Transcript rimini slides - Purdue University

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:[email protected]
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
P rimary Antibody:
murine monoclonal
antibody
Fab
Second Antibody:
fluoresceinated
goat anti-mouse
IgG F(ab') 2
FcR
A
epitope
B
C
D
ISOTYP E 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
10
C
D
1
9
3
10
2
10
CD19 -->
3
10
2
10
1
1
10
10
4
10
4
10
B 19
18
CD19 -->
3
1
10
2
10
3
CD7 -->
CD7
5
10
20
4
21
10
1
10
2
10
3
10
4
CD2 -->
C
4
6
3
C
D
7
4
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
10
4
CD7
-->
CD7
Inte rpr e ting Co-e xpre s s ion: The patte r n of e xpre s s ion of tw o or thre e m ark e r s
pr ovide s the m e ans for as s ignm e nt of co-e xpr e s s ion. The pos s ibilities are illus tr ate d
in this Figur e . The antibody com bination CD7CD19CD2 w as use d and in A, B and C the
s am e s pe cim e n is s how n. C D19 is hom oge ne ous ly e xpr e s s e d on the ce lls , as s how n in
A, but CD7 is e xpr e s s e d in a he ter oge ne ous m anne r , as s how n by a continuum of CD7
e xpre s s ion fr om ne gative (cyan) to pos itive (gr e e n). Thus , a pr oportion of the CD19+
ce lls e xpre s s C D7. In B, a s m all population of CD19+CD2+ ce lls (viole t) is s how n.
This could be he te roge ne ous e xpr e s s ion of CD2 on the C D19 population, a dis tinct
s ubs e t that e xpre s s e s both m ar k e r s , or an ar tifact. Be caus e of this unce r tainty, w he n
the fr e que ncy of ce lls co-e xpre s s ing tw o or m or e m ark e r s is s m all, and be caus e the
le ve l of confide nce incre as e s w ith incr e as e d fr e que ncy, w e have s et a thr e s hold of
10% above w hich w e des ignate co-e xpr e s s ion as re al and be low w hich it is unce rtain.
In this cas e, the fre que ncy w as be low 10% of the C D19+ population and w e do not
k now its s ignificance . In C, a distinct s ubs e t of CD7+CD2+ ce lls (blue ) is s how n
w hich do not e xpre s s CD19 (A and B): the s e ar e nor m al T-ce lls . Us ing a s pe cim e n
from anothe r patie nt, in D, non-s pe cific binding is ofte n character ize d by e ve nts on a
45 de gr ee angle (re d) as the antibodie s bind e qually to all ce lls . Populations
e xhibiting this patte rn ar e not include d in the inter pr e tation e ve n though the y m ay be
gr e ate r than 10%.
4
10
1
2
A
10
3
C
D
1
9
4
B
5
6
C
D
2
0
10
2
10
R1
FL2-Height -->
1
10
2
10
R5
FL1-Height -->
4
10
1
10
2
10
3
7
1
10
D
2
4
10
3
2
10
3
C
D
2
2
2
10
10
Fluorescence Two Height -- >
3
10
2
4
4
10
1
10
10
3
Fluorescence
One Height -- >
CD5
10
E
5
6
2
10
4
10
L
A
M
B
D
A
10
2
8
10
3
Fluorescence Three Height -->
CD20
10
F
1
R1
Fluorescence Two Height -- >
3
4
2
10
10
10
4
2
10
1
10
10
1
10
3
3
1
7
4
10
KAPPA
Fluorescence One Height -- >
4
2
FL1-Height -->
R5
R1
1
3
10
3
10
10
R1
CD22
1
1
10
FL3-Height -->
CD5
C
D
1
9
2
8
10
FL1-Height -->
10
10
10
4
10
3
1
10
3
C2
1
FL2-Height -->
1
10
4
10
L
A
M
B
D
A
3
4
10
1
10
2
4
10
3
10
4
Fluorescence One Height -- >
KAPPA
Typical B-ce ll Lym phom a (A,B,C) vs Re active Lym phoid Hype r plas ia (D,E,F): The
m os t com m only found phe notype in B-ce ll lym phom a is the e xpr e s sion of CD19
(A), CD20 (B), CD22 (B) and one of the tw o light chains (C). CD5 e xpr e s s ion is
variable , typically lik e that s how n in A. In C, Kappa vs Lam bda coe xpr e ss ion w ith
CD19 is s how n; this lym phom a is Kappa light chain r e s tricte d. The cor re s ponding
dis plays for B-ce lls fr om a node w ith r e active lym phoid hype rplas ia are s how n in
D, E and F.
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
KAPPA -->
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
KAPPA -->
10
3
10
4
8
10
1
10
2
CD2
CD19 -->
10
3
10
4
CD19 -->
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
7KAPPA -->
10
10
2
2
10
10
1
1
1
10
10
10
3
4
10
1
10
2
CD3
KAPPA -->
10
3
10
F
R5
7
4
6
3
L AMBDA -->
R1
5
10
1
10
2
CD8
10
3
10
4
CD19 -->
8
10
1
10
2
CD2
10
3
10
4
CD19 -->
Reactive Lymph Node
Typical T-cell Lymphoma or Reactive Lymph Node: The hallma rk of
T-cell malignancy is inappropriate marker expression. This ca n
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 ex pression on the lymphoma cells (A) is more varia ble
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.