the abc’s of hla - ucsf medical educations_.pdfthe abc’s of hla: beginners to advanced...
TRANSCRIPT
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The ABC’s of HLA:Beginners to Advanced
�Rajalingam Raja, Ph.D, D(ABHI)�Professor of Clinical Surgery�Director of Immunogenetics and Transplantation Laboratory�University of California, San Francisco
� Phone: 415-476-0647� Email: [email protected]
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HLA class I
Cell
HLA Antibody
23%
In peripheral blood
CytotoxicT cell
TCR
CD8
Every cell expresses a HLA to present antigens to T lymphocytes
Infection
Perforine, Granzyme, Granulysin
HLA class II
APC
Helper T cell
TCR
CD4
48%
B cell
BCR
11%
IL2, IFN-γ
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HLA is the Challenging Barrier to Transplantation
HLA mismatched Allograft
Recipient
• Transplantation• Pregnancy• Transfusion
Antibody DepletionPlasmaphoresis
Antibody BlockingIVIGAnti-C5a
Unacceptable Antigens HLA antibodies
Plasma cell
Rejection Lymphocytes
TTTT
TTTT
TTTT
TTTT
TTTT
NKNKNKNK
BBBB
BBBB
BBBB
NKNKNKNK
NKNKNKNK
MaintenanceTherapy
ImmunosuppressionCyclosporine MMFSteroids
InductionTherapy
Lymphocytes DepletionAnti-Thymoglobulin → T & NK cellsAnti-CD3 → T cellsAnti-CD25 → Activated T cellsAnti-CD52 → mature lymphocytesAnti-CD20 → B cells
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Consequences of Pre-formed Donor-Specific HLA Antibodies
• Hyperacute rejection• Delayed graft function• Accelerated acute rejection• Chronic rejection• Prolonged waiting times• No transplantation
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Means of HLA Antibody-Mediated Rejection
Donor-Specific HLA Antibodies
1. Activation of complement cascade
DSA
C1q
2. Antibody-dependent Cell-mediated cytotoxicity (ADCC)
FcR
NK Cell
3. Opsonization & increased antigen presentation
FcR
APCAPC4. Activation of
Endothelial Cell
OrganAllograftEndothelium
HLA Class I
HLA Class II
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Histocompatibility Testing for Solid Organ Transpla ntation
DonorRecipient
Pre-Transplant
Post-Transplant
HLA Typing HLA Typing HLA Mismatch
HLA Antibodies Preformed-DSA
Crossmatch Compatibility
Serum Cells
HLA Antibodies Donor-specific Antibodies (DSA)
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Complement
Donor Recipient
Lymphocytes Serum+
Complement Dependent Cytotoxicity (CDC) Crossmatch
Fluorescein Diacetate + Ethidium Bromide
Live cellsDead cells
Positive Negative
Membrane Attack Complex (MAC)
Paul Terasaki
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CDC xM(n=225)
Hyperacute or Accelerated
Rejection
Functional Graft
Positive(n=30)
24 6
Negative(n=195)
8 187Specificity Problem
Sensitivity Problem
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Flow Cytometry CrossmatchDonor Recipient
Lymphocytes Serum+
Anti-human IgG F(ab’)2 FITC
• Median Chanel Shift (MCS) –a quantitative readout (Ag+Ab)
• Detects only IgG antibodies• Non-specific reactivity can be
reduced by Pronase digestion
Measure FITC intensity by flow cytometry
T cell MCS > 50B cell MCS > 120
Negative Control
Patient Serum
T cell B cell
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Flow Crossmatch - problems
• ~8% of flow crossmatches are false positive – unneccessary exclusion
• ~7% of flow crossmatches are false negative – risk to patient
Flow Cytometry Crossmatch
Donor Recipient
Lymphocytes Serum
Anti-human IgG F(ab’)2 FITC
+
• Median Chanel Shift (MCS) –a quantitative readout (Ag+Ab)
• Detects only IgG antibodies• Non-specific reactivity can be
reduced by Pronase digestion
Measure FITC intensity by flow cytometry
T cell MCS > 50B cell MCS > 120
Negative Control
Patient Serum
T cell B cell
Virtual
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Donor Recipient
Lymphocytes Serum
Virtual Crossmatch - Essentials
HLA Typing
HLA Antibody Testing
A2, A24, B7, B18, DR1, DR4
Anti HLA-A2 antibodies
VirtualCrossmatchPositive
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Crossmatch Methods
Crossmatch method
Sensitivity Specificity Cost(US $)
Turnaroundtime
CDC Low Low 600 3.5 hours
Flow Intermediate Intermediate 600 5 hours
Pronase >Intermediate >intermediate 600 6.5 hours
Virtual 100% 100% 0 10 min
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Virtual Crossmatch - Advantages
• Eliminates the physical crossmatch- Saves 4-6 hours – cut downs cold ischemic time - No samples required - Reduces laboratory & OPO workload - Reduces laboratory, OPO, and Tx program cost
• Adds precision to actual crossmatch- CDC/flow XM prediction- DSA identification
• Improves allocation efficiency• Increased rate of transplantation for sensitized
patients• Risk of memory response can be accounted:
Previous transplants & pregnancies
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Virtual crossmatch by listing Unacceptable Antigens in UNet
X
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1 36 7 44 9 17 4 9
2 24 7 18 1 10 5 52 29 13 51 8 14 4 8
2 68 39 71 15 16 5 6
2 34 57 61 11 14 2 4
2 25 39 65 9 17 4 9 2 23 44 45 13 18 7 81 2 8 62 4 17 4 7
Candidate:
Potential Donors, >12,000
A B DR DQ
+ anti-DR4 61% cPRA
+ anti-DQ5 76% cPRA
Unacceptable HLA Antigens & Virtual Crossmatch
1 68 8 13 4 15 2 5
69 74 55 60 4 7 7 8 3 24 18 39 1 4 4 4
24 43 27 45 4 8 4 8
66 68 27 39 4 15 8 5
23 26 49 62 1 17 2 5
11 33 51 64 15 18 5 7
3 29 35 44 1 11 7 6
48% cPRAanti-A2
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• Increase priority for sensitized candidates/CPRA sliding scale
• Replace SCD/ECD with KDPI• Add longevity matching• Include pre ‐registration dialysis time• Incorporate A2/A2B to B• Base pediatric priority on KDPI• Remove payback system• Remove variances
KAS: Major Allocation Components
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CPRA (%) KAS Priority PointsOld New
0–19 020–29 030–39 040–49 050–59 060–69 070–74 075–79 080–84 485–89 490–94 4
95 496 497 498 499 4100 4
Regional SharingNational Sharing
Priority points for CPRA>19%
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Distribution of CPRA scores in UCSF Kidney Transpla nt Waitlist (n=5461)
100%99%98%85-97%20-84%0-19%
CPRA5%4%
1.6%0.8%
15.9%
72.7%
Points
202.150.0924.44.05-17.30.08-2.460
#Patients
21685462738673974
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Sequence-Specific Oligonucleotide ( SSO) Hybridization Method
Polystyrene MicrospheresPolystyrene Microspheres
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• 100 Color-coded Polystyrene beads using a blend of different fluorescent intensities of two dyes• Each bead is conjugated with oligonucleotide probe specific for a HLA allele (s)
Luminex technologyA2
A1
A11
A3
A23
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PCR amplificationusing florochrome-taggedlocus-specific primers
Cell
DNA
*
Amplified DNA
Denatured PCR
products
Detection & Interpretation
Luminex: rSSO Method
A2 A1
A11
A3
A23
A2 A1
A11
A3
A23
Hybridization
+
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Single Antigen Bead-based HLA Antibody Testing: Lum inex Technology
Polystyrene MicrospheresPolystyrene Microspheres
A2
A66B55
Single HLA Antigen Beads
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Detection & Interpretation
A2
A66B55
patient’s serum
A2A66
B55+
Single Antigen beads
Single Antigen Bead-based HLA Antibody Testing: Lum inex Technology
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HLA class I antibody test results: Antibodies to A2 CREG
• Specificities: A2, A68, A69, B57, B58• CPRA: 62%• One Antibody
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α1-domain α2-domain
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Public and Private Epitopes (antigenic determinants)
Public Epitopes
A36A1B58B57A69A68A2
Private Epitopes
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• Specificities: A2, A68, A69, B57, B58• CPRA: 62%• One Antibody
CPRA: 62%
HLA class I antibody test results: Antibodies to A2 CREG
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Recipient
A1, A1, B7, B8
A28A23
A69
A68
B57
A24 B58
No antibodies to self-HLA are made.
Allograft
A1, A2, B7, B8
Individuals alloimmunized by a specific HLA type ca n make antibodies to many HLA types.
Anti-A2
Cross-REactive groups (CREG)
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Cross-REactive Groups (CREG)CREG HLA Specificities CPRA value
A1 A1,A3,A11,A29,A30,A31,A36,A80 65%
A2 A2,A23,A24,A68,A69,B57,B58 75%
A10 A25,A26,A32,A33,A34,A43,A66,A74 22%
Bw4 A23,A24,A25,A32,Bw4 74%
B5 B18,B35,B46,B49,B50,B51,B52,B53,B62,B63,B71,B72,B73,B75,B76,B77,B78 56%
Bw6 Bw6 85%
B7 B7,B8,B13,B27,B41,B42,B47,B48,B54,B55,B56,B59,B60,B61,B67,B81,B82 59%
B8 B8,B18,B38,B39,B59,B64,B65,B67 36%
B12 B13,B37,B41,B44,B45,B47,B49,B50,B60,B61 48%
C1 Cw1,Cw7,Cw8,Cw9,Cw10,Cw12,Cw14,Cw16,B46,B73 77%
C2 Cw2,Cw4,Cw5,Cw6,Cw15,Cw17,Cw18 66%
DR1 DR1,DR10,DR103 21%
DR51 DR51,DR15,DR16 29%
DR52 DR52,DR11,DR12,DR13,DR14,DR17,DR18 62%
DR53 DR53,DR4,DR7,DR9 50%
DQ1 DQ5,DQ6 64%
DQ2 DQ2 37%
DQ3 DQ7,DQ8,DQ9 56%
DQ4 DQ4 10%
DP1c DP2,DP3,DP4,DP6,DP9,DP10,DP11,DP14,DP17,DP18.DP20,DP28 ----
DP2c DP1,DP5,DP13,DP15,DP19,DP23 ----
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Bw6 Antibodies
CPRA=85%
A B Cw DR DR DQ2 13(Bw4) 10 15 51 533 38(Bw4) 7 16 51 5
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Spouse HLAA2-B61(Bw6)-DR4A2-B39(Bw6)-DR4
Women alloimmunized by Bw6 motif can make antibodies to 2/3 of HLA-B types
B7, B8, B14, B18, B22, B35, B39, B40, B4005, B41, B42, B45, B46, B48, B50, B54, B55, B56, B60, B61, B62, B64, B65, B67, B70, B71, B72, B73, B75, B76, B78, B81, B82
A2-B44(Bw4)-DR4A2-B52(Bw4)-DR4
Self HLA
CPRA=85%
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Weak Bw6 Antibodies – Risk of memory response
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Bw4 Antibodies
CPRA=61%
A B Cw DR DR DQ2 35(Bw6) 4 4 53 831 35(Bw6) 4 11 52 7
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A B Cw DR DR DQ2 46 1 9 53 92 46 1 14 52 5
Bw4 & Bw6 Antibodies
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Antibodies to all HLA except to self-HLA
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Allele-specific Antibodies
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LABXpress™ Pipettor HLA Antibody Report
Page-1 of 2 Page-2 of 2
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HLA Lab
HLA lab updates VXM & PXM qualification weekly
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VXM orPXM
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Antibody Binding Sites on HLA (epitopes)
Peptide+HLAepitope
Conformational epitope
Peptide epitope
Linear epitope
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- Protein Miss fold- Denatured Antigens- Cryptic Epitope- Loss of Epitope- High Sensitivity- Variable Densities- Not all Alleles are Covered
Problems with Single Antigen Assay(False Positive/Negative Reactions)
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Female – 1 st Tx (n=1522)Female – Re-Tx (n=209)Male – 1st Tx (n=2109)Male – Re-Tx (n=259)
HLA Antibody Profile in Kidney Waiting List Candida tes (n=5281)
Negative (n=1182)
HLA Antibody Testing by Single Antigen Beads
Positive (n=4099)
HLA Antibody Screen by Mixed Beads/Phenotype Beads
4099 candidates X 123 antibodies = 504,177 antibodies with MFI
Female – 1 st Tx (n=364)Female – Re-Tx (n=26)Male – 1st Tx (n=751)Male – Re-Tx (n=41)
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Re-Tx FemaleN= 197
Re-Tx MaleN= 237
1st Tx FemaleN=1241
1st Tx MaleN=1537
A DPDQDRCB
Candidate
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� Locus-specific (<Cw)� Allele-specific (<B44)� Tissue-specific (<neuronal tissue)� Cytokine-induced (IFN- γ)� Down regulation by viral infection
and tumor transformation.
HLA Expression Variation
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Single Class I DSA MFI vs. T cell Crossmatch MCS
2000
50
51
Single Class II DSA MFI vs. B cell Crossmatch MCS
2000
120
0
50
100
150
200
250
300
350
400
450
500
0 5000 10000 15000 20000 25000 30000
MCS
MFI
DPB1DRB1DQB1DQA1DRB345
Few / well defined HLA-A,B,C, DR, DQB and/or DQAAntibodies only
- Typically >1000 MFI- CREG with any MFI
VXM- (DSA-)
HLA lab Protocol for Deceased Donor Kidney Transpla ntationHLA lab Protocol for Deceased Donor Kidney Transpla ntation
Single antigen bead HLA antibody identification (At least 2 sera are tested that are drawn within a year)Single antigen bead HLA antibody identification (At least 2 sera are tested that are drawn within a year)
HLA Antibodies Negative
HLA Antibodies Positive
None
VXM- (DSA-)
Unacceptable HLA Antigens
Crossmatch
All VXM are retrospectively confirmed by FXM
PXM - Call Lab/ Director PXM - Call Lab/ Director
Well defined antibodies and/or• DPβ, DPα Ab• Allele-specific Ab• Unstable Ab• Too many weak Ab
>2000 MFI
VXM FXM (pronase)
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Tray List ; quarterly sera
~900 Active Candidates• with total points of >7• all AB-blood group• consented for KDPI
>85%
Virtual XMcandidate Update
Unacceptable Antigens in UNet
Transplant
Antibody by Single HLA Beads
Physical XMcandidate
every 3 months
every 3 months
Waiting List (n=5416)
New Kidney Allocation System
New Candidate
HLA Typing
Antibodies by Single HLA Beads
& List Unacceptable Antigens;Receives points per CPRA
<20% CPRA
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5.5%17.9%
31.7% 28.8%
61.2%37.6%
74.8%60% 62.5%
33%56.9%
7.3% 8.3% 8.7% 5.8%
0%
20%
40%
60%
80%
100%
CPRA 100%(n=209)
99%(n=80)
95-98%(n=120)
80-94%(n=151)
0-79%(n=1159)
% of
cand
idates
Re-Tx candidates with total points >7 (n=1719)
32.6% (n=560)
Male
Female
Re-
tx1
st-t
x
55
0
20
40
60
80
100
DRB DPDQ
CA
DR53
DR51
DR52 DRB DPDQ
CA
DR53
DR51
DR52 DRB DPDQ
CA
DR53
DR51
DR52 DRB DPDQ
CA
DR53
DR51
DR52 DRB DPDQ
CA
DR53
DR51
DR52
Antibody profile of candidates with total points >7 (n=1719)
% of
cand
idates
CPRA 100%(n=209)
99%(n=80)
95-98%(n=120)
80-94%(n=151)
0-79%(n=1159)
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85.4%(n=108)
14.6%(n=44)
FXM
VXM
Most transplants are preformed using VXM approach in new KAS era
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0
10
20
30
40
50
60
70
80
90
Pre-KAS (1/1/2014 to 12/3/2014) n=235Post-KAS (12/4/2014 to 7/31/2015) n=152
CPRA 100%99%95-98%80-94%0-79%
% of
Tran
splan
tsPre- vs. Post-KAS: Transplant rate
58
59
99%n=60
100%n=202
98%n=49
80-97%n=276
20-79%n=939
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
1 3 5 7 9 110%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
1 3 5 7 9 110%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
1 3 5 7 9 11
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
1 3 5 7 9 110%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
1 3 5 7 9 11
CPRA
% F
requ
ency
of C
andi
date
s
Frequency of CREG Antibodies in Kidney waitlist can didates with different CPRA Groups
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Most 100% CPRA candidates are sensitized to large number of HLA antigens
Candidate#2:A :1 2 11 24 25 26 29 30 31 32 33 34 36 43 66 68 69 74B :13 18 27 37 38 39 41 42 44 45 46 47 49 51 52 53 54 55 56 57
58 59 61 62 63 64 65 67 7 71 72 73 75 76 77 78 8 81 82Cw :1 2 5 7 8 9 10 12 14 15 16 18DR :1 4 7 8 9 10 11 12 13 15 16 103 14:02DR :51 53DQ :4 6 7 8 9DQA:02 03DP :2 3 6 9 10 14 17 18 20 28 04:02
Candidate#1:DR :4 7 8 11 12 13 14 15 16 17 18 103DRw:51 52DQ :6 7 8 9
100%CPRA
100%CPRA
97%CPRA
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100
79.5
51.5
32.527.5
8.5
19.5
15.516.5
3.5
6
10.5
5.5
8.5
23
41.5
50.5
0
10
20
30
40
50
60
70
80
90
100
current >2000 >5000 >8500 >10000
CPRA
MFI Cutoff
% W
aitli
st C
andi
date
s (n
=200
)
<97%
98%
99%
100%
MFI Cutoff and CPRA
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Am
ount
of A
ntib
odie
s -
MF
I
De Nova DSA
CDC xMFlow xMDSA
+++
–++
––+
–––
Memory Response
HLA Antibodies and Risks of Antibody-Mediated Rejec tion
Accelerated Rejection
HyperacuteRejection
Chronic Rejection
63
45
179
21
16
367
136
Living Donor 147Deceased Donor 220
(Wait list=5198)
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Adult=69Pediatric=67
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Number of Transplants Performed in UCSF in 2015 (n= 779)
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Waiting List
University of California San Francisco (UCSF)Kidney Transplant Program (7/1/2014-6/30/2015)
Transplants
95.1% (n=105,743) 98% (n=17,425)
2.0% (n=359)4.9% (n=5,198) UCSF
All other
centers
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CIBMTR monitoring of 1-year Overall Survival for First Allogeneic HCT (performed 2011-2013) suggests that, based on the complexity of the HCTs performed at UCSF:
Our predicted OS rate should be 78.9% (95% CI: 71.5-86.6%)
Our actual OS was 85.4% (N = 103)
UCSF Tops North America in Pediatric Hematopoietic Stem Cell Transplant Outcome
Slide: Christopher C. Dvorak
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Immunogenetics and Transplantation LaboratoryDepartment of Surgery, University of California, San Francisco
Thank You