Continuing Education Webinar Series Program Handouts... · Presentation Recording •Session will...

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 CLIA

requirements 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



• 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

https://www.surveymonkey.com/r/HLAImmucor


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


• 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

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 serum Cells 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)

Complement-dependent Cytotoxicity

+

30 min

Add complement

60 min

Y

Y

Y

Y

Y

Y

Cell death

Y

Magnetic separation HLA antibody in wells PBLs

ASHI Scoring System

1 (0-10%)

2 (11-20%)

4 (21-50%)

6 (51-80%)

8 (81-100%)

Determination of Significance

+ -

+

a

TP

+/+

b

FP

-/+

-

c

FN

+/-

d

TN

-/-

Correlation Coefficient (r)

r = a*d - b*c

SQRT (A*B*C*D)

Chi square (c2)

c2 = r 2 * T

Serum

reactivity

Antigen

A = a + b

B = c + d

C = a + c D = b + d

T = Total #

p value depends on degrees of freedom

2 x 2 tables have 1 degree of freedom

c2 2.7055 3.8415 6.6349 7.8794

p 0.1 0.05 0.01 0.005

+ -

+ 14 3

- 1 21

A2

Serum

reactions

r = a*d - b*c

SQRT (A*B*C*D)

r = 14*21 - 3*1

SQRT (17*22*15*24)

r = 0.793

c2 = 0.7932 * 39

c2 = 24.52

p < 0.0001

c2 = 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

• 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 3,000 What is your cut-off MFI? Well, that MFI is too low to matter. MFI STOP!!!!!!! MFI MFI

Single Antigen PRA Results

0

10

20

30

40

50

60

70

1 2

Antigen

Time 1

Time 2

Time 3

Time 4

Single Antigen PRA Results

Assay Classification

Class Result Standardization

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 standard curve

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

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 Lab Challenges 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.

Affinity vs Avidity

Polyclonal vs Monoclonal

Roitt IM, Brostoff JB, Male DK. Immunology – Fifth Edition.

Mosby Internation Ltd. 1998.

Inhibitors

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

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

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

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)

http://upload.wikimedia.org/wikipedia/commons/e/ef/Flag_of_Hawaii.svg

http://upload.wikimedia.org/wikipedia/commons/5/5c/Flag_of_Greece.svg

CREG Epitope Locations

B7 CREG appears safe

30-86-48

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

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(B

64)

B*1

402(B

65)

B*1

501(B

62)

B*1

502(B

75)

B*1

503(B

72)

B*1

512(B

76)

B*1

513(B

77)

B*1

516(B

63)

B*1

518(B

71)

B*1

801

B*2

703

B*2

705

B*2

708

B*3

501

B*3

701

B*3

801

B*3

901

B*4

001(B

60)

B*4

002(B

61)

B*4

101

B*4

201

B*4

402

B*4

501

B*4

601

B*4

701

B*4

801

B*4

901

B*5

001

B*5

101

B*5

201

B*5

301

B*5

401

B*5

501

B*5

601

B*5

701

B*5

802

B*7

301

B*7

801

B*8

101

B*8

202

Cw

*0102

Cw

*0210

Cw

*0303

Cw

*0304

Cw

*0401

Cw

*0403

Cw

*0501

Cw

*0602

Cw

*0701

Cw

*0702

Cw

*0704

Cw

*0801

Cw

*0802

Cw

*1202

Cw

*1402

Cw

*1502

Cw

*1601

Cw

*1701

Cw

*1801

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

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

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(B

64)

B*1

402(B

65)

B*1

501(B

62)

B*1

502(B

75)

B*1

503(B

72)

B*1

512(B

76)

B*1

513(B

77)

B*1

516(B

63)

B*1

518(B

71)

B*1

801

B*2

703

B*2

705

B*2

708

B*3

501

B*3

701

B*3

801

B*3

901

B*4

001(B

60)

B*4

002(B

61)

B*4

101

B*4

201

B*4

402

B*4

501

B*4

601

B*4

701

B*4

801

B*4

901

B*5

001

B*5

101

B*5

201

B*5

301

B*5

401

B*5

501

B*5

601

B*5

701

B*5

802

B*7

301

B*7

801

B*8

101

B*8

202

Cw

*0102

Cw

*0210

Cw

*0303

Cw

*0304

Cw

*0401

Cw

*0403

Cw

*0501

Cw

*0602

Cw

*0701

Cw

*0702

Cw

*0704

Cw

*0801

Cw

*0802

Cw

*1202

Cw

*1402

Cw

*1502

Cw

*1601

Cw

*1701

Cw

*1801

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

Class II – DQ Abs pop out

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

CO

N1

CO

N2

CO

N3

DP

B1*0

101/D

PA

*0103

DP

B1*0

101/D

PA

*0201

DP

B1*0

101/D

PA

*0202

DP

B1*0

101/D

PA

*0301

DP

B1*0

201/D

PA

*0103

DP

B1*0

301/D

PA

*0103

DP

B1*0

301/D

PA

*0201

DP

B1*0

401/D

PA

*0103

DP

B1*0

401/D

PA

*0201

DP

B1*0

401/D

PA

*0202

DP

B1*0

401/D

PA

*0301

DP

B1*0

402/D

PA

*0103

DP

B1*0

402/D

PA

*0201

DP

B1*0

402/D

PA

*0301

DP

B1*0

501/D

PA

*0103

DP

B1*0

501/D

PA

*0201

DP

B1*0

501/D

PA

*0202

DP

B1*0

501/D

PA

*0301

DP

B1*1

301/D

PA

*0103

DP

B1*1

301/D

PA

*0201

DP

B1*1

401/D

PA

*0103

DP

B1*1

401/D

PA

*0201

DP

B1*1

701/D

PA

*0201

DP

B1*1

801/D

PA

*0103

DP

B1*1

801/D

PA

*0201

DP

B1*1

901/D

PA

*0103

DP

B1*1

901/D

PA

*0201

DP

B1*1

901/D

PA

*0301

DP

B1*2

801/D

PA

*0103

DP

B1*2

801/D

PA

*0201

DP

B1*2

801/D

PA

*0202

DQ

B1*0

202/D

QA

*0302

DQ

B1*0

202/D

QA

*0201

DQ

B1*0

402/D

QA

*0302

DQ

B1*0

402/D

QA

*0401

DQ

B1*0

401/D

QA

*0401

DQ

B1*0

301/D

QA

*0302

DQ

B1*0

301/D

QA

*0501

DQ

B1*0

302/D

QA

*0302

DQ

B1*0

302/D

QA

*0201

DQ

B1*0

303/D

QA

*0302

DQ

B1*0

303/D

QA

*0401

DQ

B1*0

501/D

QA

*0104

DQ

B1*0

601/D

QA

*0104

DR

B1*0

101

DR

B1*0

103

DR

B1*0

401

DR

B1*0

701

DR

B1*0

801

DR

B1*0

901

DR

B1*1

001

DR

B1*1

101

DR

B1*1

201

DR

B1*1

301

DR

B1*1

401

DR

B1*1

501

DR

B1*1

601

DR

B1*0

301 (

17)

DR

B1*0

303 (

18)

DR

B3*0

101 (

52)

DR

B3*0

202 (

52)

DR

B4*0

101 (

53)

DR

B5*0

101 (

51)

Pt. always called DR 52 (previous MM)

DQ specificity confounded by DR 52 and 53

CREG Epitope Locations

HLA-A Cross Reactivities

HLA-B Cross Reactivities

HLA-DR Cross Reactivities

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

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 evidence

MICA 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.

????


Vimentin 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


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

A receptor like AT1R

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.


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• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:


• 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 CLIA

requirements 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

Continuing Education Webinar Series Program Handouts... · Presentation Recording •Session will...

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