Flowcytometry training

YOUR SCIENCE – OUR FOCUS

NovoCyte™ Quanteon

Charlotte SvendsenField Application SpecialistFlowcytometry & Cell AnalysisAH Diagnostics

Flowcytometry training

Agenda

• Features in the NovoCyte Quanteon• Software demonstration

Cytometry – in a quick overview

3

Flow Cell

Data collection• Non fluorescent parameters

• FSC = relative size• SSC = cellular complexity

• Fluorescent parameters• Antibodies conjugated with

fluorophore • Fluorescent probes and

sensors• Expression tags

One or more lasers• See specific emission lights from

each individual excitation lasers• See various molecules

simultaneously

Sheath

FSC

SSCFluo

Sheath

Automated and High Throughput Sampling

Intuitive Software

Consistent and Reliable

Superior Sensitivity

27-Parameter Flexibility

The Cell Analysis Company (5)

Expand your capability with 4 lasers, 25 fluorescence channel options

Lasers 405 nm (100mW) 488 nm (100mW) 561 nm (100mW) 637 nm (100mW)

Dete

cto

rs

445/45 nm •

530/30 nm • •

586/20 nm • • •

615/20 nm • • •

660/20 nm • • • •

695/40 nm • • • •

725/40 nm • • • •

780/60 nm • • • •

Fluorescence Channels 8 7 6 4

Optical Detection Capability 25 channels

FSC/SSC Off 561nm laser - 0.4µm FSC; 0.1µm SSC


Lasers

80 um

80 um Central axis of the collection optics (half-ball lens)

561 nm

488 nm

637 nm80 um

405 nm

Sample Core Stream

Y

Z

X

Beam size at flow cell center:

10 (Y) X 60 (X) um (to be finalized)


“Smart” optical filters

Correct configuration

Incorrect configuration

Expand your capability with 4 lasers, 25 fluorescence channel options

NovoCyte™ Training

PhotoMultiplier Tubes

PMTs

Detection of the Cell and Fluorescent Label

NovoCyte™ Training

An alternative to PMTs,

SiPM/MPPC


The premier photodetector –Silicon photomultiplier (SiPM)

What is a SiPM?Silicon photomultipliers (SiPM) are solid-state, photon-level-sensitive devices.

SiPM’s strengths:• Superior photon detection sensitivity• High gain and quantum efficiency• Instantaneous warm up and fast response• Robust and long life-span• High durability

•Wide, 7-log dynamic range• No need for routine detector adjustment

# o

f p

ho

toel

ectr

on

s

Time

Array

Principle of MPPC

• MPPC: Multi-Pixel Photon Counter. also call Silicon PhotoMultiplier (Si-PM).

• The MPPC is a two-pin semiconductor photodetector capable of detecting even single photons in the 320-900 nm spectral range. The detector consists of a rectangular array of pixels that are all connected in parallel. Each pixel is a series combination of an avalanche photodiode (APD) operating in Geiger mode and a resistor, referred to as a "quenching resistor (RQ).”

Solid > No vacuumSingle photon resolutionHigh quantum efficiencyAllows weak signals detectionCompact sizeHigh mechanical robustness


FSC and SSC resolution

FSC: 0.4µm SSC: 0.1µm

Using 100mW 561nm laser

Particle size standard beads

background

Both 100nm and 150nm beads can be distinguished from 200nm beads and background/debris/noise clearly.


SSC detection using APD

Apogee beads on NovoCyte

PhotodiodePMT

Internal testQuanteonAPD SSC


Consistent and Reliable


Automated and High Throughput Sampling

Intuitive Software


Optimized and Fixed Detector Voltage

What does it mean; fixed vs adjustable?

24

Your sample = Paris

No settings to applyNo lost partOnly zoom in/out and display your dataMulti-area analysis possible


SiPM sensitivity: 8 peak beads separation

48

8n

m

56

1n

m

PE PE-Texas Red PE-Cy5 PE-Cy5.5 PE-Alexa Fluor 700 PE-Cy7

40

5n

m

63

7n

m


SiPM sensitivity: Stain Index from beads

Stain Index (SI)(MedPos - MedNeg)/(2*rSDNeg)

Slide adapted from Yoav Altman, SBP Flow CoreCYTO 2018 presentation


SiPM sensitivity: Stain Index from beads

Rainbow Peak 6 Stain Index


Quanteon

Cytometer 1

Cytometer 2


SiPM sensitivity: qNORM

qNORM (quantitative Normalized Overlap Ratio Metric)

Adapted from Ryan Duggan

• Median bound Ab of population whose 10%ile overlaps with 90%ile of a negative population.

• Lower qNORM value indicates higher sensitivity.


SiPM sensitivity: qNORM

qNORM (quantitative Normalized Overlap Ratio Metric)

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f A

B’s

th

at c

an b

e re

solv

edLower qNORM value indicates higher sensitivity


Quanteon

Cytometer 1

Cytometer 2

Resolution Limit

Note: Unstained Beads were used in this study



Automated and High Throughput Sampling Consistent and Reliable

Intuitive Software


Sampling

Opto-coupler to detect the height of the SIP and safety provide warning for any abnormality of the SIP position.

SIP Washing Apparatus

New SIP

Sensor

New molded plastic parts. High accuracy dimension control and less debris.

1. Tapered needle. Stem O.D. = 2.0 mmTip O.D. = 1.0 mmI.D. = 0.5 mmLength = 139.0 + 3.0 + 3.0 mm

2. Cutoff at the tip end to achieve low dead volume during sample aspiration (see figure below).


Fluidics

New Valve Module

Sheath (FMI) Pump

Damper

Sheath Fluid in-Line Filter

Pressure Sensor (new)

Syringe Pump

New Pressure Sensor: ceramic membrane. Could resist the contact of sheath/rinse/clean solution. → no need to disconnect tubing when maintaining.

Hydrodynamic Focusing in the NovoCyte

Small Core Diameter

Large Core Diameter

• Sheath flow rate: 6.5 ml/min, driven by ceramic pump• Flow Cell: 5 – 120 µL/min, driven by precision syringe pump• Flow Cell (X x Z): 170 x 290 um• Core diameter: ~ 5 to >20 um (elliptical shape)• Real-time monitoring of fluidic status


Exceptional Consistency and Reliability

0%

10%

20%

30%

FITC-H PE-H APC-H PacB-H

10 µl/min 60 µl/min

0,5

0,6

0,7

0,8

0,9

1

FITC-H PE-H APC-H PacB-H


0,5

0,6

0,7

0,8

0,9

1


0%

10%

20%

30%

FITC-H PE-H APC-H PacB-H10 µl/min 60 µl/min

NovoCyt

e

Quanteo

n

Competito

r

MFI Ratio to Low Flow

RateCV’s measured with various flow

rates

CV’s measured with various flow

rates

Sample flow consistency MF

I

C

V

MFI Ratio to Low Flow

Rate

Peri

stalt

ic

Pu

mp

Syri

ng

e

Pu

mp


Exceptional Consistency and Reliability

LinearityChicken Erythrocyte Nuclei (CEN)

• Enables Direct Absolute Cell/Particle Counts removing the need to use

expensive reference beads

• Provides consistent results between sample runs

Absolute Counting

The precise volumetric syringe pump provides

highly accurate sample volume aspiration

36

Event Rate50,000 events/sec!


3 running modes

Mode:- Standard

Fixed setting: 120uL/min sample flow rate; 1 mixing per well; 1 rinse.- HT

Fixed setting: 66uL/min sample flow rate; 1 mixing per well; no rinse. - Custom (default)

Custom setting.

Absolute Count:- If checked, the absolute counting result will be more accurate. - Minimum sample volume: 10uL- Sampling Overhead: 30uL (instead of 10uL)

Mode Standard HT

Sample Flow Rate (µL/min) 66 120

Sampling Volume (µL)384: 5 uL

Others: 10 uL384: 5uL

Others: 10 uLMixing (Cycle) 1 1

Rinse (Cycle) 1 0

Mixing Speed (rpm) 1500 1500

Mixing Duration (s) 10 10

Acceleration (s) 1 1

Absolute Counting ? No No


Fluidic procedures: duration and consumption

Step Abs. Count mode Other Mode

Preparing sample acquisition time 12.6 s 7.5 s

Reset and rinse sample tubing time (Select “Rinse after sampling”) 9.4 s

Reset time (Not select “Rinse after sampling”) 2.5 s

Procedure Function Time Reagent Consumption

Start-up Automatic system cleaning and debubble during start-up. 6 minNovoFlow: 23.5 mL NovoRinse: 10.3 mL NovoClean: 0.0 mL

Shut downAutomatic system cleaning and decontaminationduring shutdown.

8 minNovoFlow: 32.2 mLNovoRinse: 1.4 mLNovoClean: 11.3 mL

Enter sleepIf there is no operation of NovoCyte Quanteon in one hour, the

instrument will enter sleep. This procedure will rinse the instrument automatically.

0.5 minNovoFlow: 3.3 mL NovoRinse: 0.0 mLNovoClean: 0.0 mL

Exit sleep User operates the instrument when instrument is sleeping. 1 minNovoFlow: 6.5 mLNovoRinse: 0.0 mLNovoClean: 0.0 mL


Fluidic procedures: duration and consumption

Maintenance Function When to Conduct Time Reagent Consumption

Debubble

1. Remove bubble from flow chamber2. Remove bubble from sampling pump's chamber3. Remove bubble from sampling tubing4. Remove bubble from sheath pump's inner chamber

► No data comes out for a period of time after sample acquiring start► Instrument is not be used over one week► The CV of the data is larger than normal► QC test failed

6 minNovoFlow: 23.5 mL NovoRinse: 10.3 mL NovoClean: 0.0 mL

Cleaning

1. Clean flow cell's inner surface2. Clean sampling tubing's inner surface3. Clean waste tubing4. Clean SIP's outer surface

► Instrument is infected by biological dye► SIP's outer surface is dirty► The CV of the data is larger than normal► QC test failed


Priming1. Prime all instrument's tubing2. Prime sheath fluid in-line filter

► After adding instrument reagents► After replacing fluidic system consumables

5 minNovoFlow: 23.7 mL NovoRinse: 7.9 mLNovoClean: 6.0 mL

Unclog1. Unclog SIP and sampling tubing2. Unclog flow cell3. Unclog waste tubing

► Fewer events are detected than expected► Perform a large amount of tests in one day



Fluidics Auto recovering procedure

Error Error Code Reason Recover Procedure

Collision of sample injection probe. 0x0001Sample injection probe hits something when it moves toward sample.

1. Stop current test 2. SIP and all pumps and valves reset3. Rinse all sampling tubings4. Instrument is ready

Pressure is out of limit. 0x0014System's fluidic pressure is abnormal.

1. Instrument will diagnose itself automatically2. NovoExpress will tell user where is clogged and instruct user to deal it3. Instrument will diagnose itself again after unclog4. If pressure is normal, clear error and instrument is ready. Otherwise, keep error.

Sample injection probe reset failed. 0x001C

Pump's motor or optical coupler is abnormal.

1. Reset sample injection probe again2. If reset is succeed, clear error and instrument is ready. Otherwise, keep error.

Syringe pump reset failed. 0x001D None. User must power off the instrument.

Sheath pump reset failed. 0x0021 None. User must power off the instrument.


Fluidics Auto recovering procedure

Pressure out of Limit Error



Automated and High Throughput sampling Consistent and

Reliable

Intuitive Software


NovoExpress® softwareImproved versatility and ease of operation

Streamlined layout, easy report functions, drag and drop functionality

Reporting:• Fully

customized• Automated• Batch reporting


NovoExpressTM Software User Interface

Toolbars

Experiment

Setting

Sampling

Status

Data-Analysis

(Plots & Statistics)

Experiment

Manager

Status Bar

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Gate ManagerPopulation hierarchy


NovoExpress® softwareImproved versatility and ease of operation Batch Analysis

Batch Statistics

Batch Reports



Automatic Levey-Jennings tracking


New functions with QC Test

1

2

3


New beads lot?



Multiple Plot options Cell Proliferation Modeling Heat Map Displays

Automatic cell cycle analysis modules:-Watson -Dean-Jett-Fox (DJF)

DJF model can modify S-phase fitting as either:• Rectangle• Trapezoid• Polynomial

Useful for non-standard cell cycle distribution which is typical after drug treatment that affects the cell cycle progression


Cell proliferation modeling


Heatmap


Dean-Jett-Fox (DJF) cell cycle algorithm


Bivariate plots

Bivariate Plot to create a matrix of plots


Export to LIS



Automated and High Throughput Sampling Consistent and

Reliable

Intuitive Software


Automate Sample Loading To Your Specifications

• Automated calibration eliminates the needs for any

manual alignment and calibration

• Automated X,Y, and Z coordinates calibration during installation

• Automated plate calibration with well bottom depth

measurement and mapping for various plate types

• Versatile loading mode and increased throughput utilizing various sample formats (40 tube rack,

24/48/96/384 well plates) including customizable plates

• The barcode reader automatically and instantaneously

identifies plate information for keeping track of numerous samples

in high throughput experiments.

• Lab automation friendly with open architecture and developer-

ready API


Automate Sample Loading To Your Specifications

• Rapid and high-throughput reading as fast as <20

mins for a 96-well plate and <80 mins for a 384-well plate

• Efficient mixing to maintain cells in suspension for the

duration of the plate

Where Exceptional Performance Meets Simplicity

Flowcytometry training

Documents

Transcript of Flowcytometry training