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

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
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
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 6 22 12 13 1 26 18 39 21
Effective
polymorphism

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

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)

17. 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

18. 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

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

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

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

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

23. Antibody Mediated Rejection:

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

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

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

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

28. 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

29. Effect of HLA matching on renal transplant outcomes

30. 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

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

32. 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)

33. 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

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

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

36. SSOP typing by Luminex HLA-A locus Patient’s DNA 1 1 2 2 3 3 4 4 5 5 67 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 HLA-A locus Patient’s DNA 1 1 2 2 3 3 4 4 5 5 67 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

38. SSOP typing by Luminex Biotinilated PCR products 1 1 2 2 3 3 4 4 5 5 67 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

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

40. SSOP typing by Luminex Strpeptavidin-PE 1 1 3 3 4 4 5 5 7 7 8 8 9 9 102 6
A*24
A*02
Strpeptavidin-PE

41. SSOP typing by Luminex 1 1 3 3 4 4 5 5 7 7 8 8 9 9 10 10 A*01 A*032 6
A*24
A*02

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

43. 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

44. 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

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

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

47. Complement mediated cytotoxicity (CDC) crossmatchLy

48. Complement mediated cytotoxicity (CDC) crossmatchLy

49. Complement mediated cytotoxicity (CDC) crossmatchLy

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

51. Complement mediated cytotoxicity (CDC) crossmatchLy
Red dye

52. Complement mediated cytotoxicity (CDC) crossmatch
Cell death Ly

53. Complement mediated cytotoxicity (CDC) crossmatchLy

54. Anti-human globulin (AHG-CDC) crossmatchLy

55. Anti-human globulin (AHG-CDC) crossmatchLy

56. Anti-human globulin (AHG-CDC) crossmatch
Complement Ly

57. Anti-human globulin (AHG-CDC) crossmatch
Cell death Ly
Red dye

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

60. 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)

61. 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

62. 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