1. Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky
Massively Multi-parametric Flow Cytometry with Mass Spectrometer Detection
Scott D. Tanner,
Vladimir I. Baranov, Dmitry R. Bandura, Olga Ornatsky

2. Flow Cytometer with (elemental) Mass Spectrometer Detection
advantage
metal-labeled tags
bulk (average) assay
cytometry (individual cell) assay
bead assay Au

3. Fluorophores: signal overlap limits multiplex capability10 20 30 40 50 60 70 80 90
100
380
403
426
449
472
495
518
541
564
587
610
633
656
679
702
725
748
771
794
wavelength
intensity
Conventional single cell assays by flow cytometry use fluorescent tags. Overlap of the emission spectra limit the method to about 4-plex (typically; efforts are being made to extend this to >10-plex, but with inherent compensation problems).

4. Fluorophores: dynamic range challenge20 40 60 80
100
120
140
380
403
426
449
472
495
518
541
564
587
610
633
656
679
702
725
748
771
794
wavelength
intensity
2.0
5.0
Fluorophore spectra scaled to the relative peak amplitudes shown.
1.0
0.8
0.6
0.5
0.4
0.3

5. the ideal reporter tag non-degradable non-reactive non-interfering
many “colors”
completely independent detection channels
quantitative …
a close approximation:
Ch 10
Ch 20
Ch 30
Ch 40
Ch 50
Ch 60
Log (signal)
stable isotope metal atoms
measured by mass spectrometry
10-6 abundance sensitivity
dynamic range of 108

6. ICP-MS detection of element-tagged cells
~ 7500 K
~ 1015 e-/cm3
 sun’s surface
rf energy in
sample
auxiliary gas
plasma gas
Our approach is to use elements (of the periodic table) as tags, and to atomize and ionize the contents of each cell. The elemental signature of the cell includes the element tags introduced with the antibodies. When chosen to be uncommon in biological samples, the presence of the tag element indicates that the antibody found and bound the target biomarker, and the intensity of the signal is directly proportional to the copy-count (absolute quantification).
element
labeled
reagents

7. Potentially suitable elements and (unique mass) stable isotopes
13 lanthanides: 37 unique isotopes
24 elements: 67 isotopes La 1 Hf 5 Re 2 Ru 6 Rh Ir Pd 4 Pt Ag Au In Ce 1 Pr Nd 5 Sm 6 Eu 2 Gd Tb Dy 4 Ho Er Tm Yb Lu

8. synthesis and use of metal-labeled tags
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
linker and antibody
isotope tag

9. Antibody labeling protocol

10. 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)

11. Cellular Enumeration Using Rh DNA Intercalator
[Rh(phi)2bpy]3+

12. For bulk (solution) analysis:
Analytical Protocol
triplicate tubes
CD45-Eu
1) wash PBS
3) fix 3.7% formaldehyde
30 min
2) centrifuge
4) stain metal-containing DNA intercalator
live cells
For cytometric analysis:
5) individual cell injection
For bulk (solution) analysis:
5) digest 37% HCl
6) add 1.00 ppb Ir
Analyze by conventional ICP-MS
Normalize signals to Ir and Rh
Analyze by fast ICP-MS
Normalize signals Rh

13. Individual and 5-plex bulk assays
KG1a
KG1a, a primitive progenitor cell, shows 500x lower expression of CD33 than THP-1, a more differentiated cell type
tagged antibodies
CD54-Tm
CD45-Eu
CD38-Ho (THP-1 higher expression)
CD34-Tb (THP-1 lower expression)
CD33-Pr (KG-1a low expression)
THP-1
Angew. Chem. Int. Ed., 46, 1–5 (2007)

14. 10 surface markers + Rh intercalator for cell number normalization
Cell Differentiation with (PMA, 72h) 
DMSO
50 ng/ml PMA
KG1a
x400
20mm
THP-1
Cell differentiation with PMA. THP-1 representative of promonocytic AML-M5, K562 chronic myeloid leukemia cells, and KG1a (early myeloblasts, AML-MO) were treated with PMA for 72 hr. After treatment cells were washed and immunolabeled with a mixture of 10 element-tagged antibodies or for background control – element-tagged mouse IgG for 30 min RT. Washed cells were fixed in formaldehyde and DNA stained with Rh(III)-containing metallointercalator. Finally, cells were washed several times and dissolved in HCl. Polar diagrams in logarithmic scale represent data fro triplicate samples normalized to internal standard (1ppb Ir). Notice that DNA-Rh is identical for DMSO or PMA treated cells. For THP-1 differentiation, the cells change morphology and increase cell surface attachment (microphotograph), this is reflected by upregulation in the expression of adhesion receptors: a 10-fold increase in ICAM-1 (intercellular adhesion molecule , CD54) and 2-fold increase in the hyaluronan (CD44) and vitronectin receptors (CD51); while tyrosine phosphatase content (CD45), CD33 (myeloid precursor marker), and receptor for fibrinogen (CD41) are unchanged, while CD38 (NAD glycohydrolase and ADP ribosylcyclase) is decreased. THP-1 does not express significant levels of CD34 (similar low levels in DMSO and PMA).
As an interesting control to the assay is the case of KG1a cells which are known to be completely unresponsive to PMA stimulation, unlike their parent clone KG1.
10 surface markers + Rh intercalator for cell number normalization
x400
20mm
PMA, 4α-Phorbol 12-Myristate 13-Acetate

15. characteristic of undifferentiated cells
KG1a
FAB M0
20-plex cell surface marker
2 leukemic cell lines and
one patient sample
THP-1
FAB M5
BCLQ
Patient M5a
147Sm
145Nd
142Nd
174Yb
146Nd
139La
170Er
141Pr
165Ho
151Eu
144Nd
176Yb
169Tm
164Dy
159Tb
171Yb
152Sm
153Eu
166Er
156Gd
HLA-DR
CD14
CD20
CD4
CD15
CD123
CD64
CD117
CD3
CD7
CD19 10 1 2 3
CD56
CD45
CD33
CD36
CD38
CD34
CD44
CD49d
CD13
characteristic of undifferentiated cells
isotopically enriched tags

16. Flow - ICP-TOF-MS
every cell ? or
pre-selected cells ?

17. photo (“rear”) of Research Prototype CyTOF™ instrument

18. Phase 0 CyTOF™ Prototype

19. 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)
m/z
scan number

20. 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

21. 2-D plots for DTPA-metal labeled beads measured with ICP-MS-based cytometer for mixture of two bead types.
151Eu
153Eu
Pr,Eu,Tb beads (total 461 events)
159Tb
Ho, Tm beads (total 327 events)
DTPA conjugated 1.8 um Amine-modified Beads
Differential labelling of beads with two (Tm, Ho) and three (Tb, Pr, Eu) lanthanide mixtures .
165Ho
169Tm
141Pr
151Eu
153Eu
159Tb
165Ho

22. Next: massively multiplexed bead assay
(RT-PCR)
Cell mRNA cDNA cDNA-Au
(labeling)
(labeling)
Ru Rh
mRNA-Au Au Au
Polystyrene beads
Gene A Dy O
m/z
Proposed metal-tagged gene expression assay using metal-imbibed multiplexed beads. Antisense oligos are linked to beads labeled to differentiate the target gene; PCR can be arranged to add an oligo linker tail (e.g., polyA as for mRNA) that can be linked to an antisense metal-tagged oligo, or can be coded to incorporate metals (already done for fluorophores); concomitant signals in cytometric analysis determine the target genes.
Probe A Ru Au Au Ce
Gene B Gd O
m/z
Probe B
0.8-3 mm
30,000 distinguishable beads =
30-fold redundancy assay for kHz = 30 seconds

23. http:/www/uhnres.utoronto.ca/studies/stemspec
MAXPAR™ reagents
CyTOF™ analyzer

24. 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