CyFlow® Counter

Instrument Operating Manual

 CyFlow® Counter Operating Manual

Table of Contents

Table of Contents ................................................................................................................................................2

Foreword ..............................................................................................................................................................3
Theory ..................................................................................................................................................................4
Introduction to Flow Cytometry .......................................................................................................................4
Flow Cuvette and Light Signals ......................................................................................................................4
Optical Standard Setup – Parameters and Filters.........................................................................................5
True Volumetric Absolute Counting................................................................................................................7
Traditional Flow Cytometric Techniques for Absolute Counting...............................................................7
Operation .............................................................................................................................................................9
Daily Start-Up Procedure ................................................................................................................................9
Performing a Measurement ..........................................................................................................................11
Daily Shut-Down Procedure..........................................................................................................................12
Detailed description of Software: CyView........................................................................................................13
Software Menu ...............................................................................................................................................13
Touch Panel ...................................................................................................................................................16
Keyboard ........................................................................................................................................................16
External mouse and keyboard ......................................................................................................................16
Software Functions ........................................................................................................................................17
Config-Script...............................................................................................................................................17
Measure-Script ...........................................................................................................................................17
Regions.......................................................................................................................................................17
Trigger/L-L ..................................................................................................................................................18
Gain.............................................................................................................................................................19
Sample Speed............................................................................................................................................19
Save ............................................................................................................................................................20
Print.............................................................................................................................................................20
Troubleshooting .................................................................................................................................................21
What to do if… ...............................................................................................................................................21
Emergency Cleaning of the Flow Cuvette ...................................................................................................21
Appendix ............................................................................................................................................................22
Installation Requirements..............................................................................................................................22
Setup of the CyFlow® Counter .....................................................................................................................22
Basic connections ......................................................................................................................................22
Maintenance and Service..............................................................................................................................23
Maintenance ...............................................................................................................................................23
Service ........................................................................................................................................................23
Transport and Storage...............................................................................................................................23
Disposal ......................................................................................................................................................23
Laser Safety ...................................................................................................................................................24
CyFlow® Counter – Technical Specifications .............................................................................................25

For in vitro diagnostic use with Partec recommended IVD reagents.

The Partec CyFlow® Counter flow cytometer complies with the European IVD Directive 98/79/EC and is
therefore marked with the CE sign.

© 2008 Partec GmbH

Otto-Hahn-Str. 32
48161 Münster
Germany

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 CyFlow® Counter Operating Manual

Foreword
The CyFlow® Counter is a Flow Cytometry System (FCM) of Partec. It is equipped with a green
(532 nm) solid-state laser and three optical parameters for the detection of side scatter (SSC), orange
(FL2) and red (FL3) fluorescence signals. The instrument is dedicated to monitoring the immune status of
+
HIV/AIDS patients (HIV Monitoring). It offers the possibility for absolute counting of CD4 positive (CD4 )
+ +
T-cells, the determination of CD4 T-cells among lymphocytes (CD4%) and absolute values of CD3 and
+
CD8 T-cells. The determination of CD4 percent is, according to the world health organization (WHO), the
clinically relevant parameter to determine initiation of the HAART therapy for children.

The CyFlow® Counter Operating Manual provides a short overview of the method of Flow Cytometry and
the physical phenomena. For further details please refer to the literature (e.g. Howard M. Shapiro:
Practical Flow Cytometry, Wiley, 2002). The other chapters explain the operation and provide further
details about the software.

For more details about the reagent kits suitable for use with the CyFlow® Counter please refer to the
respective product data sheets. There are also several Application Notes available.

If you have questions please contact your local distributor, one of the Partec subsidiaries or Partec in
Germany. Further details and addresses can be found on our website at:

www.partec.com/distributors

supportcenter@partec.com

Each request should be accompanied by the following information:

-Serial no. of the CyFlow® Counter

-Your complete contact address

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Theory

Introduction to Flow Cytometry

The term Flow Cytometry describes a technique for the analysis of cells – or particles – in aqueous
suspension, which is now used routinely. Cells or other biological particles pass in a fluid, single cell
stream through a measuring apparatus. In the most common scenario, one or more lasers interrogate
each particle and, at a minimum, the system measures the degree and direction of scattered light –
indicators of the particle’s size, shape and structure. If particles have been stained with one or more
fluorescent dyes – known as fluorochromes – the light source excites these dyes to provide additional
biological information about each particle, such as the presence of specific surface markers. The unique
advantage of Flow Cytometry is that they can rapidly and quantitatively measure multiple simultaneous
parameters on individual live cells.

Flow Cuvette and Light Signals

1. Flow Cuvette

The main element of the CyFlow® Counter Flow Cytometer is a flow cuvette. This is made of
quartz glass which contains a capillary with a diameter of 200 x 350 µm.

2. Fluidics

The fluidic system of a flow cytometer is used to transport particles from a random three
dimensional sample suspension to an orderly stream of particles travelling past one or more
illuminating beams. The fluidic system uses air pressure regulation to ensure stable operation
and consists of a sheath fluid line and a sample line feeding into the flow cell. As the sample
enters the flow cell chamber, the outer, faster flowing sheath fluid hydrodynamically focuses this
sheath fluid into a narrow core region within the jet and presents a single file of particles to
excitation sources. This geometry provides increased positioning accuracy at the laser
interrogation point for consistent excitation irradiance and greatly reduced particle blockage of the
flow cell.

3. Scatter signals

In the CyFlow® Counter cells pass in a single file through the laser beam focused onto the
capillary. This causes scattering of light providing information about different cellular
characteristics. The intensity of the forward scattered light (FSC) correlates with the size of the
cell. Smaller cells generate a weaker signal, larger cells a stronger signal. The side scattered light
(side scatter, SSC) is emitted in an angle of 90° and gives information about the “granularity” of
the cell. Cells with a high granularity create a stronger signal in the SSC. This happens because
of the high refraction index of the granules.

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4. Fluorescence signals

Fluorescence occurs when a molecule excited by light of one wavelength returns to a lower state
by emitting light of a longer wavelength. Cells can be sub typed by selecting epitope specific
fluorochrome labelled antibodies.

Optical Standard Setup – Parameters and Filters

The CyFlow® Counter is equipped with three optical parameters for the detection of side scattered light
(SSC), orange (FL2) and red fluorescence (FL3). The side scattered light and the fluorescence light are
collected through a Microscope lens in an angle of 90°. The light is then subdivided into different
wavelength by optical filters. (See picture below.)

1. Signal processing with Photomultiplier Tubes (PMT)

In the next step, the Photomultiplier collects the different wavelengths of light, which generate an
electronic impulse. The amplitude is proportional to the number of incoming photons. Following
linear or logarithmic amplification, the impulses are then converted into electronic signals by an
Analog/Digital-Converter (ADC). For CD4- and CD4%-measurements, only the logarithmic
amplification is applied. As a particle of interest passes through the focus, fluoresces and is
detected by a photodetector, an electric pulse is generated and presented to the signal
processing electronics. The instrument is triggered when this signal exceeds a predefined
threshold level. The threshold is primarily used to reject non particle events such as debris or
noise from optical and electronic sources.

2. Optical filter

Optical filter only allow the transmission of certain wavelength, while the rest of the light is
absorbed. Three types of filter can be distinguished:

a. Band pass filter

Allows the transmission of light within a small wavelength range.

b. Long pass filter

Allows the transmission of light above a certain wavelength.

c. Short pass filter

Allows the transmission of light below a certain wavelength.

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3. Display of a measurement

The data is displayed as either a 1-Parameter Histogram (CD4) or 2-Parameter Dot Plot (CD4%).

4. Gain

Gain is a measure of the amplification applied to a signal. In Flow Cytometry it occurs between
the time of the initial detection of a light pulse and the final output that is stored as the channel on
an ADC. For a weak light pulse, the gain can be increased by the use of an amplifier, increasing
the electrical signal after it leaves the photodetector, or it can be increased by altering the voltage
directly applied to a photomultiplier tube (For practical information see also chapter ”Gain”.).

5. Regions and Gates

Regions are drawn to define a certain cell population. In the 1-Parameter Histogram this is done
by setting the Range, within the 2-Parameter Dot Plot a Polygon is used (For practical information
see also chapter „Regions“). A gate is a restriction placed on the flow cytometric data that will be
included in subsequent analysis.

6. Fluorecence staining

For a further characterization of cells they are often marked with a fluorescent marker.
Leukocytes can be determined in subpopulations because of their CD-Markers (CD: Cluster of
Differentiation). For this specific antibodies are used which recognize a specific epitope on the
cell of interest. Because of the link between the antibody and the specific fluorescent dye (e.g.
R-PE: Phycoerythrin) the cell which carries the antibody on its cell surface emits an orange
fluorescence light when exited by a 532 nm Laser. This fluorescence light is detected in the FL2
channel of the CyFlow® Counter and assigned to a specific cell.

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True Volumetric Absolute Counting

The determination of cell counts or subsets of cells, also called Absolute Counting, can be of significant
interest for medical diagnostics or monitoring the status of cells in cell cultures or biotechnological
processes.

Traditional Flow Cytometric Techniques for Absolute Counting

1. Dual Platform Technique

To determine the number of cells per ml, Flow Cytometers (FCM) are frequently used together
with Haematology Analyzers. By multiplying the result of cell percentages from the FCM with the
count result from the Haematology Counter, the concentration of any cell subpopulation can be
determined. Obviously, this technique relies on the assumption, that the number of cells seen by
the FCM and counted by the Haematology Analyzer are identical.

2. Single Platform Technique with Reference Beads

Another way to analyze cell concentrations with a single FCM, is to add micro beads with a
“known” number or concentration to the cell suspension. By comparing the cell counts with the
bead count, the cell concentration can be calculated. Another variation of this method uses beads
of “known” concentration to recalibrate the fluidic system from time to time. This technique,
however, depends on the precision of the specified bead concentration.

3. Partec True Volumetric Absolute Counting TVAC

To overcome the drawbacks of the Dual- and traditional Single-Platform Technique, Partec
instruments offer an alternative way of absolute cell counting, which is based directly on the basic
definition of a concentration c

c=N/V

by precisely counting the number N of cells suspended in a purely mechanically defined volume
V.

a. Precise Counting: the Determination of N

For True Volumetric Counting, precise counting of the number N of cells is essential. This
requires fast recognition and analysis of the events by both the electronics and computer. All
Partec instruments are specifically designed to minimize counting losses by providing direct
connection between the computer and electronics, which avoids dead-times involved in
traditional FCM designs and instrument interfaces. Partec systems can handle event rates up
to 15,000 events/s. This reduces the probability of a counting loss for event rates below 2%.

b. Electrode-Principle: Determination of V

The method for True Volumetric Absolute Counting supported by the CyFlow® Counter is
based on the precise measurement of a fixed sample volume by means of two electrodes.

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While being analyzed by the FCM, at a certain time the sample disconnects from the upper
electrodes, thus generating a digital START signal for counting. The disconnection from the lower
electrodes generates a STOP signal. The volume V of the sample analyzed between START and
STOP is physically defined by the distance of the two electrodes and the diameter of the sample
tube. Effects of the sample meniscus in the tube, disconnecting from the electrodes only a certain
time after the sample level passes by, are eliminated: They occur for the START and STOP
condition in a similar way, since START and STOP electrodes are arranged symmetrically. The
sample conductivity does not influence the volumetric measurement, as long as the fluid can be
detected electrically.

In some cases, larger or smaller counting volumes V are advantageous, either to improve the
counting statistics (e.g. for rare events < 10/ml) or to decrease the analyzing time. This can be
accomplished by replacing the standard 200 µl volumetric sample port by one with a different
volume.

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Operation
Daily Start-Up Procedure
1. SHEATH and WASTE bottle

Make sure the Sheath bottle is filled with Sheath Fluid (Partec Code No. 04-4007) and with the
blue screw cap tightly closed. Tilt Sheath bottle in order to release air bubbles trapped under the
yellow inline filter unit. The Waste bottle should be empty and the red lid tightly closed.

Note: In resource poor settings Partec recommends the use of the Sheath Fluid Production Kit
(Partec Code No. 04-4020) for preparation of good quality Sheath Fluid.

2. Switching on the instrument

The CyFlow® Counter is operated with 240 V AC. Switch on the main power at the back of the
instrument, then push the green button on the left of the Partec logo located at the front.

3. Acquisition Software CyView

The software CyView starts automatically after the computer has booted. Then the Config-Script
and the Measure-Script (see: Performing a Measurement) for the different analysis (Count Check
Beads green, CD4 or CD4% analysis) has to be selected.

a. Load the Config-Script: Scripts  Load Config-Script  CountCheckBeadsGreen

b. The respective Measure-Script is automatically selected.

4. Cleaning

Plug a sample tube with 1.6 ml Cleaning Solution (Partec Code No. 04-4009) into the sample
port. Press the Start button to start the measurement. Wait until the measurement and
the subsequent cleaning procedure have stopped automatically.

Repeat with 1.6 ml of Sheath Fluid (Partec Code No. 04-4007) in order to remove
residual Cleaning Solution.

5. Quality Control

Plug a sample tube with 850 µl Count Check Beads green (Partec Code No. 05-4011)
into the sample port and press the Start button to begin the measurement. Wait until the
measurement and the subsequent cleaning procedure have stopped automatically. Data
are stored and printed on request.

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The result of the Count Check Beads green measurement is indicated in the Results
area. Compare with the lot specific number indicated on the bottle and check if the count
result ALL_1 is within the 10% range. If yes, the CyFlow Counter® is now ready for
further analysis. If no, shake bottle intensively and repeat measurement (See also
chapter: Troubleshooting.).

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Performing a Measurement
1. Check that the CyFlow® Counter is ready for measurement (See chapter: Daily Start-Up
Procedure).

2. For CD4 absolute counting (CD4% counting) stain a blood sample with the CD4 easy count kit
(CD4% easy count kit) as indicated in the product data sheet.

3. Load the respective Config-Script for CD4 absolute (CD4%) measurement which contains the
correct histogram and dot plot layout for analysis.

a. Load the Config-Script: Scripts  Load Config-Script  CD4 (CD4percent)

b. The respective Measure-Script is automatically selected.

CD4 CD4percent

4. Attach the sample tube with your sample to the sample port.

a. Click START to start the measurement. The instrument performs certain processes,
which are shown in the lower part of the screen:

i. PRERUN :Creates a temporary, fast sample flow

ii. RUN :The particles are measured and classified
iii. MEASURE :A certain volume gets measured and counted

b. First adjust, if necessary, the position of the CD4 T-cell peak in the histogram (the
position of the CD45 peak) with the GAIN bar. Then check the lower level threshold to cut
off unwanted background signals on the left side of the histogram if necessary. However,
do not cut off the main peak of interest. Please refer to the figures below.

5. During the COUNT Modus, indicated on the right green status bar, do not change Speed, Gain,
LL or other software parameters.

6. Wait until the measurement and a subsequent cleaning procedure have stopped automatically.
During this process FLUSH is indicated at the right sight.

7. If necessary adjust the region to mark the cell or particle population of interest (See Chapter:
Regions).

8. Enter or change the patient name.

9. Then save (See Chapter: Save) or print (See Chapter: Print) the result.

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CD4 CD4percent

10. The result of the measurement is shown in the windows “Regions” and “Results”. In “Regions” the
absolute number of the indicated cell population – analyzed in the 200 µl counting volume – is
displayed. “Results” shows the concentration of the respective cell population within the original
blood volume.

Daily Shut-Down Procedure

Flow Cytometers are complex but robust instruments, which can be cleaned and decontaminated in a few
easy steps. For this procedure the Partec Cleaning Solution and Decontamination Solution are used.

The following steps should be performed at the end of each day that instrument is used:

1. Plug a sample tube with 1.6 ml Decontamination Solution into the sample port and press the
START button. Run for 15 seconds and pinch the sheath fluid tube. Stop the instrument by
pressing STOP, release the pinched tube and incubate for 15 minutes. Re-start the system by
pressing START and wait until the measurement and a subsequent cleaning procedure have
stopped automatically.

2. Repeat with 1.6 ml Cleaning Solution without pinching the tube and incubation step.

3. Plug a sample tube with 1.6 ml sheath fluid to the sample port and press the START button. After
2 minutes of measurement press the STOP button and keep the sample tube with Sheath Fluid
attached to the sample port.

4. Switch off the instrument.

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Detailed description of Software: CyView

Software Menu
The following chapter gives an overview of the software parameter functions.

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

In the dropdown menu the following options are available:

Reload!: 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

Help: Program help: A help menu can be opened

About OEMP: Shows the version number of the software

Language: The menu language can be chosen (English or German)

By clicking on the respective button on the left side of the display, the following parameters can be
chosen and modified or functions are activated:

Change the threshold level of the trigger parameter. (L-L)

Insert/change a patient’s name. (Patient)

Change the settings for the sample flow. (Speed)

Choose between the parameters SSC, CD4 and CD45 by clicking several times on this
button. Change high voltage of the chosen parameter. (Gain)

Save the measurement as FCS data.

Print the measurement as report.

Clicking on one of the above displayed buttons opens a status bar on the right side of the display which
can be used for modification of the activated parameter.

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L-L: Set the threshold level of the trigger parameter by

choosing a value between 0-0.7.

Patient: Change the patient’s name. This function can be

found by pressing the L-L button.

Speed: Change the speed of the sample flow between 0.2

and 20 µl/sec.

Gain: Change the gain value of the parameter chosen (SSC,

CD4, CD45) which is the high voltage of the PMT;
value between 0-1000.

After clicking on the layout window the following options can be chosen by pressing the upper button on
the right sight of the display several times:

LogX: Switch between Log.- and Lin.-scaling (histogram

and x-axis of dot plot)

LogY: Switch between Log.- and Lin.-scaling (y-axis of

dot plot)

Base: Change the z-level of a dot plot; value 1-10.

Const: Apply a z-level of value 0 or 1 to a dot plot.

Clicking on one of the regions activates the following functions:

DEL: Delete a region if not used for display of result

NEW: Create a new region, choose name, region colour

and set points

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START: Starts a measurement when a sample is attached.

BREAK: Interrupts a measurement, which can be continued later

STOP: Stops a measurement

BOTTLES/LEVEL: Shows you the fill level of the contents of the bottles (Sheath/Waste) and the
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

LEVEL: Green indicates sample full, red indicates sample empty. Green/red blinking
indicates that sample is between two electrodes.

In the layout window different histograms and dot plots, depending on the ConfigScript that is used, are
shown.

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Touch Panel
CyView has been optimized for the use of a Touch panel with a pointer. Alternatively you can also use a
mouse. Most inputs can be recognized with the Touch panel and the Software keyboard. A “double-click”
is possible.

Keyboard
Inputs with the software keyboard are achieved by pressing ENTER and the keyboard closes
automatically afterwards.

External mouse and keyboard

Alternative you can use an external mouse or keyboard.

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Software Functions

Config-Script

The Config-Script defines the layout which includes number, labeling and display of histograms, dot plots,
regions and results windows. The Config-Scripts are stored in the Config-Folder and need to be loaded
before you start a measurement:

Scripts  Load Config-Script  CountCheckBeadsGreen (CD4, CD4percent)

For each measurement choose the corresponding Config-Script:

Measurement Reagent Kit Config-Script

Quality Control Count Check Beads green CountCheckBeadsGreen
CD4 absolute CD4 easy count kit CD4
CD4% and absolute CD4% easy count kit CD4percent

CountCheckBeadsGreen CD4 CD4percent

Measure-Script

The Measure-Script contains default settings for the Trigger parameter, Gain values, Speed and Regions.
It is loaded automatically with the respective Config-Script and can be changed before or during a
measurement.

Regions

A region separates an area of particles (cluster) from the Plot. A region belonging to a histogram has two
borders (Range), one belonging to a Dot Plot can have between 3 and 20 points (Polygon). A region can
be created/deleted during or after a measurement.

The following regions are predefined for each measurement and can be adjusted but not deleted:

Measurement Reagent Kit Predefined regions

Quality Control Count Check Beads green ALL_0, ALL_1, ALL_2
CD4 absolute CD4 easy count kit CD4
CD4% and absolute CD4% easy count kit CD4, LYM

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Range Polygon

How to add or change a region:

Change a region already set:

1. Click REGIONS until the region to be changed is selected

2. Click DO IT!

3. Draw a new region within the Plot (Histogram/Dot Plot)

a. Histogram: First set the left line then set the right line of the region

b. Dot Plot: Click for every corner point, to draw a Polygon around the
signal population. A maximum of 20 points is allowed.

4. Click DO IT! The new region is set

Create a new region:

1. Click REGION

2. Click DEL. The window NEW appears. Click DO IT!

3. Choose a name. You can do this with the software keyboard. Also a colour can be chosen. Click
NEXT

4. Draw a region within the Plot (Histogram/Dot Plot)

a. Histogram: First set the left line then the right line of the region

b. Dot Plot: Click for every corner point, to draw a Polygon around the
signal population. A maximum of 20 points is allowed

5. Click NEXT. The new region is set

Trigger/L-L

The trigger parameter for each measurement is saved within the Measure-Script and cannot be changed.

Measurement Reagent Kit Trigger parameter

Quality Control Count Check Beads green CD4 (FL2, orange fluorescence)
CD4 absolute CD4 easy count kit CD4 (FL2, orange fluorescence)
CD4% and absolute CD4% easy count kit CD45 (FL3, red fluorescence)

For an accurate measurement it might be necessary adjusting the default value of the L-L which is the
threshold level of the trigger parameter.

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L-L

Open the LL-tool by clicking on the L-L/Patient button on the left side of the display. Change the L-L value
by moving the bar up and down. The chosen value between 0-0.7 is displayed in the left green field.

Gain

The Gain value is the high voltage HV of the photomultiplier tube and describes the signal amplification of
each measured particle. The value can be set between 0 and 999. An increase of the amplification shifts
the signals/peaks to the right, a lowering shifts them to the left. With the Gain value the signals/peaks
within the histogram/Dot Plot can be optimized.

Gain

Choose the parameter (SSC, CD4, CD45) for which you would like to change the gain value by clicking
on the middle button on the left side until the desired parameter name appears. Then change the Gain
value by moving the bar up and down. The value between 0-999 is displayed in the left green field.

Sample Speed

With the Speed function, the sample speed can be adjusted from 0.2 - 20 µl/sec. A lower/higher sample
speed causes a lower/higher accuracy, but also expends/reduces the time needed for a measurement.
For a standard measurement like Count Check Beads green, CD4 absolute and CD4%, the default speed
is 3 µl/sec.

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Speed

To change the sample speed, activate the parameter by clicking on the µl/s button on the left side. Then
change the Speed value by moving the bar up and down. The value between 0.2-20 µl/sec is displayed in
the left green field.

Save

The measurement is saved on request by pressing the button FCS. The default file name is:
<patient>_<date>_<time>.

Print

The histograms and dot plots along with the results are printed on request by pressing the Print button.

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Troubleshooting

What to do if…
1. The result of the Count Check Beads green measurement is not within 10% range of the lot specific
concentration stated on the bottle.
• Shake bottle vigorously and repeat measurement.
• Perform an Emergency Cleaning Procedure.

2. During analysis sheath fluid flows back into your sample tube.
• The sample tube is not attached properly to the sample port. Make sure that you hear a “click”
sound when you plug the sample tube to the sample port. Repeat measurement with a new
sample.
• The sample tube is cracked. Repeat measurement with a new sample.
• Change the sealing of the BioSafety Sample Port.

3. Sample flow is very slow or is not running. Counting sometimes takes up to 5 min even with high
speed.
• Flow cuvette is dirty and partially blocked. Perform an Emergency Cleaning Procedure.
• Change the sealing of the BioSafety Sample Port.
• Check that the Waste bottle is closed properly and that the bottle is not cracked.

4. Sample run does not start automatically.

• Clean the two electrodes of the sample port carefully by wiping with a soft tissue soaked in
Decontamination Solution.

5. There is not histogram/dot plot visible during the RUN phase

• Check that you have loaded the correct ConfigScript for your measurement.
• Increase Speed value. If this works, then you should perform an Emergency Cleaning Procedure
• Check that the laser is functioning. Carefully open the cover of the instrument and check if the
green light of the laser is visible. Please refer to the chapter: Laser Safety.

6. There is not good separation between the specific peak/cell cluster and the background signals.
• Change the Sheath Fluid.
• Check the CyFlow® Counter with Count Check Beads green.
• Repeat the analysis (EDTA-blood from healthy donor).
• Change inline filter of Sheath bottle.

7. The peaks are very broad and separation between the peaks/cell clusters is bad.
• Check the blood sample (blood collection system, storage, preparation procedure).
• Check for an air bubble inside the flow cuvette.

Emergency Cleaning of the Flow Cuvette

Please perform the following steps:

1. Plug a sample tube with 1.6 ml Hypochlorite Solution into the sample port and press the START
button. Run for 15 seconds and pinch the sheath fluid tube. Stop the instrument by pressing
STOP, release the pinched tube and incubate for 15 minutes. Re-start the system by pressing
START and wait until the measurement and a subsequent cleaning procedure have stopped
automatically.

2. Repeat with 1.6 ml Decontamination Solution. The incubation time can also be extended
overnight.

3. Plug a sample tube with 1.6 ml Sheath Fluid to the sample port and press the START button.
Wait until the measurement and a subsequent cleaning procedure have stopped automatically.

4. Run a Count Check Beads green measurement to check the performance of the CyFlow®
Counter.

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Appendix

Installation Requirements
Power supply 100/240 VAC
Car battery / Solar panel 12 V DC (only in combination with
voltage converter)

Size W 325 mm x H 330 mm x D 265 mm

Weight: 11.5 kg

Operating Environment

The CyFlow® Counter should be placed on a solid base e.g. a laboratory table. It must be placed
horizontally. Reduce smoke, dust, vibrations, direct sunlight and any source of direct heat. The installation
room must be well ventilated and dry.

Temperature 15-30˚C
Humidity 20-85% relative (non-condensing)
Room Clean environment. Direct sun light should be avoided.
Air condition, if necessary.

Minimum Recommended Work Space

Table dimensions and space on top W 575 mm x H 350 mm x D 600 mm

Setup of the CyFlow® Counter

Basic connections

• Plug the mains line cable to the UPS (uninterruptible power supply) input.
• Connect the UPS with the CyFlow® Counter.
• Connect the waste tubing and air tubing to the respective connector (see labels) on the rear side
of the instrument
• Close the red screw cap of the waste bottle tightly.
• Connect the sheath tubing to the connector (see label) on the rear side of the instrument.
• Fill the Sheath Fluid bottle with Sheath Fluid, close the lid, and remove any air bubbles which
might be trapped below the inline filter by carefully tilting the bottle.

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Maintenance and Service

Maintenance

Carefully clean the CyFlow® Counter casing on a regular base with a soft cloth. Water must not enter
the CyFlow® Counter or UPS, or come into contact with electric connections and switches. When
cleaning the screen, always use special screen cleaner and soft cloth.

Do not use any organic solvents, nitro thinner, benzol, alcohol, highly concentrated bleach etc.!

Regularly empty the Waste bottle and clean with washing-up liquid and a brush.

Clean the Sheath bottle with bottled drinking water and a clean brush. Flush with bottled drinking water
several times. Remember that cleanliness of Sheath bottle is critical for proper operation.

If the CyFlow® Counter is not be used for a longer period of time, make sure that you leave a sample
tube with Sheath Fluid attached to the sample port after performing a daily shut-down procedure.

Service

All servicing must be undertaken by an authorized service engineer. Please contact your local supplier or
Partec (supportcenter@partec.com).

Transport and Storage

To transport the system to a different location it is necessary to disconnect all external tubings as well as
data and power supply connections. When using with potentially biohazardous material, please see
Partec Standard Operating Procedure (SOP) for decontamination. The system should be carried in
upright position. During transport or storage please ensure that the system will be stored under the
following conditions.

Temperature: 5-50°C
Humidity: 20-85% relative (non-condensing)
Room: Clean environment, no direct sunlight

Disposal

In case of product disposal, please proceed according to the Partec Standard Operating Procedure
(SOP) for decontamination. After decontamination, the system has to be disposed of according to the
local regulations and laws.

For further information, please contact your local distributor or Partec.

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Laser Safety
The CyFlow® Counter is a class I laser product according to the EN 60825-1.

Warning:

Laser light can be emitted if the protection cover for the laser beam is removed and the beam shutter is
opened. Therefore, the system is marked with the following laser safety labels:

Warning: laser radiation

Additional explanation

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 CyFlow® Counter Operating Manual

CyFlow® Counter – Technical Specifications

Note: Due to fast technological improvements, specifications herein are subject to change. For details,
please ask your supplier.

1. CyFlow® Counter System

Size W 325 mm x H 330 mm x D 265 mm

Weight 11.5 kg

Maximum sound power level < 70 dBA

Installation/overvoltage category 2/II

Degree of protection IP 20

Operating Environment Temperature 15-30°C

Humidity 20-85% relative (non-condensing)
Room Clean environment. Direct sun light should be
avoided.

Applications HIV/AIDS Patient Monitoring, Immunophenotyping, 2 Colour

Analysis and Determination of Particle Concentrations with TVAC.

TVAC True Volumetric Absolute Counting. Based on the precise counting

and mechanical fluid volume measurement. No need for reference
sample or reference beads.

Instrument Check Count Check Beads green (Partec Code No. 05-4011)

Setup Time Max. 5 minutes

Parameters 3 optical parameters: SSC, FL2, FL3

Particle Size Range 0.5 – 50 µm (standard cuvette)

Maximum Acquisition Speed 15,000 events/sec

Acquisition Stop Time volume-based

Trigger FL2 (CD4 count)

FL3 (CD4 percent)

Data Resolution 65,536 (16 bit)

Graphics Resolution 256 channels resolution for 1P histogram

256/256 channels resolution for 2P dot plot

Service Please contact your local supplier or Partec

Warranty 12 months on all parts except filters, mirrors, other quartz or glass
parts, disposables and cuvettes as long as not otherwise stated

2. CyFlow® Counter Optics

Laser/Output Green NdYAG: 30 mW at 532 nm

Detectors 3 (SSC, FL2, FL3)

Filters Standard setup and filters for SSC, FL2 and FL3
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Excitation Optics Elliptical 15 µm x 100 µm at 488 nm

3. CyFlow® Counter Fluidics

Flow Cuvette Synthetic quartz flow cuvette with acentric flow channel (350 x 200
µm) for laminar sample transport with sheath fluid for scatter and
fluorescence light detection.

Sample Delivery Computer controlled precision syringe pump for contamination-free

sample transport. Built-in vacuum pump (400 mbar) for Sheath
Fluid and sample transport.

Sampling Volume Continuous up to 2000 µl

At least 840 µl for True Volumetric Absolute Counting (TVAC)

Flow Rates Sample volume speed adjustable continuously between 0.2 and 20
µl/sec

Fluidics Volume 2 x 1l glass bottle (explosion protected) for Sheath Fluid and waste

4. CyFlow® Counter Electronics and Computer

Electronics Signal processing

Amplifiers
16 bit ADC
Trigger

Computer Built-in Computer

Intel Celeron computer processor
8.4’’ TFT colour touchscreen (SVGA)
Keyboard (optional)
Mouse (optional)
Printer (built-in)

5. CyView Software

Software Windows™ Software

FCS 2.0 standard listmode format
Real-time acquisition
Report (automatic calculation)

Acquisition Gating Lower level hardware thresholds for event triggering parameters,
adjusted by software
Region (up to 20 spots)
Range

Gating On- and offline gating, gating stored with ConfigScript

ConfigScript Saves Layout of the Measurement

MeasureScript Saves Gain, speed, etc as default. Loaded with respective

ConfigScript

Report Automatic calculation of CD4 and CD4%

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