Table 1.

MRD levels in HSC-B collections from AML patients with 3- and 8-color technique

3-color FC technique / 8-color technique
No / Diagnosis FAB / MAb comba / MRD% in HSC-B
collection / MAb combb / MRD% in HSC-B
collection / Outcome
1 / M4 / CD2-117-34 / 0.04 / No 4 / 0.10 / CR
2 / M2 / CD34-7-19 / 0.06 / No 5 / 0.06 / CR
3 / M2 / CD65-34-33 / < 0.1 / No 4 / 0.02 / R - BMT
4 / M2 / CD33-13-38-34 / < 0.1 / No 2 / 0.01 / CR
5 / M0 / CD7-2-34 / 0.02 / No 4 / 0.02 / R/D
6 / M0 / CD33-13-34 / < 0.1 / No 2 / 0.02 / R - BMT

Hematopoietic Stem Cells in Blood(HSC-B), Bone Marrow Transplantation (BMT), Complete Remission (CR), Relapse(R), Death (D),HLADR (DR), subpopulation (s).

a.3-color technique combinations were tailored depending on phenotypes at diagnosis.

pat no1:positive for CD34(s), CD15, CD13, CD2(s), TdT, DR, dimCD45, negative for CD117 and CD33,

pat no2: positive for CD34, CD7, CD33, CD13, CD19, dimCD45, negative for CD117, pat no 3: positive for CD34, CD65, CD13, strongDR, CD38, dimCD45, negative for CD117 and CD33,

pat no 4:positive for CD34, CD13, CD15, CD123, DR, CD38, dimCD4(s), dimCD45, negative for CD33 and 117,

pat no 5: positive for CD34, CD117, CD7, CD13, CD15(s), DR, CD38, TdT, dimCD45, CD2 (s), negative for CD33,

pat no 6: positive for CD34, CD13, DR(s) dimCD4, dimCD45, negative for CD33 and CD117

b.The best combination detecting MRD with the standard 8-color MAb combinations panel (Table 2).

Table 2. Five 8-color MAb combinations used to delineate CD34+ cell subsets

Combination / FITC / PE / PerCP5.5a or
PE-Cy5b / PE-Cy7 / APC / APC-Cy7 / PA / AM
1 / CD38 / CD90 / CD34b / CD33 / CD133 / HLADR / CD45RA / CD45
2 / CD13 / CD133 / CD33a / CD117 / CD34 / HLADR / CD4 / CD45
3 / CD38 / CD45RO / CD34b / CD13 / CD117 / HLADR / CD45RA / CD45
4 / CD15 / CD45RO / CD33a / CD34 / CD117 / CD14 / CD45RA / CD45
5 / TdT / CD7 / CD61a / CD13 / CD34 / HLADR / CD19 / CD45

MAbs directly conjugated with Fluoresceinisothiocyanate (FITC), Phycoerythrin (PE), Peridin chlorophyll protein 5.5 (PerCP5.5) or PE-cyanine 5 (PE-Cy5), PE-cyanine 7 (PE-Cy7), Allophycocyanine (APC), Allophycocyanine 7(APC-Cy7), Pacific Blue (PA), AmCyan (AM)

Antibody source: CD133PE and CD133APC from MACS, Milteyi Biotec, Germany, CD13F and CD38F from Dako, Glostrup, Denmark, CD7PE, CD34 PECy5, CD45ROPE and CD117APC from Immunotech, Marseille, France,CD117PECy-7, CD19PA and CD45RAPA from Biolegand, San Diego, USA and

all other antibodies fromBecton Dickinson (BD), San Jose, CA, USA.

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Table 3. Frequenciesaof various CD34 subpopulations in HSC-B performed with a standard 8-color panel.

Comb.1 / Total CD34+ / CD34+DR+CD133+ / CD34+DR+
CD133neg / CD34+DRneg
CD133neg / CD34+DRneg’
CD133+ / CD34+DR+CD133+
CD38+CD45RAd+ / CD34+DR+CD133+
CD38+CD45RAneg / CD34+DR+CD133+
CD38negCD45RAneg / CD34+DR+CD133+
CD38negCD45RAd+
Non AML
N=8 / 2.2
(0.62, 3.2)
[0.1-7.43] / 2.2 (0.55, 3.1)
[0.08-8.1] / 0.1 (0.03, 0.18)
[0-0.64] / <0.01
no cluster / <0.01
no cluster / 1.89 (0.53, 2.7)
[0.08-6.45] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.35
(0.25, 0.30)
[0.07-0.83] / 0.34 (0.20, 0.28)
[0.06-0.85] / 0.01 (0.01, 0.01)
[0-0.03] / <0.01
no cluster / <0.01
no cluster / 0.32 (0.23, 0.29)
[0.06-0.78] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
Comb.2 / Total CD34+ / CD34+DR+CD133+ / CD34+DR+
CD133neg / CD34+DRneg
CD133neg / CD34+DRneg
CD133+ / CD34+DR+CD133+
CD13+CD33+ / CD34+DR+CD133+
CD13+CD33neg / CD34+DR+CD133+
CD13negCD33neg / CD34+DR+CD133+
CD13negCD33+
Non AML
N=8 / 2.3
(0.57, 3.4)
[0.11-8.34] / 2.2 (0.55, 3.1)
[0.08-8.1] / 0.1 (0.03, 0.18)
[0-0.64] / <0.01
no cluster / <0.01
no cluster / 0.33 (0.33, 0.09)
[0.2-0.45] / 0.07 (0.07, 0.04)
[0.02-0.12] / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.37
(0.21, 0.32)
[0.11-0.94] / 0.34 (0.20, 0.28)
[0.06-0.85] / 0.01 (0.01, 0.01)
[0-0.03] / <0.01
no cluster / <0.01
no cluster / 1.8 (0.33, 2.8)
[0.09-7.3] / 0.45 (0.17, 0.67)
[0.01-2] / <0.01
no cluster / <0.01
no cluster
Comb.3 / Total CD34+ / CD34+DR+CD38+ / CD34+DRneg
CD38+ / CD34+DRneg
CD38neg / CD34+DR+
CD38neg / CD34+DR+CD38+
CD45ROd+
CD45RAd+ / CD34+DR+CD38+
CD45ROd+
CD45RAneg / CD34+DR+CD38+
CD45Roneg
CD45RAneg / CD34+DR+CD38+
CD45ROneg+
CD45RAd+
Non AML
N=8 / 2.37
(0.63, 3.5)
[0.08-9.4] / 2.3 (0.58, 3.5)
[0.08-9.35] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster / 2.0 (0.54, 3.0)
[0.07-8.06] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.26
(0.24, 0.17)
[0.05-0.5] / 0.25 (0.23, 0.17)
[0.05-0.49] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster / 0.19 (0.16, 0.13)
[0.05-0.41] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
Comb.4 / Total CD34+ / CD34+CD15neg
CD33+ / CD34+15neg
CD33neg / CD34+CD15+
CD33+ / CD34+CD15+
CD33neg / CD34+CD15neg
CD133+
CD45ROd+
CD45RAd+ / CD34+CD15neg
CD38neg
CD45RO+d
CD45Raneg / CD34+CD15+CD33+
CD45Roneg
CD45RAneg / CD34+CD15+CD33neg
CD45ROneg+
CD45RAd+
Non AML
N=8 / 1.8
(0.68, 2.1)
[0.39-5.6] / 1.0 (0.29, 1.5)
[0.01-4.1] / 0.23 (0.11, 0.29)
[0.02-0.99] / 0.31 (0.13, 0.36)
[0.02-0.99] / 0.07 (0.04, 0.06)
[0.01-0.16] / 0.99 (0.28, 1.5)
[0.01-3.9] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.36
(0.29, 0.25)
[0.09-0.78] / 0.18 (0.17, 0.11)
[0.06-0.35] / 0.015 (0.005, 0.027)
[0-0.07] / 0.13 (0.13, 0.11)
[0.01-0.3] / 0.02 (0.02, 0.02)
[0-0.06] / 0.17 (0.16, 0.11)
[0.05-0.33] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
Comb.5 / Total CD34+ / CD34+CD13+DR+ / CD34+CD13negDR+ / CD34+CD13neg
DRneg / CD34+CD13+
DRneg / CD34+CD13+DR+
CD7+CD61neg / CD34+CD13+DR+
CD7negCD61neg / CD34+CD13+DR+
CD7negCD61+ / CD34+CD13+DR+
CD7+CD61+
Non AML
N=8 / 2.1
(1.0, 1.9)
[0.5-5.4] / 1.7 (0.92, 1.6)
[0.4-4.6] / 0.37 (0.12, 0.42)
[0.09-1.2] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster / 1.7 (0.9, 1.5)
[0.39-4.5] / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.74
(0.53, 0.55)
[0.15-1.7] / 0.47 (0.40, 0.34)
[0.06-0.98] / 0.23 (0.12, 0.28)
[0.07-0.8] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster / 0.46 (0.40, 0.33)
[0.06-0.97] / <0.01
no cluster / <0.01
no cluster

a. All values in percentage (% ) of total cells and presented as: mean (median, SD) [range].

Table 3 ctd. Frequenciesa of various CD34 subpopulations in HSC-B performed with a standard 8-color panel.

Comb. 1 / CD34+DR+CD133+
CD38+CD45RAdim
CD90d+CD33+ / CD34+DR+CD133+
CD38+CD45RAdim
CD90d+CD33neg / CD34+DR+CD133+
CD38+CD45RAdim
CD90negCD33neg / CD34+DR+CD133+
CD38+CD45RAdim
CD90negCD33+
Non AML
N=8 / 1.7 (0.4, 2.5)
[0.08-6] / <0.05
no cluster / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.29 (0.22, 0.26)
[0.06-0.72] / <0.05
no cluster / <0.01
no cluster / <0.01
no cluster
Comb. 2 / CD34+DR+CD133+
CD13+CD33+
CD4d+CD117+ / CD34+DR+CD133+
CD13+CD33+
CD4negCD117+ / CD34+DR+CD133+
CD13+CD33+
CD4negCD117neg / CD34+DR+CD133+
CD13+CD33+
CD4d+CD117neg
Non AML
N=8 / 1.2 (0.27, 1.9)
[0.06-5,2] / 0.38 (0.06, 0.60)
[0.02-1.4] / 0.04 (0.08, 0.005)
[0 – 0.23] / 0.11 (0.27 , 0.18)
[0.01-0.51]
AML
N=6 / 0.20
(0.16, 0.16)
[0.05-0.47] / 0.05 (0.03, 0.06)
[0.01-0.18] / <0.05
no cluster / <0.05
no cluster
Comb. 3 / CD34+DR+CD38+
CD45ROd+45RAd+
CD13+CD117+ / CD34+DR+CD38+
CD45ROd+45RAd+
CD13+CD117neg / CD34+DR+CD38+
CD45ROd+45RAd+
CD13negCD117neg / CD34+DR+CD38+
CD45ROd+45RAd+
CD13negCD117+
Non AML
N=8 / 1.4 (0.42, 2.0)
[0.05-4.9] / 0.54 (0.1, 1.0)
[0.02-3,0] / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.13 (0.10, 0.1)
[0.03-0.26] / 0.04 (0.01, 0.06)
[0-0.015] / <0.01
no cluster / <0.01
no cluster
Comb. 4 / CD34+CD15neg
CD33+
CD45ROd+45RAd+
CD14negCD117+ / CD34+CD15neg
CD33+
CD45ROd+45RAd+
CD14negCD117neg / CD34+CD15neg
CD33+
CD45ROd+45RAd+
CD14d+CD117neg / CD34+CD15negCD33+
CD45ROd+45RAd+
CD14d+CD117+
Non AML
N=8 / 1.0 (0.27, 1.4)
[0.01-3.8] / 0.04 (0.21, 0.07)
[0.08-0.37] / <0.01
no cluster / 0.32 (0.28, 0.14)
[0.11-0.58]
AML
N=6 / 0.15 (0.16, 0.08)
[0.05-0.25] / <0.01
no cluster / <0.01
no cluster / 0.17 (0.16, 0.09)
[0.07-0.31]
Comb. 5 / CD34+CD13+DR+
CD7negCD61neg
CD19neg
TdTneg/d+ / CD34+CD13+DR+
CD7negCD61neg
CD19+TdTneg/d+ / CD34+CD13+DR+
CD7negCD61neg
CD19+TdT+ / CD34+CD13+DR+
CD7negCD61neg
CD19negTdT+
Non AML
N=8 / 1.7 (0.9, 1.5)
[0.38-4.44] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster
AML
N=6 / 0.45 (0.39, 0.33)
[0.06-0.96] / <0.01
no cluster / <0.01
no cluster / <0.01
no cluster

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