Transcript Slide 1

Massively Multi-parametric Flow Cytometry
with Mass Spectrometer Detection
Scott D. Tanner,
Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky
Flow Cytometer with (elemental) Mass Spectrometer Detection
•advantage
•metal-labeled tags
O
N
O
•bulk (average) assay
•cytometry (individual cell) assay
12
•bead assay
10
Ho165
8
Au
6
4
2
0
0
2
4
6
Eu153
8
10
12
O
N
O
Fluorophores: signal overlap limits multiplex capability
100
90
80
60
50
40
30
20
wavelength
794
771
748
725
702
679
656
633
610
587
564
541
518
495
472
449
426
0
403
10
380
intensity
70
Fluorophores: dynamic range challenge
140
2.0
5.0
120
80
60
1.0
0.8
40
0.6
0.5
0.4
20
wavelength
794
771
748
725
702
679
656
633
610
587
564
541
518
495
472
449
426
403
0
0.3
380
intensity
100
the ideal reporter tag
stable isotope metal atoms
measured by mass spectrometry
 10-6 abundance sensitivity
 dynamic range of 108
Ch 60
Ch 50
Ch 40
Ch 30
Ch 20
Log (signal)
a close approximation:
Ch 10
non-degradable
non-reactive
non-interfering
many “colors”
completely independent detection channels
quantitative
…
ICP-MS detection of
element-tagged cells
plasma gas
auxiliary gas
sample
element
labeled
reagents
rf energy in
~ 7500 K
~ 1015 e-/cm3
 sun’s surface
Potentially suitable elements and
(unique mass) stable isotopes
13 lanthanides: 37 unique isotopes
24 elements: 67 isotopes
Ru Rh Pd Ag
6
1
4
2
La
1
Hf
5
Re
2
Ce Pr Nd
1
1
5
Ir
2
In
2
Pt Au
4
1
Sm Eu Gd Tb Dy Ho Er Tm Yb Lu
6
2
5
1
4
1
4
1
5
1
synthesis and use of metal-labeled tags
SC(=S)Ph
chelator
SH
polymer produced in collaboration with
Prof. Mitch Winnik, Dep’t Chemistry, U of T
linker and antibody
O
O
N
chelator produced in collaboration with
Prof. Mark Nitz, Dep’t Chemistry, U of T
N
O
O
isotope tag
O
N
O
O
N
O
Antibody labeling protocol
ligands for
*
Ln 3+
n
antibody
O
O
O
N
Ln(III)
R
N
O
reduce –S-S- groups
attach polymer
*
n
O
O
S
N
O
R
N
O
metal-tagged antibody
add Ln 3+
ions
comparison tagging reagents
Primary anti-CD33 antibodies labeled with lab. tag-Eu and
reacted with MBA-4 cells
MAXPAR™ (primary tags)
commercial (secondary tags)
blank (commercial 20 tags)
Cellular Enumeration Using Rh DNA Intercalator
[Rh(phi)2bpy]3+
b
Rh+ Au+ cell 1
3
Rh+ Au+ cell 6
Rh+ Au+ cell 2
Rh+ Au+ cell 7
Rh+ Au+ cell 3
Rh+ Au+ cell 8
Rh+ Au+ cell 4
Buffer after
Au cells
Rh Signal /Volts
Rh+ Au+ cell 5
0.4
average of 4
Rh- Au+ cells
2
0.2
1
0
0
100
200
300
Time /s
400
500
0
100
200
300
Time /s
400
0.0
500
Au Signal /Volts
a
Analytical Protocol
triplicate
tubes
CD45-Eu
1) wash PBS
30 min
2) centrifuge
3) fix 3.7% formaldehyde
4) stain metalcontaining DNA
intercalator
live cells
For bulk (solution) analysis:
5) digest 37% HCl
6) add 1.00 ppb Ir
For cytometric analysis:
5) individual cell injection
Analyze by conventional ICP-MS
Analyze by fast ICP-MS
Normalize signals to Ir and Rh
Normalize signals Rh
Individual and 5-plex bulk assays
KG1a
KG1a
KG1a, a primitive progenitor cell,
shows 500x lower expression of
CD33 than THP-1, a more
differentiated cell type
CD54
CD45
CD38
CD34
CD33
mix
CD33 Pr
CD34 Tb
CD38 Ho
CD45 Eu
CD54 Tm
A
.
10-1
tagged antibodies
CD54-Tm
CD45-Eu
CD38-Ho (THP-1 higher expression)
CD34-Tb (THP-1 lower expression)
CD33-Pr (KG-1a low expression)
100
101
102
normalized response
(Ir, Rh, bkgd subtracted)
THP-1
THP-1
CD54
CD45
CD38
CD34
CD33
mix
CD33 Pr
CD34 Tb
CD38 Ho
CD45 Eu
CD54 Tm
B
10-1
Angew. Chem. Int. Ed., 46, 1–5 (2007)
100
101
102
normalized response
(Ir, Rh, bkgd subtracted)
Cell Differentiation with (PMA, 72h)
DMSO
50 ng/ml PMA
KG1a
x400
20m
THP-1
10 surface markers + Rh intercalator for cell number normalization
x400
20m
PMA, 4α-Phorbol 12-Myristate 13-Acetate
KG1a FAB M0
THP-1 FAB M5
BCLQ Patient M5a
142Nd
174Yb
146Nd
145Nd
CD14
147Sm
HLA-DR
20-plex cell surface marker
2 leukemic cell lines and
one patient sample
156
Gd
CD20
CD4
CD15
166Er
153Eu
CD123
CD64
152Sm
CD117
139La
CD3
CD7
10
0
10
1
10
170Er CD56
2
10
CD19 171Yb
3
CD45
141Pr CD33
CD36
165HoCD38
CD34
CD44
151Eu
isotopically enriched tags
CD13
144Nd
CD49d
176Yb
159Tb
164Dy
169Tm
characteristic of
undifferentiated
cells
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 acquisition
KG-1a cells
fixed, Ir- intercalator
CD7-139La
CD13-144Nd
CD44-151Eu
CD45-159Tb
CD38-165Ho
CD34-169Tm
Time Of Flight (mass)
scan number (@12s)
m/z
139 144
151
159
165
169
191 193
KG-1a cells
multiplex detection
August 3, 2007
research bread-board
Yb 175.9
fixed, Ir- intercalator
CD7-139La,
CD13-144Nd,
CD44-151Eu,
CD45-159Tb,
CD38-165Ho,
CD34-169Tm
CD49d-176Yb
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
Yb 175.9
2-D plots for DTPA-metal labeled beads
measured with ICP-MS-based cytometer for
mixture of two bead types.
12
10
151Eu
Eu151
8
6
4
2
0
0
2
4
6
8
10
12
12
12
10
10
8
8
Eu153
6
6
4
4
2
2
0
2
4
6
8
10
0
12
2
4
10
8
8
8
6
Tb159
12
10
Tb159
6
6
4
4
4
2
2
2
0
0
2
4
6
8
10
0
0
12
Ho, Tm beads
(total 327 events)
2
4
6
8
10
0
12
2
4
6
8
10
12
Eu153
Eu151
12
12
10
10
10
8
8
8
8
6
Ho165
12
10
Ho165
12
6
Ho165
6
6
4
4
4
4
2
2
2
2
0
0
0
2
4
6
8
10
0
12
2
4
6
8
10
0
0
12
2
4
6
8
10
12
0
2
4
Eu153
Eu151
Pr141
6
8
10
12
Tb159
12
12
12
10
10
10
10
8
8
8
8
8
6
Tm169
12
10
Tm169
12
6
6
Tm169
Tb159
Ho165
12
12
0
Tm169
10
10
Pr141
169Tm
8
12
0
165Ho
6
Eu151
Pr141
159Tb
Pr,Eu,Tb beads
(total 461 events)
0
0
Tm169
153Eu
Eu153
Pr141
6
6
4
4
4
4
4
2
2
2
2
2
0
0
0
0
0
2
4
6
8
Pr141
141Pr
10
12
0
2
4
6
8
Eu151
151Eu
10
12
0
2
4
6
8
10
Eu153
153Eu
12
0
0
2
4
6
8
Tb159
159Tb
10
12
0
2
4
6
8
Ho165
165Ho
10
12
Next: massively multiplexed bead assay
Cell
mRNA
(RT-PCR)
cDNA
(labeling)
cDNA-Au
Ru Rh
mRNA-Au
Polystyrene beads
Gene A
Au
Au
Dy
O
m/z
Probe A
Ru
Au
Gene B
Ce
Au
Gd
O
Probe B
0.8-3 mm
m/z
30,000 distinguishable beads =
30-fold redundancy assay for
1000 genes@ 1kHz = 30 seconds
http:/www/uhnres.utoronto.ca/studies/stemspec
http://www.DVSsciences.com
[email protected]
MAXPAR™ reagents
CyTOF™ analyzer
Acknowledgements – funding
Genome Canada through the Ontario Genomics Institute
Ontario Cancer Research Network
Materials and Manufacturing Ontario
National Institutes of Health
University of Toronto
MDS Sciex
DVS Sciences Inc.
Cytopeia
Parker Life Sciences
Leybold Vacuum GmBH
ETP division of SGE
CCR/MKS Process Products