Flow Cytometric Opsonophagocytic Assays
description
Transcript of Flow Cytometric Opsonophagocytic Assays
Flow Cytometric Opsonophagocytic Assays
Joseph E. MartinezCDC, 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
Data.011
100 101 102 103 104
FL2-Height
M1
Opsonophagocytic Assays (OPA)
•Two approaches for OP measurement- Killing assays (Steiner, et al.,
others)- Uptake assays (Martinez; Jansen)
•Opsonophagocytic Assay
Fre
q
Fluorescence
FALS Sensor
Fluorescence detector
(PMT3, PMT4 etc.)
Flow Opsonophagocytic AssaysData.011
0 200 400 600 800 1000SSC-Height
R1
Data.011
100 101 102 103 104
FL2-Height
M1
Data.107
100 101 102 103 104
FL2-Height
M1
Phagocytic cells Complement Ctl
Positive Rx
Example OPA Graph
Reciprocal Dilution
8 16 32 64 128 256 5121024
Per
cen
t U
pta
ke
0
20
40
60
80
100
Pt Sample
Complement uptake
50% Maximum Uptake
Reported Titer
OPA Graph
Bacteria or Microspheres
S. pneumoniae Microspheres
uptake
Multiplexed Flow OPAExample OPA Graph
Reciprocal Dilution
8 16 32 64 128 256 5121024
Per
cent
Upt
ake
0
20
40
60
80
100
Pt Sample
Complement uptake
50% Maximum Uptake
Reported Titer
Single-plex
uptake
uptake
Uptake
uptake
Multiplexed Bead Based OPA
Log2 Reciprocol Dilution
2 4 6 8 10
Per
cent
Upt
ake
by H
L60
PM
Ns
5
10
15
20
25
30
35
40
45
Serotype 14, GreenSerotype 9V, PinkSerotype 6B, RedSerotype 4, Far Red
Multiplex
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 (<0.001) 0.61 (0.04) 0.86 (<0.001) 0.80 (0.002) 0.85 (<0.001)
6B 0.77 (0.003) 0.87 (<0.001) 0.88 (<0.001) 0.86 (<0.001) 0.77 (<0.001)
9V 0.53 (0.08)1 0.79 (0.006) 0.80 (<0.001) 0.73 (0.008) 0.88 (0.002)
14 0.54 (0.04)1 0.75 (0.005) 0.85 (<0.001) 0.63 (0.03) 0.92 (<0.001)
18C 0.77 (0.003) 0.91 (<0.001) 0.71 (,0.001) 0.74 (0.005) 0.92 (<0.001)
19F 0.95 (<0.001) 0.91(<0.001) 0.68 (0.01) 0.91 (<0.001) 0.87 (<0.001)
23F 0.83 (<0.001) 0.78 (0.003) 0.68 (,0.001) 0.88 (<0.001) 0.88 (<0.001)
1Different strains used
Correlations between reference OPA and flow OPA
Technical Considerations
Targets 1. Bacteria
-Variable label
uptake
Variability of Labeled Bacteria
Negative Cells
Positive Cells
Technical Considerations
Targets 1. Bacteria
-Variable label 2. Beads
-Standardized label
uptake
Beads Provided a Standard Fluorescent Target
CDC, Sero 4-Y Beads FA, Sero 4-Y Beads
Technical Considerations
Effector Cells 1. Donor PMNs 2. HL60 PMNs
Log2 HL60 PMN Titers
0 2 4 6 8 10 12
Log
2 D
ono
r P
MN
Tite
rs
0
2
4
6
8
10
12
Serotype 4, r = 0.96, p < 0.001Serotype 6B, r = 0.92, p < 0.001Serotype 9V, r = 0.94, p < 0.001Serotype 14, r = 0.96, p < 0.001Serotype 18C, r = 0.86, p < 0.001Serotype 19F, r = 0.92, p < 0.001Serotype 23F, r = 0.89, p < 0.001
Comparison of PMN and HL60 PMN Derived OPA Titers
Technical Considerations Cont.
Effector Cells 1. Donor PMNs 2. HL60 PMNs
a. Culture maintenance
Effects of Different Culture Conditions
HL60 Stock Published Induction Protocol
Modified InductionProtocol
38% S and G2/M 16% S and G2/M 3% S and G2/M
18%3% 57%
Technical Considerations
Effector Cells 1. Donor PMNs 2. HL60 PMNs
a. Culture maintenance
b. Induction protocol
Effects of Culture Conditions on HL60 Differentiation and Function
Normal Culture Method
Roller Culture Method
Data Collection
• Gating– Minimize overlap of “dead” cells within
gate– Ensure gate contains a cells with
ingested targets
M1
Gating Effects
Data Collection
• Gating– Minimize overlap of “dead” cells within
gate– Ensure gate contains a cells with
ingested targets
• Compensation– Critical in multiplexed assays
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
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 run2Four hundred eighty files3Does not require constant attention
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
Assay Comparisons
*Overnight incubation required
Advantages and Disadvantages1Multiplexed killing assay2Instrument/technician costs
Reference1 Flow Multiplex
Specificity ++++ ++++ ++++
Technically difficult
++ ++ ++++
Infectious Target ++++ - -
Multiplexed ++++ ++ ++++
Commercial NA ++ ++++
Automation ++ ++++ ++++
Cost ++ ++2 +2
Flow Cytometric OPA Technology
• Can be applied to other bacteria cleared through an OP mechanism
• S. aureus (Vernachio)
Beads Only Guinea Pig C' T1-2 (1ug/ml) Human IgG (1ug/ml)0
20406080
100120140160180200
Ph
ag
oc
yti
c P
rod
uc
t
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.
Summary
• Non-infectious targets• Correlate well with reference OP
method• High throughput• Automation can further increase
relative throughputs• Meet GLP guidelines