ANALYSIS OF
IMMUNOFLUORESCENCE
AND MULTIPARAMETER DATA
Carleton C. Stewart, PhD
and Sigrid J. Stewart
RP
L
OSWE L
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:stewart@sc3101.med.buffalo.edu
CELLULAR ANTIGENS
Sensory
Metabolic
Adhesion
ONE COLOR IMMUNOPHENOTYPING
Antibodies Labeled with Fluorescein
SINGLE COLOR IMMUNOFLUORESCENCE
1. Ig Block MAB
•
2. Ig Block B-MAB
•
3. Ig Block FL-MAB
•
FL-second antibody F(ab')
2
FL-Avidin
• = wash
•
•
CORRELATED (LIST MODE)
DATA ACQUISITION
Entry No. Value
1
2
3
4
5
6
7
8
k
80
100
40
20
90
120
100
110
50
75
110
120
Cell Number Parameter
1
1
1
1
2
2
2
2
n
n
n
n
FSC
SSC
Green
Red
FSC
SSC
Green
Red
FSC
SSC
Green
Red
A
REGION A
B
C
forward scatter
CD4 fluorescence
REGION B
CD4 fluorescence
REGION C
WAYS ANTIBODIES
BIND TO CELLS
Specific:
Fab to epitope
Fc 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 divided
by the protein
concentration.
SA = {F(ab')2}
(protein)
Reasons antibodies do not
bind to cells:
1.
2.
3.
4.
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 in ascitic fluid
immediately
DETERMINING CORRECT
ANTIBODY TITER
I.C.
number
of
cells
noise
signal
fluorescence units
too much antibody
I.C.
noise
number
of
cells
signal
fluorescence units
correct antibody
ISOTYPE CONTROL
SIGNAL = NIL
NOISE =62
S/N=NONE
0
256
PE CD21 ->
041895029
512
768
1024
3 µg REAGENT
SIGNAL = 454
NOISE =183
S/N=2.48
0
256
PE CD21 ->
041895025
512
768
1024
1 µg REAGENT
SIGNAL = 404
NOISE =214
S/N=1.88
0
256
512
768
1024
PE CD21 ->
041895026
0.3 µg REAGENT
SIGNAL = 459
NOISE =209
S/N=2.20
0
256
PE CD21 ->
041895027
512
768
1024
0.1 µg REAGENT
SIGNAL = 454
NOISE =149
S/N=5.24
0
256
PE CD21 ->
041895028
512
768
1024
BLOCKING IS IMPORTANT
INDIRECT IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Fc
Primary Antibody:
murine monoclonal
antibody
Fab
Second Antibody:
fluoresceinated
goat anti-mouse
IgG F(ab') 2
FcR
A
epitope
B
C
D
ISOTYPE CONTROL- myeloma protein
E
F
G
F
AUTOFLUORESCENCE CONTROL
BLOCKING WITH GOAT IgG
goat IgG
add Mab
Fab
add fluoresceinated goat anti-mouse IgG F(ab')2
VERIFICATION OF BLOCK
• FcR and non-specific binding
FL-MAB + PE-mIgG
gIgG + FL-MAB + PE-mIgG
EFFECT OF BLOCKING ON MAB BINDING
TO MONONUCLEAR CELLS
L
O
G
F
L
U
O
R
E
S
C
E
N
C
E
CELL VOLUME
R
E
L
A
T
I
V
E
N
U
M
B
E
R
UNBLOCKED
1µg
3 µg
O
F
C
E
L
L
S
9 µg
0
64
128
192
CHANNEL NUMBER
256
SECOND REAGENT QUALITY
log fluorescence
F(ab’)2 of anti IgG
anti
IgG
cell volume
VARIATION IN GAMMA 1 MYELOMA
PROTEIN BINDING TO
MACROPHAGES
60
PERCENT POSITIVE
50
40
30
20
10
0
0
17
39
4
23
13
21
DEAD CELLS CAN BE A
PROBLEM
• They bind antibodies nonspecifically
• They masquerade as specific subsets
• They cause data misinterpretation
ANTIBODIES BIND NON-SPECIFICALLY
TO DEAD CELLS
VIABLE CELLS
ALL CELLS
4
4
PE-LAMBDA
10
10
10
A
dead cells
3
10
3
2
2
10
10
1
1
10
10
10 0 0
10
B
1
10
10
2
10
3
4
10
0
10 0
10
1
10
FL-KAPPA
10
2
10
3
4
10
EMA PROCEDURE
1
lysed, washed
cells
+ 5 µg EMA
18 cm.
2
10 min.
WASH, FIX, AND ANALYSE
3
EVALUATING VIABILITY
EMA
WITH ETHIDIUM MONOAZIDE
forward scatter
TWO COLOR IMMUNOPHENOTYPING
Antibodies labeled with fluorescein
Antibodies labeled with phycoerythrin
COMPENSATION
FITC
+
gain=1
FITC-PE
-
PE
+
gain=1
-
PE-FITC
uncompensated
fluorescence 2
4
4
10
10
10
3
10
2
10
1
1
10
0
3
2
10
10 0
10
compensated
10
1
10
10
2
10
3
4
10
0
10 0
10
1
10
fluorescence 1
10
2
10
3
4
10
COMPENSATION IS
INTENSITY DEPENDENT
partially
compensated
uncompensated
fluorescence 2
4
4
10
10
10
3
10
2
2
10
10
1
1
10
0
10 0
10
3
10
1
10
10
2
10
3
0
10 0
10
4
10
1
10
10
fully
compensated
4
10
10
3
2
10
1
10
0
10 0
10
1
10
10
2
10
3
4
10
fluorescence 1
2
10
3
4
10
TWO COLOR IMMUNOFLUORESCENCE
• 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 •
1. Ig Block + MAB
COMBINED INDIRECT AND DIRECT
IMMUNOFLUORESCENCE STAINING
NO BLOCKING
Primary Antibody: mMAB
Second Antibody: FGAM
PE-mMAB
4
4
10
10
3
21%
10
NO BLOCK
10
BLOCK
3
12%
2
2
10
10
0
10 0
10
49%
6%
1
10
1
10
10
2
10
PE-CD4
3
42%
1
10
4
10
0
10 0
10
1
10
10
2
10
PE-CD4
3
4
10
VERIFICATION OF BLOCK
Second Reagent Block
• FL-GAM • PE-mIg
• FL-GAM • mIg + PE-mIg
gIg + MAB
gIg + MAB
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
number of
cells
SINGLE COLOR HISTOGRAMS
0
10
1
10
2
10
CD3
3
10
4 0
1010
1
10
2
10
CD4
3
10
4 0
1010
1
10
2
10
CD8
3
10
4
10
TWO COLOR PATTERN
4
10
A
3
10
CD
4
10
4
10
2
10
1
1
10
0
3
2
10
10 0
10
B
10
1
10
10
2
CD3
10
3
4
10
0
10 0
10
1
10
10
2
CD3
10
3
4
10
THREE COLOR PATTERN
ALL CELLS
ALL CELLS
4
10
A
SSC
10
B
3
2
10
1
10
0
10 0
10
FSC
ALL CELLS
10
4
C
CD
8
10
2
10
3
4
10
CD3+ CELLS
D
3
2
10
10
1
1
10
0
2
10
3
10 0
10
10
CD3
4
10
1
10
10
1
10
10
2
CD4
10
3
4
10
0
10 0
10
1
10
10
2
CD4
10
3
4
10
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
Low
low-intermediate
high
FLUOROCHROME
phycoerythrin
tandem
fluorescein
COMPENSATE INSTRUMENT
USING STAINED CELLS
1. Adjust PMT voltages using
unstained cells
2. Adjust compensation
for each fluorochrome
THREE COLOR COMPENSATION
4
10
half
each
side
3
10
PE-CD4
PE-CD4
10
4
10
2
10
1
0
3
2
10
10
10 0
10
half
each
side
1
10
1
10
10
2
10
3
FL-CD45
4
10
0
10 0
10
1
10
10
2
10
3
TC-CD8
4
10
CELLULAR COMPENSATION STANDARD
CURRENT
4
4
10
10
10
3
10
2
2
10
10
1
1
PE-CD4
10
0
10 0
10
3
10
1
10
10
2
10
3
4
10
0
10 0
10
1
10
10
2
10
3
4
10
PREVIOUS
4
4
10
10
10
3
10
2
2
10
10
1
1
10
0
10 0
10
3
10
1
10
10
2
10
3
FL-CD45
4
10
0
10 0
10
1
10
10
2
10
3
TC-CD8
4
10
THREE COLOR SOP
50 µl
washed, and
blocked*
whole blood or
bone marrow
FL-mab
+
PE-mab
+
TC-mab
lyse,
centrifuge,
decant,
blot,
and
resuspend
pellet
15 min. on ice
*add 10 µl mIg (10 mg/ml) to 1 ml washed whole blood.
wash,
fix,
and
analyse
THIRD COLOR REAGENT
PROPERTIES TO CONSIDER
• monocyte binding
PE-CY5
• light sensitivity
PE-CY5 and PerCP
• batch variation
PE-TR and PE-CY5
LEUKEMIA GATE USING CD45
NORMAL BONE MARROW
0
256
512
768
1024
102
103
104
SSC ->
/6/05133061
100
101
HLADr ->
/6/05133061
LEUKEMIA GATE USING CD45
LEUKEMIC (TALL) BONE
MARROW
0
256
512
768
1024
SSC ->
/7/06064121
10 0
HLADr ->
/7/06064121
10 1
10 2
10 3
10 4
4
10
C
D
1
9
A2
1
4
10
C
D
1
9
3
10
2
10
CD19 -->
B 19
18
3
10
2
10
CD19 -->
1
1
10
10
3
4
10
1
10
2
10
3
10
20
4
21
10
1
10
CD7 -->
4
10
C
4
6
3
C
D
7
10
3
10
4
CD2 -->
CD7
5
2
10
C
D
1
9
10
2
10
CD7 -->
CD2
1
D2
3
10
2
10
CD19 -->
R1
1
1
10
10
7
8
10
1
10
2
CD2 -->
CD2
10
3
10
4
3
4
10
1
10
2
10
3
CD7
-->
CD7
Interpreting Co-expression: The pattern of expression of two or three markers
provides the means for assignment of co-expression. The possibilities are illustrated
in this Figure. The antibody combination CD7CD19CD2 was used and in A, B and C the
same specimen is shown. CD19 is homogeneously expressed on the cells, as shown in
A, but CD7 is expressed in a heterogeneous manner, as shown by a continuum of CD7
expression 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 distinct
subset that expresses both markers, or an artifact. Because of this uncertainty, when
the frequency of cells co-expressing two or more markers is small, and because the
level of confidence increases with increased frequency, we have set a threshold of
10% 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 not
know its significance. In C, a distinct subset of CD7+CD2+ cells (blue) is shown
which do not express CD19 (A and B): these are normal T-cells. Using a specimen
from another patient, in D, non-specific binding is often characterized by events on a
45 degree angle (red) as the antibodies bind equally to all cells. Populations
exhibiting this pattern are not included in the interpretation even though they may be
greater than 10% .
10
4
4
10
1
2
A
10
3
C
D
1
9
4
B
5
6
C
D
2
0
10
2
10
FL2-Heig ht -->R1
10
2
10
FL1-Heig ht -->
1
4
10
1
10
2
10
3
1
10
2
10
3
D
4
10
3
C
D
2
2
2
10
4
10
1
10
10
Fluoresce nce Two Height -->
3
E
5
6
2
10
10
3
Fluoresce nce
One Height -->
CD5
10
2
8
10
3
Fluoresce nce Three He ight -->
CD20
4
10
F
1
R1
Fluoresce nce Two Height -->
3
4
2
10
10
10
10
2
10
1
3
10
1
10
10
3
1
7
4
4
10
L
A
M
B
D
A
10
Fluoresce nce One Height -->
4
2
KAPPA
R5
R1
2
4
FL1-Heig ht -->
3
10
10
3
10
CD22
2
1
10
FL3-Heig ht -->
1
10
2
8
10
CD5
1
10
1
7
FL1-Heig ht -->
C
D
1
9
3
10
4
10
C2
1
FL2-Heig ht -->R1
10
3
10
R5
1
10
4
10
L
A
M
B
D
A
3
4
10
1
10
2
4
10
3
10
Fluoresce nce One Height -->
KAPPA
Typical B-cell Lymphoma (A,B,C) vs Reactive Lymphoid Hyperplasia (D,E,F):
The
most 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 is
variable, typically like that shown in A. In C, Kappa vs Lambda coexpression with
CD19 is shown; this lymphoma is Kappa light chain restricted. The corresponding
displays for B-cells from a node with reactive lymphoid hyperplasia are shown in
D, E and F.
4
4
10
1
4
A2
3
C
D
4
4
B
10
10
3
C
D
4
10
2
10
L AMBDA -->
2
10
2
10
KA PPA -->
1
1
10
10
10
4
10
1
10
2
CD3
10
3
R5
7
10
6
10
1
3
C
3
C
D
7
10
L AMBDA -->
R1
5
4
10
1
10
2
CD8
KA PPA -->
10
3
10
4
8
10
1
10
2
CD2
CD 19 -->
10
3
10
4
CD 19 -->
Lymphoma
4
10
1
D
2
3
C
D
4
4
4
10
10
E
3
10
C
D
4
2
10
L AMBDA -->
C
D
7KA PPA -->
10
10
2
2
10
10
1
1
1
10
10
10
3
4
10
1
10
2
CD3
KA PPA -->
10
3
10
F
R5
7
4
6
3
L AMBDA -->
R1
5
10
1
10
2
CD8
10
3
10
4
CD 19 -->
8
10
1
10
2
CD2
10
3
10
4
CD 19 -->
Reactive Lymph Node
Typical T-cell Lymphoma or Reactive Lymph Node: The hallmark of
T-cell malignancy is inappropriate marker expression. This can
present itself as either an abnormal epitope density or as the absence
of a marker. In this example, CD4 expression in a lymphoma (green in
A and B) is not different from that found on normal T-cells (green in D
and E), but CD3 expression on the lymphoma cells (A) is more variable
than on normal T-cells (D). The differences are the absence of CD7 and
the increased density of CD2 (rust) expression in the lymphoma cells
(C). Note that normal T-cells (blue in C or F) are easily distinguished
from the lymphoma cells (rust in C). The CD7-CD2+ cells (rust) in F
are normal NK cells.