Optimization of flow cytometric crossmatch assay to expedite pre-transplant immunologic risk assessment
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1 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 Department of Pathology and Laboratory Medicine Dalhousie University, Halifax, Canada
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4 2 Outline Introduction to flow cytometric crossmatch FCXM Standard 3-colour FCXM technique Development of Rapid Optimized FCXM assay (ASHI 2011) Canadian Standardization of FCXM National FCXM proficiency testing data (ASHI 2012) Nation-wide Evaluation of Rapid Optimized FCXM assay (ASHI 2012)
5 HLA testing... is similar to pretransfusion testing.
6 HLA testing... is similar to pretransfusion testing. ABO, D antigen typing HLA typing Antibody screen HLA antibody screen Identify red cell antigen alloantibody specificity HLA antibody specificity identification Red cell Crossmatch final compatibility check before transfusion Lymphocyte Crossmatch final compatibility check before transplant
7 Significance of a positive crossmatch test in kidney transplantation Patel and Terasaki NEJM 1969 Graft rejection Functioning graft itive 24 6 ative crossmatch
8 3-Color Flow Cytometric Crossmatch Recipient serum Donor lymphocytes T cell B cell Bray, Lebeck and Gebel. Transplantation 48: , 1989.
9 3-Color Flow Cytometric Crossmatch T cell B cell Bray, Lebeck and Gebel. Transplantation 48: , 1989.
10 3-Color Flow Cytometric Crossmatch T cell B cell Bray, Lebeck and Gebel. Transplantation 48: , 1989.
11 3-Color Flow Cytometric Crossmatch FITC-a-IgG T cell B cell Bray, Lebeck and Gebel. Transplantation 48: , 1989.
12 3-Color Flow Cytometric Crossmatch FITC-a-IgG FITC-a-IgG T cell B cell Bray, Lebeck and Gebel. Transplantation 48: , 1989.
13 3-Color Flow Cytometric Crossmatch PE-a-CD19 PerCP-a-CD3 FITC-a-IgG FITC-a-IgG T cell B cell Bray, Lebeck and Gebel. Transplantation 48: , 1989.
14 3-Color Flow Cytometric Crossmatch FITC-a-IgG FITC-a-IgG PerCP-a-CD3 T cell PE-a-CD19 B cell Detect fluorescent labels by flow cytometry Bray, Lebeck and Gebel. Transplantation 48: , 1989.
15 3-Color Flow Cytometric Crossmatch T cell X-match B cell X-match ative Gating strategy Weak positive Strong positive FITC-a-IgG FITC-a-IgG
16 ASHI Lab Manual Procedure Hamrick and Lebeck Incubate 5.0x105 cells (30 ml) and serum 30 ml (4oC) 30 min. Wash x3 (5 min/spin) 15 min. Incubate with 100 ml anti-igg-fitc/cd3/cd19 (4oC) 30 min. Wash x2 (5min/spin) 10 min. Total assay time Evidence for: incubation times/temperature? cell number/serum volume/wash times? 1h 25 min. 2h
17 An Optimized Flow Cytometric Crossmatch Assay Expedites Pre-Transplant Immunologic Risk Assessment. Robert Liwski, Geoff Adams, Geoff Peladeau, David Eckels, Robert Bray and Howard Gebel. ASHI 2011
18 Objectives To develop an expeditious flow crossmatch procedure without compromising the sensitivity of the assay (good signal to noise ratio). Investigate the effects of: Assay platform (96 well plate vs Tube) Serum incubation time anti-igg-fitc incubation time Incubation temperature Cell number Serum volume
19 Acknowledgements Geoff Adams and Geoff Peladeau
20 Assay platform 96-well tray vs Tube Reduce centrifugation time from 5 min to 1 min (total 20 minutes saved) Tray flick vs decanting/aspirating supernatants Reduces buffer and reagent delivery time Liwski et al ASHI 2011
21 Yay, another crossmatch!!! Geoff Adams
22 I ll definitely be done before lunch.
23 Do you really want me to do it this way?
24 Did I do this tube already?
25 zzzzzzzzz.
26 Beep, Beep, Beep.
27 Delta MCF 96-well Plate vs Tube Platform T cell X-match B cell X-match Tube Plate 1 Tube Plate 2 Tube PC PC PC 1/100 1/200 1/400 Tube PC 1/50 Plate 1 Plate 2 PC 1/50 Serum dilution PC PC PC 1/100 1/200 1/400 Liwski et al ASHI 2011
28 Assay platform 96-well tray vs Tube Reduce centrifugation time from 5 min to 1 min (total 20 minutes saved) Tray flick vs decanting/aspirating supernatants Reduces buffer and reagent delivery time Approximately minute time saving! (depending on sample number) Liwski et al ASHI 2011
29 Effects of serum incubation time Time course experiment. Incubation with different dilutions of PC serum for 3, 5, 10, 15 or 30 minutes. All groups were incubated with anti-iggfitc for 30 minutes. Liwski et al ASHI 2011
30 Effects of serum incubation time (3-30 min), T cell X-match Delta MCF Time (min) 200 Serum 3 Serum Serum 10 Serum 15 Serum PC 1/50 PC 1/100 PC 1/200 Serum dilution PC 1/400 Liwski et al ASHI 2011
31 Effects of serum incubation time (3-30 min), B cell X-match Delta MCF Time (min) 250 Serum Serum 5 Serum Serum Serum PC 1/50 PC 1/100 PC 1/200 Serum dilution PC 1/400 Liwski et al ASHI 2011
32 Effects of anti-igg-fitc incubation time Time course experiment. Incubation with different dilutions of PC for 30 minutes. anti-igg-fitc incubation time course (5, 10, 15 or 30 minutes). Liwski et al ASHI 2011
33 Effects of anti-igg-fitc incubation time (5-30 min), T cell X-match 300 Delta MCF 250 Time (min) 200 FITC 5 FITC FITC FITC PC 1/50 PC 1/100 PC 1/200 Serum dilution PC 1/400 Liwski et al ASHI 2011
34 Effects of anti-igg-fitc incubation time (5-30 min), B cell X-match Delta MCF 300 Time (min) 250 FITC FITC 10 FITC FITC PC 1/50 PC 1/100 PC 1/200 Serum dilution PC 1/400 Liwski et al ASHI 2011
35 Effects of incubation temperature Liwski et al ASHI 2011
36 Effects of incubation temperature (4oC vs RT), T cell X-match Delta MCF 350 Temperature 300 4o C 250 RT R PC 1/25 PC 1/50 PC 1/100 PC 1/200 Serum dilution PC 1/400 cutoff Liwski et al ASHI 2011
37 Effects of incubation temperature (4oC vs RT), B cell X-match Delta MCF 400 Temperature 350 4o C 300 RT PC 1/25 PC 1/50 PC 1/100 PC 1/200 Serum dilution PC 1/400 cutoff Liwski et al ASHI 2011
38 Effects of serum volume Liwski et al ASHI 2011
39 Effects of serum volume T cell X-match 350 Delta MCF Serum/Cell Volume Ratio 200 1: : PC 1:50 PC 1:100 PC 1:200 Serum dilution PC 1:400 Liwski et al ASHI 2011
40 Effects of serum volume B cell X-match Delta MCF Serum/Cell Volume Ratio 200 1: : PC 1:50 PC 1:100 PC 1:200 Serum dilution PC 1:400 Liwski et al ASHI 2011
41 Effects of cell number Liwski et al ASHI 2011
42 Effects of cell number T cell X-match Delta MCF Cell Number Serum dilution Liwski et al ASHI 2011
43 Effects of cell number B cell X-match Delta MCF Cell Number Serum dilution Liwski et al ASHI 2011
44 Summary Marginal effect of serum incubation beyond 15 minutes. ligible effect of anti-igg-fitc incubation beyond 5 minutes. Slight increase in delta MCF with increased incubation temperature. Slight increase in delta MCF with increased serum volume. Increase in delta MCF with decreased cell number. Liwski et al ASHI 2011
45 Rapid Optimized Flow Crossmatch Protocol Incubate 5x105 cells (30 ml) and serum 30 ml (4oC) 2.5x105 cells (25 ml) 50 ml (RT) Wash x3 (5 min/spin) (1 min/spin) Incubate with 100 ml anti-igg-fitc/cd3/cd19 (4oC) (RT) Wash x2 (5min/spin) (1 min/spin) Total assay time 30 min 20 min 15 min 3 min 30 min 10 min 10 min 2 min 1h 25 min 35 min
46 Rapid Optimized FXM Protocol Validation Compare standard vs optimized protocols with respect to: ative control sera Predicted negative patient sera 3 SD cut-offs itive control sera Predicted positive patient sera Liwski et al ASHI 2011
47 ative Control Comparison MCF MESF N=15/20 Method Standard Optimized Liwski et al ASHI 2011
48 ative Patient Sera Comparison MCF MESF N=90/140 Method Standard Optimized Liwski et al ASHI 2011
49 MCF MESF Average itive Cut-off Method Standard Optimized Liwski et al ASHI 2011
50 itive Control Comparison Standard MESF Optimized PC 1/50 PC 1/100 PC 1/200 PC cutoff PC 1/50 PC 1/400 1/100 T cell X-match PC 1/200 PC cutoff 1/400 B cell X-match N=10 Liwski et al ASHI 2011
51 Delta Channels From itive Cut-off itive Patient Sera Comparison (MCF) T cell XM B cell XM Method Serum# Standard Optimized Liwski et al ASHI 2011
52 Conclusion We can reduce the time it takes to perform crossmatch without compromising assay sensitivity. Significant time and cost saving. Impact on ischemia time? Liwski et al ASHI 2011
53 Going with the Flow, Canadian Crossmatch Standardization Robert Liwski, Denise Pochinco, Kathryn Tinckam, Howard Gebel, Patricia Campbell and Peter Nickerson On behalf of the Nationwide Laboratory Oversight Committee & Canadian Blood Services Liwski et al ASHI 2012
54 Introduction Expeditious FXM assay giving consistent inter-lab results is critical for successful and safe national organ sharing. In order to understand the degree and nature of variability in FXM testing across Canada, national proficiency testing (PT) program was established. PT surveys are designed and organized by the National PT Committee which has representation from all 13 Canadian HLA Laboratories. 3 PT surveys organized since September Liwski et al ASHI 2012
55 Objectives To determine the extent of variability in FXM procedure/results across Canada. To identify factors that influence FXM results. Liwski et al ASHI 2012
56 PT Survey 1 design 13 labs asked to perform flow crossmatches: Two Donor Cells Three Test Sera FXM interpretation and raw data were collected and analyzed. Questionnaire, specific information regarding FXM assay. Liwski et al ASHI 2012
57 T Cell XM Cell 1 Ser 1 Ser 2 Ser3 Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6 Lab 7 Lab 8 Lab 9 Lab 10 Lab 11 Lab 12 Lab 13 Consensus A33 2.5K B65 2.5K B13 7K Liwski et al ASHI 2012 Wk pos Wk pos B Cell XM Cell 2 Cell 1 Ser 1 Ser 2 Ser 3 Ser 1 Ser 2 Ser 3 B7 9K Wk pos A24 1.5K B65 2.5K Wk pos Wk pos Wk pos A33 2.5K B65 2.5K B13 7K DP17 5K DR7 11K DR53 8K DQ2 12K
58 FXM Result Variability (Cell 1 vs Serum 1) Delta MCF T cell X-match B cell X-match Neat Class I DSA: Class II DSA: 1/2 1/4 A33 (2.5K), B13 (7K) DP17 (5K) Neat 1/2 1/4 Liwski et al ASHI 2012
59 Effect of Pronase (Cell 1 vs Serum 1) Delta MCF T cell X-match B cell X-match Neat Class I DSA: Class II DSA: 1/2 1/4 A33 (2.5K), B13 (7K) DP17 (5K) Neat 1/2 1/4 No pronase Liwski et al ASHI 2012
60 Effect of cell number and serum volume T cell FXM MESF ratio Serum/ Control K 250K Cell 1 500K 250K Cell 2 Liwski et al ASHI 2012
61 Conclusions, PT Survey 1 Significant variability in FXM results Differences in FXM protocols. Factors that contribute to variability +/-Pronase Cell numbers and sera/cell volume ratios Washing technique Liwski et al ASHI 2012
62 Questions Can we improve reproducibility of FXM results through standardization of FXM protocol/reagents? Which protocol to choose as a gold standard Liwski et al ASHI 2012
63 FXM Result Variability (Cell 1 vs Serum 1) Delta MCF T cell X-match B cell X-match Neat Class I DSA: Class II DSA: 1/2 1/4 A33 (2.5K), B13 (7K) DP17 (5K) Neat 1/2 1/4 Liwski et al ASHI 2012
64 PT survey 2, Objectives To determine if standardization of FXM protocol/reagents can improve reproducibility of FXM results. Evaluate performance of ROFXM protocol nationally. Liwski et al ASHI 2012
65 PT Survey 2 design Perform crossmatches using Lab method with the following modifications: Pronase (reagent and protocol provided) Standardized cell#, cell suspension volume and serum volume Perform crossmatches using the ROFXM protocol Instructions + video Common crossmatch reagents Liwski et al ASHI 2012
66 98 How many labs does it take to validate this method?
67 Comparison of FXM result interpretation CD12-A T cell Lab Method Serum 1 Serum 2 Serum 3 ROFXM Serum 1 Serum 2 Serum 3 CD12-A B cell Lab Method Serum 1 Serum 2 Serum 3 ROFXM Serum 1 Serum 2 Serum 3 ZWP GUS ANG NIZ ZZP STZ GUN STP LUK KIJ STK ROP ZWP GUS ANG NIZ ZZP STZ GUN STP LUK KIJ STK ROP Fail CD12-B T cell ZWP Lab Method Serum 1 Serum 2 Serum 3 Serum 1 ROFXM Serum 2 Serum 3 CD12-B B cell ZWP Lab Method Serum 1 Serum 2 Serum 3 ROFXM Serum 1 Serum 2 Serum 3 GUS ANG NIZ ZZP STZ GUN STP LUK KIJ STK ROP STP GUS ANG NIZ ZZP STZ GUN KIJ STK ROP 88 LUK Fail 18.5
68 T cell FXM comparison Delta MCF Modified Lab Method ROFXM Protocol Neat 1/2 1/4 1/8 1/16 0 Neat 1/2 1/4 1/8 1/16 Moderate class I HLA DSA Liwski et al ASHI 2012
69 B cell FXM comparison Delta MCF Modified Lab Method ROFXM Protocol Neat 1/2 1/4 1/8 1/16 Neat 1/2 1/4 1/8 1/16 Moderate class I HLA DSA Liwski et al ASHI 2012
70 FXM signal/noise ratio Lab vs ROFXM 400 T cell XM *** 350 Delta MCF B cell XM * Lab ROFXM *** ns ** 450 *** Lab 400 ** 350 ** ROFXM 300 Lab 200 HFX protocol ns Lab HFX protocol ns ns ns vs A 2 vs A 3 vs A 1 vs B 2 vs B 3 vs B Paired Student s t-test, 2 tail analysis 1 vs A 2 vs A 3 vs A 1 vs B 2 vs B 3 vs B *p < **p < 0.01, ***p < Liwski et al ASHI 2012
71 FXM Precision Relative Standard Error (%) (Relative standard error) T cell XM B cell XM Lab Lab ROFXM ROFXM 8 6 Lab HFX protocol vs 2 vs 3 vs 1 vs 2 vs 3 vs A A A B B B 7.0+/-1.3 vs 4.0+/-1.5 p< /-2.2 vs 5.9+/-2.2 p=0.058 Liwski et al ASHI 2012
72 FXM time Time (minutes) FXM time per lab Average FXM time Lab ROFXM *** Lab ROFXM Paired Student s t-test, 2 tail analysis ***p < Liwski et al ASHI 2012
73 Conclusions Protocol/reagent standardization can greatly improve precision of FXM. ROFXM compared favourably with conventional lab FXM methods used across Canada Significantly improved signal/noise ratio Significantly improved precision Significantly decreased TAT National implementation of ROFXM protocol will expedite pre-transplant testing and facilitate equitable national organ sharing. Liwski et al ASHI 2012
74 Proof is in the pudding. Canada 12 USA Germany 2 Switzerland 11 1 Australia Poland 2 UK 2 and 9 France 2 1 Brazil 2 and commencing national evaluation
75 Acknowledgements Collaborators Howard Gebel, Robert Bray and David Eckels Co-authors: Denise Pochinco Kathryn Tinckam Patricia Campbell Peter Nickerson National PT Testing Committee Canadian Blood Services Halifax Lab Technologists Geoff Adams Geoff Peladeau Sandra Lee Labs Vancouver Edmonton Calgary Saskatoon Winnipeg Toronto Ottawa Hamilton London Montreal Laval Quebec City Halifax
76 107
77 108 Liwski et al ASHI 2012
78 Shukran
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