COULTER CORPORATION
COULTER EPICS XL-MCL™ Flow Cytometer System II Software Reference
Reference
110 Pages
![Reference COULTER® EPICS® XL™ Flow Cytometer COULTER EPICS XL-MCL™ Flow Cytometer SYSTEM II™ Software CYTOMETER READY FS ON AUX SS LASER SAMPLE FLOW FL1 FL2 LOW MED HIGH FL3 FL4 ® TM PN 4237298B (Dectember 1998) COULTER CORPORATION Miami, Florida 33196 ®](https://images.bioclinicalservices.com.au/wgpnmQrpLVH3s4v6nNe4YPFa/320w/COULTER%20CORPORATION%20-%20COULTER%20EPICS%20XL-MCL%E2%84%A2%20Flow%20Cytometer%20System%20II%20Software%20Reference.png)
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LEGAL NOTICES
READ ALL PRODUCT MANUALS AND CONSULT WITH COULTER-TRAINED PERSONNEL BEFORE ATTEMPTING TO OPERATE INSTRUMENT.
HAZARDS AND OPERATIONAL PRECAUTIONS AND LIMITATIONS WARNINGS, CAUTIONS, and IMPORTANTS alert you as follows: WARNING CAUTION IMPORTANT
-
Might cause injury. Might cause damage to the instrument. Might cause misleading results.
CAUTION System integrity might be compromised and operational failures might occur if:
This equipment is used in a manner other than specified. Operate the instrument as instructed in the Product Manuals.
You introduce software that is not authorized by Coulter into your computer. Only operate your system’s computer with software authorized by Coulter.
You install software that is not an original copyrighted version. Only use software that is an original copyrighted version to prevent virus contamination.
Coulter Corporation urges its customers to comply with all national health and safety standards such as the use of barrier protection. This may include, but it is not limited to, protective eyewear, gloves, and suitable laboratory attire when operating or maintaining this or any other automated laboratory analyzer. IMPORTANT If you purchased this product from anyone other than Coulter or an authorized Coulter distributor, and, if it is not presently under a Coulter service maintenance agreement, Coulter cannot guarantee that the product is fitted with the most current mandatory engineering revisions or that you will receive the most current informational bulletins concerning the product. If you purchased this product from a third party and would like further information concerning this topic, call Coulter Corporation or its distributor.
REVISION STATUS
Initial Issue, 1/96 SYSTEM II Software Version 1.0. Initial issue for customer distribution. Issue B, 12/98 Complete revision. SYSTEM II Software, Version 3.0. Includes new quality control features, patient report export feature, and user-defined run time reports.
This document applies to the latest software listed and higher versions. When a subsequent software version changes the information in this document, a new issue will be released.
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CONTENTS
LEGAL NOTICES, ii REVISION STATUS, iii INTRODUCTION, xi USING YOUR XL AND XL-MCL FLOW CYTOMETER MANUALS, xi ABOUT THIS MANUAL, xii CONVENTIONS, xiii Description of Reporting Units, xiii 1
USE AND FUNCTION, 1 1.1 INTENDED USE, 1 1.2 SYSTEM COMPONENTS, 2 Cytometer, 2 Power Supply, 3 Workstation, 3 1.3 OPTIONS, 3 Hardware Options, 3 Multi-tube Carousel Loader (MCL), 3 Printer, 4 Bar-Code Printer, 4 Bar-Code Scanner, 4 Data Storage, 5 Network Kits, 5 Workstation/Network Server, 5 Fourth FL Sensor, 5 RAM Upgrade Kits, 5 Software Options, 5 tetraONE™ System, 5 reticONE™ System, 5 MULTICYCLE® Software, 6 Artisoft® LANtastic® Software, 6 Sybase® SQL Anywhere™ Software, 6 1.4 REAGENTS AND QUALITY CONTROL MATERIALS, 6 Sheath Fluid, 6 Cleaning Agent, 6 Quality Control Materials, 7 1.5 MATERIAL SAFETY DATA SHEETS (MSDS), 7
2
INSTALLATION, 9 2.1 DELIVERY INSPECTION, 9 2.2 SPECIAL REQUIREMENTS, 9 Space and Accessibility, 9 Electrical Input, 10 Ambient Temperature and Humidity, 10
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CONTENTS
Heat Dissipation, 10 Drainage, 10 2.3 SYSTEM CONNECTIONS, 10 Power and Signal Cables, 10 Waste and Pneumatic Tubing, 12 2.4 INSTALLING SOFTWARE VERSION 3.0, 13 3
OPERATION PRINCIPLES, 15 3.1 WHAT THIS CHAPTER EXPLAINS, 15 3.2 SAMPLE FLOW, 15 Sample Loading, 15 Manual, 15 Automated, 15 Hydrodynamic Focusing, 16 3.3 LASER BEAM SHAPING, 17 3.4 CELL ILLUMINATION, 18 Forward Scatter, 18 Side Scatter and Fluorescent Light, 18 3.5 LIGHT COLLECTION, SEPARATION AND MEASUREMENT, 18 Forward Scatter Collection, 18 Neutral Density Filter, 20 Side Scatter and Fluorescent Light Collection, 20 Side Scatter, 20 Fluorescent Light, 20 3.6 SIGNAL PROCESSING, 23 Voltage Pulse Signals, 23 Peak Signal, 23 Integral Signal, 23 Amplification, 25 Signals Generated, 25 3.7 PARAMETERS, 26 AUXiliary Parameter, 26 When to Use the AUX Parameter, 26 TIME Parameter, 26 RATIO Parameter, 27 PRISM Parameter, 27 3.8 AUTOSTANDARDIZATION PROTOCOLS, 28 _A Protocols, 28 _C Protocols, 29 _Q Protocols, 29 Examples of Autostandardization Protocols and Panels, 30 3.9 QUALITY CONTROL (QC) FEATURES, 30 QC Templates, 30 Levey-Jennings Graphs, 30 Data Table, 31 Westgard Rules, 31 Compare Screen, 32
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CONTENTS
Maintenance Screen, 32 3.10
4
HISTOGRAM DISPLAY, 32 Regions, 32 Gating, 33 Data Storage, 33 Histogram Statistics, 33 Linear Region Statistics, 33 Log Region Statistics, 34
SPECIFICATIONS/CHARACTERISTICS, 37 4.1 SAMPLE REQUIREMENTS, 37 4.2 INSTRUMENT SPECIFICATIONS, 37 Dimensions, 37 Installation Category, 37 Cytometer, 37 Flow Cell, 37 Flow Rate, 38 Laser, 38 Beam-Shaping Optics, 38 Laser Beam Spot Size, 38 Optical Filters, 38 Sensors, 38 Signal Processing, 38 Workstation, 40 Computer, 40 Data Storage, 40 Interfaces, 40 Input Devices, 40 Monitor, 40 4.3 SOFTWARE SPECIFICATIONS, 40 Data Output and Compatibility, 40 Reporting Units, 41 Two-Digit Date Format, 41 For Non Date-of-Birth Dates, 41 For Date-of-Birth Dates, 41 Acquisition, 41 Regions, 42 Listmode Analysis, 42 Multigraph Analysis, 42 4.4 PERFORMANCE SPECIFICATIONS, 42 Carryover, 42 Data Acquisition Throughput, 42 Doublet Discrimination, 42 Precision for Surface Markers, 43 Resolution, 43 Forward Scatter, 43 Fluorescence, 43 Sensitivity, 43 Scatter, 43 Fluorescence, 43 Stability, 43 Day-To-Day, 43 Within Day, 43
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CONTENTS
4.5 PERFORMANCE CHARACTERISTICS, 44 4.6 BAR-CODE SPECIFICATIONS, 44 Bar-Code Labels, 44 Acceptable Bar Codes, 44 Bar-Code Label Optical Characteristics at 670 nm ±10%, 45 NE Width, 45 WE/NE Ratio, 45 Printing Methods, 45 MCL Bar-Code Reader, 45 Hand-Held Bar-Code Reader, 46 Bar-Code Decoder, 46 Check Sum Algorithm, 46 5
PRECAUTIONS AND HAZARDS, 47 5.1 LASER SAFETY, 47 5.2 WARNINGS, 47 5.3 WARNING LABELS, 47 APPENDIX A LOG SHEETS, 53 APPENDIX B PREDEFINED PANELS, PROTOCOLS, AND QC TEMPLATES, 69 GLOSSARY, 77 INDEX, 85 TRADEMARKS
ILLUSTRATIONS Figure 1 EPICS XL-MCL Flow Cytometry System, 3 Figure 2 MCL Carousel Bar-Code Labels, 4 Figure 3 Power and Signal Cable Connections, 11 Figure 4 Waste and Pneumatic Tubing Connections, 12 Figure 5 Installing the Software Diskette, 13 Figure 6 Flow Cell (Hydrodynamic Focusing), 16 Figure 7 Laser Beam Shaping, 17 Figure 8 Optical System, 19 Figure 9 Filter Configuration, Three Fluorescence Sensors, 21 Figure 10 Filter Configuration, Four Fluorescence Sensors, 22 Figure 11 Voltage Pulse Formation, Peak Signal, 24 Figure 12 Integral and Peak Pulses, 25 Figure 13 Prism Histogram, 28 Figure 14 Laser Labels on the Sensing Compartment Cover, 48 Figure 15 Laser Labels in the Optical Area, Side View, 48 Figure 16 Laser Labels in the Optical Area, Front View, 49 Figure 17 Laser Labels on the Laser Head, 49 Figure 18 Laser Label on the Cytometer Back Panel, 50
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CONTENTS
Figure 19 Laser Labels on the MCL Probe Housing Cover, 50 Figure 20 Laser Labels on the MCL Bar-Code Reader, 51
TABLES 1 Applications for the Instrument, 1 2 Code-Related Specifications, 45 3 General Use Panels and Protocols, 69 4 Two-Color Application Panels and Protocols, 69 5 Three-Color Application [4 PMT System] Panels and Protocols, 71 6 Three-Color Application [3PMT System] Panels and Protocols, 74 7 Four-Color Application Panels and Protocols, 76 8 DNA Application Panels and Protocols, 76
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CONTENTS
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INTRODUCTION
This introductory section contains the following topics:
Using your COULTER EPICS XL and XL-MCL Flow Cytometer manuals
About this Manual
Conventions
Icons.
USING YOUR XL AND XL-MCL FLOW CYTOMETER MANUALS Use the Reference manual for in-depth information on the principles of flow cytometry, information about what your instrument does, the methods it uses, its specifications, and information on installation, safety, and system options. Use the Getting Started manual to become familiar with the controls and indicators for your system and to learn about protocols, regions, panels, and the basic skills you need to operate the system. This manual also has an overview of the software. Use the Operator’s Guide for the day-to-day running of your instrument. Go through the detailed step-by-step procedures of startup, quality control (QC), running samples, analyzing data, printing reports, reviewing QC data, and shutdown. Use the Data Management manual for instructions on how to export, save, copy, move, archive, and delete files. It also has information about the types of files your system creates and uses, instructions for working with QC features, and instructions for setting up the report template that you need to create your patient reports. Use the Special Procedures and Troubleshooting manual to clean, replace, or adjust a component of the instrument. The Troubleshooting tables and error messages appear at the back of the manual. Use the Operating Summary as a quick reference for basic procedures. Use the Master Index to easily locate a topic in any of your manuals. Use the User's Comment Card in the Reference manual to give us your comments about the manual and ways to improve it.
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INTRODUCTION
ABOUT THIS MANUAL Your EPICS XL and XL-MCL Flow Cytometer Reference manual provides in-depth information about what the instrument does, the methods it uses, its specifications, and information on installation, safety and software features. This information is organized as follows:
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Chapter 1, Use and Function Contains the intended use of the instrument, the reagents used, and a short description of the major components and options.
Chapter 2, Installation Contains instrument requirements, and diagrams of the interunit cable connections.
Chapter 3, Operation Principles Contains a description of flow cytometry, the normal sample flow through the instrument, how light collection and signal processing are accomplished and how the parameters are derived.
Chapter 4, Specifications/Characteristics Details the instrument and performance specifications, the performance characteristics and interfering substances.
Chapter 5, Precautions and Hazards Describes laser safety precautions and the location of the laserrelated labels.
Appendices The appendices provide reference material on the following topics: Log Sheets Protocols
Glossary The glossary has definitions for many of the words and terms used in flow cytometry.
Index Use the Index to easily locate specific information in this manual.
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CONVENTIONS
CONVENTIONS This manual uses the following conventions: Throughout this manual your EPICS XL or XL-MCL flow cytometer is referred to as the system. Italics indicate screen messages. Bold indicates a menu item.
Courier font indicates text you have to type using the keyboard. indicates a key (such as Enter ). + indicates that the two keys listed (such as Alt + F2 ) are linked for a specific function and must be pressed in this sequence: 1.
Press down on the first key listed and while continuing to press it, press down on the second key listed.
2.
Release both keys at the same time.
indicates to press and release the first key listed then press and release the next key listed. For example, Y Enter .
F1
File Save
Select the Save item on the File menu.
[OKAY]
Use the mouse to click on the screen button labeled [OKAY].
through F12
Special function keys.
Description of Reporting Units Unless otherwise stated, all parameter units are shown in the US unit format (cells/µL) throughout the manuals.
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INTRODUCTION
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USE AND FUNCTION 1
1.1
INTENDED USE
IMPORTANT The use of the data generated by this instrument will depend upon the regulatory status of the reagents you use. If the reagent is labeled by the manufacturer “For Research Use Only. Not for use in diagnostic procedures,” federal law prohibits the use of the data for diagnosis. If, however, the reagent subsystem is labeled “For in vitro diagnostic use,” the entire system can be considered a medical device for generating data to be used for diagnosis.
The instrument can simultaneously measure forward scatter, side scatter, and three or, optionally, four fluorescent dyes using a single laser at 488 nm. Therefore, the instrument can do correlated multiparameter analyses of individual cells. Table 1 lists the major applications for the instrument. In addition to human cells, other cell types can be analyzed, such as:
Plant cells Animal cells
Marine plankton Bacteria
Table 1 Applications for the Instrument
Applications Cell surface antigens
Cell Types Whole blood Buffy coats Mononuclear cells Dissociated tissue Platelets Bone Marrow
Sample Preparation (refer to the reagent's package insert) ImmunoPrep™ reagents with the Q-PREP™ Workstation, the Multi-Q-Prep™ Workstation or the TQ-Prep™ Workstation Whole blood lysing reagent kit Ficol Fluorescent-labeled antibodies
Measurements Cell size and granularity FITC, RD1, ECD, and PC5
Parameters Log/Linear Forward and side scatter Log/Linear fluorescence one, two, three, and four (optional) Prism Ratio Time AUX
continued
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1 USE AND FUNCTION
The XL and XL-MCL flow cytometers are systems for the qualitative and quantitative measurement of biological and physical properties of cells and other particles. These properties are measured when the cells pass through a laser beam in single-file.
USE AND FUNCTION
1 USE AND FUNCTION
Table 1 Applications for the Instrument
Applications
Cell Types
Sample Preparation (refer to the reagent's package insert)
Measurements
Parameters
continued Nucleic acids
Kinetics
Whole blood Dissociated tissue Frozen sections Paraffin sections Body fluids
Whole blood Buffy coats Mononuclear cells Tissue culture cells
Various staining methods, including:
Cell size and granularity
Forward and side scatter
Ethidium bromide Propidium iodide DNA Prep reagents
Green, red, and orange fluorescence
Log/Linear Fluorescence one, two, and three (four is optional). Ratio, AUX
Various methods, including:
Cell size and granularity
Forward and side scatter
Fluo 3 fluorescence
Green fluorescence Time
Fluorescence one Ratio Time
Cell function
Reticulocytes
1.2
Whole blood Buffy coats Mononuclear cells Tissue culture cells Whole blood
Various methods and dyes, including:
Cell size and granularity
Forward and side scatter
DCFH-DA DiOC5(3) FDA
Green fluorescence
Fluorescence one
Thiazole orange coriphosphine O (CPO)
Cell size and granularity
Log/Linear Forward and Side Scatter
Green fluorescence
Log Fluorescence one
Red fluorescence
Log Fluorescence four
SYSTEM COMPONENTS The system components are shown in Figure 1.
Cytometer This unit analyzes the sample. It contains the sheath fluid and cleaning agent bottles. A fourth fluorescence sensor and MCL (Multi-tube Carousel Loader) are optional.
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OPTIONS
Power Supply This unit provides dc power, pressure, and vacuum to the Cytometer. A 4-L waste bottle sits in a bracket on the right side of the Power Supply.
1 USE AND FUNCTION
Workstation The Workstation runs the software that enables you to control the instrument. It displays sample results and other information. It consists of:
A monitor A computer with data storage devices A keyboard and a mouse. COMPUTER WORKSTATION
CYTOMETER CYTOMETER
READY
FS
ON
AUX
SS
LASER
SAMPLE FLOW
FL1 FL2
LOW MED HIGH
FL3 FL4
®
MCL OPTION
TM
TM
POWER SUPPLY MODULE
®
7029077D
Figure 1 EPICS XL-MCL Flow Cytometry System
1.3
OPTIONS Hardware Options Multi-tube Carousel Loader (MCL) The MCL is an automated sample loader for the instrument. It uses a carousel that holds thirty-two 12- x 75-mm test tubes. The MCL reads the following bar-code types:
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Codabar Code 128
Code 39® bar code Interleave 2-of-5. 3
USE AND FUNCTION
1 USE AND FUNCTION
For additional information on the bar-code specifications, see Heading 4.6, Bar-Code Specifications. The MCL mixes each sample before analysis. You can interrupt the MCL to manually run Stat samples, and then have the MCL return to where it left off. A Worklist and MCL carousel report can be displayed or printed. Figure 2 shows the location of the carousel number, tube position, and sample tube bar-code labels on the MCL carousel.
SAMPLE BAR CODE LABEL
5968035C
TUBE POSITION BAR CODE LABEL
CAROUSEL ID BAR CODE
Figure 2 MCL Carousel Bar-Code Labels
Printer Provides a printout of sample results and other information. The Printers listed here are subject to change, so contact your Coulter Representative for the correct model selection:
HP LaserJet® V HP 1200C and 1600C color printers HP 560C color printer.
Bar-Code Printer Used to print bar-code labels from the Worklist for identification of sample tubes in the MCL carousel. Bar-Code Scanner A hand-held scanner/reader used to read specimen collection bar-code labels. A fast, accurate method for inputting specimen and sample tube information and tube ID into the software database.
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OPTIONS
Data Storage 2.6-GB optical drive 650-GB external, recordable CD-ROM drive Iomega® Zip™ external 100-MB drive (parallel)
1 USE AND FUNCTION
Network Kits Single and multi-node network kits used to connect an XL or XL-MCL system to an existing workstation. Workstation/Network Server A COULTER FlowCentre™ Multimedia Workstation can be used as an off-line analysis workstation and network server for data and worklist transfer. The standard workstation that runs the flow cytometer can be upgraded to a FlowCentre Multimedia Workstation. Fourth FL Sensor This optional kit contains a photo-multiplier tube with a 200- to 800-nm spectral range and a 675-BP dichroic filter that are used to generate the following signals (parameters):
FL4 (can be assigned to the AUX parameter) FL4 LOG FL4 PEAK (must be assigned to the AUX parameter).
RAM Upgrade Kits Kits to upgrade 8 MB RAM systems to 16 MB RAM.
Software Options tetraONE™ System Software and reagent kits for performing automated four-color immunofluorescence analysis on the instrument. reticONE™ System Software and reagent kits for performing automated reticulocyte enumeration on the instrument.
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USE AND FUNCTION
MULTICYCLE® Software
1 USE AND FUNCTION
DNA analysis software. Artisoft® LANtastic® Software Network operating system software. Sybase® SQL Anywhere™ Software Allows access to the System II database for extended queries.
1.4
REAGENTS AND QUALITY CONTROL MATERIALS Coulter recommends these reagents or their equivalents. All stated performance characteristics in this manual are based on the use of the XL and XL-MCL system with the following reagents.
Sheath Fluid In the Cytometer, sample is guided into a stream of sheath fluid to make the sample cells flow single file through the laser beam. IsoFlow™ sheath fluid, a nonfluorescent, balanced electrolyte solution, is made for this purpose. IsoFlow sheath fluid has the following characteristics:
Filtered to 0.2 µm
Transparent and nonfluorescent to 488-nm laser light
Low background, bacteriostatic, fungistatic
Compatible with the characteristics of the sample being measured (such as pH, osmolality, conductivity).
The sheath container has a working capacity of about 2 L. The amount of sheath fluid the container holds beyond the working capacity is for pressurization and level sensing.
Cleaning Agent When the Cytometer is in the Cleanse mode, COULTER CLENZ® cleaning agent flushes sample tubing and helps to reduce protein buildup and particles in the instrument. Each cleanse cycle uses about 15 mL of cleaning agent. The cleaning agent container has a working capacity of about 500 mL. That is enough cleaning agent to use the Cleanse mode once a day for one month. The amount of cleaning agent the container holds beyond the working capacity is for pressurization and level sensing.
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MATERIAL SAFETY DATA SHEETS (MSDS)
Read the container’s label for more information on the cleaning agent.
Quality Control Materials
Flow-Check™ Fluorospheres
Fluorospheres used to check the stability of the optical and fluidic systems.
Flow-Set™ Fluorospheres
Fluorospheres used to standardize light scatter and fluorescence intensity.
CYTO-TROL™ Control Cells
Lyophilized lymphocytes with assay values for specific surface antigens. Used to:
Assess monoclonal antibody function. Verify proper flow cytometer setup.
Immuno-Trol™ Cells Stabilized erythrocytes and leukocytes with a known quantity of surface antigens. Used to verify monoclonal antibody performance as well as verify the process of sample staining, lysing, and analysis.
1.5
CYTO-COMP™ Cell Kit
Lyophilized lymphocytes used with the CYTO-COMP Reagent Kit to adjust color compensation settings for multicolor analysis.
CYTO-COMP Reagent Kit
Four two-color fluorescent reagents used with the CYTO-COMP Cell Kit to adjust color compensation settings for multicolor analysis.
MATERIAL SAFETY DATA SHEETS (MSDS) To obtain an MSDS for COULTER reagents used on the XL or XL-MCL flow cytometer: 1.
In the USA, either call Coulter Customer Operations (800-5267694) or write to: Coulter Corporation Attention: MSDS Requests P.O. BOX 169015 Miami, FL 33116-9015
2.
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Outside the USA, contact your Coulter Representative.
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1 USE AND FUNCTION
The quality control materials available from Coulter are:
1 USE AND FUNCTION
USE AND FUNCTION
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