STANDARD OPERATING PROCEDURE

Partec CyFlow® Counter

Introduction:
The Partec CyFlow® Counter is a desk top single platform technology Flow Cytometry
System (FCM) in which CD4 T lymphocytes can be enumerated by ‘True
Volumetric Absolute Counting TVAC’..It is equipped with either a single 532 nm
green solid-state laser used for one fluorescence parameter or two lasers with a mercury
arc lamp applicable for 2 or 3-color analyses
Principle: Please refer the chapter on Principles of Flow cytometry
Purpose :
To enumerate the CD 4 T-lymphocytes in HIV/AIDS affected individuals as absolute
CD4 T Lymphocyte count and CD4 percentage
Operation:
I Daily Start Up procedure
1. Fill the Sheath Bottle with 800 ml – 900 ml of Sheath Fluid (Partec Code No. 04-
4007) and close the blue screw cap tightly. Tilt the Sheath Bottle in order to release
air bubbles trapped under the yellow inline filter unit.
2. The Waste Bottle should be empty and the red lid tightly closed.
3. Handle all the glass bottles carefully. Rotate only the Bottles to uncap do not rotate
the caps.
4. Switch on the Main supply. Switch on the UPS and then switch on the main power at
the back of the instrument.
5. The software Cyview window opens automatically.(see below )
6. Press green button on the left of the Partec logo located at the front; wait for the
main display which takes about 3 min.
7. Cleaning 1 : Pipette 1.6 ml of Cleaning Solution (Partec Code No. 04-4009) into a
Sample Tube (Partec Code No. 04-2000) and plug it to the sample port until you hear
the ‘click’ sound.
Press ‘Start’ and then wait till “Searching for zero- ready” is displayed on the
screen
8. Cleaning 2 : Pipette 1.6 ml of Sheath Fluid (Partec Code No. 04-4007) into a Sample
Tube (Partec Code No. 04-2000) and plug it to the sample port until you hear the
‘click’ sound.
Press ‘Start’ and then wait till “Searching for zero- ready” is displayed on the
screen.
II Quality Control with Count Check Beads (CCB) green
1. Press ‘Scripts Load config-script Count Check Beads –Green in the
dropdown menu Wait till it loads.
2. Bring the Count Check Beads (CCB) green (Partec Code No. 05-4011) to room
temperature and shake the Count Check Beads green bottle up and down intensively
ten times before use.
3. Remove the cap of CCB bottle and pipette 850 µl of Count Check Beads green into a
new Sample Tube (Partec Code No. 04-2000), plug to the sample port until you hear
the ‘click’ sound.

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Partec CyFlow® Counter

Press ‘Start’ & wait till “Searching for zero- ready” is displayed on the screen.

Note:The result must be within +10 % of the concentration mentioned on the label of
CCB bottle. If the result is not within +10 % of the concentration mentioned on the
label of the bottle, shake the bottle vigorously ten times and repeat the step 3.

4. Enter the name of the quality control -CCB run( by pressing minimum 3 letters on
the key board as illustrated below. Click ‘ent’ to close the Software Keyboard.
5. Press FCS button to save the data
6. To print the results data, press Print button and wait until it prints the results
completely.

III Sample preparation : (using CD4 easy count kit (Partec Code No. 05-8401) – this
contains 2000 µl CD4 mAb PE and 100 ml no lyse buffer)
1. Mix the CD4 mAb PE reagent bottle gently by inverting the bottle three times. Pipette
20 µl CD4 mAb PE with a fresh pipette tip (Use 20 µl fixed pipette) at the bottom of
the sample tube (Partec Code No. 04-2000) without touching the walls.Mix by
tapping the sample tube.
2. Add 20 µl EDTA whole blood sample (Use 20 µl fixed pipette and fresh tip) at
bottom of the tube without touching the walls .

Caution: All used tips and sample tubes should be disposed off in a separate
container filled with 0.1 % sodium hypochlorite solution (1000 p.p.m chlorine
prepared from commercially available 5% concentrate..

3. Incubate for 15 minutes at room temperature in the dark (protected from light).After the
incubation is over, add 800 µl of “no lyse buffer” into the sample tube (use100- 1000 µl
variable pipette) and mix it by tapping the tube 10 times.

Note: Always add the ‘no lyse buffer’ before running the processed samples on the
instrument. The prepared sample should be run within one hour after addition of
“no lyse buffer”.The incubated samples after adding the “no lyse buffer” can be
stored at 2-8 degree centigrade in the dark for maximum up to 48 hours.

IV Measurement of CD4 counts .

1. Press ‘Scripts’‘ Load config-script CD4 in the dropdown menu Wait
till it loads.
2. Take the sample tube and plug it to the sample port.
3. Press ‘Start’ & the instrument will start automatically analyzing the sample. After
counting the sample, the instrument stops automatically.
Wait till “Searching for zero- ready” is displayed on the screen.

4. See the histogram, check the CD4 region (Gating) and adjust if necessary.

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Partec CyFlow® Counter

To adjust, touch inside the CD 4 histogram marked red once, then press ‘Do it’
on the right side of the screen.
Touch Point 1 and then Point 2; then press ‘Do it’ (available on the right side of the
screen) to confirm the region.
7. Enter the name of the patient/identifiers ( by pressing minimum 3 letters on the key
board as illustrated below .Click ‘ent’ to close the Software Keyboard.
8. Press FCS to save the data
9. To print the results data, press Print button and wait until it prints the results
completely.
10. Repeat from Step 2 in order to analyse other samples.

Note: The system performs an automatic cleaning cycle after each sample runs. After
running all the samples proceed with the shut down procedure.

V Shut down procedure. (after all samples have been analyzed )

1. Pipette 1.6 ml of the Decontamination Solution (Partec Code No. 04-4010) into a
sample tube and insert in the sample port, press ‘‘Start’ and wait till “Searching
for zero- ready” is displayed on the screen. .
2. Pipette 1.6 ml of the Cleaning Solution (Partec Code No. 04-4009) into a sample tube
and insert in the sample port, press ‘Start’ and wait till “Searching for zero- ready”
is displayed on the screen.
3. Pipette 1.6 ml of the Sheath Fluid Solution (Partec Code No. 04-4007) into a sample
tube and insert in the sample port, press ‘Start’.
4. After 2 minutes of measurement press ‘Stop’ button and keep the sample tube with
Sheath Fluid attached to the sample port so that the probe and electrodes are kept
under wet condition.
5. Close the window using stylus and click on icon ‘Stop ‘(available in the top right
corner of the screen).
6. Switch off the Instrument using Back side ON/OFF switch.
7. Switch off the UPS. Leave the Mains in ON condition for charging the UPS Battery

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Partec CyFlow® Counter

8. Add 40 -50 ml of concentrated sodium hypochlorite solution (Commercially available

0.5 % wt. /volume available chlorine) to the Waste Bottle, incubate for 30 minutes
to neutralize the waste and discard
.
Caution: Always keep a Sample Tube with 1 ml Sheath Fluid attached to the sample
port even if the instrument is not in use.

CyView : Software Menu

The title contains information about program name and version, name of Config- and
Measure-Script,status of the software and language chosen.

Drop down menu

Reload ǃ Load a saved measurement
Scripts: Load Config-Script:A layout can be loaded
Save Config-Script: A layout can be saved
Load Measure-Script: The parameter setup can be loaded
Run Cleaning: An automatic cleaning cycle can be initiated
Language: The menu language can be chosen (English or German)

Left side Keys Right side Keys

Change the threshold level of the
trigger parameter. (L-L);
Insert/change a patient’s name.
(Thresh hold button)
Change the settings for the sample
flow. ( Sample Flow button)
Set the threshold level of the trigger
parameter by choosing a value
between 0-0.7. (LL button)
Change the patient’s name. This
function can be found by pressing the
L-L button.
(Patient button)

Choose between the parameters
SSC, CD4 and CD45 by clicking
several times on this button.Change
high voltage of the chosen para-
meter. (Parameter button)
Save the measurement as FCS data
(FCS button)
Change the speed of the sample flow
between 0.2 and 20 μl/sec.
(Speed button)
Change the gain value of the parameter
chosen (SSC,CD4, CD45) which is the
high voltage of the PMT; value
between 0-1000 (Gain button)

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Partec CyFlow® Counter

Print the measurement as report Close button

(Print button)

Start: Starts a measurement when a sample is attached.

Break Interrupts a measurement, which can be continued later
Stop: Stops a measurement
Bottles/ Shows you the fill level of the contents of the bottles (Sheath/Waste) and the
Level: sample tube. Red indicates almost empty sheath or almost full waste bottle.
Fluids: Shows in turn the amount of sheath (μl/sec) and sample fluid (particles/sec)
kdata: Shows the memory capacity and deletes a histogram

Keyboard (touch screen ) appears while operating the Threshold//patient key on the left &
LL or Patient change keys on the right. The keyboard automatically closes by pressing ‘ent’
on the keyboard

References :

1. Partec CyFlow® Counter Instrument Operating Manual

2. Standard operating procedure for Partec cyflow counter –Diagnova division ,RFCL Limited