Future Webinars. Future Webinars 3/19/ April Post-Haematopoietic Stem Cell Transplant Chimerism Testing and Engraftment Monitoring

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Continuing Education Webinar Series Future Webinars 12 April Post-Haematopoietic Stem Cell Transplant Chimerism Testing and Engraftment Monitoring featuring Dr Anil Handoo, Sr.

1 Continuing Education Webinar Series Future Webinars 12 April Post-Haematopoietic Stem Cell Transplant Chimerism Testing and Engraftment Monitoring featuring Dr Anil Handoo, Sr. Consultant and Director Pathology BLK Super Specialty Hospital New Delhi, India Link to register: All Content 215 Immucor, Inc. Future Webinars All Content 215 Immucor, Inc. 1

Handouts http://www.immucor.com/en us/pages/educational Program Handouts.aspx All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. Continuing Education ABHI, PACE, Florida and California DHS 1.

2 Handouts us/pages/educational Program Handouts.aspx All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. Continuing Education ABHI, PACE, Florida and California DHS 1. Contact Hours Each attendee must register to receive CE at: Registration deadline is April 13, 218 Certificates will be sent via only to those who have registered by April 27, 218 All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. Presentation Recording Session will be recorded and posted. Access information will be sent to each registrant when the recording becomes available CE credits will be issued to anyone who listens to the recording within one year of the original presentation date (today). Learn website: learn.immucor.com All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. 2

learn.immucor.com All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. You are all muted Type in questions Questions? All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. Course content is for information and illustration purposes only.

3 learn.immucor.com All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. You are all muted Type in questions Questions? All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented, and this information is not to be used for clinical or maintenance evaluations. The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. All Content 215 Immucor, Inc. All Content 215 Immucor, Inc. 3

4 Optimization of HLA Antibody Testing. Dr. Robert Liwski, MD, PhD, FRCPC Medical Director, HLA Typing Laboratory Interim Head, Division of Hematopathology Professor, Department of Pathology Dalhousie University, Halifax Disclosure Nothing significant to disclose..still waiting for attractive offers Single Antigen Bead () Luminex assay Used by most HLA labs for HLA antibody testing. Revolutionized HLA antibody identification and virtual crossmatching. Number of advantages compared to Flow and ELISA. number of analytes tested simultaneously High throughput Rapid analysis Still, the procedure is time intensive and is not optimal for use in urgent cases. Friday afternoons come to mind Some important limitations: Susceptible to interfering substances ( prozone effect). 4

5 Outline Optimization of HLA antibody testing Rapid Optimized Single Antigen Bead (ROB) protocol for LABScreen (Human Immunology 217). Development Validation Multicenter evaluation Enhanced and ROB protocols for LIFECODES LSA. Multicenter evaluation Development of a novel, prozone resistant Dual Antibody Rapid Test (DART) protocol for LABScreen (ASHI Quarterly 217). Single antigen bead () Luminex LABScreen and LIFECODES LSA protocols Incubate beads (5 l) and serum 2 l (RT) 3 min. 4 l 1 l Wash x3 (5 min/spin) 15 min. Filter plate 5 min. Incubate with 1 l anti IgG PE, 1:1 dilution (RT) 3 min. 5 l 1:1 Wash x2 (5min/spin) 1 min. No wash min. Total assay time 1h 25 min. Evidence for incubation time/reagent concentration? wash times? 1h 5 min. 2h Transfusion Medicine Red cell antibody testing (IAT) How long does it take? 25 3 minutes!!! Can assay be optimized and expedited? 5

6 Objectives To develop a rapid single antigen bead LABScreen protocol 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 Effect of reduced spin time (1 vs 5 min) on bead counts Class I beads Class II beads Bead count Bead number N=3 6

7 Effect of reduced spin time Standard 5 washes x 5 min = 25 min Rapid 5 washes x 1 min = 5 min 13 x g 18 x g No impact on bead counts or overall results 2 minutes saved! Effects of reduced incubation times Serum incubation time Anti IgG PE incubation time Effects of reduced incubation time ¼ PPC, HLA class I Bead number 7

8 Effects of reduced incubation time ¼ PPC, HLA class I Bead number Effects of reduced incubation time ¼ PPC, HLA class I Bead number Effects of reduced incubation time ¼ PPC, HLA class I Bead number 8

Effects of reduced incubation time NC and PC beads Small Effect on NC bead (#1)

9 Effects of reduced incubation time ¼ PPC, HLA class II Bead number Effects of reduced incubation time Negative control serum Class I Class II Bead number Effects of reduced incubation time NC and PC beads Small Effect on NC bead (#1) background PC bead (#2) Significant Effect on IgG binding Serum/IgG-PE incubation time 9

10 Conclusion Reduction in incubation time with serum and/or anti IgG PE results in decreased values. Negligible impact on LSNC and NC bead reactivity. The degree of decrease when incubation time with anti IgG PE was reduced was surprising. IgG PE concentration appears to be sub optimal? Effects of increasing IgG PE concentration ¼ PPC, HLA class I Bead number Effects of increasing IgG PE concentration ¼ PPC, HLA class II Bead number 1

PE concentration from 1:1 to 1:5 increases in the standard assay including PC bead.

11 Effects of increasing IgG PE concentration Negative control serum Class I Class II Bead number Effects of increasing IgG PE concentration NC and PC beads NC bead (#1) PC bead (#2) Serum/IgG-PE incubation time Conclusion Increasing the anti IgG PE concentration from 1:1 to 1:5 increases in the standard assay including PC bead. Negligible effect on background (LSNC and NC bead). Can we compensate for reduced values in the 15/5 min protocol by optimizing the concentration of anti IgG PE? 11

12 Effects of increasing IgG PE concentration on in 15/5 protocol ¼ PPC, HLA class I Bead number Effects of increasing IgG PE concentration on in 15/5 protocol ¼ PPC, HLA class I Bead number Effects of increasing IgG PE concentration on in 15/5 protocol ¼ PPC, HLA class I Bead number 12

13 Effects of increasing IgG PE concentration on in 15/5 protocol ¼ PPC, HLA class I Bead number Effects of increasing IgG PE concentration on in 15/5 protocol ¼ PPC, HLA class I Bead number Effects of increasing IgG PE concentration on in 15/5 protocol ¼ PPC, HLA class II Bead number 13

14 Conclusion Increasing concentration of anti IgG PE compensates for the reduction in incubation times. IgG PE concentration of 1:1 closely matches obtained with the standard assay. ROB LABScreen Protocol Incubate beads (5 l) and serum 25 l (RT) 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 2 l anti IgG PE, 1:1 dilution (RT) 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. ROB LABScreen Protocol Incubate beads (5 l) and serum 25 l (RT) 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 2 l anti IgG PE, 1:1 dilution (RT) 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. 7% time reduction! 14

Reactivity Standard vs ROB protocol AC 463 Class I AC 463 Class II Bead

15 Standard vs ROB protocol, correlation 8 patient, 9 ASHI PT, 3 ABH PT sera ROB protocol Class I Class II Standard Assay Representative Serum Reactivity Standard vs ROB protocol AC 463 Class I AC 463 Class II Bead number Discrepant reactions Cut off rxn/panel 44 rxn/4 panels rxn 15

16 Patient serum CF, class I ROB Standard Patient serum CF, class I ROB Standard Conclusion We can reduce the time it takes to perform LABScreen Luminex assay without compromising assay sensitivity. Correlation between the Standard and ROB protocols is excellent. No significant impact on test results when using ROB protocol. Significant time savings. ROB protocol allows for rapid testing of urgent patient sera. Ex. testing during deceased donor work up. 16

17 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 214 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 214 Design 214 ASHI PT sera AC Tested by LABScreen Luminex assay Standard lab method ROB protocol Same lot of class I and class II beads Result analysis: comparison CV Pearson s correlation (R 2 ) Specificity assignment Pos/Neg ctrl beads (signal vs noise) Liwski et al ASHI

18 AC464 class I Individual lab comparison Standard ROB Bead number Liwski et al ASHI 214 AC464 class I Average lab and CV comparison %CV Bead number Liwski et al ASHI 214 AC46 class II Individual lab comparison Standard ROB Bead number Liwski et al ASHI

19 AC46 class II Average lab and CV comparison %CV Bead number Liwski et al ASHI 214 Overall mean correlation Class I Class II ROB Standard Liwski et al ASHI 214 Class I Average CV Standard vs ROB protocol Class II % CV Serum Liwski et al ASHI

20 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 214 2

21 LIFECODES LSA Assay Evaluation Objectives 2 well characterized and challenging sera Standard LIFECODES LSA vs ROB protocol Single Antigen Bead () Luminex Assay ROB and Standard LIFECODES LSA protocols Incubate beads (1 l) and serum 25 l (RT) 15 min. 4 l 1 l 3 min. Wash x3 (1 min/spin) 3 min. Filter plate 1 min. Incubate with 2 l anti IgG PE, 1:2 dilution (RT) 5 min. 5 l 1:1 3 min. Wash (1 min/spin) 1 min. No wash min. Total assay time 25 min. 1h 21

22 22 Case 5, low titer DSA A*1:1 A*2:3 A*11:1 A*23:1 A*24:3 A*26:1 A*3:1 A*32:1 A*33:3 A*36:1 A*66:1 A*68:1 A*69:1 A*8:1 B*8:1 B*14:1 B*15:1 B*15:3 B*15:12 B*15:16 B*27:5 B*35:1 B*38:1 B*4:1 B*41:1 B*44:2 B*45:1 B*47:1 B*49:1 B*51:1 B*53:1 B*55:1 B*57:1 B*59:1 B*73:1 B*81:1 C*1:2 C*3:3 C*4:1 C*6:2 C*8:1 C*14:2 C*16:1 C*18:2 A1, A11 DSA B8 DSA Standard Case 5, low titer DSA A*1:1 A*2:3 A*11:1 A*23:1 A*24:3 A*26:1 A*3:1 A*32:1 A*33:3 A*36:1 A*66:1 A*68:1 A*69:1 A*8:1 B*8:1 B*14:1 B*15:1 B*15:3 B*15:12 B*15:16 B*27:5 B*35:1 B*38:1 B*4:1 B*41:1 B*44:2 B*45:1 B*47:1 B*49:1 B*51:1 B*53:1 B*55:1 B*57:1 B*59:1 B*73:1 B*81:1 C*1:2 C*3:3 C*4:1 C*6:2 C*8:1 C*14:2 C*16:1 C*18:2 A1, A11 DSA B8 DSA Standard ROB Case 7, low titer Aw4/Bw A*1:1 A*2:1 A*2:3 A*3:1 A*11:1 A*11:2 A*23:1 A*24:2 A*24:3 A*25:1 A*26:1 A*29:2 A*3:1 A*31:1 A*32:1 A*33:1 A*33:3 A*34:2 A*36:1 A*43:1 A*66:1 A*66:2 A*68:1 A*68:2 A*69:1 A*74:1 A*8:1 B*7:2 B*8:1 B*13:2 B*14:1 B*14:2 B*15:1 B*15:2 B*15:3 B*15:1 B*15:12 B*15:13 B*15:16 B*18:1 B*27:5 B*27:8 B*35:1 B*37:1 B*38:1 B*39:1 B*4:1 B*4:2 B*41:1 B*42:1 B*44:2 B*44:3 B*45:1 B*46:1 B*47:1 B*48:1 B*49:1 B*5:1 B*51:1 B*52:1 B*53:1 B*54:1 B*55:1 B*56:1 B*57:1 B*58:1 B*59:1 B*67:1 B*73:1 B*78:1 B*81:1 B*82:1 C*1:2 C*2:2 C*3:3 C*3:4 C*4:1 C*5:1 C*6:2 C*7:2 C*8:1 C*12:3 C*14:2 C*15:2 C*16:1 C*17:1 C*18:2 A23, 24, 25, 32 (Aw4) Bw4 Standard

23 23 Case 7, low titer Aw4/Bw A*1:1 A*2:1 A*2:3 A*3:1 A*11:1 A*11:2 A*23:1 A*24:2 A*24:3 A*25:1 A*26:1 A*29:2 A*3:1 A*31:1 A*32:1 A*33:1 A*33:3 A*34:2 A*36:1 A*43:1 A*66:1 A*66:2 A*68:1 A*68:2 A*69:1 A*74:1 A*8:1 B*7:2 B*8:1 B*13:2 B*14:1 B*14:2 B*15:1 B*15:2 B*15:3 B*15:1 B*15:12 B*15:13 B*15:16 B*18:1 B*27:5 B*27:8 B*35:1 B*37:1 B*38:1 B*39:1 B*4:1 B*4:2 B*41:1 B*42:1 B*44:2 B*44:3 B*45:1 B*46:1 B*47:1 B*48:1 B*49:1 B*5:1 B*51:1 B*52:1 B*53:1 B*54:1 B*55:1 B*56:1 B*57:1 B*58:1 B*59:1 B*67:1 B*73:1 B*78:1 B*81:1 B*82:1 C*1:2 C*2:2 C*3:3 C*3:4 C*4:1 C*5:1 C*6:2 C*7:2 C*8:1 C*12:3 C*14:2 C*15:2 C*16:1 C*17:1 C*18:2 A23, 24, 25, 32 (Aw4) Bw4 Standard ROB Case 2 prozone effect, interfering substance A*1:1 A*2:3 A*11:1 A*23:1 A*24:3 A*26:1 A*3:1 A*32:1 A*33:3 A*36:1 A*66:1 A*68:1 A*69:1 A*8:1 B*8:1 B*14:1 B*15:1 B*15:3 B*15:12 B*15:16 B*27:5 B*35:1 B*38:1 B*4:1 B*41:1 B*44:2 B*45:1 B*47:1 B*49:1 B*51:1 B*53:1 B*55:1 B*57:1 B*59:1 B*73:1 B*81:1 C*1:2 C*3:3 C*4:1 C*6:2 C*8:1 C*14:2 C*16:1 C*18:2 Standard Case 2 prozone effect, interfering substance A*1:1 A*2:3 A*11:1 A*23:1 A*24:3 A*26:1 A*3:1 A*32:1 A*33:3 A*36:1 A*66:1 A*68:1 A*69:1 A*8:1 B*8:1 B*14:1 B*15:1 B*15:3 B*15:12 B*15:16 B*27:5 B*35:1 B*38:1 B*4:1 B*41:1 B*44:2 B*45:1 B*47:1 B*49:1 B*51:1 B*53:1 B*55:1 B*57:1 B*59:1 B*73:1 B*81:1 C*1:2 C*3:3 C*4:1 C*6:2 C*8:1 C*14:2 C*16:1 C*18:2 Standard ROB

24 Case 2 prozone effect, interfering substance A*1:1 A*2:3 A*11:1 A*23:1 A*24:3 A*26:1 A*3:1 A*32:1 A*33:3 A*36:1 A*66:1 A*68:1 A*69:1 A*8:1 B*8:1 B*14:1 B*15:1 B*15:3 B*15:12 B*15:16 B*27:5 B*35:1 B*38:1 B*4:1 B*41:1 B*44:2 B*45:1 B*47:1 B*49:1 B*51:1 B*53:1 B*55:1 B*57:1 B*59:1 B*73:1 B*81:1 C*1:2 C*3:3 C*4:1 C*6:2 C*8:1 C*14:2 C*16:1 C*18:2 Standard ROB ROB EDTA Summary Good correlation between ROB and Standard LIFECODES LSA protocol in many cases. ROB protocol exhibits enhanced enhances weak reactivity with low titer DSA. enhances reactivity with low titer abs directed against CREGs. Standard LSA protocol is less susceptible to the prozone effect compared with the ROB protocol. Treatment with EDTA resolves the prozone Differences are likely due to serum dilution in the Standard protocol. Enhanced LIFECODES LSA Protocol Immucor developed an enhanced LSA protocol to generate higher values. Motivation was based feedback from the worldwide HLA community. Clinical correlations with have been established In order to encourage more widespread adoption of the LIFECODES LSA kits, values need to be in line with what clinicians are used to seeing. Enhanced LSA protocol uses 2 l instead of 1 l of serum per reaction to increase the values. 24

Multicenter Evaluation of the Rapid Optimized Single Antigen Bead (ROB) Protocol and Enhanced LIFECODES LSA Protocol. Participating Centers Dalhousie University, Halifax, NS, Canada Dr.

25 Multicenter Evaluation of the Rapid Optimized Single Antigen Bead (ROB) Protocol and Enhanced LIFECODES LSA Protocol. Participating Centers Dalhousie University, Halifax, NS, Canada Dr. Rob Liwski Institut Armand Frappier, Laval, QC, Canada Dr. Claude Daniel Universite Laval, Quebec, QC, Canada Dr. Eric Wagner University of Toronto, Toronto, ON, Canada Drs. Kathryn Tinckam/Neal denhollander Western University, London, ON, Canada Dr. Qingyong Xu University of Manitoba, Winnipeg, MB, Canada Dr. Peter Nickerson University of Alberta, Edmonton, AB, Canada Drs. Patricia Campbell/Luis Hidalgo University Of British Columbia, Vancouver, BC, Canada Drs. Paul Keown/Lenka Allan Thomas Jefferson University Hospital, Philadelphia, PA Dr. Anthony Nizio Johns Hopkins University, Baltimore, MD Dr. Annette Jackson University of Pittsburgh Medical Center, Pittsburgh, PA Drs. Adriana Zeevi/Massimo Mangiola Wake Forest School of Medicine, Winston Salem, NC Dr. Michael Gautreaux Gift of Life Michigan, Ann Arbor, MI Dr. Sam Ho University of Utah, Salt Lake City, UT Dr. Julio Delgado Southwest Immunodiagnostics Inc. Lab, San Antonio, TX Dr. Cathi Murphy Queen Mary Hospital, Hong Kong Dr. Janette Kwok Participating Centers Dalhousie University, Halifax, NS, Canada Dr. Rob Liwski Institut Armand Frappier, Laval, QC, Canada Dr. Claude Daniel Universite Laval, Quebec, QC, Canada Dr. Eric Wagner University of Toronto, Toronto, ON, Canada Drs. Kathryn Tinckam/Neal denhollander Western University, London, ON, Canada Dr. Qingyong Xu University of Manitoba, Winnipeg, MB, Canada Dr. Peter Nickerson University of Alberta, Edmonton, AB, Canada Drs. Patricia Campbell/Luis Hidalgo University Of British Columbia, Vancouver, BC, Canada Drs. Paul Keown/Lenka Allan Thomas Jefferson University Hospital, Philadelphia, PA Dr. Anthony Nizio Johns Hopkins University, Baltimore, MD Dr. Annette Jackson University of Pittsburgh Medical Center, Pittsburgh, PA Drs. Adriana Zeevi/Massimo Mangiola Wake Forest School of Medicine, Winston Salem, NC Dr. Michael Gautreaux Gift of Life Michigan, Ann Arbor, MI Dr. Sam Ho University of Utah, Salt Lake City, UT Dr. Julio Delgado Southwest Immunodiagnostics Inc. Lab, San Antonio, TX Dr. Cathi Murphy Queen Mary Hospital, Hong Kong Dr. Janette Kwok 25

26 Design Class I and II HLA LIFECODES LSA kits, filter trays and high speed rotators (provided by Immucor). Technical support provided by Immucor (Dusanka D Atri) to labs not using Immucor kits routinely. 9ASHI PT sera (provided by Immucor) ( surveys). AC46, 464, 469, 47, 474, Design Tested by LIFECODES LSA Luminex assay Standard protocol (4:1) Side by side Enhanced protocol (4:2) ROB protocol Result analysis: Pos/Neg ctrl beads (signal to noise differential) comparison Mean and SD Pearson s correlation (R 2 ) Specificity assignment Negative/Positive Control Beads Signal to noise differential Negative control (probe 1) Positive control (probe 77) S1 S2 S3 S4 S5 S6 S7 S8 S9 S1 S1 S2 S3 S4 S5 S6 S7 S8 S9 S1 Standard Enhanced ROB 26

27 Serum 1 Class I (no HLA abs) 5 4 Standard Adjusted Enhanced ROB Probe # Serum 1 Class II (weak abs) 5 4 Standard Adjusted Enhanced na ROB 1or Probe # 5 4 Serum 1 Class II (weak abs) Standard Enhanced ROB Mean 3 2 Adjusted SD Probe # 27

28 28 Serum 4 Class I (weak/moderate abs) Adjusted Standard Enhanced ROB Probe # 1or 1or Serum 4 Class I (weak/moderate abs) Adjusted Probe # Mean SD Standard Enhanced ROB Serum 2 Class II (weak/moderate abs) Adjusted Standard Enhanced ROB Probe # 1or 1na

29 25 2 Serum 2 Class II (weak/moderate abs) Standard Enhanced ROB Mean 15 1 Adjusted SD Probe # Pearson s correlation, class I HLA Standard vs Enhanced Standard vs ROB Pearson s correlation, class II HLA Standard vs Enhanced Standard vs ROB 29

30 Pearson s correlation Enhanced vs ROB Class I HLA Class II HLA Class I HLA comparison Courtesy Dr. Bryan Ray, Immucor Class II HLA comparison Courtesy Dr. Bryan Ray, Immucor 3

31 CV comparison Class I HLA Class II HLA Courtesy Dr. Bryan Ray, Immucor Class I HLA, comparison to ASHI PT Consensus Number of Positive Antigens Percentage of Sites Reporting Positive Specificity ROB 2 µl 1 µl ASHI SPS Protocol Courtesy Dr. Bryan Ray, Immucor < Class II HLA, comparison to ASHI PT Consensus 14 Percentage of Sites Reporting Positive Specificities Number of Positive Antigens < ROB 2µL 1µL ASHI SPS Protocol Courtesy Dr. Bryan Ray, Immucor 31

32 Conclusion Enhanced and ROB protocols increase sensitivity ( values) in LIFECODES LSA assay (Enhanced > ROB). Improve signal to noise differential with no significant impact on background reactivity. Good overall correlation between all three protocols (best between Enhanced and ROB). Enhanced and ROB protocol show improved concordance of and results. ROB protocol confers significant time saving allows for rapid testing of urgent patient sera. Ex. testing during deceased donor work up. Development of a novel, prozone resistant Dual Antibody Rapid Test (DART) Protocol for LABScreen assay. Anna Greenshields, Robert Bray, Howard Gebel and Robert Liwski Department of Pathology, Dalhousie University Halifax, Nova Scotia, Canada Interfering Substances Prozone Effect 32

33 IgG neat IgG neat IgG 1:1 Prozone effect 33

34 Prozone effect Low titer Non C fixing Ab Prozone effect Low titer Non C fixing Ab Prozone effect Low titer Non C fixing Ab 34

35 Prozone effect Low titer Non C fixing Ab Prozone effect High titer C fixing Ab Prozone effect High titer C fixing Ab 35

36 Prozone effect High titer C fixing Ab C1q binds Prozone effect High titer C fixing Ab C1q binds C1r & C1s recruited Prozone effect High titer C fixing Ab C1q binds C1r & C1s recruited C4 converted to C4b Ca 2+ 36

37 Prozone effect High titer C fixing Ab C1q binds Ca 2+ C1r & C1s recruited C4 converted to C4b C4b binds HLA-Ab complex Prozone effect High titer C fixing Ab C1q binds Ca 2+ C1r & C1s recruited C4 converted to C4b C4b binds HLA-Ab complex C2 converted to C2a Prozone effect High titer C fixing Ab C1q binds Ca 2+ C1r & C1s recruited C4 converted to C4b C4b binds HLA-Ab complex C2 converted to C2a C2a binds C4b (C3 convertase) 37

38 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 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 C3b binds HLA-Ab complex and C4b2a (C5 convertase) 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 C3b binds HLA-Ab complex and C4b2a (C5 convertase) 38

39 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 C3b binds HLA-Ab complex and C4b2a (C5 convertase) Binding of anti-igg-pe is blocked HLA antibody not detected 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 C3b binds HLA-Ab complex and C4b2a (C5 convertase) Binding of anti-igg-pe is blocked HLA antibody not detected Solutions: Heat treatment (56 o C), destroys C1q and other C Serum dilution, dilutes out complement DTT, breaks C1q EDTA, chelates Ca 2+ Objectives To develop a protocol that is resistant to the prozone effect without serum treatment.. Greenshields et al ASHI Quarterly

40 Anti-IgG-PE 4

41 Anti-C -PE 41

42 Rapid Optimized (ROB) LABScreen Protocol Incubate beads and serum 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 2 l anti IgG PE, 1:1 dilution 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. 7% time reduction! Dual Antibody Rapid Test (DART) LABScreen Protocol Incubate beads and serum 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 2 l anti IgG PE and anti C PE 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. 7% time reduction! Study design 2 prozone positive sera, 1 class I and 1 class II Tested by : Anti IgG PE Anti IgG PE with EDTA Anti C PE DART, Anti IgG PE + anti C PE Comparison of 42

43 Serum 1, Class I HLA IgG Serum 1, Class I HLA IgG IgG EDTA Serum 1, Class I HLA IgG IgG EDTA 43

44 Serum 1, Class I HLA IgG IgG EDTA C Serum 1, Class I HLA IgG IgG EDTA C IgG/C DART Serum 2, Class II HLA IgG 44

45 Serum 2, Class II HLA IgG IgG EDTA Serum 2, Class II HLA IgG IgG EDTA Serum 2, Class II HLA IgG IgG EDTA C 45

46 Serum 2, Class II HLA IgG IgG EDTA C IgG/C DART > 1, < 1, N=737 No Prozone N=739 Slight 3-5K N=51 Moderate 5-1K N=95 Marked >1K N=38 IgG EDTA IgG IgG/C DART C > 1, < 1, N=737 No Prozone N=739 Slight 3-5K N=51 Moderate 5-1K N=95 Marked >1K N=38 IgG EDTA IgG IgG/C DART C 46

47 > 1, < 1, N=737 No Prozone N=739 Slight 3-5K N=51 Moderate 5-1K N=95 Marked >1K N=38 IgG EDTA IgG IgG/C DART C IgG EDTA no prozone, 3K IgG DART IgG/C N=5 IgG EDTA no prozone, 3K IgG DART IgG/C N=5 47

48 IgG Serum 3, Class I HLA IgG Serum 3, Class I HLA IgG EDTA IgG Serum 3, Class I HLA IgG EDTA 48

49 IgG Serum 3, Class I HLA IgG EDTA IgG Serum 3, Class I HLA IgG EDTA C IgG Serum 3, Class I HLA IgG EDTA C IgG/C DART 49

Conclusions DART protocol is resistant to the prozone effect. Eliminates the necessity to treat sera thus avoiding potential interference with HLA antibody testing.

50 Conclusions DART protocol is resistant to the prozone effect. Eliminates the necessity to treat sera thus avoiding potential interference with HLA antibody testing. Improved correlation for prozone negative specificities compared with EDTA. Dual Antibody Rapid Test (DART) Final thoughts Value of protocol optimization Impact on test quality, TAT and clinical patient care Should be an integral part of any assay validation Significant variability in antibody testing protocols and results Impact on patient care Impact on transplantation research Assay standardization improves concordance of results 5

51 Acknowledgements Collaborators Drs. Howard Gebel, Robert Bray, Cathi Murphey Halifax HLA lab Dr. Anna Greenshields Geoff Adams Geoff Peladeau Kelly Heinstein Immucor Team Dr. Bryan Ray Dr. Masako Osada Dusanka D Atri Kelly Cousins 51

Acknowledgements IMMUCOR Enhanced/ROB Evaluation Study Dr. Claude Daniel Dr. Eric Wagner Drs. Kathryn Tinckam/Neal denhollander Dr. Qingyong Xu Dr. Peter Nickerson Drs.

52 Acknowledgements IMMUCOR Enhanced/ROB Evaluation Study Dr. Claude Daniel Dr. Eric Wagner Drs. Kathryn Tinckam/Neal denhollander Dr. Qingyong Xu Dr. Peter Nickerson Drs. Patricia Campbell/Luis Hidalgo Drs. Paul Keown/Lenka Allan Dr. Anthony Nizio Dr. Annette Jackson Drs. Adriana Zeevi/Massimo Mangiola Dr. Michael Gautreaux Dr. Sam Ho Dr. Julio Delgado Dr. Cathi Murphy Dr. Janette Kwok ROB Protocol Evaluation Study Dr. Patricia Campbell Dr. Adriana Colovai Dr. Deborah Crowe Dr. Anne Halpin Dr. Ronald Kerman Dr. Dong Li John Lunz Dr. Cathi Murphey Dr. Peter Nickerson Denise Pochinco Dr. Sandra Rosen Bronson Dr. Olga Timofeeva Dr. Paul Warner Dr. Adriana Zeevi You are all muted Type in questions Questions? All Content 215 Immucor, Inc. 52

We like you! Like us on social media! All Content 215 Immucor, Inc. You are all muted Type in questions Questions? All Content 215 Immucor, Inc. Continuing Education ABHI, PACE, Florida and California DHS 1.

53 We like you! Like us on social media! All Content 215 Immucor, Inc. You are all muted Type in questions Questions? All Content 215 Immucor, Inc. Continuing Education ABHI, PACE, Florida and California DHS 1. Contact Hours Each attendee must register to receive CE at: Registration deadline is April 13, 218 Certificates will be sent via only to those who have registered by April 27, 218 All Content 215 Immucor, Inc. 53

Future Webinars 12 April Post-Haematopoietic Stem Cell Transplant Chimerism Testing and Engraftment Monitoring featuring Dr Anil Handoo, Sr.

54 Future Webinars 12 April Post-Haematopoietic Stem Cell Transplant Chimerism Testing and Engraftment Monitoring featuring Dr Anil Handoo, Sr. Consultant and Director Pathology BLK Super Specialty Hospital New Delhi, India Link to register: All Content 215 Immucor, Inc. Future Webinars All Content 215 Immucor, Inc. Thank you! All Content 215 Immucor, Inc. 54

Optimization of flow cytometric crossmatch assay to expedite pre-transplant immunologic risk assessment

1 Optimization of flow cytometric crossmatch assay to expedite pre-transplant immunologic risk assessment Dr. Robert Liwski, MD, PhD, FRCPC Medical Director, HLA Laboratory Division of Hematopathology