Assays to detect and identify HLA antibodies An overview Howard M. - - PowerPoint PPT Presentation

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Assays to detect and identify HLA antibodies An overview Howard M. - - PowerPoint PPT Presentation

Dec 02, 2023 493 likes •784 views

Assays to detect and identify HLA antibodies An overview Howard M. Gebel, Ph.D., D. (ABHI) Professor of Pathology Emory University Hospital Atlanta, GA 1 Howard Gebel, Ph.D., D(ABHI) Professor of Pathology Emory University Hospital

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Assays to detect and identify HLA antibodies

An overview

Howard M. Gebel, Ph.D., D. (ABHI) Professor of Pathology Emory University Hospital Atlanta, GA

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Howard Gebel, Ph.D., D(ABHI) Professor of Pathology Emory University Hospital Atlanta, GA.

No financial relationships related to this presentation AND The presentation does not include discussion of “off-label” or “investigational” use.

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Problems

Sensitivity not optimal (false negatives) Specificity (false positives) Panel composition Viability Typically restricted to class I antibodies

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Howard M. Gebel and Robert A. Bray

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  • Cumbersome cell-

based assays for XM, anibody ID and HLA typing.

  • Highly sensitive and

specific bead-based assays for antibody ID and Molecular- based HLA typing.

Solid Phase Assays Cytotoxicity 5

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Adapted from Gebel and Bray. Transplantation Reviews 20: 189-194, 2006 Anti-IgG HLA alloantibody Anti-IgG-FITC/PE

Flow Cytometry Suspension Arrays

Solid Phase HLA antibody detection

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AHG+ FCXM >300 FCXM <300 XM - Cl1/II DSA >10,000 Cl1/II DSA>5,000<10,000 Cl1/II DSA<5,000 NO DSA

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1) Vendors 2) Antigen source/type a) Native b) Recombinant 3) Antigen expression a) Conformationally correct b) Amount 4) Interfering factors 5) Reagents 6) Tech-tech variation 7) Protocols 8) Assay conditions

Why laboratories don’t get identical results when testing the same sample

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Interfering Factors

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PE PE PE PE

PE ~250,000 d

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PE PE PE PE

C1s, C1r and C3d

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Class I Class II

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Transplantation 2015; 99:1151-1155

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What are these? Assay concerns Reagents Sensitivity Cross reactivity

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Early view

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Gebel et al., Current Opin Organ Transplant 18:455-462, 2013

7 CREG

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Adapted from Gebel and Bray, Am J Transplant 14:1964-1975, 2014

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"It was the best of times, it was the worst of times; it was the age of wisdom, it was the age of foolishness; it was the epoch of belief, it was the epoch of incredulity; it was the season of Light, it was the season of Darkness; it was the spring of hope, it was the winter of despair; we had everything before us, we had nothing before us; we were all going directly to Heaven, we were all going the other way."

  • - Charles Dickens
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"It was the best of tests, it was the worst of tests; it was the test of wisdom, it was the test of foolishness; it was the test of belief, it was the test of incredulity; it was the test of Light, it was the test of Darkness; it was the test of hope, it was the test of despair; we had everything before us, we had nothing before us; we were all going directly to Heaven, we were all going the other way."

  • - with apologies to Charles Dickens