SYSMEX CyFlow Cube 8 User manual

Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com
CyFlow®Cube 8
Instrument Operating Manual
Revision:
Rev-27_2015-07-02
Issued by:
Sysmex Partec GmbH

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 2 / 43
Imprint
Copyright 2014 by
Sysmex Partec GmbH
Am Flugplatz 13
02828 Görlitz
Germany
Phone: [phone]
Fax: [phone]
Mail: [e-mail]
Web: www.sysmex-partec.com

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 3 / 43
Table of content
Table of content................................................................................................................3
1Introduction................................................................................................................5
2Presentation...............................................................................................................6
2.1 Basics..................................................................................................................6
What is the Sysmex Partec CyFlow®Cube 8?............................................................6
What are the applications for which the CyFlow® Cube 8 can be used? .....................6
What are topics covered by this manual?...................................................................6
What other manuals are available?............................................................................6
What should I know before operating the CyFlow®Cube 8?.......................................6
2.2 In flow cytometry, what is... ..................................................................................7
… a parameter? .........................................................................................................7
… a one-parameter histogram?..................................................................................7
… a histogram channel? ............................................................................................7
… the count in a histogram?.......................................................................................7
… a peak? ..........................................................................................................7
… background in a histogram?...................................................................................7
3. Instrument starting procedure.....................................................................................10
3.1 Sheath fluid level control and/or refill ...................................................10
3.2 Switching on the CyFlow®Cube 8........................................................10
3.3 User levels...........................................................................................12
4. CyViewTM Controls......................................................................................................13
4.1 CyView™ Instrument Setting Controls .................................................14
4.1.1 1D/2D PLOTS–display options ...........................................................14
4.1.2 RESULTS............................................................................................15
4.1.3 REGIONS............................................................................................16
4.1.4 GENERAL - properties of the instrument.............................................17
4.1.5 ACQUISITION –instrument control .....................................................17
4.1.6 MEASURE –definition of measure modes...........................................18
4.1.7 Measure modes...................................................................................19
5. CyViewTM Instrument/Measurement Settings.......................................................22
5.1 Priming / Initialization process..............................................................22
5.2 Work process.......................................................................................24
5.3 Shut down process..............................................................................24
5.4 Intermediate cleaning process.............................................................25
5.5 Sheath and Waste bottle......................................................................25
6. Measurement parameters...........................................................................................26

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 4 / 43
6.1Trigger properties (P)...........................................................................26
6.2 Instrument setting properties ...............................................................26
6.3 1D/2D Plot properties ..........................................................................27
6.4Region/ROI properties.........................................................................28
6.5 Compensation .....................................................................................31
7. Typical Sample Analysis.............................................................................................32
8. Keyboard/Mouse combinations ..................................................................................34
9. Appendix....................................................................................................................35
9.1 Biohazards.........................................................................................................35
9.2 Maintenance ......................................................................................................36
9.2.1 Service ................................................................................................36
9.2.2 Transport and Storage.........................................................................36
9.2.3 Disposal...............................................................................................37
9.3 Laser Safety.......................................................................................................38
9.4 Technical Specifications.....................................................................................39
9.4.1 Example for Optical Standard Setup....................................................39
9.4.2 CyFlow® Cube 8 System......................................................................40
9.4.3 CyFlow® Cube 8 Optics........................................................................41
9.4.4 CyFlow® Cube 8 Fluidics......................................................................41
Notes..............................................................................................................................42

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 5 / 43
1 Introduction
The CyFlow®Cube 8 Instrument Operating Manual is aimed for a large spectrum of users,
from beginner up to the most skilled flower. The beginner or casual user will find the key
functions and concepts to use the Cube 8 and its software. The confirmed flower will find
an in-depth detail of the inner working and parameters of the Cube 8 to customize its use
and obtain optimal performances.
CyView™for Cube 8 is the instrument operation software for the CyFlow®Cube 8.
Sysmex- Partec is continuously working on CyView™to better fulfill your demands. If you
have questions concerning this manual or the software, find problems associated with
CyView™or you have a good suggestion to be included in a new version, please let us
know by sending an email or a note to Sysmex Partec GmbH.
The present manual is valid as of CyView™ software version 1.5.
For more details about the reagent kits, suitable for use with the CyFlow®Cube 8,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 Sysmex Partec
subsidiaries, or Sysmex Partec in Germany ([e-mail]).
Further details and addresses can be found on our website at:
www.sysmex-partec.com
Please do not forget to add in your request the following information:
Serial number (serial No.) of the CyFlow® Cube 8
Your complete contact address

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 6 / 43
2 Presentation
2.1 Basics
What is the Sysmex Partec CyFlow®Cube 8?
The Sysmex Partec CyFlow®Cube 8 is a fully equipped desktop Flow Cytometer (FCM).
CyFlow®Cube 8 features a modular optical concept. This allows using different lasers as
light sources and the detection of up to 8 optical channels (parameters). The
CyFlow®Cube 8 allows easy optimization of the optics for any application by simple
exchange of optical filters and mirrors. The CyFlow®Cube 8 runs with an internal PC.
Data acquisition, instrument control, and data analysis are controlled and performed by
the CyView™software.
What are the applications for which the CyFlow® Cube 8 can be used?
Together with the software, the CyFlow®Cube 8 offers automation for routine use and
flexibility for research use for practically any flow cytometric application. The applications
cover:
Routine multi-color-immuno-phenotyping
Blood Cell Analysis/HIV monitoring (e.g. CD4 cell count)
Leukocyte Counting/Rare Event Analysis
Microorganism Analysis
Fermentation Control
Particle Concentration Analysis
True Volumetric Absolute Counting
Particle Size and Fluorescence Distribution Analysis
What are topics covered by this manual?
The CyFlow®Cube 8 Instrument Operating Manual covers the basic operation and
maintenance of the CyFlow®Cube 8 instrument. This manual also covers details related
to the software.
What other manuals are available?
Application Notes and Service Manuals are available to get started. They contain hints
to achieve the best results.
What should I know before operating the CyFlow®Cube 8?
This manual assumes that you have basic knowledge on flow cytometry. In the best case
a well experienced "flower" is around - so let her/him help you. Basic books are available
about flow cytometry which may help you as well (e.g. Howard M. Shapiro, Practical Flow
Cytometry. Wiley 2002).

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 7 / 43
2.2 In flow cytometry, what is...
… a parameter?
In flow cytometry, parameter denotes a measured property of
the particles. Frequently, a parameter is synonymous to an
optical channel. E.g. an instrument with 6 parameters is
equipped with 6 optical detectors.
… a one-parameter histogram?
A one- parameter histogram displays the distribution of cells
among a specific property, e.g. how many cells contain a given
quantity of DNA or bind a given number of antibody molecules.
… a histogram channel?
The measured signal intensity is assigned to one of 65536 (16bit) quantity classes or
channels. In a one-parameter histogram the channels are represented on the x-axis.
… the count in a histogram?
The number of cells being assigned to a given channel is referred to as channel content
or simply count. In a one-parameter histogram, the count is shown on the y-axis.
… a peak?
All cells having about equal characteristics among the analysed cell property (e.g. content
of a specific constituent like DNA) form a peak. In the case of a typical DNA histogram
one peak represents the G1 and another peak (with twice the channel value) represents
the G2/M phase of the cell cycle.
In case of immunolabelled cells often one peak for unlabelled (negative) and one peak for
labelled (positive) cells can be detected. Peaks can be analysed by identifying them with
region markers.
… background in a histogram?
Histograms sometimes show undesired signals in the lower channels, frequently called
´noise´ or ´background´. These signals may originate from cell fragments or other

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 8 / 43
particles resulting from sample preparation. In case of high signal amplification,
background can also be caused by particle contaminated sheath fluid.
The lower level (L-L) or threshold?
The lower level (L-L) threshold is a mean to suppress background signals. Signals below
the lower level are rejected from the signal acquisition. To exclude noise from a histogram
already acquired, a region-gate can be used.
Example of a histogram
A histogram represents a distribution of measured signals
(events) over 1 dimension. Data can be presented on the
dimensions of relative particle size or optical particle
structure (Forward Scatter (FCS) or Side Scatter (SSC)),
resp. or on their relative fluorescence intensities in
different light colors (fluorescence parameters FL1 to FL6).
In this example, the dimension represented is the relative
size (FSC) on a logarithmic scale in X, and the number of
events on a linear scale in Y. Two peaks are visible.
Example of a dot plot
A 2D dot plot presents correlated data over 2 dimensions. In the image on the right a
sample of leukocytes (after lyse of the red blood cells) is plotted with their relative light
scattering (SSC) property against the intensity of the CD45 antigen.
The Z value represents the number of events that have the same coordinates. 1 event will
be represented by a black point; if 10 events have the same coordinates, the point
representing them will be grey. It will be yellow if more than 20 points are overlaying one
another. The Z scale is dynamic and will adapt during the measurement to a scale of 1
(black) to the maximum overlaying event coordinate color-coded in red.

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 9 / 43
Histogram and dot plot in immunology
Lysed blood 2D dot plot
Lysed blood is represented in a dot plot presenting the FSC in X axis and SSC in Y axis.
Both axes are in a linear scale.
Three distinct groups are visible; they represent the granulocytes (G), monocytes (M) and
lymphocytes (L).
Histogram and dotplot of immunological staining
This histogram represents the spectrum of the cells presented in the previous dot plot
stained with antibodies anti-CD45 conjugated to Phycoerythrin (PE) Dy647. The X axis
displays the fluorescence in a 4-dec logarithmic scale and the Y axis displays the number
of events in a linear scale.
This dot plot presents the cells fluorescence in X on a logarithmic
scale (CD45 PE-Dy647) versus SSC in Y on a linear scale.
This data display allows an easier interpretation of 2 parametric
data compared to the histogram. Concluded from this example,
the lymphocytes are strongly stained with the anti-CD45 antibody,
the monocytes are slightly stained and the population of
granulocytes is negative.

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 10 / 43
3. Instrument starting procedure
3.1 Sheath fluid level control and/or refill
Before switching on the machine, it is recommended to
check the levels of the sheath fluid and waste bottles.
They can be found at the back-left of the apparatus in a
sliding compartment.
Make sure SHEATH bottle is filled with 700 ml of clean,
filtered, and degassed sheath fluid and is closed with
the screw top.
Please notice: Higher level of sheath fluid could lead to
unstable sample flow.
In order to guarantee highest quality of the
measurements we highly recommend use Sysmex Partec Sheath Fluid (order no. 04-4007).
It is recommended to replace the sheath fluid at least once a week or before any daily
use.
When filling up the sheath fluid bottle make sure no air bubbles are trapped in the yellow
filter unit inside the bottle!
Make sure that the WASTE bottle is empty and the screw top is tightly closed.
The waste bottle must be emptied after and before each user session. When using bio-
hazardous samples, a volume of 50 ml of hypochlorite 0.5% (Order No 04-4012) should
be introduce into the empty waste bottle for initial disinfection.
3.2 Switching on the CyFlow®Cube 8
The power supply switch is found at the back of the Cube next to the main supply cable.
The Cube is, in default, set in a stand-by mode. The full activation of the Cube requires
pressing lightly the on/off tactile button on the top of the machine. The display screen
must be first lift up to access it.
This will start the embedded computer, automatically start the CyView™software and load
the last employed configuration.
Casual/medium expertise user:
Once the Cube started no further steps
are necessary. You can directly start your
measurements!

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 11 / 43
Lost your login details?
Important: On each new instrument a Main
User login is already established
Login: USER (case sensitive)
Password: Cube1 (case sensitive)
Login as a standard USER:
The code will be given to you by the main
user(s) of the instrument. The main user
has the rights to define or delete users.
Main CyView™login window
The log in window allows to start the software at the USER, MAIN USER or SERVICE level.
During start of the instrument the automated self-testing procedures are processed:
Cleaning up and XML-configuration:displays
the correct loading of the setting files
Searching Device: display of the correct
recognition of the connection between the
computer and the embedded electronics
Please verify that all operations are confirmed
by a green tick.

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 12 / 43
3.3 User levels
Three different user levels exist:
SERVICE/ADMINSTRATOR:
Restricted to authorized Sysmex Partec trained persons and for service purposes
only
MAIN USER:
Complete functionality of the instrument, method development
Main users can create new accounts of the Main User and User level
USER:
Applying standardized methods only
Users cannot create new accounts.
As a main user in order to create new accounts type in your login name and password
and select “User Administration”.
To create a new MAIN USER account type in name and password and activate “Main
user” followed by “Build new account”.
To create a new USER account type in name and password followed by “Build new
account”. (Main user should be deactivated).
As User the own password can be changed by selecting the name and placing a new
password followed by “Change my password”.
As Main user the own password can be changed by selecting the name and placing a
new password followed by “Change my password”. As Main user any user account can
be deleted by selecting the name followed by “Delete this account”. The Main user does
not require the respective password to delete any user account.
The default user account (Name: USER; Password: Cube1) can be deleted when logged
in as Main user. Please make sure at least one Main User remains in the user list in order
to guarantee complete functionality of the software.
To enter the CyView™software from the user administration level select “Back to login”,
login with your personal Login name followed by “Work with CyView™.

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 13 / 43
4. CyViewTM Controls
CyView™Main Page –after successful log-in
The main window will be your interface to acquire, save, re-load and analyse your
data
Instrument real time display of workload (L), on-board memory status (M), analysis
volume (V) and analysis duration (D).

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 14 / 43
4.1 CyView™Instrument Setting Controls
Overview of instrument settings
4.1.1 1D/2D PLOTS–display options
The 1D/2D Plots register defines properties of the graphical plots. The basic layout in
terms of number of plots, type of plots (histograms, dot plots) and position of the plots is
defined in the used Configuration File.
As example: 12Pl →6 Histograms + 6 dot plots
Plots are named H1 –Hx for histograms and
P1 –Px for dotplots. A specific plot is
selected with the arrow keys.
Define a Comment characterizing the plot e.g. FSC
Select X-axis channel and Y-axis channel in dot plots
or X-axis channel only for histograms
Switch between Lin and Log scale
for Xand Yaxis and change the Zaxis
(scaling)
Define an Erosion level for dots to be displayed (z-axis
level), e.g. an Erosion level of 2 shows only dots
representing 3 and more signals
Select Dotplot (DP)-Mode
(Color Mode, Contour Mode or Color + Contour Mode)
Select histogram resolution as BitRange,
(values 16bit to 12bit)
Select CR –Mode to show “All events” or “Regions
only”
Confirm all modifications by pressing Accept

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 15 / 43
4.1.2 RESULTS
The RESULTS register defines properties of calculated results displayed in the RESULTS
table.
Properties of result calculations
It is possible to set up calculations with the COUNT of individual regions according to the
specified formula:
NumReg1 (+ - x / ) NumReg2
--------------------------------------------------x Scale
DenomReg1 (+ - x / ) DenomReg2
NumRegion1/2 defines numerators
DenomRegion1/2 defines denominators
NumOperator defines operator between 2
numerators „+“, „-“, „x“ or „/“
DenomOperator defines operator between 2
denominators „+“, „-“, „x“ or „/“
Unit allows to add text to the result table
Scale introduces a factor to the formula
Counter results on refers to the result of a
Volumetric counting
Create a result by pressing “New” or delete a
result by pressing “Delete”
Confirm all modifications by pressing Accept
Result will be display in the “Results table” next to the “Region statistics”

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 16 / 43
4.1.3 REGIONS
Home Plot Name defines the plot the
region refers to
Color RGB defines the region´s color
and its color in color gating
Max Count defines a maximum count for
an “Cells in Region” particle limit
(see also Pages 17-19)
Sorter Region On activates region as
sorter region (only in CyFlow® Cube Sorter)
Color Gating On activates/deactivates
the region
Use Delete and New to delete and
create new regions
Use arrow keys to switch between regions
Confirm all modifications by pressing
Accept

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 17 / 43
4.1.4 GENERAL - properties of the instrument
The active Configuration File
The instruments Serial Number
Total Operating Time of the instrument
Measure Number shows total number of measure-
ments
Version of the instrument
Modification of the instrument
Activates Sorter function
Activates Lowpasses
Defines a factor for sample Dilution
Clinic/Customer display specific user information
Defines sample port electrode Volume in µl
µlPerSec/mBar defines a sheath fluid flow parameter
SW-Version specifies current software version
Rescale LSB shows area of automatic scaling
Storemode defines FCS file format
4.1.5 ACQUISITION –instrument control
Select the trigger parameter
multiple trigger parameters are logical “or” connections
Gain values to change the voltage of the individual
optical parameters
(0 volt - 999 volts) defines PMT signal amplification
Threshold defines the trigger signals cut-off level
(on 4 dec log scale)
Flow defines the speed of sample injection in µl/s
Lights on switches light sources on/off

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 18 / 43
4.1.6 MEASURE –definition of measure modes
Measmode allows selection of:
Analyze all:
run sample until its levels reached the stop electrode
Volumetric counting (VC) with volume
a volume can be pre-selected
Volumetric counting (VC) with electrodes:
START and STOP electrodes of the sample port are
used to define a fixed sample volume of 200µl
Cells in region
a particle number can be pre-selected
Speed values and Volume values can be edited and will be stored upon saving a
configuration file.
Autoflags allows activation of specific functions:
Autostart: automatic start of the measurement
Activate the AutoStart flag
Press start (only once to confirm the process)
Connect a sample tube
Autostart:
With each new sample tube the next measurement will be started until the STOP button is
pressed.
AutoPA:
automatic peak analysis, at the end of the measurement →only for Ploidy
Autosave:
Automatic storage at the end of the measurement. Autosave does not function when the
measurement is finished by the “end” button.

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 19 / 43
Schematic overview of the acquisition process for all measure modes
Green and red colored bars refer to particle count indicator of CyView.
Please note: In the measure mode “VC of Volume” or “Cells in Regions” there is no cleaning
cycle. Subsequently the data can be stored. In the measure modes “VC electrodes” and “Analyze
all” the cleaning cycle will occur automatically as soon as the sample is finished.
No cleaning cycle will be started if the measurement is terminated by the “end” button.
Beware: for all measure modes the sample analysis automatically stops when the sample is empty
(the stop electrode is reached) even if the selected end criterion is not yet realized.
4.1.7 Measure modes
The following measure modes can be selected prior to start of an analysis:
Analyze all
Volumetric counting (VC) of volume
Volumetric counting (VC) electrodes
Cells in region

Instrument Operating Manual CyFlow® Cube 8 Rev-27_2015-07-02
Sysmex Partec GmbH
Am Flugplatz 13, 02828 Görlitz, Germany · Phone [phone] · Fax [phone] · [e-mail] · www.sysmex-partec.com 20 / 43
Analyze all (default selection)
This default mode will allow you to run your sample until its levels reached the stop
electrode.
Volumetric Counting with Volume
In the measure mode volumetric counting with volume the counting volume is flexible
and can be pre-selected by the user. In a first analysis phase the sample is acquired
normally as in continuous acquisition mode. Reaching the pre-selected volume the data
are cleared and the volumetric counting phase starts and the pre-selected volume will be
analyzed. The volume can be used as the basis for concentration determination.
Volumetric Counting with Electrodes
This measuring mode uses the START and STOP electrodes of the sample port to define
a fixed sample volume. In a standard sample port this “counting volume” is 200µl. In a
pre-counting phase the sample is acquired normally as in continuous acquisition mode.
Reaching the START electrode the data are cleared and the volumetric counting phase
starts. Reaching the STOP electrode the counting procedure will be terminated and a
system cleaning cycle will be initiated automatically.
Cells in Region
The Events in Region measure mode allows to define a number of particles within a
specified region to operate as STOP condition (select the respective MaxCount function
in the REGIONS register).
Working principle of the Absolute Volumetric Counting with Electrodes