ANALYSIS OF IMMUNOFLUORESCENCE

ANALYSIS OF IMMUNOFLUORESCENCE

AND MULTIPARAMETER DATACarleton C. Stewart, PhD

DEPARTMENT OF HEALTH, STATE OF NEW YORKELM AND CARLTON STREETS

BUFFALO, NY 14263phone 716-845-8471FAX 716-845-8806

email:stewart@sc3101.med.buffalo.edu

R ARK LLOSWEP

L Cancer Instituteaboratory

Flowof

Cytometry

and Sigrid J. Stewart

CELLULAR ANTIGENS

Sensory AdhesionMetabolic

ONE COLOR IMMUNOPHENOTYPINGAntibodies Labeled with Fluorescein

SINGLE COLOR IMMUNOFLUORESCENCE

1. Ig Block MAB •FL-second antibody F(ab')2 •

2. Ig Block B-MAB • FL-Avidin •3. Ig Block FL-MAB •

• = wash

CORRELATED (LIST MODE) DATA ACQUISITION

Entry No. Value Cell Number Parameter12345678---k

80100402090

1201001105075

110120

11112222nnnn

FSCSSCGreenRedFSCSSCGreenRedFSCSSCGreenRed

forward scatter CD4 fluorescence

CD4 fluorescence

A

BC

REGION A

REGION CREGION B

NUM

BER

OF C

ELLS

WAYS ANTIBODIES BIND TO CELLS

Specific:Fab to epitopeFc to Fc receptor

binding is high affinity and saturable

Non Specific:binding is low affinity and not saturable

Specific Activity is the concentration of

bindable antibody to its epitope dividedby the protein concentration.

SA = {F(ab')2} (protein)

Reasons antibodies do notbind to cells:1. overconjugation 2. not purified

3. degradation of binding site4. aggregation

Storing of antibodies:

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

Use sodium azideUse highly purified albumin or

gelatin as carrierPurify antibodies in ascitic fluid

immediately

numberof

cells

numberof

cells

DETERMINING CORRECT ANTIBODY TITER

fluorescence unitstoo much antibody

noiseI.C. signal

fluorescence unitscorrect antibody

noiseI.C.

signal

041895029PE CD21 ->0 256 512 768 1024

041895025PE CD21 ->0 256 512 768 1024

041895026PE CD21 ->0 256 512 768 1024

041895027PE CD21 ->0 256 512 768 1024

041895028PE CD21 ->0 256 512 768 1024

3 µg REAGENTSIGNAL = 454NOISE =183S/N=2.48

1 µg REAGENTSIGNAL = 404NOISE =214S/N=1.88

0.3 µg REAGENTSIGNAL = 459NOISE =209S/N=2.20

0.1 µg REAGENTSIGNAL = 454NOISE =149S/N=5.24

ISOTYPE CONTROLSIGNAL = NILNOISE =62S/N=NONE

BLOCKING IS IMPORTANT

INDIRECT IMMUNOFLUORESCENCE STAININGNO BLOCKING

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

IgG F(ab')2Fab

epitope

FcR

ISOTYPE CONTROL- myeloma protein

AUTOFLUORESCENCE CONTROL

A B C D

E F G F

Fc

BLOCKING WITH GOAT IgG

goat IgG

add Mab

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

Fab

VERIFICATION OF BLOCK• FcR and non-specific binding

FL-MAB + PE-mIgGgIgG + FL-MAB + PE-mIgG

EFFECT OF BLOCKING ON MAB BINDINGTO MONONUCLEAR CELLS

CELL VOLUME

LOG FLUORESCENCE

RELATIVE

NUMBER

OF

CELLS

CHANNEL NUMBER0 64 128 192 256

UNBLOCKED

1µg

3 µg

9 µg

SECOND REAGENT QUALITY

F(ab’)2 of anti IgG

anti IgG

cell volume

log

fluor

esce

nce

0

10

20

30

40

50

60

0 17 39 4 23 13 21

VARIATION IN GAMMA 1 MYELOMA PROTEIN BINDING TO

MACROPHAGES

PERC

ENT

POSI

TIVE

DEAD CELLS CAN BE A PROBLEM

• They bind antibodies nonspecifically

• They masquerade as specific subsets

• They cause data misinterpretation

ANTIBODIES BIND NON-SPECIFICALLYTO DEAD CELLS

PE-L

AMBD

A

FL-KAPPA

A BALL CELLS VIABLE CELLS

10

103

104

101

102

0

103

104

101

102

01010

310

410

110

2100

103

104

101

102

100

dead cells

lysed, washedcells

+ 5 µg EMA10 min.

18 cm.

EMA PROCEDURE

WASH, FIX, AND ANALYSE

1

3

2

EVALUATING VIABILITYWITH ETHIDIUM MONOAZIDE

EMA

forward scatter

TWO COLOR IMMUNOPHENOTYPING

Antibodies labeled with fluorescein

Antibodies labeled with phycoerythrin

COMPENSATION

FITC

FITC-PE

PE-FITC

-

-

+

+gain=1

gain=1

PE

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

compensateduncompensated

fluor

esce

nce

2

fluorescence 1

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

partiallycompensateduncompensate

d

fluor

esce

nce

2

fluorescence 1

103

104

101

102

01010

310

410

110

210

0

fullycompensated

COMPENSATION IS INTENSITY DEPENDENT

TWO COLOR IMMUNOFLUORESCENCE

1. Ig Block + MAB • FL-second antibody F(ab’) •Ig Block + PE-MAB •

2. Ig Block + B-MAB + FL -MAB • PE-Avidin •3. Ig Block + FL-MAB + PE-MAB •

COMBINED INDIRECT AND DIRECTIMMUNOFLUORESCENCE STAINING

NO BLOCKING

Primary Antibody: mMAB Second Antibody: FGAM

PE-mMAB

103

104

101

102

010

PE-CD4

CD8

+ F

GAM

NO BLOCK

103

104

101

102

100

49%6%

21%103

104

101

102

010

PE-CD4

CD8

+ F

GAM

BLOCK

103

104

101

102

100

42%

12%

VERIFICATION OF BLOCK

Second Reagent Block

gIg + MAB • FL-GAM • PE-mIg

gIg + MAB • FL-GAM • mIg + PE-mIg

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 ofperidinin and chlorophyll a protein

103 104101 102100 103 104101 102100 103 104101 102100

num

ber

of

cells

CD3 CD4 CD8

SINGLE COLOR HISTOGRAMS

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

TWO COLOR PATTERN

CD3

CD3

CD 8CD 4

A B

103

104

101

102

01010

310

410

110

210

0

THREE COLOR PATTERN

FSC CD3

CD 4SSC

A B

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

CD4

CD4

CD 8CD 8

C D

ALL CELLS ALL CELLS

ALL CELLS CD3+ CELLS

POPULATIONS RESOLVED BY THREE ANTIBODIES

up to 8 populations can be resolved for each additional panel

FL-Ab PE-Ab TC-Ab+ + ++ + -+ - ++ - -

FL-Ab PE-Ab TC-Ab- + +- + -- - +- - -

THREE COLOR IMMUNOFLUORESCENCE

1. Ig Block + MAB •

B- second antibody F(ab')2 • Ig Block + FL- MAB + PE-MAB + TC- Avidin • 2. Ig Block + FL-MAB + PE-MAB + B-MAB •

TC-Avidin •

3. Ig Block + FL-MAB + PE-MAB + TC-MAB •

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

STRATEGY FOR SELECTING FLUOROCHROME:

EPITOPE DENSITY FLUOROCHROMELow phycoerythrinlow-intermediate tandemhigh fluorescein

COMPENSATE INSTRUMENT USING STAINED CELLS

1. Adjust PMT voltages using unstained cells

2. Adjust compensation for each

fluorochrome

THREE COLOR COMPENSATION

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

PE-C

D4

PE-C

D4

TC-CD8FL-CD45

halfeachside

halfeachside

CELLULAR COMPENSATION STANDARD

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

PE-C

D4

TC-CD8FL-CD45

103

104

101

102

01010

310

410

110

210

0

103

104

101

102

01010

310

410

110

210

0

CURRENT

PREVIOUS

FL-mab+

PE-mab+

TC-mab

50 µl washed, and

blocked*whole blood orbone marrow

15 min. on ice

lyse,centrifuge,

decant,blot,and

resuspendpellet wash,

fix,and

analyse

THREE COLOR SOP

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

THIRD COLOR REAGENTPROPERTIES TO CONSIDER

• monocyte bindingPE-CY5

• light sensitivityPE-CY5 and PerCP

• batch variationPE-TR and PE-CY5

0 256 512 768 1024

/6/05133061SSC ->

LEUKEMIA GATE USING CD45NORMAL BONE MARROW

10 10 10 10 100 1 2 3 4

/6/05133061HLADr ->

0 256 512 768 1024

/7/06064121SSC ->

10 10 10 10 100 1 2 3 4

/7/06064121HLADr ->

LEUKEMIA GATE USING CD45LEUKEMIC (TALL) BONE

MARROW

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

CD7Interpreting Co-expression: The pattern of expression of two or three markersprovides the means for assignment of co-expression. The possibilities are illustratedin this Figure. The antibody combination CD7CD19CD2 was used and in A, B and C thesame specimen is shown. CD19 is homogeneously expressed on the cells, as shown inA, but CD7 is expressed in a heterogeneous manner, as shown by a continuum of CD7expression from negative (cyan) to positive (green). Thus, a proportion of the CD19+cells express CD7. In B, a small population of CD19+CD2+ cells (violet) is shown.This could be heterogeneous expression of CD2 on the CD19 population, a distinctsubset that expresses both markers, or an artifact. Because of this uncertainty, whenthe frequency of cells co-expressing two or more markers is small, and because thelevel of confidence increases with increased frequency, we have set a threshold of10% above which we designate co-expression as real and below which it is uncertain.In this case, the frequency was below 10% of the CD19+ population and we do notknow its significance. In C, a distinct subset of CD7+CD2+ cells (blue) is shownwhich do not express CD19 (A and B): these are normal T-cells. Using a specimenfrom another patient, in D, non-specific binding is often characterized by events on a45 degree angle (red) as the antibodies bind equally to all cells. Populationsexhibiting this pattern are not included in the interpretation even though they may begreater than 10%.

10 1 10 2 10 3 10 4

FL1-Height -->

10

1

10

2

10

3

10

4

FL2-Height --> R1

1 2

3 410 1 10 2 10 3 10 4

FL3-Height -->

10

1

10

2

10

3

10

4

FL1-Height --> R5

5 6

7 810 1 10 2 10 3 10 4

FL1-Height -->

10

1

10

2

10

3

10

4

FL2-Height --> R1

1 2

3 4

10 1 10 2 10 3 10 4

Fluorescence One Height -->

10

1

10

2

10

3

10

4

Fluorescence Two Height -->

R1

1 2

3 410 1 10 2 10 3 10 4

Fluorescence Three Height -->

10

1

10

2

10

3

10

4

Fluorescence One Height -->

R5

5 6

7 810 1 10 2 10 3 10 4

Fluorescence One Height -->

10

1

10

2

10

3

10

4

Fluorescence Two Height -->

R1

1 2

3 4

A

D E

B

F

C

CD19

CD19

CD20

CD22

LAMBDA

LAMBDA

CD22

CD20CD5

KAPPA

KAPPA

CD5

Typical B-cell Lymphoma (A,B,C) vs Reactive Lymphoid Hyperplasia (D,E,F): Themost commonly found phenotype in B-cell lymphoma is the expression of CD19(A), CD20 (B), CD22 (B) and one of the two light chains (C). CD5 expression isvariable, typically like that shown in A. In C, Kappa vs Lambda coexpression withCD19 is shown; this lymphoma is Kappa light chain restricted. The correspondingdisplays for B-cells from a node with reactive lymphoid hyperplasia are shown inD, E and F.

10 1 10 2 10 3 10 4

KAPPA -->

10

1

10

2

10

3

10

4

LAMBDA --> R1

1 2

3 410 1 10 2 10 3 10 4

CD19 -->

10

1

10

2

10

3

10

4

LAMBDA -->

10 1 10 2 10 3 10 4

CD19 -->

10

1

10

2

10

3

10

4

KAPPA -->

R5

5 6

7 8

10 1 10 2 10 3 10 4

KAPPA -->

10

1

10

2

10

3

10

4

LAMBDA --> R1

1 2

3 410 1 10 2 10 3 10 4

CD19 -->

10

1

10

2

10

3

10

4

LAMBDA -->

10 1 10 2 10 3 10 4

CD19 -->

10

1

10

2

10

3

10

4

KAPPA -->

R5

5 6

7 8

CD2

CD2CD8

CD8CD3

CD3

CD7

CD7

CD4

CD4

CD4

CD4

Typical T-cell Lymphoma or Reactive Lymph Node: The hallmark ofT-cell malignancy is inappropriate marker expression. This canpresent itself as either an abnormal epitope density or as the absenceof a marker. In this example, CD4 expression in a lymphoma (green inA and B) is not different from that found on normal T-cells (green in Dand E), but CD3 expression on the lymphoma cells (A) is more variablethan on normal T-cells (D). The differences are the absence of CD7 andthe increased density of CD2 (rust) expression in the lymphoma cells(C). Note that normal T-cells (blue in C or F) are easily distinguishedfrom the lymphoma cells (rust in C). The CD7-CD2+ cells (rust) in Fare normal NK cells.

A

FED

CB

Lymphoma

Reactive Lymph Node