Continuing Education Webinar Series

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1 Continuing Education Webinar Series

2 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 2015 Immucor, Inc.

3 Future Webinars All Content 2015 Immucor, Inc.

4 Handouts us/pages/educational Program Handouts.aspx All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

5 Continuing Education ABHI, PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is April 13, 2018 Certificates will be sent via only to those who have registered by April 27, 2018 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

6 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 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

7 learn.immucor.com All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

8 Questions? You are all muted Type in questions All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

9 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 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

10 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

11 Disclosure Nothing significant to disclose..still waiting for attractive offers

12 Single Antigen Bead (SAB) Luminex assay Used by most HLA labs for HLA antibody testing. Revolutionized HLA antibody identification and virtual crossmatching. Number of advantages compared to Flow SAB 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).

13 Outline Optimization of HLA antibody testing Rapid Optimized Single Antigen Bead (ROB) protocol for LABScreen (Human Immunology 2017). 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 2017).

14 Single antigen bead (SAB) Luminex LABScreen and LIFECODES LSA protocols Incubate beads (5 l) and serum 20 l (RT) 30 min. Wash x3 (5 min/spin) 15 min. Filter plate Incubate with 100 l anti IgG PE, 1:100 dilution (RT) 30 min. Wash x2 (5min/spin) 10 min. No wash 40 l 10 l 50 l 1:10 Total assay time 1h 25 min. Evidence for incubation time/reagent concentration? wash times? 5 min. 0 min. 1h 5 min. 2h

15 Transfusion Medicine Red cell antibody testing (IAT) How long does it take? minutes!!! Can SAB assay be optimized and expedited?

16 Liwski et al Hum. Immunol. 2017

17 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 Liwski et al Hum. Immunol. 2017

18 Effect of reduced spin time (1 vs 5 min) on bead counts Class I beads Class II beads Bead count Liwski et al Hum. Immunol Bead number N=3

19 Effect of reduced spin time Standard Rapid 5 washes x 5 min = 25 min 5 washes x 1 min = 5 min 1300 x g 1800 x g No impact on bead counts or overall results 20 minutes saved! Liwski et al Hum. Immunol. 2017

20 Effects of reduced incubation times Serum incubation time Anti IgG PE incubation time Liwski et al Hum. Immunol. 2017

21 Effects of reduced incubation time ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

22 Effects of reduced incubation time ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

23 Effects of reduced incubation time ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

24 Effects of reduced incubation time ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

25 Effects of reduced incubation time ¼ PPC, HLA class II MFI Liwski et al Hum. Immunol Bead number

26 Effects of reduced incubation time Negative control serum Class I Class II MFI Liwski et al Hum. Immunol Bead number

27 Effects of reduced incubation time NC and PC beads Small Effect on NC bead (#1) background PC bead (#2) Significant Effect on IgG binding MFI Liwski et al Hum. Immunol Serum/IgG-PE incubation time

28 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

29 Effects of increasing IgG PE concentration ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

30 Effects of increasing IgG PE concentration ¼ PPC, HLA class II MFI Liwski et al Hum. Immunol Bead number

31 Effects of increasing IgG PE concentration Negative control serum Class I Class II MFI Liwski et al Hum. Immunol Bead number

32 Effects of increasing IgG PE concentration NC and PC beads NC bead (#1) PC bead (#2) MFI Liwski et al Hum. Immunol Serum/IgG-PE incubation time

33 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). 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

34 Effects of increasing IgG PE concentration on MFI in 15/5 protocol ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

35 Effects of increasing IgG PE concentration on MFI in 15/5 protocol ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

36 Effects of increasing IgG PE concentration on MFI in 15/5 protocol ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

37 Effects of increasing IgG PE concentration on MFI in 15/5 protocol ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

38 Effects of increasing IgG PE concentration on MFI in 15/5 protocol ¼ PPC, HLA class I MFI Liwski et al Hum. Immunol Bead number

39 Effects of increasing IgG PE concentration on MFI in 15/5 protocol ¼ PPC, HLA class II MFI Liwski et al Hum. Immunol Bead number

40 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

41 ROB LABScreen Protocol Incubate beads (5 l) and serum 25 l (RT) 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 20 l anti IgG PE, 1:10 dilution (RT) 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. Liwski et al Hum. Immunol. 2017

42 ROB LABScreen Protocol Incubate beads (5 l) and serum 25 l (RT) 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 20 l 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

43 Standard vs ROB protocol, MFI correlation 8 patient, 9 ASHI PT, 3 ABH PT sera ROB protocol MFI Class I Class II Liwski et al Hum. Immunol Standard Assay MFI

44 Representative Serum Reactivity Standard vs ROB protocol AC 463 Class I AC 463 Class II MFI Liwski et al Hum. Immunol Bead number

45 Discrepant reactions Cut off 2000 MFI 1.1 rxn/panel 44 rxn/40 panels MFI Liwski et al Hum. Immunol rxn

46 Patient serum CF, class I ROB Standard Liwski et al Hum. Immunol. 2017

47 Patient serum CF, class I ROB Standard Liwski et al Hum. Immunol. 2017

48 Conclusion We can reduce the time it takes to perform LABScreen SAB 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. Liwski et al Hum. Immunol. 2017

49 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

50 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

51 Design 2014 ASHI PT sera AC 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 (R 2 ) Specificity assignment Pos/Neg ctrl beads (signal vs noise) Liwski et al ASHI 2014

52 AC464 class I Individual lab MFI comparison Standard MFI ROB Bead number Liwski et al ASHI 2014

53 AC464 class I Average lab MFI and CV comparison MFI %CV Bead number Liwski et al ASHI 2014

54 AC460 class II Individual lab MFI comparison Standard MFI ROB Bead number Liwski et al ASHI 2014

55 AC460 class II Average lab MFI and CV comparison MFI %CV Bead number Liwski et al ASHI 2014

56 Overall mean MFI correlation Class I Class II ROB Standard Liwski et al ASHI 2014

57 Average CV Class I Standard vs ROB protocol Class II % CV Serum Liwski et al ASHI 2014

58

59 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

60

61 LIFECODES LSA SAB Assay Evaluation

62 Objectives 20 well characterized and challenging sera Standard LIFECODES LSA vs ROB protocol

63 Single Antigen Bead (SAB) Luminex Assay ROB and Standard LIFECODES LSA protocols Incubate beads (10 l) and serum 25 l (RT) 15 min. Wash x3 (1 min/spin) 3 min. Filter plate 40 l 10 l Incubate with 20 l anti IgG PE, 1:2 dilution (RT) 5 min. 50 l 1:10 Wash (1 min/spin) 1 min. No wash Total assay time 25 min. 1h 30 min. 1 min. 30 min. 0 min.

64 Case 5, low titer DSA A1, A11 DSA B8 DSA MFI A*01:01 A*02:03 A*11:01 A*23:01 A*24:03 A*26:01 A*30:01 A*32:01 A*33:03 A*36:01 A*66:01 A*68:01 A*69:01 A*80:01 B*08:01 B*14:01 B*15:01 B*15:03 B*15:12 B*15:16 B*27:05 B*35:01 B*38:01 B*40:01 B*41:01 B*44:02 B*45:01 B*47:01 B*49:01 B*51:01 B*53:01 B*55:01 B*57:01 B*59:01 B*73:01 B*81:01 C*01:02 C*03:03 C*04:01 C*06:02 C*08:01 C*14:02 C*16:01 C*18:02 Standard

65 Case 5, low titer DSA A1, A11 DSA B8 DSA MFI A*01:01 A*02:03 A*11:01 A*23:01 A*24:03 A*26:01 A*30:01 A*32:01 A*33:03 A*36:01 A*66:01 A*68:01 A*69:01 A*80:01 B*08:01 B*14:01 B*15:01 B*15:03 B*15:12 B*15:16 B*27:05 B*35:01 B*38:01 B*40:01 B*41:01 B*44:02 B*45:01 B*47:01 B*49:01 B*51:01 B*53:01 B*55:01 B*57:01 B*59:01 B*73:01 B*81:01 C*01:02 C*03:03 C*04:01 C*06:02 C*08:01 C*14:02 C*16:01 C*18:02 Standard ROB

66 Case 7, low titer Aw4/Bw A*01:01 A*02:01 A*02:03 A*03:01 A*11:01 A*11:02 A*23:01 A*24:02 A*24:03 A*25:01 A*26:01 A*29:02 A*30:01 A*31:01 A*32:01 A*33:01 A*33:03 A*34:02 A*36:01 A*43:01 A*66:01 A*66:02 A*68:01 A*68:02 A*69:01 A*74:01 A*80:01 B*07:02 B*08:01 B*13:02 B*14:01 B*14:02 B*15:01 B*15:02 B*15:03 B*15:10 B*15:12 B*15:13 B*15:16 B*18:01 B*27:05 B*27:08 B*35:01 B*37:01 B*38:01 B*39:01 B*40:01 B*40:02 B*41:01 B*42:01 B*44:02 B*44:03 B*45:01 B*46:01 B*47:01 B*48:01 B*49:01 B*50:01 B*51:01 B*52:01 B*53:01 B*54:01 B*55:01 B*56:01 B*57:01 B*58:01 B*59:01 B*67:01 B*73:01 B*78:01 B*81:01 B*82:01 C*01:02 C*02:02 C*03:03 C*03:04 C*04:01 C*05:01 C*06:02 C*07:02 C*08:01 C*12:03 C*14:02 C*15:02 C*16:01 C*17:01 C*18:02 A23, 24, 25, 32 (Aw4) Bw4 Standard MFI

67 Case 7, low titer Aw4/Bw A*01:01 A*02:01 A*02:03 A*03:01 A*11:01 A*11:02 A*23:01 A*24:02 A*24:03 A*25:01 A*26:01 A*29:02 A*30:01 A*31:01 A*32:01 A*33:01 A*33:03 A*34:02 A*36:01 A*43:01 A*66:01 A*66:02 A*68:01 A*68:02 A*69:01 A*74:01 A*80:01 B*07:02 B*08:01 B*13:02 B*14:01 B*14:02 B*15:01 B*15:02 B*15:03 B*15:10 B*15:12 B*15:13 B*15:16 B*18:01 B*27:05 B*27:08 B*35:01 B*37:01 B*38:01 B*39:01 B*40:01 B*40:02 B*41:01 B*42:01 B*44:02 B*44:03 B*45:01 B*46:01 B*47:01 B*48:01 B*49:01 B*50:01 B*51:01 B*52:01 B*53:01 B*54:01 B*55:01 B*56:01 B*57:01 B*58:01 B*59:01 B*67:01 B*73:01 B*78:01 B*81:01 B*82:01 C*01:02 C*02:02 C*03:03 C*03:04 C*04:01 C*05:01 C*06:02 C*07:02 C*08:01 C*12:03 C*14:02 C*15:02 C*16:01 C*17:01 C*18:02 A23, 24, 25, 32 (Aw4) Bw4 Standard ROB MFI

68 Case 2 prozone effect, interfering substance A*01:01 A*02:03 A*11:01 A*23:01 A*24:03 A*26:01 A*30:01 A*32:01 A*33:03 A*36:01 A*66:01 A*68:01 A*69:01 A*80:01 B*08:01 B*14:01 B*15:01 B*15:03 B*15:12 B*15:16 B*27:05 B*35:01 B*38:01 B*40:01 B*41:01 B*44:02 B*45:01 B*47:01 B*49:01 B*51:01 B*53:01 B*55:01 B*57:01 B*59:01 B*73:01 B*81:01 C*01:02 C*03:03 C*04:01 C*06:02 C*08:01 C*14:02 C*16:01 C*18:02 Standard MFI

69 Case 2 prozone effect, interfering substance A*01:01 A*02:03 A*11:01 A*23:01 A*24:03 A*26:01 A*30:01 A*32:01 A*33:03 A*36:01 A*66:01 A*68:01 A*69:01 A*80:01 B*08:01 B*14:01 B*15:01 B*15:03 B*15:12 B*15:16 B*27:05 B*35:01 B*38:01 B*40:01 B*41:01 B*44:02 B*45:01 B*47:01 B*49:01 B*51:01 B*53:01 B*55:01 B*57:01 B*59:01 B*73:01 B*81:01 C*01:02 C*03:03 C*04:01 C*06:02 C*08:01 C*14:02 C*16:01 C*18:02 Standard ROB MFI

70 Case 2 prozone effect, interfering substance A*01:01 A*02:03 A*11:01 A*23:01 A*24:03 A*26:01 A*30:01 A*32:01 A*33:03 A*36:01 A*66:01 A*68:01 A*69:01 A*80:01 B*08:01 B*14:01 B*15:01 B*15:03 B*15:12 B*15:16 B*27:05 B*35:01 B*38:01 B*40:01 B*41:01 B*44:02 B*45:01 B*47:01 B*49:01 B*51:01 B*53:01 B*55:01 B*57:01 B*59:01 B*73:01 B*81:01 C*01:02 C*03:03 C*04:01 C*06:02 C*08:01 C*14:02 C*16:01 C*18:02 Standard ROB ROB EDTA MFI

71 Summary Good correlation between ROB and Standard LIFECODES LSA protocol in many cases. ROB protocol exhibits enhanced MFI 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.

72 Enhanced LIFECODES LSA Protocol Immucor developed an enhanced LSA protocol to generate higher MFI values. Motivation was based feedback from the worldwide HLA community. Clinical correlations with MFI have been established In order to encourage more widespread adoption of the LIFECODES LSA kits, MFI values need to be in line with what clinicians are used to seeing. Enhanced LSA protocol uses 20 l instead of 10 l of serum per reaction to increase the MFI values.

73 Multicenter Evaluation of the Rapid Optimized Single Antigen Bead (ROB) Protocol and Enhanced LIFECODES LSA Protocol.

74 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

75 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

76 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). AC460, 464, 469, 470, 474,

77 Design Tested by LIFECODES LSA SAB Luminex assay Standard protocol (40:10) Enhanced protocol (40:20) ROB protocol Side by side Result analysis: Pos/Neg ctrl beads (signal to noise differential) MFI comparison Mean and SD Pearson s correlation (R 2 ) Specificity assignment

78 Negative/Positive Control Beads Signal to noise differential Negative control (probe 1) Positive control (probe 77) MFI MFI S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Standard Enhanced ROB

79 Serum 1 Class I (no HLA abs) Adjusted MFI Standard Enhanced ROB Probe #

80 Serum 1 Class II (weak abs) Standard Adjusted MFI Enhanced na ROB 1or Probe #

81 Serum 1 Class II (weak abs) Standard Enhanced ROB Mean Adjusted MFI SD Probe #

82 Serum 4 Class I (weak/moderate abs) Adjusted MFI Standard Enhanced ROB Probe # 1or 1or

83 Serum 4 Class I (weak/moderate abs) Adjusted MFI Probe # Mean SD Standard Enhanced ROB

84 Serum 2 Class II (weak/moderate abs) Standard Adjusted MFI Enhanced na ROB 1or Probe #

85 Serum 2 Class II (weak/moderate abs) Standard Enhanced ROB Mean Adjusted MFI SD Probe #

86 Pearson s correlation, class I HLA Standard vs Enhanced Standard vs ROB

87 Pearson s correlation, class II HLA Standard vs Enhanced Standard vs ROB

88 Pearson s correlation Enhanced vs ROB Class I HLA Class II HLA

89 Class I HLA MFI comparison Courtesy Dr. Bryan Ray, Immucor

90 Class II HLA MFI comparison Courtesy Dr. Bryan Ray, Immucor

91 CV comparison Class I HLA Class II HLA Courtesy Dr. Bryan Ray, Immucor

92 Class I HLA, comparison to ASHI PT Consensus Number of Positive Antigens Percentage of Sites Reporting Positive Specificity < ROB 20 µl 10 µl ASHI SPS Protocol Courtesy Dr. Bryan Ray, Immucor

93 Class II HLA, comparison to ASHI PT Consensus 140 Percentage of Sites Reporting Positive Specificities Number of Positive Antigens < ROB 20µL 10µL ASHI SPS Protocol Courtesy Dr. Bryan Ray, Immucor

94 Conclusion Enhanced and ROB protocols increase sensitivity (MFI 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 MFI and results. ROB protocol confers significant time saving allows for rapid testing of urgent patient sera. Ex. testing during deceased donor work up.

95 Development of a novel, prozone resistant Dual Antibody Rapid Test (DART) Protocol for LABScreen SAB assay. Anna Greenshields, Robert Bray, Howard Gebel and Robert Liwski Department of Pathology, Dalhousie University Halifax, Nova Scotia, Canada

96 Interfering Substances Prozone Effect

97 IgG SAB neat

98 IgG SAB neat IgG SAB 1:10

99 Prozone effect SAB HLA-A2

100 Prozone effect Low titer Non C fixing Ab SAB HLA-A2

101 Prozone effect Low titer Non C fixing Ab SAB HLA-A2

102 Prozone effect Low titer Non C fixing Ab SAB HLA-A2

103 Prozone effect Low titer Non C fixing Ab SAB HLA-A2

104 Prozone effect High titer C fixing Ab SAB HLA-A2

105 Prozone effect High titer C fixing Ab SAB HLA-A2

106 Prozone effect High titer C fixing Ab C1q binds SAB HLA-A2

107 Prozone effect High titer C fixing Ab C1q binds C1r & C1s recruited SAB HLA-A2

108 Prozone effect High titer C fixing Ab C1q binds C1r & C1s recruited C4 converted to C4b Ca 2+ SAB HLA-A2

109 Prozone effect High titer C fixing Ab C1q binds Ca 2+ C1r & C1s recruited C4 converted to C4b C4b binds HLA-Ab complex SAB HLA-A2

110 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 SAB HLA-A2

111 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) SAB HLA-A2

112 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

113 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) SAB HLA-A2

114 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) SAB HLA-A2

115 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) SAB HLA-A2 Binding of anti-igg-pe is blocked HLA antibody not detected

116 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) SAB HLA-A2 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+

117 Objectives To develop a SAB protocol that is resistant to the prozone effect without serum treatment.. Greenshields et al ASHI Quarterly 2017

118 SAB HLA-A2

119 SAB HLA-A2 SAB HLA-A2

120 Anti-IgG-PE SAB HLA-A2 SAB HLA-A2

121 SAB HLA-A2 SAB HLA-A2

122 Anti-C -PE SAB HLA-A2 SAB HLA-A2

123 SAB HLA-A2 SAB HLA-A2

124 Rapid Optimized SAB (ROB) LABScreen Protocol Incubate beads and serum 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 20 l anti IgG PE, 1:10 dilution 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. 70% time reduction!

125 Dual Antibody Rapid Test (DART) LABScreen Protocol Incubate beads and serum 15 min. Wash x3 (1 min/spin) 3 min. Incubate with 20 l anti IgG PE and anti C PE 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. 70% time reduction!

126 Study design 20 prozone positive sera, 10 class I and 10 class II Tested by SAB: Anti IgG PE Anti IgG PE with EDTA Anti C PE DART, Anti IgG PE + anti C PE Comparison of MFI

127 IgG Serum 1, Class I HLA

128 Serum 1, Class I HLA IgG IgG EDTA

129 Serum 1, Class I HLA IgG IgG EDTA

130 Serum 1, Class I HLA IgG IgG EDTA C

131 Serum 1, Class I HLA IgG IgG EDTA C IgG/C DART

132 IgG Serum 2, Class II HLA

133 Serum 2, Class II HLA IgG IgG EDTA

134 Serum 2, Class II HLA IgG IgG EDTA

135 Serum 2, Class II HLA IgG IgG EDTA C

136 Serum 2, Class II HLA IgG IgG EDTA C IgG/C DART

137 > 1,000 MFI < 1,000 MFI N=737 No Prozone N=739 Slight 3-5K N=51 Moderate 5-10K N=95 Marked >10K N=308 IgG EDTA IgG IgG/C DART C

138 > 1,000 MFI < 1,000 MFI N=737 No Prozone N=739 Slight 3-5K N=51 Moderate 5-10K N=95 Marked >10K N=308 IgG EDTA IgG IgG/C DART C

139 > 1,000 MFI < 1,000 MFI N=737 No Prozone N=739 Slight 3-5K N=51 Moderate 5-10K N=95 Marked >10K N=308 IgG EDTA IgG IgG/C DART C

140 IgG EDTA no prozone, 3K IgG DART IgG/C N=50

141 IgG EDTA no prozone, 3K IgG DART IgG/C N=50

142 IgG Serum 3, Class I HLA

143 Serum 3, Class I HLA IgG IgG EDTA

144 Serum 3, Class I HLA IgG IgG EDTA

145 Serum 3, Class I HLA IgG IgG EDTA

146 Serum 3, Class I HLA IgG IgG EDTA C

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

148 Conclusions DART protocol is resistant to the prozone effect. Eliminates the necessity to treat sera thus avoiding potential interference with HLA antibody testing. Improved MFI correlation for prozone negative specificities compared with EDTA.

149 Dual Antibody Rapid Test (DART)

150 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

151

152

153 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

154 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

155

156 You are all muted Type in questions Questions? All Content 2015 Immucor, Inc.

157 We like you! Like us on social media! All Content 2015 Immucor, Inc.

158 You are all muted Type in questions Questions? All Content 2015 Immucor, Inc.

159 Continuing Education ABHI, PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is April 13, 2018 Certificates will be sent via only to those who have registered by April 27, 2018 All Content 2015 Immucor, Inc.

160 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 2015 Immucor, Inc.

161 Future Webinars All Content 2015 Immucor, Inc.

162 Thank you! All Content 2015 Immucor, Inc.