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 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 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 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 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 22 12 13 1 26 18 39 21 Effective polymorphism
Class II Class I maternal paternal DP DQ DR B C A b1 a1 b1 a1 b3,4,5
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)
Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove) Top view Side views HLA-A b2 microglobulin HLA-B HLA-C
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 Normal Courtesy Dr. Jennifer Merrimen
Antibody mediated rejection (low power) Hyperacute rejection Courtesy Dr. Jennifer Merrimen
Antibody mediated rejection (high power) Hyperacute rejection 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
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 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 A*01
SSOP typing by Luminex HLA-A locus Patient’s DNA 1 1 2 2 3 3 4 4 5 5 6 7 7 8 8 9 9 10 10 A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30 HLA-A locus Maternal Patient’s DNA Paternal
SSOP typing by Luminex HLA-A locus Patient’s DNA 1 1 2 2 3 3 4 4 5 5 6 7 7 8 8 9 9 10 10 A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30 HLA-A locus Maternal Patient’s DNA Paternal
SSOP typing by Luminex Biotinilated PCR products 1 1 2 2 3 3 4 4 5 5 6 7 7 8 8 9 9 10 10 A*01 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30 Biotinilated PCR products
SSOP typing by Luminex 1 1 3 3 4 4 5 5 7 7 8 8 9 9 10 10 A*01 A*03 A*11 A*23 A*25 A*26 A*29 A*30 2 6 A*24 A*02 Biotinilated PCR products reacts with microspheres coated with a specific probe
SSOP typing by Luminex Strpeptavidin-PE 1 1 3 3 4 4 5 5 7 7 8 8 9 9 10 2 6 A*24 A*02 Strpeptavidin-PE
SSOP typing by Luminex 1 1 3 3 4 4 5 5 7 7 8 8 9 9 10 10 A*01 A*03 2 6 A*24 A*02
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 Ly
Complement mediated cytotoxicity (CDC) crossmatch Membrane attack complex (MAC) Ly
Complement mediated cytotoxicity (CDC) crossmatch Ly Red dye
Complement mediated cytotoxicity (CDC) crossmatch Cell death Ly
Complement mediated cytotoxicity (CDC) crossmatch Ly
Anti-human globulin (AHG-CDC) crossmatch Ly
Anti-human globulin (AHG-CDC) crossmatch Ly
Anti-human globulin (AHG-CDC) crossmatch Complement Ly
Anti-human globulin (AHG-CDC) crossmatch Cell death Ly Red dye
Panel Reactive Antibodies (PRA) to predict likelihood of a positive crossmatch and identify HLA antibody specificity Frozen Cell Tray (FCT) Method
1 1 8 1 1 8 1 8 1 1 8 1 1 8 1 1 8 1 1 1 8 1 1 8 1 1 1 1 8 PRA = 36% (21/58)
1 1 8 1 1 8 1 8 1 1 8 1 1 8 1 1 8 1 1 1 8 1 1 8 1 1 1 1 8 PRA = 36% (21/58) Anti-A11
1 1 8 1 1 8 1 8 1 1 8 1 1 8 1 1 8 1 1 1 8 1 1 8 1 1 1 1 8 PRA = 36% (21/58) Anti-A11