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