gérald grégori, ph.d purdue university cytometry laboratories hansen life sciences research...
TRANSCRIPT
![Page 1: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/1.jpg)
Gérald Grégori, Ph.D Purdue University Cytometry Laboratories
Hansen Life Sciences Research Building, B050201 S. University Street,
West Lafayette, IN 47907-2064,USAPh: +1(765) 494-0757Fax: +1(765) 494-0517
e-mail: [email protected]
Quality Controls: Quality Controls:
Get your instruments Get your instruments under control! under control!
![Page 2: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/2.jpg)
In flow cytometry scatter and fluorescence scales are in arbitrary units
Scatter and Fluorescence values = function of set up (Voltage and gain of PMT)
- +FS-PMT Voltage or gain
Example : beads 10 m
Gre
en F
luor
esce
nce
(au)
Forward Scatter (au)Forward Scatter (au)
Arbitrary units?
![Page 3: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/3.jpg)
Confidence in the instrument performance&
Confidence in the results
Use of a Standard
Quality Control Tests
Daily basis
![Page 4: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/4.jpg)
In theory : • A standard = a reference (defined by a user, a laboratory, or any aknowledged authority)
• Properties accurately known (i.e., provided by the manufacturer)
In practice : • A manufactured particle (fluorescent beads: several sizes and excitation or emission wavelengths)
• A biological particle (i.e., chicken and trout erythrocytes DNA measurements)
• Used as an absolute reference for qualitative and quantitative comparisons
What’s a Standard?
![Page 5: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/5.jpg)
• Check the alignment of the optical pathway
• Check the stability of the machine (Quality Control)
• Test the capacities of the cytometer (flow rate)
• Set up the Sorting (define the delay)
Quality Control of Instruments
![Page 6: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/6.jpg)
0 200 400 600 800 1000FL1 Lin
020
040
060
080
010
00N
umbe
r
1.931.93
0 200 400 600 800 1000FL2 Lin
020
040
060
080
010
0012
0014
00N
umbe
r
1.991.99
0 200 400 600 800 1000FS Lin
020
040
060
080
010
0012
00N
umbe
r 1.371.37CVs < 2% expected for
beads (1-10 μm)
If CVs > 2% check the alignment (flow cell or laser position,
dichroic mirrors)
Very useful on sorters
Beads
Parameter intensity (au)
Num
ber
of e
vent
sTo check the alignment
of the cytometer
Intensity is constant with
time
![Page 7: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/7.jpg)
Example of bad alignment
CV=4.5
![Page 8: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/8.jpg)
0 200 400 600 800 1000FL3 Lin
02
00
40
06
00
80
01
00
01
20
0N
um
be
r 1.62
R4
0 200 400 600 800 1000FL1 Lin
02
00
40
06
00
80
01
00
0N
um
be
r
1.93R2
0 200 400 600 800 1000FL2 Lin
02
00
40
06
00
80
01
00
01
20
01
40
0N
um
be
r
1.99
R3
0 200 400 600 800 1000FS Lin
02
00
40
06
00
80
01
00
01
20
0N
um
be
r 1.37R1
Once a protocol is defined for a particular standard bead solution
Beads must always be plotted in the same regions
Daily quality control
Parameter intensity (au)
Num
ber
of e
vent
s
Internal quality control
Check the stability of the cytometer over time
![Page 9: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/9.jpg)
Many possible problems :
• Leak in the fluidics modified flow rates; instability
• Check for bubbles
• Dirty flow cell
• Laser dying beam intensity decreases
• Bead solution too old or damaged photobleaching
Problem of stability of the cytometer?
![Page 10: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/10.jpg)
For example : Determination of the “dead time” of a Cytoron Absolute (Ortho Diagnostic Systems) Value furnished by the manufacturer = 2000 events s-1
Dilution factor
1
10
100
1000
10000
1 10 100 1000
Measured concentration(beads mm-3)
Sample flow rate = 1mm3 s-1
Test the capacities of the cytometer
Analysis of 1 μm fluorescent bead solutions (dilutions in cascade)
![Page 11: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/11.jpg)
laser
Interrogation point
Flowcell
Last attached undulation
Delay(s)
Piezoelectric Transducer Input
Break-off pointFirst droplet
Electric pulse
++
++
+
++
++
+
Delay determined using
fluorescent beads
Droplet formation and timing
![Page 12: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/12.jpg)
Sort 50 beads on a slide with different delays (ex : from 29 to 35)
•If you achieve 50 out of 50 beads, set the delay to that setting (ex: 31).•If beads are split between 2 drops, adjust the flow cell vertically
3332313029
SlideDrop
Sorting process on the Altra (Coulter, USA)
Green Fluorescence (au)
For
war
d S
catt
er (
au)
beads
Beads (10 m)
Delay value
Count beads(epifluorescent
Microscope)
Set up the delay with fluorescent beads
![Page 13: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/13.jpg)
The necessary step to convert arbitrary units into absolute physical values
Calibration
Calibration = adjustment of an instrument in order to express the results in some accurate physical measure.
Calibrator = a material known to have accurate measured values for one or several characteristics
![Page 14: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/14.jpg)
Example :Quantitation of antibody binding
capacity of cell populations by flow cytometry
Quantum Simply Cellular (QSC)
![Page 15: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/15.jpg)
Identical microbeads with various calibrated binding capacities of goat-anti-mouse IgG on their surface :
Eve
nts
Mean fluorescence intensity (au)
1 2
3 4
Blank
QSC, Cat. No. 815Bangs Laboratories, Inc.www.bangslabs.com
Antibody binding capacity (ABC) provided by the manufacturer :
Blank. 0 MESF
1. 6851 MESF
2. 23379 MESF
3. 58333 MESF
4. 213369 MESF
bead
Ab site
MESF=Molecules of equivalent soluble fluorochrome
QSC Beads (Quantum Simply Cellular)
![Page 16: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/16.jpg)
y = 32790x - 3926.1
R2 = 0.9981
0
50000
100000
150000
200000
250000
0 1 2 3 4 5 6 7
mean fluorescence- HLA-DR-FITC (au)
Mol
ecu
les
of e
qu
ival
ent
solu
ble
fl
uor
och
rom
e (M
ES
F)
Courtesy of K. Rhageb
measure by FCM
corresponding MESF
Binding capacity of cells in your sample
Quantum Simply Cellular Beads Calibration Curve
![Page 17: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/17.jpg)
Absolute Counts Determination of cell concentration
by flow cytometry
Get the control of the volume analyzed
Four different methods
![Page 18: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/18.jpg)
Sample
Cell concentration(# of events / volume)
CYTORON ABSOLUTE(Ortho Diagnostic Systems)
I. Direct Absolute Count
To analyze a bead solution of known concentration Control of the fluidic
![Page 19: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/19.jpg)
Sample
Weigh the sample before analysis
(Weight W0)
Weigh the sample afteranalysis (Weight W1)
Analysis(Flow Cytometer)
Volume analyzed V = (W0 – W1)/
Cell concentration = N/V
cell number (N)
density
DisadvantagesDisadvantages• Time consuming• Less accurate (back flow of sheath fluid in the sample)• Analysis in one run
II. Weigh a Sample
![Page 20: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/20.jpg)
Bead solution (known concentration)•count by microscopy•TruCountTM beads (BD)
Sample
Add a very accurate volume
calculate the bead concentration in the sample (1-10% of the
expected density of the target cells)
Flow Cytometer
cell number (N)
bead numberVolume
analyzed (V)
Cell concentration = N/V
Stewart & Steinkamp, 1982, Cytometry 2 : 238-243
III. Add Beads in the Sample
![Page 21: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/21.jpg)
Hypothesis: flow rate (µl/s) through a flow cytometer = constantBergeron et al, 2003, Cytometry 52B:37-39
Conclusion: volume (V) analyzed in a fixed time = constantNo need to add beads in the samples.
If N events are analyzed by the cytometer in the fixed time
then cell concentration = N/V (cells/µl)
Hint!• Analyzes must be done with the same flow rate
• Volume accurately determined (microscopy, TruCountTM beads) and controlled
• Beads not necessary in the sample, but can be used as internal standard
IV. Determination of the Flow Rate
![Page 22: Gérald Grégori, Ph.D Purdue University Cytometry Laboratories Hansen Life Sciences Research Building, B050 201 S. University Street, West Lafayette, IN](https://reader034.vdocuments.us/reader034/viewer/2022050809/56649e125503460f94afe3d5/html5/thumbnails/22.jpg)
Quality Control Tests are mandatory … To assess the alignment To assess and control instrument performance
for quantitative and reproducible applications on any flow cytometer.
ReferencesReferencesR.A. Hoffman, Current Protocols in Cytometry, 1997 : 1.3.1-1.3.19J.C.S. Wood, Current Protocols in Cytometry, 1997 : 1.4.1-1.4.12Cytometry, Volume 33, Number 2, 1998 :
Conclusion