combined slides patterns program... · laboratory’s recurrent “non-specific” patterns •...
TRANSCRIPT
5/17/2019
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2019 Transplant Webinars
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Link to register: https://immucor.webinato.com/register
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learn.immucor.com
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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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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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2019 Transplant Webinars
Link to register: https://immucor.webinato.com/register
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Questions?
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• Q&A following session - Type in questions
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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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