COMBINED Slides Patterns Program... · laboratory’s recurrent “non-specific” patterns • Tested 40 different specimens using OneLamda SAB and compared to Immucor’s LSA: •

5/17/2019

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2 All Content © Immucor, Inc.

Handouts

http://www.immucor.com/en‐us/Pages/Educational‐Program‐Handouts.aspx


2019 Transplant Webinars

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

5/17/2019

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learn.immucor.com


Continuing Education

• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/PatternsEverywhere

• Registration deadline is June 7, 2019

• Certificates will be sent via email only to those who have registered June 21, 2019

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Questions?

• You are all muted

• Q&A following session - Type in questions

H . C L I F F S U L L I V A N , M . D . , D ( A B H I )

ANTIBODY INTERPRETATION: PATTERNS, PATTERNS EVERYWHERE

PATTERNS FROM AN EARLY AGE

https://www.youtube.com/watch?v=sTu-btx1y1I

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WHERE’S WALDO?

https://www.google.com/search?q=where%27s+waldo&rlz=1C1GCEU_enUS820US820&source=lnms&tbm=isch&sa=X&ved=0ahUKEwjj44PQyOnhAhUumuAKHcurDVYQ_AUIDigB&biw=1877&bih=946#imgrc=4fs2YI-Yo0L6QM:

WHAT’S THE PATTERN

https://www.khanacademy.org/math/cc-third-grade-math/cc-3rd-mult-div-topic/cc-3rd-grade-properties-patterns/e/patterns

ANATOMIC PATHOLOGY

https://www.webpathology.com/image.asp?case=966&n=77

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CLINICAL PATHOLOGY

HLA ANTIBODY DETECTION

Evolution of HLA Antibody DetectionCytotoxicity Cytotoxicity

Ly

Ly

C1

Dye

Membrane AttackComplex

Ly

Anti-HLA Antibody

Ly

Ly

Ly

Ly

Membrane AttackComplex

Dye

Anti-Human Globulin

Enhanced Cytotoxicity Enhanced Cytotoxicity

Flow Cytometer

Ly

Ly

Ly

CD19 CD3or

FluorescenatedAnti-Human Globulin

Flow Cytometry Flow Cytometry

Bray et al. Immunol. Res 3:41-53,2004

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EBV Transformed Cell LineHLA Transfected Cell Line

HLA Antigens extracted

Class I/Class II coated microparticles

Purified HLA AntigensPhenotypessingle alleles

Class I/Class II

conjugation

Adapted from Gebel and Bray. Transplantation Reviews 20: 189-194, 2006

Solid Phase HLA Antigen Specific Assays

PHENOTYPIC BEADS

SINGLE ANTIGEN BEADS

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PUBLIC EPITOPES

CREG GROUPS

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Likely have 2 antibodies:1.Stronger anti-DQA1*05/*06 (share common

epitope)2.Weaker anti-DQ7

Ellis, T.

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HLA STRUCTURE: CRYSTALLOGRAPHY

Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC. The foreign antigen binding site and T cell recognition regions of class Ihistocompatibility antigens. Nature. 1987 Oct 8-14;329(6139):512-8.

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ANTIGEN VS. EPITOPE

EPITOPE VS. EPLET?

Lim WH, Wong G, Heidt S, Claas FHJ. Novel aspects of epitope matching and practical application in kidney transplantation. Kidney Int. 2018 Feb;93(2):314-324.

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EPITOPE MATCHING:

CLINICAL RELEVANCE

IN RENAL TRANSPLANT

Sapir-Pichhadze R, Tinckam K, Quach K, Logan AG, Laupacis A, John R, Beyene J, Kim SJ. HLA-DR and -DQ eplet mismatches and transplant glomerulopathy: a nested case-control study. Am J Transplant. 2015 Jan;15(1):137-48. doi:10.1111/ajt.12968.

Dankers MK, Witvliet MD, Roelen DL, de Lange P, Korfage N, Persijn GG, Duquesnoy R, Doxiadis II, Claas FH. The number of amino acid triplet differences between patient and donor is predictive for the antibody reactivity against mismatched human leukocyte antigens. Transplantation. 2004 Apr 27;77(8):1236-9.

Duquesnoy RJ, Takemoto S, de Lange P, Doxiadis II, Schreuder GM, Persijn GG, Claas FH. HLAmatchmaker: a molecularly based algorithm for histocompatibility determination. III. Effect of matching at the HLA-A,B amino acid triplet level on kidney transplant survival. Transplantation. 2003 Mar 27;75(6):884-9.

Duquesnoy RJ, Howe J, Takemoto S. HLAmatchmaker: a molecularly based algorithm for histocompatibility determination. IV. An alternative strategy to increase the number of compatible donors for highly sensitized patients. Transplantation. 2003 Mar 27;75(6):889-97.

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Major Epitopes of HLA DPB1

Hypervariable Regions* Codons Amino acids Alleles Present on SABs**

A 8 , 9 VY 01, 03, 06, 11, 13, 15, 18, 20LF 02, 04, 05, 19, 23, 28VH 09, 10, 14, 17

B 34, 35 FV 02, 03, 04:02, 06, 09, 10, 14, 17, 18, 19, 20YA 01, 11, 13, 15FA 04:01, 28LV 05, 23

C 55 - 57 AAE 01, 04:01, 11, 13, 15, 23DED 03, 06, 09, 14, 17, 20DEE 02, 04:02, 10, 18, 28EAE 5,19

D 65 - - - 69 I - - - K 01, 04, 05, 09, 18, 23I - - - E 02, 10, 13, 17, 19L - - - K 03, 14, 20, 28L - - - E 06,L - - - R 11, 15

V 01, 03, 09, 10, 14E 76 M 02, 04, 05, 06, 11, 15, 17, 18, 20, 23, 28

I 13, 19

DEAV 01, 03, 05, 06, 09, 10, 11, 13, 14, 17, 19, 20F 84 - - 87 V / GGPM 02, 04, 15(V), 18(V), 23, 28(V)

** Alleles taken from: LS2A001, Lot# 10.*Adapted from Billen; et al. Tissue Antigens 75;278-285. 2009.

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Patient: DPB1*02:01 / *04:01GGPM (84--87)

Antibody: DEAV (84--87)

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Donor: DPB1*02:01 / *85:01 (1.8% of AA- DDs*)

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Patient: DPB1*02:01 / *04:01GGPM (84--87)

Antibody: DEAV (84--87)

Donor: *85:01 has DEAV (84--87)

Therefore: any DEAV+ allele wouldbe considered “Incompatible”

NOT SO OBVIOUS EPITOPES

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Donor: C*04:01, XX

MFI = TiterMFI = Titer

HLA Antibody Quantitation

• Luminex‐based test was NOT approved as a quantitative assay by the FDA.

• MFI values reflect a given bead's relative fluorescence without reference to a standard

• Relative fluorescence can be affected by many variables• At best, MFI is semi-quantitative surrogate of antibody level

Sullivan HC, Gebel HM, Bray RA. Understanding solid-phase HLA antibody assays and the value of MFI. Hum Immunol. 2017 Jul - Aug;78(7-8):471-480. doi: 10.1016/j.humimm.2017.05.007. Epub 2017 May 30. Review.

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THE “PEANUT BUTTER” EFFECT

7 CREG

HLA B type Flow Cytometry Crossmatch

T cells B cells

Donor A B7(w6) B8(w6) POS POS

Donor B B44(w4) B62(w6) NEG NEG

Donor C B27(w4) B50(w6) POS POS

Donor D B42(w6) B60(w6) POS POS

Titers

HLA ANTIBODY DETECTION: FOR YOUR CONSIDERATION

"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

"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

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CLASS I PAN-REACTIVE PATTERN

ASSOCIATED CLASS I FLOWPRA SCREENS

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• 105 (1.8%) samples (58 patients) with class I pattern • MFI range: 0-17,345 (avg: 1,153)

• 29% had no history of sensitization• e.g. blood transfusion, pregnancy, transplant

• 62% with SLE diagnosis • 62% women

• 83% had reactivity against self-antigen with 1 or more of the suspect alleles • 45% HLA-C*07:02 most common

OF ALL CLASS I SAB PERFORMED IN 2017 FOR RENAL TRANSPLANT RECIPIENTS:

XM n (11)

Avg B dMESF(range)

# w/ pattern Ag

Avg MFI (range)

B-/T- 11 n/a 10 1,693 (0-4,535)

B+/T-

0 n/a n/a n/a

B+/T+

0 n/a 0 n/a

Allogeneic XM: Non-SLE patients

XM n (23)

Avg B dMESF(range)

Avg T dMESF(range)

# w/ pattern

Ag

AvgMFI

(range)

B-/T-

10 n/a n/a 9 2,463 (0‐9,102)

B+/T-

10 27,632(1,972‐79,836)

n/a 10 2,690 (383‐8,224)

B+/T+

3 35,632(21,298‐45,988)

2,625(1,354‐3,713)

1 1427 (1,389‐1,465)

Allogeneic XM: SLE patients

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CLASS II PAN-REACTIVE PATTERN

CLASS II PAN-REACTIVE PATTERN: NEW LOT

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SURROGATE CROSSMATCHES

T-ce

llB-

cell

Negative Control Patient Sample

Denatured Antigen

Denatured Antigen

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DENATURED ANTIGEN

Native Class I antigen

Denatured Class I antigen

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• Used Immucor’s LSA to evaluate some of our laboratory’s recurrent “non-specific” patterns

• Tested 40 different specimens using OneLamda SAB and compared to Immucor’s LSA:• Using manufacturer protocol• Using our protocol and Immucor beads and secondary

detection antibody

POTENTIAL UTILITY OF SECOND SINGLE ANTIGEN ASSAY

C1,12,15 PATTERN

DP19,DR16 PATTERN

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DP1,5 PATTERN

EA5220

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DR53 PATTERN

01:01

01:03

Negative on LM2Q DRB4*01:01

DQ7,8,9 PATTERN

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1000 MFI on OL

800 MFI on OL

25,000 MFI on OL

19,000 MFI on LMQ219,000 MFI and 11,000on LMQ2

SUMMARY

• Utility of pattern recognition in HLA antibody testing• Determine antibody specificity• Recognize potentially clinically relevant HLA antibodies

despite low MFI values. • Detect recurrent denatured/non-specific antibodies

• Importance of using >1 method for antibody detection• Screening assay• Dilution studies/titrations• Surrogate crossmatches• Phenotypic beads• Second single antigen assay

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Questions?




2019 Transplant Webinars



Questions?



5/17/2019

29


Continuing Education

• ABHI, PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at: https://www.surveymonkey.com/r/PatternsEverywhere

• Registration deadline is June 7, 2019

• Certificates will be sent via email only to those who have registered June 21, 2019


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Documents

COMBINED Slides Patterns Program... · laboratory’s recurrent “non-specific” patterns • Tested 40 different specimens using OneLamda SAB and compared to Immucor’s LSA: •