handouts - immucor, inc. program handouts/lsa... · 2018-02-13 · michael d. gautreaux, ph.d.,...

2/13/2018

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Continuing Education Webinar Series

All Content © 2015 Immucor, Inc.

22 February Donor Selection of Solid Organ Transplant: Is Virtual Xmatch better than real?

28 February Proficiency, Competency, and QC: A practical approach to CLIArequirements and AABB, CAP, and Joint Commission Expectations

22 March Clinical Significance of HLA Antibodies in Solid Organ Transplantations

Future Webinars

Link to register: https://immucor.webinato.com/register

All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.

Handouts

http://www.immucor.com/en‐us/Pages/Educational‐Program‐Handouts.aspx

2/13/2018

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Continuing Education

• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:https://www.surveymonkey.com/r/HLAImmucor

• Registration deadline is March 2, 2018

• Certificates will be sent via email only to those who have registered by March 16, 2018


Presentation Recording

• Session will be recorded and posted. – Access information will be sent to each

registrant when the recording becomes available

• CE credits will be issued to anyone who listens to the recording within one year of the original presentation date (today).

Learn website: learn.immucor.com


Questions?

• You are all muted

• Type in questions

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• Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations.

• The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor.

Testing for HLA Antibodies:We’re Not the Chemistry Lab

Michael D. Gautreaux, Ph.D., D.ABHI

Impact of HLA Ab in Transplantation

•Pre- and peri-transplant– Access to transplantation– Short-term survival (hyperacute, accelerated)

•Post-transplant– Survival

» Acute and chronic rejection– Marker for rejection response

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PRA

• Panel Reactive Antibody• Percent Reactive Antibody

• Intended to be an assessment of transplantability.

• 20% PRA means that a recipient should crossmatch positive with 20% of donors

• DO NOT CONFUSE WITH TITER

Basis of Antibody Testing

+Y Y

Y

Y

Y

Patient’s serumCells or beads

Reaction No reaction

Y

Y

Y

Y

Y

Y

PRA Techniques

•Complement-dependent cytotoxicity•(original gold standard; usually augmented)

•Flow cytometry•(mostly solid phase)

•Solid Phase•(ELISA & Luminex)

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Complement-dependent Cytotoxicity

+

30 min

Add complement

60 min

Y

Y

Y

Y

Y

Y

Cell death

Y

Magnetic separation HLA antibody in wellsPBLs

ASHI Scoring System

1(0-10%)

2(11-20%)

4(21-50%)

6(51-80%)

8(81-100%)

Determination of Significance

+ -

+a

TP+/+

bFP-/+

-c

FN+/-

dTN-/-

Correlation Coefficient (r)r = a*d - b*c

SQRT (A*B*C*D)

Chi square (2)2 = r 2 * T

Serumreactivity

Antigen

A = a + b

B = c + d

C = a + c D = b + d

T = Total #p value depends on degrees of freedom2 x 2 tables have 1 degree of freedom

2 2.7055 3.8415 6.6349 7.8794p 0.1 0.05 0.01 0.005

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+ -

+ 14 3

- 1 21

A2

Serumreactions

r = a*d - b*cSQRT (A*B*C*D)

r = 14*21 - 3*1SQRT (17*22*15*24)

r = 0.793

2 = 0.7932 * 39

2 = 24.52

p < 0.0001

2 = r 2 * T

Luminex - xMAP

Luminex’s xMAP Technology combines fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver multiplexed assay capabilities that are configured to perform a wide variety of bioassays quickly, cost-effectively and accurately.

Bead Array

• Microspheres are dyed to create 100 distinct colors

• Each microsphere has ‘spectral address’ based on red/infrared content

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• Analyzer samples well• Lasers excite fluorescent dyes

– red laser for bead classification and green laser for assay result

• Multiple readings for each microsphere set

• Software reports results in real-time

• Up to 9600 results in 1 hour

xMAP Technology

_ Analysis Software

HLA Antibody Testing by Luminex

Does the MFI of anti-A2 affect the sCr? AM MFI is 2,500 but PM is 3,000What is your cut-off MFI?Well, that MFI is too low to matter.MFISTOP!!!!!!!MFIMFI

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Single Antigen PRA Results

0

10

20

30

40

50

60

70

1 2Antigen

Time 1

Time 2

Time 3

Time 4

Single Antigen PRA Results

Assay Classification

Class ResultStandardization

Method

References,Calibrators,

Controls

Qualitative Positive or Negative Single or dual 1 reference sample,positive and negative controls

Semi-quantitative Arbitrary units Single or dual Negative and bi-level positive controls; References unique for test system made from pooled patient sera

Quantitative Units related to recognized reference preparation

Multipoint standardcurve

Tri-level controls; Reference calibrated towards a qualified reference preparation

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SABs are not quantitative – why?

• Quantitative assay = result in units/volume

• Don’t have the necessary components

• Not standardized– Challenging from biologic and technical perspectives

• So says regulatory authorities – (intended use)

This ain’t the Chemistry LabChallenges for Immunoassay Standardization

• Measuring analyte • serum with multiple isotypes, subclasses and varying avidities

• No standard• Sample handling/Interfering substances• Antigen standardization• Suitable controls with defined range of Ab

Factors affecting all assays

•Affinity vs Avidity•Polyclonal vs monoclonal•Inhibitors•Accuracy vs Precision•Birthday cake!!

Yeah, you still gotta worry about all that biology, chemistry and physics stuff.

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Affinity vs Avidity

Polyclonal vs Monoclonal

Roitt IM, Brostoff JB, Male DK. Immunology – Fifth Edition. Mosby Internation Ltd. 1998.

Inhibitors

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Accuracy vs Precision

• Accuracy– The ability to measure purified amounts of a substance– Known positive sera as a control– Mixed mono-specific serum with non-specific serum

• Precision– Ability of an assay to consistently reproduce a result – Measured by Coefficient of Variation (%CV)– Good intra-assay CV ≤ 10%– Good inter-assay CV ~ 10-15%

Intra-assay %CV

Inter-assay %CV

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The Real World

• SOPs are routinely deviated– Lunch– Birthday cake!

• Ambient conditions change

• Lasers fade

Effect of Temperature on Variability

Room temperature 0C

Temperature Control

Negative Control Bead

Courtesy: Derek O’Neill, Beaumont Hospital, Dublin, Ireland

A*0201(donor Specific)

B*3701 B*1503

Temp 0C

Rxn Score

MFI Rxn Score

MFI Rxn Score

MFI

Day of transplant serum

cold 4 707 2 186 6 2077

Day 7 post-transplant serum Run 1

cold 4 1162 6 2077 6 3000

Day 7 post-transplant serum Run 2

warm 1 21 2 159 4 1032

Patient serum variability: Post-transplant monitoring

Courtesy: Derek O’Neill, Beaumont Hospital, Dublin, Ireland

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Luminex Summary

• Luminex assays should be carried out under controlled conditions

• Luminex results should be interpreted by experienced personnel

– take measurement error into account.

• SAB assays are at best semi-quantitative.

• Resist the siren call of MFI

Other PRA Testing Considerations

• Polyclonal vs. monoclonal

• CYNAP

• Monospecific vs. polyspecific

• Cross Reactive Groups (CREGS)

Antibody Specificity

•Monospecific– serum that has specificity for a single HLA antigen

•Polyspecific– serum that reacts with more than one distinct HLA antigen

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Cross-reactive epitope groups (CREGs)

• Multiply reactive sera can result from:

– multiple “private” specificity antibodies» antibodies specific for one specific determinant

– “true crossreactive” antibodies» one antibody that bind to structurally similar private

epitopes

– antibodies to “public” epitopes» one antibody that binds to a unique epitope shared

among several different HLA antigens (Bw4 or Bw6)

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CREG Epitope Locations

B7 CREG appears safe

30-86-48

02000400060008000

1000012000140001600018000200002200024000

CO

N1

CO

N2

CO

N3

CO

N4

A*0

101

A*0

201

A*0

203

A*0

206

A*0

301

A*1

101

A*1

102

A*2

302

A*2

402

A*2

403

A*2

501

A*2

601

A*2

902

A*3

001

A*3

101

A*3

201

A*3

301

A*3

303

A*3

402

A*3

601

A*4

301

A*6

601

A*6

602

A*6

801

A*6

802

A*6

901

A*7

401

A*7

405

A*8

001

B*0

702

B*0

703

B*0

801

B*1

302

B*1

401(

B6

4)B

*140

2(B

65)

B*1

501(

B6

2)B

*150

2(B

75)

B*1

503(

B7

2)B

*151

2(B

76)

B*1

513(

B7

7)B

*151

6(B

63)

B*1

518(

B7

1)B

*18

01B

*27

03B

*27

05B

*27

08B

*35

01B

*37

01B

*38

01B

*39

01B

*400

1(B

60)

B*4

002(

B6

1)B

*41

01B

*42

01B

*44

02B

*45

01B

*46

01B

*47

01B

*48

01B

*49

01B

*50

01B

*51

01B

*52

01B

*53

01B

*54

01B

*55

01B

*56

01B

*57

01B

*58

02B

*73

01B

*78

01B

*81

01B

*82

02C

w*0

102

Cw

*021

0C

w*0

303

Cw

*030

4C

w*0

401

Cw

*040

3C

w*0

501

Cw

*060

2C

w*0

701

Cw

*070

2C

w*0

704

Cw

*080

1C

w*0

802

Cw

*120

2C

w*1

402

Cw

*150

2C

w*1

601

Cw

*170

1C

w*1

801

Pro

be 7

7

Raw

MF

I-B

G

Pt. PRA consistently >90% (multispecific)

Pt. HLA-A11, -; B56, 65 (? sensitization)

B Ag specificities narrowed

45-30-96

02000400060008000

1000012000140001600018000200002200024000

CO

N1

CO

N2

CO

N3

CO

N4

A*0

101

A*0

201

A*0

203

A*0

206

A*0

301

A*1

101

A*1

102

A*2

302

A*2

402

A*2

403

A*2

501

A*2

601

A*2

902

A*3

001

A*3

101

A*3

201

A*3

301

A*3

303

A*3

402

A*3

601

A*4

301

A*6

601

A*6

602

A*6

801

A*6

802

A*6

901

A*7

401

A*7

405

A*8

001

B*0

702

B*0

703

B*0

801

B*1

302

B*1

401(

B64

)B

*140

2(B

65)

B*1

501(

B62

)B

*150

2(B

75)

B*1

503(

B72

)B

*151

2(B

76)

B*1

513(

B77

)B

*151

6(B

63)

B*1

518(

B71

)B

*180

1B

*270

3B

*270

5B

*270

8B

*350

1B

*370

1B

*380

1B

*390

1B

*400

1(B

60)

B*4

002(

B61

)B

*410

1B

*420

1B

*440

2B

*450

1B

*460

1B

*470

1B

*480

1B

*490

1B

*500

1B

*510

1B

*520

1B

*530

1B

*540

1B

*550

1B

*560

1B

*570

1B

*580

2B

*730

1B

*780

1B

*810

1B

*820

2C

w*0

102

Cw

*021

0C

w*0

303

Cw

*030

4C

w*0

401

Cw

*040

3C

w*0

501

Cw

*060

2C

w*0

701

Cw

*070

2C

w*0

704

Cw

*080

1C

w*0

802

Cw

*120

2C

w*1

402

Cw

*150

2C

w*1

601

Cw

*170

1C

w*1

801

Pro

be 7

7

Raw

MF

I-B

G

Pt tx with B44 mismatch

6/2005 66% (A68, B44)4/2006 100% (B44 strongest)

B specificities B44, 45, 76, 82

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Class II – DQ Abs pop out

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

2000022000

CO

N1

CO

N2

CO

N3

DP

B1*

0101

/DP

A*0

103

DP

B1*

0101

/DP

A*0

201

DP

B1*

0101

/DP

A*0

202

DP

B1*

0101

/DP

A*0

301

DP

B1*

0201

/DP

A*0

103

DP

B1*

0301

/DP

A*0

103

DP

B1*

0301

/DP

A*0

201

DP

B1*

0401

/DP

A*0

103

DP

B1*

0401

/DP

A*0

201

DP

B1*

0401

/DP

A*0

202

DP

B1*

0401

/DP

A*0

301

DP

B1*

0402

/DP

A*0

103

DP

B1*

0402

/DP

A*0

201

DP

B1*

0402

/DP

A*0

301

DP

B1*

0501

/DP

A*0

103

DP

B1*

0501

/DP

A*0

201

DP

B1*

0501

/DP

A*0

202

DP

B1*

0501

/DP

A*0

301

DP

B1*

1301

/DP

A*0

103

DP

B1*

1301

/DP

A*0

201

DP

B1*

1401

/DP

A*0

103

DP

B1*

1401

/DP

A*0

201

DP

B1*

1701

/DP

A*0

201

DP

B1*

1801

/DP

A*0

103

DP

B1*

1801

/DP

A*0

201

DP

B1*

1901

/DP

A*0

103

DP

B1*

1901

/DP

A*0

201

DP

B1*

1901

/DP

A*0

301

DP

B1*

2801

/DP

A*0

103

DP

B1*

2801

/DP

A*0

201

DP

B1*

2801

/DP

A*0

202

DQ

B1*

0202

/DQ

A*0

302

DQ

B1*

0202

/DQ

A*0

201

DQ

B1*

0402

/DQ

A*0

302

DQ

B1*

0402

/DQ

A*0

401

DQ

B1*

0401

/DQ

A*0

401

DQ

B1*

0301

/DQ

A*0

302

DQ

B1*

0301

/DQ

A*0

501

DQ

B1*

0302

/DQ

A*0

302

DQ

B1*

0302

/DQ

A*0

201

DQ

B1*

0303

/DQ

A*0

302

DQ

B1*

0303

/DQ

A*0

401

DQ

B1*

0501

/DQ

A*0

104

DQ

B1*

0601

/DQ

A*0

104

DR

B1*

0101

DR

B1*

0103

DR

B1*

0401

DR

B1*

0701

DR

B1*

0801

DR

B1*

0901

DR

B1*

1001

DR

B1*

1101

DR

B1*

1201

DR

B1*

1301

DR

B1*

1401

DR

B1*

1501

DR

B1*

1601

DR

B1*

0301

(17

) D

RB

1*03

03 (

18)

DR

B3*

0101

(52

) D

RB

3*02

02 (

52)

DR

B4*

0101

(53

) D

RB

5*01

01 (

51)

Pt. always called DR 52 (previous MM)

DQ specificity confounded by DR 52 and 53

CREG Epitope Locations

HLA-A Cross Reactivities

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HLA-B Cross Reactivities

HLA-DR Cross Reactivities

TABLE 1. Overview of common molecular targets for non-HLA antibody responses, allograft types with confirmedinjury phenotypes and corresponding time frames, as well as experimental evidence for pathogenicity of non-HLAantibodies with corresponding references

Antibody target Human allograft type Time-frame Experimental evidence

MICA Kidney (28–31, 35–39), heart (33), Hyperacute, acute, chronic —pancreas (31)

Vimentin Heart (52) Chronic Mouse (57), non-human primate

(55, 56)

AT1R Kidney (46) Acute Rat renal allograft (46)

ICAM-1 Heart (58) ? —

HLA, human leukocyte antigens; AT1R, angiotensin type 1 receptor; ICAM-1, intercellular adhesion molecule-1.

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MICA and MICB

MICA

MHC class I-like chain A or B

It is thought that MICA and MICB function as stress-induced antigens that are broadly recognized by NK cells and most of the subtypes of T cells.

Expressed on endothelial cells, gut epithelium, monocytes but not lymphocytes

Routine crossmatch will not detect Ab to MICA or MICB

Antibodies to MICA don’t generally fix complement

Up-regulation of MICB assoc with class II MHC expression and lymphocyte infiltration

MICB

Antibody target Human allograft type Time-frame Experimental evidenceMICA Kidney (28–31, 35–39), heart (33), Hyperacute, acute, chronic -

pancreas (31)

Vimentin

Vimentin is a member of the intermediate filament family, and is part of the cytoskeletal network.

Non-polymorphic protein found in abundance in endothelium and smooth muscle cells.

antibodies to vimentin are considered “auto-reactive”

In a monkey model, anti-vimentin antibodies are associated with chronic cardiac rejection but not kidney rejection. ????

Antibody target Human allograft type Time-frame Experimental evidenceVimentin Heart (52) Chronic Mouse (57), non-human primate (55, 56)

Angiotensin type 1 Receptor (AT1R)

• AT1 receptor has vasopressor effects and regulates aldosterone secretion. It is an important effector controlling blood pressure and volume in the cardiovascular system.

• Antibodies to AT1R have been associated with pre-eclampsia and are implicated with severe vascular rejection.

• Article describes a patient who received a 6 Ag match kidney and then had a sudden onset of malignant hypertension that mimicked the pre-eclampsia she suffered >20 years before.

Angiotensin type II

Antibody target Human allograft type Time-frame Experimental evidenceAT1R Kidney (46) Acute Rat renal allograft (46)

A receptor like AT1R

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ICAM and anti-GBM

• Antibodies to ICAM found in cardiac transplant patients.

• Antibodies to agrin and perlecan are associated with duplication of the GBM -a hallmark of transplant glomerulopathy.

Mechanisms of Injury

• Complement-mediated injury

• Antibody-dependent Cellular Cytotoxicity– for non-C’ fixing Abs (like those against MICA/B)

• May contribute to structural changes

• Signalling?

Perspective

• With exceptions of MICA and AT1R, routine testing for these antibodies are not currently available.

• Question as to whether non-HLA antibody related pathologies represent “true” rejections of the transplanted organ or organ-specific autoimmune phenomena.

• Non-HLA Abs may find application as biomarkers of immune response and herald the need for more suitable immunosuppression.

2/13/2018

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Continuing Education

• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:https://www.surveymonkey.com/r/HLAImmucor

• Registration deadline is March 2, 2018

• Certificates will be sent via email only to those who have registered by March 16, 2018


22 February Donor Selection of Solid Organ Transplant: Is Virtual Xmatch better than real?

28 February Proficiency, Competency, and QC: A practical approach to CLIArequirements and AABB, CAP, and Joint Commission Expectations

22 March Clinical Significance of HLA Antibodies in Solid Organ Transplantations

Future Webinars

Link to register: https://immucor.webinato.com/register

handouts - immucor, inc. program handouts/lsa... · 2018-02-13 · michael d. gautreaux, ph.d.,...

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