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Objectives Overview of HLA genes and their function Importance of HLA in solid organ transplantation Overview of HLA typing and histocompatibility testing.

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Presentation on theme: "Objectives Overview of HLA genes and their function Importance of HLA in solid organ transplantation Overview of HLA typing and histocompatibility testing."— Presentation transcript:

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.

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6 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) Effective polymorphism

14 ACBDRDQDP        Class IClass II ACBDRDQDP maternal paternal

15 HLA class I and class II antigens Monomer with non- covalently associated subunit (  2m) 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)

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17 HLA-A HLA-B HLA-C Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove) Top viewSide views  2 microglobulin

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”) HLADonor (“allo”) HLA

21 HLA antibody development Your (“self”) HLADonor (“allo”) HLA

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

23 Antibody Mediated Rejection:

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

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

26 Antibody mediated rejection (high power) 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 Mother Father Sib 1 Sib 2Sib 3Sib 4 A C B DR DQ 25% chance of having an HLA matched sibling 50% chance of having a haploidentical sibling Patient

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) Tells the instrument which bead is being examined Tells the instrument how much DNA is bound to the bead 2 lasers

34 SSOP typing by Luminex

35 A*01A*02A*03A*11A*23A*24A*25A*26A*29A*30 SSOP typing by Luminex

36 A*01A*02A*03A*11A*23A*24A*25A*26A*29A*30 SSOP typing by Luminex HLA-A locus Patient’s DNA Maternal Paternal

37 A*01A*02A*03A*11A*23A*24A*25A*26A*29A*30 SSOP typing by Luminex Maternal Paternal HLA-A locus Patient’s DNA

38 A*01A*02A*03A*11A*23A*24A*25A*26A*29A*30 SSOP typing by Luminex Biotinilated PCR products

39 A*01 A*02 A*03A*11A*23 A*24 A*25A*26A*29A*30 SSOP typing by Luminex Biotinilated PCR products reacts with microspheres coated with a specific probe

40 A*01 A*02 A*03A*11A*23 A*24 A*25A*26A*29A*30 SSOP typing by Luminex Strpeptavidin-PE

41 A*01 A*02 A*03A*11A*23 A*24 A*25A*26A*29A*30 SSOP typing by Luminex

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 crossmatch Significance of the positive crossmatch test in kidney transplantation Positive Negative Graft rejection Functioning graft Patel and Terasaki NEJM 1969

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

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

47 Ly Complement mediated cytotoxicity (CDC) crossmatch

48 Ly Complement mediated cytotoxicity (CDC) crossmatch

49 Ly Complement Complement mediated cytotoxicity (CDC) crossmatch

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

51 Ly Red dye Complement mediated cytotoxicity (CDC) crossmatch

52 Ly Cell death Complement mediated cytotoxicity (CDC) crossmatch

53 Ly Complement mediated cytotoxicity (CDC) crossmatch

54 Ly Anti-human globulin (AHG-CDC) crossmatch Anti-human globulin

55 Ly Anti-human globulin (AHG-CDC) crossmatch

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

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

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

59

60 PRA = 36% (21/58)

61 Anti-A11

62 PRA = 36% (21/58)Anti-A11


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