The role of HLA in Allogeneic Hematopoietic Stem Cell Transplantation and Platelet Refractoriness.
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1 The role of HLA in Allogeneic Hematopoietic Stem Cell Transplantation and Platelet Refractoriness. Robert Liwski, MD, PhD, FRCPC Medical Director HLA Typing Laboratory Department of Pathology Dalhousie University DALHOUSIE UNIVERSITY Inspiring minds
2 Introduction Hematopoietic stem cell transplant (HSCT) is a commonly used treatment for hematologic malignancies or bone marrow failure syndromes/immunodeficiency
3 Indications for Allogeneic HSCT Chronic myeloid leukemia (CML) Acute myeloid leukemia (AML) Acute lymphoblastic leukemia (ALL) Plasma cell myeloma Aplastic anemia Congenital bone marrow failure syndromes Congenital immunodeficiency (SCID) Sickle cell anemia Thalassemia Selected solid tumors
4 Allogeneic HSCT Conditioning with high dose chemotherapy and radiation therapy lethal Transplant with stem cell from healthy allogeneic donor. PBSC mobilized with G CSF BM Umbilical cord
5 Hematopoiesis
6 Allogeneic HSCT Transplant of the immune system. Immunosuppression necessary to prevent/minimize Host vs Graft (HVG) and Graft vs Host (GVH) Disease. May get cure due to Graft vs Leukemia (GVL) effect.
7 HVG, GVH,GVL T cell mediated reactions against the graft (stem cells), host tissue, or leukemia (malignancy). Due to T cell mediated recognition of either major or minor histocompatibility differences (MHC, and mhc) between the donor an recipient. In humans MHC molecules are named Human Leukocyte Antigen (HLA).
8 HLA class I and class II antigens Monomer with non Heterodimer covalently associated subunit (β2m) Presents antigenic peptides to CD4+ T Presents antigenic tge cells peptides to CD8+ T cells Restricted expression on antigen presenting Expressed dby all cells (dendritic d cells, B nucleated cells cells, macrophages) including endothelium Inducible on other cells (endothelium and epithelium)
9 Human Leukocyte Antigen (HLA) B Menu F
10
11 Class II Class I DP DQ DR B C A β 1 β 3,4,5 α maternal α 1 β 1 α 1 β 1 DP DQ DR B C A paternal
12 HLA inheritance A C B DR DQ Mother Father Sib 1 Sib 2 Sib 3 Sib 4
13 HLA inheritance A C B 4 haplotypes DR DQ Mother Father Sib 1 Sib 2 Sib 3 Sib 4
14 HLA inheritance A C B 4 haplotypes DR DQ Mother Father Sib 1 Sib 2 Sib 3 Sib 4 Approximately 25 30% chance of having an HLA matched sibling
15 Polymorphism of the Major Histocompatibility Complex in humans HLA
16 Polymorphism of the Major Histocompatibility Complex in humans HLA Effective (caucasians)
17 Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove) Top view Side views HLA-A HLA-B β2 microglobulin li HLA-C
18 Human Leukocyte Antigen (HLA) B Menu F
19 Relevance of HLA Necessary to mount immune responses against pathogens Polygenic survival advantage to individual Polymorphic survival advantage to species Transplantation Difficulty findingcompatible donors. Causes sensitization (T cell response and antibody response) Can lead to graft rejection (failure to engraft) or GVHD
20 Major mismatch/direct allorecognition Normal situation HSCT situation Self T cell Donor T cell TCR Viral antigen n (peptide) Self HLA Allo HLA Foreign viral antigen and self HLA Self antigen and allo HLA Self body cell Recipient cell 5 10% T cells able to respond to allo MHC Potent reaction
21 Your ( self ) HLA HLA antibody development
22 HLA antibody development Your ( self ) HLA allo HLA
23 HLA antibody development Your ( self ) HLA allo HLA
24 HLA antibody development Your ( self ) HLA allo HLA Sensitizing events: Transfusion Pregnancy Transplantation
25 Studies investigating the impact of HLA mismatch in HSCT
26 Lee et. al. Blood 2007
27 Lee et. al. Blood 2007
28 Donor selection for allo HSCT HLA inheritance pattern Extreme HLA polymorphism
29 Probability of finding HLA Identical Donor Sibling Parent 1:100 Cousin, uncle, etc 1:10, :4 (30% on average) Unrelated 1:1,000,000 Worldwide Registry of Bone Marrow Donors >10,000,000 donors
30 Patient workup HLA Typing A, B, C, DR, DQ New patient Siblings and/ or related donors? HLA Typing A, B, C, DR, DQ Yes 70% No Match No match 30% HLA Typing A, B, C, DR, DQ Unrelated donor search Matched donor Mismatched donor 50% 20%
31 Cases
32 Family study 1
33 Family study 1 Patient A B C 07 9 DRB DQB1 06 8
34 Family study 1 Patient brother 1 brother 2 brother 3 sister A B C DRB DQB
35 Family study 1 Patient brother 1 brother 2 brother 3 sister A B C DRB DQB Brother 1 shares 1 haplotype
36 Family study 1 Patient brother 1 brother 2 brother 3 sister A B C DRB DQB Brother 1 shares 1 haplotype
37 Family study 1 Patient brother 1 brother 2 brother 3 sister A B C DRB DQB Brother 1 shares 1 haplotype Brother 2 Is not a match th
38 Family study 1 Patient brother 1 brother 2 brother 3 sister A B C DRB DQB Brother 1 shares 1 haplotype Brother 2 Brother 3 Is not a match thshares 1 haplotype
39 Family study 1 Patient brother 1 brother 2 brother 3 sister A B C DRB DQB Brother 1 Brother 2 Brother 3 Sister shares 1 haplotype Is not a match thshares 1 haplotype is a 10/10 match th
40 Family study 1 patient brother 1 brother 2 brother 3 sister A B C DRB DQB A B C Parent 1 Parent 2 DRB1 DQB1
41 Family study 1 patient brother 1 brother 2 brother 3 sister A B C DRB DQB Parent 1 Parent 2 A B C 07 9 DRB DQB1 06 8
42 Family study 1 patient brother 1 brother 2 brother 3 sister A B C DRB DQB Parent 1 Parent 2 A B C DRB DQB
43 Family study 1 patient brother 1 brother 2 brother 3 sister A B C DRB DQB Parent 1 Parent 2 A B C DRB DQB
44 Family Study 2
45 Family study 2 Patient A B C DRB (17) DQB1 03(8) 02
46 Family study 2 Brother Patient A B C DRB (17) 03(8) 03(7) DQB1 03(8) 02
47 Family study 2 Father Mother A B C DRB (17) 03(17) 04 DQB1 03(8) (7) Brother Patient A B C DRB (17) 03(8) 03(7) DQB1 03(8) 02
48 Family study 2 Father Mother A B C DRB (17) 03(17) 04 DQB1 03(8) (7) 01 Brother Patient A B C DRB (17) 03(8) 03(7) DQB1 03(8) 02
49 Family study 2 Father Mother A Identical haplotypes B C DRB (17) 03(17) 04 DQB1 03(8) (7) 01 Brother Patient A B C DRB (17) 03(8) 03(7) DQB1 03(8) 02
50 Family study 2 Father Mother A Identical haplotypes B C DRB (17) 03(17) 04 DQB1 03(8) (7) 01 Brother Patient A B C DRB (17) 03(8) 03(7) DQB1 03(8) 02 Father and son are a 10/10 HLA match!
51 Family study 3
52 Family study 3 Patient sister 1 sister 2 sister 3 A B 44 14(65) (65) C DRB (17) 14 03(17) DQB1 03(7) 03(7) (7) 03(7) 05 Predicted parental typing Parent t1 Parent t2 A B (65) 35 C DRB (17) DQB1 03(7) 02 03(7) 05
53 Family study 3 Patient sister 1 sister 2 sister 3 A B 44 14(65) (65) C DRB (17) 14 03(17) DQB1 03(7) 03(7) (7) 03(7) 05 Predicted parental typing Parent t1 Parent t2 A B (65) 35 C DRB (17) DQB1 03(7) 02 03(7) 05 No Match Double cousins in the family!
54 GP 1 GP 2 GP 3 GP 4 Sister ¼ chance ¼ chance Parent 1 Parent 2 Brother HLA ID HLA ID Patient Sister 1 Sister 2 Sister 3 ¼ x ¼ = 1/16 (6.25%) chance of presence of all 4 haplotypes Cousin 1 Cousin 2 Double cousins ¼ X 1/16 = 1/64 (1.5%) chance of inheriting the same haplotypes as the patient
55 Family study 3 Patient sister 1 sister 2 sister 3 A B 44 14(65) (65) C DRB (17) 14 03(17) DQB1 03(7) 03(7) (7) 03(7) 05 Double cousins Cousin 1 Cousin 2 A B 44 14(65) 44 14(65) C DRB DQB1 03(7) 03(7) 03(7) 03(7) Both double cousins were matches with the patient!
56 Platelet Transfusion Refractoriness Definition a post-transfusion platelet count that is less than expected 1h corrected count increment (CCI) 9 < 5 10 x 10 9 /L percent platelet recovery (PPR) < 20% 1h CCI < 5 x 10 9 /L x2 (twice) using ABOidentical platelets 56
57 Platelet Transfusion Refractoriness Incidence 7-34% in hematology/oncology patients depending on a study and definition of refractoriness (Hod and Schwartz, 2008). 57
58 Platelet Transfusion Refractoriness - Etiology Non-immune causes fever sepsis splenomegaly DIC bleeding VOD GVHD 58
59 Immune causes Platelet Transfusion Anti-ABO antibodies Refractoriness Anti-HPA antibodies (2-11%, may be auto and/or transient) Anti-HLA antibodies (Class I HLA only, A and B) Anti HLA antibodies (Class I HLA only, A and B) account for the majority of cases of immune causes.
60 Platelet Transfusion Refractoriness - Etiology causes of alloimmunization: - transfusion (contaminating leukocytes) - pregnancy - transplantation 60
61 TRAP Study, Schlichter et. al Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997; 337:1861-9
62 Platelet Transfusion Refractoriness - Etiology Proportion of cases due to immune vs non-immune etiologies is not clear. Incidence varies depending on a study and HLA antibody detection methods. Most studies performed in the 1980s and 1990s utilizing older and less reliable HLA antibody detection techniques. 62
63 HLA antibody identification by Luminex HLA antigen coated beads (solid phase) Assay 2 lasers Tells the instrument which bead is being examined Tells the instrument how much antibody is bound to the bead
64 HLA antibody detection by Luminex assay
65 HLA antibody detection by Luminex assay A1 A2 A3 A11 A23 A24 A25 A26 A29 A30
66 HLA antibody detection by Luminex assay 8 A26 10 A30 1 A1 5 9 A29 6 A24 2 A2 4 A11 1 A1 7 A25 8 A A23 A A30 A23 2 A2 3 A3 7 A25 A3 4 A11 A24
67 HLA antibody detection by Luminex assay 8 A26 10 A30 1 A1 5 9 A29 6 A24 2 A2 4 A11 1 A1 7 A25 8 A A23 A A30 A23 2 A2 3 A3 7 A25 A3 4 A11 A24
68 HLA antibody detection by Luminex assay 8 A26 10 A30 1 A1 5 9 A29 6 A24 2 A2 4 A11 1 A1 7 A25 8 A A23 A A30 A23 2 A2 3 A3 7 A25 A3 4 A11 A24
69 HLA antibody detection by Luminex assay 8 A26 10 A30 1 A1 5 9 A29 6 A24 2 A2 4 A11 1 A1 7 A25 8 A A23 A A30 A23 2 A2 3 A3 7 A25 A3 4 A11 A24
70 HLA antibody detection by Luminex assay PE-α-IgG 8 A26 10 A30 1 A1 5 9 A29 6 A24 2 A2 4 A11 1 A1 7 A25 8 A A23 A A30 A23 2 A2 3 A3 7 A25 A3 4 A11 A24
71 HLA antibody detection by Luminex assay 8 A26 10 A30 1 A1 5 9 A29 6 A24 2 A2 4 A11 1 A1 7 A25 8 A A23 A A30 A23 2 A2 3 A3 7 A25 A3 4 A11 A24
72 HLA Class I antibody analysis Patient A3,31 31 B7,60 DR1,14 14 (52) DQB5,6
73 HLA Class I antibody analysis Patient A3 31 B7 60 DR1 14 (52) DQB5 6 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2
74 HLA Class I antibody analysis Patient A3 31 B7 60 DR1 14 (52) DQB5 6 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2
75 HLA Class II antibody analysis Patient A3 31 B7 60 DR1 14 (52) DQB5 6 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2
76 HLA Class II antibody analysis Patient A3 31 B7 60 DR1 14 (52) DQB5 6 Patient A3,31 B7,60 DR1,14 (52) DQB5,6 Donor A1, B8 DR7,17 (53,52) DQB2
77 CPRA Calculator Resources, professional resources, choose cpra calculator from options
78
79 CDHA Policy platelet count < 10 at 24 hours on two occasions following buffy coat platelet pool give single donor apheresis platelets (ABO matched) platelet count < 10 at 24 hours on two occasions following oo gsingle gedonor o apheresis es s pateets( platelets (ABO matched) give HLA matched platelets platelet count < 10 at 24 hours on two occasions following single donor apheresis platelets (HLAmatched) give HPA matched platelets 79
80 HLA Alloimmunization Rate In The Setting Of Refractoriness To Platelet Transfusion: A Single Center Study Sharpe C, Liwski R, Couban S, Sadek I, Kahwash E
81 Patients/Methods 33 platelet refractory patients identified at the QEII hospital between 01/06and06/11 06/ male 15 female 28 diagnosed with a hematological malignancy platelet refractoriness: platelet count rise of <10X10 9 /L within 24 hours post tx of ABO matched single donor platelets on at least 2 occasions. HLA antibody analysis was performed by single antigen luminex assay at the CBS laboratory in Winnipeg. PRA was calculated using OPTN cpra calculator
82 Results HLA antibodies were found in 42% of patients 4/18 males (22%) 10/15 females (67%) Degree of sensitization males were mildly sensitized with an average PRA of 33% (range 11 62%) females were highly sensitized (PRA values %) No association between the presence of HLA antibodies and either patient t diagnosis i or a history of allogeneic stem cell transplantation.
83 Conclusions/Implications We found that in most patients (60%) platelet refractoriness was due to non immunological causes. Approximately 2/3 of sensitized patients (all females) were highly hl sensitized (PRA = %) and would require HLA matched platelets. Approximately 1/3 of the sensitized patients (all males) had low PRA values and could probably be managed with using antigen negative platelets. ltlt
84 Suspect Alloimmune refractoriness ABO identical platelets Not refractory, support With standard platelets Measure increment x 2 Measure PRA Define antibody Crossmatch random platelets unit < 20% > 20% HLA ab present HLA absent HLA matched or antigen negative Consider and treat non immune causes Manage bleeding as needed Modified from Hod and Schwartz, 2008
85 Suspect alloimmune platelet refractoriness ABO identical platelets Not refractory, support With standard platelets Measure increment x 2 Measure PRA Define antibody Crossmatch random platelets unit < 20% > 20% HLA ab present HLA absent HLA matched or antigen negative Consider and treat non immune causes Manage bleeding as needed Modified from Hod and Schwartz, 2008
86 Thank you
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