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Objectives

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

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Objectives

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  1. 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

  2. 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

  3. 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

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

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

  6. HLA testing..... is similar to pre-transfusion testing. • ABO, D antigen typing and transfuse ABO, D matched blood

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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?

  13. 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 Effective polymorphism 6 22 12 13 1 26 18 39 21

  14. Class II Class I DP DQ DR B C A maternal b1 a1 b1 a1 b3,4,5 b1 a DP DQ DR B C A paternal

  15. 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)

  16. 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

  17. 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

  18. HLA antibody development Your (“self”) HLA

  19. HLA antibody development Your (“self”) HLA Donor (“allo”) HLA

  20. HLA antibody development Your (“self”) HLA Donor (“allo”) HLA

  21. HLA antibody development Your (“self”) HLA Donor (“allo”) HLA Sensitizing events: Transfusion Pregnancy Transplantation

  22. Antibody Mediated Rejection:

  23. Normal Kidney (high power) tubules glomeruli Courtesy Dr. Jennifer Merrimen

  24. Antibody mediated rejection (low power) Courtesy Dr. Jennifer Merrimen

  25. Antibody mediated rejection (high power) Courtesy Dr. Jennifer Merrimen

  26. Acute Antibody Mediated Rejection, C4d positive Courtesy Dr. Jennifer Merrimen

  27. 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 cytometrycrossmatch (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

  28. Effect of HLA matching on renal transplant outcomes

  29. Patient Sib 1 HLA inheritance A C B DR DQ Father Mother Sib 2 Sib 3 Sib 4 25% chance of having an HLA matched sibling 50% chance of having a haploidentical sibling

  30. Effect of HLA matching on deceased donor renal transplant outcomes 0 MM = 7.4%

  31. 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)

  32. 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

  33. SSOP typing by Luminex 1 2 3 4 5 6 7 8 9 10

  34. 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 A*02 A*03 A*11 A*23 A*24 A*25 A*26 A*29 A*30

  35. 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 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

  36. 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 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

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

  38. 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 6 2 A*24 A*02 Biotinilated PCR products reacts with microspheres coated with a specific probe

  39. 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 6 2 A*24 A*02 Strpeptavidin-PE

  40. 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 6 2 A*24 A*02

  41. Effect of HLA matching on deceased donor renal transplant outcomes 0 MM = 7.4%

  42. 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 cytometrycrossmatch (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

  43. Significance of the positive crossmatch test in kidney transplantation Patel and Terasaki NEJM 1969 Graft rejection crossmatch Functioning graft Positive 24 6 Negative 8 187

  44. Complement mediated cytotoxicity (CDC) crossmatch HLA Ly Donor lymphocyte

  45. Complement mediated cytotoxicity (CDC) crossmatch Recipient serum Ly Donor lymphocyte

  46. Complement mediated cytotoxicity (CDC) crossmatch Ly

  47. Complement mediated cytotoxicity (CDC) crossmatch Ly

  48. Complement mediated cytotoxicity (CDC) crossmatch Complement Ly

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

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