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 Medical Director, HLA Typing Laboratory Division of Hematopathology Department of Pathology and Laboratory Medicine Dalhousie University
[email protected]
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
HLA testing..... is similar to pretransfusion testing.
HLA testing..... is similar to pretransfusion testing.
HLA testing..... is similar to pretransfusion testing. • ABO, D antigen typing and transfuse ABO, D matched blood
HLA testing..... is similar to pretransfusion testing. • ABO, D antigen typing and transfuse ABO, D matched blood
• HLA typing and transplant HLA matched kidney or bone marrow
HLA testing..... is similar to pretransfusion 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 testing..... is similar to pretransfusion testing. • ABO, D antigen typing and transfuse ABO, D matched blood
• HLA typing and transplant HLA matched kidney or bone marrow
• RBC antigen antibody screen
• HLA antibody screen
• If screen is positive, identify specificity of allo-antibody and transfuse antigen negative RBC units
• If screen is positive, identify HLA allo-antibody specificity and transplant with organs from antigen negative donors
HLA testing..... is similar to pretransfusion testing. • ABO, D antigen typing and transfuse ABO, D matched blood
• HLA typing and transplant HLA matched kidney or bone marrow
• RBC antigen antibody screen
• HLA antibody screen
• If screen is positive, identify specificity of allo-antibody and transfuse antigen negative RBC units
• If screen is positive, identify HLA allo-antibody specificity and transplant with organs from antigen negative donors
• Red cell Crossmatch
• Lymphocyte Crossmatch
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
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?
Polymorphism of the Major Histocompatibility Complex in humans - Human Leukocyte Antigen (HLA)
28 136 35 106 3 814
1431
569
893
16 118 26 77
2 637
1165
431
681
6
1 26
18
39
21 Effective
22
12 13
polymorphism
Class II
Class I
DP
DQ
DR
b1 a1
b1 a1
b3,4,5 b1 a
DP
DQ
DR
B
C
A
maternal
B
C
A paternal
HLA class I and class II antigens •
•
•
Monomer with noncovalently associated subunit (b2m) Presents antigenic peptides to CD8+ T 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)
Expressed by all nucleated cells
Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove) Top view
Side views
HLA-A
HLA-B
HLA-C
b2 microglobulin
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
HLA antibody development
Your (“self”) HLA
HLA antibody development
Your (“self”) HLA
Donor (“allo”) HLA
HLA antibody development
Your (“self”) HLA
Donor (“allo”) HLA
HLA antibody development
Your (“self”) HLA
Donor (“allo”) HLA
Sensitizing events: Transfusion Pregnancy Transplantation
Antibody Mediated Rejection:
Normal Kidney (high power)
tubules
glomeruli
Courtesy Dr. Jennifer Merrimen
Antibody mediated rejection (low power)
Courtesy Dr. Jennifer Merrimen
Antibody mediated rejection (high power)
Courtesy Dr. Jennifer Merrimen
Acute Antibody Mediated Rejection, C4d positive
Courtesy Dr. Jennifer Merrimen
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
Effect of HLA matching on renal transplant outcomes
HLA inheritance A C B DR DQ
Mother
Patient
Father
Sib 1 Sib 2 Sib 3 25% chance of having an HLA matched sibling 50% chance of having a haploidentical sibling
Sib 4
Effect of HLA matching on deceased donor renal transplant outcomes
0 MM = 7.4%
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)
HLA typing by SSO using Luminex platform 100 types of microspheres distinguished by fluorescence emission signature Each microsphere type is coated with different sequence specific oligonucleotide (HLA allele) 2 lasers Tells the instrument which bead is being examined
Tells the instrument how much DNA is bound to the bead
SSOP typing by Luminex 1
2
3
4
5
6
7
8
9
10
SSOP typing by Luminex 1
2
3
4
5
6
7
8
9
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29
10
A*30
SSOP typing by Luminex 1
2
3
4
5
6
7
8
9
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29
10
A*30
HLA-A locus Maternal Paternal
Patient’s DNA
SSOP typing by Luminex 1
2
3
4
5
6
7
8
9
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29
10
A*30
HLA-A locus Maternal Paternal
Patient’s DNA
SSOP typing by Luminex 1
2
3
4
5
6
7
8
9
A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29
Biotinilated PCR products
10
A*30
SSOP typing by Luminex 1
3
A*01
4
5
7
A*03 A*11 A*23
8
9
A*25 A*26 A*29
2
6
A*02
A*24
10
A*30
Biotinilated PCR products reacts with microspheres coated with a specific probe
SSOP typing by Luminex 1
3
A*01
4
5
7
A*03 A*11 A*23
8
9
A*25 A*26 A*29
2
6
A*02
A*24
Strpeptavidin-PE
10
A*30
SSOP typing by Luminex 1
3
A*01
4
5
7
A*03 A*11 A*23
8
9
A*25 A*26 A*29
2
6
A*02
A*24
10
A*30
Effect of HLA matching on deceased donor renal transplant outcomes
0 MM = 7.4%
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
Significance of the positive crossmatch test in kidney transplantation
Patel and Terasaki NEJM 1969 crossmatch
Graft rejection
Functioning graft
Positive
24
6
Negative
8
187
Complement mediated cytotoxicity (CDC) crossmatch
HLA Ly
Donor lymphocyte
Complement mediated cytotoxicity (CDC) crossmatch Recipient serum
Ly
Donor lymphocyte
Complement mediated cytotoxicity (CDC) crossmatch
Ly
Complement mediated cytotoxicity (CDC) crossmatch
Ly
Complement mediated cytotoxicity (CDC) crossmatch Complement
Ly
Complement mediated cytotoxicity (CDC) crossmatch Complement
Membrane attack complex (MAC) Ly
Complement mediated cytotoxicity (CDC) crossmatch
Ly
Red dye
Complement mediated cytotoxicity (CDC) crossmatch
Ly
Cell death
Complement mediated cytotoxicity (CDC) crossmatch
Ly
Anti-human globulin (AHG-CDC)
crossmatch Anti-human globulin
Ly
Anti-human globulin (AHG-CDC)
crossmatch
Ly
Anti-human globulin (AHG-CDC)
crossmatch Complement
Ly
Anti-human globulin (AHG-CDC)
crossmatch
Ly
Red dye
Cell death
Panel Reactive Antibodies (PRA) to predict likelihood of a positive crossmatch and identify HLA antibody specificity Frozen Cell Tray (FCT) Method
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
PRA = 36% (21/58)
1 8 1 8 1 1 8 1 1 8 8 8 8 8 8 1 1 1 1 1 8 1 8 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 8 1 8 8 8 8 8 8 8 1 1 8 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 8 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
PRA = 36% (21/58)
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 8 1 8 1 1 8 1 1 8 8 8 8 8 8 1 1 1 1 1 8 1 8 1 1 1 1 1 1 1
Anti-A11
1 8 1 8 8 8 8 8 8 8 1 1 8 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 8 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
PRA = 36% (21/58)
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 8 1 8 1 1 8 1 1 8 8 8 8 8 8 1 1 1 1 1 8 1 8 1 1 1 1 1 1 1
Anti-A11
1 8 1 8 8 8 8 8 8 8 1 1 8 1 1 1 1 1 1 1 8 1 1 1 1 1 1 1 8 1
