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R. L. L. OSWE. P. ARK. L. aboratory. Cancer Institute. of . Flow. Cytometry. DEPARTMENT OF HEALTH, STATE OF NEW YORK. ELM AND CARLTON STREETS. BUFFALO, NY 14263. phone 716-845-8471. FAX 716-845-8806. email:[email protected]. ANALYSIS OF IMMUNOFLUORESCENCE. - PowerPoint PPT Presentation
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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:[email protected]
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
