COULTER EPICS XL-MCL™ Flow Cytometer System II Software Reference

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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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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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:

xii

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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