The HLA Laboratory Part I

Report
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

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