IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY.
20
IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY RPCI LFC
-
Upload
yvonne-mance -
Category
Documents
-
view
247 -
download
7
Transcript of IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER FLOW CYTOMETRY.
IMMUNOPHENOTYPING OF HEMATOPOIETIC PROGENITOR CELLS BY MULTIPARAMETER
FLOW CYTOMETRY
RPCI
LFC
PROTOCOLS FOR CD34 COUNTMain Features
Mulhouse All cells acquired, FSC vs SSC and SSC vs CD34
Milan All cells acquired, FSC vs SSC and SSC vs CD34 Nordic All cells acquired, FSC vs SSC and SSC vs CD34
ISHAGE CD45+ cells acquired,
CD45 vs SSC, CD34 vs SSC, CD45 vs SSC, FSC vs SSC
ProCount CD34 bright cells acquired, DNA vs SSC, CD45 vs SSC, DNA vs CD34
RPCI
LFC
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
10
20
30
40
50
60
70
80
90
100
120
SS
C-H
eig
ht -->
R4
10 1 10 2 10 3 10 4
FL3-Height -->
10
20
30
40
50
60
70
80
90
100
120
SS
C-H
eig
ht -->
R3
10 1 10 2 10 3 10 4
FL2-Height -->
10
20
30
40
50
60
70
80
90
100
120
SS
C-H
eig
ht -->
R2
10 1 10 2 10 3 10 4
FL3-Height -->
10
20
30
40
50
60
70
80
90
100
120
SS
C-H
eig
ht -->
R1
ISHAGE PROTOCOL
CD45
SS
C
R1
CD34
SS
C
R2
CD45
SS
C
FSC
SS
CR3 R4
gated on R1 and R2 gated on R1 and R2 and R3
gated on R1
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
10
20
30
40
50
60
70
80
90
100
120
FS
C-H
eig
ht -->
R1
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
10
110
210
310
4
FL
2-H
eig
ht -->
R2
SSC
CD
34
R2
SSC
FS
C
MILAN PROTOCOL
R1
10 20 30 40 50 60 70 80 90 100 120
SSC -->
101
102
103
104
FL
2 -->
R1R2
R10
RPCI PROTOCOL
SSC
CD
34
R1
R2
gated on R1 OR R2
10 20 30 40 50 60 70 80 90 100 120
FSC -->
1020
3040
5060
7080
90100
120
SS
C -->
R3
FSCS
SC
10 1 10 2 10 3 10 4
FL3 -->
1020
3040
5060
7080
90100
120
SS
C -->
R4 R5 R6
CD45
SS
C
gated on R1 AND R2 AND R3
R4 R5 R6R3
10 20 30 40 50 60 70 80 90 100 120
SSC -->
101
102
103
104
FL
2 -->
R1R2
R10
SSC
CD
34
R1
R2
10 20 30 40 50 60 70 80 90 100 120
FSC -->10
2030
4050
6070
8090
100120
SS
C -->
R3
FSC
SS
C
10 1 10 2 10 3 10 4
FL3 -->
1020
3040
5060
7080
90100
120
SS
C -->
R4 R5 R6
CD45
SS
C
R4 R5 R6R3
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
SID
E S
CA
TTER
FORWARD SCATTER
R1 R1
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
101
102
103
104
FL
2-H
eig
ht
-->
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL
1-H
eig
ht
-->
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
CD
14
SS
CS
SC
CD
34
CD45 FSC
SSC FSC
R3
R3
gated on R1
R1
R4
R2
R5
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
101
102
103
104
CD
34
PE
-->
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
101
102
103
104
CD
34
PE
-->
SSC
CD
34
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
101
102
103
104
FL
2-H
eig
ht
-->
MOSTLY BRIGHT AVERAGE BRIGHT MOSTLY DIM
Comparison of Gating Strategies
%CD34 positive*
RPCI 1.33 ± 1.44
ISHAGE 0.87 ± 1.30
ISHAGE (no CD45 gate) 1.35 ± 1.44
*n = 80
RPCI
LFC
What CD34 Count is Important?
Protocol to Use
• Pre or Post Thaw Count? Any
• CD34 bright? or CD34 dim? RPCI
• CD45+? CD45-? Any, except ISHAGE
RPCI
LFC
10 1 10 2 10 3 10 4
PCP CD45 -->
101
102
103
104
FIT
C C
D1
4 -
->
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
101
102
103
104
PE
CD
34
-->
CD
34
SSC
CD
14
CD45
threshold
ANTIBODY COMBINATIONS USED TO EVALUATEHEMATOPOIETIC PROGENITOR CELLS
panel antibodies purpose
1 FL-CD14PE-CD34PP-CD45
defines regions for determining the absolute CD34 count andprogenitor cell subsets
2 FL-IgG1PE-CD34TC-IgG2a
to determine the uncertainty of the CD34 count due to IgG binding
3 FL-CD71 PE-CD34 TC-CD45
defines CD45+ and CD45- progenitor cells, erythroid cells andtransferrin receptor bearing progenitor cells
4 FL-CD41PE-CD34PP-CD45
defines CD45+ and CD45- progenitor cells, megakaryocytes andplatelets
5 FL-CD45RAPE-CD34QR-CD45RO
evaluation of progenitor cell expression of the CD45 family ofmolecules
6 FL-CD38PE-CD34PP-HLADr
Class II expression on progenitor cells
7 FL-CD5PE-CD34PC-CD19
detects B and T-cells and their progenitors
8 FL-CD7PE-CD34TC-CD2
evaluation of the differentiation markers for T and NK cells onprogenitor cells
9 FL-CD15PE-CD34 TC-CD56
defines a myeloid progenitor cell subset
10 FL-CD33PE-CD34TC-CD4
defines myeloid progenitor cells
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL
1-H
eig
ht
-->
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL
1-H
eig
ht
-->
10 1 10 2 10 3 10 4
FL3-Height -->
101
102
103
104
FL
1-H
eig
ht
-->
10 20 30 40 50 60 70 80 90 100 120
FSC-Height -->
1020
3040
5060
7080
90100
120
SS
C-H
eig
ht
-->
GATED ON R1GATEDON R1 AND R3
SS
C
CD
14
GATED ON R2
CD45
R4 R5 R9
R6 R7 R8
R4 R5 R9
R6 R7 R8
R10 R11 R15
R12 R13 R14
SS
C
FSC CD45
FSC
GATED ON R2 AND R3
CD
14
R3
R3
R3
UNGATED
10 20 30 40 50 60 70 80 90 100 120
SSC-Height -->
101
102
103
104
FL
2-H
eig
ht
-->
R1
R2
CD
34
FSC
0
5
10
15
20
25
<15 16-25 26-35 36-45 46-55 56-65 >66
FREQUENCY OF PATIENTS EXPRESSING CD34 BRIGHT HPCs
CD34 BRIGHT (%TOTAL CD34+ HPCs)
PER
CEN
T O
F P
ATIE
NTS
CD45 EXPRESSION ON HPCs
0
20
40
60
80
100
CD34DIM
CD34BRIGHT
CD45B CD45D CD45N
PER
CEN
T
Proportion of Subset in CD34+ Population%
of
Su
bset
in
CD
34+
Pop
ula
tion
0
20
40
60
80
100
CD38HLADr
-/- -/+ +/- +/+ -/- -/+ +/-
CD45ROCD45RA
+/+
Proportion of Subset Cells in CD34+ Population
% o
f S
ub
set
CD
34+
0
2
4
6
8
10
12
14
16
CD15CD56
CD5CD19
CD33CD4
CD71CD45
CD41CD45
-/++/++/-
Measurement of CD34 Subset Proliferation
Hoechst Hoechst
Hoechst Hoechst
number
number
number
number
HLADr TC CD38 FITC
C-KIT RECEPTOR EXPRESSION ON HPC SUBSETS
CD117 HLADr
CD117
CD117
CD117
CD38
