Transcript No Slide Title

IMMUNOPHENOTYPING OF HEMATOPOIETIC
PROGENITOR CELLS BY MULTIPARAMETER
FLOW CYTOMETRY
RPCI
LFC
PROTOCOLS FOR CD34 COUNT
Main 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
RPCI
LFC
CD34 bright cells acquired,
DNA vs SSC, CD45 vs SSC, DNA vs CD34
ISHAGE PROTOCOL
gated on R1
120
120
R1
100
100
SSC
CD45
10
1
10
2
10
3
10
gated on R1 and R2 and R3
120
120
100
100
90
90
20
10
SSC
4
30
10
10
SSC-Height -->
20
3
40
30
SSC-Height -->
40
CD45
10
50
50
2
60
60
10
FL3-Height -->
70
70
R3
1
80
80
R3
10
4
FL2-Height -->
CD34
gated on R1 and R2
SSC
R2
10
10
4
R2
20
20
10
30
SSC-Height -->
30
3
10
FL3-Height -->
40
40
2
50
50
10
60
60
1
70
70
SSC-Height -->
10
80
80
SSC
90
90
R1
R4
10
R4
20
30
40
50
60
70
80
FSC-Height -->
FSC
90
100
120
MILAN PROTOCOL
4
10
120
R2
100
3
R2
SSC
90
100
120
10
80
20
70
30
60
R1
40
50
50
10
40
SSC-Height -->
FSC-Height -->
1
30
60
10
20
70
FSC
80
2
CD34
90
10
FL2-Height -->
10
R1
10
20
30
40
50
60
70
80
SSC-Height -->
SSC
90
100
120
RPCI PROTOCOL
gated on
R1 AND R2 AND R3
gated on
R1 OR R2
4
120
120
10
50
60
70
80
90
100
120
10
20
30
40
50
SSC -->
60
70
80
90
100
10
120
1
10
2
10
3
10
4
FL3 -->
FSC -->
SSC
R6
10
40
R6
20
R3
10
30
R5
R5
30
20
20
R4
R4
40
30
10
10
50
40
SSC -->
SSC
60
50
SSC -->
SSC
70
60
1
FL2 -->
R3
80
70
2
R2
80
R10
10
CD34
90
10
90
R1
3
100
100
R1
R2
CD45
FSC
4
100
100
90
SSC
80
90
100
120
10
20
30
SSC
R5
R6
R6
10
70
R5
20
R3
10
60
SSC -->
R4
30
20
50
R4
40
30
40
50
40
10
30
SSC -->
50
SSC -->
1
FL2 -->
20
60
60
10
10
70
2
R3
80
70
R2
R10
80
SSC
10
90
R1
3
CD34
120
120
10
R1
R2
40
50
60
70
FSC -->
FSC
80
90
100
120
10
1
10
2
FL3 -->
CD45
10
3
10
4
100
120
120
50
60
70
80
90
R1
30
40
SSC-Height -->
100
90
80
70
60
50
20
10
SSC-Height -->
40
30
20
10
SIDE SCATTER
R1
10
20
30
40
50
60
70
80
FSC-Height -->
90
100
120
10
20
30
FORWARD SCATTER
40
50
60
70
80
FSC-Height -->
90
100
120
4
120
10
70
80
SSC
60
50
10
20
10
30
40
SSC-Height -->
2
10
1
FL2-Height -->
CD34
90
10
3
100
R1
10
20
30
40
50
60
70
SSC
80
90
100
120
R3
10
20
30
40
50
60
70
FSC
80
90
100
120
FSC-Height -->
120
10
4
SSC-Height -->
R4
40
50
60
70
80
SSC
SSC-Height -->
30
1
10
2
R2
10
20
R5
R3
10
FL1-Height -->
CD14
90
10
3
100
gated on R1
10
1
10
2
CD45
FL3-Height -->
10
3
10
4
10
20
30
40
50
60
70
FSC
80
FSC-Height -->
90
100
120
MOSTLY DIM
20
30
40
50
60
70
80
SSC-Height -->
90
100
120
10
3
1
10
FL2-Height -->
2
10
CD34 PE -->
10
1
10
10
2
10
3
10
3
10
2
CD34 PE -->
10
1
10
CD34
10
10
4
4
4
MOSTLY BRIGHT AVERAGE BRIGHT
10
20
30
40
50
60
70
80
SSC-Height -->
SSC
90
100
120
10
20
30
40
50
60
70
80
SSC-Height -->
90
100
120
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
4
4
10
10
2
1
10
10
CD34
PE CD34 -->
10
3
3
10
2
CD14
FITC CD14 -->
10
1
10
10
1
10
2
CD45
PCP CD45 -->
10
3
10
4
threshold
10
20
30
40
50
60
70
80
SSC-Height -->
SSC
90
100
120
AN TIBODY COMBIN ATION S U S ED TO EVALU ATE
HEMATOPOIETIC PROGEN ITOR CELLS
panel
antibodies
purpose
1
FL-CD14
PE-CD34
PP-CD45
defines regions for determining the absolute CD34 count and
progenitor cell subsets
2
FL-IgG1
PE-CD34
TC-IgG2a
to determine the uncertainty of the CD34 count due to IgG binding
3
FL-CD71 PECD34 TCCD45
defines CD45+ and CD45- progenitor cells, erythroid cells and
transferrin receptor bearing progenitor cells
4
FL-CD41
PE-CD34
PP-CD45
defines CD45+ and CD45- progenitor cells, megakaryocytes and
platelets
5
FL-CD45RA
PE-CD34
QR-CD45RO
evaluation of progenitor cell expression of the CD45 family of
molecules
6
FL-CD38
PE-CD34
PP-HLADr
Class II expression on progenitor cells
7
FL-CD5
PE-CD34
PC-CD19
detects B and T-cells and their progenitors
8
FL-CD7
PE-CD34
TC-CD2
evaluation of the differentiation markers for T and NK cells on
progenitor cells
9
FL-CD15
PE-CD34 TCCD56
defines a myeloid progenitor cell subset
10
FL-CD33
PE-CD34
TC-CD4
defines myeloid progenitor cells
50
60
70
80
90
100
10
3
2
10
FL1-Height -->
10
1
CD14
R6
10
120
20
30
40
50
60
70
80
90
100
R7
120
10
SSC-Height -->
FSC-Height -->
FSC
R9
10
10
2
1
R2
10
40
1
10
R8
2
10
3
FL3-Height -->
CD45
10
4
FSC
120
R4
R9
3
R5
2
10
GATED
ON
R1 AND R3
20
10
30
1
10
FL1-Height -->
80
70
60
50
40
SSC-Height -->
90
100
GATED ON R1
50
60
70
80
90
100
120
R7
10
1
10
R8
2
10
3
10
R10
R11 R15
3
GATED
ON
R2 AND R3
10
FL1-Height -->
80
70
60
50
4
FL3-Height -->
GATED ON R2
10
30
1
40
SSC-Height -->
90
100
120
FSC-Height -->
R6
4
40
10
30
2
20
10
10
CD14
10
R3
20
30
R3
10
20
SSC
10
R5
3
R4
10
CD34
90
80
70
60
50
40
30
20
SSC-Height -->
FL2-Height -->
100
120
10
R1
R3
10
SSC
4
4
UNGATED
10
20
30
40
50
60
70
80
FSC-Height -->
FSC
90
100
120
R12
R13
10
1
10
R14
2
FL3-Height -->
10
3
CD45
10
4
10
4
PERCENT OF PATIENTS
FREQUENCY OF PATIENTS EXPRESSING CD34
BRIGHT HPCs
25
20
15
10
5
0
<15
16-25 26-35 36-45 46-55 56-65
>66
CD34 BRIGHT (%TOTAL CD34+ HPCs)
CD45 EXPRESSION ON HPCs
PERCENT
100
80
60
40
20
0
CD34
DIM
CD45B
CD34
BRIGHT
CD45D
CD45N
Proportion of Subset in CD34+ Population
% of Subset in CD34+
Population
100
80
60
40
20
0
-/-
-/+
+/-
CD38
HLADr
+/+
-/-
-/+
+/-
CD45RO
CD45RA
+/+
Proportion of Subset Cells in CD34+ Population
% of Subset CD34+
16
14
12
-/+
+/+
+/-
10
8
6
4
2
0
CD15
CD56
CD5
CD19
CD33
CD4
CD71
CD45
CD41
CD45
Measurement of CD34 Subset
Proliferation
n
u
m
b
e
r
Hoechst
H
L
A
D
r
T
C
n
u
m
b
e
r
Hoechst
n
u
m
b
e
r
Hoechst
CD38 FITC
n
u
m
b
e
r
Hoechst
C-KIT RECEPTOR EXPRESSION ON HPC SUBSETS
CD117
H
L
A
D
r
CD117
CD38
CD117
CD117