Prognostic Significance of monitoring
Leukemia-Associated Immunophenotypes by 8-color
flow cytometry in Adult B-Acute Lymphoblastic
Leukemia
Running title: Assessing of MRD by 8-color Flow cytometry in adult
B-ALL
X-Q Weng1,2, Y Shen1,2, Y Sheng1,2, B Chen1, J-H Wang1, J-M Li1, J-Q
Mi1, Q-S Chen1, Y-M Zhu1, C-L Jiang1, H-Yan1, X-X Zhang1, T Huang1,
Z Zhu1, Z Chen1, S-J Chen1
1
State Key Laboratory of Medical Genomics, Shanghai Institute of
Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of
Medicine, Shanghai, China;
2
These authors contributed equally to this work.
Correspondence: Sai-Juan Chen, State Key Laboratory of Medical
Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai
Jiao Tong University School of Medicine, 197 Rui Jin Er Road, Shanghai
200025, China. Email: sjchen@stn.sh.cn
Tel: +86-21-33562658
Fax: +86-21-64743206
The supplementary information includes two figures and three tables:
Supplementary figure 1 and 2 displayed a detailed process of MRD
detection for a case with B-ALL and dilution experiments for one sample;
Supplementary table 1 to 3 presented the reagents list for diagnosis, the
distribution of LAIPs combinations and their background expression in
normal BM, and detailed results of dilution experiments.
Supplementary Figure legends
Supplementary Figure 1. Dot plots of an 8-mAbs analytical matrix of
the immunophenotypic assessment of a case with B-ALL from
diagnosis to relapse in which only CD19-positive cells were presented
after excluding the doublets.
Leukemic blasts shown in gate P21 were intersection set of P14 “and”
P15 “and” P16 “and” P17 “and” P18. Red dots represent the population
with entirely same expression patterns of leukemia-associated
immunophenotypes (LAIPs) defined at diagnosis; the deep blue cluster
was identified as new abnormal population with some LAIPs shifted as
compared to initial one; dots of yellow, light blue and grey represent the
normal B-lineage cells in BM with sequential stages of differentiation.
(A-D) Leukemic population as shown by LAIPs at different time points
such as at diagnosis, the end of induction of CR, after 1 consolidation and
relapse. In (B) and (C), leukemic cells as shown in gate P21 were
undetectable, while a new abnormal cluster shown with deep blue color
was observed with an occurrence rate of 0.005% and 0.0158% displaying
some up-modulation of CD10 and／or CD20 at the end of induction and
1 consoidation, respectively. (D) Most of leukemic cells reverted to initial
aberrant patterns of LAIPs when relapse. (E) Neither red nor deep blue
dots were presented in regenerating BM sample abtained from a patient
with T-ALL of MRD-free.
Supplementary Figure 2. The percentage of LAIPs+ blasts from patient
number 5 with B-Lineage lymphoblastic leukemia (B-ALL) diluted with
MRD-free regenerating BM in different titers from 1:1 to 1:200,000.
All the dot plots were gated from CD19+CD10+CD34+ cells after
excluding the doublets. Final leukemic blasts shown in gate P8 were
intersection set of P6 “and” P7 using “and” logistic gating strategy with
the aberrant phenotype of CD38 dim expression, CD58 strong expression
and CD13 positive.
(A) The BM sample from a B-ALL patient at diagnosis without dilution.
(B-K) 1:1 to 1:200000 dilutions.
(L) MRD-free regenerating BM.
The total WBC count in (A-C), (D),(E) and (F-L) were 1.0x105, 5.0x105,
1.0x106 and 2.0x106, respectively.
Supplementary Table 1. Information and panels of mAbs for diagnosis of B ALL
mAb
mAb clone
Fluorochrome
Catalogue
Source
number
7.1
7.1
PE
MHTDT01-5
Immunotech (Marseille, France)
CD10
ALB1
APC
IM3633
Immunotech
CD11b
ICRF44
Pacific Blue
558123
Becton Dickinson ( San José,
CD13
WM15
APC
301706
Biolegend (San Diego, USA)
CD15
80H5
FITC
IM1423U
Immunotech
CD19
HIB19
Brilliant Violet
302234
Biolegend (San Diego, USA)
CA, USA)
421TM
CD20
B9E9
APC-eFluor780
85-47-0209-42
eBioscience (San Diego, USA)
CD22
SJ10.1H11
PE
IM1835
Immunotech
CD33
D3HL60.251
PE-Cy5.5
A70198
Immunotech
CD34
581
PE-Cy7
348791
Becton Dickinson ( San José,
CA, USA)
CD38
HIT2
perCP-cy5.5
551400
Becton Dickinson ( San José,
CA, USA)
CD45
HI30
Pacific Orange
MHCD4530
Invitrogen (Camarillo, USA)
CD58
AICD58
FITC
IM1218U
Immunotech
CD65
88H7
FITC
IM1654U
Immunotech
CD66c
KORSA-3544
PE
IM2357U
Becton Dickinson ( San José,
CA, USA)
HLA-DR
L243
APC-Cy7
307618
Biolegend (San Diego, USA)
TDT
HT-6
FITC
MHTDT01-5
Invitrogen (Camarillo, USA)
CD79a
HM47
PE
IM2221
Immunotech
IgM
Polyclonal
FITC
732561
Immunotech
κ/λ
Polyclonal
FITC /PE
FR48150
DAKO (Glostrup, Denmark)
Fix-and-Perm
-
-
GAS-002A-1
Invitrogen (Camarillo, USA)
kit
GAS-002B-1
Panels for diagnosis of B ALL:
Tube1 CD58 /CD66c /CD38/CD34 /CD10 /CD20 /CD19/CD45
Tube2 CD65+CD15 /CD22 /CD33 /CD13 /HLA-DR /CD11b /CD45
Tube3 TdT/CD79a / CD34 /CD10 /CD19/CD45
Tube4 cμ/anti-7.1 /CD45
Tube5 sIg-κ/sIg-λ/CD45
Supplementary Table 2. The distribution of LAIPs combinations in 120 B-ALL samples and
their background expression in normal BM
Number of
cases in
B-ALL
% LAIPs+ cells in non-hematologic
malignancy BM
(analyzed samples)
% LAIPs+ cells in
regenerating BM
(analyzed samples)
2
＜0.001 (20)
0.002 (30)
5
＜0.001 (20)
＜0.001 (30)
CD10- CD34+ CD13+ 45d
2
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD13+ CD33+ 19s
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD58s CD19s
2
＜0.001 (20)
＜0.001 (30)
CD10- CD34+ CD38d CD19s 7.1+
2
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD38d 7.1+ 45d
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD58s 7.1+ CD15+
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD33+ CD19s
3
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD38d CD19s
1
＜0.001 (20)
＜0.001 (30)
CD10- CD34+ CD38d CD66c+ CD45d
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD38d CD13+CD66c+CD33+
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34+ CD38d CD33+ CD19d
1
＜0.001 (10)
＜0.001 (15)
LAIPs combinations
CD10-1 CD34+2
3
CD10- CD34+ CD58s CD38d
4
0.002
2
CD10d CD34+ CD38d CD66c+
2
＜0.001 (10)
＜0.001 (15)
CD10d CD34+ CD38d CD13+33+ 45d
3
＜0.001 (10)
＜0.001 (15)
CD10d CD34+ CD38d CD66c+CD33+
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34- CD20- CD58s 7.1+ CD15+
4
＜0.001 (10)
＜0.001 (15)
CD10- CD34- CD20- CD58s CD38d
1
＜0.001 (20)
＜0.001 (30)
CD10+ CD34+CD 58s CD20+
2
＜0.001 (20)
＜0.001 (30)
CD10+ CD34+ CD38d CD19s 45d
5
＜0.001 (20)
＜0.001 (30)
CD10+ CD34+ CD58s
2
＜0.001 (20)
＜0.001 (30)
CD10+ CD34+ CD58s CD38d 45d
10
＜0.001 (20)
＜0.001 (30)
CD10+ CD34+ CD38d CD15+
1
＜0.001 (10)
＜0.001 (15)
0.002
(20)
0.002 (30)
CD10d CD34+ CD19s
(10)
0.007 (15)
CD10+ CD34+ CD66c+ CD19s
6
CD10+ CD34+ CD38d CD13+CD33+45d
7
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD58s CD38d CD33+
3
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD58s CD66c+ 45d
2
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD38d CD66c+ CD19s
8
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD58s CD38d CD33+CD66c+
6
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD38d CD66c+ 45d
2
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD38d CD33+CD66c+ CD19s 45d
5
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD58s CD66c+CD33+ CD19s 45d
1
＜0.001 (10)
＜0.001 (15)
CD10+ CD34+ CD58s CD66c+ CD20+ 45d
2
＜0.001 (10)
＜0.001 (15)
CD10+ CD34- CD13+ CD19d
2
0.002 (10)
0.006 (15)
CD10+ CD34- CD45-
1
＜0.001 (20)
＜0.001 (30)
CD10+ CD34- CD58s 45d
4
＜0.001 (20)
＜0.001 (30)
CD10+ CD34- CD58s 10d
2
＜0.001 (20)
＜0.001 (30)
CD10+ CD34- CD58s CD20s
3
＜0.001 (20)
＜0.001 (30)
CD10+ CD34- CD66c+ 45d
4
0.003 (10)
0.008 (15)
CD10+ CD34- CD58s CD19d 45d
1
＜0.001 (20)
＜0.001 (30)
CD10+ CD34- CD58s CD10+ CD20 CD66c+ 45d
1
＜0.001 (10)
＜0.001 (15)
CD10+ CD34- CD58s CD38d CD13+CD33+ 45d
1
＜0.001 (10)
＜0.001 (15)
CD10+ CD34- CD33+ CD20s
1
＜0.001 (10)
＜0.001 (15)
CD10- CD34- CD20+ 45d
2
0.003 (20)
0.002 (30)
Notes : -1, negative; +2, positive; s3, strong expression; d4, dim expression;  median percentage of LAIPs+ cells
Supplementary Table 3. The quantification of LAIPs+ blasts in dilution experiments
Dilution series
LAIPs+ blasts, Number (%)
Total WBC number
No.1
No.2
No.3
No.4
No.5
1×10
5
92485 (92.5)
90210 (90.2)
90433 (90.4)
85492 (85.5)
81649 (81.6)
1×10
5
40177 (40.2)
68269 (68.3)
33516 (33.5)
37129 (37.1)
60465 (60.5)
1:10
1×10
5
3590 (3.59)
9323(9.32)
3770 (3.77)
4665 (4.67)
7404 (7.40)
1:100
5×105
1371 (0.27)
3918 (0.78)
1455 (0.29)
1857 (0.37)
3357 (0.67)
1:1000
1×106
178 (0.018)
802 (0.080)
203 (0.020)
356 (0.036)
620 (0.062)
1:10000
2×106
33 (0.0017)
254 (0.013)
64 (0.0032)
64 (0.0032)
123 (0.0062)
1:20000
2×10
6
14 (0.0007)
120 (0.0060)
28 (0.0014)
38 (0.0019)
75 (0.0038)
2×10
6
7 (＜0.0005)
64 (0.0032)
10 (0.0005)
22 (0.0011)
38 (0.0019)
1:80000
2×10
6
3 (＜0.0005)
35 (0.0018)
7(＜0.0005)
12 (0.0006)
19 (0.0010)
1:100000
2×106
2 (＜0.0005)
32 (0.0016)
5(＜0.0005)
10 (0.0005)
15 (0.0008)
1:200000
2×106
2 (＜0.0005)
30 (0.0015)
6(＜0.0005)
10 (0.0005)
8(＜0.0005)
Normal BM
2×106
1 (＜0.0005)
30 (0.0015)
4(＜0.0005)
10 (0.0005)
6(＜0.0005)
Undiluted
1:1
1:40000