This document describes the gating strategy and analysis for detecting minimal residual disease (MRD) in B-cell acute lymphoblastic leukemia (B-ALL) using flow cytometry. It includes details on the antibody panel and tubes used, gating approaches on CD19+ cells and viability markers, and quantification of MRD levels in specific cell populations from two patient samples.

1. 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 Instit
Bethesda, M
Public Health

2. FL1 FL2 FL4 FL5 FL6 FL8
FITC PE
Per CP-
Cy5.5
PC7 APC
APC-Cy7
or APC-H7
Tube 1
CD20 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*
ALB6
SJ1D1
/D3HL60.2
51
8G12 J3-119 HI10a 2D1
3ml 5ml / 10ml 15ml 3ml 3ml 3ml
Tube 3
Syto-16 CD3* CD19 CD71* CD45*
S7578 SK7 J3-119 L10.1 2D1
1ml 10ml 3ml 5ml 3ml
The COG Assay Panel

3. The COG Gating Strategy for B-ALL MRD:
Time
FSC-A
0 12500 2500 0 37500 5 0000
0
65536
13 1072
19 6608
26 2144
Time 8color
CD19 PC7
SSC-A
10
2
10
3
10
4
10
5
0
65 536
131 072
196 608
262 144
FSC-A
SSC-A
0 65536 131072 1966 08 262144
0
65536
1310 72
1966 08
2621 44
CD45 AH7
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
FSC-A
FSC-H
0 65536 1 31072 196608 262 144
0
65536
131072
196608
262144
SINGLETS

4. Case 1
SF15 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 6 5536 131072 196608 26214 4
0
6 5536
1 3107 2
1 9660 8
2 6214 4
SINGLETS
SF15 1185 7aad9_viability.fcs Viability Singlets
7AAD
SSC-A
10
2
10
3
10
4
10
5
0
65 536
1 310 72
1 966 08
2 621 44
7aad SSC Viable
7AAD
Gate % of
Gated
Cells
None 100
7aad SSC 97
SF15 1185 bm_01_S-1.fcs SINGLETS
FSC-A
SSC-A
0 65 536 131072 1966 08 26 2144
0
65536
1 3107 2
1 9660 8
2 6214 4
SF15 1185 bm_04_L-1.fcs SINGLETS
CD19 PC7
SSC-A
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
SF15 1185 bm_04_L-1.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Clean L1 gate
SF15 1185 bm_05_L-2.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD19PC7 10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5

5. Quadrant # of
Events
% of
Gated
Cells
% of
All
Cells
UL 90199 16.65 9.02
UR 613 0.11 0.06
LL 305369 56.37 30.54
LR 145532 26.87 14.55
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 906160 100.00 90.62
Syto 16 positive 906160 100.00 90.62
Syto16 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
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
Syto 16 positive
SF15 1185 bm_06_L-3.fcs
Syto16 MNC
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 16.65% 0.11%
56.37% 26.87%
SF15 1185 bm_06_L-3.fcs
Syto 16 positive
FSC-A
SSC-A
26 65556 131085 196615 262144
0
65536
1310 72
1966 08
2621 44
Syto16 MNC
SF15 1185 bm_06_L-3.fcs
Syto 16 positive
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
16.55%73.09%
0.09%10.27%

6. SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD10 PE
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Case 1
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD20FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
L1 Plasma cells?
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate 1 L1
1.10%
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
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

7. Case 1
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD9 FITC
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
% of L-22 Cells ALL=
% of L-22 MNC ALL=
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD10 APC
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD13+33 PE
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD19 PC7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 Gate 2 L1
0.88%
SF15 1185 bm_05_L-2.fcs Clean L2 Gate
CD45 AH7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
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

8. Case 1
Tube 1:
845 x 90,199 x 100= 0.18
76,867 541,713
Tube 2:
806 x 90,199 x 100= 0.15
91,625 541,713

9. Case 2
SF15 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
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Clean L1 gate
SF15 657 bm_05_L-1.fcs SINGLETS
CD19 PC7
SSC-A
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
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
262144
SF15 657 7aad_viability.fcs Viability Singlets
7AAD
SSC-A
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
7AAD
7aad SSC Viable
Gate % of
Gated
Cells
None 100
7aad SSC 89
SF15 657 bm_01_S-1.fcs SINGLETS
FSC-A
SSC-A
0 65536 131072 196608 262144
0
65536
131072
196608
262144

10. Case 2
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD10 PE
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD20FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate 1 L1
2.55%
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 47182 100.00 4.72
8c B CD19 PC7 47182 100.00 4.72
Clean L1 gate 47182 100.00 4.72
Gate 1 L1 1205 2.55 0.12
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD20 FITC
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD19 PC7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD19 PC7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD19 PC7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_05_L-1.fcs Clean L1 gate
CD45 AH7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5

11. SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD10 APC
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Case 2
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD45 AH7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD45 AH7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate 1 L2
2.57%
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD13+33 PE
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 44447 100.00 4.44
Clean L2 44447 100.00 4.44
Gate 1 L2 1141 2.57 0.11
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD10 APC
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD19 PC7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD10 APC
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 657 bm_06_L-2.fcs Clean L2 Gate
CD45 AH7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5

12. Case 2
SF15 657 bm_07_L-3.fcs
Syto 16 positive
Syto 16
SSC-A
10
2
10
3
10
4
10
5
0
65 536
13 1 07 2
19 6 60 8
26 2 14 4
Syto 16 positive
SF15 657 bm_07_L-3.fcs
Syto16 MNC
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
8.24%39.24%
0.09%52.43%
SF15 657 bm_07_L-3.fcs
Syto 16 positive
FSC-A
SSC-A
26 65556 131085 196615 262144
0
65536
1 31072
1 96608
2 62144
Syto16 MNC
SF15 657 bm_07_L-3.fcs
Syto 16 positive
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 5.75% 0.04%
91.58% 2.62%
Quadrant # of
Events
Y
Geometric
Mean
% of
Gated
Cells
% of
All
Cells
UL 43565 7998.77 52.43 4.36
UR 75 4515.68 0.09 0.01
LL 32601 0.00 39.24 3.26
LR 6848 0.00 8.24 0.68
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 850029 100.00 85.00
Syto 16 positive 850029 100.00 85.00
Syto16 MNC 83089 9.77 8.31

13. Case 2
Tube 1:
1,205 x 43,565 x 100= 1.34
47,182 83,089
Tube 2:
1,141 x 43,565 x 100= 1.35
44,447 83,089

14. Case 3
SF15 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 65 536 131 072 19 660 8 2 62144
0
6 55 36
131 0 72
196 6 08
262 1 44
SINGLETS
SF15 2200 bm_01_S-1.fcs Time 8color
FSC-A
FSC-W
0 6 5 536 131 072 19 6608 2 62144
0
655 36
1310 7 2
1966 0 8
2621 4 4
SF15 2200 bm 7aad_viability.fcs Viability Singlets
7AAD
SSC-A
10
2
10
3
10
4
10
5
0
65 5 36
13 1 072
19 6 608
26 2 144
7aad SSC Viable
7AAD
Gate % of
Gated
Cells
None 100
7aad SSC 86
SF15 2200 bm_03_L-1.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Clean L1 gate SF15 2200 bm_03_L-1.fcs SINGLETS
CD19 PC7
SSC-A
10
2
10
3
10
4
10
5
0
6553 6
1 310 7 2
1 966 0 8
2 621 4 4

15. Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 4735 100.00 0.55
8c B CD19 PC7 4735 100.00 0.55
Clean L1 gate 4735 100.00 0.55
Gate 1.L1 180 3.80 0.02
Case 3
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD20FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
L1 Plasma cells?
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD20 FITC
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD19 PC7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD45 AH7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 Gate 1.L1
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD10 PE
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD19 PC7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_03_L-1.fcs Clean L1 gate
CD19 PC7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5

16. SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Case 3
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD10 APC
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD9 FITC
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
% of L-22 Cells ALL=
% of L-22 MNC ALL=
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD13+33 PE
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD34 PerCP Cy55
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD19 PC7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2200 bm_04_L-2.fcs Clean L2 Gate
CD45 AH7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 4485 100.00 0.57
8c B CD19 PC7 4485 100.00 0.57
Gate 1.L2 170 3.79 0.02
Clean L2 Gate 4485 100.00 0.57

17. Case 3
SF15 2200 bm_05_L-3.fcs
Syto16 MNC
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 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 1 31 08 5 19 66 15 2 6214 4
0
65 53 6
1 31 072
1 96 608
2 62 144
Syto16 MNC
SF15 2200 bm_05_L-3.fcs
Syto 16 positive
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
7.55%91.34%
0.44%0.68%
Quadrant # of
Events
% of
Gated
Cells
% of
All
Cells
UL 5335 6.10 0.53
UR 121 0.14 0.01
LL 56778 64.89 5.68
LR 25271 28.88 2.53
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 826111 100.00 82.61
Syto 16 positive 826111 100.00 82.61
Syto16 MNC 87505 10.59 8.75
SF15 2200 bm_05_L-3.fcs
Syto 16 positive
Syto 16
SSC-A
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
Syto 16 positive

18. Case 3
Tube 1:
180 x 5,335 x 100= 0.23
4,735 87,505
Tube 2:
170 x 5,335 x 100= 0.23
4,485 87,505

19. Case 4
SF15 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
262144F15 2455 7aad_viability.fcs Viability Singlets
7AAD
SSC-A
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
7AAD
7aad SSC Viable
Gate % of
Gated
Cells
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
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Clean L1 gate
SF15 2455 bm_04_L-1.fcs SINGLETS
CD19 PC7
SSC-A
10
2
10
3
10
4
10
5
0
65 536
131072
196608
262144

20. Case 4
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
L1 Plasma cells?
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD20 FITC
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD20FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD10 PE
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD38 PerCP Cy55
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD19 PC7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_04_L-1.fcs Clean L1 gate
CD45 AH7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate # of
Events
% of
Gated
Cells
% of
All
Cells
None 3518 100.00 0.35
8c B CD19 PC7 3518 100.00 0.35
Clean L1 gate 3518 100.00 0.35
Gate 1.L1 1331 37.83 0.13

21. 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
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD34 PerCP Cy55
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD19 PC7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate 1.L2
55.81%
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD13+33 PE
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD34 PerCP Cy55
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD19 PC7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD45 AH7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD10 APC
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD34 PerCP Cy55
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD10 APC
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
% of L-22 Cells ALL=
% of L-22 MNC ALL=
SF15 2455 bm_05_L-2.fcs Clean L2 Gatee
CD19 PC7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5

22. Case 4
SF15 2455 bm_06_L-3.fcs
Syto 16 positive
Syto 16
SSC-A
10
2
10
3
10
4
10
5
0
65536
131072
196608
262144
Syto 16 positive
SF15 2455 bm_06_L-3.fcs
Syto16 MNC
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
16.59%81.40%
0.06%1.94%
SF15 2455 bm_06_L-3.fcs
Syto 16 positive
FSC-A
SSC-A
26 6 5556 13 1085 196615 262 144
0
655 36
131072
196608
262144
Syto16 MNC
SF15 2455 bm_06_L-3.fcs
Syto 16 positive
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 0.54% 0.09%
91.81% 7.57%
Quadrant # of
Events
% of
Gated
Cells
% 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

23. 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?

24. Case 5
SF16 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 65536 1310 72 19 6608 26214 4
0
65536
131072
196608
262144
SINGLETS
SF16 043 bm_01_S-1_002.fcs Time 8color
FSC-A
FSC-W
0 65536 1310 72 19 6608 2 62144
0
65536
131072
196608
262144F16 043 7aad_viability.fcs Viability Singlets
7AAD
SSC-A
10
2
10
3
10
4
10
5
0
65 536
13 107 2
19 660 8
26 214 4
7AAD
7aad SSC Viable
Gate % of
Gated
Cells
None 100
7aad SSC Viable 93
SF16 043 bm_01_S-1_002.fcs Time 8color
FSC-A
SSC-A
0 65536 13107 2 19 660 8 262 14 4
0
65 53 6
1 31 07 2
1 96 60 8
2 62 14 4
SF16 043 bm_04_L-1_001.fcs 8c B CD19 PC7 SSC
CD45 AH7
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Clean L1 gate
SF16 043 bm_04_L-1_001.fcs SINGLETS
CD19 PC7
SSC-A
10
2
10
3
10
4
10
5
0
65 536
131072
196608
262144

25. Case 5
SF16 043 bm_06_L-3_003.fcs
Syto 16 positive
Syto 16
SSC-A
10
2
10
3
10
4
10
5
0
65 536
131 072
196 608
262 144
Syto 16 positive
SF16 043 bm_06_L-3_003.fcs
Syto16 MNC
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
25.93%44.99%
0.08%29.00%
SF16 043 bm_06_L-3_003.fcs
Syto 16 positive
FSC-A
SSC-A
26 65 556 13 10 8 5 1 9 6615 2 62 1 44
0
65 5 36
1 310 72
1 966 08
2 621 44
Syto16 MNC
SF16 043 bm_06_L-3_003.fcs
Syto 16 positive
CD3 PerCP
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5 5.91% 0.11%
86.68% 7.30%
Quadrant # of
Events
% of
Gated
Cells
% 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
% of
All
Cell
s
None 977423 100 98
Syto 16 positive 886478 91 89
Syto16 MNC 165323 17 17

26. Case 5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
L1 Plasma cells?
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD20 FITC
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD45 AH7
CD20FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD19 PC7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD38 PerCP Cy55
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD10 PE
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Gate 7
CD45 AH7
CD10PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD19 PC7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD38 PerCP Cy55
CD19PC7
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Gate 7
CD45 AH7
CD38PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate 1.L1
2.08%
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD19 PC7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_04_L-1_001.fcs Clean L1 gate
CD45 AH7
CD58APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate # of
Events
% of
Gated
Cells
None 49767 100.00
8c B CD19 PC7 49767 100.00
Clean L1 gate 49767 100.00
Gate 1.L1 1111 2.23

27. SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Case 5
L-2 9 13+33 34 19 10 45
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Gate 9
CD9 FITC
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
% of L-22 Cells ALL=
% of L-22 MNC ALL=
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD34 PerCP Cy55
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD10 APC
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD9FITC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD34 PerCP Cy55
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD13+33 PE
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD34 PerCP Cy55
CD10APC
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD45 AH7
CD13+33PE
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
SF16 043 bm_05_L-2_002.fcs Clean L2 Gate
CD19 PC7
CD34PerCPCy55
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Gate # of
Events
% of
Gated
Cells
% o
All
Cel
None 42549 100.00 4.2
8c B CD19 PC7 42549 100.00 4.2
Gate 1.L2 918 2.16 0.0
Clean L2 Gate 42549 100.00 4.2

28. Case 5
Tube 1:
1111 x 47936 x 100= 0.65
49767 165323
Tube 2:
918 x 47936 x 100= 0.63
42549 165323

29. Remember Typical Analysis Errors:
Miscalling hematogones
Overly tight gating, especially initial B cell gate
Lack of hierarchical gating
Artifacts and normal variants