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, FRCPCMedical Director, HLA Typing LaboratoryDivision of HematopathologyDepartment of Pathology and Laboratory MedicineDalhousie Universityrliwski@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 pre-transfusion testing.

HLA testing..... is similar to pre-transfusion testing.

HLA testing..... is similar to pre-transfusion testing.

• ABO, D antigen typing and transfuse ABO, D matched blood

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

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 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

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

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)

89356914313 81435 10628 136

68143111652 63726 7716 118

2139181 2612 136 22 Effective polymorphism

ACBDRDQDP

ab1a1b1a1b1 b3,4,5

Class IClass II

ACBDRDQDP

maternal

paternal

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)

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

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:TransfusionPregnancy Transplantation

Antibody Mediated Rejection:

Normal Kidney (high power)

glomeruli

tubules

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

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

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 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

1 2 3 4 8 109765

SSOP typing by Luminex

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

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

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

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

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

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

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

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

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

Ly Donor lymphocyte

HLA

Complement mediated cytotoxicity (CDC) crossmatch

Ly Donor lymphocyte

Recipient serum

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Complement

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Complement

Membrane attack complex (MAC)

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Red dye

Complement mediated cytotoxicity (CDC) crossmatch

LyCell death

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Complement mediated cytotoxicity (CDC) crossmatch

Ly

Anti-human globulin (AHG-CDC) crossmatch

Anti-human globulin

Ly

Anti-human globulin (AHG-CDC) crossmatch

Ly

Complement

Anti-human globulin (AHG-CDC) crossmatch

Ly

Red dye

Cell death

Anti-human globulin (AHG-CDC) crossmatch

Panel Reactive Antibodies (PRA)

to predict likelihood of a positive crossmatch and identify HLA antibody specificity

Frozen Cell Tray (FCT) Method

181811

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811888

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118181

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181888

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888811

111111

811111

111111

118111

111111

111181

111111

PRA = 36% (21/58)

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

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