6/19/2012

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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

25

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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June 19, 2012 55

All other antigens to which the candidate has

antibodies, not including the unacceptable

antigens

June 19, 2012 56

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 msl@jhmi.edu

J. Michael Cecka, Ph.D MCecka@mednet.ucla.edu

Ruthanne L Hanto RN, MPH ruthanne.hanto@unos.org

For more information