flowcytometry training
TRANSCRIPT
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YOUR SCIENCE – OUR FOCUS
NovoCyte™ Quanteon
Charlotte SvendsenField Application SpecialistFlowcytometry & Cell AnalysisAH Diagnostics
Flowcytometry training
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Agenda
• Features in the NovoCyte Quanteon• Software demonstration
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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
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Automated and High Throughput Sampling
Intuitive Software
Consistent and Reliable
Superior Sensitivity
27-Parameter Flexibility
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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
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The Cell Analysis Company (6)
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)
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The Cell Analysis Company (7)
“Smart” optical filters
Correct configuration
Incorrect configuration
Expand your capability with 4 lasers, 25 fluorescence channel options
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NovoCyte™ Training
PhotoMultiplier Tubes
PMTs
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Detection of the Cell and Fluorescent Label
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NovoCyte™ Training
An alternative to PMTs,
SiPM/MPPC
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The Cell Analysis Company (11)
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
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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
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The Cell Analysis Company (15)
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.
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The Cell Analysis Company (16)
SSC detection using APD
Apogee beads on NovoCyte
PhotodiodePMT
Internal testQuanteonAPD SSC
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27-Parameter Flexibility
Consistent and Reliable
Superior Sensitivity
Automated and High Throughput Sampling
Intuitive Software
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The Cell Analysis Company (24)
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
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The Cell Analysis Company (25)
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
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The Cell Analysis Company (27)
SiPM sensitivity: Stain Index from beads
Stain Index (SI)(MedPos - MedNeg)/(2*rSDNeg)
Slide adapted from Yoav Altman, SBP Flow CoreCYTO 2018 presentation
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The Cell Analysis Company (28)
SiPM sensitivity: Stain Index from beads
Rainbow Peak 6 Stain Index
Slide adapted from Yoav Altman, SBP Flow CoreCYTO 2018 presentation
Quanteon
Cytometer 1
Cytometer 2
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The Cell Analysis Company (29)
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.
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The Cell Analysis Company (30)
SiPM sensitivity: qNORM
qNORM (quantitative Normalized Overlap Ratio Metric)
# o
f A
B’s
th
at c
an b
e re
solv
edLower qNORM value indicates higher sensitivity
Slide adapted from Yoav Altman, SBP Flow CoreCYTO 2018 presentation
Quanteon
Cytometer 1
Cytometer 2
Resolution Limit
Note: Unstained Beads were used in this study
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Superior Sensitivity
27-Parameter Flexibility
Automated and High Throughput Sampling Consistent and Reliable
Intuitive Software
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The Cell Analysis Company (32)
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).
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The Cell Analysis Company (33)
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.
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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
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The Cell Analysis Company (35)
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
10 µl/min 60 µl/min
0,5
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0,9
1
10 µl/min 60 µl/min
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
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The Cell Analysis Company (36)
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
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Event Rate50,000 events/sec!
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The Cell Analysis Company (37)
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
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The Cell Analysis Company (38)
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
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The Cell Analysis Company (39)
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
8 minNovoFlow: 32.2 mLNovoRinse: 1.4 mLNovoClean: 11.3 mL
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
8 minNovoFlow: 35.0 mLNovoRinse: 1.5 mLNovoClean: 15.0 mL
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The Cell Analysis Company (40)
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.
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The Cell Analysis Company (41)
Fluidics Auto recovering procedure
Pressure out of Limit Error
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Superior Sensitivity
27-Parameter Flexibility
Automated and High Throughput sampling Consistent and
Reliable
Intuitive Software
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The Cell Analysis Company (43)
NovoExpress® softwareImproved versatility and ease of operation
Streamlined layout, easy report functions, drag and drop functionality
Reporting:• Fully
customized• Automated• Batch reporting
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The Cell Analysis Company (44)
NovoExpressTM Software User Interface
Toolbars
Experiment
Setting
Sampling
Status
Data-Analysis
(Plots & Statistics)
Experiment
Manager
Status Bar
44
Gate ManagerPopulation hierarchy
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The Cell Analysis Company (45)
NovoExpress® softwareImproved versatility and ease of operation Batch Analysis
Batch Statistics
Batch Reports
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The Cell Analysis Company (46)
NovoExpress® softwareImproved versatility and ease of operation
Automatic Levey-Jennings tracking
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The Cell Analysis Company (47)
New functions with QC Test
1
2
3
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The Cell Analysis Company (48)
New beads lot?
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The Cell Analysis Company (49)
NovoExpress® softwareImproved versatility and ease of operation
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
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The Cell Analysis Company (50)
Cell proliferation modeling
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Heatmap
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The Cell Analysis Company (52)
Dean-Jett-Fox (DJF) cell cycle algorithm
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The Cell Analysis Company (53)
Bivariate plots
Bivariate Plot to create a matrix of plots
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The Cell Analysis Company (54)
Export to LIS
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Superior Sensitivity
27-Parameter Flexibility
Automated and High Throughput Sampling Consistent and
Reliable
Intuitive Software
![Page 47: Flowcytometry training](https://reader030.vdocuments.site/reader030/viewer/2022021310/62087c4d9f12473b656826ae/html5/thumbnails/47.jpg)
The Cell Analysis Company (56)
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
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The Cell Analysis Company (57)
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
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Where Exceptional Performance Meets Simplicity