Future Webinars - Immucor, Inc. Program Handouts... · • ABHI, PACE, Florida and California DHS...
Transcript of Future Webinars - Immucor, Inc. Program Handouts... · • ABHI, PACE, Florida and California DHS...
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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
All Content © 2015 Immucor, Inc.
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
All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
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
All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
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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All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
learn.immucor.com
All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
Questions?
• You are all muted
• Type in questions
All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
• 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
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
Liwski et al Hum. Immunol. 2017
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Liwski et al Hum. Immunol. 2017
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
Liwski et al Hum. Immunol. 2017
Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class II
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of reduced incubation timeNegative control serum
Class I Class II
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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
Liwski et al Hum. Immunol. 2017
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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?
Liwski et al Hum. Immunol. 2017
Effects of increasing IgG‐PE concentration¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of increasing IgG‐PE concentration¼ PPC, HLA class II
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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Effects of increasing IgG‐PE concentrationNegative control serum
Class I Class II
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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
Liwski 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
Bead numberLiwski et al Hum. Immunol. 2017
Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class I
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
Effects of increasing IgG‐PE concentration on MFI in 15/5 protocol¼ PPC, HLA class II
MF
I
Bead numberLiwski et al Hum. Immunol. 2017
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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.
Liwski et al Hum. Immunol. 2017
ROB LABScreen® Protocol
• Incubate beads (5 l) and serum 25 l (RT) 15 min.
• Wash x3 (1 min/spin) 3 min.
• Incubate with 20 l anti‐IgG‐PE, 1:10 dilution (RT) 5 min.
• Wash x2 (5min/spin) 2 min.
• Total assay time 25 min.
Liwski et al Hum. Immunol. 2017
ROB LABScreen® Protocol
• Incubate beads (5 l) and serum 25 l (RT) 15 min.
• Wash x3 (1 min/spin) 3 min.
• Incubate with 20 l anti‐IgG‐PE, 1:10 dilution (RT) 5 min.
• Wash x2 (5min/spin) 2 min.
• Total assay time 25 min.
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
Bead numberLiwski et al Hum. Immunol. 2017
“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
Liwski et al Hum. Immunol. 2017
ROB
Patient serum CF, class I
Standard
Liwski et al Hum. Immunol. 2017
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.
Liwski et al Hum. Immunol. 2017
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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
Liwski et al ASHI 2014
Liwski et al ASHI 2014
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
Liwski et al ASHI 2014
AC464 class IAverage lab MFI and CV comparison
Bead number
MFI
%CV
Liwski et al ASHI 2014
AC460 class IIIndividual lab MFI comparison
Bead number
MFI
Standard
ROB
Liwski et al ASHI 2014
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AC460 class IIAverage lab MFI and CV comparison
Bead number
MFI
%CV
Liwski et al ASHI 2014
Overall mean MFI correlation
Standard
RO
B
Class I Class II
Liwski et al ASHI 2014
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
Liwski et al ASHI 2014
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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
• Incubate beads (10 l) and serum 25 l (RT) 15 min.
• Wash x3 (1 min/spin) 3 min.
• Incubate with 20 l anti‐IgG‐PE, 1:2 dilution (RT) 5 min.
• Wash (1 min/spin) 1 min.
• Total assay time 25 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
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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
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 ROB
MF
I
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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 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.
Participating Centers
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
Participating Centers
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
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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
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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
295
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
Standard Enhanced ROB
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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
Standard Enhanced ROB
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
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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
Standard Enhanced ROB
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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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
Courtesy Dr. Bryan Ray, Immucor
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CV comparison
Class I HLA Class II HLA
Courtesy Dr. Bryan Ray, Immucor
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
Courtesy Dr. Bryan Ray, Immucor
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
Courtesy Dr. Bryan Ray, Immucor
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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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IgG SAB neat
IgG SAB neat
IgG SAB 1:10
“Prozone” effect
SABHLA-A2
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“Prozone” effect
SABHLA-A2
Low titerNon C fixing Ab
“Prozone” effect
SABHLA-A2
Low titerNon C fixing Ab
“Prozone” effect
SABHLA-A2
Low titerNon C fixing Ab
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“Prozone” effect
SABHLA-A2
Low titerNon C fixing Ab
“Prozone” effect
SABHLA-A2
High titerC fixing Ab
“Prozone” effect
SABHLA-A2
High titerC fixing Ab
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“Prozone” effect
SABHLA-A2
High titerC fixing AbC1q binds
“Prozone” effect
SABHLA-A2
High titerC fixing AbC1q bindsC1r & C1s recruited
“Prozone” effect
SABHLA-A2
High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4b
Ca2+
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“Prozone” effect
SABHLA-A2
High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complex
Ca2+
“Prozone” effect
High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complexC2 converted to C2a
Ca2+
SABHLA-A2
“Prozone” effect
High titerC fixing AbC1q bindsC1r & C1s recruitedC4 converted to C4bC4b binds HLA-Ab complexC2 converted to C2aC2a binds C4b (C3 convertase)
Ca2+
SABHLA-A2
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“Prozone” effect
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
“Prozone” effect
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)
“Prozone” effect
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)
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“Prozone” effect
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)
Binding of anti-IgG-PE is blockedHLA antibody not detected
“Prozone” effect
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)
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
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SABHLA-A2
SABHLA-A2
SABHLA-A2
SABHLA-A2
SABHLA-A2
Anti-IgG-PE
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SABHLA-A2
SABHLA-A2
SABHLA-A2
SABHLA-A2
Anti-C’-PE
SABHLA-A2
SABHLA-A2
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Rapid Optimized SAB (ROB) LABScreen® Protocol
• Incubate beads and serum 15 min.
• Wash x3 (1 min/spin) 3 min.
• Incubate with 20 l anti‐IgG‐PE, 1:10 dilution 5 min.
• Wash x2 (5min/spin) 2 min.
• Total assay time 25 min.
70% time reduction!
Dual Antibody Rapid Test (DART) LABScreen Protocol
• Incubate beads and serum 15 min.
• Wash x3 (1 min/spin) 3 min.
• Incubate with 20 l anti‐IgG‐PE and anti‐C’‐PE 5 min.
• Wash x2 (5min/spin) 2 min.
• Total assay time 25 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
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IgG
Serum 1, Class I HLA
IgG
IgG EDTA
Serum 1, Class I HLA
IgG
IgG EDTA
Serum 1, Class I HLA
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IgG
C’
IgG EDTA
Serum 1, Class I HLA
IgG
C’
IgG EDTA
IgG/C’DART
Serum 1, Class I HLA
IgG
Serum 2, Class II HLA
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IgG
IgG EDTA
Serum 2, Class II HLA
IgG
IgG EDTA
Serum 2, Class II HLA
IgG
C’
IgG EDTA
Serum 2, Class II HLA
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IgG
C’
IgG EDTA
IgG/C’DART
Serum 2, Class II HLA
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’
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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’
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IgG
Serum 3, Class I HLA
IgG
IgG EDTA
Serum 3, Class I HLA
IgG
IgG EDTA
Serum 3, Class I HLA
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IgG
IgG EDTA
Serum 3, Class I HLA
IgG
C’
IgG EDTA
Serum 3, Class I HLA
IgG
C’
IgG EDTA
IgG/C’DART
Serum 3, Class I HLA
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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
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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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• 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
All Content © 2015 Immucor, Inc.
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