Comparison between ChipCytometryand Flow Cytometry for Biomarkers and Immunophenotyping Applications

Rong Zeng, Ph.D

OncoMed Pharmaceuticals

10th WRIB (Orlando, FL)

April 22, 2016

OncoMed 2

Overview

• Introduction

• Technology

• Case studies– Immunophenotyping

– Biomarkers

• Summary and discussion

OncoMed 3

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

OncoMed 4

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)

OncoMed 5

Comparison of next generation single-cell analysis platforms

Zellkraftwerk

OncoMed 6

Overview

• Introduction

• Technology

• Case studies– Immunophenotyping

– Biomarkers

• Summary and discussion

OncoMed 7

Chipcytometry workflow

Load Chip

1

2 3

ZellScanner™ Instrument

Start

Instrument

Stain

Image

Switch-offLong-term

Storage

Data Processing >> FCS

4

Zellkraftwerk

OncoMed 8

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

OncoMed 9

Maroz, et al, 2014

Step-wise staining of the same cells again and again

OncoMed 10

Data processing

raw image quality control dot plot

FCS

csv

patient stratification cluster analysis heat map

Zellkraftwerk

OncoMed 11

Instruments: ZellScannerONE and CYTOBOT

Zellkraftwerk

OncoMed 12

Overview

• Introduction

• Technology

• Case studies– Immunophenotyping

– Biomarkers

• Summary and discussion

OncoMed 13

Human whole blood immunophenotyping

Zeng, et al. 2015

OncoMed 14

Stability

• The fluorescence signal of all 21 protein markers measured by chipcytometry did not change significantly after 4-week storage

Zeng, et al. 2015

OncoMed 15

Precision

• The mean coefficient of variation (CV) for 9 representative markers was 6 - 22%

Zeng, et al. 2015

OncoMed 16

Comparable cytometric profiles of T cells assessed by Chip vs. Flow

Maecker, et al. 2012 Zeng, et al. 2015

OncoMed 17

Comparable cytometric profiles of B cells assessed by Chip vs. Flow

Maecker, et al. 2012 Zeng, et al. 2015

OncoMed 18

Comparable cytometric profiles of neutrophils and monocytes assessed by Chip vs. Flow

Zeng, et al. 2015Maecker, et al. 2012

OncoMed 19

Overview

• Introduction

• Technology

• Case studies– Immunophenotyping

– Biomarkers

• Summary and discussion

OncoMed 20

Active IL-17A producers in inflamed tonsils

Welzenbach, 2015

OncoMed 21

Deep immunophenotyping at single cell level

Welzenbach, 2015

OncoMed 22

Characterization of BAL-derived basophils of asthmatics

Dijkstra, et al, 2014

OncoMed 23

Functional defects of B cells from a patient with Primary humoral immunodeficiencies (PHID)

Hennig, et al, 2009

OncoMed 24

Overview

• Introduction

• Technology

• Case studies– Immunophenotyping

– Biomarkers

• Summary and discussion

OncoMed 25

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)

OncoMed 26

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