B-ALL MRD Maryalice Stetler-Stevenson, M.D., Ph.D. Flow Cytometry Unit, Laboratory of Pathology, CCR, NCI, NIH DEPARTMENT OF HEALTH & HUMAN SERVICES
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B-ALL MRD
Maryalice Stetler-Stevenson, M.D., Ph.D. Flow Cytometry Unit, Laboratory of Pathology, CCR, NCI, NIH
DEPARTMENT OF HEALTH & HUMAN SERVICES
National Institutes of Health Bethesda, Maryland 20892
Public Health Service
FL1 FL2 FL4 FL5 FL6 FL8
FITC PE Per CP-Cy5.5 PC7 APC
APC-Cy7 or APC-
H7
Tube 1CD20 CD10 CD38* CD19 CD58 CD45*B9E9 J5 HIT2 J3-119 N901 2D1
3ml 10ml 5ml 3ml 10ml 3ml
Tube 2
CD9 CD13/ CD33 CD34* CD19 CD10* CD45*
ALB6SJ1D1
/D3HL60.251
8G12 J3-119 HI10a 2D1
3ml 5ml / 10ml 15ml 3ml 3ml 3ml
Tube 3Syto-16 CD3* CD19 CD71* CD45*S7578 SK7 J3-119 L10.1 2D1
1ml 10ml 3ml 5ml 3ml
The COG Assay Panel
The COG Gating Strategy for B-ALL MRD:
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
CD19 PC7
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
FSC-A
SSC
-A
0 65536 131072 196608 262144
0
65536
131072
196608
262144
CD45 AH7
CD
19 P
C7
102
103
104
105
102
103
104
105
FSC-A
FSC
-H
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SINGLETS
Case 1SF15 1185 bm_06_L-3.fcs
No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 1185 bm_04_L-1.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 1185 bm_05_L-2.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 1185 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-H
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SINGLETS
SF15 1185 7aad9_viability.fcs Viability Singlets
7AAD
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
7aad SSC Viable
7AAD
Gate % of GatedCells
None 1007aad SSC 97
SF15 1185 bm_01_S-1.fcs SINGLETS
FSC-A
SSC
-A
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SF15 1185 bm_04_L-1.fcs SINGLETS
CD19 PC7
SSC-
A
102 103 104 105
0
65536
131072
196608
262144
SF15 1185 bm_04_L-1.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD
19 P
C7
102
103
104
105
102
103
104
105
Clean L1 gateSF15 1185 bm_05_L-2.fcs 8c B CD19 PC7 SSC
CD45 AH7C
D19
PC
710
210
310
410
5
102
103
104
105
Quadrant # of Events
% of GatedCells
% of All Cells
UL 90199 16.65 9.02UR 613 0.11 0.06LL 305369 56.37 30.54LR 145532 26.87 14.55
Gate # of Events
% of Gated Cells
% of All Cells
None 906160 100.00 90.62Syto 16 positive 906160 100.00 90.62Syto16 MNC 541713 59.78 54.17
Syto16 + Syto16 + MNC
Case 1
SF15 1185 bm_06_L-3.fcs Syto 16 positive
Syto 16
SSC
-A
102 103 104 10 5
0
65536
131072
196608
262144
Syto 16 positive
SF15 1185 bm_06_L-3.fcs Syto16 MNC
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105 16.65% 0.11%
56.37% 26.87%
SF15 1185 bm_06_L-3.fcs Syto 16 positive
FSC-A
SSC
-A
26 655 56 1310 85 196615 262144
0
655 36
131072
196608
262144
Syto16 MNC
SF15 1185 bm_06_L-3.fcs Syto 16 positive
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105
16.55%73.09%
0.09%10.27%
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD10 PE
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
19 P
C7
102
103
104
105
102
103
104
105
Case 1
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
20 F
ITC
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
L1 Plasma cells?SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
10 P
E
102
103
104
105
102
103
104
105
Gate 1 L11.10%
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD
58 A
PC
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% of All Cells
None 76867 100.00 7.69
8c B CD19 PC7 76867 100.00 7.69
Clean L1 gate 76867 100.00 7.69
Gate 1 L1 845 1.10 0.08
Case 1SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD9 FITC
CD
13+3
3 PE
102
103
104
105
102
103
104
105
% of L-22 Cells ALL=
% of L-22 MNC ALL=
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
9 FI
TC10
210
310
410
5
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD10 APC
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
10 A
PC
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD13+33 PEC
D10
APC
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD
10 A
PC
102
103
104
105
102
103
104
105 Gate 2 L1
0.88%
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD
10 A
PC
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% of All Cells
None 91625 100.00 9.16
8c B CD19 PC7 91625 100.00 9.16
Clean L2 Gate 91625 100.00 9.16
Gate 2 L1 806 0.88 0.08
Case 1
Tube 1: 845 x 90,199 x 100= 0.1876,867 541,713
Tube 2: 806 x 90,199 x 100= 0.1591,625 541,713
Case 2SF15 657 7aad_viability.fcs
No Gate
Time
FSC
-A
0 5000 10000 15000 20000
0
65536
131072
196608
262144
Time 4color
SF15 657 bm_07_L-3.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 657 bm_05_L-1.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 657 bm_06_L-2.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 657 bm_05_L-1.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD
19 P
C7
102
103
104
105
102
103
104
105 Clean L1 gate
SF15 657 bm_05_L-1.fcs SINGLETS
CD19 PC7
SSC
-A
102
103
104
105
0
6553 6
1310 72
1966 08
2621 44
SF15 657 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-H
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SINGLETS
SF15 657 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-W
0 65536 131072 196608 262144
0
65536
131072
196608
262144SF15 657 7aad_viability.fcs Viability Singlets
7AAD
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
7AAD
7aad SSC Viable
Gate % of Gated Cells
None 1007aad SSC 89
SF15 657 bm_01_S-1.fcs SINGLETS
FSC-A
SSC
-A
0 65536 131072 196608 262144
0
65536
131072
196608
262144
Case 2
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD10 PE
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD
20 F
ITC
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD
10 P
E
102
103
104
105
102
103
104
105
Gate 1 L12.55%
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD
10 P
E
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% of All Cells
None 47182 100.00 4.728c B CD19 PC7 47182 100.00 4.72Clean L1 gate 47182 100.00 4.72Gate 1 L1 1205 2.55 0.12
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD
19 P
C7
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD19 PC7
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD19 PC7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD19 PC7
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD10 APC
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD
10 A
PC
102
103
104
105
102
103
104
105
Case 2
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD45 AH7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD45 AH7
CD
10 A
PC
102
103
104
105
102
103
104
105
Gate 1 L22.57%
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD13+33 PE
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% ofAll Cells
None 44447 100.00 4.44Clean L2 44447 100.00 4.44Gate 1 L2 1141 2.57 0.11
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD10 APC
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD10 APC
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD45 AH7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
Case 2SF15 657 bm_07_L-3.fcs
Syto 16 positive
Syto 16
SSC
-A
102
103
104
105
0
65536
13 1072
19 6608
26 2144
Syto 16 positive
SF15 657 bm_07_L-3.fcs Syto16 MNC
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105
8.24%39.24%
0.09%52.43%
SF15 657 bm_07_L-3.fcs Syto 16 positive
FSC-A
SSC
-A
26 6555 6 131 085 196 615 262 144
0
65536
131 072
196 608
262 144
Syto16 MNC
SF15 657 bm_07_L-3.fcs Syto 16 positive
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105 5.75% 0.04%
91.58% 2.62%
Quadrant # of Events
Y GeometricMean
% of Gated Cells
% of All Cells
UL 43565 7998.77 52.43 4.36UR 75 4515.68 0.09 0.01LL 32601 0.00 39.24 3.26LR 6848 0.00 8.24 0.68
Gate # of Events
% of Gated Cells
% of All Cells
None 850029 100.00 85.00Syto 16 positive 850029 100.00 85.00Syto16 MNC 83089 9.77 8.31
Case 2
Tube 1: 1,205 x 43,565 x 100= 1.3447,182 83,089
Tube 2: 1,141 x 43,565 x 100= 1.3544,447 83,089
Case 3SF15 2200 bm 7aad_viability.fcs
No Gate
Time
FSC
-A
0 5000 10000 15000 20000
0
65536
131072
196608
262144
Time 4color
SF15 2200 bm_05_L-3.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 2200 bm_03_L-1.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 2200 bm_04_L-2.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 2200 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-H
0 65536 131072 19660 8 262 14 4
0
65536
131072
196608
262144
SINGLETS
SF15 2200 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-W
0 655 36 131 072 196608 262144
0
65536
131 07 2
196 60 8
262 14 4SF15 2200 bm 7aad_viability.fcs Viability Singlets
7AAD
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
7aad SSC Viable
7AAD
Gate % of GatedCells
None 100
7aad SSC 86
SF15 2200 bm_03_L-1.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD19
PC
7
102
103
104
105
102
103
104
105
Clean L1 gate SF15 2200 bm_03_L-1.fcs SINGLETS
CD19 PC7
SSC
-A
102 103 10 4 10 5
0
65536
13107 2
19660 8
26214 4
Gate # of Events
% of Gated Cells
% of All Cells
None 4735 100.00 0.558c B CD19 PC7 4735 100.00 0.55Clean L1 gate 4735 100.00 0.55Gate 1.L1 180 3.80 0.02
Case 3SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD
20 F
ITC
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD
19 P
C7
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
L1 Plasma cells?SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD19 PC7
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD
10 P
E
102
103
104
105
102
103
104
105 Gate 1.L1
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD10 PE
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD19 PC7
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD19 PC7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD
10 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD
10 A
PC
102
103
104
105
102
103
104
105
Case 3
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD10 APC
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD9 FITC
CD
13+3
3 PE
102
103
104
105
102
103
104
105
% of L-22 Cells ALL=
% of L-22 MNC ALL=
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD13+33 PE
CD
10 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
10 A
PC
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% of All Cells
None 4485 100.00 0.578c B CD19 PC7 4485 100.00 0.57Gate 1.L2 170 3.79 0.02Clean L2 Gate 4485 100.00 0.57
Case 3
SF15 2200 bm_05_L-3.fcs Syto16 MNC
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105 6.10% 0.14%
64.89% 28.88%
SF15 2200 bm_05_L-3.fcs Syto 16 positive
FSC-A
SSC
-A
26 655 56 131 08 5 19 661 5 262 14 4
0
65 536
13 107 2
19 660 8
26 214 4
Syto16 MNC
SF15 2200 bm_05_L-3.fcs Syto 16 positive
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105
7.55%91.34%
0.44%0.68%
Quadrant # of Events
% of Gated Cells
% of All Cells
UL 5335 6.10 0.53UR 121 0.14 0.01LL 56778 64.89 5.68LR 25271 28.88 2.53
Gate # of Events
% of Gated Cells
% of All Cells
None 826111 100.00 82.61Syto 16 positive 826111 100.00 82.61Syto16 MNC 87505 10.59 8.75
SF15 2200 bm_05_L-3.fcs Syto 16 positive
Syto 16
SSC
-A
102 103 104 105
0
65536
131072
196608
262144
Syto 16 positive
Case 3
Tube 1: 180 x 5,335 x 100= 0.234,735 87,505
Tube 2: 170 x 5,335 x 100= 0.234,485 87,505
Case 4SF15 2455 7aad_viability.fcs
No Gate
Time
FSC
-A
0 5000 10000 15000 20000
0
65536
131072
196608
262144
Time 4color
SF15 2455 bm_06_L-3.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 2455 bm_04_L-1.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 2455 bm_05_L-2.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF15 2455 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-H
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SINGLETS
SF15 2455 bm_01_S-1.fcs Time 8color
FSC-A
FSC
-W
0 65536 131072 196608 262144
0
65536
131072
196608
262144SF15 2455 7aad_viability.fcs Viability Singlets
7AAD
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
7AAD
7aad SSC Viable
Gate % of GatedCells
None 100
7aad SSC Viable 93
SF15 2455 bm_01_S-1.fcs SINGLETS
FSC-A
SSC
-A
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SF15 2455 bm_04_L-1.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD
19 P
C7
102
103
104
105
102
103
104
105
Clean L1 gate
SF15 2455 bm_04_L-1.fcs SINGLETS
CD19 PC7
SSC
-A
102
103
104
105
0
65536
131072
196608
262144
Case 4
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD
10 P
E
102 103 104 10 5
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
19 P
C7
102
103
104
105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
10 P
E
102
103
104
105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
38 P
erC
P C
y55
102 103 104 105
102
103
104
105L1 Plasma cells?
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD
58 A
PC
102
103
104
105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
20 F
ITC
10 2 10 3 104 10 5
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
10 P
E
102 10 3 10 4 105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD10 PE
CD
58 A
PC
102 103 10 4 105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD
58 A
PC
102 103 10 4 105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
10 P
E
10 2 10 3 104 105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD
38 P
erC
P C
y55
102 10 3 104 105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
38 P
erC
P C
y55
102 103 104 10 5
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD
58 A
PC
10 2 10 3 104 105
102
103
104
105
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD
58 A
PC
102
103
104
105
102
103
104
105Gate # of
Events% of Gated Cells
% of All Cells
None 3518 100.00 0.358c B CD19 PC7 3518 100.00 0.35Clean L1 gate 3518 100.00 0.35
Gate 1.L1 1331 37.83 0.13
Case 4
Gate # of Events
% of Gated Cells
% of All Cells
None 4582 100.00 0.46
8c B CD19 PC7 4582 100.00 0.46
Gate 1.L2 2557 55.81 0.26
Clean L2 Gate 4582 100.00 0.46
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD
10 A
PC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD
19 P
C7
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD
10 A
PC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD34 PerCP Cy55
CD
10 A
PC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD19 PC7
CD
34 P
erC
P C
y55
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD
13+3
3 PE
102 103 104 105
102
103
104
105 Gate 1.L255.81%
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD13+33 PE
CD
10 A
PC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD34 PerCP Cy55
CD
13+3
3 PE
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD19 PC7
CD
13+3
3 PE
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD
9 FI
TC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD10 APC
CD
9 FI
TC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD34 PerCP Cy55
CD
9 FI
TC
102 103 104 105
102
103
104
105
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD10 APC
CD
13+3
3 PE
102 103 104 105
102
103
104
105
% of L-22 Cells ALL=% of L-22 MNC ALL=
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD19 PC7
CD
9 FI
TC
102 103 104 105
102
103
104
105
Case 4SF15 2455 bm_06_L-3.fcs
Syto 16 positive
Syto 16
SSC
-A
102
103
104
105
0
6 553 6
1 310 72
1 966 08
2 621 44
Syto 16 positive
SF15 2455 bm_06_L-3.fcs Syto16 MNC
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105
16.59%81.40%
0.06%1.94%
SF15 2455 bm_06_L-3.fcs Syto 16 positive
FSC-A
SSC
-A
26 65556 131085 196615 262144
0
65536
131072
196608
262144
Syto16 MNC
SF15 2455 bm_06_L-3.fcs Syto 16 positive
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105 0.54% 0.09%
91.81% 7.57%
Quadrant # of Events
% of GatedCells
% of All Cells
UL 4588 1.94 0.46
UR 152 0.06 0.02
LL 192527 81.40 19.25
LR 39240 16.59 3.92
Gate # of Events
% of Gated Cells
% of All Cells
None 877716 100.00 87.77
Syto 16 positive 877716 100.00 87.77
Syto16 MNC 236507 26.95 23.65
Case 4
Tube 1: 1331 x 4588 x 100= 0.73 3518 236507
Tube 2: 2557 x 4588 x 100= 1.1 4582 236507
Which tube had better isolation of ALL from Normal?
Case 5SF16 043 7aad_viability.fcs
No Gate
Time
FSC
-A
0 5000 10000 15000 20000
0
65536
131072
196608
262144
Time 4color
SF16 043 bm_06_L-3_003.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF16 043 bm_04_L-1_001.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF16 043 bm_05_L-2_002.fcs No Gate
Time
FSC
-A
0 12500 25000 37500 50000
0
65536
131072
196608
262144
Time 8color
SF16 043 bm_01_S-1_002.fcs Time 8color
FSC-A
FSC
-H
0 6 5536 131072 196608 262 144
0
65536
13 1072
19 6608
26 2144
SINGLETS
SF16 043 bm_01_S-1_002.fcs Time 8color
FSC-A
FSC
-W
0 655 36 131 072 196 608 26 2144
0
6 5536
131072
196608
262144SF16 043 7aad_viability.fcs Viability Singlets
7AAD
SSC
-A
102
103
104
105
0
65 536
1 31072
1 96608
2 62144
7AAD
7aad SSC Viable
Gate % of GatedCells
None 1007aad SSC Viable 93
SF16 043 bm_01_S-1_002.fcs Time 8color
FSC-A
SSC
-A
0 65536 13 1072 19 6608 262144
0
65536
13 10 72
19 66 08
26 21 44
SF16 043 bm_04_L-1_001.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD
19 P
C7
102
103
104
105
102
103
104
105
Clean L1 gateSF16 043 bm_04_L-1_001.fcs SINGLETS
CD19 PC7
SSC
-A
102
103
104
105
0
6553 6
13107 2
19660 8
26214 4
Case 5SF16 043 bm_06_L-3_003.fcs
Syto 16 positive
Syto 16
SSC
-A
102
103
104
105
0
655 36
1310 72
1966 08
2621 44
Syto 16 positive
SF16 043 bm_06_L-3_003.fcs Syto16 MNC
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105
25.93%44.99%
0.08%29.00%
SF16 043 bm_06_L-3_003.fcs Syto 16 positive
FSC-A
SSC
-A
26 65 55 6 13 1 08 5 19 66 15 26 21 44
0
655 3 6
1 31 07 2
1 96 60 8
2 62 14 4
Syto16 MNC
SF16 043 bm_06_L-3_003.fcs Syto 16 positive
CD3 PerCP
CD
19 P
C7
102
103
104
105
102
103
104
105 5.91% 0.11%
86.68% 7.30%
Quadrant # of Events
% of GatedCells
% of All Cells
UL 47936 29.00 4.79
UR 140 0.08 0.01
LL 74375 44.99 7.44
LR 42872 25.93 4.29
Gate # of Events
% of Gated Cells
% ofAll Cells
None 977423 100 98Syto 16 positive 886478 91 89Syto16 MNC 165323 17 17
Case 5SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD
19 P
C7
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD
10 P
E
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
L1 Plasma cells?SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD
58 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD45 AH7
CD
20 F
ITC
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD19 PC7
CD
10 P
E
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD38 PerCP Cy55
CD
10 P
E
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD10 PE
CD
58 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Gate 7
CD45 AH7
CD
10 P
E
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD19 PC7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD38 PerCP Cy55
CD
19 P
C7
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Gate 7
CD45 AH7
CD
38 P
erC
P C
y55
102
103
104
105
102
103
104
105 Gate 1.L1
2.08%
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD19 PC7
CD
58 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD45 AH7
CD
58 A
PC
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% of All Cells
None 49767 100.00 4.52
8c B CD19 PC7 49767 100.00 4.52
Clean L1 gate 49767 100.00 4.52
Gate 1.L1 1111 2.23 0.10
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD
10 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD
10 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
Case 5L-2 9 13+33 34 19 10 45
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Gate 9
CD9 FITC
CD
13+3
3 PE
102
103
104
105
102
103
104
105
% of L-22 Cells ALL=% of L-22 MNC ALL=
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD10 APC
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD
9 FI
TC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD13+33 PE
CD
10 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD34 PerCP Cy55
CD
10 A
PC
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD
13+3
3 PE
102
103
104
105
102
103
104
105
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD
34 P
erC
P C
y55
102
103
104
105
102
103
104
105
Gate # of Events
% of Gated Cells
% of All Cells
None 42549 100.00 4.25
8c B CD19 PC7 42549 100.00 4.25
Gate 1.L2 918 2.16 0.09
Clean L2 Gate 42549 100.00 4.25
Case 5
Tube 1: 1111 x 47936 x 100= 0.6549767 165323
Tube 2: 918 x 47936 x 100= 0.6342549 165323
Remember Typical Analysis Errors:
Miscalling hematogones Overly tight gating, especially initial B cell gate Lack of hierarchical gating Artifacts and normal variants
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