Partec CyFlow Cube 6 User manual

CyFlow®Cube 6
Instrument Operating Manual
CyFlow®Cube 6

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For in vitro diagnostic use with Partec recommended IVD reagents.
The PartecCyFlow®Cube Flow Cytometer complies with the European IVD Directive 98/79/EC and is therefore CE marked.
IVD
Contact Information:
Partec GmbH • Otto-Hahn-Straß3 32 •D-48161 Münster •Germany
Tel [phone] • Fax [phone]
E-mail: [e-mail]
© 2012Partec GmbH

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Table of content
FOREWORD ................................................................................................................... 5
PRESENTATION............................................................................................................. 6
BASICS ........................................................................................................................................................................................... 6
What is the Partec CyFlow®Cube?....................................................................................................................................... 6
What are the applications for which the CyFlow® Cube 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? ....................................................................................... 6
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
Example of a histogram............................................................................................................................................................. 8
Example of a dotplot ................................................................................................................................................................... 8
Histogram and dotplot in immunology.............................................................................................................................. 9
INSTRUMENT STARTING PROCEDURE.....................................................................................................................................10
Sheath fluid level control and/or refill .............................................................................................................................10
MAIN CYVIEW™LOGIN WINDOW.............................................................................................................................................11
USER LEVELS...............................................................................................................................................................................12
CYVIEW™MAIN PAGE –AFTER SUCCESSFUL LOG-IN..........................................................................................................13
CYVIEW™CONTROLS................................................................................................................................................................13
1D/2D PLOTS–display options............................................................................................................................................14
RESULTS .........................................................................................................................................................................................15
REGIONS .........................................................................................................................................................................................16
GENERAL - properties of the instrument.........................................................................................................................16
ACQUISITION –instrument control ...................................................................................................................................17
MEASURE –definition of measure modes .......................................................................................................................17
Measure modes ............................................................................................................................................................................18
Analyze all (default selection) ..............................................................................................................................................18
Volumetric Counting with Volume .....................................................................................................................................18
Volumetric Counting with Electrodes ...............................................................................................................................19
Cells in Region ..............................................................................................................................................................................19
CYVIEW®INSTRUMENT/MEASUREMENT SETTINGS.........................................................................................................20
PRIME/WORK/CLEAN .............................................................................................................................................................20
Priming / Initialization process...........................................................................................................................................20
Work process ................................................................................................................................................................................22
Shut down process......................................................................................................................................................................22
INTERMEDIATE CLEANING PROCESS .......................................................................................................................................23
SHEATH AND WASTE BOTTLE..................................................................................................................................................23
MEASUREMENT PARAMETERS.................................................................................................................................................24

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Trigger properties (P) ..............................................................................................................................................................24
Threshold settings (T) ..............................................................................................................................................................24
Flow speed (S) ..............................................................................................................................................................................24
Light source (L) ...........................................................................................................................................................................24
1D/2D PLOT PROPERTIES.......................................................................................................................................................25
Plot name........................................................................................................................................................................................25
X Log On/Y Log On.....................................................................................................................................................................25
Erosion levels................................................................................................................................................................................25
X/Y Channel...................................................................................................................................................................................25
BitRange .........................................................................................................................................................................................25
CR-Mode..........................................................................................................................................................................................25
REGION/ROI PROPERTIES .......................................................................................................................................................26
Create a polygonal region ......................................................................................................................................................26
Region properties .......................................................................................................................................................................26
Region name .................................................................................................................................................................................26
Home Plot name..........................................................................................................................................................................26
Color RGB........................................................................................................................................................................................26
Max count.......................................................................................................................................................................................26
Sorter region.................................................................................................................................................................................26
Moving of regions within a histogram..............................................................................................................................26
Create a vertical or horizontal histogram splitter......................................................................................................27
Create a quadrant assembly..................................................................................................................................................27
Change layout for regions.......................................................................................................................................................27
Applying regions to other plots  Gating Function...................................................................................................27
COMPENSATION .........................................................................................................................................................................28
TYPICAL SAMPLE ANALYSIS.....................................................................................................................................................29
KEYBOARD/MOUSE COMBINATIONS....................................................................... 31
APPENDIX .................................................................................................................... 32
BIOHAZARDS...............................................................................................................................................................................32
MAINTENANCE ...........................................................................................................................................................................33
Service..............................................................................................................................................................................................33
Transport and Storage.............................................................................................................................................................34
Disposal ...........................................................................................................................................................................................34
LASER SAFETY............................................................................................................................................................................35
TECHNICAL SPECIFICATIONS ...................................................................................................................................................36
Optical Standard Setup ........................................................................................................................................................36
NOTES .......................................................................................................................... 39

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Foreword
The CyFlow®Cube 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 and its software. The confirmed flower will find an in-depth detail of the inner working and parameters
of the Cube to customize its use and obtain optimal performances.
CyView™for Cube 6 is the instrument operation software for the CyFlow®Cube 6 (CY-S-3060). 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 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,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 ([e-mail]).
Further details and addresses can be found on our website at:
www.partec.com/distributors
Please do not forget to add in your request the following information:
Serial number (serial No.) of the CyFlow®Cube
Your complete contact address
This manual contains references to names and products from Partec and other companies which
are registered trademarks or protected by copyright.
Partec GmbH, CyFlow®Cube6, CyView™for Cube6, Robby®.
Microsoft®Corp.: Windows, Word, Excel, PowerPoint, Paint.
Hewlett Packard®: Deskjet Laserjet.

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Presentation
Basics
What is the Partec CyFlow®Cube?
The PartecCyFlow®Cube is a fully equipped desktop Flow Cytometer (FCM). CyFlow®Cube features a
modular optical concept. This allows using different lasers as light sources and the detection of up to 8
(CyFlow®Cube 8) or of up to 6 (CyFlow®Cube 6) optical channels (parameters). The CyFlow®Cube allows
easy optimization of the optics for any application by simple exchange of optical filters and mirrors. The
CyFlow®Cube 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 can be used?
Together with the software, the CyFlow®Cube 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 Instrument Operating Manual covers the basic operation and maintenance of the
CyFlow®Cube 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?
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).

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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 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.

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Peaks
Number of events
Threshold
of lower
limit
Linear representation of SSC
Log representation
for fluorescence
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 dotplot
A 2D dotplot 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.

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G
M
L
Histogram and dotplot in immunology
Lysed blood 2D dotplot
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
PhycoErytrin (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.

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Rear side of CyFlow®Cube showing the
main power switch and the sheath fluid and
waste bottle compartment
Instrument starting procedure
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 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 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.
Switching on the CyFlow®Cube
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 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!

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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.
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.

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User levels
Three different user levels exist:
SERVICE/ADMINSTRATOR: Restricted to authorized 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™.

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Buttons allowing to load and save data files
(.fcs data files).
Buttons dedicated to saving and retrieving
instrument settings (configuration files).
Button for the generation of a PDF report of
acquired data.
Buttons to select “Prime”, “Work” or “Clean” batch
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).
Console
PC/control
board link
Fileoperations
Real time
instrument
work load
Region statistics
Results table
Instrument settings
L
M
Measurement
interface and
indicators
V
D
Status of sample (high, counting phase, empty)
and of waste and sheath fluid bottles levels.
Start and Stop measure buttons.
“Meas” button opens the PROCESS register
Compensation button and random bias button.
Clear button –deletes all data during a
measurement

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CyView™Controls
Overview of instrument settings
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 Dotplots
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 6bit to 12bit)
Select CR –Mode to show “All events” or “Regions only”
Confirm all modifications by pressing Accept

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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”

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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
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 measurements
Version of the instrument
Modification of the instrument
Activates Sorter function (only in CyFlow® Sorter)
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

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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
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 were
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 confirmation file.
Autoflags allows activation of specific functions:
Autostart: automatic start of the measurement
Activate the AutoStart flag
Press start (only ones to confirm the processes)
Connect a sample tube
With each new connection a sample tube the next measurement will be initialled until the button is
pressed.
AutoPA: automatic peak analyse, 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.

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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 criteria is not yet realized.
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
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.

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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 200 µl. 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).
Schematic overview of the measure modes with a flash indicates clearing of the data. User selectable
criteria in green, software-defined values in red
Working principle of the Absolute Volumetric Counting with Electrodes

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CyView® Instrument/Measurement Settings
Prime/Work/Clean
There are 3 different possible modes available: The “PRIME” mode for a priming/initialization process, the
“WORK” mode for measurements and the “CLEAN” mode for cleaning of the system.
Prime, Work and Clean mode
Priming / Initialization process
There is a PRIME mode available as a Start Up process for the CUBE instrument. This process includes
cleaning, filling the tubes with sheath fluid and control of the instrument set up. If a new original CFG-file will
be loaded the PRIME mode is always selected. User defined configuration files can start in PRIME or
WORK mode, depend on the status during saving.
In the following cases the PRIME mode should be performed:
Starting the instrument for the first time → Daily initialization
After the Sheath-fluid bottle was filled
As Trouble-Shooting procedure (no/bad signals, blocking, etc.)
To start the priming/initialization process, please follow the instructions below:
Open a configuration file and select the initialization modus by clicking on the Initialization batch
symbol (Partec Master Cfg-file: Prime mode is preset).
If you will perform the daily check-up please select the “Calibration Beads”configuration-file. Only in
this configuration file you will find the correct settings for the calibration beads. These settings are
crucial for a correct verification of the instrument performance.
Initialization batch, Work batch and Clean batch
After pressing Start the system will start the priming process automatically and guide through
the program.
Connect a sample tube with Decontamination Solution (violet solution, Order No. 04-4010), press
“Continue” and wait until the system finished.