It s about time. The Development of a Rapid Optimized Single Antigen Bead (ROB) LABScreen Protocol to Expedite HLA Antibody Testing.

Transcription

1 It s about time. The Development of a Rapid Optimized Single Antigen Bead (ROB) LABScreen Protocol to Expedite HLA Antibody Testing. Robert Liwski, Jorge Neumann 2, Geoff Peladeau, Kelly Heinstein, Roxanne Sperry, Robert Bray 3 and Howard Gebel 3. Department of Pathology, Dalhousie University, Halifax NS, Canada 2 Lab of Transplant Immunology, Santa Casa Hospital, Porto Alegre, Brazil 3 Department of Pathology, Emory University, Atlanta, USA

2 LABScreen single antigen bead (SAB) Luminex assay Uses the Lambda Array Beads Multi-analyte System. Number of advantages compared to FlowPRA SAB and ELISA. number of analytes tested simultaneously High throughput Rapid analysis (Fusion software) Still, the procedure is time intensive and is not optimal for use in urgent cases. Friday afternoons come to mind

3 LABScreen single antigen bead (SAB) Luminex protocol Incubate beads (5 µl) and serum 2 µl (RT) 3 min. Wash x3 (5 min/spin) 5 min. Incubate with µl anti-igg-pe, : dilution (RT) 3 min. Wash x2 (5min/spin) min. Total assay time h 25 min. Evidence for incubation time/reagent concentration? 2h wash times?

4 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 Anti-IgG-PE concentration

5 Effect of reduced spin time Standard Rapid 5 washes x 5 min = 25 min 5 washes x min = 5 min 3 x g 8 x g

6 Effect of reduced spin time on bead counts Bead count Class I beads min min Class II beads min min Bead number N=3

7 Effect of reduced spin time Standard Rapid 5 washes x 5 min = 25 min 5 washes x min = 5 min 3 x g 8 x g No impact on bead counts or overall results 2 minutes saved!

8 Effects of reduced incubation times Serum incubation time Anti-IgG-PE incubation time

9 Effects of reduced incubation time ¼ PPC, HLA class I 25 2 MFI 5 3/3 min Bead number

10 Effects of reduced incubation time ¼ PPC, HLA class I 25 2 MFI 5 3/3 min 5/3 min Bead number

11 Effects of reduced incubation time ¼ PPC, HLA class I 25 2 MFI 5 3/3 min 5/3 min 3/5 min Bead number

12 Effects of reduced incubation time ¼ PPC, HLA class I 25 2 MFI 5 3/3 min 5/3 min 3/5 min 5/5 min Bead number

13 Effects of reduced incubation time ¼ PPC, HLA class II 25 2 MFI 5 3/3 min 5/3 min 3/5 min 5/5 min Bead number

14 Effects of reduced incubation time Negative control serum Class I Class II 6 6 MFI 4 3/3 min 5/3 min 3/5 min 5/5 min 4 3/3 min 5/3 min 3/5 min 5/5 min Bead number

15 MFI Effects of reduced incubation time NC bead NC and PC beads Small Effect on background 3/3 min 5/3 min 3/5 min 5/5 min PC bead Serum/IgG-PE incubation time Significant Effect on IgG binding 3/3 min5/3 min 3/5 min 5/5 min

16 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?

17 Effects of increasing IgG-PE concentration ¼ PPC, HLA class I 3 25 MFI 2 5 PE /5 PE / PE /2 PE /5 PE / Bead number

18 Effects of increasing IgG-PE concentration ¼ PPC, HLA class II 3 25 MFI 2 5 PE /5 PE / PE /2 PE /5 PE / Bead number

19 Effects of increasing IgG-PE concentration Negative Control Serum Class I 6 Class II 6 MFI 4 PE /5 PE / PE /2 PE /5 PE / 4 PE /5 PE / PE /2 PE /5 PE / Bead number

20 MFI Effects of increasing IgG-PE concentration NC and PC beads NC bead PC bead PE / PE /5 PE /2 PE / PE / Serum/IgG-PE incubation time PE / PE /5 PE /2 PE / PE /5

21 Anti-IgG-PE titration conclusion: Increasing the anti-igg-pe concentration from : to :5 increases MFI in the standard assay including PC bead MFI. Negligible effect on background. Can we compensate for reduced MFI values in the 5/5 min protocol by optimizing the concentration of anti-igg-pe?

22 Effects of increasing IgG-PE concentration on MFI in 5/5 protocol PPC, HLA class I 25 2 MFI 5 3/3 min PE / Bead number

23 Effects of increasing IgG-PE concentration on MFI in 5/5 protocol PPC, HLA class I 25 2 MFI 5 5/5 min PE / 3/3 min PE / Bead number

24 Effects of increasing IgG-PE concentration on MFI in 5/5 protocol PPC, HLA class I 25 2 MFI 5 5/5 min PE /2 5/5 min PE / 3/3 min PE / Bead number

25 Effects of increasing IgG-PE concentration on MFI in 5/5 protocol PPC, HLA class I 25 2 MFI 5 5/5 min PE / 5/5 min PE /2 5/5 min PE / 3/3 min PE / Bead number

26 Effects of increasing IgG-PE concentration on MFI in 5/5 protocol PPC, HLA class I 25 2 MFI 5 5 5/5 min PE /5 5/5 min PE / 5/5 min PE /2 5/5 min PE / 3/3 min PE / Bead number

27 Effects of increasing IgG-PE concentration on MFI in 5/5 protocol PPC, HLA class II 25 2 MFI 5 5 5/5 min PE /5 5/5 min PE / 5/5 min PE /2 5/5 min PE / 3/3 min PE / Bead number

28 Conclusion Increasing concentration of anti-igg-pe compensates for the reduction in incubation times. IgG-PE concentration of : closely matches MFI obtained with the standard assay.

29 Rapid Optimized SAB (ROB) LABScreen Protocol Incubate beads (2.5 µl) and serum 25 µl (RT) 5 min. Wash x3 ( min/spin) 3 min. Incubate with 2 µl anti-igg-pe, : dilution (RT) 5 min. Wash x2 (5min/spin) 2 min. Total assay time 25 min. 7% time reduction!

30 Standard vs ROB protocol, MFI correlation 8 patient, 9 ASHI PT, 3 ABH PT sera ROB protocol MFI Class I y =.3x R² =.998 Class II y =.256x R² = Standard Assay MFI

31 Representative Serum Reactivity Standard vs ROB protocol MFI AC-463 Class I Standard ROB AC-463 Class II Standard ROB Bead number

32 Average MFI ratio ROB vs Standard Protocol MFI Ratio

33 4 35 Discrepant reactions Standard Cut-off 2 MFI ROB. rxn/panel 44 rxn/4 panels 3 MFI rxn

34 Patient serum CF, class I ROB Standard

35 Patient serum CF, class I ROB Aw4/Bw4 Standard Aw4/Bw4

36 Class I Porto Alegre Laboratory Standard vs ROB MFI correlation, May 24 ABH PT sera Class II 3 3 ROB protocol MFI y =.9x R² = y =.235x R² = Standard Assay MFI

37 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 and cost saving. ROB protocol allows for rapid testing of urgent patient sera. Ex. testing during deceased donor work up. LBP