Massively Multiparametric Flow Cytometry with Mass Spectrometer Detection

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 • bulk (average) assay • cytometry (individual cell) assay • bead assay Au

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-/cm 3 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 5 1

synthesis and use of metal-labeled tags SC(=S)Ph chelator polymer produced in collaboration with Prof. Mitch Winnik, Dep’t Chemistry, U of T linker and antibody chelator produced in collaboration with Prof. Mark Nitz, Dep’t Chemistry, U of T isotope tag SH

Antibody labeling protocol

comparison tagging reagents Primary anti-CD 33 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)2 bpy]3+

Analytical Protocol triplicate tubes CD 45 -Eu 1) wash PBS 3) fix 3. 7% formaldehyde 30 min 2) centrifuge 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 KG 1 a, a primitive progenitor cell, shows 500 x lower expression of CD 33 than THP-1, a more differentiated cell type tagged antibodies CD 54 -Tm CD 45 -Eu CD 38 -Ho (THP-1 higher expression) CD 34 -Tb (THP-1 lower expression) CD 33 -Pr (KG-1 a low expression) Angew. Chem. Int. Ed. , 46, 1– 5 (2007) THP-1

Cell Differentiation with (PMA, 72 h) DMSO 50 ng/ml PMA KG 1 a x 400 20 mm THP-1 10 surface markers + Rh intercalator for cell number normalization x 400 20 mm PMA, 4α-Phorbol 12 -Myristate 13 -Acetate

KG 1 a FAB M 0 THP-1 FAB M 5 BCLQ Patient M 5 a 142 Nd 174 Yb 146 Nd 145 Nd CD 14 147 Sm HLA-DR 20 -plex cell surface marker 2 leukemic cell lines and one patient sample 156 CD 20 CD 4 Gd CD 15 166 Er 153 Eu CD 123 CD 64 152 Sm CD 117 139 La CD 3 CD 7 10 0 10 170 Er CD 56 2 10 CD 19 171 Yb 3 CD 45 141 Pr CD 33 CD 36 165 Ho. CD 38 CD 34 CD 44 151 Eu isotopically enriched tags CD 13 144 Nd CD 49 d 176 Yb 159 Tb 164 Dy 169 Tm characteristic of undifferentiated cells

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

photo (“rear”) of Research Prototype Cy. TOF™ instrument

Phase 0 Cy. TOF™ Prototype

real time screen capture during data acquisition KG-1 a cells fixed, Ir- intercalator CD 7 -139 La CD 13 -144 Nd CD 44 -151 Eu CD 45 -159 Tb CD 38 -165 Ho CD 34 -169 Tm Time Of Flight (mass) scan number (@12 s) m/z 139 144 151 159 165 169 191 193

KG-1 a cells multiplex detection August 3, 2007 research bread-board Yb 175. 9 fixed, Ir- intercalator CD 7 -139 La, CD 13 -144 Nd, CD 44 -151 Eu, CD 45 -159 Tb, CD 38 -165 Ho, CD 34 -169 Tm CD 49 d-176 Yb 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. 151 Eu 153 Eu Pr, Eu, Tb beads (total 461 events) 159 Tb Ho, Tm beads (total 327 events) 165 Ho 169 Tm 141 Pr 151 Eu 153 Eu 159 Tb 165 Ho

Next: massively multiplexed bead assay Cell m. RNA (RT-PCR) (labeling) c. DNA-Au lin be (la g) Ru Rh m. RNA-Au Polystyrene beads Gene A Au Au Dy O m/z Probe A Ru Au Gene B O Probe B 0. 8 -3 mm Au Ce Gd m/z 30, 000 distinguishable beads = 30 -fold redundancy assay for 1000 genes@ 1 k. Hz = 30 seconds

http: /www/uhnres. utoronto. ca/studies/stemspec http: //www. DVSsciences. com sd. tanner@utoronto. ca MAXPAR™ reagents Cy. TOF™ 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 Gm. BH ETP division of SGE CCR/MKS Process Products