Comparison between ChipCytometryand Flow Cytometry for Biomarkers and Immunophenotyping Applications
Rong Zeng, Ph.D
OncoMed Pharmaceuticals
10th WRIB (Orlando, FL)
April 22, 2016
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Overview
• Introduction
• Technology
• Case studies– Immunophenotyping
– Biomarkers
• Summary and discussion
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Issues with conventional flow cytometry
• Flow cytometry is a platform for analyzing cellular heterogeneity and identifying biomarkers in clinical settings, widely used for immunomonitoring, biomarker discovery, and disease diagnostics
• However, the samples have to be run real-time (within 1-3 days) and discarded post-analysis
• Costly sample handling for multi-center clinical trials
• Impossible for reanalysis of the same sample
• Limited parameters/colors for unravelling the complex immune system
• Cumbersome color compensation
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ChipCytometry: alternative cytometric platform
• Image-based cytometric system for high-parameter biomarker analysis/re-analysis of the same sample
• Cells or tissue sections are immobilized on microfluidic chips for long-term storage
• Over 80 cell surface/intracellular markers are validated
• Two systems are developed and commercialized by Zellkraftwerk(Hannover, Germany):– ZellScannerONE (benchtop instrument)
– CYTOBOT (automated analyzer)
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Comparison of next generation single-cell analysis platforms
Zellkraftwerk
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Overview
• Introduction
• Technology
• Case studies– Immunophenotyping
– Biomarkers
• Summary and discussion
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Chipcytometry workflow
Load Chip
1
2 3
ZellScanner™ Instrument
Start
Instrument
Stain
Image
Switch-offLong-term
Storage
Data Processing >> FCS
4
Zellkraftwerk
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Microfluidic chips
Product ZellSafe™ Cells ZellSafe™ Rare ZellSafe™ TissueSpecimen cell suspension rare cells (<0.02%) sectionsLoading capacity 40-100µl 40-300µl 6 biopsies or 2x1cm sectionTotal cell number typically 250,000 typically 1,000,000 tissue-dependent
Sample inlet
Outlet
++++++- - - - - -
Zellkraftwerk
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Maroz, et al, 2014
Step-wise staining of the same cells again and again
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Data processing
raw image quality control dot plot
FCS
csv
patient stratification cluster analysis heat map
Zellkraftwerk
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Instruments: ZellScannerONE and CYTOBOT
Zellkraftwerk
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Overview
• Introduction
• Technology
• Case studies– Immunophenotyping
– Biomarkers
• Summary and discussion
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Human whole blood immunophenotyping
Zeng, et al. 2015
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Stability
• The fluorescence signal of all 21 protein markers measured by chipcytometry did not change significantly after 4-week storage
Zeng, et al. 2015
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Precision
• The mean coefficient of variation (CV) for 9 representative markers was 6 - 22%
Zeng, et al. 2015
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Comparable cytometric profiles of T cells assessed by Chip vs. Flow
Maecker, et al. 2012 Zeng, et al. 2015
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Comparable cytometric profiles of B cells assessed by Chip vs. Flow
Maecker, et al. 2012 Zeng, et al. 2015
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Comparable cytometric profiles of neutrophils and monocytes assessed by Chip vs. Flow
Zeng, et al. 2015Maecker, et al. 2012
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Overview
• Introduction
• Technology
• Case studies– Immunophenotyping
– Biomarkers
• Summary and discussion
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Active IL-17A producers in inflamed tonsils
Welzenbach, 2015
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Deep immunophenotyping at single cell level
Welzenbach, 2015
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Characterization of BAL-derived basophils of asthmatics
Dijkstra, et al, 2014
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Functional defects of B cells from a patient with Primary humoral immunodeficiencies (PHID)
Hennig, et al, 2009
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Overview
• Introduction
• Technology
• Case studies– Immunophenotyping
– Biomarkers
• Summary and discussion
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ChipCytometry: very promising but not perfect
• Advantages:– Major immune cell populations exhibit comparable cytometric profiles (frequencies)
when assessed by ChipCytometry vs. flow cytometry (alternative/complementary cytometric platform)
– Deep phenotyping at single-cell level with dozens of cell surface and intracellular protein biomarkers without spillover or color compensation (from Multi-PLEX to Extreme-PLEX)
– Reanalyze the same precious samples after long-term storage (from real time to weeks/months)
– Being employed in preclinical and clinical trials
• Challenges:– More mature for PBMCs, exploratory for other sample types
– Further optimization/validation needed for current and additional biomarkers
– Long sample preparation and analysis time, getting better
– Single vendor (instruments, chips, software, contract research services)
– Not GLP-compliant / CLIA-certified (so far)
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References
• Zellkraftwerk: http://www.zellkraftwerk.com. Sample preparation instructions: https://www.youtube.com/watch?v=X1bXN2rbyTU
• Maroz A, Stachorski L, Emmrich S, Reinhardt K, Xu J, Shao Z, Käbler S, Dertmann T, Hitzler J, Roberts I, Vyas P, Juban G, Hennig C, Hansen G, Li Z, Orkin S, Reinhardt D, Klusmann JH. GATA1s induces hyperproliferation of eosinophil precursors in Down syndrome transient leukemia. Leukemia 28: 1259–70. 2014
• Zeng R, Yang L, Patnaik M, Spitz S, Robbie G, Wu Y, Roskos L, White WI. Comparison of human whole blood immunophenotyping by ChipCytometry and Flow Cytometry: Potential applications for biomarker identification and immunomonitoring in clinical studies. 2015 AAPS National Biotechnology Conference. San Francisco, CA. June 8-10, 2015
• Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nature Reviews Immunology 12: 191-200, 2012
• Welzenbach K. ChipCytometry identifies presence of uncommon B cell subset in inflamed tonsils associated to autoimmunity. CYTO 2015. Glasgow, Scotland. June 26-30, 2015
• Dijkstra D, Hennig C, Hansen G, Biller H, Krug N, Hohlfeld JM. Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge. Cytometry A 85:580-7, 2014
• Hennig C, Adams N, Hansen G. A versatile platform for comprehensive chip-based explorative cytometry. Cytometry A 75: 362-70, 2009