1. Flow Cytometric Opsonophagocytic Assays Joseph E. Martinez CDC, Atlanta Multiplexed OPA work supported in part by a non-restricted CRADA with Flow Applications, Inc. and covered under US patent 6,815,172 S. pneumoniae

2. Opsonophagocytic Assays (OPA ) Two approaches for OP measurement - Killing assays (Steiner, et al., others) - Uptake assays (Martinez; Jansen)

3. Opsonophagocytic Assay

4. Fluorescence FALS Sensor Fluorescence detector (PMT3, PMT4 etc.)

5. Flow Opsonophagocytic Assays Phagocytic cellsComplement Ctl Positive Rx OPA Graph

6. Bacteria or Microspheres S. pneumoniaeMicrospheres

7. Multiplexed Flow OPA Single-plex Multiplex

8. Serotype Reference OPA vs. Single Flow OPA r Values (p value) Reference OPA vs. Three Color Flow OPA r Values (p value) Reference OPA vs. Multiplexed Flow OPA r Values (p value) Single Flow OPA vs. Three Color Flow OPA r Values (p value) Single Flow OPA vs. Multiplexed Flow OPA r Values (p value) 40.88 (<0.001)0.61 (0.04)0.86 (<0.001) 0.80 (0.002)0.85 (<0.001) 6B0.77 (0.003)0.87 (<0.001)0.88 (<0.001) 0.86 (<0.001)0.77 (<0.001) 9V0.53 (0.08) 1 0.79 (0.006)0.80 (<0.001) 0.73 (0.008)0.88 (0.002) 140.54 (0.04) 1 0.75 (0.005)0.85 (<0.001) 0.63 (0.03)0.92 (<0.001) 18C0.77 (0.003)0.91 (<0.001)0.71 (,0.001) 0.74 (0.005)0.92 (<0.001) 19F0.95 (<0.001)0.91(<0.001)0.68 (0.01) 0.91 (<0.001)0.87 (<0.001) 23F0.83 (<0.001)0.78 (0.003)0.68 (,0.001) 0.88 (<0.001) 1 Different strains used Correlations between reference OPA and flow OPA

9. Technical Considerations Targets 1. Bacteria -Variable label

10. Variability of Labeled Bacteria Negative Cells Positive Cells

11. Technical Considerations Targets 1. Bacteria -Variable label 2. Beads -Standardized label

12. Beads Provided a Standard Fluorescent Target CDC, Sero 4-Y BeadsFA, Sero 4-Y Beads

13. Technical Considerations Effector Cells 1. Donor PMNs 2. HL60 PMNs

14. Comparison of PMN and HL60 PMN Derived OPA Titers

15. Technical Considerations Cont. Effector Cells 1. Donor PMNs 2. HL60 PMNs a. Culture maintenance

16. Effects of Different Culture Conditions HL60 Stock Published Induction Protocol Modified Induction Protocol 38% S and G2/M16% S and G2/M3% S and G2/M 18% 3% 57%

17. Technical Considerations Effector Cells 1. Donor PMNs 2. HL60 PMNs a. Culture maintenance b. Induction protocol

18. Effects of Culture Conditions on HL60 Differentiation and Function Normal Culture Method Roller Culture Method

19. Data Collection Gating –Minimize overlap of “dead” cells within gate –Ensure gate contains a cells with ingested targets

20. Gating Effects

21. Data Collection Gating –Minimize overlap of “dead” cells within gate –Ensure gate contains a cells with ingested targets Compensation –Critical in multiplexed assays

22. Data Analysis-Automation Flow cytometric OPA can be automated –Sample handler –Batch file analysis of raw data files Attractors FlowJo –Curve fitting software to determine titer Statlia GLP compatible

23. Automation Efficiencies Published Single-plex Automated Multiplexed Data collection 1 4hrs3.5hrs 3 Data processing 2 2hrs3min Titer Determination 30min15min Total time required 6.5hr3.6hr or 0.9hr per serotype 1 Average for 5 plate run 2 Four hundred eighty files 3 Does not require constant attention

24. ReferenceSingle-plexMultiplex Setup time30min Assay time12-15hr*1.5 hr Instrument time/plate 2-3min1hr0.7hr Data Analysis15min30sec Tests/plate5520 Daily (8hrs) throughput 15*30120 Assay Comparisons *Overnight incubation required

25. Advantages and Disadvantages 1 Multiplexed killing assay 2 Instrument/technician costs Reference 1 FlowMultiplex Specificity++++ Technically difficult ++ ++++ Infectious Target++++-- Multiplexed++++++++++ CommercialNA++++++ Automation++++++ Cost++++ 2 +2+2

26. Flow Cytometric OPA Technology Can be applied to other bacteria cleared through an OP mechanism S. aureus (Vernachio) Figure 1. Opsonization of ClfA-coated fluorescent beads. PMNs were incubated with unopsonized beads, complement opsonized beads, T1-2 plus complement opsonized beads, and non-specific human IgG1 complement opsonized beads. The phagocytic product (PP) represents the mean beads per cell multiplied by the percent fluorescent PMNs, as determined via flow cytometric analysis.

27. Summary Non-infectious targets Correlate well with reference OP method High throughput Automation can further increase relative throughputs Meet GLP guidelines