Where to Flow?Challenges, Solutions & Innovations in modern Flow Cytometry

1st Pan Arab Flow Cytometry Working Group Workshop

Friday, October 14th, 2016

Cairo, Egypt

Dr. Gerald Pfister, PhD, CCyManager Flow Cytometry Core Facility

Qatar Biomedical Research Institute, Doha, Qatar

Web: www.qbri.org.qa / E-mail: gpfister@qf.org.qa

The Qatar Biomedical Research Institute QBRI

http://www.qbri.org.qa

The Qatar Biomedical Research Institute QBRI

http://www.qbri.org.qa

What is Flow Cytometry?

From: www.keokimed.com

What is Flow Cytometry good for?

http://www.accessexcellence.org/RC/VL/GG/

2015

15000

Why is Flow Cytometry important?

Published papers containing the key word „Flow Cytometry“

What are the Challenges & Innovations in Flow Cytometry?

LaboratoryInstrumentationHardware & SoftwarePerformance & QC

ApplicationsCell CultureCell Preparation & CollectionBiosafety

Data AnalysisData Management & StorageInterpretation of ResultsEducation & DevelopmentLab & User ManagementTroubleshooting

What are the Challenges & Innovations in Flow Cytometry?

LaboratoryInstrumentationHardware & SoftwarePerformance & QC

What Instrumentation is available and needed?

Basic

High-End

What Instrumentation is available and needed?

$

$$$

Instrumentation – Lasers & Optics

Instrumentation – Lasers & Optics

From the webinar „Fluorescent Proteins in Flow Cytometry“, June 26th 2015© CYTO University, by Teresa Hawley & Bill Telford

Instrumentation – Optimization, Performance Tracking & QC

Optimization of PMT-V enhances resolution of signals

T cells:SSClow / CD45high / SSClow / CD3high

B cells:SSClow / CD45high / CD3neg / CD19high

Monocytes:SSC / CD45interm / CD3neg / CD14high

PMT-V PE: 350V 400V 450V 500V550V

CD4 PE (0.1 ml / test)

Instrumentation – Recent Technologies

The ImageStreamX Imaging Flow Cytometer from Amnis / MilliporeCombines flow cytometry with microscopy for multiple applications

Source: http://www.amnis.com

Instrumentation – Recent Technologies

The ImageStreamX Imaging Flow Cytometer from Amnis / MilliporeMorphology: Shape change in primary monocytes

Source: http://www.amnis.com

Chemoattractant

Migration to sites

of inflammation

Reduced inflammatory response Autoimmune drugs

Instrumentation – Recent Technologies

Sorting examples: BD FACSAria FusionTM with Class II Biosafety Cabinet

Instrumentation – Recent Technologies

BD FACSAriaTM Fusion:Sorting of 100-160-200-240 nm beads (nanoparticles)

Scatter thresholding - 4-way sort

BioCytex Gigamix beads (Mixture of Megamix-Plus SSC and Megamix-Plus FSC) were prepared according to manufacturer’s instruction.

240 nm200 nm

160 nm

PRE-SORT

POST-SORT

530/30-H

SSC

-H

100 nm

900 nm

500 nm300 nm

240 nm200 nm160 nm

100 nm

Instrumentation – Recent Technologies

Small Particles: Analysis and Sort of Extracellular Vesicles (<300nm) on the BD InfluxTM

Instrumentation – Recent Technologies

The Sony SP6800 Spectral AnalyzerCollects all 500-800nm emitted light from sample and focus it onto 32-channel PMT

No optical filters and no conventional compensation needed

Up to 15 colors with two lasers, allows for use of high-spectral-overlap-dyes in one sample

Instrumentation – Recent Technologies

Analysis of marine water samples by the Sony SP6800 Spectral Analyzer

Source: https://precym.mio.univ-amu.fr/spip.php?article232

Instrumentation – Recent Technologies

Mass Cytometry – the CyTOF2™, Fluidigm, Inc.Use of antibodies coupled to isotopically purified mass tagsQuantified in individual cells by an Inductively-Coupled Plasma Mass Spectrometer (ICP-MS)

About 40-simultaneous antigens can be quantified at a rate of about 500-1000 cells/second

Source: http://cytof.scilifelab.se/

What are the Challenges & Innovations in Flow Cytometry?

Cell CultureCell Preparation & CollectionApplicationsBiosafety

Cell Preparation & Collection

Sample quality & cell viability

Sample concentration

Fluorochrome selection

Cell Preparation & Collection

Sample quality & cell viabilitySample concentration

Fluorochrome selection

G I G Oarbage arbagen ut

Filter to avoid aggregates

Treat cells gently Check cell viability

dead

alive

Cell Preparation & Collection

Sample quality & cell viability

Sample concentration

Fluorochrome selection

• Instrument ConfigurationSelect fluorochromes according to optical configuration

• Fluorochrome brightnessPut bright fluorochromes to dim markers and vice versa

• Spectral SpilloverAvoid high spillover from markers on same cells

• Stability of tandem dyesDegradation due to light, fixatives, temperature, pH…

• Non-specific bindingConsider unspecific binding to FC receptors

• Size of fluorochromeHardly influences intracellular & extracellular staining

Applications – Sort for Single Cell RNA Sequencing

Single cell isolation techniques

Applications – Sort for Single Cell RNA Sequencing

Direct sort onto the PCR plate by the Fuidigm C1 system

Single Cell Transcriptomics, etc...

Advantages of Microfluidics:

AutomationHigh ThroughputLower risk of contamination

Indi

vidu

al c

ells

Individual genes

© Dr .Vasiliki Chini, QBRI

Biosafety – The Two Fundamentals

Risk ManagementProtective instrumentation and engineering controls

Personal protective equipment and clothing (PPE)

Safe laboratory procedures (SOP)

Laboratory design for containment

Risk AssessmentIdentification of hazards

Measurement of the risk that something will happen as a result of that risk

Takes into account the Risk Group and procedures performed

Biosafety – General Rules for Laboratory Work

European Directive 2000/54/ECNo eating and drinking in a lab

No mouth pipetting

Appropriate cleaning procedures

Lab design (floor, tables, cupboards)

Good laboratory practice (GLP) & standard operation protocols (SOP)

What are the Challenges in Innovative Flow Cytometry?

Data AnalysisData Management & StorageInterpretation of ResultsEducation & DevelopmentLab & User ManagementTroubleshooting

Data Analysis

Example: Influence of sample size and plot type on data display

Source: Practical Flow Cytometry 4th Edition, HM Shapiro, Wiley-Liss Press

Data Analysis

Example: Influence of number of events recorded on data accuracy

Increasing Variability of data

Number of events

Data Analysis – Statistics in Population Analysis

Quantification of the average intensity of a parameter in a population

M F Iean luorescence ntensity

Arithmetic mean ?Geometric mean ?

Median ? Mode ?

Data Analysis – Statistics in Population Analysis

Median Fluorescence Intensity is often the best choice for biological samples

Data Analysis – Statistics in Population Analysis

Coefficient of Variation (CV)

Data Analysis – Statistics in Population Analysis

Effect of high CV’s on resolving populations

Bad resolution

Good resolution

Data Analysis – What do we see on our Plots?

http://www.emdmillipore.com

Events (not cells!)

Data Analysis – Gating Strategy

1: Flow stability gatingTo capture events once the flow stream has stabilized, eliminating effects of clogging, back-

pressure, and other instrument issues

2: Pulse geometry gatingTo remove doublets from the dataset

3: Forward and side scatter gatingTo remove debris and other events of non-interest while preserving cells based on size and or complexity

4: Subsetting gatingTo rely on expression of markers and what they identify. Using viability dyes and dump

channels further narrow to the cells of interest. This is where Fluorescence Minus One (FMO) controls become critical in defining the populations of interest

5: BackgatingTo provide visualization of cells in final gate at higher level

http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp

Data Analysis – Gating Strategy

Flow stability gating

http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp

Data Analysis – Gating Strategy

Pulse geometry gating (Doublet exclusion)

http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp

Data Analysis – Gating Strategy

Forward and side scatter gating

http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp

Data Analysis – Gating Strategy

Subsetting gating

http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp

Data Analysis – Gating Strategy

Backgating

http://expertcytometry.com/gating-strategies-get-your-flow-cytometry-data-published/hp

Flow Resources: Possibilities, Possibilities, Possibilities...

Questions?

E-mail: gpfister@qf.org.qa