clinical trial support
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Immunology: Clinical Trial Support
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Conventional Flow Cytometry ChipCytometry
Sample Stability 1-3 days > 24 months
Markers/Sample 8-16 > 70
Re-interrogate SampleX
NEW!
Cell Storage (fixed cells)
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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
++++++- - - - - -
Compatible Specimens
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Cell Suspensions:• PBMC / whole blood• bone marrow• CSF• bronchoalveolar lavage (BAL)• cell lines• sorted cells• digested tissues (lung, gut, tonsil, spleen, liver)
Tissue / Sections• lung• gut• bone• cancer biopsy• skin• bone sections
Sample Analysis Workflow
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Load Chip
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2 3
ZellScanner™ Instrument
StartInstrument
Stain
Image
Switch-offLong-term
Storage
Data Processing >> FCS
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Central Lab Services
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- Fully automated analyzer- 24/7 operation- 4,000 data points/week- “Spirit of GLP”
Predictive Biomarkers
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Outcome:- Predictive biomarker
candidate identified- Prospective study
pending
- Disease: Neonate Sepsis
- Study: Clinical
- Patients: 370
- Sample: PBMC
Respiratory
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CD3 CD4 CD8 CD45R0 CD45RACD19 CD56 HLA-DR CD16 CD14
Alveolar macrophages
Neutrophils
T cells
Th cells
B cells
- Disease: Primary Immunodeficiency
- Study: Exploratory
- Sample: BAL
Immuno-Oncology
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Outcome:Infiltrating cell types identified and quantified
- Disease: Colon Carcinoma
- Study: Translational
- Sample: Cryosections
By courtesy of
Translational Immunology
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PX4: Germinal center B-cells: CD19(+), CD86+, CD10+, CD38+, CD11c+/-CD14(+)
PX4DC
MZ: Marginal zone B-cells: CD19++, CD86-, CD10+, CD38-, CD11c-,IgD+
MZ
PX4
Outcome:- CD11c+ cells identified
and localized By courtesy of
Validated Markers
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CCR3 * CD25 CD68 IgMCCR4 * CD27 CD69 * IL10CCR5 * CD272 (BTLA) CD71 IL12CCR6 * CD279 (PD-1) CD8 IL17ACCR7 * CD3 CD86 IL17FCD10 CD30 CTLA-4 IL1bCD115 CD31 CXCR3 * IL8CD11b CD38 CXCR4 * kappa light chainCD11c CD4 CXCR5 * lambda light chainCD14 CD40 FoxP3 pan CytokeratinCD141 CD45 GM-CSF PD-1 (CD279)CD16 CD45RA Helios ROR gtCD163 CD45RO HLA-DR T-betCD19 CD5 IFNy TNFaCD20 CD54 IgA Zap-70CD206 CD56 IgD *= Markers to be analyzed on live
cells only.CD24 CD64 IgG
Deep Immunophenotyping
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LEVEL 1 Phenotyping• T-cells (CD3+CD56-) • B-cells (CD19+) • NK-cells (CD56+CD3-) • NKT-cells (CD3+CD56+) • Monocytes (CD14+CD4dimCD3-) • Neutrophils (CD16+CD14-) • Basophils (CD123+HLA-DR-CD3-
CD4-CD8,CD14-,CD19-CD56-) • mDC (CD11c+HLA-DR+CD123-CD3-
CD4-CD8,CD14-CD19-CD56-) • pDC (CD11c+HLA-DR+CD123-CD3-
CD4-CD8,CD14-CD19-CD56-)
LEVEL 2 Advanced Phenotyping• T-cells: CD4/CD8/DN/DP (CD4, CD8) • T-cells: Central/Peripheral Effector/Memory
(CD27, CD45RO, CD45RA) • B-cells: Memory, Naive, Transitional (IgD,
CD27, CD38)
LEVEL 3 Functional Phenotyping• T-cells: activation Status (CD25,MHC2) • B-cells: activation Status (CD86, CD25, AID,
CD27, CD38) • Monocyte/Neutrophil activation Status
(MHC2 CD64) • Upon request: Cell-Cycle analysis, Cytokine
production, Homing markers
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Preparing Clinical Trials
Candidate Sites 1, 2, 3, 4,..
2. Site Qualification Kit
1. SOP, Training Video
Typical Milestone-based Project
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2. Load- Load sample, wait 5‘- Fix (4% PFA), wait 15‘- rinse, seal chip
1. Prep- 1 ml blood- CPT, Ficoll or RBC lysis- total time: 40’
3. Store- store on-site- temperature 4°C- up to several months
4. ShipGDP Logistics (Good Distribution Practice)
5. Bioanalysis“Spirit of GLP” clinical trial support (SOP, QA)
6. Re-interrogateTest additional markers over >24 months
Selected Primary Publications
• Happle, Science Translational, 2014
• Dijkstra, Nature Medicine 2012
• Hennig, Br. J. Hematology 2010
• Woelbeling, Immunobiology 2011
• Polte, JACI 2008
System Validation according to:
• O'Hara, JIM 2011
• Green, JIM 2011
• Cunliffe, Pharm. Res. 2009
• Lee, Pharm. Res. 2006
• Bioanalytical Method Validation, FDA, 2013
Thank you for your attention!
“Precision Immunology” References
A B C Control Diseased Groups Group
Focus on:- Translational Immunology- Mode of Action(MoA)- Drug Safety- Patient Stratification
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