optimizing solid phase assays - afdt.org
TRANSCRIPT
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Dr. Robert Liwski, MD, PhD, FRCPCMedical Director, HLA Typing LaboratoryHead, Division of HematopathologyProfessor, Department of PathologyDalhousie University, HalifaxMedical Director, HLA Laboratory, CBS, [email protected]
Optimizing Solid Phase Assays
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Evolution of HLA antibody testing
Gebel and Bray Curr. Opin. Nephrol. Hypertens. 2010.
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HLA antibody identification by LABScreen®Single Antigen Bead Luminex Assay
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HLA antibody analysisPatient with a history of transplant
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Amico et al, Transplantation 2009.
Significant decrease in graft survival in patients with pre-transplant DSA
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Significant decrease in graft survival in patients with pre-transplant DSA
6Lefaucher et al, JASN 2010.
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Calculated Panel Reactive Antibody (cPRA)
Tinckam, Liwski, Pochinco, et al, AJT 2015.
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Highly sensitized
Tinckam, Liwski, Pochinco, et al, AJT 2015.
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Non-HLA abs
False pos FCXM
Prozone
FP 3.1%
FN 14%
Some allele specific (non-DSA)
Some weak DSA
Tambur et al. AJT 9:1886, 2009
Sensitivity = 93.1%
Specificity = 85.3%
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10 fold decrease in positive XM
Improved allocation
Cecka et al. AJT 11:719, 2011
OPTN/UNOS
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2 fold increase in HSP transplant
OPTN/UNOS
11Cecka et al. AJT 11:719, 2011
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Baxter-Lowe et al. AJT, 14:1592, 2014
National Kidney Registry
91% concordance between virtual and FCXM
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13Paramesh et al. AJT, 17:2139, 2017
17/114 (15%) unexpected positive FCXM
Variability in listing unacceptable antigens
Allele specific abs
DQA and DP abs
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Challenges with HLA antibody testing and virtual crossmatching
• Virtual crossmatch is only as good, as current, as accurate, and
as complete as the HLA antibody and HLA typing information
• Reproducibility
• Interfering substances, “prozone” effect
• Antibodies against denatured HLA epitopes
• Interpretation
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Reproducibility
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Reed et al. Am. J. Transplant 13:1859, 2013.
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LABScreen® single antigen bead (SAB) Luminex protocol
• Incubate beads (5 ml) and serum 20 ml (RT) 30 min.
• Wash x3 (5 min/spin) 15 min.
• Incubate with 100 ml 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?
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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
Liwski et al Hum. Immunol. 2017
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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
Liwski et al Hum. Immunol. 2017
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Effect of reduced spin time (1 vs 5 min) on bead counts
Class I beads Class II beads
Be
ad
co
un
t
Bead number
N=3
Liwski et al Hum. Immunol. 2017
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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 results
20 minutes saved!
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation times
• Serum incubation time
• Anti-IgG-PE incubation time
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class I
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation time¼ PPC, HLA class II
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of reduced incubation timeNegative control serum
Class I Class II
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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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 binding
Small 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
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Effects of increasing IgG-PE concentration¼ PPC, HLA class I
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of increasing IgG-PE concentration¼ PPC, HLA class II
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of increasing IgG-PE concentrationNegative control serum
Class I Class II
MF
I
Bead number
Liwski et al Hum. Immunol. 2017
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Effects of increasing IgG-PE concentrationNC and PC beads
NC bead (#1) PC bead (#2)
MF
I
Serum/IgG-PE incubation time
Negligible effect on background Significant effect on positive rxns
Liwski et al Hum. Immunol. 2017
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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).
• Improved signal to noise delta
• Can we compensate for reduced MFI values in the 15/5 min
protocol by optimizing the concentration of anti-IgG-PE?
Liwski 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 number
Liwski 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 number
Liwski 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 number
Liwski 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 number
Liwski 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 number
Liwski et al Hum. Immunol. 2017
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Effects of increasing IgG-PE concentration on MFI in 15/5 protocol¼ PPC, HLA class II
MF
I
Bead number
Liwski 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
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ROB LABScreen® Protocol
• Incubate beads (5 ml) and serum 25 ml (RT) 15 min.
• Wash x3 (1 min/spin) 3 min.
• Incubate with 20 ml 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
co
l M
FI
Standard Assay MFI
Liwski et al Hum. Immunol. 2017
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Representative Serum ReactivityStandard vs ROB protocol
AC-463 Class I AC-463 Class II
MF
I
Bead number
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
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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
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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)
Liwski et al ASHI 2014
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AC464 class I
Individual lab MFI comparison
Bead number
MFI
Standard
ROB
Liwski et al ASHI 2014
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AC464 class IAverage lab MFI and CV comparison
Bead number
MFI
%CV
Liwski et al ASHI 2014
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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
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Overall mean MFI correlation
Standard
RO
B
Class I Class II
Liwski et al ASHI 2014
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Average CVStandard vs ROB protocol
Class I Class II
% C
V
Serum
Liwski et al ASHI 2014
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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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Interfering Substances “Prozone” Effect
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IgG SAB neatWould you transplant this patient?
A2 DSA
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IgG SAB neatWould you transplant this patient?
A2 DSA
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IgG SAB neat
IgG SAB 1:10
Would you transplant this patient?
A2 DSA
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Dithiotreitol (DTT)
Hypotonic dialysis ?IgM
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Kosmoliaptsis et al. Human Immunology 71:45, 2010
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Schnaidt et al. Transplantation 92:510, 2011
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Schnaidt et al. Transplantation 92:510, 2011
C1 inhibition?
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Visentin et al. Transplantation 98, 625, 2014
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“Prozone” effect
SAB
HLA-A2
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“Prozone” effect
SAB
HLA-A2
Low titer
Non C fixing Ab
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“Prozone” effect
SAB
HLA-A2
Low titer
Non C fixing Ab
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“Prozone” effect
SAB
HLA-A2
Low titer
Non C fixing Ab
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“Prozone” effect
SAB
HLA-A2
Low titer
Non C fixing Ab
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“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
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“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
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“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
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“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
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“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
Ca2+
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“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
Ca2+
![Page 77: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/77.jpg)
“Prozone” effect
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
Ca2+
SAB
HLA-A2
![Page 78: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/78.jpg)
“Prozone” effect
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
C2a binds C4b (C3 convertase)
Ca2+
SAB
HLA-A2
![Page 79: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/79.jpg)
“Prozone” effect
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
C2a binds C4b (C3 convertase)
C3 converted to C3b
SAB
HLA-A2
Ca2+
![Page 80: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/80.jpg)
“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
C2a binds C4b (C3 convertase)
C3 converted to C3b
C3b binds HLA-Ab complex
and C4b2a (C5 convertase)
Ca2+
![Page 81: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/81.jpg)
“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
C2a binds C4b (C3 convertase)
C3 converted to C3b
C3b binds HLA-Ab complex
and C4b2a (C5 convertase)
Ca2+
![Page 82: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/82.jpg)
“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
C2a binds C4b (C3 convertase)
C3 converted to C3b
C3b binds HLA-Ab complex
and C4b2a (C5 convertase)
Binding of anti-IgG-PE is
blocked
HLA antibody not detected
Ca2+
![Page 83: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/83.jpg)
“Prozone” effect
SAB
HLA-A2
High titer
C fixing Ab
C1q binds
C1r & C1s recruited
C4 converted to C4b
C4b binds HLA-Ab complex
C2 converted to C2a
C2a binds C4b (C3 convertase)
C3 converted to C3b
C3b binds HLA-Ab complex
and C4b2a (C5 convertase)
Binding of anti-IgG-PE is
blocked
HLA antibody not detected
Ca2+
Solutions:
Heat treatment (56oC), destroys C1q and other C
Serum dilution, dilutes out complement
DTT, breaks C1q
EDTA, chelates Ca2+
![Page 84: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/84.jpg)
“Prozone” effect
EDTA Ca2+X
SAB
HLA-A2
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“Prozone” effect
EDTA Ca2+X
SAB
HLA-A2
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“Prozone” effect
EDTA Ca2+X
SAB
HLA-A2
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IgG SAB neat IgG SAB 1:10
IgG SAB EDTA C1q
“prozone” effect
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Zhang and Reinsmoen Hum. Immunol. 2017
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Zhang and Reinsmoen Hum. Immunol. 2017
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Zhang and Reinsmoen Hum. Immunol. 2017
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Treatment of sera with EDTA
• Simple and effective solution to prevent “prozone”.
• Anecdotal evidence that the procedure is not effective.
• The protocol for serum treatment with EDTA published by Schnaidt et. al. is very ambiguous and impossible to follow.
• “5mL EDTA solution (6%) + 95mL of serum”
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Treatment of sera with EDTA protocol
• Schnaidt et al: “5mL EDTA solution (6%) + 95mL of serum”
• Does not indicate which EDTA salt is used:
• Disodium EDTA, MW = 292.24
• Dipotassium EDTA, MW = 368.42
• Dipotassium EDTA dihydrate, MW = 404.47
• Does not indicate if solid or solution used.
• Does not indicate whether 6% means v/v or w/v
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Divalent Metal Ions in
Blood
Normal Range (mmol/L)
Calcium (Total) 2.18-2.58
Calcium (Ionized) 1.05-1.3
Magnesium 0.75-0.95
Iron 0.011-0.032
Copper 0.011-0.025
Divalent Metal Ion, Reference Ranges
• EDTA forms a 1:1 complex with divalent metal ions
• Required amount of EDTA should “mop up” all divalent metal
ions in serum (ionized calcium and magnesium), > 1.8 - 2.5
mmol/L.
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Experimental design
• Six “prozone” positive sera (3 class I and 3 class II) were
selected based on dilution studies.
• Sera were treated with different concentrations of EDTA or
PBS control.
• Disodium EDTA, 0.5M stock solution, Sigma-Aldrich
• Final EDTA concentrations: 2mM, 3mM and 6mM
• Tested sera using LABScreen Single antigen bead assay
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Class I HLA, representative serum
PBS
3mM EDTA
2mM EDTA
6mM EDTA
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Class II HLA, representative serum
PBS
3mM EDTA
2mM EDTA
6mM EDTA
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Class I HLA “Prozone” positive specificities
MF
I
EDTA
Specificities
(sorted in order of MFI for no EDTA)
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MF
I
Specificities
(sorted in order of MFI for no EDTA)
EDTA
Class II HLA “Prozone” positive specificities
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Conclusions
• EDTA treatment of serum is an effective way to eliminate “prozone” effect.
• Concentration of EDTA is important when treating serum.
• Minimal dose of EDTA needed to prevent “prozone” is ≥ 3mM.
• This concentration of EDTA is necessary to chelate all divalent metal ions in blood.
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Questions
•How common is prozone?
•Propensity to prozone?
•Degree of prozone?
•Mechanism of prozone and relative contribution of IgM vs C3d?
•Loci affected?
![Page 101: Optimizing Solid Phase Assays - afdt.org](https://reader033.vdocuments.us/reader033/viewer/2022042410/625ea35116e70c49a4653fa4/html5/thumbnails/101.jpg)
• Studied 129 class I and 85 class II patient sera
• Complement interference is common
• 30% class I HLA sera
• 46% class II HLA sera
Guidicelli et al. HLA. 2018
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• 30 highly sensitized patients (cPRA ≥ 95%) on active renal waitlist
• 18 female (60%), 12 male (40%)
• Female patient sensitization history:
• 6 pregnancy (+/- transfusion 50%)
• 6 transplant (+/- transfusion 50%)
• 6 transplant + pregnancy (+/- transfusion 67%)
• Male patient sensitization history:
• 12 transplant (+/- transfusion 67%)
Greenshields and Liwski Hum. Immunol. 2019
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Greenshields and Liwski Hum. Immunol. 2019
Highly sensitized patients (n=30): cPRA ≥ 95%
0
20
40
60
80
100
120
140
Class I Class II Class I/II
% c
PR
A
Female Male All
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• LABScreen SAB assay
• IgG +/- EDTA to identify prozone, +/- dilution
• C3d, IgM
• Total of 6049 specificities (3,104 class; 2,945 class II) analysed
• IgG negative: n = 3700 (61%)
• IgG positive: n = 2349 (39%)
• No prozone (DMFI EDTA < 3K): n = 1690 (72%)
• Prozone (DMFI EDTA > 3K): n = 659 (28%)
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Greenshields and Liwski Hum. Immunol. 2019
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Greenshields and Liwski Hum. Immunol. 2019
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Greenshields and Liwski Hum. Immunol. 2019
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MFI
MFI
EDTA IgG-PE
IgG-PE
MFI
MFI
C3d-PE
IgM-PE
A)
Mild prozone (n=88)
IgG-PE DMFI=3,000-4,999
B)
Moderate prozone (n=131)
IgG-PE DMFI=5,000-9,999
C)
Marked prozone (n=297)
IgG-PE DMFI≥10,000
C3d +ve = 87.5%
MFI=5,878+/-2,462
IgM +ve = 13.6%
MFI = 2,847+/-1,515
C3d +ve = 100%
MFI=9,593+/-1,972
IgM +ve = 29.8%
MFI = 2,898+/-1,330
C3d +ve = 100%
MFI=15,572+/-2,945
IgM +ve = 64.0%
MFI = 4,794+/-2,965
Greenshields and Liwski Hum. Immunol. 2019
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MFI
MFI
EDTA IgG-PE
IgG-PE
MFI
MFI
C3d-PE
IgM-PE
A)
Mild prozone (n=88)
IgG-PE DMFI=3,000-4,999
B)
Moderate prozone (n=131)
IgG-PE DMFI=5,000-9,999
C)
Marked prozone (n=297)
IgG-PE DMFI≥10,000
C3d +ve = 87.5%
MFI=5,878+/-2,462
IgM +ve = 13.6%
MFI = 2,847+/-1,515
C3d +ve = 100%
MFI=9,593+/-1,972
IgM +ve = 29.8%
MFI = 2,898+/-1,330
C3d +ve = 100%
MFI=15,572+/-2,945
IgM +ve = 64.0%
MFI = 4,794+/-2,965
Greenshields and Liwski Hum. Immunol. 2019
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MFI
MFI
EDTA IgG-PE
IgG-PE
MFI
MFI
C3d-PE
IgM-PE
A)
Mild prozone (n=88)
IgG-PE DMFI=3,000-4,999
B)
Moderate prozone (n=131)
IgG-PE DMFI=5,000-9,999
C)
Marked prozone (n=297)
IgG-PE DMFI≥10,000
C3d +ve = 87.5%
MFI=5,878+/-2,462
IgM +ve = 13.6%
MFI = 2,847+/-1,515
C3d +ve = 100%
MFI=9,593+/-1,972
IgM +ve = 29.8%
MFI = 2,898+/-1,330
C3d +ve = 100%
MFI=15,572+/-2,945
IgM +ve = 64.0%
MFI = 4,794+/-2,965
Greenshields and Liwski Hum. Immunol. 2019
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C3d+ IgM+
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MFI%
po
sit
ive
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B)
D EDTA-untreated C3d IgM
Greenshields and Liwski Hum. Immunol. 2019
C3d MFI ≥ 4,000 predicts prozonesensitivity = 95.2%specificity = 97.2%
Degree of prozonecorrelates with C3d deposition
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Greenshields and Liwski Hum. Immunol. 2019
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I
A)
Dilution did not reveal additional mechanisms of “prozone”.
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Greenshields and Liwski Hum. Immunol. 2019
Frequency and severity of prozone in HSP
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Greenshields and Liwski Hum. Immunol. 2019
32%
62%
6%
B) Prozone by locus, Class I
A B C
14%
68%
18%
C) Prozone by locus, Class II
DR DQ DP
70%
30%
A) Prozone by HLA class
Class I Class II
D)
Frequency of prozone by HLA class and locus
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Greenshields and Liwski Hum. Immunol. 2019
Degree of prozone by sensitizing event
0
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50
60
70
80
90
100
Pregnancy Transplant All
None
Mild
Mod/Marked
Prozone
% p
atients
Sensitization
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How can we identify sera that exhibit prozone?
Positive control bead?Negative control bead?
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MFI
Positive control bead reactivity
Greenshields and Liwski Hum. Immunol. 2019
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MFI
Negative control bead reactivity
Greenshields and Liwski Hum. Immunol. 2019
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0
5000
10000
15000
20000
25000
30000
1
73
86
79
16
87
48
13
71
94
54
14
83 9
77
47
23
98
19
68
49
52
33
43
78
HFX Tx
0
5000
10000
15000
20000
25000
30000
1
73
86
79
16
87
48
13
71
94
54
14
83 9
77
47
23
98
19
68
49
52
33
43
78
No Tx
Class I, Serum 2
EDTA
No TX
0
5000
10000
15000
20000
25000
30000
1
73
86
79
16
87
48
13
71
94
54
14
83 9
77
47
23
98
19
68
49
52
33
43
78
HFX Tx
0
5000
10000
15000
20000
25000
30000
1
73
86
79
16
87
48
13
71
94
54
14
83 9
77
47
23
98
19
68
49
52
33
43
78
No Tx
EDTA
No TX
Class II, Serum 1
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Summary
• The “prozone” effect is very common among the highly sensitized
patients (84%; 75% marked).
• Main risk factor: history of previous transplant
• Class I more commonly affected than class II
• Most commonly affected loci: HLA-A, B and DQ
• Due to complement mediated interference.
• Contribution from IgM?
• Degree of interference is proportional to the amount of complement
deposition
• EDTA is effective at minimizing complement mediated interference
Greenshields and Liwski Hum. Immunol. 2019
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Denatured HLA epitopes
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Patient DD kidney
A 24 29 01 02B 44:03 48 44:02 57C 08 16 05 06
DRB1 07 12 01 07DRB3/4/5 52 53 Blank 53NullDQB1 02:02 03:01 03:03 05DQA1 02:01 05 01 02:01
Class I SAB analysis
Unacceptable antigens
B8, B37, B41, B42, B44(44:02), B45, B82
Patient CaseWould you transplant this patient?
Previous cardiac transplant
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FCXM resultsT cell B cell
Neg control
Patient
Pos control
T cell B cell
Neg Control 178 229
Patient 166 201
Pos Control 493 500
Allo X-match
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AA sequence alignment
Unacceptable antigens
B8, B37, B41, B42, B44(44:02), B45, B82
156D
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El-Awar et al. Hum. Immunol.
70:844, 2009
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Eapen et al. Transplant Immunology 25:77, 2011
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Acid treatment denatures class I HLA antigens
Untreated
Acid treated
Positive Control Serum
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Acid treatment, patient serum
Untreated Acid Tx
A31 & A33
Denatured epitope, 73I
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• Studied a cohort of 323 renal wait list patients
– 40% with class I HLA cPRA of > 85%
– 12% with class I HLA cPRA = 0
– 91% experienced at least one sensitizing event
• Sera tested by standard SAB, acid denatured SAB and iBead SAB for class I HLA antibodies
• Correlation with FCXM
• Impact on cPRA
Visentin et al. Transplantation 98:738 2014
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Visentin et al. Transplantation 98:738 2014
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83% FCXM positive
16% FCXM positive
Visentin et al. Transplantation 98:738 2014
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Visentin et al. Transplantation 98:738 2014
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• Retrospective study of 837 renal transplant recipient transplanted with negative CDC T/B XM
• Pre-transplant sera were retrospectively tested by standard SAB, acid denatured SAB and iBead SAB
– DSA classified into native vs denatured epitope specific
• 156 patients were found to have class I HLA DSA by standard SAB, 11% had class I DSA directed against denatured epitopes only.
• Correlation with graft survival.
Otten et al. Clin. Exp. Immun. 173:536, 2013
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Denatured epitope
Native epitope (iBeads)
Otten et al. Clin. Exp. Immun. 173:536, 2013
No DSA
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• Clinical significance is questionable
• Prevalence
• 20-40% of patients (Visentin et al.), 11% of DSA (Otten et al.)
• Significant effect on cPRA and allocation (Visentin et al.)
• Solutions:
• Acid treatment (class I)
• iBeads (class I), no longer available
• Epitope analysis, pattern recognition.
• Correlation with FCXM +/- absorbtion elution studies
Antibodies against denatured HLA epitopes
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Loupy et al. NEJM. 2013
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< 6,000> 6,000
Loupy et al. NEJM. 2013
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Prozone?< 6,000> 6,000
Loupy et al. NEJM. 2013
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Prozone?
Denatured?
< 6,000> 6,000
Loupy et al. NEJM. 2013
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Requirements for successful and safe virtual crossmatching
• Accurate patient sensitization history, including immunosuppression
• HLA typing
• All loci
• SABR resolution
• Up to date SAB HLA antibody information
• On call SAB testing and vXM
• Reproducible SAB and FCXM
• Serum treatment with EDTA/DTT/Heat to eliminate the prozone effect
• HLA antibody assignment
• Epitope analysis
• Denatured epitopes/non-specific reactivity
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Thank you!
Questions?