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Arbitrary units?
In flow cytometry scatter and fluorescence scales are in arbitrary units
Scatter and Fluorescence values = function of set up (Voltage and gain of PMT)
Example : beads 10 m
Use of a Standard
Whats a Standard?
In theory :
A standard = a reference (defined by a user, a laboratory, or any
aknowledged authority)
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
1200
1000
800
1000
Beads
600
1.37
Number
0
200
200
400
1.93
200
400
600
800
1000
200
400
600
FL1 Lin
800
1000
1400
FS Lin
Number
600
800
1000
1200
1.99
200
400
Intensity is
constant with
time
Number of events
1.93
400
Number
600
1.37
200
400
600
FL2 Lin
800
1000
CV=4.5
600
800
800
1000
1200
1000
Number
1.93
200
400
200
0
0
200
400
600
800
1000
200
400
600
FL1 Lin
800
1000
1.99
200
200
400
400
R3
R4
1.62
Number
600
Number
600
800
800
1000
1000
1200
1200
1400
FS Lin
Number of events
R2
400
Number
600
1.37
200
400
600
FL2 Lin
800
1000
200
400
600
FL3 Lin
800
1000
1000
Measured concentration
(beads mm-3)
100
10
1
1
10
100
Dilution factor
1000
Piezoelectric Transducer
Input
laser
Interrogation
point
Last attached
undulation
First droplet
Delay
(s)
+
+ +
++
+
+ +
++
Break-off point
Delay determined
using
fluorescent beads
beads
Green Fluorescence (au)
Beads (10 m)
Slide
Drop
Delay value
29
30
31
32 33
Count beads
(epifluorescent
Microscope)
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
Sorting process on the Altra (Coulter, USA)
Calibration
The necessary step to convert arbitrary units
into absolute physical values
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)
1.
0 MESF
6851 MESF
Ab site
2.
23379 MESF
3.
58333 MESF
4.
213369 MESF
Events
bead
Blank.
34
1 2
Blank
Mean fluorescence intensity (au)
250000
y = 32790x - 3926.1
2
R = 0.9981
200000
corresponding MESF
150000
100000
measure by FCM
50000
0
0
Binding capacity
of cells in your sample
Courtesy of K. Rhageb
Absolute Counts
Determination of cell concentration
by flow cytometry
Cell concentration
(# of events / volume)
Sample
CYTORON ABSOLUTE
(Ortho Diagnostic Systems)
Sample
Disadvantages
Time consuming
Less accurate
(back flow of sheath fluid
in the sample)
Analysis in one run
Analysis
(Flow Cytometer)
Volume analyzed
V = (W0 W1)/
density
Cell concentration
= N/V
bead number
Volume
analyzed (V)
Cell concentration
= N/V
Conclusion
Quality Control Tests are mandatory
To assess the alignment
To assess and control instrument
performance
for quantitative and reproducible applications on any
flow cytometer.
References
R.A. Hoffman, Current Protocols in Cytometry, 1997 : 1.3.1-1.3.19
J.C.S. Wood, Current Protocols in Cytometry, 1997 : 1.4.1-1.4.12
Cytometry, Volume 33, Number 2, 1998 :