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!
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?
Confidence in the instrument performance&
Confidence in the results
Use of a Standard
Quality Control Tests
Daily basis
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?
• 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
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
Example of bad alignment
CV=4.5
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
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?
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)
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
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
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
Example :Quantitation of antibody binding
capacity of cell populations by flow cytometry
Quantum Simply Cellular (QSC)
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)
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
Absolute Counts Determination of cell concentration
by flow cytometry
Get the control of the volume analyzed
Four different methods
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
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
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
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
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