Partec CyFlow Cube Series, CyFlow Cube 6, CyFlow Cube 8, CyFlow Cube 8 Sorter Instrument Operating Manual

CyFlow® Cube
Instrument Operating Manual

CyFlow® Cube 6 │ CyFlow® Cube 8 │ CyFlow® Cube 8 Sorter

CyFlow® Cube Instrument Operating Manual

© 201

2 Partec GmbH

For in vitro diagnostic use with Partec recommended IVD reagents.

The Partec CyFlow® Cube Flow Cytometer complies with the European IVD Directive 98/79/EC and is therefore CE marked.

For in vitro diagnostic use with Partec recommended IVD reagents.

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Partec GmbH • Am Flugplatz 13 • D-02828 Görlitz • Germany

Tel [phone] • Fax [phone]

Contact Information:

E-mail: [e-mail]

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IVD

CyFlow® Cube Instrument Operating Manual

Table of Content

FOREWORD ................................................................................................................... 5

PRESENTATION ............................................................................................................. 6

B

ASICS

........................................................................................................................................................................................... 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

I

N FLOW CYTOMETRY, WHAT IS

… 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? ............................................................................................................................................... 8

The lower level (L-L) or threshold? ..........................................................................................................................................................................8

Example of a histogram ............................................................................................................................................................. 9

Example of a dotplot ................................................................................................................................................................... 9

Histogram and dotplot in immunology ............................................................................................................................ 10

... ............................................................................................................................................. 7

GETTING STARTED ..................................................................................................... 10

I

NSTRUMENT STARTING PROCEDURE

..................................................................................................................................... 11

Sheath fluid level control and/or refill ............................................................................................................................. 11

Switching on the CyFlow® Cube .............................................................................................................................................................................. 11

M

AIN CYVIEW™ LOGIN WINDOW

U

SER LEVELS

CYV

IEW™ MAIN PAGE

............................................................................................................................................................................... 12

.............................................................................................................................................................. 14

............................................................................................................................................. 12

Presentation .................................................................................................................................................................................. 14

CyView™ Controls ....................................................................................................................................................................... 15

CHANNELS – parameter definition ....................................................................................................................................................................... 15

PLOTS – display options ............................................................................................................................................................................................. 15

PROCCESS – instrument control .............................................................................................................................................................................. 17

TYPICAL SAMPLE ANALYSIS .................................................................................... 19

S

TARTING A MEASURE

M

EASURE MODES

Continous (default selection) ................................................................................................................................................ 20

Volumetric Counting with Electrodes ............................................................................................................................... 20

Volumetric Counting with Volume ..................................................................................................................................... 20

Events in Region .......................................................................................................................................................................... 20

C

LEANING PROCEDURES

Cleaning procedure during Cube running ....................................................................................................................... 21

Cleaning procedure for switching off ................................................................................................................................ 21

No cross contamination between samples ...................................................................................................................... 21

............................................................................................................................................................... 19

....................................................................................................................................................................... 20

........................................................................................................................................................... 21

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CyFlow® Cube Instrument Operating Manual

I

NSTRUMENT SETTINGS

............................................................................................................................................................ 22

Measurement parameters ...................................................................................................................................................... 22

Gains settings .................................................................................................................................................................................................................... 22

Threshold settings (T) .................................................................................................................................................................................................. 22

Light source (L) ............................................................................................................................................................................................................... 22

Trigger properties (P) .................................................................................................................................................................................................. 22

Flow speed (S) .................................................................................................................................................................................................................. 22

Plot name ............................................................................................................................................................................................................................ 23

Plot properties.............................................................................................................................................................................. 23

X Log On/Y Log On ......................................................................................................................................................................................................... 23

Erosion levels ................................................................................................................................................................................................................... 23

X/Y Channel ....................................................................................................................................................................................................................... 23

Mode ..................................................................................................................................................................................................................................... 23

BitRange .............................................................................................................................................................................................................................. 23

CR-Mode .............................................................................................................................................................................................................................. 23

Region name ...................................................................................................................................................................................................................... 24

Home Plot name .............................................................................................................................................................................................................. 24

Colour RGB ......................................................................................................................................................................................................................... 24

Max count ........................................................................................................................................................................................................................... 24

Sorter region ..................................................................................................................................................................................................................... 24

Colour Gating On ............................................................................................................................................................................................................. 24

Moving of regions within a histogram .................................................................................................................................................................. 24

Create a polygonal region ........................................................................................................................................................................................... 24

Region properties ........................................................................................................................................................................................................... 24

Create a vertical histogram splitter ....................................................................................................................................................................... 25

Create a vertical dotplot splitter ............................................................................................................................................................................. 25

Create a horizontal dotplot splitter ....................................................................................................................................................................... 25

Create a quadrant assembly ...................................................................................................................................................................................... 25

Change layout for regions ........................................................................................................................................................................................... 25

Applying regions to other plots  Gating Function ...................................................................................................................................... 26

Keyboard/Mouse combinations ........................................................................................................................................... 26

APPENDIX .................................................................................................................... 28

B

IOHAZARDS

M

AINTENANCE

............................................................................................................................................................................... 28

........................................................................................................................................................................... 29

Service .............................................................................................................................................................................................. 29

Transport and Storage ............................................................................................................................................................. 30

Disposal ........................................................................................................................................................................................... 30

L

ASER SAFETY

T

ECHNICAL SPECIFICATIONS

............................................................................................................................................................................ 31

................................................................................................................................................... 32

Optical Standard Setup ............................................................................................................................................................ 32

The Parameters ............................................................................................................................................................................................................... 32

NOTES .......................................................................................................................... 35

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CyFlow® Cube Instrument Operating Manual

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 8 is the instrument operation software for the CyFlow® Cube 8 (CY-S-3068). New
software versions and you profit from your requests: new features and software improvements. Partec is
continuously working on CyView™ to better fulfil 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.

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 This manual contains references to names and products from Partec and other companies
which are registered trademarks or protected by copyright.

Partec GmbH, CyFlow® Cube8, CyView™ for Cube8, Robby®.

®

Microsoft
Hewlett Packard®: Deskjet Laserjet.

Corp.: Windows, Word, Excel, PowerPoint, Paint.

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CyFlow® Cube Instrument Operating Manual

Presentation

Basics

What is the Partec CyFlow® Cube?

The Partec CyFlow® 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
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-colour 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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CyFlow® Cube Instrument Operating Manual

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 quantity classes or channels. In a one­parameter 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 a of 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.

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CyFlow® Cube Instrument Operating Manual

… 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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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
colours (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.

CyFlow® Cube Instrument Operating Manual

Number of events

Peaks

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 red 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 colour coded in red.

Threshold of
lower limit
(L-L)

Log representation
for fluorescence

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Linear
representation
of channels

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CyFlow® Cube Instrument Operating Manual

Histogram and dotplot in immunology

Lysed blood 2D dotplot

G

M

L

Histogram and dotplot of immunological staining

Neg.

Background

Slightly pos.

Pos.

Getting Started

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).

This histogram represents the spectrum of the
cells presented in the previous dot plot stained
with antibodies anti-CD16 conjugated to
PhycoErytrin (PE). 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 FITC) versus SSC
in Y on a linear scale.

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G

M

L

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.

This example is a simplification of an immune­staining analysis by flow cytometry. It is
directed to new users, and aims to link the
histogram to the dotplot representation. To
complete the analysis, gating and statistics
are required.

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Instrument starting procedure

switch

fluid

On/Standby

measurements!

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 800 ml of
clean, filtered, and degassed sheath fluid and is
closed with the screw top. 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.

CyFlow® Cube Instrument Operating Manual

Main

power

Rear side of CyFlow® Cube showing the main
power switch and the sheath fluid and waste
bottle compartment.

Waste and

sheath

The waste bottle must be emptied after and
before each user session. When using bio­hazardeous 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 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

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