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The role of HLA in Allogeneic Hematopoietic Stem Cell Transplantation and Platelet p
Refractoriness.
Robert Liwski, MD, PhD, FRCPCMedical Director
HLA Typing Laboratory
Department of Pathology
Dalhousie University
DALHOUSIE UNIVERSITYInspiring minds
IntroductionIntroduction
• Hematopoietic stem cell transplant (HSCT) is aHematopoietic stem cell transplant (HSCT) is a commonly used treatment for hematologic malignancies or bone marrow failuremalignancies or bone marrow failure syndromes/immunodeficiency
Indications for Allogeneic HSCTIndications for Allogeneic HSCT
– Chronic myeloid leukemia (CML)Chronic myeloid leukemia (CML)– Acute myeloid leukemia (AML)– Acute lymphoblastic leukemia (ALL)Plasma cell myeloma– Plasma cell myeloma
– Aplastic anemia– Congenital bone marrow failure syndromes– Congenital immunodeficiency (SCID)– Sickle cell anemia– ThalassemiaThalassemia– Selected solid tumors
Allogeneic HSCTAllogeneic HSCT
• Conditioning with high dose chemotherapy and g g pyradiation therapy – lethal
• Transplant with stem cell from healthy allogeneic donor.
PBSC mobili ed with G CSF– PBSC mobilized with G‐CSF – BM– Umbilical cord
Allogeneic HSCTAllogeneic 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.
HVG, GVH,GVLHVG, GVH,GVL
• T cell mediated reactions against the graft (stemT 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 differencesmajor or minor histocompatibility differences (MHC, and mHC) between the donor an recipient.
• In humans MHC molecules are named Human• In humans MHC molecules are named Human Leukocyte Antigen (HLA).
HLA class I and class II antigens
• Monomer with non‐ • Heterodimercovalently associated subunit (β2m)
• Presents antigenic
• Presents antigenic peptides to CD4+ T cellsese ts a t ge c
peptides to CD8+ T cells
E d b ll
ce s
• Restricted expression on antigen presenting ll (d d iti ll B• Expressed by all
nucleated cells including endothelium
cells (dendritic cells, B cells, macrophages)
• Inducible on other cells (endothelium and epithelium)
HLA inheritanceACB 4 haplotypes
DRDQ
Mother Father
Sib 1 Sib 2 Sib 3 Sib 4
Approximately 25‐30% chance of having an HLA matched sibling
Polymorphism of the Major Histocompatibility Complex in humans HLAin humans ‐ HLA
89356914313 81435 10628 136 89356933 835 068 36
68143111652 63726 7716 118
Polymorphism of the Major Histocompatibility Complex in humans HLAin humans ‐ HLA
89356914313 81435 10628 136 89356933 835 068 36
68143111652 63726 7716 118
2139181 2612 136 22 Effective(caucasians)
Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove)
Top view Side views
HLA-A
β2 i l b liHLA-B β2 microglobulin
HLA-C
Relevance of HLARelevance of HLA
• Necessary to mount immune responses against hpathogens
– Polygenic ‐ survival advantage to individual– Polymorphic ‐ survival advantage to speciesy p g p
• Transplantation– Difficulty finding compatible donorsDifficulty finding compatible donors.– Causes sensitization (T cell response and antibody response)
– Can lead to graft rejection (failure to engraft) orCan lead to graft rejection (failure to engraft) or GVHD
Major mismatch/direct allorecognition
Self T cell Donor T cell
Normal situation HSCT situation
Vi l nti n
TCR
Self HLA
Viral antigen(peptide)
Allo HLA
Foreign viral antigenand self HLA
Self antigenand allo HLA and self HLA and allo HLA
Self body cell Recipient cell
5‐10% T cells able to respond to allo‐MHCPotent reaction
HLA antibody development
Your (“self”) HLA “allo” HLA
Sensitizing events:TransfusionPregnancy Transplantation
Donor selection for allo HSCTDonor selection for allo HSCT
• HLA inheritance pattern
• Extreme HLA polymorphism
Probability of finding HLA‐Identical DonorProbability of finding HLA Identical Donor
• Sibling 1:4 (30% on average)Sibling 1:4 (30% on average)
• Parent 1:100
C i l 0 000• Cousin, uncle, etc 1:10,000
• Unrelated 1:1,000,000
• Worldwide Registry of Bone Marrow DonorsWorldwide Registry of Bone Marrow Donors
• >10,000,000 donors
Patient workupPatient workup
New patientHLA TypingA, B, C, DR, DQ, , , , Q
Siblings and/or related donors?
Yes NoHLA TypingA, B, C, DR, DQ
70%
Match No match
70%
Unrelated donor searchHLA TypingA, B, C, DR, DQ
30%
Matched donor Mismatched donor
50% 20%
Patient brother 1 brother 2 brother 3 sister
Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Patient brother 1 brother 2 brother 3 sister
Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Brother 1 shares 1 haplotype
Patient brother 1 brother 2 brother 3 sister
Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Brother 1 h 1 h l tshares 1 haplotype
Patient brother 1 brother 2 brother 3 sister
Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Brother 1 h 1 h l t
Brother 2 I t t hshares 1 haplotype Is not a match
Patient brother 1 brother 2 brother 3 sister
Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Brother 1 h 1 h l t
Brother 2 I t t h
Brother 3 h 1 h l tshares 1 haplotype Is not a match shares 1 haplotype
Patient brother 1 brother 2 brother 3 sister
Family study 1A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Brother 1 h 1 h l t
Brother 2 I t t h
Brother 3 h 1 h l t
Sisteri 10/10 t hshares 1 haplotype Is not a match shares 1 haplotype is a 10/10 match
patient brother 1 brother 2 brother 3 sister
Family study 1p
A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Parent 1 Parent 2
A
BB
C
DRB1DRB1
DQB1
patient brother 1 brother 2 brother 3 sister
Family study 1p
A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Parent 1 Parent 2
A 02 24B 07 62B 07 62C 07 9
DRB1 13 04DRB1 13 04
DQB1 06 8
patient brother 1 brother 2 brother 3 sister
Family study 1p
A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Parent 1 Parent 2
A 02 02 24B 07 50 62B 07 50 62C 07 06 9
DRB1 13 17 04DRB1 13 17 04
DQB1 06 02 8
patient brother 1 brother 2 brother 3 sister
Family study 1p
A 02 24 02 24 01 02 02 01 02 24B 07 62 50 62 08 50 07 08 07 62C 07 9 06 9 07 06 07 07 07 9C 07 9 06 9 07 06 07 07 07 9
DRB1 13 04 17 04 17 17 13 17 13 04
DQB1 06 8 02 8 02 02 06 02 06 8
Parent 1 Parent 2
A 02 02 24 01B 07 50 62 08B 07 50 62 08C 07 06 9 07
DRB1 13 17 04 17DRB1 13 17 04 17
DQB1 06 02 8 02
Family study 2
Brother Patient02 02 A 02 0127 44 B 27 0802 05 C 02 0704 04 DRB1 04 03(17)03(8) 03(7) DQB1 03(8) 0203(8) 03(7) DQB1 03(8) 02
Family study 2
Father MotherA 02 01 01 02B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)
Brother Patient02 02 A 02 0127 44 B 27 0802 05 C 02 0704 04 DRB1 04 03(17)03(8) 03(7) DQB1 03(8) 0203(8) 03(7) DQB1 03(8) 02
Family study 2
Father MotherA 02 01 01 02B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)
01
Brother Patient02 02 A 02 0127 44 B 27 0802 05 C 02 0704 04 DRB1 04 03(17)03(8) 03(7) DQB1 03(8) 0203(8) 03(7) DQB1 03(8) 02
Family study 2
Father MotherA 02 01 01 02Identical haplotypes
B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)
01
Brother Patient02 02 A 02 0127 44 B 27 0802 05 C 02 0704 04 DRB1 04 03(17)03(8) 03(7) DQB1 03(8) 0203(8) 03(7) DQB1 03(8) 02
Family study 2
Father MotherA 02 01 01 02Identical haplotypes
B 27 08 08 44C 02 07 07 05DRB1 04 03(17) 03(17) 04DQB1 03(8) 02 02 03(7)
01
Brother Patient02 02 A 02 0127 44 B 27 0802 05 C 02 0704 04 DRB1 04 03(17)03(8) 03(7) DQB1 03(8) 0203(8) 03(7) DQB1 03(8) 02
Father and son are a 10/10 HLA match!
Family study 3Patient sister 1 sister 2 sister 3
A 02 68 01 11 01 68 02 11B 44 14(65) 08 35 08 14(65) 44 35C 05 08 07 04 07 08 05 04C 05 08 07 04 07 08 05 04DRB1 04 13 03(17) 14 03(17) 13 04 14DQB1 03(7) 03(7) 02 05 02 03(7) 03(7) 05
Predicted parental typing
P t 1 P t 2Parent 1 Parent 2A 02 01 68 11B 44 08 14(65) 35C 05 07 08 04DRB1 04 03(17) 13 14DQB1 03(7) 02 03(7) 05
Family study 3Patient sister 1 sister 2 sister 3
A 02 68 01 11 01 68 02 11B 44 14(65) 08 35 08 14(65) 44 35C 05 08 07 04 07 08 05 04C 05 08 07 04 07 08 05 04DRB1 04 13 03(17) 14 03(17) 13 04 14DQB1 03(7) 03(7) 02 05 02 03(7) 03(7) 05
Predicted parental typing
P t 1 P t 2Parent 1 Parent 2A 02 01 68 11B 44 08 14(65) 35C 05 07 08 04DRB1 04 03(17) 13 14DQB1 03(7) 02 03(7) 05
No MatchDouble cousins in the family!
GP 1 GP 2 GP 3 GP 4
Sister Parent 1 Parent 2 Brother¼ chance HLA ID
¼ chance HLA ID
Patient Sister 1 Sister 2 Sister 3
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 2Double cousins
¼ X 1/16 = 1/64 (1.5%) chance of inheriting the same haplotypes as the patient
Family study 3Patient sister 1 sister 2 sister 3
A 02 68 01 11 01 68 02 11B 44 14(65) 08 35 08 14(65) 44 35C 05 08 07 04 07 08 05 04C 05 08 07 04 07 08 05 04DRB1 04 13 03(17) 14 03(17) 13 04 14DQB1 03(7) 03(7) 02 05 02 03(7) 03(7) 05
Double cousins
Cousin 1 Cousin 2A 02 68 02 68B 44 14(65) 44 14(65)C 05 08 05 08C 05 08 05 08DRB1 04 13 04 13DQB1 03(7) 03(7) 03(7) 03(7)
Both double cousins were matches with the patient!
Platelet Transfusion fRefractoriness
• Definition“a post-transfusion platelet count that is less than expected” 1h corrected count increment (CCI)
9< 5 – 10 x 109/Lpercent platelet recovery (PPR) < 20%1h CCI < 5 x 109/L x2 (twice) using ABO-identical platelets
56
Platelet Transfusion fRefractoriness
• Incidence7-34% in hematology/oncology patients depending on a study and definition of refractoriness (Hod and Schwartz, 2008).
57
Platelet Transfusion fRefractoriness - Etiology
• Non-immune causesfeversepsissplenomegalyDICDICbleedingVODGVHDGVHD
58
Platelet Transfusion fRefractoriness
• Immune causesImmune causes
Anti-ABO antibodies
Anti-HPA antibodies (2-11%, may be auto and/or transient)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.
Platelet Transfusion fRefractoriness - Etiology
causes of alloimmunization: transfusion (contaminating leukocytes)- transfusion (contaminating leukocytes)
- pregnancy - transplantation- transplantation
60
TRAP Study, Schlichter et. al. 1997TRAP Study, Schlichter et. al. 1997
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusionsprevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997; 337:1861-9
Platelet Transfusion fRefractoriness - Etiology
Proportion of cases due to immune vs non immune etiologies is not clearnon-immune etiologies is not clear.
Incidence varies depending on a study and HLAIncidence varies depending on a study and HLA antibody detection methods.
Most studies performed in the 1980s and 1990s utilizingMost studies performed in the 1980s and 1990s utilizing older and less reliable HLA antibody detection techniques.
62
HLA antibody identification by Luminex (solid phase) Assay(solid phase) Assay
HLA antigen coated beads
Tells the instrument which bead is being examined
2 lasers
Tells the instrument how much antibody is bound to the bead
HLA antibody detection by Luminex assay
9
A29
78
A26
1810
A29
A30
4
A11 5
6
A24
A25
910
A301
3 65
A1A26A30
2
A2
1
A1
5
A23
A24A29
A30
24
7
6
A3
A11
A23 A24
A25
A2
3
A3A2
A11A3
HLA antibody detection by Luminex assay
9
A29
78
A26
1810
A29
A30
4
A11 5
6
A25A26
910
A301
3 65
A1A26A30
2
A2
1
A1
5
A23
A24A29
A30
24
7
6
A3
A11
A23 A24
A25
A2
3
A3A2
A11A3
HLA antibody detection by Luminex assay
9
A29
78
A26
1810
A29
A30
4
A11 5
6
A25A26
910
1
3 65
A1A26A30
2
A2
1
A1
5
A23
A24A29
A30
2
3
47
65
A3
A11
A23 A24
A25
A2
3
A3A2
A11A3
HLA antibody detection by Luminex assay
9
A29
78
A26
1810
A29
A30
4
A11 5
6
A24
A25A26
910
A301
3 65
A1A26A30
2
A2
1
A1
5
A23
A24A29
A30
24
7
6
A3
A11
A23 A24
A25
A2
3
A3A2
A11A3
HLA antibody detection by Luminex assay
PE-α-IgG
9
A29
78
A26
1810
A29
A30
4
A11 5
6
A24
A25A26
910
A301
3 65
A1A26A30
2
A2
1
A1
5
A23
A24A29
A30
24
7
6
A3
A11
A23 A24
A25
A2
3
A3A2
A11A3
HLA antibody detection by Luminex assay
9
A29
78
A26
1810
A29
A30
4
A11 5
6
A24
A25
910
A301
3 65
A1A26A30
2
A2
1
A1
5
A23
A24A29
A30
24
7
6
A3
A11
A23 A24
A25
A2
3
A3A2
A11A3
HLA Class I antibody analysis
Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6
HLA Class I antibody analysis
Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class I antibody analysis
Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class II antibody analysis
Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2
HLA Class II antibody analysis
Patient A3 31 B7 60 DR1 14 (52) DQB5 6Patient A3,31 B7,60 DR1,14 (52) DQB5,6Donor A1, B8 DR7,17 (53,52) DQB2
CPRA Calculator
http://optn.transplant.hrsa.gov/ Resources, professional resources, choose cPRA calculator from options
CDHA PolicyCDHA Policy• platelet count < 10 at 24 hours on two occasions following buffy coat platelet pool ‐ give single donorfollowing buffy coat platelet pool ‐ give single donor apheresis platelets (ABO‐matched)
• platelet count < 10 at 24 hours on two occasions following single donor apheresis platelets (ABO‐o o g s g e do o ap e es s p ate ets ( Omatched) ‐ give HLA‐matched platelets
• platelet count < 10 at 24 hours on two occasions following single donor apheresis platelets (HLA‐
79
matched) ‐ give HPA‐matched platelets
HLA Alloimmunization Rate In The Setting OfHLA Alloimmunization Rate In The Setting Of Refractoriness To Platelet Transfusion: A
Single Center StudySingle Center Study
Sharpe C, Liwski R, Couban S, Sadek I, Kahwash E
Patients/MethodsPatients/Methods• 33 platelet refractory patients identified at the QEII hospital
between 01/06 and 06/11between 01/06 and 06/11.
– 18 male
– 15 female
– 28 diagnosed with a hematological malignancy
• platelet refractoriness: platelet count rise of <10X109/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• HLA antibody analysis was performed by single antigen luminex assay at the CBS laboratory in Winnipeg.
• PRA was calculated using OPTN cPRA calculator
Results• HLA antibodies were found in 42% of patients
– 4/18 males (22%)4/18 males (22%)
– 10/15 females (67%)
• Degree of sensitization– males were mildly sensitized with an average PRA of 33% y g(range 11‐62%)
– females were highly sensitized (PRA values 95‐100%)
• No association between the presence of HLA tib di d ith ti t di i hi tantibodies and either patient diagnosis or a history
of allogeneic stem cell transplantation.
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 hi hl iti d (PRA 95 100%) d ld i HLAhighly sensitized (PRA = 95‐100%) 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 ti ti l t l tantigen negative platelets.
Suspect Alloimmune refractoriness
ABO identical platelets
Measure increment x 2Not refractory, supportWith standard platelets
Define antibodyMeasure PRA Crossmatchrandom platelets unitp
HLA ab present HLA absent< 20% > 20%
HLA matched or antigen negative
Consider and treat non immune causesManage bleeding as needed
Modified from Hod and Schwartz, 2008
Suspect alloimmune platelet refractoriness
ABO identical platelets
Measure increment x 2Not refractory, supportWith standard platelets
Define antibodyMeasure PRA Crossmatchrandom platelets unitp
HLA ab present HLA absent< 20% > 20%
HLA matched or antigen negative
Consider and treat non immune causesManage bleeding as needed
Modified from Hod and Schwartz, 2008