MULTIPARAMETER IMMUNOFLUORESCENCE

Carleton C. Stewart, Ph.D. & Sigrid J. Stewart

R LLOSWEARKP

L Cancer Instituteaboratory

Flowof

CytometryDepartment of Health, State of New York

Elm and Carlton Streets

Buffalo, New York 14263

phone: 716-648-5480

fax: 716-648-8806

email: stewart@sc3101.med.buffalo.edu

ANTIBODY BINDING TO CELLS

RPCI

LFC

Kf

Kr

Ab + Ep AbEp

Kf range is usually ~ 106

Kr range is usually ~ 10-3

Ka = Kf/Kr = 106/10-3 = 109

Kf =AbEpAb

EpKr = AbEp

Ab

Ep

THE LAW OF MASS ACTION

Specific:Fab to epitopeFc to Fc receptorbinding is high affinity and saturable

Non Specific:binding is low affinity and not saturable

WAYS ANTIBODIES BIND TO CELLS

Specific Activity is the concentration

of bindable antibody to its epitope

divided by the protein concentration.

2

[F(ab') ] SA =

(protein)

Reasons Antibodies do not bind to cells:

•overconjugation•not purified•degradation of binding site•aggregation

STORING OF ANTIBODIES :

Proteases destroy antibodies in:• ascitic fluid• serum• bacteria

Use sodium azide

Use highly purified albumin or gelatin as carrier

Purify antibodies immediately

TITERING ANTIBODIESRPCI

LFC

SPECIFIC ANTIBODY

NON-SPECIFIC ANTIBODY

CONCENTRATION

AM

OU

NT B

OU

ND

3

3 µgs/n = 2.5

1 µgs/n = 2.1

0.3 µgs/n = 2.4

0.1 µgs/n = 4.1

0.03 µgs/n = 4.8

0.01 µgs/n = 4.6

0.003 µgs/n = 3.5

0.001 µgs/n = 3.2

auto

8765432102

3

4

5

Dilution

Sig

nal to

Nois

eTITER

101 102 103 104101 102 103 104101 102 103 104

1 µg S/NAb 278IC 5.8

isotypecontrol

antibody

cytokeratin

.3 µg S/NAb 100IC 3.6

101 102 103 104

.01 µg S/NAb 25.7IC 2.6

nu

mb

er

65432100

10

20

30

40

50

60

Dilution

Sig

nal to

Nois

e

TITER

10 410 4 10 1 10 2 10 3 10 410 1 10 2 10 3 10 4

PE-CD4 TC-CD810 1 10 2 10 310 1 10 2 10 3

FITC-CD3

nu

mb

er

10 1 10 2 10 3 10 410 1 10 2 10 3 10 4

VERIFICATION OF ANTIBODY COMBINATIONS

101 102 103 104

10

1

10

2

10

3

10

4

R1

PE

-CD

4

FITC-CD3

x = 69y = 94

PE

- C

D4

101 102 103 104

10

1

10

2

10

3

10

4

R2

TC-CD8

x = 98

NEW BATCH

R1

101 102 103 104

10

1

10

2

10

3

10

4

PE

-CD

4

FITC-CD3

x = 62 faily = 96 pass R2

PE

- C

D4

TC-CD81 2 3 4

10

1

10

2

10

3

10

4

10 10 10 10

x = 97 pass

CONTROL BATCHCONTROL BATCH

BLOCKING IS IMPORTANT

INDIRECT IMMUNOFLUORESCENCE STAINING

NO BLOCKING

Primary Antibody: Second Antibody:murine monoclonal fluoresceinatedantibody goat anti-mouse

IgG F(ab')2Fab

epitope

FcR

A B C D

Fc

ISOTYPE CONTROL- myeloma protein

AUTOFLUORESCENCE CONTROL

E F G H

BLOCKING WITH GOAT IgG

goat IgG

add MabFab

add fluoresceinated goat anti-mouse IgG F(ab')2

VERIFICATION OF BLOCK

1. FcR and non-specific binding

FL-MAB + PE-mIgG

2. gIgG + FL-MAB + PE-mIgG

EFFECT OF BLOCKING ON ANTIBODY BINDING TO MONONUCLEAR CELLS

LO

G F

LU

OR

ES

CEN

CE

CELL VOLUME

nu

mb

er

TOTALA

B

C

D

channel number

variation in gamma 1 myeloma protein binding to macrophages

0

510

1520

25

3035

4045

50

0 17 39 4 23 13 21

mopc myeloma protein

Second Reagent Qualitylo

g fl

uore

scen

ce

cell volume

F(ab’)2 of anti IgG anti IgG

DEAD CELLS CAN BE A PROBLEM

• They bind antibodies non-specifically• They masquerade as specific subsets• They cause data misinterpretation

ANTIBODIES BIND NON-SPECIFICALLY TO DEAD CELLS

FL-KAPPA

PE-L

AM

BD

A

dead cells

ALL CELLS VIABLE CELLS

A B

lysed, washedcells

+ 5 µg EMA

10 min.

18 cm.

EMA PROCEDURE

WASH, FIX, AND ANALYZE

1

3

2

Evaluating Viability with Ethidium Monoazide

FSC

SS

C

10 20 30 40 50 60 70 80 90 100 120

Forward Scatter Height -->

10

20

30

40

50

60

70

80

90

100

120

Sid

e S

catte

r Heig

ht -->

R9

FSC

SS

C

10 20 30 40 50 60 70 80 90 100 120

Forward Scatter Height -->

10

110

210

310

4

Fluore

scence

Thre

e H

eig

ht -->

R2

FSC

FL3-E

MA

10 20 30 40 50 60 70 80 90 100 120

Forward Scatter Height -->

10

20

30

40

50

60

70

80

90

100

120

Sid

e S

catte

r Heig

ht -->

R9R9

% dead = 5%

R2

% dead in gate = 1%

R9

ONE COLOR IMMUNOPHENOTYPING

RPCI

LFC

1. IgG BlockMAB wash FL-second antibody F(ab')2 wash

2. IgG BlockB-MAB wash FL-Avidin wash

3. IgG BlockFL-MAB wash

SINGLE COLOR IMMUNOPHENOTYPING

CORRELATED (LIST MODE) DATA ACQUISITION

Entry No. Value Cell Number Parameter1 80 1 FSC2 100 1 SSC3 40 1 Green4 20 1 Red5 90 2 FSC6 120 2 SSC7 100 2 Green8 110 2 Red- 50 n FSC- 75 n SSC- 110 n Greenk 120 n Red

Analysis of List Mode Data

CD4 FLUORESCENCE

nu

mb

er

of

cells

forward scatter CD4 fluorescence

A

B

C

region A

region B region C

STRATEGIES IN MULTICOLOR FLOW CYTOMETRY

RPCI

LFC

CELLULAR ANTIGENS

AdhesionReceptors

Metabolic

cytokines

structureenzymes

courtesy of Jim Bender

FLOW CYTOMETRY VS MICROSCOPIC IMAGING

so rt ing c e llsm o le c ular quant it at io nant ige n c o e xpre ss io nrare c e lls de t e c t io n

c e llu lar int e rac t io nspac ial re lat io nshipsc o m part m e nt alizat io ns ignal t o no ise re je c t io n

TWO COLOR IMMUNOPHENOTYPING

Antibodies labeled with fluorescein

Antibodies labeled with phycoerythrin

RPCI

LFC

TWO COLOR IMMUNOPHENOTYPING

1. IgG Block + MAB wash FL- second antibody F(ab')2 wash

IgG Block + PE-MAB wash

2. IgG Block + B-MAB + FL-MAB wash PE-Avidin wash

3. IgG Block + FL-MAB + PE- MAB wash

COMBINED INDIRECT AND DIRECT

IMMUNOFLUORESCENCE STAINING

NO BLOCKING

Primary Antibody: mMAB Second Antibody: FGAM PE-mMAB

NO BLOCKC

D8 +

FG

AM

PE-CD4

21%

6%

49%

VERIFICATION OF BLOCK

• Second Reagent Block

gIgG + MAB wash FL-GAM wash PE-mIgG

gIgG + MAB wash FL-GAM wash mIgG + PE-mIgG

COMBINED INDIRECT AND DIRECT IMMUNOFLUORESCENCE STAINING

BLOCKING

Primary Antibody: mMAB Second Antibody: FGAM PE-mMAB

BLOCKEDC

D8 +

FG

AM

PE-CD4

42%

12%

THREE COLOR IMMUNOPHENOTYPING

Antibodies labeled with Fluorescein

Antibodies labeled with Phycoerythrin

Antibodies labeled with Tandem Complex to Avidin

Tandem Complexes are Texas Red or CY 5 coupled to Phycoerythrin

Per CP is a natural Tandem Complex of peridinin and chlorophyll a protein

SINGLE COLOR HISTOGRAMS

TWO COLOR PATTERN

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 -

->

FL1-CD3

FL2-C

D4 color

CD3-CD4- black

CD3+CD4- blue

CD3-CD4+ cyan

CD3+CD4+green

THREE COLOR PATTERN

CD3C

D4

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 -

->

CD3

CD

4

CD8C

D8

10 1 10 2 10 3 10 4

CD8 -->

101

102

103

104

CD

4 -

->10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

8 -

->

FOUR COLOR SINGLE LASER IMMUNOPHENOTYPING

Antibodies labeled with fluorescein

Antibodies labeled with phycoerythrin (PE)

Antibodies labeled with PE/Texas Red

Antibodies labeled with PE/CY5 or PerCP

FOUR COLOR DUAL LASER IMMUNOPHENOTYPING

Antibodies labeled with fluorescein

Antibodies labeled with phycoerythrin (PE)

Antibodies labeled with PE/CY5 or PerCP

Antibodies labeled with APC, CY5 or CY7

10 1 10 2 10 3 10 4

CD56 -->

101

102

103

104

CD

4 -

->

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

4 --

>

CD3CD3 CD310 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

56 -

->

10 1 10 2 10 3 10 4

CD3 -->

101

102

103

104

CD

8 -

->

CD5610 1 10 2 10 3 10 4

CD56 -->

101

102

103

104

CD

8 --

>

CD56 CD810 1 10 2 10 3 10 4

CD8 -->

101

102

103

104

CD

4 -

->

FOUR COLOR PATTERN

CD

4

CD

8

CD

56

CD

8

CD

4

CD

4

FL1 FL3 FL3

FL2

FL2

FL1

FL1

FL1

FL2

FL2

FL3

FL3

VISUALIZING EACH POPULATION

BIVARIATE DOT PLOTS CAN BE USED TO DISPLAY THE PATTERNS GENERATED BY MULTIPARAMETER DATA.

FL1

FL4

FL2

FL4

FL3

FL4

FL1

FL3

FL2

FL3

FL1

FL2

4 COLOR

3 COLOR

2 COLOR

UNDERSTANDING BINARY LOGIC IS USEFUL

Note that in binary logic the 1st parameter is sequenced as -/+, two parameters as --/++, 3 parameters as ----/++++, etc.. for as many parameters that are measured. The problem encountered after 3 parameters is...how does one visualize the multiple populations?

P1 P2 P3 P4- - - -+ - - - P1- + - -+ + - - P2- - + -+ - + -- + + -+ + + - P3- - - ++ - - +- + - ++ + - +- - + ++ - + +- + + ++ + + + P4

Ab2

Ab1 Ab3 Ab3

Ab2

Ab1

Ab1 Ab2 Ab3 color Ab1 Ab2 Ab3 colorneeg.(-) neg.(-) neg.(-) grey neg.(-) neg.(-) pos.(+) rustos.(+) neg.(-) neg.(-) yellow pos.(+) neg.(-) pos.(+) blueneg.(-) pos.(+) neg.(-) cyan neg.(-) pos.(+) pos.(+) violetnos.(+) pos.(+) neg.(-) green pos.(+) pos.(+) pos.(+) red

COLOR PATTERNS USED TO VIEW COEXPRESSION

R1 R2 R3 R4 boolean colorAb1 Ab2 Ab3 Ab4

- - - - not(R1 or R2 or R3 or R4 ) grey+ - - - R1 and not (R2 or R3 or R4 ) ye llow- + - - R2 and not (R1 or R3 or R4 ) cyan+ + - - R1 and R2 and not (R3 or R4 ) green- - + - R3 and not (R1 or R2 or R4 ) rust+ - + - R1 and R3 and not (R2 or R4 ) blue- + + - R2 and R3 and not (R1 or R4 ) v io le t+ + + - R1 and R2 and R3 and not R4 red- - - + R4 and not (R1 or R2 or R3 ) grey+ - - + R4 and R1 and not (R2 or R3 ) ye llow- + - + R4 and R2 and not (R1 or R3 ) cyan+ + - + R4 and R1 and R2 and not R3 green- - + + R4 and R3 and not (R1 or R2 ) rust+ - + + R4 and R1 and R3 and not R2 blue- + + + R4 and R2 and R3 and not R1 vio le t+ + + + R4 and R2 and R3 and R1 red

Ab4 is t he gat ing ant ibody

Boolean Logic Table For Four Color Immunophenotyping

A B C

D E F

Ab1 Ab1 Ab1

Ab1 Ab1 Ab1

Ab

2A

b2

Ab

2A

b2

Ab

2A

b2

Types of Antigen Expression

10 1 10 2 10 3 10 4

CD7 -->

10

1

10

2

10

3

10

4

CD19 -->

1 2

3 410 1 10 2 10 3 10 4

CD2 -->

10

1

10

2

10

3

10

4

CD19 -->

18 19

20 21

10 1 10 2 10 3 10 4

CD2 -->

10

1

10

2

10

3

10

4

CD7 -->

5 6

7 810 1 10 2 10 3 10 4

CD7 -->

10

1

10

2

10

3

10

4

CD19 -->R1

1 2

3 4

A B

DC

CD19

CD19

CD19

CD2

CD7

CD7

CD2

CD7

INTERPRETING COEXPRESSION

THREE OR MORE COLOR IMMUNOPHENOTYPING

1. IgG Block + MAB wash Biotin- second antibody F(ab')2

wash IgG Block + FL- MAB + PE-MAB + TC- Avidin wash

2. IgG Block + FL-MAB + PE-MAB + B-MAB wash TC-Avidin wash

3. IgG Block + FL-MAB + PE-MAB + TC-MAB wash

TC (third color) = PE/TR or PE/CY5 tandem or PerCP

STRATEGY FOR SELECTING FLUOROCHROME:

EPITOPE DENSITY FLUOROCHROMELow phycoerythrin, APClow-intermediate tandem, CY5high fluorescein, PerCP

STAINING SOP50 µl

washed, andblocked*

whole blood orbone marrow

15 min. on ice

lyse,centrifuge,

decant,blot,and

resuspendpellet wash,

fix,and

analyse

*add 10 µl mIg (10 mg/ml) to 1 ml washed whole blood.

Add antibody combination

TANDEM COMPLEX PROPERTIES TO CONSIDER

• monocyte binding PE-CY5

• light sensitivity PE-CY5

• batch variation PE-TR and PE-CY5

Non-specific Binding to Monocytes

A B C

PerCP-CD4 PE-CY5-CD4 FSC

SS

C

CD

25

CD

25

TC-CD45 TC-CD3

Effect of Light Exposure on PE-CY5 Tandem Fluorescence

PE-fl

uore

scen

ce

EFFECT OF BATCH VARIATION

PE-fl

uore

scen

ce

FSC

SS

C10 20 30 40 50 60 70 80 90 100 120

FSC-H -->

1020

3040

5060

7080

90100

120

SS

C-H

-->

CD16

CD

32

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

CD

32

CD6410 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

1-H

-->

CD

16

CD64

Fc Receptor Expression on Blood Leukocytes

CD16 CD64

All Cells

CD16 CD64

neutrophils

T-cells

eosinophils basophils

monocytes NK cells

B-cells

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL1-H -->

101

102

103

104

FL

2-H

-->

10 1 10 2 10 3 10 4

FL3-H -->

101

102

103

104

FL

2-H

-->

CD

32

CD

32

CD

32

CD

32