INTERPRETATION OF FLOW CYTOMETRIC DATA

• Three positive ?

• Two positive ?• Two positive ?

• One positive ?

• No positive ?

SPECTRAL OVERLAPAUTOFLUORESCENCE UNDESIRED Ab BINDING

• antibody specificity• cell type• cell activation/death• physiological conditions• clone/affinity

• binding through fluorochrome

• optical configuration

• excitation source

• spectral compensation

• fluorescence intensity

• optical configuration

• excitation source

• biological conditions• cell type• cell activation/cell death

Background Fluorescence

• binding through fluorochrome• choice of fluorochrome

• binding through Fc region

• labeling conditions• Ab amount• Ab concentration

• fluorescence intensity• antigen expression level• choice of fluorochrome

• cell type• cell activation/cell death

• physiological conditions• labeling conditions• sample prep conditions

Hulspas R, O’Gorman MRG, Wood BL, Gratama, JW, Sutherland DR.

Considerations for the control of background fluorescence in clinical flow cytometry.

Cytometry Part B 2009;76B:355–364

5 x brighter than negative2,500 binding sites

488nm excitation

10% binding sites

20% binding sites

Autofluorescence

• Different excitation wavelengths

result in different levels of

autofluorescence.

CD4-PE

11 x brighter than negative2,500 binding sites

528.7nm excitation

CD4-PE

10% binding sites

20% binding sites

• Naturally occurring cellular

components (Flavins, NADPH).

• Biological and physiological

conditions.

# c

ells

lymphocytes

Autofluorescence Due to Sample Preparation Procedure

• Fixation procedure can cause

increase in autofluorescence

585/42

# c

ells

# c

ells

monocytes

• Increase most pronounced

when measured around 530 nm

• Cell type specificfixedlife

Spectral Overlap

• Narrow bandpass

emission filters

• Spectrally separated

PE

-Cy5 PE

PE-Cy5

PE-Cy7

blank

• Spectrally separated

fluorochromes

• Individual excitation

wavelengths for

‘each’ dyes

PE-Cy7

PE-Cy7

PE

-Cy5

COMPENSATED

Epitope D1 = C1

Undesired Ab Binding

SPECIFIC BINDING

Epitope C1

antigen d

(not of interest)

antigen b

(Fc-receptor)

antigen c

(antigen of interest)

fluorochrome-conjugated

anti-”C”(epitope C1)

Epitope C1

Epitope C3

Undesired Ab BindingNON-SPECIFIC BINDING

antigen a

(not of interest)

Epitope C1

antigen b

(Fc-receptor)

antigen c

(antigen of interest)

fluorochrome-conjugated

anti-”C”(epitope C1)

Antibody titration

6 ng/ml 60 ng/ml 300 ng/ml 600 ng/mlunstained

Side Light Scatter

PE

-CD

3 inte

nsity

• Typical manufacturer’s recommendations:

X µµµµL per 1E6 cells (in 0.5 ml).

• Background increases with increasing number of Ab molecules.

1000

10000

rela

tive f

luo

res

cen

ce in

ten

sit

y

303 30060

antibody amount (ng)

1000

10000

rela

tive f

luo

res

cen

ce in

ten

sit

y

0.55 0.050.1

assay volume (mL)

1

Minimizing non-Specific Binding

rel.

flu

ore

sc

en

ce

in

ten

sit

y

1

10

100

1 10 100 1000

antibody concentration (ng/ml)

rela

tive f

luo

res

cen

ce in

ten

sit

y

1

10

100

1 10 100 1000

antibody concentration (ng/ml)

rela

tive f

luo

res

cen

ce in

ten

sit

y

Antibody concentration (ng/mL)

10 100 1000 10 100 10001 1

rel.

flu

ore

sc

en

ce

in

ten

sit

y

Non-specific binding of

low affinity antibody

Increasing antibody amount in set assay volume (0.5 mL)

10 ng/mL 20 ng/mL 100 ng/mL 200 ng/mL 1 µg/mLno antibody

SSC

SSC

Decreasing assay volume with set antibody amount (10 ng)

Labeling one Billion Cells

• Many more cells can

be labeled with

‘standard’ amount of

antibody

5 µµµµg/ml

10 µµµµg/ml

100

1000

rela

tiv

e P

E i

nte

nsit

y (

me

dia

n)

1 µµµµg/ml1 µµµµg total1 µµµµg/ml

mouse IgG anti-human CD4

rel.

flu

ore

sc

en

ce

in

ten

sit

y

antibody

0.1 µµµµg/ml:T=150’T=30’

1

10

1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09

total cells in test

rela

tiv

e P

E i

nte

nsit

y (

me

dia

n)

1E6 1E7 1E8 1E9

total number of cells in assay

1 µµµµg total1 µµµµg/ml0.1 µµµµg total

1 µµµµg/ml0.01 µµµµg total

rel.

flu

ore

sc

en

ce

in

ten

sit

y

The IgG isotype as ligand

FcγRI (CD64) high

FcγRIIA (CD32) low

FcγRIIB1 (CD32) low

Monocytes

Dendritic cells

MacrophagesFcγRIIB1 (CD32) low

FcγRIIB2 (CD32) low

FcγRIIIA (CD16a) low

FcγRIIIB (CD16b) low

FcRn

Neutrophils

Eosinophils

Mast cells

Platelets

B cells

NK cells

unstained control

non-specific bindingIsotype control

Isotype control

no Fc-receptor binding

Isotype control

(specific) Fc-receptor binding

Isotype control

PE-conjugated mouse IgG1

anti-human CD34 (clone 581)

0 µµµµg/ml

0.25 µµµµg/ml 2.5 µµµµg/ml

manufacturer A

0.20 µµµµg/ml 2.0 µµµµg/ml

SS

C

585/42

manufacturer B

Fc-receptor binding (specific !)

SPECTRAL OVERLAPAUTOFLUORESCENCE UNDESIRED Ab BINDING

• antibody specificity• cell type• cell activation/death• physiological conditions• clone/affinity

• binding through fluorochrome• choice of fluorochrome

• optical configuration• excitation source• spectral compensation• fluorescence intensity

• antigen expression level• choice of fluorochrome

• optical configuration• excitation source• biological conditions

• cell type• cell activation/cell death

• physiological conditions• labeling conditions

Background Fluorescence

• binding through fluorochrome• choice of fluorochrome

• binding through Fc region• labeling conditions

• Ab amount• Ab concentration

• choice of fluorochrome• physiological conditions• labeling conditions• sample prep conditions

- unlabeled controls

- FMO controls

- (single) positive controls

- unlabeled controls

- internal negative controls

- isotype controls

- isoclonic controls

- unlabeled controls

Hulspas R, O’Gorman MRG, Wood BL, Gratama, JW, Sutherland DR. Considerations for the control of background fluorescence in clinical flow cytometry. Cytometry Part B 2009;76B:355–364

Multi-parameter analysis

unstained human PBMCs