• 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

Dr. Robert Liwski, MD, PhD, FRCPC
Medical Director, HLA Typing Laboratory
Division of Hematopathology
Department of Pathology and Laboratory Medicine
Dalhousie University rliwski@dal.ca

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
• HLA typing and transplant HLA matched kidney or bone marrow
• RBC antigen antibody screen
• If screen is positive, identify specificity of allo-antibody and transfuse antigen negative RBC units

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
– 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
– 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
6 22 12 13
2 637
1 26
1165 431
18 39
681
21 Effective polymorphism

DP b
1 a
1
Class II
DQ b
1 a
1
DR b
3,4,5 b
1 a
B C
Class I
A maternal
DP DQ DR B C A paternal

• Monomer with noncovalently associated subunit ( b
2m)
• 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)

Polymorphic residues on Class I HLA molecules
(polymorphisms are concentrated around peptide binding groove)
Top view Side views
HLA-A
HLA-B b
2 microglobulin
HLA-C

• 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

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 Father
Patient 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

1 2 3 4 5 6 7 8 9 10

A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30

A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
Maternal
Paternal
HLA-A locus
Patient’s DNA

A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
Maternal
Paternal
HLA-A locus
Patient’s DNA

A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30
Biotinilated PCR products

A*01
2
A*02
A*03 A*11 A*23
6
A*24
A*25 A*26 A*29 A*30
Biotinilated PCR products reacts with microspheres coated with a specific probe

A*01
2
A*02
A*03 A*11 A*23
6
A*24
A*25 A*26 A*29 A*30
Strpeptavidin-PE

A*01
2
A*02
A*03 A*11 A*23
6
A*24
A*25 A*26 A*29 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
Positive
Negative
Graft rejection
24
8
Functioning graft
6
187

Complement mediated cytotoxicity (CDC) crossmatch
Ly
HLA
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
Ly
Membrane attack complex (MAC)

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
Cell death
Red dye

Panel Reactive Antibodies (PRA) to predict likelihood of a positive crossmatch and identify HLA antibody specificity
Frozen Cell Tray (FCT) Method

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
8
1
1
1
1
8
1
8
8
1
1
1
1
1
8
1
8
8
8
8
8
1
1
1
1
1
1
1
1
1
1
1
8
1
1
1
1
1
8
1
1
1
8
8
8
8
1
8
1
8
8
8
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
8
1
1
1
1
8
1
8
8
1
1
1
1
1
8
1
8
8
8
8
8
1
1
1
1
1
1
1
Anti-A11
1
1
1
1
8
1
1
1
1
1
8
1
1
1
8
8
8
8
1
8
1
8
8
8
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
8
1
1
1
1
8
1
8
8
1
1
1
1
1
8
1
8
8
8
8
8
1
1
1
1
1
1
1
Anti-A11
1
1
1
1
8
1
1
1
1
1
8
1
1
1
8
8
8
8
1
8
1
8
8
8
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