This document discusses new technologies for single cell detection and analysis using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). It describes a method for labelling T cells with gadolinium for tracking cell-based therapies. A new laser ablation system design is presented that improves speed and sensitivity for single cell analysis by using a small inner chamber and single diameter transport tubing to minimize sample loss. The system enables detection of labelled cells for over 10 days both in vitro and in mouse studies.

1. New Technologies for Single Cell
Detection
1
Amy J. Managh
Centre for Analytical Science, Department of Chemistry,
Loughborough University, UK
A.J.Managh@lboro.ac.uk

2. Presentation Outline
 Why do we need single cell detection?
 Application to healthcare monitoring
 The drive to develop new technology
2

3. Why analysis at the single cell level?
 The ONE Study - a major collaborative project to evaluate the use of
regulatory immune cells in kidney transplantation.
 Monitoring the survival and tissue distribution of administered cells is
crucial to understanding their immunomodulatory effects in vivo.
 Tracking cell based therapies is problematic:
 The administered cells are indistinguishable from the patients
own cells using conventional detection methods.
 The cells are massively infrequent compared to the patients
own cells.
3

4. 3 Laser ablation
Sampling
2
1 Administration of labelled cells to patients
Au
ICP-MS45 Data processing
4
Cell tracking methodology

5. 5
0.E+00
1.E+08
2.E+08
3.E+08
4.E+08
0 2 4 6 8
Gdatomspercell
Dose / mg Gd per ml
Gd-DOTA (Dotarem)
Gd-DTPA-BMA (Omniscan)
T cell labelling
Managh et al., Analytical Chemistry. 2013, 85, 10627−10634.

6. 6
T cell labelling
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
0 1 2 4 8 16
Gdatomspercell
Incubation time / h
Gd-DOTA (Dotarem)
Gd-DTPA (Omniscan)
Managh et al., Analytical Chemistry. 2013, 85, 10627−10634.

7.  100% cell detection efficiency at day 1 in vitro.
 98.8% cell detection efficiency at day 10 in vitro. 7
Single cell laser ablation
7

8. Single cell tracking in mice
8Managh et al., Analytical Chemistry. 2013, 85, 10627−10634.
 Labelled cells administered IP
to mice.
 Peritoneal lavage at 3, 6 and
10 days.
 Cells successfully detected at 10 days post-administration!
 Experiments at University of Oxford (Andrew Bushell) indicate that the
labelling does not affect the ability of T reg to regulate rejection.

9. 0
50
100
150
200
Heart Kidney Liver Lung Spleen Brain
Auperorgan/ng
Control
24 hour
7 day
Organ analysis by solution ICP-MS
0.0
0.5
1.0
Heart Kidney Liver Lung Spleen Brain
Auperorgan/ng
Expanded
view
Managh et al., Journal of Immunology. 2014, 193, 2600-2608. 9

10. Laser ablation imaging
10

11. Laser ablation imaging
11
Lung – 24 h Liver – 7 d
Managh et al., Journal of Immunology. 2014, 193, 2600-2608.

12. The drive to develop new technology
 Speed – Long particle residence times = long analysis times for spot resolved
data.
 Current wash out times: 700 ms for commercial systems, lower for
research cells.
 Sensitivity – Analysis of small sample masses, e.g. single cell analysis,
requires high transport efficiency.
 Uniform response – Spatially dependent transport efficiencies may impair
image resolution and signal response.
121) S. Shuttleworth, www.cetac.com/pdfs/PST025.pdf.

13. sample
He
ablation
gas in
A standard laser ablation system
(1) Large chamber volume:
plume expansion + loss of
sample on walls
(2) Loss of sample on
walls of tubing
Laser beam
13

14. Tubing can be in excess of 1 m in length!
(3) Peak broadening
14

15. Argon
(4) Turbulent
mixing of gas flows
(5) Disruption of gas flows due to
the change in diameter of the
transport conduit
(6) Loss of sample on
mass spec interface
15
Argon
RF coil

16. Overview of the New Design
Magnetically coupled
sample tray
Laser
The “Sniffer” – a low
volume inner cell
16
Argon RF coil

17. The Outer “Enterprise” Chamber
17

18. The Outer “Enterprise” Chamber
The
Sniffer
18

19. Laser
The Inner “Sniffer” Cell
Fused silica
capillary
0.005cm3 inner volume
19

20. Overview of the New Design
Laser
Single diameter fused silica conduit
(250 µm i.d. and typically 65 cm length).
Integrated design. No valves or T-pieces.
Dual Concentric Injector (DCI) extends beyond the
turbulent region at the base of the plasma
Argon
20

21. The Dual Concentric Injector (DCI)
Inner channel
21

22. Results
22
Fused silica Injector12 mm
extension
6 mm
extension
3 mm
extension
0 mm
extension
• Peaks now ~10 ms – a 50 fold reduction!
• Up to 6-10 fold increase in sensitivity.

23. Conclusion and outlook
23
NWR imaging platform
 Single cell analysis is set to enable monitoring of cell based therapies
in patient trials.
 Improvements to LA-ICP-MS technology dramatically improves the
speed and sensitivity of tissue and cell imaging.
 The data presented here was
acquired using a prototype
system.
 LU are currently collaborating
with an industrial partner to
integrate the technology into
their ablation system.

24. 24
University of Oxford
Andrew Bushell
Sheldon Edwards
Kathryn Wood
University Hospital Regensburg
James Hutchinson
Ed Geissler
Nantes University Hospital
Aurelie Moreau
Cristina Cuturi
Electro Scientific Industries
Rob Hutchinson
The Analytical research group at
Loughborough University
Acknowledgements