Flow Cytometric Opsonophagocytic Assays

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Flow Cytometric Opsonophagocytic Assays
Joseph E. Martinez CDC, Atlanta
S. pneumoniae
Multiplexed OPA work supported in part by a
non-restricted CRADA with Flow Applications, Inc.
and covered under US patent 6,815,172
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• Opsonophagocytic Assays (OPA)
• Two approaches for OP measurement
• - Killing assays (Steiner, et al., others)
• - Uptake assays (Martinez Jansen)

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• Opsonophagocytic Assay

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(No Transcript)
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Flow Opsonophagocytic Assays
Phagocytic cells
Complement Ctl
Positive Rx
OPA Graph
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Bacteria or Microspheres
S. pneumoniae
Microspheres
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Multiplexed Flow OPA
 
Single-plex
Multiplex
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Correlations between reference OPA and flow OPA
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)
4 0.88 (lt0.001) 0.61 (0.04) 0.86 (lt0.001) 0.80 (0.002) 0.85 (lt0.001)
6B 0.77 (0.003) 0.87 (lt0.001) 0.88 (lt0.001) 0.86 (lt0.001) 0.77 (lt0.001)
9V 0.53 (0.08)1 0.79 (0.006) 0.80 (lt0.001) 0.73 (0.008) 0.88 (0.002)
14 0.54 (0.04)1 0.75 (0.005) 0.85 (lt0.001) 0.63 (0.03) 0.92 (lt0.001)
18C 0.77 (0.003) 0.91 (lt0.001) 0.71 (,0.001) 0.74 (0.005) 0.92 (lt0.001)
19F 0.95 (lt0.001) 0.91(lt0.001) 0.68 (0.01) 0.91 (lt0.001) 0.87 (lt0.001)
23F 0.83 (lt0.001) 0.78 (0.003) 0.68 (,0.001) 0.88 (lt0.001) 0.88 (lt0.001)
1Different strains used
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Technical Considerations
Targets 1. Bacteria -Variable label

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Variability of Labeled Bacteria
Positive Cells
Negative Cells
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Technical Considerations
Targets 1. Bacteria -Variable label
2. Beads -Standardized label
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Beads Provided a Standard Fluorescent Target
CDC, Sero 4-Y Beads
FA, Sero 4-Y Beads
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Technical Considerations
Effector Cells 1. Donor PMNs 2. HL60
PMNs
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Comparison of PMN and HL60 PMN Derived OPA Titers
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Technical Considerations Cont.
Effector Cells 1. Donor PMNs 2. HL60
PMNs a. Culture maintenance
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Effects of Different Culture Conditions
38 S and G2/M
16 S and G2/M
3 S and G2/M
Published Induction Protocol
Modified Induction Protocol
HL60 Stock
3
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Technical Considerations
Effector Cells 1. Donor PMNs 2. HL60
PMNs a. Culture maintenance b. Induction
protocol
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Effects of Culture Conditions on HL60
Differentiation and Function
Normal Culture Method
Roller Culture Method
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Data Collection

• Gating
• Minimize overlap of dead cells within gate
• Ensure gate contains a cells with ingested targets

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Gating Effects
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Data Collection

• Gating
• Minimize overlap of dead cells within gate
• Ensure gate contains a cells with ingested
  targets
• Compensation
• Critical in multiplexed assays

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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

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Automation Efficiencies
Published Single-plex Automated Multiplexed
Data collection1 4hrs 3.5hrs3
Data processing2 2hrs 3min
Titer Determination 30min 15min
Total time required 6.5hr 3.6hr or 0.9hr per serotype
1Average for 5 plate run 2Four hundred eighty files 3Does not require constant attention 1Average for 5 plate run 2Four hundred eighty files 3Does not require constant attention 1Average for 5 plate run 2Four hundred eighty files 3Does not require constant attention
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Assay Comparisons
Reference Single-plex Multiplex
Setup time 30min 30min 30min
Assay time 12-15hr 1.5 hr 1.5 hr
Instrument time/plate 2-3min 1hr 0.7hr
Data Analysis 15min 30sec 30sec
Tests/plate 5 5 20
Daily (8hrs) throughput 15 30 120
Overnight incubation required
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Advantages and Disadvantages
1Multiplexed killing assay 2Instrument/technician costs Reference1 Flow Multiplex
Specificity
Technically difficult
Infectious Target - -
Multiplexed
Commercial NA
Automation
Cost 2 2
 
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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.
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Summary

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