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KPD Webinar Series: Part 3 Demystifying the OPTN Kidney Paired Donation Pilot Program
Unacceptable HLA antigens:
The key to finding a compatible donor for your patient
M. Sue Leffell, Ph.D. J. Michael Cecka, Ph.D.
Facilitator: Ruthanne L Hanto RN, MPH
Histocompatibility Lab Director
Other Lab Personnel
Physician or Surgeon
Transplant Coordinator
Mixed group
Other
Who is in the room today?
1 Clueless
2 Novice
3 Basic understanding
4 Good at some stuff
5 Expert – ask me anything
What is your level of understanding of Donor Antigens and Candidate Unacceptables in KPD?
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Goal
Provide an understanding of the importance of defining unacceptable antigens for successful KPD matches.
Provide an understanding of the importance of donor antigen reporting in KPD paired donors
•This is an Exchange •There are 4 Matches in this Exchange •If the Match between Donor A and Candidate B is declined due to positive crossmatch – all other Matches in the Exchange also fall apart
20% Data are incomplete
40% other matches in the exchange fell through
Of the remaining (40%) of refused matches:
33% refused due to positive crossmatch or unacceptable antigens
7% due to “candidate involved in a pending exchange”
60% due to a variety of other donor and candidate reasons
OPTN KPDPP Match Offer Refusal Reasons
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Define appropriate candidate unacceptable antigens for successful KPD transplants.
Accurately report donor antigens for successful KPD donation.
Demonstrate data entry into the KPD application of UNet™
Objectives
Unacceptable HLA Antigens:
The key to finding a compatible donor for
your patient in KPD
Donor Antigens
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• J. Michael Cecka, Ph.D., Professor Emeritus
UCLA Immunogenetics Center
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UNOS Kidney Paired Donation
Recommendations
• Laboratory recommendations
•Molecular HLA typing HLA-A,B,C,Bw4,Bw6, DRB1-5, DQA, DQB
• DQA required for donors only
•Solid-phase tests for antibody (single antigen)
• Two levels of stringency – Unacceptable antigens + All others
•Update antibody profile whenever
• Antibodies change on quarterly screens
• A sensitizing event occurs
• A patient is inactive >3 mo
• An unexplained positive crossmatch occurs
•Histocompatibility Committee review of positive crossmatches in real time.
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Virtual Crossmatch
Patient
A2, A23, A24,A68,A69,B57,B58
Donor 1
A1, A2, B8, B57, Bw4, Bw6, DR4, DR17, DR52, DR53, DQA1*03, DQA1*05, DQ2, DQ7, DP2, DP6
Donor 2
Anti-HLA Antibodies
HLA Type
A1, A3, B7, B13, Bw4, Bw6, DR1, DR7, DR53,DQA1*01, DQA1*02, DQ2, DQ5, DP402
HLA Type
Incompatible
Compatible
• Accurate and realistic identification of patient antibodies that define unacceptable HLA antigens
• Accurate and complete identification AND reporting of donor HLA types
• If the donor antigens don’t match the patient’s antibody specificities there will be mistakes
• If the donor HLA type is not complete, there will be mistakes
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Critical Elements of the virtual crossmatch
Most virtual crossmatch failures are due
to:
a. Inability to identify all unacceptable HLA antigens
b. Lack of allele-level HLA typing of the donor
c. Data entry errors
d. Failure to type the donor for HLA-DP
e. Sleeping during the webinar
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HLA antigens are complex
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HLA Antigens and Alleles
Antigens WHO Nomenclature Committee 2010
Locus A
26
B
47
C
17
DR
15
DQ
7
Alleles 965 1,543 626 762 107
Some HLA antigens are common
14
49
60 47 27
46
71
44 45
46 47 57
20
66
31
28
58 43
43
HLA-A2
Some HLA antigens have a different
distribution
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17 30 22
3 18 4
6
0
9 16 2
8
0
15
13
2 0
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HLA-B8
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Some HLA antigens are rare and limited
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0 0
0 0 0
0
0
0
0 0 0
0
0
0
0
10 14
0
HLA-B54
HLA Antigens were defined by antibodies:
Parent Antigens and Splits
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HLA-A9
HLA-A23 HLA-A24
HLA-B14
HLA-B64 HLA-B65
Alleles
Antigens
A*23:01 A*24:02 B*14:02
A*23:02 A*24:03 A*23:03 A*24:04 A*23:04 A*24:06
B*14:01
B*14:03 B*14:04 B*14:05
Don’t report broad HLA Antigens or
allele groups
•Broad Ag Antigen •A9 23,24
•A10 25,26,34,66
•A19 29-33,74
•A28 68,69
•B12 44,45
•B16 38,39
•B17 57,58
•B21 49,50
•B22 54,55,56
•Allele Groups Antigen •B14 64,65
•B15 62,63,71 75,76,77
•B40 60,61
•Cw3 9,10
•DRB1*03 17,18
•DQB1*01 5,6
•DQB1*03 7,8,9
18 B*14 B*15 B*40 DRB1*03 DQB1*03 are NOT antigens
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• A*11:01
• A*11:02 (~10% of 11s)
• A*30:01
• A*30:02 (~50% of 30s)
• A*66:01
• A*66:02 (~20% of 66s)
• B*27:05
• B*27:08 (~1% of 27s)
• B*15:02 (75)
• B*15:11 (~5% of 75s)
• DRB1*04
• DRB1*04:05 (1% of 4s)
• DRB1*12:01
• DRB1*12:02 (~5% of 12s)
• DRB1*15:01
• DRB1*15:02 (~10% of 15)
Some common uncommon alleles
The promiscuous habits of HLA-DQ alpha
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DQB1*02:01 DQB1*02:02 DQB1*03:03 DQB1*04:01 DQB1*02:01
DQA1*04:01 DQA1*02:01 DQA1*02:01 DQA1*02:01 DQA1*02:01
DQ2 DQ2 DQ2 DQ9 DQ4
(-) (+) (+) (+) (+)
DQA1*02 associated with DR7…Surrogate CPRA=23%
HLA-DP B1 Allele Frequencies
21 Petersdorf, E…Blood 2007; 110(13)4560
98% of 12,000 un-related HSCT donors and recipients had common HLA-DP alleles (>2%) 75% both common 23% one common
0401 42%
0201 12%
0402 12%
0301 10%
0101 6%
0501 3%
1101 2%
1301 2%
1701 2%
601 2%
1001 2%
other 5%
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Based on the following HLA profile, what
would you enter into the KPD database?
• A 2 ,11
• B 12, 35
• Bw 4 pos; Bw6 pos
• Cw 5,4
• DR 4, 103
• DR51 neg; DR52 neg; DR53 pos
• DQ 5, 7
• DQA 01, 03
• DP undetermined
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Base on the B 12, 35 HLA prolife, you
would enter B 12 and B 35 into the KPD
database? • True
• False
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Based on the following HLA profile, what
would you enter into the KPD database?
• A 2 ,11
• B 12, 35 (Split B12 toB44)
• Bw 4 pos; Bw6 pos
• Cw 5,4
• DR 4, 103 (Report DR1 DR4)
• DR51 neg; DR52 neg; DR53 pos
• DQ 5, 7
• DQA 01, 03
• DP undetermined (Perform DP typing)
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Summary
• Make a close friend in the HLA lab…preferably the director
• Check the donor HLA type for accuracy, correctness and completeness prior to activating the pair…then have the lab check again
• Do not enter antigens that are not entered as unacceptable antigens (Parent, alleles)
• Do not forget Bw4,6 and DR51-53 DQ alpha
• Type donors for DP even though it is not required
• Use the preselect option…review with the laboratory
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Unacceptable HLA Antigens: The
key to finding a compatible donor
for your patient in KPD
Unacceptable HLA antigens
M. Sue Leffell, Ph.D.
Professor of Medicine
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Why is the definition of unacceptable
antigens so important for KPD?
• Candidates who come to KPD are usually sensitized (>60% have CPRAs >80)
• Compatible KPD matches are based on the presence
of no or low levels of donor specific antibody (DSA)
• Unacceptable antigens (UAs):
– Antigens considered a contraindication to Tx
– Usually based on antibodies to potential donors
• Accurate UA definition optimizes planning for
compatible exchanges
• Failure to accurately assess unacceptable
antigens (UAs) disrupts exchanges
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What are the effects of inaccurately entering
unacceptable antigens on exchanges?
a. There are no effects
b. Your individual match is declined but
the rest of the exchange can still move
forward
c. The entire exchange could be
terminated
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Steps for UA Definition
• Development of center specific criteria for
acceptable risk for KPD and/or KPD with
desensitization
• Correlation of solid phase antibody assays
(SPI) with crossmatch tests and center’s risk
criteria
• Analysis of the antibody specificities for
candidates to define corresponding UAs
• Re-evaluation of antibodies periodically and
following any sensitizing event
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Center Specific Criteria
• Dependent upon clinical protocols and the
level, if any, of donor specific antibody (DSA)
that is acceptable
• Factors to consider:
– Are any repeat mismatches acceptable?
– Are DSA levels that are XM - acceptable?
– Is desensitization an option?
• These factors determine the stringency that
must be applied to UA definition
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Negative Positive
T-CDC XM vs. Phenotype Panel MFI
Cutoff: 9,000 MFI correlation: r = 0.92 correct prediction: 96%
Zachary AA, et al. Hum Imm. 70: 574, 2009
Correlation of SPI with Crossmatch Results
Negative Positive
T cell FCXM vs. Phenotype Panel
Cutoff: 5300 MFI, correlation: r = 0.87 correct prediction: 98%
Zachary AA, et al. Hum Imm. 70: 574, 2009
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T-CDC XM v. Single Antigen Panel
Zachary AA, et al. Hum Imm. 70: 574, 2009
Positive Negative
Cutoff: 5300 MFI correlation r = 0.68 correct prediction: 84%
Acceptable Pretransplant DSA Levels After
Desensitization Therapy: Luminex SFI vs Flow TXM MCS
y = 3154.6e0.0157x
R2 = 0.6888
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
0.0 50.0 100.0 150.0 200.0 250.0 300.0
SF
I N
orm
alized
MCS
57,182 mSFI
Flow XM Pos
Flow XM Neg
CDC XM Pos
Class II DSA > 105
AMR
7,966 mSFI 101,244 mSFI
Reinsmoen et al, Transplantation 86:820, 2008
Considerations for SPI: Crossmatch
Correlations
• Correlations must be assay and antigen
specific
– HLA-Cw, DQ, DP concentrations are enhanced
compared to HLA-A,B,DR (Zachary, et al. Meth Mol Biol.2012;
Zachary, Reinsmoen, Cur Opin Organ Transplant. 2011)
– Individual HLA-A,B,DR concentrations may vary
• Correlations should be on-going to account
for:
– Lot-to-lot variability
– Changes in phenotypes and/or single antigen
panels 36
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Considerations for Individual
Candidate’s Antibody Analysis
• Define the level of DSA that represents a
contraindication or UA for each candidate –
– Any DSA?
– DSA+, but “virtual” CDC XM or FCXM
negative?
– Depends upon your center’s criteria
• Should account for day-to-day assay
variability
• Should consider possible test interference
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“Cut Off” MFI Values for Positive
Antibody Levels
• There are no generally accepted “cut off” MFI
values
• Reported MFI “cut offs” range from 500 to
3000
• Often what is considered “positive” is lower
than what is unacceptable
• Values should be antigen specific
– E.g., DQ values are higher than for DR UAs
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High Risk vs. Low Risk UAs
• High risk – when DSA is at a level that is
absolute contraindication at your center
– List as UA
• Low risk – DSA is present but at lower levels
and could be positive in combination with
other donor antigens or when target antigen
is homozygous
– Try to identify antigen combinations that could
reach unacceptable risk level
– Consider these combinations in evaluating
prospective KPD exchanges 39
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0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
4/1
2
4/1
9
4/2
6
5/3
5/1
0
5/1
7
5/2
4
5/3
1
6/7
6/1
4
6/2
1
6/2
8
7/5
7/1
2
7/1
9
7/2
6
MFI
A3 C15 B51 B60 cI Pos con
Interpretation of Results: Positive Control Variation
SPI: Test Interference
• Substances inherent to the serum
– High IgM levels
– Immune complexes
– Antibody to plastic
– Complement: Ab binding of C1q in high titered
sera (Schnaidt M, et al. Transplantation 92: 510, 2011)
• Extrinsic factors
– Therapeutic Abs: thymoglobulin, eculizumab, high
dose IVIg
– Bortezomib
*
*
*
*
Lowest reacting B7: 3578 MFI
Untreated
IgM Depleted
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Methods to Deal with Interference
and/or prozone
• Dilution
• Hypotonic dialysis (Zachary, et al. Human
Immunol.2009)
• Dithiothreitol (DTT) (Kosmoliaptsis, et al. Human
Immunol.2010)
• Ethylenediaminetetraacetic acid (EDTA) (Schnaidt M, et al. Transplantation.2011)
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Frequency of Antibody Testing
• Once at initial evaluation is NOT enough
• Specificity should be confirmed on at least two
samples, preferably three to assess antibody
trend
• Regular testing is necessary while the candidate
is on the wait list to be sure UAs are current
• Test after any sensitizing event
– Transfusions
– Infection, other inflammatory events
0
20
40
60
80
100
120
Baseline Time of Event
GT
I S
co
re
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
Baseline Time of Infection
GT
I S
co
re
A
B
Locke JE, et al. Am J Transplant.2009;9:2136
• 65 renal transplant
candidates with
proinflammatory event
c/in 1 month of aby
testing
A. 35 with culture+
infection; 97.1% had
significant increase in
aby
B. 30 with proimflamm.
events
– Surgery
– Myocardial infarction
– AV line placement
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Recommended Best Practices (Guidelines from the OPTN/UNOS Histocompatibility Committee and
a recent national KPD Consensus Conference)
• UAs should be based on Center specific risk criteria
• Correlation of antibody assays with risk criteria is
essential
• Antibody definition should be done by at least two
methods, one of which should be a solid phase assay
– Crossmatch confirmation should be cell based
• Specificity should be confirmed using a single antigen
assay for:
– HLA-A,B,C, DRB1, DRB3-5, DQA1, DQB1, DPB1
• Antibody interpretation may require high resolution
typing of both recipient and donor alleles
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Best Practices (con’t.)
• Antibodies and corresponding UAs should be
confirmed on at least two samples, updated at
least quarterly, after any sensitizing event, and
after any unexpected positive crossmatch
• Both high and low risk UAs should be considered
• Labs should achieve > 95% accuracy in virtual
crossmatch prediction
• Histocompatibility evaluation should be used for
preapproval of exchange matches
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Effective UA
Definition:
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Recipient DSA Analysis
No Risk Acceptable
Low Risk
Possibly Acceptable
Center Decision
Preliminary - Final XM
High Risk Unacceptable
UA Definition
Potential Donor Mismatches
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Given the following results, what would
you list for UAs?
HLA Class I Abs
Normalized MFI Specificity
16,952 A2
16,582 A69
12,951 A68
4364 B57
3108 A1
3102 B58
HLA Class II Abs
Normalized MFI Specificity
22,025 DR1
19,248 DR51 (B5*02:02)
18,564 DR9
17,438 DR51 (B5*01:01)
14,800 DQ6
12,204 DQ5
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Specificity A68 with a normalized
MFI 12,951would be listed as an
UA? • True
• False
50 June 19, 2012
Given the following results, what would
you list for UAs?
HLA Class I Abs
Normalized MFI Specificity
16,952 A2
16,582 A69
12,951 A68
4364 B57
3108 A1
3102 B58
HLA Class II Abs
Normalized MFI Specificity
22,025 DR1
19,248 DR51 (B5*02:02)
18,564 DR9
17,438 DR51 (B5*01:01)
14,800 DQ6
12,204 DQ5
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Answer: At JHMI, UAs are based on Abs that are CDC XM+:
Class I: A2, A69, A68
Class II: DR1, DR51, DR9
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June 19, 2012 53
Only unacceptable antigens that are highly
likely to cause a positive crossmatch
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All other antigens to which the candidate has
antibodies, not including the unacceptable
antigens
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Summary
Accurate identification of both donor HLA and candidate unacceptable antibodies is critical to successful matching
Transplant centers and HLA labs must work together to identify, enter, and verify appropriate data into the KPD system
Declined Matches affect all other Matches in an Exchange
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M. Sue Leffell, Ph.D [email protected]
J. Michael Cecka, Ph.D [email protected]
Ruthanne L Hanto RN, MPH [email protected]
For more information