Download - ICSH Differential Project
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ICSH Differential Project
Progress Update December 2019
Sèvres, France
JCTLM meeting
Marie C Béné, Nantes
On behalf of Brent Wood, Seattle
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Intent
• Replace morphology (CLSI H20-A2) with flow
cytometry as the reference method for performing
nucleated cell identification in blood
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Intended Use• Reference method for the validation of new automated
hematology instrumentation
– Manufacturers
– Validation of high precision clinical studies
– Value assignment to controls
• NOT Intended for routine clinical use to:
– Clarify problematic differential following morphologic evaluation
– Replace morphologic smear examination
– Replace current differential on automated hematology analyzers
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Populations
Core
Lymphocytes
Monocytes
Neutrophils
Immature
myeloids
Eosinophils
Basophils
Blasts
nRBCs
Miscellaneous
Lymphocytes
B cells
T cells
NK cells
Reactive
Cytotoxic
Plasma cells
Dendritic cells
CD16+ Monocytes
Mast cells
ICSH 10/2007
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Reference WBC diff working project:
Proposal for 2nd Phase Study
Syto16 Nucleic acid binding dye
CD45-KO Leukocytes
CD3-APC T Lymphocytes
CD19-APC B Lymphocytes
CD7-PE NK cells
CD123-PerCP-Cy5.5 Basophils, pDendritic Cells
CD14-PE-Cy7 Monocytes
CD16-v450 or Pacific Blue Neutrophils, immature myeloid cells, NK, monocyte subset
CD11b-APC-A750 Myeloid cells, monocytes, basophils, NK
Panel of antibodies
- Lyse, no wash with counting beads
- Syto-16 provides nucleated cell identification
- 6 requested populations are defined by positive markers
- Blasts are identified by exclusion, since phenotypes are widely divergent
- Nucleated RBCs with no positive antigens, but are identifiable
- Redundancy: most cells are defined by multiple markers
- Secondary objective: additional cell type would be detectable if abnormal
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ExampleNormal Example
10/31/20188
Monos
Beads DendriticNucleated
T/NK
Viable
NK NC Monos
Grans Eo IG Eo
Baso/pDCB/T/NK
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ICSH WBC differential working project
Validation Protocol
Validation performed in Melbourne and Seattle, including:
Sensitivity – 3 samples by serial dilution in triplicate
Specificity – 3 samples vs. H20-A2
Reproducibility
Within run – 3 samples in triplicate
Between run – Immunotrol over 5 days
Stability – 5 samples at 0, 24 and 48 hours
Linearity – 3 samples serially diluted 7 times run in triplicate
Accuracy and Robustness – 150 samples (30 normals) at 5 sites vs. H20-A2
London, Canada (Michael Keeney, Ben Hedley) Completed
Nantes, France (Marie Christine Bene) Incomplete
Melbourne, Australia (Peter Gambell) Complete
Beijing, China (Chenxue Qu) Complete
Seattle, USA (Brent Wood) Complete
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Accuracy• Intent
– Real world implementation
• Reagents
• Procedure (SOP)
• Example of data analysis
• Reality
– 4 of 5 sites executed procedure successfully
• 1 site had technical difficulties with cytometer programming
– Representative instrumentation
• Hematology analyzers – BC, Sysmex, Abbott
• Cytometers – BD, BC
– Data submitted for central review
• Variability in content
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WBC Count
N = 616
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WBC Count
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Neutrophil %R² 0.93 R² 0.89
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Neutrophils %
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Neutrophils %
Page 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-233-04891 Flow Diff MP00003 00002308 425.LMD
Date of data set acquisit ion 21-Aug-18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 8,479
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 8,773
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b
APC
A750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlets
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 200 400
CD
45 K
rO
103
104
105
106
Real
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Baso
pDC
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
LymphsPage 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-233-04891 Flow Dif f MP00003 00002308 425.LMD
Date of data set acquisit ion 21-Aug-18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 8,479
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 8,773
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlets
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 200 400
CD
45 K
rO
103
104
105
106
Real
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b
AP
C A
750
0
103
104
105
106
Baso
pDC
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
Lymphs
Page 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-173-06160 Flow Diff MP00003 00002166 283.LMD
Date of data set acquisit ion 22-Jun-18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 5,822
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 6,016
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b
AP
C A
750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlets
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 100 200 300
CD
45 K
rO
103
104
105
106
Real
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
Lymphs
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b
APC
A750
0
103
104
105
106
Baso
pDC
Page 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-173-06160 Flow Diff MP00003 00002166 283.LMD
Date of data set acquisit ion 22-Jun-18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 5,822
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 6,016
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b
AP
C A
750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlets
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 100 200 300
CD
45 K
rO
103
104
105
106
Real
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
Lymphs
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b
AP
C A
750
0
103
104
105
106
Baso
pDC
Page 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-229-00133 Flow Diff MP00003 00002301 418.LMD
Date of data set acquisit ion 17-Aug-18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 400
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 421
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlets
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 10 20 30 40
CD
45 K
rO
103
104
105
106
Real
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b
APC
A750
0
103
104
105
106
Baso
pDC
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
Lymphs
Page 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-285-01156 Flow Diff MP00003 00002420 537.LMD
Date of data set acquisit ion 12-Oct -18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 1,877
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 1,962
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlet s
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 50 100
CD
45 K
rO
103
104
105
106
Real
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Baso
pDC
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
Lymphs
Page 1 of 3
[Nucleated cells]
FS INT
0 500 1000
(x 103)
SS I
NT
0
500
1000
(x 1
03)
Viable
[Viable]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
W B Cs
B last
[WBCs]
SS INT
0 500 1000
(x 103)
CD
7 P
E
0
103
104
105
106
Name of the data f ile containing the data set 18-285-01156 Flow Diff MP00003 00002420 537.LMD
Date of data set acquisit ion 12-Oct -18
BCI Navios EX LHSC
[Beads]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Bead singlets: 1,877
[Blast ]
CD14 PC7
0 103 104 105 106
CD
16 V
450
0
103
104
105
106
Blast .1
[Blast .1]
CD11b APC A750
0 103 104 105 106
CD
45 K
rO
0
103
104
105
106
Blasts.1
[Singlets]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Beads: 1,962
CD123
[Blasts.1]
CD123 PerCp Cy5.5
0 103 104 105 106
CD
3/C
D19 A
PC
0
103
104
105
106
Blasts
[Mono]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E
0
103
104
105
106
Mono.1
[Not Beads.]
FS INT
0 500 1000
(x 103)
Syto
16
103
104
105
106
Nucleated cells
[Mono.1]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
Mono.2
[Not Eos]
CD16 V450
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Granulocytes
Immature Grans
[Real]
FS INT
0 500 1000
(x 103)
FS P
EA
K
0
100
200
(x 1
03)
Singlets
[Mono.2]
CD16 V450
0 103 104 105 106
CD
14 P
C7
0
103
104
105
106
Monos
[Eo]
CD123 PerCp Cy5.5
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eos
[Lymphs]
CD3/ CD19 APC
0 103 104 105 106
CD
7 P
E0
103
104
105
106
NK cellsT cells
B cells
[Ungated]
TIME
0 50 100
CD
45 K
rO
103
104
105
106
Real
[Grans.]
CD16 V450
103 104 105 106
CD
45 K
rO
0
103
104
105
106
Eo
[Not monos]
CD16 V450
0 103 104 105 106
CD
123 P
erC
p C
y5.5
0
103
104
105
106
pDC+Baso
[WBCs]
SS INT
0 500 1000
(x 103)
CD
14 P
C7
0
103
104
105
106
Mono
[pDC+Baso]
CD45 KrO
0 103 104 105 106
CD
11b A
PC
A750
0
103
104
105
106
Baso
pDC
[Not monos]
SS INT
0 500 1000
(x 103)
CD
16 V
450
0
103
104
105
106
Grans.
[NOT CD123/ Monos/ Grans]
SS INT
0 500 1000
(x 103)
CD
45 K
rO
0
103
104
105
106
Lymphs
![Page 14: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/14.jpg)
Neutrophils Absolute
R² 0.81
![Page 15: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/15.jpg)
Lymphocytes %
R² 0.96 R² 0.84
![Page 16: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/16.jpg)
Lymphocytes %
R² 0.93 - 0.96
![Page 17: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/17.jpg)
Lymphocytes Abs
R² 0.97
![Page 18: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/18.jpg)
Monocytes %R² 0.82 R² 0.48
![Page 19: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/19.jpg)
Monocytes %
R² 0.79 – 0.87
![Page 20: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/20.jpg)
Monocyte Abs
R² 0.70 R² 0.52
![Page 21: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/21.jpg)
Eosinophils %
R² 0.64 – 0.60
![Page 22: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/22.jpg)
Eosinophils Abs
R² 0.28
![Page 23: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/23.jpg)
Basophils %
R² 0.35 – 0.18
![Page 24: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/24.jpg)
Basophils Abs
R² 0.98 – 0.93
![Page 25: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/25.jpg)
IG %
R² 0.32
![Page 26: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/26.jpg)
IG %
R² 0.51
![Page 27: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/27.jpg)
IG Abs
R² 0.91
![Page 28: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/28.jpg)
IG Abs
22 %
19.9 %2.5 %
0.3 %
![Page 29: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/29.jpg)
Accuracy SummaryPopulation Morphology Hematology Flow Cytometry
WBC count - Good Good
Neutrophils Good Good Good
Lymphocytes Good Subset poor Good
Monocytes Good Subset poor Good
Eosinophils Fair Fair Fair
Basophils Poor Poor Good
IG Fair Poor Fair
![Page 30: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/30.jpg)
Accuracy Summary
Flow reference method at least
equivalent to morphology
Discrepancies with analyzers to
investigate
![Page 31: ICSH Differential Project](https://reader030.vdocument.in/reader030/viewer/2022032610/6239460d5accb11902574ac6/html5/thumbnails/31.jpg)
Timeline
• Complete Sample Acquisition
– Missing data from Nantes to complete
• Harmonize Reported Data
– Raw data from Beijing
– Complete reporting of all parameters
• Perform Outlier Analysis
– Identify technical errors
– Refine gating strategy / cutoff for Eos, Igs, blasts
• Write Draft Manuscript for Review