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Quality Controls:

Get your instruments


under control!
Grald Grgori, Ph.D
Purdue University Cytometry Laboratories
Hansen Life Sciences Research Building, B050
201 S. University Street,
West Lafayette, IN 47907-2064,USA
Ph: +1(765) 494-0757
Fax: +1(765) 494-0517
e-mail: gregori@flowcyt.cyto.purdue.edu

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

Green Fluorescence (au)

FS-PMT Voltage or gain

Forward Scatter (au)


Forward Scatter (au)

Quality Control Tests


Daily basis

Confidence in the instrument performance


&
Confidence in the results

Use of a Standard

Whats a Standard?
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

Quality Control of Instruments


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)

1200

1000

To check the alignment


of the cytometer
800

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

If CVs > 2% check the alignment


(flow cell or laser position,
dichroic mirrors)
1.99

1.99

200

400

Intensity is
constant with
time

Number of events

CVs < 2% expected for


beads (1-10 m)

1.93

400

Number
600

1.37

200

400

600
FL2 Lin

800

Parameter intensity (au)

1000

Very useful on sorters

Example of bad alignment

CV=4.5

Once a protocol is defined for a


particular standard bead solution

600

800

800

1000

1200

1000

Check the stability of the


cytometer over time
R1

Number

1.93

200

400

200
0
0

200

400

600

800

1000

200

400

600
FL1 Lin

800

1000

Internal quality control

1.99

200

200

400

400

R3

Daily quality control

R4

1.62

Number
600

Number
600
800

800

1000

1000

1200

1200

1400

FS Lin

Number of events

Beads must always be plotted


in the same regions

R2

400

Number
600

1.37

200

400

600
FL2 Lin

800

1000

200

Parameter intensity (au)

400

600
FL3 Lin

800

1000

Problem of stability of the


cytometer?
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

Test the capacities 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
Analysis of 1 m fluorescent bead solutions (dilutions in cascade)
10000

Sample flow rate


= 1mm3 s-1

1000

Measured concentration
(beads mm-3)
100

10

1
1

10

100

Dilution factor

1000

Droplet formation and timing


Electric pulse
Flow
cell

Piezoelectric Transducer
Input

laser

Interrogation
point
Last attached
undulation
First droplet

Delay
(s)

+
+ +
++
+
+ +
++

Break-off point

Delay determined
using
fluorescent beads

Forward Scatter (au)

Set up the delay with fluorescent beads

Sort 50 beads on a slide with


different delays (ex : from 29 to 35)

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)

QSC Beads (Quantum Simply Cellular)


Identical microbeads with various calibrated binding capacities
of goat-anti-mouse IgG on their surface :
Antibody binding capacity (ABC) provided
by the manufacturer :

1.

0 MESF
6851 MESF

Ab site
2.

23379 MESF

3.

58333 MESF

4.

213369 MESF

MESF=Molecules of equivalent soluble fluorochrome

Events

bead

Blank.

QSC, Cat. No. 815


Bangs Laboratories, Inc.
www.bangslabs.com

34
1 2
Blank
Mean fluorescence intensity (au)

Quantum Simply Cellular Beads


Calibration Curve
Molecules of equivalent soluble
fluorochrome (MESF)

250000

y = 32790x - 3926.1
2

R = 0.9981

200000

corresponding MESF

150000

100000

measure by FCM

50000

0
0

mean fluorescence- HLA-DR-FITC (au)

Binding capacity
of cells in your sample
Courtesy of K. Rhageb

Absolute Counts
Determination of cell concentration
by flow cytometry

Get the control of the volume analyzed


Four different methods

I. Direct Absolute Count

Cell concentration
(# of events / volume)
Sample

CYTORON ABSOLUTE
(Ortho Diagnostic Systems)

To analyze a bead solution of known concentration


Control of the fluidic

II. Weigh a Sample


Weigh the sample
before analysis
(Weight W0)

Sample

Disadvantages
Time consuming
Less accurate
(back flow of sheath fluid
in the sample)
Analysis in one run

Analysis
(Flow Cytometer)

cell number (N)

Weigh the sample after


analysis (Weight W1)

Volume analyzed
V = (W0 W1)/
density

Cell concentration
= N/V

III. Add Beads in the Sample


Bead solution (known concentration)
count by microscopy
TruCountTM beads (BD)
Add a very
accurate volume

calculate the bead concentration


in the sample (1-10% of the
expected density of the target cells)
Flow Cytometer

bead number

Volume
analyzed (V)

cell number (N)


Sample

Stewart & Steinkamp, 1982, Cytometry 2 : 238-243

Cell concentration
= N/V

IV. Determination of the Flow Rate


Hypothesis: flow rate (l/s) through a flow cytometer = constant
Bergeron et al, 2003, Cytometry 52B:37-39

Conclusion: volume (V) analyzed in a fixed time = constant


No 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/l)
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

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 :