Continuing Education Webinar Series

Transcription

1 Continuing Education Webinar Series

2 Future Webinars 22 February Donor Selection of Solid Organ Transplant: Is Virtual Xmatch better than real? 28 February Proficiency, Competency, and QC: A practical approach to CLIA requirements and AABB, CAP, and Joint Commission Expectations 22 March Clinical Significance of HLA Antibodies in Solid Organ Transplantations Link to register: All Content 2015 Immucor, Inc.

3 Handouts Program-Handouts.aspx All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

4 Continuing Education ABHI, PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is March 2, 2018 Certificates will be sent via only to those who have registered by March 16, 2018 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc.

5 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.

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

7 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.

8 Testing for HLA Antibodies: We re Not the Chemistry Lab Michael D. Gautreaux, Ph.D., D.ABHI

9 Impact of HLA Ab in Transplantation Pre- and peri-transplant Access to transplantation Short-term survival (hyperacute, accelerated) Post-transplant Survival» Acute and chronic rejection Marker for rejection response

10 PRA Panel Reactive Antibody Percent Reactive Antibody Intended to be an assessment of transplantability. 20% PRA means that a recipient should crossmatch positive with 20% of donors DO NOT CONFUSE WITH TITER

11 Y Y Basis of Antibody Testing + Y Y Y Y Cells or beads Patient s serum Y Y Y Y Y Reaction No reaction

12 PRA Techniques Complement-dependent cytotoxicity (original gold standard; usually augmented) Flow cytometry (mostly solid phase) Solid Phase (ELISA & Luminex)

13 Y Complement-dependent Cytotoxicity + Y Y Y Y PBLs Magnetic separation HLA antibody in wells 30 min Y Y 60 min Cell death Add complement

14 ASHI Scoring System 1 (0-10%) 2 (11-20%) 4 (21-50%) 6 (51-80%) 8 (81-100%)

15 Determination of Significance Antigen Correlation Coefficient (r) Serum reactivity a TP +/+ c FN +/- b FP -/+ d TN -/- A = a + b B = c + d r = a*d - b*c SQRT (A*B*C*D) Chi square (c 2 ) c 2 = r 2 * T C = a + c D = b + d T = Total # p value depends on degrees of freedom 2 x 2 tables have 1 degree of freedom c p

16 A Serum reactions r = a*d - b*c SQRT (A*B*C*D) r = 14*21-3*1 SQRT (17*22*15*24) r = c 2 = r 2 * T c 2 = * 39 c 2 = p <

17 Luminex - xmap Luminex s xmap Technology combines fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver multiplexed assay capabilities that are configured to perform a wide variety of bioassays quickly, cost-effectively and accurately.

18 Bead Array Microspheres are dyed to create 100 distinct colors Each microsphere has spectral address based on red/infrared content

19 xmap Technology Analyzer samples well Lasers excite fluorescent dyes red laser for bead classification and green laser for assay result Multiple readings for each microsphere set Software reports results in real-time Up to 9600 results in 1 hour

20 _ Analysis Software

21 HLA Antibody Testing by Luminex MFI Does Well, AM the What MFI that MFI is MFI is 2,500 of your is anti-a2 MFI too but cut-off low PM affect to is MFI? 3,000 matter. the scr? STOP!!!!!!!

22 Single Antigen PRA Results Antigen Time 1 Time 2 Time 3 Time 4

23 Single Antigen PRA Results

24 Assay Classification Class Result Standardization Method References, Calibrators, Controls Qualitative Positive or Negative Single or dual 1 reference sample, positive and negative controls Semi-quantitative Arbitrary units Single or dual Negative and bilevel positive controls; References unique for test system made from pooled patient sera Quantitative Units related to recognized reference preparation Multipoint standard curve Tri-level controls; Reference calibrated towards a qualified reference preparation

25 SABs are not quantitative why? Quantitative assay = result in units/volume Don t have the necessary components Not standardized Challenging from biologic and technical perspectives So says regulatory authorities (intended use)

26 This ain t the Chemistry Lab Challenges for Immunoassay Standardization Measuring analyte serum with multiple isotypes, subclasses and varying avidities No standard Sample handling/interfering substances Antigen standardization Suitable controls with defined range of Ab

27 Factors affecting all assays Yeah, you still gotta worry about all that biology, chemistry and physics stuff. Affinity vs Avidity Polyclonal vs monoclonal Inhibitors Accuracy vs Precision Birthday cake!!

28 Affinity vs Avidity

29 Polyclonal vs Monoclonal Roitt IM, Brostoff JB, Male DK. Immunology Fifth Edition. Mosby Internation Ltd

30 Inhibitors

31 Accuracy vs Precision Accuracy The ability to measure purified amounts of a substance Known positive sera as a control Mixed mono-specific serum with non-specific serum Precision Ability of an assay to consistently reproduce a result Measured by Coefficient of Variation (%CV) Good intra-assay CV 10% Good inter-assay CV ~ 10-15%

32 Intra-assay %CV

33 Inter-assay %CV

34 The Real World SOPs are routinely deviated Lunch Birthday cake! Ambient conditions change Lasers fade

35 Effect of Temperature on Variability Room temperature 0 C Temperature Control Negative Control Bead Courtesy: Derek O Neill, Beaumont Hospital, Dublin, Ireland

36 Patient serum variability: Post-transplant monitoring Day of transplant serum Day 7 posttransplant serum Run 1 Day 7 posttransplant serum Run 2 Temp 0 C Rxn Score A*0201 (donor Specific) MFI Rxn Score B*3701 B*1503 MFI Rxn Score MFI cold cold warm Courtesy: Derek O Neill, Beaumont Hospital, Dublin, Ireland

37 Luminex Summary Luminex assays should be carried out under controlled conditions Luminex results should be interpreted by experienced personnel take measurement error into account. SAB assays are at best semi-quantitative. Resist the siren call of MFI

38 Other PRA Testing Considerations Polyclonal vs. monoclonal CYNAP Monospecific vs. polyspecific Cross Reactive Groups (CREGS)

39 Antibody Specificity Monospecific serum that has specificity for a single HLA antigen Polyspecific serum that reacts with more than one distinct HLA antigen

40 Cross-reactive epitope groups (CREGs) Multiply reactive sera can result from: multiple private specificity antibodies» antibodies specific for one specific determinant true crossreactive antibodies» one antibody that bind to structurally similar private epitopes antibodies to public epitopes» one antibody that binds to a unique epitope shared among several different HLA antigens (Bw4 or Bw6)

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43 CREG Epitope Locations

44 CON1 CON2 CON3 CON4 A*0101 A*0201 A*0203 A*0206 A*0301 A*1101 A*1102 A*2302 A*2402 A*2403 A*2501 A*2601 A*2902 A*3001 A*3101 A*3201 A*3301 A*3303 A*3402 A*3601 A*4301 A*6601 A*6602 A*6801 A*6802 A*6901 A*7401 A*7405 A*8001 B*0702 B*0703 B*0801 B*1302 B*1401(B64) B*1402(B65) B*1501(B62) B*1502(B75) B*1503(B72) B*1512(B76) B*1513(B77) B*1516(B63) B*1518(B71) B*1801 B*2703 B*2705 B*2708 B*3501 B*3701 B*3801 B*3901 B*4001(B60) B*4002(B61) B*4101 B*4201 B*4402 B*4501 B*4601 B*4701 B*4801 B*4901 B*5001 B*5101 B*5201 B*5301 B*5401 B*5501 B*5601 B*5701 B*5802 B*7301 B*7801 B*8101 B*8202 Cw*0102 Cw*0210 Cw*0303 Cw*0304 Cw*0401 Cw*0403 Cw*0501 Cw*0602 Cw*0701 Cw*0702 Cw*0704 Cw*0801 Cw*0802 Cw*1202 Cw*1402 Cw*1502 Cw*1601 Cw*1701 Cw*1801 Probe 77 RawMFI-BG B7 CREG appears safe Pt. PRA consistently >90% (multispecific) Pt. HLA-A11, -; B56, 65 (? sensitization)

45 CON1 CON2 CON3 CON4 A*0101 A*0201 A*0203 A*0206 A*0301 A*1101 A*1102 A*2302 A*2402 A*2403 A*2501 A*2601 A*2902 A*3001 A*3101 A*3201 A*3301 A*3303 A*3402 A*3601 A*4301 A*6601 A*6602 A*6801 A*6802 A*6901 A*7401 A*7405 A*8001 B*0702 B*0703 B*0801 B*1302 B*1401(B64) B*1402(B65) B*1501(B62) B*1502(B75) B*1503(B72) B*1512(B76) B*1513(B77) B*1516(B63) B*1518(B71) B*1801 B*2703 B*2705 B*2708 B*3501 B*3701 B*3801 B*3901 B*4001(B60) B*4002(B61) B*4101 B*4201 B*4402 B*4501 B*4601 B*4701 B*4801 B*4901 B*5001 B*5101 B*5201 B*5301 B*5401 B*5501 B*5601 B*5701 B*5802 B*7301 B*7801 B*8101 B*8202 Cw*0102 Cw*0210 Cw*0303 Cw*0304 Cw*0401 Cw*0403 Cw*0501 Cw*0602 Cw*0701 Cw*0702 Cw*0704 Cw*0801 Cw*0802 Cw*1202 Cw*1402 Cw*1502 Cw*1601 Cw*1701 Cw*1801 Probe 77 RawMFI-BG B Ag specificities narrowed Pt tx with B44 mismatch 6/ % (A68, B44) 4/ % (B44 strongest) B specificities B44, 45, 76, 82

46 CON1 CON2 CON3 DPB1*0101/DPA*0103 DPB1*0101/DPA*0201 DPB1*0101/DPA*0202 DPB1*0101/DPA*0301 DPB1*0201/DPA*0103 DPB1*0301/DPA*0103 DPB1*0301/DPA*0201 DPB1*0401/DPA*0103 DPB1*0401/DPA*0201 DPB1*0401/DPA*0202 DPB1*0401/DPA*0301 DPB1*0402/DPA*0103 DPB1*0402/DPA*0201 DPB1*0402/DPA*0301 DPB1*0501/DPA*0103 DPB1*0501/DPA*0201 DPB1*0501/DPA*0202 DPB1*0501/DPA*0301 DPB1*1301/DPA*0103 DPB1*1301/DPA*0201 DPB1*1401/DPA*0103 DPB1*1401/DPA*0201 DPB1*1701/DPA*0201 DPB1*1801/DPA*0103 DPB1*1801/DPA*0201 DPB1*1901/DPA*0103 DPB1*1901/DPA*0201 DPB1*1901/DPA*0301 DPB1*2801/DPA*0103 DPB1*2801/DPA*0201 DPB1*2801/DPA*0202 DQB1*0202/DQA*0302 DQB1*0202/DQA*0201 DQB1*0402/DQA*0302 DQB1*0402/DQA*0401 DQB1*0401/DQA*0401 DQB1*0301/DQA*0302 DQB1*0301/DQA*0501 DQB1*0302/DQA*0302 DQB1*0302/DQA*0201 DQB1*0303/DQA*0302 DQB1*0303/DQA*0401 DQB1*0501/DQA*0104 DQB1*0601/DQA*0104 DRB1*0101 DRB1*0103 DRB1*0401 DRB1*0701 DRB1*0801 DRB1*0901 DRB1*1001 DRB1*1101 DRB1*1201 DRB1*1301 DRB1*1401 DRB1*1501 DRB1*1601 DRB1*0301 (17) DRB1*0303 (18) DRB3*0101 (52) DRB3*0202 (52) DRB4*0101 (53) DRB5*0101 (51) Class II DQ Abs pop out Pt. always called DR 52 (previous MM) DQ specificity confounded by DR 52 and 53

47 CREG Epitope Locations

48 HLA-A Cross Reactivities

49 HLA-B Cross Reactivities

50 HLA-DR Cross Reactivities

51 TABLE 1. Overview of common molecular targets for non-hla antibody responses, allograft types with confirmed injury phenotypes and corresponding time frames, as well as experimental evidence for pathogenicity of non-hla antibodies with corresponding references Antibody target Human allograft type Time-frame Experimental evidence MICA Kidney (28 31, 35 39), heart (33), Hyperacute, acute, chronic pancreas (31) Vimentin Heart (52) Chronic Mouse (57), non-human primate (55, 56) AT 1 R Kidney (46) Acute Rat renal allograft (46) ICAM-1 Heart (58)? HLA, human leukocyte antigens; AT 1 R, angiotensin type 1 receptor; ICAM-1, intercellular adhesion molecule-1.

52 MICA and MICB MHC class I-like chain A or B It is thought that MICA and MICB function as stressinduced antigens that are broadly recognized by NK cells and most of the subtypes of T cells. Expressed on endothelial cells, gut epithelium, monocytes but not lymphocytes Routine crossmatch will not detect Ab to MICA or MICB Antibodies to MICA don t generally fix complement Up-regulation of MICB assoc with class II MHC expression and lymphocyte infiltration MICA MICB Antibody target Human allograft type Time-frame Experimental evidence MICA Kidney (28 31, 35 39), heart (33), Hyperacute, acute, chronic - pancreas (31)

53 Vimentin Vimentin is a member of the intermediate filament family, and is part of the cytoskeletal network. Non-polymorphic protein found in abundance in endothelium and smooth muscle cells. antibodies to vimentin are considered auto-reactive In a monkey model, anti-vimentin antibodies are associated with chronic cardiac rejection but not kidney rejection.???? Antibody target Human allograft type Time-frame Experimental evidence Vimentin Heart (52) Chronic Mouse (57), non-human primate (55, 56)

54 Angiotensin type 1 Receptor (AT 1 R) AT 1 receptor has vasopressor effects and regulates aldosterone secretion. It is an important effector controlling blood pressure and volume in the cardiovascular system. Antibodies to AT 1 R have been associated with pre-eclampsia and are implicated with severe vascular rejection. Article describes a patient who received a 6 Ag match kidney and then had a sudden onset of malignant hypertension that mimicked the pre-eclampsia she suffered >20 years before. Angiotensin type II A receptor like AT 1 R Antibody target Human allograft type Time-frame Experimental evidence AT 1 R Kidney (46) Acute Rat renal allograft (46)

55 ICAM and anti-gbm Antibodies to ICAM found in cardiac transplant patients. Antibodies to agrin and perlecan are associated with duplication of the GBM - a hallmark of transplant glomerulopathy.

56 Mechanisms of Injury Complement-mediated injury Antibody-dependent Cellular Cytotoxicity for non-c fixing Abs (like those against MICA/B) May contribute to structural changes Signalling?

57 Perspective With exceptions of MICA and AT 1 R, routine testing for these antibodies are not currently available. Question as to whether non-hla antibody related pathologies represent true rejections of the transplanted organ or organ-specific autoimmune phenomena. Non-HLA Abs may find application as biomarkers of immune response and herald the need for more suitable immunosuppression.

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

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

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

61 Continuing Education ABHI, PACE, Florida and California DHS 1.0 Contact Hours Each attendee must register to receive CE at: Registration deadline is March 2, 2018 Certificates will be sent via only to those who have registered by March 16, 2018 All Content 2015 Immucor, Inc.

62 Future Webinars 22 February Donor Selection of Solid Organ Transplant: Is Virtual Xmatch better than real? 28 February Proficiency, Competency, and QC: A practical approach to CLIA requirements and AABB, CAP, and Joint Commission Expectations 22 March Clinical Significance of HLA Antibodies in Solid Organ Transplantations Link to register: All Content 2015 Immucor, Inc.