massively multi-parametric flow cytometry with mass spectrometer detection scott d. tanner, vladimir...
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Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection
Scott D. Tanner, Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky
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Eu153
Ho
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Flow Cytometer with (elemental) Mass Spectrometer Detection
•advantage
•metal-labeled tags
•bulk (average) assay
•cytometry (individual cell) assay
•bead assay
NN
O
O O
O
Au
Fluorophores: signal overlap limits multiplex capability
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449
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541
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748
771
794
wavelength
inte
nsity
Fluorophores: dynamic range challenge
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794
wavelength
inte
nsity
1.0
5.0
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0.8
2.0
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the ideal reporter tagnon-degradablenon-reactivenon-interferingmany “colors”completely independent detection channelsquantitative …
a close approximation:
Ch
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Ch
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Ch
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Ch
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Ch
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Ch
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Log
(sig
nal)
stable isotope metal atomsmeasured by mass spectrometry
10-6 abundance sensitivity
dynamic range of 108
ICP-MS detection of element-tagged cells
sample
auxiliary gas
plasma gas
~ 7500 K~ 1015 e-/cm3
sun’s surface
element labeledreagents
rf energy in
Potentially suitable elements and (unique mass) stable isotopes
24 elements: 67 isotopes
La1
Hf5
Re2
Ru6
Rh1
Ir2
Pd4
Pt4
Ag2
Au1
In2
Ce1
Pr1
Nd5
Sm6
Eu2
Gd5
Tb1
Dy4
Ho1
Er4
Tm1
Yb5
Lu1
13 lanthanides: 37 unique isotopes
polymer produced in collaboration with Prof. Mitch Winnik, Dep’t Chemistry, U of T
SC(=S)Ph
chelator produced in collaboration with Prof. Mark Nitz, Dep’t Chemistry, U of T
chelator
SH
NN
O
O O
O
isotope tag
linker and antibody
NN
O
O O
O
synthesis and use of metal-labeled tags
Antibody labeling protocol
reduce –S-S- groups
add Ln3+
ions
N
O
O
NOO
*
R
n
metal-tagged antibody
Ln(III)
ligands for Ln3+
attach polymer
S N
O
O
R N
O
O
*
n
antibody
reduce –S-S- groups
add Ln3+
ions
N
O
O
NOO
*
R
n
N
O
O
NOO
*
R
n
metal-tagged antibody
Ln(III)
ligands for Ln3+
attach polymer
S N
O
O
R N
O
O
*
n
SS N
O
O
R N
O
O
*
n
N
O
O
R N
O
O
*
n
antibody
Primary anti-CD33 antibodies labeled with lab. tag-Eu and reacted with MBA-4 cells
comparison tagging reagents
MAXPAR™ (primary tags)
commercial (secondary tags)
blank (commercial 20 tags)
Time /s0 100 200 300 400 500
Rh
Sig
nal /
Vol
ts
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1
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Rh+ Au+ cell 1
Rh+ Au+ cell 2
Rh+ Au+ cell 3
Rh+ Au+ cell 4
Rh+ Au+ cell 5
average of 4Rh- Au+ cells
Time /s0 100 200 300 400 500
Au
Sig
nal /
Vol
ts
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0.4
Rh+ Au+ cell 6
Rh+ Au+ cell 7
Rh+ Au+ cell 8
Buffer after Au cells
a bCellular Enumeration Using Rh DNA Intercalator
[Rh(phi)2bpy]3+
Analytical Protocol
triplicate tubes
live cells
CD45-Eu
30 min
1) wash PBS
2) centrifuge
3) fix 3.7% formaldehyde
4) stain metal-containing DNA intercalator
For bulk (solution) analysis:5) digest 37% HCl6) add 1.00 ppb Ir
Analyze by conventional ICP-MS
Normalize signals to Ir and Rh
For cytometric analysis:5) individual cell injection
Analyze by fast ICP-MS
Normalize signals Rh
KG1anormalized response (Ir, Rh, bkgd subtracted)10-1100101102mixCD33CD34CD38CD45CD54
CD33 PrCD34 TbCD38 HoCD45 EuCD54 Tm
THP-1normalized response (Ir, Rh, bkgd subtracted)10-1100101102mixCD33CD34CD38CD45CD54
CD33 PrCD34 TbCD38 HoCD45 EuCD54 Tm
A
B
.tagged antibodies
CD54-TmCD45-EuCD38-Ho (THP-1 higher expression)CD34-Tb (THP-1 lower expression)CD33-Pr (KG-1a low expression)
Individual and 5-plex bulk assaysKG1a
THP-1
KG1a, a primitive progenitor cell, shows 500x lower expression of
CD33 than THP-1, a more differentiated cell type
Angew. Chem. Int. Ed., 46, 1–5 (2007)
x400 20m
Cell Differentiation with (PMA, 72h)
PMA, 4α-Phorbol 12-Myristate 13-Acetate
10 surface markers + Rh intercalator for cell number normalization
x400 20m
DMSO 50 ng/ml PMA
THP-1
KG1a
147Sm145Nd
142Nd
174Yb
146Nd
139La
170Er
141Pr
165Ho
151Eu144Nd
176Yb
169Tm
164Dy
159Tb
171Yb
152Sm
153Eu
166Er
156Gd
CD19
CD3
CD64
CD15
CD20HLA-DR
CD14
CD4
CD123
CD117
CD7
CD56
CD33
CD38
CD44CD13
CD49d
CD34
CD36
CD45
10 0 10 1 10 2 10 3
20-plex cell surface marker2 leukemic cell lines and
one patient sample
characteristic of undifferentiated
cells
THP-1 FAB M5
KG1a FAB M0
BCLQ Patient M5a
isotopically enriched tags
Flow - ICP-TOF-MS
every cell ?
or pre-selected cells ?
photo (“rear”) of Research Prototype CyTOF™ instrument
Phase 0 CyTOF™ Prototype
real time screen capture during data acquisitionKG-1a cellsfixed, Ir- intercalator CD7-139La CD13-144NdCD44-151EuCD45-159TbCD38-165Ho CD34-169Tm
m/z 139 144 151 159 165 169 191 193
Time Of Flight (mass)
scan
nu
mb
er (@12
s)
Yb
175.
9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
fixed, Ir- intercalatorCD7-139La, CD13-144Nd,CD44-151Eu,CD45-159Tb,CD38-165Ho, CD34-169TmCD49d-176Yb
KG-1a cells multiplex detectionAugust 3, 2007research bread-board
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Pr141
Eu
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Pr141
Eu
153
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Pr141
Tb
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Pr141
Ho
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Pr141
Tm
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Eu151
Eu
153
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Eu151
Tb
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Eu151
Ho
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Eu151
Tm
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0 2 4 6 8 10 12
Eu153
Tb
159
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Eu153
Ho
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0 2 4 6 8 10 12
Eu153
Tm
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Tb159
Ho
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Tb159
Tm
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Ho165
Tm
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151Eu
153Eu
159Tb
165Ho
169Tm
2-D plots for DTPA-metal labeled beads measured with ICP-MS-based cytometer for mixture of two bead types.
Ho, Tm beads(total 327 events)
Pr,Eu,Tb beads(total 461 events)
151Eu 153Eu 159Tb 165Ho141Pr
m/z
m/z
O
AuGene A
Probe A
O
AuGene B
Probe B
Cell mRNA cDNA cDNA-Au(RT-PCR) (labeling)
(labeling)
mRNA-Au
0.8-3 mm
Polystyrene beads
Ru Rh
Dy
Ru
Ce
Gd
Au
Au
Next: massively multiplexed bead assay
30,000 distinguishable beads = 30-fold redundancy assay for
1000 genes@ 1kHz = 30 seconds
http:/www/uhnres.utoronto.ca/studies/stemspec
MAXPAR™ reagents CyTOF™ analyzer
http://www.DVSsciences.com
Acknowledgements – funding
Genome Canada through the Ontario Genomics InstituteOntario Cancer Research NetworkMaterials and Manufacturing OntarioNational Institutes of HealthUniversity of TorontoMDS SciexDVS Sciences Inc.CytopeiaParker Life Sciences Leybold Vacuum GmBHETP division of SGECCR/MKS Process Products