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1 Dr. Robert Liwski, MD, PhD, FRCPC Medical Director, HLA Typing Laboratory Head, Division of Hematopathology Professor, Department of Pathology Dalhousie University, Halifax Medical Director, HLA Laboratory, CBS, Ottawa [email protected] Optimizing Solid Phase Assays

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Page 1: Optimizing Solid Phase Assays - afdt.org

1

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

Page 2: Optimizing Solid Phase Assays - afdt.org

Evolution of HLA antibody testing

Gebel and Bray Curr. Opin. Nephrol. Hypertens. 2010.

Page 3: Optimizing Solid Phase Assays - afdt.org

HLA antibody identification by LABScreen®Single Antigen Bead Luminex Assay

Page 4: Optimizing Solid Phase Assays - afdt.org

HLA antibody analysisPatient with a history of transplant

Page 5: Optimizing Solid Phase Assays - afdt.org

Amico et al, Transplantation 2009.

Significant decrease in graft survival in patients with pre-transplant DSA

Page 6: Optimizing Solid Phase Assays - afdt.org

Significant decrease in graft survival in patients with pre-transplant DSA

6Lefaucher et al, JASN 2010.

Page 7: Optimizing Solid Phase Assays - afdt.org

Calculated Panel Reactive Antibody (cPRA)

Tinckam, Liwski, Pochinco, et al, AJT 2015.

Page 8: Optimizing Solid Phase Assays - afdt.org

Highly sensitized

Tinckam, Liwski, Pochinco, et al, AJT 2015.

Page 9: Optimizing Solid Phase Assays - afdt.org

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%

Page 10: Optimizing Solid Phase Assays - afdt.org

10 fold decrease in positive XM

Improved allocation

Cecka et al. AJT 11:719, 2011

OPTN/UNOS

Page 11: Optimizing Solid Phase Assays - afdt.org

2 fold increase in HSP transplant

OPTN/UNOS

11Cecka et al. AJT 11:719, 2011

Page 12: Optimizing Solid Phase Assays - afdt.org

Baxter-Lowe et al. AJT, 14:1592, 2014

National Kidney Registry

91% concordance between virtual and FCXM

Page 13: Optimizing Solid Phase Assays - afdt.org

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

Page 14: Optimizing Solid Phase Assays - afdt.org

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

Page 15: Optimizing Solid Phase Assays - afdt.org

Reproducibility

Page 16: Optimizing Solid Phase Assays - afdt.org

Reed et al. Am. J. Transplant 13:1859, 2013.

Page 17: Optimizing Solid Phase Assays - afdt.org

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?

Page 18: Optimizing Solid Phase Assays - afdt.org

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

Page 20: Optimizing Solid Phase Assays - afdt.org

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

Page 22: Optimizing Solid Phase Assays - afdt.org

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

Page 24: Optimizing Solid Phase Assays - afdt.org

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

Page 26: Optimizing Solid Phase Assays - afdt.org

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

Page 30: Optimizing Solid Phase Assays - afdt.org

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

Page 31: Optimizing Solid Phase Assays - afdt.org

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

MF

I

Bead number

Liwski et al Hum. Immunol. 2017

Page 32: Optimizing Solid Phase Assays - afdt.org

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

Page 34: Optimizing Solid Phase Assays - afdt.org

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

Page 36: Optimizing Solid Phase Assays - afdt.org

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

Page 40: Optimizing Solid Phase Assays - afdt.org

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

Page 44: Optimizing Solid Phase Assays - afdt.org

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

Page 47: Optimizing Solid Phase Assays - afdt.org

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

Page 49: Optimizing Solid Phase Assays - afdt.org

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

Page 54: Optimizing Solid Phase Assays - afdt.org

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

Page 58: Optimizing Solid Phase Assays - afdt.org

IgG SAB neatWould you transplant this patient?

A2 DSA

Page 59: Optimizing Solid Phase Assays - afdt.org

IgG SAB neatWould you transplant this patient?

A2 DSA

Page 60: Optimizing Solid Phase Assays - afdt.org

IgG SAB neat

IgG SAB 1:10

Would you transplant this patient?

A2 DSA

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Dithiotreitol (DTT)

Hypotonic dialysis ?IgM

Page 62: Optimizing Solid Phase Assays - afdt.org

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?

Page 65: Optimizing Solid Phase Assays - afdt.org

Visentin et al. Transplantation 98, 625, 2014

Page 66: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

Page 67: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

Low titer

Non C fixing Ab

Page 68: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

Low titer

Non C fixing Ab

Page 69: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

Low titer

Non C fixing Ab

Page 70: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

Low titer

Non C fixing Ab

Page 71: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

High titer

C fixing Ab

Page 72: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

High titer

C fixing Ab

Page 73: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

High titer

C fixing Ab

C1q binds

Page 74: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

High titer

C fixing Ab

C1q binds

C1r & C1s recruited

Page 75: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

SAB

HLA-A2

High titer

C fixing Ab

C1q binds

C1r & C1s recruited

C4 converted to C4b

Ca2+

Page 76: Optimizing Solid Phase Assays - afdt.org

“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

“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

“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

“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

“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

“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

“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

“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

“Prozone” effect

EDTA Ca2+X

SAB

HLA-A2

Page 85: Optimizing Solid Phase Assays - afdt.org

“Prozone” effect

EDTA Ca2+X

SAB

HLA-A2

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“Prozone” effect

EDTA Ca2+X

SAB

HLA-A2

Page 87: Optimizing Solid Phase Assays - afdt.org

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”

Page 92: Optimizing Solid Phase Assays - afdt.org

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

Page 93: Optimizing Solid Phase Assays - afdt.org

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.

Page 94: Optimizing Solid Phase Assays - afdt.org

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

Page 95: Optimizing Solid Phase Assays - afdt.org

Class I HLA, representative serum

PBS

3mM EDTA

2mM EDTA

6mM EDTA

Page 96: Optimizing Solid Phase Assays - afdt.org

Class II HLA, representative serum

PBS

3mM EDTA

2mM EDTA

6mM EDTA

Page 97: Optimizing Solid Phase Assays - afdt.org

Class I HLA “Prozone” positive specificities

MF

I

EDTA

Specificities

(sorted in order of MFI for no EDTA)

Page 98: Optimizing Solid Phase Assays - afdt.org

MF

I

Specificities

(sorted in order of MFI for no EDTA)

EDTA

Class II HLA “Prozone” positive specificities

Page 99: Optimizing Solid Phase Assays - afdt.org

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.

Page 100: Optimizing Solid Phase Assays - afdt.org

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

• 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

Page 102: Optimizing Solid Phase Assays - afdt.org

• 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

Page 103: Optimizing Solid Phase Assays - afdt.org

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

Page 104: Optimizing Solid Phase Assays - afdt.org

• 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%)

Page 105: Optimizing Solid Phase Assays - afdt.org

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

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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

Page 111: Optimizing Solid Phase Assays - afdt.org

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C3d MFI ≥ 4,000 predicts prozonesensitivity = 95.2%specificity = 97.2%

Degree of prozonecorrelates with C3d deposition

Page 112: Optimizing Solid Phase Assays - afdt.org

Greenshields and Liwski Hum. Immunol. 2019

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Page 113: Optimizing Solid Phase Assays - afdt.org

Greenshields and Liwski Hum. Immunol. 2019

Frequency and severity of prozone in HSP

Page 114: Optimizing Solid Phase Assays - afdt.org

Greenshields and Liwski Hum. Immunol. 2019

32%

62%

6%

B) Prozone by locus, Class I

A B C

14%

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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

Page 115: Optimizing Solid Phase Assays - afdt.org

Greenshields and Liwski Hum. Immunol. 2019

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Page 116: Optimizing Solid Phase Assays - afdt.org

How can we identify sera that exhibit prozone?

Positive control bead?Negative control bead?

Page 117: Optimizing Solid Phase Assays - afdt.org

MFI

Positive control bead reactivity

Greenshields and Liwski Hum. Immunol. 2019

Page 118: Optimizing Solid Phase Assays - afdt.org

MFI

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Greenshields and Liwski Hum. Immunol. 2019

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Page 120: Optimizing Solid Phase Assays - afdt.org

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

Page 121: Optimizing Solid Phase Assays - afdt.org

Denatured HLA epitopes

Page 122: Optimizing Solid Phase Assays - afdt.org

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

Page 123: Optimizing Solid Phase Assays - afdt.org

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

Page 124: Optimizing Solid Phase Assays - afdt.org

AA sequence alignment

Unacceptable antigens

B8, B37, B41, B42, B44(44:02), B45, B82

156D

Page 125: Optimizing Solid Phase Assays - afdt.org

El-Awar et al. Hum. Immunol.

70:844, 2009

Page 126: Optimizing Solid Phase Assays - afdt.org

Eapen et al. Transplant Immunology 25:77, 2011

Page 127: Optimizing Solid Phase Assays - afdt.org

Acid treatment denatures class I HLA antigens

Untreated

Acid treated

Positive Control Serum

Page 128: Optimizing Solid Phase Assays - afdt.org

Acid treatment, patient serum

Untreated Acid Tx

A31 & A33

Denatured epitope, 73I

Page 129: Optimizing Solid Phase Assays - afdt.org
Page 130: Optimizing Solid Phase Assays - afdt.org

• 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

Page 131: Optimizing Solid Phase Assays - afdt.org

Visentin et al. Transplantation 98:738 2014

Page 132: Optimizing Solid Phase Assays - afdt.org

83% FCXM positive

16% FCXM positive

Visentin et al. Transplantation 98:738 2014

Page 133: Optimizing Solid Phase Assays - afdt.org

Visentin et al. Transplantation 98:738 2014

Page 134: Optimizing Solid Phase Assays - afdt.org

• 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

Page 135: Optimizing Solid Phase Assays - afdt.org

Denatured epitope

Native epitope (iBeads)

Otten et al. Clin. Exp. Immun. 173:536, 2013

No DSA

Page 136: Optimizing Solid Phase Assays - afdt.org

• 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

Page 137: Optimizing Solid Phase Assays - afdt.org

Loupy et al. NEJM. 2013

Page 138: Optimizing Solid Phase Assays - afdt.org

< 6,000> 6,000

Loupy et al. NEJM. 2013

Page 139: Optimizing Solid Phase Assays - afdt.org

Prozone?< 6,000> 6,000

Loupy et al. NEJM. 2013

Page 140: Optimizing Solid Phase Assays - afdt.org

Prozone?

Denatured?

< 6,000> 6,000

Loupy et al. NEJM. 2013

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Page 142: Optimizing Solid Phase Assays - afdt.org

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

Page 143: Optimizing Solid Phase Assays - afdt.org

Thank you!

Questions?