Continuing Education Webinar Series

Transcription

1 Continuing Education Webinar Series

2 Future Webinars February 14 February 22 February 28 Testing for HLA Antibodies: We re NOT the Chemistry Lab Donor Selection of Solid Organ Transplant: Is Virtual Xmatch better than real? Proficiency, Competency, and QC: A practical approach to CLIA requirements and AABB, CAP, and Joint Commission Expectations Link to register:

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5 Continuing Education ABHI, PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is February 23, 2018 Certificates will be sent via only to those who have registered by March 9, 2018

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).

7 Questions? You are all muted Q&A following session - Type in questions

8 Prozone effect can be specific to single antigen bead kit manufacturers Dr Richard Battle PhD FRCPath SNBTS, Edinburgh

9 SAB Assay HLA ab detection Single antigen bead (SAB) assays for the detection of HLA antibody Highly sensitive Accurate analysis of complex HLA specific antibody profiles Important questions raised regarding sensitivity Various reports of blocking mechanisms which can produce false negative Hidden specificities reportedly revealed after dilution

10 Previous observations Dilution of samples reveals masked HLA antibodies DTT resolves and suspected cause IgM Identify complement component as key blocking mechanism Complement Component C3 Activation: The Leading Cause of the Prozone Phenomenon Affecting HLA Antibody Detection on Single-Antigen Beads Elisabeth Schwaiger,1 Markus Wahrmann,1 Gregor Bond,1 Farsad Eskandary,1 and Georg A. Bo hmig1,2

11 So what s going on? Assay interference has been attributed to Blocking by complement factors (C1, C3, C4) IgM antibodies Prozone / High Dose hook effect Note blocking by IgM and Complement blocking are not a true Prozone effect! Prozone blocking by high titre antibody

12 Complement interference Major cause - mimic s a prozone effect and often reported as such Antibodies undetectable in neat sample become detectable after dilution Complement C1 split products prevents binding of secondary IgG Commonly eliminated through addition of EDTA Represents the most common mechanism of blocking interference in the literature

13 Proposed mechanism complement blocking Secondary conjugate HLA-A1 Anti-HLA-A1 Single antigen bead

14 Proposed mechanism complement blocking Secondary conjugate HLA-A1 Anti-HLA-A1 Single antigen bead

15 EDTA complement blocking Addition of agents known to interfere with C1 integrity increase MFI in affected sera Secondary conjugate HLA-A1 Anti-HLA-A1 Single antigen bead

16 EDTA complement blocking Some researchers have demonstrated a role for C3 proposing that C1 complex initiates classical cascade resulting in dense C3b/d deposition (Schwaiger et al, 2014 Transplantation) In this study cobra venom was used to fragment C3 while leaving C1 integrity unaffected Prozoning was removed

17 Is complement to blame for all blocking effects? A few reports in the literature describing a blocking effect when EDTA and other agents targeting complement were used. Tambar et al AJT 2015 We further showed that EDTA treatment (6%) does not always remove all inhibitory factors compared with titration studies Schwaiger et al Remarkably, DTT treatment and heat inactivation also uncovered a second population of low-level SA reactions that also fulfilled our defining criteria of prozoning

18 Blocking effects and MFI levels

19 Dilution to monitor HLA Epitopes

20 Edinburgh HLA antibody specificity analysis Use two suppliers kits Immucor, Lifecodes Single Antigen and Onelamdba SAB. Alternate between kits Different HLA epitopes profiles are present in the panel composition of these kits Helps resolve HLA epitopes in reactive sera The ratio of beads to serum within the Immucor and One Lambda kits is different Effectively this allows two test dilutions neat and diluted to be set up.

21 Edinburgh HLA antibody specificity analysis

22 Different HLA epitope composition Some currently defined HLA epitopes are absent from the panels of both kits. Using two kits covers all currently described epitopes Increases ability to define epitopes / awareness of intra /Inter locus epitopes Light grey = OL, Dark grey = Immu

23 Hidden HLA antibody reactivity Case report When alternating manufacturers we observed a prozone effect which was manufacturer specific The case a renal transplant patient with a failing graft (due to AMR) Monitoring HLA antibodies during graft failure / for relisting Consistent approach when monitoring so one manufacturer is used (i.e. Immucor or One Lambda) One Lambda detected HLA specificities seemed to disappear within 8 weeks of testing

24 Hidden HLA antibody reactivity A genuine change in the profile seemed unlikely Patient was sensitised to specific mismatches on the failing allograft Reactivity towards these seemed to be increasing on previous testing No known change to treatment strategy IS had previously been withdrawn and no Plasma ex had been commenced Further testing performed Immucor assay set up Dilution on One Lambda Rpt sample requested

25 MFI value Reactivity profile Battle et al. AJT Kit 1 (Neat) Kit 1 (1:5) Kit 2 (Neat) Kit 1 = OL, Kit 2 = Immu EDTA routinely added to test serum

26 True Prozone effect? Proposed mechanism relates to the high density on the beads. (a) at a low density only univalent binding is possible (b) High antigen density enables bivalent binding increases antigen/antibody stability Weinstock et al IJI (c) Very high density may lead to tightly packed antibodies which interfere with binding of secondary antibody

27 Summary Other causes Cannot exclude IgM blocking as EDTA treatment does not fragment IgM The increasing level of antibody over time is suggestive of a true prozone effect It is clear that addition of EDTA does not remove all blocking mechanisms

28 Summary Alternating between suppliers kits for SAB has two key advantages Increases the panel of antigens testing panels epitope composition are different Cost effective way to incorporate a dilution effect into routine testing

29 Thanks Questions?

30 CONQUERING THE PROZONE EFFECT Masako Osada, Ph.D. Director, Commercial Scientific Affairs, Immucor February 7 th,

31 SNBTS Presentation: Key Conclusions Prozone effect can cause false negatives when performing Single Antigen testing False negatives could result in a poor donor-recipient match Prozone effect is caused by high titer antibodies due to complement interfering with the binding of the reporter antibody. Diluting the sample 1:5 resolves some, but not all, prozone effect Addition of EDTA does not remove all blocking mechanisms In several case studies, Immucor s LIFECODES Single Antigen assay resolved prozone effect without requiring a dilution The LIFECODES Single Antigen assay is a cost effective way to avoid prozone effect without manual dilutions 31

32 ADDITIONAL PUBLICATIONS ASHI P EFI P ASHI P Immucor Data 32

33 Prozone effect is a common form of interference ASHI 2017 P274 : Discrepancy Between DSA and HLA Flow Cross Match Associated with High Frequency of Prozone Phenomenon in Solid Phase Assay in Organ Transplantation Conclusions: Our results demonstrate the large incidence of prozone effect in solid phase assay. It is therefore strong recommended to treat the serum -in our cases dilution- for prozone effect always and whenever there is no correlation between strong positive FXM and weak or negative DSA. Reference: ASHI 2017 P274 Discrepancy Between DSA and HLA Flow Cross Match Associated with High Frequency of Prozone Phenomenon in Solid Phase Assay in Organ Transplantation. Ahmad Bokhari et al, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia 33

34 EFI 2016 Abstract P12: Prozone effect makes One Lambda SAB results variable EFI 2016 P12: Comparison of One Lambda and Immucor For the Detection of Anti HLA Antibodies in Patients Desensitized for a Kidney Transplantation: Is there A Better Choice? Dominique Masson et al, EFS Rhone Alpes, La Tronche, France, Nephrologie dialyse et transplantation d'organe CHU de Toulouse, Toulouse, France, Toulouse hospital Toulouse, France Conclusions: With the tests using Immucor, the MFIs decreased for all the patients during IA. But for the tests using One Lambda, for two patients, the MFIs increased during IA, then decreased or increased, which possibly reflects the prozone effect. The apparent absence of the prozone effect with Immucor is in favor of this provider. 34

35 EFI P12 Results and Conclusion: One Lambda LabScreen More Susceptible to Prozone Samples: Three sensitized patients desensitized to receive living organ transplantation Testing: Before desensitization, at desensitization, at transplantation Positive criteria: OL MFI >1000, LIFECODES Software assignment Concordance between OL and LC: Class I 92%, Class II 94% Results: LIFECODES LSA - MFI decreased during desensitization for all samples One Lambda LabScreen - MFI increased during desensitization then decreased or increased after - Possibility of prozone effect 35

36 EFI P12 Results and Conclusion: One Lambda LabScreen More Susceptible to Prozone Samples: Three sensitized patients desensitized to receive living organ transplantation Testing: Before desensitization, at desensitization, at transplantation Positive criteria: OL MFI >1000, LIFECODES Software assignment Concordance between OL and LC: Class I 92%, Class II 94% Results: LIFECODES LSA - MFI decreased during desensitization for all samples One Lambda LabScreen - MFI increased during desensitization then decreased or increased after - Possibility of prozone effect Conclusions: With the tests using Immucor, the MFIs decreased for all the patients during IA. But for the tests using One Lambda, for two patients, the MFIs increased during IA, then decreased or increased, which possibly reflects the prozone effect. The apparent absence of the prozone effect with Immucor is in favor of this provider. EFI 2016 P12: Comparison of One Lambda and Immucor For the Detection of Anti HLA Antibodies in Patients Desensitized for a Kidney Transplantation: Is there A Better Choice? Dominique Masson et al, EFS Rhone Alpes, La Tronche, France, Nephrologie dialyse et transplantation d'organe CHU de Toulouse, Toulouse, France, Toulouse hospital Toulouse, France 36

37 ASHI 2016 Abstract P008 One Lambda LabScreen More Susceptible to Prozone ASHI 2016 P008: Case Scenario, Performance Differences of the Two Commercially Available Solid Phase Single Antigen Reagents. Fadi Al Zayer et al, King Faisal Specialist hospital and Research Center, Riyadh, Saudi Arabia Conclusion: Significantly, higher prozone effect was observed with OL single antigen panel than in IC. Serum to bead-volume ratio may have contributed to the lower correlation between OL versus IC. Testing patients sera by more than one manufacturer is an advantageous in determination of antibody strength and improves correlation with the cross match results. 37

38 ASHI 2016 Abstract P008 Results and Conclusion: One Lambda LabScreen More Susceptible to Prozone Samples: Results: Serum from two patients CDC-Strong positive FXM-Strong positive OL LabScreen positive DSA DQB1*06:03 (2,510 MFI) A2 (891 MFI) B41(1,631 MFI) LIFECODES LSA positive DSA DQB1*06:03 (17,946 MFI) A2 (10,500 MFI) B41(2,863 MFI) 38

39 ASHI 2016 Abstract P008 Results and Conclusion: One Lambda LabScreen More Susceptible to Prozone Samples: Results: Serum from two patients CDC-Strong positive FXM-Strong positive OL LabScreen positive DSA DQB1*06:03 (2,510 MFI) A2 (891 MFI) B41(1,631 MFI) OL LabScreen positive DSA (1:4 dilution) DQB1*06:03 (10,503 MFI) increased from 2,510 A2 (3,048 MFI) increased from 891 B41(2,863 MFI) increased from 1,631 LIFECODES LSA positive DSA DQB1*06:03 (17,946 MFI) A2 (10,500 MFI) B41(2,863 MFI) 39

40 ASHI 2016 Abstract P008 Results and Conclusion: One Lambda LabScreen More Susceptible to Prozone Samples: Results: Serum from two patients CDC-Strong positive FXM-Strong positive OL LabScreen positive DSA DQB1*06:03 (2,510 MFI) A2 (891 MFI) B41(1,631 MFI) OL LabScreen positive DSA (1:4 dilution) DQB1*06:03 (10,503 MFI) increased from 2,510 A2 (3,048 MFI) increased from 891 B41(2,863 MFI) increased from 1,631 LIFECODES LSA positive DSA DQB1*06:03 (17,946 MFI) A2 (10,500 MFI) B41(2,863 MFI) Conclusion: Significantly, higher prozone effect was observed with OL single antigen panel than in IC. Serum to bead-volume ratio may have contributed to the lower correlation between OL versus IC. Testing patients sera by more than one manufacturer is an advantageous in determination of antibody strength and improves correlation with the cross match results. ASHI 2016 P008 Case Scenario, Performance Differences of the Two Commercially Available Solid Phase Single Antigen Reagents. Fadi Al Zayer et al, King Faisal Specialist hospital 40 and Research Center, Riyadh, Saudi Arabia

41 Prozone Effect Internal Analysis Run 1: Sample run according to the Immucor SAB product insert; no additional dilution to the sample Sample run according to the One Lambda SAB product insert; no additional dilution to the sample Results: Immucor Result: Top Ranked bead MFI of 21K One Lambda Result: Top Ranked bead MFI of 2K (Prozone Effect Suspected) 41

42 Prozone Effect Internal Analysis Run 1: Sample run according to the Immucor SAB product insert; no additional dilution to the sample Sample run according to the One Lambda SAB product insert; no additional dilution to the sample Results: Immucor Result: Top Ranked bead MFI of 21K One Lambda Result: Top Ranked bead MFI of 2K (Prozone Effect Suspected) Run 2: Sample run according to the Immucor SAB product insert; sample diluted 1:16 Sample run according to the One Lambda SAB product insert; sample diluted 1:16 Results: Immucor Result: Top Ranked bead MFI of 7K One Lambda Result: Top Ranked bead MFI of 16K (Prozone Effect Confirmed) 42

43 Single Antigen Competitive Comparison Single Antigen Assays Immucor 10:40 Immucor 20:40 One Lambda Serum to Bead Ratio 1:4 1:2 4:1 MFI Values Lower High Higher Prozone/Interference Effect Lowest Lower* Known Issues False Positives from Denatured Antigen Lower Lower Known Issues * Theoretical, not tested. 43

44 Single Antigen Recommendations Laboratory Evaluation If a lab runs both One Lambda and Immucor s SAB assays to compare results, the great majority of results will be concordant, but there will be some discrepant results. The lab needs to investigate further to determine which is correct. Single Antigen Assays One Lambda Positive One Lambda Negative Immucor Positive Immucor Negative True Positive Denatured Antigen? Denature the beads and rerun sample Test on PRA Beads Run mab for denatured antigen. Crossmatch Prozone or Interference? Serial dilutions Treat samples Crossmatch True Negative 44

45 Single Antigen Recommendations Laboratory Evaluation If a lab runs both One Lambda and Immucor s SAB assays to compare results, the great majority of results will be concordant, but there will be some discrepant results. The lab needs to investigate further to determine which is correct. Single Antigen Assays One Lambda Positive One Lambda Negative Immucor Positive Immucor Negative True Positive Denatured Antigen? Denature the beads and rerun sample Test on PRA Beads Run mab for denatured antigen. Crossmatch Prozone or Interference? Serial dilutions Treat samples Crossmatch True Negative 45

46 Single Antigen Recommendations Laboratory Evaluation If a lab runs both One Lambda and Immucor s SAB assays to compare results, the great majority of results will be concordant, but there will be some discrepant results. The lab needs to investigate further to determine which is correct. Single Antigen Assays One Lambda Positive One Lambda Negative Immucor Positive Immucor Negative True Positive Denatured Antigen? Denature the beads and rerun sample Test on PRA Beads Run mab for denatured antigen. Crossmatch Prozone or Interference? Serial dilutions Treat samples Crossmatch True Negative 46

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48 Continuing Education ABHI, PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is February 23, 2018 Certificates will be sent via only to those who have registered by March 9, 2018

49 Future Webinars February 14 February 22 February 28 Testing for HLA Antibodies: We re NOT the Chemistry Lab Donor Selection of Solid Organ Transplant: Is Virtual Xmatch better than real? Proficiency, Competency, and QC: A practical approach to CLIA requirements and AABB, CAP, and Joint Commission Expectations Link to register:

50 Thank you!