Future Webinars - Immucor, Inc. Program Handouts... · • ABHI, PACE, Florida and California DHS...

3/19/2018

1

Continuing Education Webinar Series

All Content © 2015 Immucor, Inc.

12 April Post-Haematopoietic Stem Cell Transplant Chimerism Testing and Engraftment Monitoring

featuring

Dr Anil Handoo, Sr. Consultant and Director Pathology BLK Super Specialty HospitalNew Delhi, India

Future Webinars

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


Future Webinars

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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


Continuing Education

• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:https://www.surveymonkey/OptimizeHLAwithImmucor

• Registration deadline is April 13, 2018

• Certificates will be sent via email only to those who have registered by April 27, 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

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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.

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Dr. Robert Liwski, MD, PhD, FRCPCMedical Director, HLA Typing LaboratoryInterim Head, Division of HematopathologyProfessor, Department of PathologyDalhousie University, [email protected]

Optimization of HLA Antibody Testing.

Disclosure

• Nothing significant to disclose

…..still waiting for attractive offers

• Used by most HLA labs for HLA antibody testing.

• Revolutionized HLA antibody identification and virtual crossmatching.

• Number of advantages compared to Flow SAB and ELISA.– ↑ number of analytes tested simultaneously– High throughput– Rapid analysis

• Still, the procedure is time intensive and is not optimal for use in urgent cases…. – Friday afternoons come to mind……

• Some important limitations:– Susceptible to interfering substances (“prozone” effect).

Single Antigen Bead (SAB) Luminex assay

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Outline

• Optimization of HLA antibody testing

– Rapid Optimized Single Antigen Bead (ROB) protocol for LABScreen (Human Immunology 2017).

• Development

• Validation

• Multicenter evaluation

– Enhanced and ROB protocols for LIFECODES LSA.• Multicenter evaluation

– Development of a novel, prozone‐resistant Dual Antibody Rapid Test (DART) protocol for LABScreen (ASHI Quarterly 2017).

Single antigen bead (SAB) LuminexLABScreen and LIFECODES LSA protocols

• Incubate beads (5 l) and serum 20 l (RT) 30 min.

• Wash x3 (5 min/spin) 15 min.

• Incubate with 100 l anti‐IgG‐PE, 1:100 dilution (RT) 30 min.

• Wash x2 (5min/spin) 10 min.

• Total assay time 1h 25 min.

Evidence for incubation time/reagent concentration?wash times?

2h

50 l 1:10

40 l 10 l

1h 5 min.

Filter plate 5 min.

No wash 0 min.

Transfusion Medicine

• Red cell antibody testing (IAT)

• How long does it take?

• 25‐30 minutes!!!

• Can SAB assay be optimized and expedited?

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Liwski et al Hum. Immunol. 2017


Objectives

• To develop a rapid single antigen bead LABScreenprotocol without compromising the sensitivity of the assay.

• Investigate the effects of:

– Centrifugation time

– Serum incubation time

– Anti‐IgG‐PE incubation time

– Serum volume

– Anti‐IgG‐PE concentration

Effect of reduced spin time (1 vs 5 min) on bead counts

Class I beads Class II beads

Bea

d c

ou

nt

Bead numberN=3


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Effect of reduced spin time

• Standard

5 washes x 5 min = 25 min

1300 x g

• Rapid

5 washes x 1 min = 5 min

1800 x g

No impact on bead counts or overall results20 minutes saved!

Effects of reduced incubation times

• Serum incubation time

• Anti‐IgG‐PE incubation time


Effects of reduced incubation time¼ PPC, HLA class I

MF

I

Bead numberLiwski et al Hum. Immunol. 2017

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MF

I



MF

I



MF

I


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Effects of reduced incubation time¼ PPC, HLA class II

MF

I


Effects of reduced incubation timeNegative control serum

Class I Class II

MF

I


Effects of reduced incubation timeNC and PC beads

NC bead (#1) PC bead (#2)

MF

I

Serum/IgG-PE incubation time

Significant Effect on IgG bindingSmall Effect on

background


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Conclusion

• Reduction in incubation time with serum and/or anti‐IgG‐PE results in decreased MFI values.

• Negligible impact on LSNC and NC bead reactivity.

• The degree of MFI decrease when incubation time with anti‐IgG‐PE was reduced was surprising.

• IgG‐PE concentration appears to be sub‐optimal?


Effects of increasing IgG‐PE concentration¼ PPC, HLA class I

MF

I


Effects of increasing IgG‐PE concentration¼ PPC, HLA class II

MF

I


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Effects of increasing IgG‐PE concentrationNegative control serum

Class I Class II

MF

I


Effects of increasing IgG‐PE concentrationNC and PC beads

NC bead (#1) PC bead (#2)

MF

I

Serum/IgG-PE incubation timeLiwski et al Hum. Immunol. 2017


Conclusion

• Increasing the anti‐IgG‐PE concentration from 1:100 to 1:5 increases MFI in the standard assay including PC bead MFI.

• Negligible effect on background (LSNC and NC bead).

• Can we compensate for reduced MFI values in the 15/5 min protocol by optimizing the concentration of anti‐IgG‐PE?

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Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class I

MF

I



MF

I



MF

I


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MF

I



MF

I


Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class II

MF

I


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Conclusion

• Increasing concentration of anti‐IgG‐PE compensates for the reduction in incubation times.

• IgG‐PE concentration of 1:10 closely matches MFI obtained with the standard assay.


ROB LABScreen® Protocol





• Total assay time 25 min.


ROB LABScreen® Protocol






70% time reduction!Liwski et al Hum. Immunol. 2017

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Standard vs ROB protocol, MFI correlation8 patient, 9 ASHI PT, 3 ABH PT sera

Class I Class II

RO

B p

roto

col M

FI

Standard Assay MFILiwski et al Hum. Immunol. 2017

Representative Serum ReactivityStandard vs ROB protocol

AC‐463 Class I AC‐463 Class II

MF

I


“Discrepant” reactionsCut‐off 2000 MFI

MF

I

1.1 rxn/panel44 rxn/40 panels

rxnLiwski et al Hum. Immunol. 2017

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ROB

Patient serum CF, class I

Standard


ROB

Patient serum CF, class I

Standard


Conclusion

• We can reduce the time it takes to perform LABScreen®

SAB Luminex assay without compromising assay sensitivity.

• Correlation between the Standard and ROB protocols is excellent.

• No significant impact on test results when using ROB protocol.

• Significant time savings.

• ROB protocol allows for rapid testing of urgent patient sera.– Ex. testing during deceased donor work up.


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Multicenter Evaluation of the Rapid Optimized Single Antigen Bead (ROB) LABScreen® Protocol.

Robert Liwski, Patricia Campbell, Adriana Colovai, Deborah Crowe, Anne Halpin, Ronald Kerman, Dong Li, John Lunz, Cathi Murphey, Peter Nickerson, Denise Pochinco, Sandra Rosen‐Bronson, Olga Timofeeva, Paul Warner, Adriana Zeevi

Liwski et al ASHI 2014

Participating Centers

Dalhousie University, Halifax, NS, Canada

University of Alberta, Edmonton, AB, Canada

Montefiore‐Einstein Transplant Center, Bronx, NY

Dialysis Clinic Inc. (DCI) Laboratory, Nashville, TN

Baylor College of Medicine, Houston, TX

Medstar Georgetown University Hospital, Washington, DC

University of Pittsburgh Medical Center, Pittsburgh, PA

Southwest Immunodiagnostics Inc. Laboratory, San Antonio, TX

University of Manitoba, Winnipeg, MB, Canada

Puget Sound Blood Center, Seattle, WA



Design

• 2014 ASHI PT sera– AC460‐464

• Tested by LABScreen SAB Luminex assay– Standard lab method – ROB protocol– Same lot of class I and class II beads

• Result analysis:– MFI comparison– CV– Pearson’s correlation (R2)– Specificity assignment– Pos/Neg ctrl beads (signal vs noise)

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AC464 class IIndividual lab MFI comparison

Bead number

MFI

Standard

ROB


AC464 class IAverage lab MFI and CV comparison

Bead number

MFI

%CV


AC460 class IIIndividual lab MFI comparison

Bead number

MFI

Standard

ROB


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AC460 class IIAverage lab MFI and CV comparison

Bead number

MFI

%CV


Overall mean MFI correlation

Standard

RO

B

Class I Class II


Average CVStandard vs ROB protocol

Class I Class II

% C

V

Serum Liwski et al ASHI 2014

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http://stats.stackexchange.com/questions/423/what-is-your-favorite-data-analysis-cartoon

Conclusion

• Confirmed excellent correlation between the Standard and ROB protocols.

• Confirmed that there is no significant impact on test results when using ROB protocol.

• ROB protocol appears to improve precision of the results


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LIFECODES LSA SAB Assay Evaluation

Objectives

• 20 well characterized and challenging sera

• Standard LIFECODES LSA vs ROB protocol

Single Antigen Bead (SAB) Luminex AssayROB and Standard LIFECODES LSA protocols




• Wash (1 min/spin) 1 min.


50 l 1:10

40 l 10 l

1h

Filter plate 1 min.

No wash 0 min.

30 min.

30 min.

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Case 5, low titer DSA

0

1000

2000

3000

4000

5000

6000

7000

8000

A*01:01

A*02:03

A*11:01

A*23:01

A*24:03

A*26:01

A*30:01

A*32:01

A*33:03

A*36:01

A*66:01

A*68:01

A*69:01

A*80:01

B*0

8:01

B*1

4:01

B*1

5:01

B*1

5:03

B*1

5:12

B*1

5:16

B*2

7:05

B*3

5:01

B*3

8:01

B*4

0:01

B*4

1:01

B*4

4:02

B*4

5:01

B*4

7:01

B*4

9:01

B*5

1:01

B*5

3:01

B*5

5:01

B*5

7:01

B*5

9:01

B*7

3:01

B*8

1:01

C*01:02

C*03:03

C*04:01

C*06:02

C*08:01

C*14:02

C*16:01

C*18:02

A1, A11 DSA

B8 DSA

MF

I

Standard

Case 5, low titer DSA

0

1000

2000

3000

4000

5000

6000

7000

8000

A*01:01

A*02:03

A*11:01

A*23:01

A*24:03

A*26:01

A*30:01

A*32:01

A*33:03

A*36:01

A*66:01

A*68:01

A*69:01

A*80:01

B*0

8:01

B*1

4:01

B*1

5:01

B*1

5:03

B*1

5:12

B*1

5:16

B*2

7:05

B*3

5:01

B*3

8:01

B*4

0:01

B*4

1:01

B*4

4:02

B*4

5:01

B*4

7:01

B*4

9:01

B*5

1:01

B*5

3:01

B*5

5:01

B*5

7:01

B*5

9:01

B*7

3:01

B*8

1:01

C*01:02

C*03:03

C*04:01

C*06:02

C*08:01

C*14:02

C*16:01

C*18:02

A1, A11 DSA

B8 DSA

MF

I

Standard ROB

Case 7, low titer Aw4/Bw4

0

1000

2000

3000

4000

5000

6000

A*01:01

A*02:01

A*02:03

A*03:01

A*11:01

A*11:02

A*23:01

A*24:02

A*24:03

A*25:01

A*26:01

A*29:02

A*30:01

A*31:01

A*32:01

A*33:01

A*33:03

A*34:02

A*36:01

A*43:01

A*66:01

A*66:02

A*68:01

A*68:02

A*69:01

A*74:01

A*80:01

B*07:02

B*08:01

B*13:02

B*14:01

B*14:02

B*15:01

B*15:02

B*15:03

B*15:10

B*15:12

B*15:13

B*15:16

B*18:01

B*27:05

B*27:08

B*35:01

B*37:01

B*38:01

B*39:01

B*40:01

B*40:02

B*41:01

B*42:01

B*44:02

B*44:03

B*45:01

B*46:01

B*47:01

B*48:01

B*49:01

B*50:01

B*51:01

B*52:01

B*53:01

B*54:01

B*55:01

B*56:01

B*57:01

B*58:01

B*59:01

B*67:01

B*73:01

B*78:01

B*81:01

B*82:01

C*01:02

C*02:02

C*03:03

C*03:04

C*04:01

C*05:01

C*06:02

C*07:02

C*08:01

C*12:03

C*14:02

C*15:02

C*16:01

C*17:01

C*18:02

A23, 24, 25, 32 (Aw4)

Bw4Standard

MF

I

3/19/2018

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Case 7, low titer Aw4/Bw4

0

1000

2000

3000

4000

5000

6000

A*01:01

A*02:01

A*02:03

A*03:01

A*11:01

A*11:02

A*23:01

A*24:02

A*24:03

A*25:01

A*26:01

A*29:02

A*30:01

A*31:01

A*32:01

A*33:01

A*33:03

A*34:02

A*36:01

A*43:01

A*66:01

A*66:02

A*68:01

A*68:02

A*69:01

A*74:01

A*80:01

B*07:02

B*08:01

B*13:02

B*14:01

B*14:02

B*15:01

B*15:02

B*15:03

B*15:10

B*15:12

B*15:13

B*15:16

B*18:01

B*27:05

B*27:08

B*35:01

B*37:01

B*38:01

B*39:01

B*40:01

B*40:02

B*41:01

B*42:01

B*44:02

B*44:03

B*45:01

B*46:01

B*47:01

B*48:01

B*49:01

B*50:01

B*51:01

B*52:01

B*53:01

B*54:01

B*55:01

B*56:01

B*57:01

B*58:01

B*59:01

B*67:01

B*73:01

B*78:01

B*81:01

B*82:01

C*01:02

C*02:02

C*03:03

C*03:04

C*04:01

C*05:01

C*06:02

C*07:02

C*08:01

C*12:03

C*14:02

C*15:02

C*16:01

C*17:01

C*18:02

A23, 24, 25, 32 (Aw4)

Bw4Standard ROB

MF

I

Case 2 “prozone” effect, interfering substance

0

5000

10000

15000

20000

25000

A*01:01

A*02:03

A*11:01

A*23:01

A*24:03

A*26:01

A*30:01

A*32:01

A*33:03

A*36:01

A*66:01

A*68:01

A*69:01

A*80:01

B*0

8:01

B*1

4:01

B*1

5:01

B*1

5:03

B*1

5:12

B*1

5:16

B*2

7:05

B*3

5:01

B*3

8:01

B*4

0:01

B*4

1:01

B*4

4:02

B*4

5:01

B*4

7:01

B*4

9:01

B*5

1:01

B*5

3:01

B*5

5:01

B*5

7:01

B*5

9:01

B*7

3:01

B*8

1:01

C*01:02

C*03:03

C*04:01

C*06:02

C*08:01

C*14:02

C*16:01

C*18:02

Standard

MF

I


0

5000

10000

15000

20000

25000

A*01:01

A*02:03

A*11:01

A*23:01

A*24:03

A*26:01

A*30:01

A*32:01

A*33:03

A*36:01

A*66:01

A*68:01

A*69:01

A*80:01

B*0

8:01

B*1

4:01

B*1

5:01

B*1

5:03

B*1

5:12

B*1

5:16

B*2

7:05

B*3

5:01

B*3

8:01

B*4

0:01

B*4

1:01

B*4

4:02

B*4

5:01

B*4

7:01

B*4

9:01

B*5

1:01

B*5

3:01

B*5

5:01

B*5

7:01

B*5

9:01

B*7

3:01

B*8

1:01

C*01:02

C*03:03

C*04:01

C*06:02

C*08:01

C*14:02

C*16:01

C*18:02

Standard ROB

MF

I

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0

5000

10000

15000

20000

25000

A*01:01

A*02:03

A*11:01

A*23:01

A*24:03

A*26:01

A*30:01

A*32:01

A*33:03

A*36:01

A*66:01

A*68:01

A*69:01

A*80:01

B*0

8:01

B*1

4:01

B*1

5:01

B*1

5:03

B*1

5:12

B*1

5:16

B*2

7:05

B*3

5:01

B*3

8:01

B*4

0:01

B*4

1:01

B*4

4:02

B*4

5:01

B*4

7:01

B*4

9:01

B*5

1:01

B*5

3:01

B*5

5:01

B*5

7:01

B*5

9:01

B*7

3:01

B*8

1:01

C*01:02

C*03:03

C*04:01

C*06:02

C*08:01

C*14:02

C*16:01

C*18:02

Standard ROB ROB EDTA

MF

I

Summary

• Good correlation between ROB and Standard LIFECODES LSA protocol in many cases.

• ROB protocol exhibits enhanced MFI– enhances weak reactivity with low titer DSA.

– enhances reactivity with low titer abs directed against CREGs.

• Standard LSA protocol is less susceptible to the “prozone” effect compared with the ROB protocol.– Treatment with EDTA resolves the “prozone”

• Differences are likely due to serum dilution in the Standard protocol.

• Immucor developed an enhanced LSA protocol to generate higher MFI values.

• Motivation was based feedback from the worldwide HLA community.– Clinical correlations with MFI have been established

– In order to encourage more widespread adoption of the LIFECODES LSA kits, MFI values need to be in line with what clinicians are used to seeing.

• Enhanced LSA protocol uses 20 l instead of 10 l of serum per reaction to increase the MFI values.

Enhanced LIFECODES LSA Protocol

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Multicenter Evaluation of the Rapid Optimized Single Antigen Bead (ROB) Protocol and Enhanced LIFECODES LSA Protocol.


Dalhousie University, Halifax, NS, Canada Dr. Rob Liwski

Institut Armand‐Frappier, Laval, QC, Canada Dr. Claude Daniel

Universite Laval, Quebec, QC, Canada Dr. Eric Wagner

University of Toronto, Toronto, ON, Canada Drs. Kathryn Tinckam/Neal denHollander

Western University, London, ON, Canada Dr. Qingyong Xu

University of Manitoba, Winnipeg, MB, Canada Dr. Peter Nickerson

University of Alberta, Edmonton, AB, Canada Drs. Patricia Campbell/Luis Hidalgo

University Of British Columbia, Vancouver, BC, Canada Drs. Paul Keown/Lenka Allan

Thomas Jefferson University Hospital, Philadelphia, PA Dr. Anthony Nizio

Johns Hopkins University, Baltimore, MD Dr. Annette Jackson

University of Pittsburgh Medical Center, Pittsburgh, PA Drs. Adriana Zeevi/Massimo Mangiola

Wake Forest School of Medicine, Winston‐Salem, NC Dr. Michael Gautreaux

Gift of Life Michigan, Ann Arbor, MI Dr. Sam Ho

University of Utah, Salt Lake City, UT Dr. Julio Delgado

Southwest Immunodiagnostics Inc. Lab, San Antonio, TX Dr. Cathi Murphy

Queen Mary Hospital, Hong Kong Dr. Janette Kwok


Dalhousie University, Halifax, NS, Canada Dr. Rob Liwski

Institut Armand‐Frappier, Laval, QC, Canada Dr. Claude Daniel

Universite Laval, Quebec, QC, Canada Dr. Eric Wagner

University of Toronto, Toronto, ON, Canada Drs. Kathryn Tinckam/Neal denHollander

Western University, London, ON, Canada Dr. Qingyong Xu

University of Manitoba, Winnipeg, MB, Canada Dr. Peter Nickerson

University of Alberta, Edmonton, AB, Canada Drs. Patricia Campbell/Luis Hidalgo

University Of British Columbia, Vancouver, BC, Canada Drs. Paul Keown/Lenka Allan

Thomas Jefferson University Hospital, Philadelphia, PA Dr. Anthony Nizio

Johns Hopkins University, Baltimore, MD Dr. Annette Jackson

University of Pittsburgh Medical Center, Pittsburgh, PA Drs. Adriana Zeevi/Massimo Mangiola

Wake Forest School of Medicine, Winston‐Salem, NC Dr. Michael Gautreaux

Gift of Life Michigan, Ann Arbor, MI Dr. Sam Ho

University of Utah, Salt Lake City, UT Dr. Julio Delgado

Southwest Immunodiagnostics Inc. Lab, San Antonio, TX Dr. Cathi Murphy

Queen Mary Hospital, Hong Kong Dr. Janette Kwok

3/19/2018

26

Design

• Class I and II HLA LIFECODES LSA kits, filter trays and high speed rotators (provided by Immucor).

• Technical support provided by Immucor (Dusanka D’Atri) to labs not using Immucor kits routinely.

• 9 ASHI PT sera (provided by Immucor) (2014‐2016 surveys).

– AC460, 464, 469, 470, 474, 480‐484

• Tested by LIFECODES LSA SAB Luminex assay

– Standard protocol (40:10)

– Enhanced protocol (40:20)

– ROB protocol

• Result analysis:– Pos/Neg ctrl beads (signal to noise differential)

– MFI comparison

– Mean and SD

– Pearson’s correlation (R2)

– Specificity assignment

Design

Side by side

Negative/Positive Control BeadsSignal to noise differential

Negative control (probe 1) Positive control (probe 77)

MFI MFI

Standard Enhanced ROB

0

200

400

600

800

1000

1200

1400

1600

1800

2000

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

0

5000

10000

15000

20000

25000

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10

3/19/2018

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Serum 1 Class I (no HLA abs)

‐1000

0

1000

2000

3000

4000

5000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

‐1000

0

1000

2000

3000

4000

5000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

‐1000

0

1000

2000

3000

4000

5000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

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297

299

Ad

jus

ted

MF

I

Standard

Enhanced

ROB

Probe #

Serum 1 Class II (weak abs)

‐1000

0

1000

2000

3000

4000

5000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

‐1000

0

1000

2000

3000

4000

5000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

‐1000

0

1000

2000

3000

4000

5000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

Ad

jus

ted

MF

I

Standard

Enhanced

ROB

Probe #

1or

1na

Serum 1 Class II (weak abs)

‐1000

0

1000

2000

3000

4000

5000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

‐1000

0

1000

2000

3000

4000

5000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

Ad

jus

ted

MF

I

Probe #

Mean

SD


3/19/2018

28

Serum 4 Class I (weak/moderate abs)

‐5000

0

5000

10000

15000

20000

25000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

‐5000

0

5000

10000

15000

20000

25000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

‐5000

0

5000

10000

15000

20000

25000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

Ad

jus

ted

MF

I

Standard

Enhanced

ROB

Probe #

1or

1or

Serum 4 Class I (weak/moderate abs)

‐5000

0

5000

10000

15000

20000

25000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

‐5000

0

5000

10000

15000

20000

25000

205

207

209

211

213

215

217

219

221

223

225

227

229

231

233

235

237

239

241

243

245

247

249

251

253

255

257

259

261

263

265

267

269

271

273

275

277

279

281

283

285

287

289

291

293

295

297

299

Ad

jus

ted

MF

I

Probe #

Mean

SD


Serum 2 Class II (weak/moderate abs)

‐5000

0

5000

10000

15000

20000

25000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

‐5000

0

5000

10000

15000

20000

25000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

‐5000

0

5000

10000

15000

20000

25000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

Ad

jus

ted

MF

I

Standard

Enhanced

ROB

Probe #

1or

1na

3/19/2018

29

Serum 2 Class II (weak/moderate abs)

‐5000

0

5000

10000

15000

20000

25000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

‐5000

0

5000

10000

15000

20000

25000

108110112114116118120122124126128130132134136138140142144146148150152154156158160162164166168170172174176178180182184186188 77

Ad

jus

ted

MF

I

Probe #

Mean

SD


Pearson’s correlation, class I HLA

Standard vs Enhanced Standard vs ROB

Pearson’s correlation, class II HLA

Standard vs Enhanced Standard vs ROB

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30

Pearson’s correlation Enhanced vs ROB

Class I HLA Class II HLA

Class I HLA MFI comparison

Courtesy Dr. Bryan Ray, Immucor

Class II HLA MFI comparison


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31

CV comparison

Class I HLA Class II HLA


0

10

20

30

40

50

60

70

80

90

100

ROB 20 µL 10 µL ASHI SPS

Number of Positive Antigens

Protocol

Percentage of Sites Reporting Positive Specificity

<50

50‐79

80‐98

99‐100

Class I HLA,comparison to ASHI PT Consensus


0

20

40

60

80

100

120

140

ROB 20µL 10µL ASHI SPS

Number of Positive Antigens

Protocol

Percentage of Sites Reporting Positive Specificities

< 50

50‐79

80‐98

99‐100

Class II HLA,comparison to ASHI PT Consensus


3/19/2018

32

Conclusion

• Enhanced and ROB protocols increase sensitivity (MFI values) in LIFECODES LSA assay (Enhanced > ROB).

• Improve signal to noise differential with no significant impact on background reactivity.

• Good overall correlation between all three protocols (best between Enhanced and ROB).

• Enhanced and ROB protocol show improved concordance of MFI and results.

• ROB protocol confers significant time saving allows for rapid testing of urgent patient sera.• Ex. testing during deceased donor work up.

Development of a novel, prozone‐resistant Dual Antibody Rapid Test (DART) Protocol

for LABScreen SAB assay.

Anna Greenshields, Robert Bray, Howard Gebel and Robert Liwski

Department of Pathology, Dalhousie UniversityHalifax, Nova Scotia, Canada

Interfering Substances“Prozone” Effect

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33

IgG SAB neat

IgG SAB neat

IgG SAB 1:10

“Prozone” effect

SABHLA-A2

3/19/2018

34


SABHLA-A2

Low titerNon C fixing Ab


SABHLA-A2



SABHLA-A2


3/19/2018

35


SABHLA-A2



SABHLA-A2

High titerC fixing Ab


SABHLA-A2

High titerC fixing Ab

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36


SABHLA-A2

High titerC fixing AbC1q binds


SABHLA-A2

High titerC fixing AbC1q bindsC1r & C1s recruited


SABHLA-A2

High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4b

Ca2+

3/19/2018

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SABHLA-A2

High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complex

Ca2+


High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complexC2 converted to C2a

Ca2+

SABHLA-A2


High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complexC2 converted to C2aC2a binds C4b (C3 convertase)

Ca2+

SABHLA-A2

3/19/2018

38


High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complexC2 converted to C2aC2a binds C4b (C3 convertase)C3 converted to C3b

SABHLA-A2


SABHLA-A2

High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complexC2 converted to C2aC2a binds C4b (C3 convertase)C3 converted to C3bC3b binds HLA-Ab complex and C4b2a (C5 convertase)


SABHLA-A2


3/19/2018

39


SABHLA-A2


Binding of anti-IgG-PE is blockedHLA antibody not detected


SABHLA-A2


Binding of anti-IgG-PE is blockedHLA antibody not detected

Solutions:Heat treatment (56oC), destroys C1q and other CSerum dilution, dilutes out complement DTT, breaks C1qEDTA, chelates Ca2+

Objectives

• To develop a SAB protocol that is resistant to the “prozone” effect without serum treatment……..

Greenshields et al ASHI Quarterly 2017

3/19/2018

40

SABHLA-A2

SABHLA-A2

SABHLA-A2

SABHLA-A2

SABHLA-A2

Anti-IgG-PE

3/19/2018

41

SABHLA-A2

SABHLA-A2

SABHLA-A2

SABHLA-A2

Anti-C’-PE

SABHLA-A2

SABHLA-A2

3/19/2018

42

Rapid Optimized SAB (ROB) LABScreen® Protocol

• Incubate beads and serum 15 min.


• Incubate with 20 l anti‐IgG‐PE, 1:10 dilution 5 min.



70% time reduction!

Dual Antibody Rapid Test (DART) LABScreen Protocol

• Incubate beads and serum 15 min.


• Incubate with 20 l anti‐IgG‐PE and anti‐C’‐PE 5 min.



70% time reduction!

Study design

• 20 “prozone” positive sera, 10 class I and 10 class II

• Tested by SAB:– Anti‐IgG‐PE

– Anti‐IgG‐PE with EDTA

– Anti‐C’‐PE

– DART, Anti‐IgG‐PE + anti‐C’‐PE

• Comparison of MFI

3/19/2018

43

IgG

Serum 1, Class I HLA

IgG

IgG EDTA


IgG

IgG EDTA


3/19/2018

44

IgG

C’

IgG EDTA


IgG

C’

IgG EDTA

IgG/C’DART


IgG

Serum 2, Class II HLA

3/19/2018

45

IgG

IgG EDTA


IgG

IgG EDTA


IgG

C’

IgG EDTA


3/19/2018

46

IgG

C’

IgG EDTA

IgG/C’DART


IgG EDTA

IgG

< 1,000 MFIN=737

No ProzoneN=739

Slight 3-5KN=51

Moderate 5-10KN=95

Marked >10KN=308

> 1,000 MFI

IgG/C’DART

C’

IgG EDTA

IgG

< 1,000 MFIN=737

No ProzoneN=739

Slight 3-5KN=51

Moderate 5-10KN=95

Marked >10KN=308

> 1,000 MFI

IgG/C’DART

C’

3/19/2018

47

IgG EDTA

IgG

< 1,000 MFIN=737

No ProzoneN=739

Slight 3-5KN=51

Moderate 5-10KN=95

Marked >10KN=308

> 1,000 MFI

IgG/C’DART

C’

IgG EDTA

IgG

DARTIgG/C’

no prozone, 3K

N=50

IgG EDTA

IgG

no prozone, 3K

N=50

DARTIgG/C’

3/19/2018

48

IgG


IgG

IgG EDTA


IgG

IgG EDTA


3/19/2018

49

IgG

IgG EDTA


IgG

C’

IgG EDTA


IgG

C’

IgG EDTA

IgG/C’DART


3/19/2018

50

Conclusions

• DART protocol is resistant to the “prozone” effect.

• Eliminates the necessity to treat sera thus avoiding potential interference with HLA antibody testing.

• Improved MFI correlation for “prozone” negative specificities compared with EDTA.

Dual Antibody Rapid Test (DART)

Final thoughts

• Value of protocol optimization

– Impact on test quality, TAT and clinical patient care

– Should be an integral part of any assay validation

• Significant variability in antibody testing protocols and results

– Impact on patient care

– Impact on transplantation research

• Assay standardization improves concordance of results

3/19/2018

51

Acknowledgements

• Collaborators

– Drs. Howard Gebel, Robert Bray, Cathi Murphey

• Halifax HLA lab

– Dr. Anna Greenshields

– Geoff Adams

– Geoff Peladeau

– Kelly Heinstein

• Immucor Team

• Dr. Bryan Ray

• Dr. Masako Osada

• Dusanka D’Atri

• Kelly Cousins

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52

• IMMUCOR Enhanced/ROB Evaluation Study

Dr. Claude Daniel

Dr. Eric Wagner

Drs. Kathryn Tinckam/Neal denHollander

Dr. Qingyong Xu

Dr. Peter Nickerson

Drs. Patricia Campbell/Luis Hidalgo

Drs. Paul Keown/Lenka Allan

Dr. Anthony Nizio

Dr. Annette Jackson

Drs. Adriana Zeevi/Massimo Mangiola

Dr. Michael Gautreaux

Dr. Sam Ho

Dr. Julio Delgado

Dr. Cathi Murphy

Dr. Janette Kwok

• ROB Protocol Evaluation Study

– Dr. Patricia Campbell

– Dr. Adriana Colovai

– Dr. Deborah Crowe

– Dr. Anne Halpin

– Dr. Ronald Kerman

– Dr. Dong Li John Lunz

– Dr. Cathi Murphey

– Dr. Peter Nickerson

– Denise Pochinco

– Dr. Sandra Rosen‐Bronson

– Dr. Olga Timofeeva

– Dr. Paul Warner

– Dr. Adriana Zeevi

Acknowledgements


Questions?



3/19/2018

53


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