D antigen typing and transfuse ABO, D matched blood
• HLA typing and transplant HLA matched kidney or bone marrow
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HLA testing..... is similar to pre-transfusion testing.
• ABO, D antigen typing and transfuse ABO, D matched blood
• RBC antigen antibody screen
• If screen is positive, identify specificity of allo-antibody and transfuse antigen negative RBC units
• HLA typing and transplant HLA matched kidney or bone marrow
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HLA testing..... is similar to pre-transfusion testing.
• ABO, D antigen typing and transfuse ABO, D matched blood
• RBC antigen antibody screen
• If screen is positive, identify specificity of allo-antibody and transfuse antigen negative RBC units
• HLA typing and transplant HLA matched kidney or bone marrow
• HLA antibody screen
• If screen is positive, identify HLA allo-antibody specificity and transplant with organs from antigen negative donors
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HLA testing..... is similar to pre-transfusion testing.
• ABO, D antigen typing and transfuse ABO, D matched blood
• RBC antigen antibody screen
• If screen is positive, identify specificity of allo-antibody and transfuse antigen negative RBC units
• Red cell Crossmatch
• HLA typing and transplant HLA matched kidney or bone marrow
• HLA antibody screen
• If screen is positive, identify HLA allo-antibody specificity and transplant with organs from antigen negative donors
• Lymphocyte Crossmatch
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Red cell antigens vs HLA antigens
• Red cell antigens– ABO– Rh (D, c, C, e, E)– Kell (k, K)– Duffy (Fya, Fyb)– Kidd (Jka, Jkb)– S (S, s)– M (M, m)– N (N,n)– Many others
• HLA antigens– Class I
• HLA-A, HLA-B, HLA-C– Class II
• HLA-DR, HLA-DQ, HLA-DP
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Red cell antigens vs HLA antigens
• Red cell antigens– ABO– Rh (D, c, C, e, E)– Kell (k, K)– Duffy (Fya, Fyb)– Kidd (Jka, Jkb)– S (S, s)– M (M, m)– N (N,n)– Many others
• HLA antigens– Class I
• HLA-A, HLA-B, HLA-C– Class II
• HLA-DR, HLA-DQ, HLA-DP
–Simple?
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Polymorphism of the Major Histocompatibility Complex in humans - Human Leukocyte Antigen (HLA)
89356914313 81435 10628 136
68143111652 63726 7716 118
2139181 2612 136 22 Effective polymorphism
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ACBDRDQDP
ab1a1b1a1b1 b3,4,5
Class IClass II
ACBDRDQDP
maternal
paternal
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HLA class I and class II antigens
• Monomer with non-covalently associated subunit (b2m)
• Presents antigenic peptides to CD8+ T cells
• Expressed by all nucleated cells
• Heterodimer
• Presents antigenic peptides to CD4+ T cells
• Restricted expression on antigen presenting cells (dendritic cells, B cells, macrophages)
• Inducible on other cells (endothelium and epithelium)
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HLA-A
HLA-B
HLA-C
Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove)
Top view Side views
b2 microglobulin
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Functional relevance of HLA
• Necessary to initiate T cell mediated immune responses against pathogens– polygenic – survival advantage to individual– polymorphic-survival advantage to species
• Transplantation– Causes sensitization (T cell response and B
cell/antibody response)– Can lead to graft rejection
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HLA antibody development
Your (“self”) HLA
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HLA antibody development
Your (“self”) HLA Donor (“allo”) HLA
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HLA antibody development
Your (“self”) HLA Donor (“allo”) HLA
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HLA antibody development
Your (“self”) HLA Donor (“allo”) HLA
Sensitizing events:TransfusionPregnancy Transplantation
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Antibody Mediated Rejection:
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Normal Kidney (high power)
glomeruli
tubules
Courtesy Dr. Jennifer Merrimen
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Antibody mediated rejection (low power)
Courtesy Dr. Jennifer Merrimen
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Antibody mediated rejection (high power)
Courtesy Dr. Jennifer Merrimen
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Acute Antibody Mediated Rejection, C4d positive
Courtesy Dr. Jennifer Merrimen
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Strategies used to avoid/minimize transplant rejection
• HLA typing and matching of recipient/donor pairs
• Detection of donor specific HLA antibodies.– Lymphocyte crossmatch
• Complement dependent cytotoxicity (CDC) crossmatch.• Flow cytometry crossmatch (newer technique, much more sensitive)
– Virtual crossmatch• Identification of HLA antibodies in recipient serum by solid phase assay• HLA typing of the donor (and recipient)• Correlation of recipient HLA antibodies and donor/recipient typing
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Effect of HLA matching on renal transplant outcomes
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HLA inheritance
Mother Father
Sib 1 Sib 2 Sib 3 Sib 4
ACB
DRDQ
25% chance of having an HLA matched sibling50% chance of having a haploidentical sibling
Patient
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Effect of HLA matching on deceased donor renal transplant outcomes
0 MM = 7.4%
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HLA typing• Typing at the HLA-A, B, C, DR, DQ, DP
• Serological techniques (being phased out for routine testing)
• Molecular techniques– Sequence specific priming (SSP)– Sequence specific oligonucleotide probe (SSOP)
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HLA typing by SSO using Luminex platform100 types of microspheres distinguished by fluorescence emission signature
Each microsphere type is coated with different sequence specific oligonucleotide (HLA allele)
Tells the instrument which bead is being examined
Tells the instrument how much DNA is bound to the bead
2 lasers
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1 2 3 4 8 109765
SSOP typing by Luminex
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1 2 3 4 8 1097651 2 3 4 8 109765
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
SSOP typing by Luminex
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1 2 3 4 8 1097651 2 3 4 8 109765
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
SSOP typing by Luminex
HLA-A locus
Patient’s DNAMaternal Paternal
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1 2 3 4 8 1097651 2 3 4 8 109765
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
SSOP typing by Luminex
Maternal Paternal
HLA-A locus
Patient’s DNA
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1 2 3 4 8 1097651 2 3 4 8 109765
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
SSOP typing by Luminex
Biotinilated PCR products
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1 3 4 8 109751
2
3 4 8 1097
6
5
A*01
A*02
A*03 A*11 A*23
A*24
A*25 A*26 A*29 A*30
SSOP typing by Luminex
Biotinilated PCR products reacts with microspheres coated with a specific probe
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1 3 4 8 109751
2
3 4 8 1097
6
5
A*01
A*02
A*03 A*11 A*23
A*24
A*25 A*26 A*29 A*30
SSOP typing by Luminex
Strpeptavidin-PE
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1 3 4 8 109751
2
3 4 8 1097
6
5
A*01
A*02
A*03 A*11 A*23
A*24
A*25 A*26 A*29 A*30
SSOP typing by Luminex
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Effect of HLA matching on deceased donor renal transplant outcomes
0 MM = 7.4%
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Strategies used to avoid/minimize transplant rejection
• HLA typing and matching of recipient/donor pairs
• Detection of donor specific HLA antibodies.– Lymphocyte crossmatch
• Complement dependent cytotoxicity (CDC) crossmatch.• Flow cytometry crossmatch (newer technique, much more sensitive)
– Virtual crossmatch• Identification of HLA antibodies in recipient serum by solid phase assay• HLA typing of the donor (and recipient)• Correlation of recipient HLA antibodies and donor/recipient typing
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crossmatch
Significance of the positive crossmatch test in kidney transplantation
Positive
Negative
Graft rejection Functioning graft
24 6
8 187
Patel and Terasaki NEJM 1969
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Ly Donor lymphocyte
HLA
Complement mediated cytotoxicity (CDC) crossmatch
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Ly Donor lymphocyte
Recipient serum
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Complement
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Complement
Membrane attack complex (MAC)
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Red dye
Complement mediated cytotoxicity (CDC) crossmatch
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LyCell death
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Complement mediated cytotoxicity (CDC) crossmatch
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Ly
Anti-human globulin (AHG-CDC) crossmatch
Anti-human globulin
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Ly
Anti-human globulin (AHG-CDC) crossmatch
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Ly
Complement
Anti-human globulin (AHG-CDC) crossmatch
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Ly
Red dye
Cell death
Anti-human globulin (AHG-CDC) crossmatch
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Panel Reactive Antibodies (PRA)
to predict likelihood of a positive crossmatch and identify HLA antibody specificity
Frozen Cell Tray (FCT) Method
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181811
111111
811888
111111
888111
111111
118181
111111
111111
111111
181888
111111
888811
111111
811111
111111
118111
111111
111181
111111
PRA = 36% (21/58)
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181811
111111
811888
111111
888111
111111
118181
111111
111111
111111
181888
111111
888811
111111
811111
111111
118111
111111
111181
111111
PRA = 36% (21/58) Anti-A11
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181811
111111
811888
111111
888111
111111
118181
111111
111111
111111
181888
111111
888811
111111
811111
111111
118111
111111
111181
111111
PRA = 36% (21/58) Anti-A11