Beckman Coulter ACT User guide

BECKMAN COULTER

™

AC•T™ 5diff Hematology Analyzer

Operator’s Guide

PN 4237615B (July 2000)
COULTER CORPORATION

A Beckman Coulter Company
Miami, Florida 33196-2500 USA

READ ALL PRODUCT MANUALS AND CONSULT WITH BECKMAN COULTER-TRAINED PERSONNEL

BEFORE ATTEMPTING TO OPERATE INSTRUMENT.

HAZARDS AND OPERATIONAL PRECAUTIONS AND LIMITATIONS

WARNINGS, CAUTIONS, and IMPORTANTS alert you as follows:

WARNING - Might cause injury.
CAUTION - Might cause damage to the instrument.
IMPORTANT - Might cause misleading results.

CAUTION System integrity might be compromised and operational failures might occur if:

r This equipment is used in a manner other than specified. Operate the instrument as instructed in the Product

Manuals.

r You introduce software that is not authorized by Beckman Coulter into your computer. Only operate your system’s

computer with software authorized by Beckman Coulter.

r You install software that is not an original copyrighted version. Only use software that is an original copyrighted

version to prevent virus contamination.

Beckman Coulter, Inc. 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.

WARNING Risk of operator injury if all covers are not secured in place prior to instrument operation or you attempt to

replace a part without carefully reading the replacement instructions. Do not attempt to replace any component until you
carefully read the instructions for replacing the component.

IMPORTANT If you purchased this product from anyone other than Beckman Coulter or an authorized Beckman Coulter

distributor, and, if it is not presently under a Beckman Coulter service maintenance agreement, Beckman Coulter cannot
guarantee that the product is fitted with the most current mandatory engineering revisions or that you will receive the most
current information bulletins concerning the product. If you purchased this product from a third party and would like
further information concerning this topic, call your Beckman Coulter Representative.

Initial Issue, 03/00

Software version 0.11

Issue B, 07/00

Software version 1.0

REVISION STATUS

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.

PN 4237615B

iii

REVISION STATUS

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REVISION STATUS

INTRODUCTION, xvii

C

HOW TO USE YOUR A

•T 5diff HEMATOLOGY ANALYZER MANUALS, xvii

ABOUT THIS MANUAL, xvii

CONVENTIONS, xix

GRAPHICS, xix

SYMBOLS, xx

Safety Symbols, xx
Tab Symbols, xx

MENU TREE, xxi

1 USE AND FUNCTION, 1-1

1.1 INTENDED USE, 1-1
General, 1-1
Purpose, 1-1
Instrument Description, 1-1

Control Panel, 1-3
Back Panel, 1-4

Warning and Caution Labels, 1-4
Modes, 1-5
Parameters, 1-5

CBC Mode, 1-5

CBC/DIFF Mode, 1-6
Features, 1-6
Reports, 1-6

CONTENTS

1.2 CONTROLS AND CALIBRATORS, 1-7
Cell Controls, 1-7
Calibrator, 1-7

1.3 REAGENTS, 1-7

C

A

•T 5diff Diluent, 1-8

C

A

•T 5diff Fix, 1-8

C

A

•T 5diff WBC Lyse, 1-8

C

A

•T 5diff Hgb Lyse, 1-8

C

A

•T 5diff Rinse, 1-8

Waste Handling Procedures, 1-9

Neutralizing the Waste and Treating for Biohazards, 1-9
Handling Expired Reagents, 1-10

1.4 PRINTER, 1-10

1.5 RANGES, 1-10

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CONTENTS

1.6 WORKING WITH THE SOFTWARE, 1-11
Moving the Cursor, 1-11
Selecting Menu Items, 1-11
Erasing Saved Text, 1-12
Selecting/De-selecting Software Fields, 1-12

1.7 PRESENTING SAMPLES TUBES (OR VIALS) AND STARTING ANALYSIS, 1-13

1.8 ORDERING MATERIAL SAFETY DATA SHEETS (MSDS), 1-13

2 OPERATION PRINCIPLES, 2-1

2.1 OVERVIEW, 2-1

2.2 MEASUREMENT PRINCIPLES, 2-1
Coulter Principle, 2-1
Aperture Sensor System, 2-1
Applying the Coulter Principle, 2-2

2.3 A

C

V TECHNOLOGY, 2-3
Dual Focused Flow (DFF), 2-3
Flowcell, 2-3
Focused Flow Impedance, 2-3
Absorbance Cytochemistry, 2-4
Signal Processing, 2-4

Thresholds, 2-4

2.4 WBC/BASO METHODOLOGY, 2-5

2.5 SAMPLE ANALYSIS OVERVIEW, 2-5
Aspiration, 2-5
Dilution, 2-6

CBC Mode, 2-7
CBC/DIFF Mode, 2-7

Delivery, 2-7

2.6 SAMPLE ANALYSIS, 2-8
RBC and Platelet Analysis, 2-8

Parameter Results Obtained from the RBC/Plt Dilution, 2-9
Hgb Measurement, 2-9
WBC Count and Differential, 2-10

Parameter Results Obtained from the WBC/BASO Dilution, 2-11

Differential, 2-11

Parameter Results Obtained from the DIFF Dilution, 2-12
Dilution Summary, 2-13

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2.7 PARAMETER DEVELOPMENT, 2-13
RBC Parameters, 2-13

RBC Count, 2-13
RBC Histogram, 2-14
Parameter Results Obtained Using the RBC Histogram, 2-14
MCH and MCHC Calculations, 2-15

Plt Parameters, 2-15

Interference on the Lower End of the Platelet Distribution Curve, 2-15
Microcytic Interferences on the Upper End of the Platelet Distribution Curve, 2-16

Parameter Results Obtained Using the Plt Histogram, 2-16
Hgb Determination, 2-17
WBC Count, BASO Count, and DiffPlot Development, 2-17

WBC Count, 2-17

BASO Count, 2-17

DiffPlot Development, 2-18

3 SPECIFICATIONS/CHARACTERISTICS, 3-1

3.1 INSTRUMENT SPECIFICATIONS, 3-1
Dimensions and Weight, 3-1
Power, 3-1

Supply, 3-1
Consumption, 3-1

Installation Category, 3-1
Grounding Requirements, 3-1
Temperature, Ambient Operating, 3-1
Altitude Range, 3-2
Recommended Location, 3-2
Electromagnetic Environment Check, 3-2
Recommended Reagents, 3-2
Recommended Controls, 3-2
Recommended Calibrator, 3-2
Recommended Anticoagulant, 3-2
Sample Volume Aspirated, 3-2
Dilution Ratios, 3-2
Throughput, 3-3
Sample Stability, 3-3
Sample Identification, 3-3
Output, 3-3
Measurements and Computation, 3-4
Counting Aperture Diameters, 3-4
Reagent Consumption, 3-4
Environmental Protection, 3-4

CONTENTS

3.2 PERFORMANCE SPECIFICATIONS, 3-5
Reproducibility, 3-5
Linearity, 3-5
Accuracy, 3-5
Carryover, 3-6
Reportable Range, 3-6

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CONTENTS

3.3 PERFORMANCE CHARACTERISTICS, 3-7
Reproducibility, 3-7
Accuracy, 3-8
Carryover, 3-8

3.4 LIMITATIONS, 3-8
Maintenance, 3-8
Blood Specimens, 3-8

3.5 INTERFERING SUBSTANCES, 3-9

4 PRECAUTIONS/HAZARDS, 4-1

4.1 DEFINITIONS, 4-1
Warnings, 4-1
Cautions, 4-1
Importants, 4-1
Attention, 4-1

4.2 SAFETY PRECAUTIONS, 4-1
Electronic, 4-1
Biological, 4-1
Moving Parts, 4-1

4.3 OPERATIONAL HAZARDS, 4-2

5 RUNNING SAMPLES, 5-1

5.1 BEFORE ANALYSIS, 5-1
Waste Container Level Check, 5-1
Printer Check, 5-1
Startup, 5-2

Startup During Power Up, 5-2

Startup After Power Up, 5-3
Specimen Collection and Mixing, 5-5
Running Cell Controls to Verify Calibration, 5-5

5.2 ANALYSIS, 5-8
Running Whole-Blood Samples, 5-8

5.3 AFTER ANALYSIS, 5-10
Results, 5-10
Printing Results for Last Sample Analyzed, 5-10
Auto-Clean, 5-11

5.4 SHUTDOWN, 5-11

5.5 ENTERING THE SAMPLE IDENTIFICATION (ID), 5-11
Auto-Numbering, 5-11
Manual Sample ID, 5-12
Scanning the Sample ID with the Barcode Reader, 5-13

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6 REVIEWING RESULTS, 6-1

6.1 GENERAL, 6-1

6.2 FLAGS AND INTERPRETIVE MESSAGES, 6-1
Flags, 6-1

Definition, 6-1
Types of Flags, 6-1
Types of Flag Printout Formats, 6-1

Interpretive Messages, 6-2

Definition, 6-2

6.3 FLAGS GENERATED BY THE INSTRUMENT, 6-2
Results Exceeding Instrument Capacity, 6-2
Hemoglobin Errors, 6-2

Hgb Blank Error, 6-2

Hgb Read Error, 6-2
Voteout Flag, 6-3
WBC Count Flag, 6-3
DiffPlot Flags, 6-3
CBC Flags, 6-8
Suspect or Detailed Flag Format, 6-11

Suspect Flag Format, 6-11

Detailed Flag Format, 6-12
Patient Ranges and Action Ranges, 6-12

CONTENTS

6.4 INTERPRETIVE MESSAGES, 6-12
WBC Interpretive Messages, 6-13
RBC Interpretive Messages, 6-13
Plt Interpretive Messages, 6-14
Combination WBC/RBC/Plt Interpretive Messages, 6-14

7CALIBRATION,7-1

7.1 GENERAL, 7-1
Recommended Calibration Conditions, 7-1
When to Calibrate, 7-1
When to Verify Calibration, 7-1

7.2 PRE-CALIBRATION CHECKS, 7-1

7.3 AUTO-CALIBRATION, 7-3
Calibration Setup, 7-3
Running Calibration, 7-5
Interpreting Calibration Results, 7-9

Forced Calibration, 7-9

7.4 MANUAL CALIBRATION FACTOR ADJUSTMENT, 7-11

7.5 PRINTING CALIBRATION FACTORS, 7-14

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CONTENTS

8 DIAGNOSTICS, 8-1

8.1 GENERAL MAINTENANCE, 8-1

8.2 MAINTENANCE SCHEDULE, 8-1

8.3 CLEANING PROCEDURES, 8-2
Cleaning the Outside of the Instrument, 8-2
Cleaning the Inside of the Instrument, 8-2
Extended Cleaning Procedure, 8-2
Auto-Clean, 8-4
Shutdown, 8-5
System Cleaning, 8-5

8.4 SYSTEM RESET CYCLE, 8-9

8.5 COMPONENT LOCATIONS, 8-10

8.6 SYSTEM TROUBLESHOOTING PROCEDURES, 8-12
Diluter System, 8-12

Backflush, 8-12

Bath and Flowcell Rinse, 8-13
Draining the Baths and/or the Diluent Reservoir, 8-14
Hardware System, 8-15

Hardware Reset, 8-15
Checking the Valves, 8-15
Checking the Motors, 8-15

8.7 REPLACEMENT PROCEDURES, 8-15
Replacing Reagents, 8-15

Viewing Reagent Levels, 8-16
Replacing the Diluent Reagent, 8-17
Replacing Fix, WBC Lyse, Hgb Lyse, and Rinse Reagents, 8-21

Priming the Reagents, 8-26
Replacing the Waste Container, 8-27
Replacing the Flowcell Lamp, 8-29

8.8 SYSTEM ERRORS, 8-34
What Error Messages Mean, 8-34

8.9 TROUBLESHOOTING GUIDES, 8-36

A INSTRUMENT SETUP, A-1

A.1 INSTALLATION, A-1

A.2 DEFAULT CONFIGURATION, A-1

A.3 CHANGES TO INSTRUMENT SETUP, A-2

A.4 LANGUAGE AND USA FIELD SELECTION, A-2

A.5 PASSWORD SETUP, A-3

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PN 4237615B

A.6 DATE/TIME SETUP, A-4

Date Setup, A-4

Selecting the Date Format, A-4
Selecting the Time Format, A-5
Setting a New Date and Time, A-5

A.7 REPORTING UNIT SELECTION, A-7

A.8 LABORATORY LIMITS SETUP, A-8

Patient Ranges, A-8

Changing CBC Patient Ranges, A-9
Changing DIFF Patient Ranges, A-11

Action Ranges, A-13

Changing CBC Action Ranges, A-13
Changing DIFF Action Ranges, A-15

A.9 SETTING FLAG SENSITIVITY AND THRESHOLDS, A-17

A.10 PRINTER CONFIGURATION, A-18

Configuring the Instrument’s Printer Settings, A-18

Printing Options, A-20

CONTENTS

A.11 ENTERING/EDITING THE INSTITUTIONAL HEADER, A-21

A.12 PRINTING A SYSTEM SETUP REPORT, A-22

A.13 CALIBRATION SETUP, A-23

Changing CV% Limits, A-23
Defining the Operator, A-25

A.14 SELECTING THE SAMPLE IDENTIFICATION (ID) MODE, A-27

A.15 DISPLAYING DIFF # OR DIFF %, A-28

A.16 ENABLING ATL, IMM, PCT, AND PDW, A-28

A.17 RESETTING THE MANUAL SAMPLE ID NUMBER AND INSTRUMENT SEQUENCE

NUMBER TO “1”,A-29

A.18 SELECTING BARCODE WITH CHECKSUM, A-30

A.19 AUTO-CLEAN FREQUENCY SETTING, A-31

A.20 CHANGING THE DAILY WORKLOAD, A-32

A.21 REAGENT VOLUMES SETUP, A-33

A.22 VIEWING THE CYCLE COUNT, A-34

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xi

CONTENTS

B LOG SHEETS, B-1

ACTION LOG, B-2

MAINTENANCE LOG, B-3

REAGENT LOG, B-4

CMANUAL CALIBRATION,C-1

C.1 ANALYSIS PROCEDURE, C-1

C.2 CALCULATIONS PROCEDURE, C-2

C.3 CALCULATING NEW CALIBRATION FACTORS, C-3

Calibration Worksheet, C-4

D TROUBLESHOOTING FLOWCHART, D-1

D.1 TROUBLESHOOTING FLOWCHART, D-1

E TRAINING CHECKLIST, E-1

E.1 INSTALLATION, E-1

E.2 GENERAL, E-1

E.3 SAMPLE HANDLING, E-1

E.4 INSTRUMENT COMPONENTS, E-1

E.5 SOFTWARE MENU, E-1

E.6 REAGENTS, E-1

E.7 INSTRUMENT SETUP/CUSTOMIZATION, E-2

E.8 CALIBRATION, E-2

E.9 CONTROLS, E-2

E.10 SYSTEM OPERATION OVERVIEW, E-2

E.11 DAILY PROCEDURES, E-2

E.12 SPECIAL PROCEDURES, E-3

E.13 MAINTAINING AND SERVICING THE INSTRUMENT, E-3

E.14 PAPERWORK, E-3

F BARCODE SPECIFICATIONS, F-1

F.1 OVERVIEW, F-1

Definition, F-1

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PN 4237615B

F.2 BARCODE LABELS, F-1

Symbologies, F-1

F.3 BARCODE SPECIFICATIONS, F-1

F.4 BARCODE LABEL TEST PAGES, F-3

F.5 BARCODE SCANNER CONFIGURATION, F-4

F.6 CODE 39 AND CODABAR BARCODE SCANNER OPTIONS, F-5

F.7 I 2-OF-5 PROGRAMMING OPTIONS AND TEST LABELS, F-7

REFERENCES, REFERENCES-1

LIST OF REFERENCES, REFERENCES-1

GLOSSARY, GLOSSARY-1

DEFINITIONS, GLOSSARY-1

CONTENTS

ABBREVIATIONS, ABBREVIATIONS-1

LIST OF ABBREVIATIONS, ABBREVIATIONS-1

INDEX, INDEX-1

TRADEMARKS

ILLUSTRATIONS

1.1 AC•T 5diff Analyzer, 1-1

1.2 Outside View of the Instrument, 1-2

1.3 Control Panel Buttons, 1-3

1.4 Back Panel, 1-4

1.5 Warning and Caution Labels on the Instrument, 1-4

2.1 Coulter Principle, 2-2

2.2 Dual Focused Flow Process, 2-3

2.3 Signal Processing, 2-4

2.4 BASO Thresholds, 2-5

2.5 Sample Partitions Inside the Probe - CBC/DIFF Mode, 2-6

2.6 Sample Partitions Inside the Probe - CBC Mode, 2-6

2.7 Bath Assembly, 2-6

2.8 Sample Delivery Using Tangential Flow, 2-7

2.9 Bath Assembly, 2-8

2.10 Bath Assembly, 2-10

2.11 Flowcell Operation, 2-11

2.12 DiffPlot Regions, 2-12

2.13 Typical RBC Histogram, 2-14

2.14 Typical Plt Histogram, 2-15

2.15 Area of the Plt Histogram Used to Determine the PDW Parameter Result, 2-16

2.16 Areas Used to Determine WBC and BASO Parameter Results, 2-17

2.17 DiffPlot Regions, 2-18

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xiii

CONTENTS

3.1 Instrument Dimensions and Weight, 3-1

3.2 Sample Report, 3-3

5.1 Sample Report, 5-10

6.1 WBC/BASO Histogram Flags: CBC Mode, 6-8

6.2 WBC/BASO Histogram Flags: CBC/DIFF Mode, 6-8

6.3 MICRO and MACRO Regions on RBC Histogram, 6-9

6.4 Plt Flags, 6-10

6.5 Mobile Threshold Positioned in the Standard Regions (Between 18fL and 25fL), 6-10

6.6 Mobile Threshold Cannot Be Positioned in the Standard Region, 6-10

6.7 Mobile Threshold Cannot Be Positioned, 6-10

7.1 Out of Range Calibration Factors, 7-9

8.1 View of the Pneumatics Area (Right Side), 8-10

8.2 Bath Assembly, 8-11

8.3 View Behind Motherboard (Left Side), 8-11

8.4 Motherboard, 8-12

8.5 Reagent Bottle Location, 8-16

8.6 Waste Sensor Alarm Unit Location, 8-27

A.1 Sample Results Report: Areas Defined, A-19
D.1 Troubleshooting Flowchart, D-1

TABLES

1.1 CBC Parameters, 1-5

1.2 CBC/DIFF Parameters, 1-6

2.1 A

2.2 Technical Characteristics for Obtaining RBC and Platelet Counts, 2-8

2.3 Technical Characteristics for the Measurement of the Hemoglobin, 2-9

2.4 Characteristics Required to Obtain WBC/BASO Results, 2-10

2.5 Technical Characteristics for Acquisition of the DiffPlot, 2-12

2.6 Summary of Dilutions, 2-13

2.7 DiffPlot Regions Defined, 2-19

2.8 Immature White Blood Cells, 2-20

3.1 Reagent Consumption by Cycle in mL, 3-4

3.2 Reproducibility Specifications, 3-5

3.3 Linearity Specifications, 3-5

3.4 Accuracy Specifications, 3-5

3.5 Carryover Specifications, 3-6

3.6 Reportable Range, 3-6

3.7 Reproducibility Characteristics From a Normal Sample with a Low Normal WBC Count, 3-7

3.8 Reproducibility Characteristics From a Normal Sample with a High Normal WBC Count, 3-7

3.9 Accuracy Characteristics, 3-8

3.10 Carryover Characteristics, 3-8

3.11 Interfering Substances, 3-9

6.1 Definition of DIFF Flags, 6-4

6.2 CBC Histogram Flags, 6-8

6.3 Patient Range and Action Range Flags, 6-12

6.4 WBC Interpretive Messages from Action Ranges, 6-13

6.5 WBC Interpretive Messages from DiffPlot, 6-13

6.6 RBC Interpretive Messages from Action Ranges, 6-13

6.7 RBC Interpretive Messages from Flag Sensitivity, 6-14

C

•T 5diff Analyzer: Measurement Technologies, 2-1

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PN 4237615B

6.8 Plt Interpretive Messages from Action Ranges, 6-14

6.9 Plt Interpretive Messages from the Plt Histogram, 6-14

6.10 Interpretive Messages from a Combination of WBC/RBC/Plt Action Ranges, 6-14

6.11 NRBCs and PLATELET AGGREGATES Interpretive Messages, 6-15

7.1 Calibration Factors Range, 7-13

8.1 Maintenance Schedule, 8-1

8.2 Error Messages, 8-34

8.3 Troubleshooting Guide, 8-36
A.1 Instrument Default Settings, A-1
A.2 Reporting Unit Format, A-7
A.3 CBC Default Patient Ranges, A-9
A.4 DIFF Default Patient Ranges, A-11
A.5 CBC Default Action Ranges, A-13
A.6 DIFF Default Action Ranges, A-15
A.7 Default CV Limits, A-23
A.8 Daily Workload Runs by Mode, A-32
A.9 Default Reagent Volumes, A-33
F.1 Default Barcode Settings, F-2
F.2 Test Labels With the Check Digit (Checksum), F-3
F.3 Test Labels Without the Check Digit, F-3
F.4 Barcode Scanner Configuration Sheet, F-4
F.5 Code 39 Barcode Scanner Options, F-5
F.6 Codabar Barcode Scanner Options, F-6
F.7 Interleaved 2-of-5 Options With Fixed Length Characters Test Labels, F-7

CONTENTS

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xv

CONTENTS

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PN 4237615B

This introductory section contains the following topics:

r HOW TO USE YOUR AC•T 5diff HEMATOLOGY ANALYZER MANUALS,
r ABOUT THIS MANUAL,
r CONVENTIONS,
r GRAPHICS
r SYMBOLS, and
r MENU TREE.

HOW TO USE YOUR AC•T 5diff HEMATOLOGY ANALYZER MANUALS

Use this Operator’s Guide to find information about:

r Getting started,
r Running your instrument,
r Reviewing results,
r Performing special procedures, such as cleaning, replacing, or adjusting an instrument

component,

r Troubleshooting problems,

INTRODUCTION

r Determining what the instrument does,
r Understanding how to safely operate the instrument,
r Powering up the instrument,
r Customizing the setup, and
r Running controls and samples.

Use the Host Transmission Specification manual (PN 4277065) to find out information about
interfacing your A

ABOUT THIS MANUAL

The information in this manual is organized as follows:

r Chapter 1, USE AND FUNCTION

Contains the intended use of the instrument, a brief history of the methods used by the
instrument, the reagents, calibrators, and controls used, a brief description of the major
components, and how to work with the software.

r Chapter 2, OPERATION PRINCIPLES

Contains the descriptions for cell counting and voting and how the parameters are
derived.

r Chapter 3, SPECIFICATIONS/CHARACTERISTICS

Details instrument specifications, characteristics, and interfering substances.

C

•T 5diff analyzer to your laboratory’s host computer.

PN 4237615B

r Chapter 4, PRECAUTIONS/HAZARDS

Provides information about key safety issues and contains information on biological
hazards and hazards pertaining to moving parts.

r Chapter 5, RUNNING SAMPLES

Provides information on how to run patient blood samples.

xvii

INTRODUCTION

ABOUT THIS MANUAL

r Chapter 6, REVIEWING RESULTS

Provides information on reviewing flagged sample results.

r Chapter 7, CALIBRATION

Provides procedures for calibrating the instrument, including manually adjusting the
calibration factors.

r Chapter 8, DIAGNOSTICS

Provides information about special procedures and troubleshooting procedures for the
instrument. Includes topics such as a maintenance schedule, cleaning and replacement
procedures, and what error messages mean.

r Appendix A, INSTRUMENT SETUP

Provides procedures on customizing the instrument’s settings, such as date/time,
reporting units, laboratory limits, and others.

r Appendix B, LOG SHEETS

Contains log sheets for your laboratory’s use.

r Appendix C, MANUAL CALIBRATION

Provides a procedure for manually calibrating the instrument.

r Appendix D, TROUBLESHOOTING FLOWCHART

Provides supplemental troubleshooting information.

r Appendix E, TRAINING CHECKLIST

Summarizes what must be done after the instrument is installed.

r Appendix F, BARCODE SPECIFICATIONS

Defines the specifications that barcode labels must meet for use with the instrument.

r REFERENCES

Lists references used in this manual.

r GLOSSARY

Defines terminology used in this manual.

r ABBREVIATIONS

Defines abbreviations used in this manual.

r INDEX

Provides page numbers for indexed information.

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PN 4237615B

CONVENTIONS

This manual uses the following conventions:

r Main Menu refers to the initial menu

r When instructed to make a menu selection, the text appears in bold with two symbols

INTRODUCTION

CONVENTIONS

displayed on the instrument after

16:0512/07/99MAIN MENU

Startup.

1 - RUN SAMPLES

2 - CALIBRATION
3 - REAGENTS
4 - DIAGNOSTICS
5 - SETUP

to distinguish the menu path. For example, if instructed to choose Calibration, then
Autocalibration, the text will appear as

CALIBRATION

AUTOCALIBRATION.

tt

GRAPHICS

r

Bold font

indicates a menu option, such as

SETUP

.

r Italics font indicates screen text displayed on the instrument, such as

Calibration Passed.

Bold, italics font

r

be instructed to do the Startup procedure, which would appear as “Do

r Instrument refers to the A

indicates a heading name within this document. For example, you may

Startup

”.

C

•T 5diff hematology analyzer.

r A Note contains information that is important to remember or helpful when

performing a procedure.

r Motherboard refers to the main card (board) in the instrument.
r RBC bath is sometimes referred to as RBC/Plt bath.

C

r A

r A

r A

r A

r A

•T 5diff Rinse reagent is sometimes referred to as Rinse.

C

•T 5diff Fix reagent is sometimes referred to as Fix.

C

•T 5diff Hgb Lyse reagent is sometimes referred to as Hgb Lyse.

C

•T 5diff WBC Lyse reagent is sometimes referred to as WBC Lyse.

C

•T 5diff Diluent reagent is sometimes referred to as Diluent.

All graphics, including screens and printouts, are for illustration purposes only and must not
be used for any other purpose.

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xix

INTRODUCTION

SYMBOLS

SYMBOLS

Safety Symbols

Safety symbols alert you to potentially dangerous conditions. These symbols, together with
text, apply to specific procedures and appear as needed throughout this manual.

Symbol Warning Condition

Biohazard

!

!

!

(specimens, reagents, controls, and
calibrators, and so forth) and areas
these materials come into contact
with as being potentially infectious.

Probe hazard.

may contain biohazardous materials,
such as controls and calibrators.

Electrical shock hazard

electrical shock when instrument is
plugged in to the power source.

.

Consider all materials

The probe is sharp and

.

Possibility of

Action

Wear standard laboratory attire and
follow safe laboratory procedures
when handling any material in the
laboratory.

Avoid any unnecessary contact with
the probe and probe area.

Before continuing, unplug the

C

A

•T 5diff analyzer from the

electrical outlet.

Tab Symbols

Tabs divide this document into four sections: reference, operation, special procedures and
troubleshooting, and appendices. Each tab reflects a unique symbol.

Symbol Definition

Identifies the reference section.

xx

Identifies the operating instructions section.

Identifies the special procedures and troubleshooting section.

Identifies the appendices section.

PN 4237615B

MENU TREE

The functions of the instrument are programmed into its software.

MENU TREE

MAIN MENU

1 - RUN SAMPLES

2 - CALIBRATION
3 - REAGENTS
4 - DIAGNOSTICS
5 - SETUP

SETUP

1 - DATE / TIME

2 - UNITS
3 - LAB. LIMITS
4 - HOST SETUP
5 - PRINTER
6 - OTHERS

* Consult a Beckman Coulter
representative before selecting this
option.

Service password required

CALIBRATION

1 - AUTOCALIBRATION

2 - CAL FACTORS
3 - PRINT CAL FACTORS
4 - REPRODUCIBILITY

REAGENTS

1 - LEVEL-CHANGE

2 - DAILY WORKLOAD
3 - PRIME

DIAGNOSTICS

1-SYSTEM RESET CYCLE

2-MINI PRIME
3-DILUTER SYSTEMS
4-HARDWARE SYSTEMS
5-SERVICE

SERVICE

PASSWORD

REQUIRED

PRIME

1- DILUENT

2 - FIX
3 - WBC LYSE
4 - HGB LYSE
5 - RINSE
6 - ALL REAGENTS
7 - UNPRIME ALL

DILUTER SYSTEMS

1-BACKFLUSH

2-RINSE
3-DRAIN BATHS
4-EXTENDED CLEANING

HARDWARE SYSTEMS

1-HARDWARE RESET

2-MOTORS *
3-VALVES *
4-TRAVERSE SERVICE POSITION

SERVICE

1 - DILUTION

2 - MEASUREMENT
3 - HEATING SYSTEMS
4 - MIXING
5 - SENSOR CHECK
6 - VACUUM CHECK
7 - BURN-IN
8 - FLOWCELL WBC CALIBRATION
9 - OTHERS

SET DATE / TIME

1 - TIME FORMAT

2 - DATE FORMAT
3 - SET DATE & TIME

LAB. LIMITS

1 - PATIENT RANGES

2 - ACTION RANGES
3 - FLAGS SENSITIVITY
4 - THRESHOLDS

HOST SETUP

1 - HOST SETUP CONFIGURATION

2 - SENDING CONFIGURATION
3 - SENDING OPTIONS
4 - VARIABLE FORMAT SETUP
5 - SEND LATEST RESULT

PRINTER

1 - PRINTER CONFIGURATION

2 - INSTITUTIONNAL HEADER
3 - PRINT LATEST RESULT

OTHERS

1 - CALIBRATION

2 - IDENTIFICATION MODE
3 - AUTOCLEAN FREQUENCY
4 - CHANGE PASSWORD
5 - LANGUAGE
6 - REAGENT VOLUMES
7 - CYCLE COUNTS
8 - PRINT SYSTEM SETUP
9 - SEND SETUP

RINSE

1 - BATHS

2 - FLOWCELL

DRAIN BATHS

1 - RINSE

2 - FIRST DILUTION
3 - DIFF
4 - WBC / BASO
5 - RBC / PLT
6 - ALL BATHS
7 - DILUENT RESERVOIR

MOTORS

1 - SAMPLING PROBE

2 - TRAVERSE
3 - SAMPLING SYRINGE
4 - DRAINING SYRINGE
5 - COUNTING SYRINGE
6 - FLOWCELL SYRINGES
7 - DILUTION SYRINGES

VALVES

1 - 1 TO 11

2 - 12 TO 16
3 - 17 AND 18
4 - 20 TO 26
5 - 27 TO 31

PATIENT RANGES

1 - CBC

2 - DIFF

ACTION RANGES

1 - CBC

2 - DIFF

VARIABLE FORMAT SETUP

1 - NUMERICAL RESULTS

2 - FLAGS AND MESSAGES
3 - HISTOGRAMS AND THRESHOLDS
4 - PATIENT FILE

CALIBRATION

1 - CV% L I M I TS

2 - DEFINE OPERATOR

LANGUAGE

1 - ENGLISH

2 - FRENCH
3 - GERMAN
4 - SPANISH
5 - ITALIAN

MEASURMENT

1 - HGB BLANK ADJUSTMENT

2 - APERTURE CURRENT
3 - RBC / PLT GAIN
4 - WBC / BASO GAIN
5 - DIFF ADJUSTMENT
6 - PULSE ADJUSTMENT

HEATING SYSTEMS

1 - HEATING COIL

2 - BATH ENCLOSURE

SENSOR CHECK

1 - DRAINING

2 - DIFF TRANSFER

VACUUM CHECK

1 - COUNTING

2 - DRAINING

BURN - IN

1 - BURN - IN CYCLES

2 - ANALYSIS CYCLES

FLOWCELL WBC CALIBRATION

1 - AUTOCALIBRATION

2 - CAL FACTORS

OTHERS

1 - USER MODE

2 - CYCLE COUNTS
3 - PARK SYRINGES
4 - RESET TO DEFAULT VALUES

HEATING COIL

1 - ADJUSTMENT

2 - REFERENCE

BATH ENCLOSURE

1 - ADJUSTMENT

2 - REFERENCE

7615022B

PN 4237615B

xxi

MENU TREE

xxii

xxii

PN 4237615B

1.1 INTENDED USE

General

USE AND FUNCTION

1

1

C

The Beckman Coulter A
hematology analyzer (Figure 1.1) is a
26-parameter, fully automated hematology
analyzer, including a five-part leukocyte
differential counter.

Of the 26 reported parameters:

r 20 parameters are

Diagnostic Use

MCV, MCH, MCHC, RDW, Plt, MPV,
NE%, NE#, LY%, LY#, MO%, MO#,
EO%, EO#, BA%, and BA#.

r 6 parameters are qualitative and are

For Research Use Only. Not for use in
diagnostic procedures

IMM%, IMM#, ATL%, and ATL#.

•T 5diff

For In Vitro

: WBC, RBC, Hgb, Hct,

.: Pct, PDW,

Figure 1.1 AC•T 5diff Analyzer

Purpose

The purpose of the AC•T 5diff hematology analyzer is to identify normal patient results with
all normal system-generated parameters and to flag or identify patient results that require
additional studies.

PN 4237615B

Instrument Description

r Figure 1.2 shows the outside of the instrument.
r Figure 1.3 shows the control panel.
r Figure 1.4 shows the back panel.
r Figure 1.5 shows the warning and caution labels on the instrument.

WARNING

operate the instrument. Ensure that all covers and doors are closed and secured before operating the
instrument.

Risk of operator injury when covers and doors are not closed and secured in place before you

1-1

USE AND FUNCTION

g

1

INTENDED USE

Figure 1.2 Outside View of the Instrument

)

b

c

d

e

LCD (liquid crystal display) screen

b

Control panel: allows you to

c

interface with the instrument. See

Control Panel

Door to reagents: allows you to

d

access the reagent bottles on
board.

Top cover

e

Door to pneumatics: allows you to

f

access the hydraulic parts for
maintenance procedures.

: The system will not operate

Note

when this door is open.
Aspirate switch: allows you to

g

start an analysis cycle.

for details.

Aspirate (sample) probe: aspirates

h

sample or control material from
tubes or vials.

Green LED (light-emitting diode):

i

f

j

i

indicates the instrument is ready.
Red LED: indicates the instrument

j

is busy.
ON/OFF switch

1)

h

1-2

PN 4237615B

USE AND FUNCTION

Control Panel

Use the control panel buttons (Figure 1.3) to setup and operate the instrument.

INTENDED USE

1

Figure 1.3 Control Panel Buttons

bcd efg

2

ESC

RANGE

1

4

7

0

3

5

6

8

CBC
DIFF

9

DEL

hi

2

1

4

7

0

3

5

6

8

9

j

2

1

4

7

0

1)

3

5

6

8

9

1!

7615006A

b

procedure, followed by a background count.

performs a prime and rinsing

Startup

c

Shutdown

typically done at the end of the day. The
instrument remains in stand-by mode with
the Rinse.

d

ESC

Escape

executing it and goes to the previous screen.

CBC

e

DIFF

Mode

CBC/DIFF modes.

performs a cleaning,

exits a function without

allows you to select CBC and

f

allows you to print the last sample

Print

result, calibration results, laboratory limits,
and so forth.

g

DEL

deletes the entered information.

Delete

h

executes a function or enters

Enter

data.

i

RANGE

used.

Cursor keys

j

and allow you to scroll through the alphabet
when entering information.

selects the flagging range to be

Range

move the cursor on the screen

PN 4237615B

Numeric keypad

1)

allows you to enter
numbers for dates, values, limits, sample
IDs, and to select menu items.

. Allows you to enter the decimal

Period

1!

number separator and to select/de-select
software options.

1-3

USE AND FUNCTION

INTENDED USE

Back Panel

Figure 1.4 shows the instrument’s back panel.

Figure 1.4 Back Panel

MOD
NO.

ASSY

S/N

xxxxxx

xxxxxx

NO.

50/60100-240

VOLTS

HZ AMPS

MANUFACTURED BY COULTER CORPORATION
A BECKMAN COULTER COMPANY

B

ECKMAN

11800 SW 147 AVENUE, MIAMI, FLORIDA 33196-2500 U.S.A.

OULTER

C

PATTENTS ISSUED AND/OR PENDING

AUTOMATED DIFFERENTIAL CELL COUNTER

FOR IN VITRO DIAGNOSTIC USE

CAUTION:

TO REDUCE THE RISK OF ELECTRICAL SHOCK DO NOT REMOVE THE COVER
OR BACK.
REFER SERVICING TO QUALIFIED SERVICE PERSONNEL.
ELECTRIC SHOCK HAZARD. DISCONNECT UNIT FROM POWER SOURCE
PRIOR TO SERVICING.
FOR CONTINUED PROTECTION AGAINSTR FIRE HAZARD, REPLACE ONLY
WITH SAME TYPE AND RATING OF FUSE.
FOR SAFETY REASONS, EQUIPMENT REQUI4RES CONNECTION TO
PROTECTIVE EARTH GROUND.

Serial number label

b

Barcode reader connector

c

b

Printer connector

d

WATTS

c

d

Host RS 232 C Output connector

e

Power supply cord connector

f

Waste output connector

g

e

Diluent input connector

h

h

g

Warning and Caution Labels

Pay close attention to the labels on the instrument (Figure 1.5).

Figure 1.5 Warning and Caution Labels on the Instrument

MOD
NO.

ASSY

xxxxxx

NO.

VOLTS

B

ECKMAN

C

OULTER

AUTOMATED DIFFERENTIAL CELL COUNTER

FOR IN VITR O D IAG N O ST IC US E

CAUTION:

TO REDUCE THE RISK OF ELECTRICAL SHOCK DO NOT REMOVE THE COVER
OR BACK.
REFER SERVICING TO QUALIFIED SERVICE PERSONNEL.
ELECTRIC SHOCK HAZARD. DISCONNECT UNIT FROM POWER SOURCE
PRIOR TO SERVICING.
FOR CONTINUED PROTECTION AGAINSTR FIRE HAZARD, REPLACE ONLY
WITH SA M E TYPE A ND RA T IN G OF FUS E.
FOR SAFETY REASONS, EQUIPMENT REQUI4RES CONNECTION TO
PROTECTIVE EARTH GROUND.

f

MOD

c

T 5diff

A

•

S/N

xxxxxx

50/60100-240

WATTS

HZ AMPS

MANUFACTURED BY COULTER CORPORATION
A BECKMAN COULTER COMPANY
11800 SW 147 AVENUE, MIAMI, FLORIDA 33196-2500 U.S.A.
PATTENTS ISSUED AND/OR PENDING

NO.

ASSY

xxxxxx

NO.

VO LTS

CAUTIO N:

T O R E D U C E TH E R IS K O F E LE C T R IC A L S H O C K D O N O T R E M O V E T H E C O V E R
OR BACK.
REFER SERVICING TO QUALIFIED SERVICE PERSONNEL.
ELECTRIC SHOCK HAZARD. DISCONNECT UNIT FROM POW ER SOURCE
PRIO R TO SERVICING .
FOR CONTINUED PROTECTION AG AINSTR FIRE HAZARD, REPLACE O NLY
W IT H S A M E T Y P E A N D R A T IN G O F F U S E .
FOR SA FETY REASO NS, EQUIPMEN T REQU IRES CONNECTIO N TO
PROTECTIVE EARTH G ROUND.

50-60100-240

B

ECKMAN

C

OULTER

AUTOMATED DIFFERENTIA L CELL CO UNTER

F O R IN V IT R O D IA G N O S T IC U S E

S/N

xxxxxx

0.9-2.0 200

HZ AMPS

MANUFACTURED FOR BECKMAN COULTER INC.
11800 SW 147 AVENUE, MIAMI, FLORIDA 33196-2500 U.S.A.
PATENTS ISSUED AND/OR PENDING
MADE IN FRANCE

WATTS

1-4

T H IS A R E A M A Y C O N T A IN
B IO H A Z A R D O U S M A T E R IA L
REFER TO PRODUICT REFERENCE
MANUAL FOR PROPER HANDLING

ALL CO VER S/PANELS M UST BE
SECUR ED IN PLACE PRIO R TO
INSTRUM ENT OPER ATIO N.
REFER TO PRODUCT REFERENCE
MANUAL FOR PROPER INSTALLATIO N.

2429555

CAUTIO N

PN 4237615B

USE AND FUNCTION

Modes

The instrument has two modes of analysis: CBC and CBC/DIFF. For information on the
parameters of each mode, see

Parameters

.

Parameters

CBC Mode

Table 1.1 lists the 12 parameters analyzed in the CBC mode.

Table 1.1 CBC Parameters

Parameter Definition

WBC White Blood Cell or leukocyte count
RBC Red Blood Cell or erythrocyte count
Hgb Hemoglobin concentration
Hct Hematocrit (relative volume of erythrocytes within the whole-blood

sample)

INTENDED USE

1

MCV Mean Corpuscular (erythrocyte) Volume
MCH Mean Corpuscular (erythrocyte) Hemoglobin
MCHC Mean Corpuscular (erythrocyte) Hemoglobin Concentration
RDW Red Cell (erythrocyte) Distribution Width
Plt Platelet or thrombocyte count
MPV Mean Platelet Volume

†

PDW

†

Pct

†

Pct and PDW are derived parameters and are For Research Use Only. Not for use in diagnostic procedures.

Platelet Distribution Width
Plateletcrit

PN 4237615B

1-5

USE AND FUNCTION

INTENDED USE

CBC/DIFF Mode

Table 1.2 lists the 26 parameters analyzed in the CBC/DIFF mode:

Table 1.2 CBC/DIFF Parameters

Parameter Definition

WBC White Blood Cell or leukocyte count

NE%: Neutrophil percentage
NE#: Neutrophil number,
LY%: Lymphocyte percentage,
LY#: Lymphocyte number,
MO%: Monocyte percentage,
MO#: Monocyte number
EO%: Eosinophil percentage,
EO#: Eosinophil number,
BA%: Basophil percentage,
BA#: Basophil number

†

IMM%
IMM#
ATL%
ATL#

: Immature cell percentage

†

: Immature cell number

†

: Atypical lymphocyte percentage

†

: Atypical lymphocyte number
RBC Red Blood Cell or erythrocyte count
Hgb Hemoglobin concentration
Hct Hematocrit (relative volume of erythrocytes within the whole-blood sample)
MCV Mean Corpuscular (erythrocyte) Volume
MCH Mean Corpuscular (erythrocyte) Hemoglobin
MCHC Mean Corpuscular (erythrocyte) Hemoglobin Concentration
RDW Red Cell (erythrocyte) Distribution Width
Plt Platelet or thrombocyte count
MPV Mean Platelet (thrombocyte) Volume
PDW
Pct

†

†

Platelet Distribution Width
Plateletcrit

†

Derived parameters are For Research Use Only. Not for use in diagnostic procedures.

Features

Features of the instrument include automated calibration, one-button aspiration with probe
wipe, 12- or 26-parameter analysis with histograms and DiffPlots, and alphanumeric or
barcode patient sample identification.

Reports

Patient sample reports are printed based on your instrument setup.

1-6

PN 4237615B

1.2 CONTROLS AND CALIBRATORS

Cell Controls

AC•T 5diff Control is available in three levels (low, normal, and high) to provide a stable
reference control for use with this instrument.

Calibrator

AC•T 5diff Cal Calibrator is a recommended alternative to the whole-blood reference method
of calibration and is traceable to reference methods and materials. Use A
Calibrator to ensure accurate instrument measurements for WBC, RBC, Plt, Hct, and Hgb.

1.3 REAGENTS

Beckman Coulter recommends these reagents:

C

r A
r A
r A
r A
r A

•T 5diff Diluent,

C

•T 5diff Fix,

C

•T 5diff WBC Lyse,

C

•T 5diff Hgb Lyse, and

C

•T 5diff Rinse.

USE AND FUNCTION

CONTROLS AND CALIBRATORS

C

•T 5diff Cal

1

These reagents are:

r Registered by the AFSSAPS (Agence Francaise de sécurité sanitaire des produits de

santé) and are For In Vitro Diagnostic Use.

r Manufactured by Coulter Corporation, Inc., Miami, Florida USA, and distributed by

Beckman Coulter France, SA 33 rue des Vanesses BP 50359 Villepinte 95942 Roissy CDG
Cedex.

All stated performance characteristics in this manual are based on the use of the A

C

•T 5diff
analyzer with the above-referenced reagents. Refer to the reagent’s bottle/container label for
detailed information, such as stability, before using the reagent.

ATTENTION:

The open container stability on the reagent labeling applies only to the reagent

when connected to the instrument with approved reagent pickups and caps.

For information on handling reagent waste, see

Waste Container

.

Waste Handling Procedures

Replacing the

and

PN 4237615B

1-7

USE AND FUNCTION

REAGENTS

AC•T 5diff Diluent

WARNING

water. Sodium azide preservative may form explosive compounds in metal drain lines. [See National
Institute for Occupational Safety and Health Bulletin: Explosive Azide Hazards (8/16/76).] When disposing
of reagents down the drain, flush with large volumes of water.

Risk of explosion if sodium azide is not properly flushed down the drain with large volumes of

Used for counting and differentiating blood cells, AC•T 5diff Diluent is clear and odorless.
Composed of stabilized saline solution containing an organic buffer and less than 0.1%
sodium azide, A

Dilutes whole-blood samples,

r

Stabilizes cell membranes for accurate counting and sizing,

r

Conducts aperture current, and

r

Rinses instrument components between analyses.

r

C

•T 5diff Diluent:

Handle as indicated in this manual. To be used at ambient temperature from 18°C to 25ºC up
to the expiration date indicated on the packaging.

AC•T 5diff Fix

Used to lyse erythrocytes, fix leukocytes, and differentially stain granules of monocytes,
neutrophils, and eosinophils, A
alcohol. A

C

•T 5diff Fix is composed of an alcohol solution containing propylene-glycol, a

formic dye, buffers, alkaline salts, wetting agents, and an aldehyde preservative.

C

•T 5diff Fix is a deep blue aqueous solution that smells like

Handle as indicated in this manual. To be used at ambient temperature from 18°C to 25ºC up
to the expiration date indicated on the packaging.

AC•T 5diff WBC Lyse

Used to lyse red blood cells for the leukocyte count and to differentiate poly-nuclear
basophils, A

C

•T 5diff WBC Lyse is a colorless, aqueous solution. It is composed of an acidic

solution containing a lytic agent.

Handle as indicated in this manual. To be used at ambient temperature from 18°C to 25ºC up
to the expiration date indicated on the packaging.

AC•T 5diff Hgb Lyse

Used to lyse blood cells and to determine hemoglobin concentration, AC•T 5diff Hgb Lyse is a
clear, aqueous solution and is composed of potassium cyanide at 0.035, a quarternary
ammonium salt, and a saline phosphate buffer containing less than 0.1% sodium azide.

Handle as indicated in this manual. To be used at ambient temperature from 18°C to 25ºC up
to the expiration date indicated on the packaging.

AC•T 5diff Rinse

Used as a rinsing agent, AC•T 5diff Rinse is a transparent liquid composed of an enzymatic
solution with proteolytic action.

1-8

Handle as indicated in this manual. To be used at ambient temperature from 18°C to 25ºC up
to the expiration date indicated on the packaging.

PN 4237615B

USE AND FUNCTION

Waste Handling Procedures

Consult the material safety data sheets (MSDS) for additional reagent information. To order
an MSDS, see

Neutralizing the Waste and Treating for Biohazards

Do this procedure before capping the waste container for disposal.

Heading 1.8, ORDERING MATERIAL SAFETY DATA SHEETS (MSDS)

.

REAGENTS

1

WARNING

neutralized before the waste container is capped.
Non-neutralized waste contents may produce
gas, which can build up pressure in a capped
container. Neutralize waste contents after
removing the waste container and before
capping it for disposal.

1

2

Risk of personal injury if waste is not

For 20L of waste liquid, add the
following to the waste container:

a. 50mL of Sodium Hydroxide

solution 200g/L to prevent gas
from forming.

b. 250mL of Sodium Hypochlorite

solution (12% available chlorine)
to treat waste for biohazards.

Cap the waste container and firmly
tighten the cap to prevent waste
contents from escaping.

PN 4237615B

Dispose of the waste container

3

according to your laboratory’s
guidelines.

1-9

USE AND FUNCTION

PRINTER

Handling Expired Reagents

Do this procedure to eliminate cyanides from expired A

1

2

For 1L of reagent, add:
a. 50mL of Sodium Hydroxide

solution 200g/L.

b. 100mL of freshly prepared

Ammonium Persulfate solution
500g/L or 50mL of Sodium
Hydroxide solution 500g/L.

c. 500mL of Sodium Hypochlorite

solution (30% available chlorine).

Dispose of expired reagents according
to your laboratory’s guidelines.

C

•T 5diff Hgb Lyse.

1.4 PRINTER

Use the printer supplied or approved by Beckman Coulter.

1.5 RANGES

The instrument provides the ability to define three separate sets of flagging criteria.

r Range 1 selects Patient Range 1 and Action Range 1.
r Range 2 selects Patient Range 2 and Action Range 2.
r Range 3 selects Patient Range 3 and Action Range 3.

This means that if you select Range 2, the sample results will be reported and flagged
according to Patient Range 2 and Action Range 2.

Remember these range associations when establishing your laboratory limits and when
selecting a range for analysis.

1-10

PN 4237615B

1.6 WORKING WITH THE SOFTWARE

When working with the instrument’s software, be sure you understand the basics of:

r Moving the Cursor,
r Selecting Menu Items,
r Erasing Saved Text, and
r Selecting/De-selecting Software Fields.

Moving the Cursor

To move the software cursor, press the appropriate cursor key: , , , or .

Selecting Menu Items

There are two ways to select a menu item:

USE AND FUNCTION

WORKING WITH THE SOFTWARE

1

r Press the number on the numeric

keypad that corresponds to the menu
item you want to select.

For example, to select
from the Main Menu, press

CALIBRATION

C

at

the numeric keypad.

OR

r Highlight the menu item by pressing

or , then press .

For example, to select

CALIBRATION

from the Main Menu,

press and .

16:0512/07/99MAIN MENU

1 - RUN SAMPLES

2 - CALIBRATION
3 - REAGENTS
4 - DIAGNOSTICS
5 - SETUP

PN 4237615B

1-11

USE AND FUNCTION

WORKING WITH THE SOFTWARE

Erasing Saved Text

There may be times when you need to erase saved text.

Move the cursor to the line of text

1

where you want to delete information.

Press .

2

The cursor becomes a flashing
underscore, which means you can now
edit the field.

Move the underscore to the character

3

you want to delete.

Press to backspace and delete.

4

Press to save the changes.

5

DEL

Selecting/De-selecting Software Fields

Some software screens allow you to select (activate) or de-select (deactivate) certain software
features.

Move the cursor to the desired field.

1

1-12

PN 4237615B

Press .

2

For example, 7001 is selected on the
Autocalibration screen shown here.

:

Note

USE AND FUNCTION

PRESENTING SAMPLES TUBES (OR VIALS) AND STARTING ANALYSIS

16:0502/27/00AUTOCALIBRATION

7001
OP2
OP3
OP4

WBC

HCT

LOT # CX294
EXP. DATE 04/05/00

TARGET VALUES

10.4

36.7

RBC
PLT

4.40
255

HGB

13.6

1

= selected.

CONTINUE

= not selected.

1.7 PRESENTING SAMPLES TUBES (OR VIALS) AND STARTING ANALYSIS

Some procedures in this manual require you to present a tube (or vial) to the instrument and
start analysis. The following information describes how.

Present the tube (or vial) to the aspirate probe.

B

Ensure that the probe is well inside the tube (or

C

b

c

vial) contents, and press the aspirate switch.
During aspiration, the red and green LEDs flash.

When the red LED remains illuminated,

r

remove the tube (or vial) from the probe.
When the green LED remains illuminated,

r

the instrument is ready for the next analysis.

1.8 ORDERING MATERIAL SAFETY DATA SHEETS (MSDS)

To obtain an MSDS for reagents used on the AC•T 5diff analyzer:

1. In the USA, either call Beckman Coulter Customer Operations (800.526.7694) or write:

Beckman Coulter, Inc.
Attn: MSDS Requests
P.O. Box 169015
Miami, FL 33116-9015

2. Outside the USA, contact a Beckman Coulter representative.

PN 4237615B

1-13

USE AND FUNCTION

ORDERING MATERIAL SAFETY DATA SHEETS (MSDS)

1-14

PN 4237615B

2.1 OVERVIEW

The AC•T 5diff analyzer is a fully automated hematology analyzer providing a complete WBC
five-part differential, which is determined simultaneously by the A
Cytochemistry and Volume) Technology and WBC/BASO methodologies.

C

The A
WBC/BASO methodology uses differential lysis, impedance technology, and differential
thresholds. See Table 2.1.

Table 2.1 AC•T 5diff Analyzer: Measurement Technologies

V Technology uses absorbance, cytochemistry, and focused flow impedance. The

Fluid Dynamics Technology Measurements Output

OPERATION PRINCIPLES

2

C

V (Absorbance

2

Dual Focused Flow

Volume aperture Differential lysis using the

Volume aperture Coulter Principle Volume and count RBC count, platelet count,

C

A

V Technology

Coulter Principle

2.2 MEASUREMENT PRINCIPLES

Coulter Principle

In the AC•T 5diff analyzer, the Coulter Principle1 is used to analyze the final RBC/Plt dilution
and the WBC/BASO dilution. This electronic method of counting and sizing particles is based
on the fact that cells, which are poor conductors of electricity, will interrupt a current flow.
The impedance variation generated by the passage of non-conductive cells through a small,
calibrated aperture is used to determine the count (number of particles) and size (volume) of
the particles passing through the aperture within a given time period.

Aperture Sensor System

The RBC/Plt aperture sensor system determines the cell count and size of red blood cells and
platelets. The WBC/BASO aperture sensor system determines the cell count and size of white
blood cells. Additionally, the differentiation between basophils and other white blood cells is
related to the A
WBC/BASO bath.

C

•T 5diff WBC Lyse-specific lytic action on the white blood cells in

Light absorbance of
cytochemically-stained
cells

Volume and count WBC count, basophil

Lymphocytes, monocytes,
neutrophils, eosinophils,
immature cells, and atypical
lymphocytes

percentage, and basophil
count

and hematocrit

PN 4237615B

To sense particles using the Coulter Principle (Figure 2.1), a current flow is established so
changes in that flow can be monitored. In this sensing system, an electrode is placed on each
side of the aperture. The most visible electrode is referred to as the counting head. These
electrodes are the conductive metallic housings attached to the front of the RBC and
WBC/BASO baths. The second electrode, referred to as the bath electrode, is not as
conspicuous. This electrode is located inside the bath. The aperture is located between the
counting head and the bath electrode.

2-1

OPERATION PRINCIPLES

V

MEASUREMENT PRINCIPLES

Figure 2.1 Coulter Principle

Solution to be analyzed

Vacuum
constant

Current
costant

olts

Pulse

Electrodes

Time

Analyzing
electronic

circuit

7616035A

When the count circuit is activated and an electronically conductive reagent is in the RBC or
WBC/BASO bath, an electric current continuously passes through the aperture. Current
moving between the two electrodes establishes the electronic flow through the aperture.

Once a sample is aspirated, an aliquot of that aspirated sample is diluted with reagent (an
electrolyte) and is delivered to the RBC or WBC/BASO bath using tangential flow, which
ensures proper mixing of the dilution. When the cells suspended in the conductive reagent
are pulled through a calibrated aperture, the electrical resistance between the two electrodes
increases proportionately with the cell volume (Figure 2.1).

The resistance creates a pulse that is sensed and counted as a particle by the instrument. The
amount of resistance (amplitude of each pulse) is directly related to the size of the particle
that produced it.

The generated pulses have a very low voltage, which the amplification circuit increases so
that the electronic system can better analyze the pulses and eliminate the background noise.

Applying the Coulter Principle

The AC•T 5diff analyzer makes several dilutions of an aspirated whole-blood sample. The
RBC/Plt dilution begins in the First Dilution/Hgb bath but is actually analyzed in the RBC
bath. The final dilution in the RBC bath is used to determine the cell count and size of red
blood cells and platelets.

The WBC/BASO aperture sensor system is directly responsible for determining the cell count
and size of white blood cells. The differentiation between basophils and other white blood
cells is also related to the A
cells.

Thresholds, which are electronically set size limits, exclude unwanted particles, such as
debris, from the analysis. Particles above the threshold are analyzed, and particles below the
threshold are excluded.

2-2

C

•T 5diff WBC Lyse-specific lytic action on these white blood

PN 4237615B

2.3 ACV TECHNOLOGY

In the DIFF bath, 25µL of whole blood is mixed with 1,000µL of AC•T 5diff Fix reagent for
12 seconds, then stabilized with 1,000µL of A
This reaction lyses the red blood cells, preserves the leukocytes at their original size, and
differentially stains the lymphocytes, monocytes, neutrophils, and eosinophils, with
eosinophils staining most intensely. The instrument maintains the reagents and reaction at a
regulated temperature of 35°C (95°F).

The lymphocytes, monocytes, neutrophils, and eosinophils each have a unique nuclear and
morphologic structure and staining intensity, and therefore, absorb light differently. Each
stained cell is individually focused by the Dual Focused Flow (DFF) system and transported
through the flowcell using sample pressure and diluent sheath flow.

Dual Focused Flow (DFF)

DFF (Figure 2.2) fluid dynamics uses a hydrodynamic focusing process to focus individual
cells or particles in a stream of diluent. The focused sample stream of the A
is about 40µm in diameter.

OPERATION PRINCIPLES

C

•T 5diff Diluent for an additional 3 seconds.

C

V TECHNOLOGY

A

C

•T 5diff analyzer

2

DFF uses the sheath fluid to surround and force cells suspended in diluent to pass one at a
time through the center of the flowcell. The first sheath flow focuses the sample through the
impedance aperture. The second sheath flow maintains the focused flow of cells as they exit
the aperture into the optical flowcell. Hydrodynamic focusing in the flowcell enables accurate
and rapid cell-by-cell measurements on a large number of individual cells.

Figure 2.2 Dual Focused Flow Process

Flowcell

Sequential analyses for cell volume (impedance) and light absorbance are performed in the
flowcell. A total of 72µL of sample is injected through the flowcell for 15 seconds. The
flowcell incorporates a 60µm aperture for cellular volume analysis and a 42µm measurement
area for light absorbance.

PN 4237615B

Focused Flow Impedance

Focused flow impedance technology measures the electrical resistance of a cell as it passes
through the aperture in the flowcell. The change in resistance is directly proportional to the
volume of the cell.

2-3

OPERATION PRINCIPLES

C

V TECHNOLOGY

A

Absorbance Cytochemistry

As a cell passes through the optical portion of the flowcell, light is scattered in all directions.
A sensor detects only forward scattered light. The optical measurement is derived as a
function of the amount of light lost due to diffraction and absorbance, as compared to full
transmission when no cell is present.

The collected signals are converted into voltage pulses and are processed. The magnitude of
the voltage pulses are proportional to the physical and chemical characteristics of the cells
being analyzed. Light absorbance is related to cellular contents (granularity, nuclear content,
and so forth) after cytochemical staining. These measurements provide the information for
lymphocytes, monocytes, neutrophils, and eosinophils, and their precursors.

Signal Processing

The signals from the flowcell aperture and from the optical measurement are correlated by a
window of time. The optical pulse must be detected within 100 to 300 microseconds of the
impedance pulse, otherwise, the signal is rejected.

The output signals from the focused flow impedance and the light absorbance measurements
are combined to define the WBC differential population clusters. See Figure 2.3.

Figure 2.3 Signal Processing

Thresholds

Most of the population partition thresholds are fixed and give the limits of the morphological
normality of leukocytes. Changes in the morphology of a population are expressed on the
DiffPlot by a shifting of the corresponding population. Volume and absorbance thresholds are
used to detect shifting populations.

2-4

PN 4237615B

2.4 WBC/BASO METHODOLOGY

In the WBC/BASO bath, 10µL of whole blood is mixed with 2,000µL of AC•T 5diff WBC Lyse
reagent. This reaction lyses the red blood cells and specifically differentiates between the
basophils and other leukocytes by volume. The instrument maintains the reagents and
reaction at a regulated temperature of 35°C (95°F).

Using a constant vacuum, the instrument then pulls the sample through an 80µm aperture.
As each cell passes through the aperture, a pulse is generated proportional to the cellular
volume. The total leukocyte count and basophil percentage are determined by specific
thresholds on the WBC/BASO histogram (Figure 2.4.).

Figure 2.4 BASO Thresholds

OPERATION PRINCIPLES

WBC/BASO METHODOLOGY

2

2.5 SAMPLE ANALYSIS OVERVIEW

Aspiration

When the sample probe is immersed in a whole-blood sample and the aspirate switch is
pressed, sample is pulled from the tube into the sample probe. Depending on the selected
mode of operation, the A
(CBC/DIFF mode) of sample.

The volume of sample aspirated into the sample probe is sufficient to make all the dilutions
needed to develop parameter results in the selected mode of operation. The aspirated sample
is then partitioned as it is distributed into the designated baths.

Figure 2.5 shows the sample partitioning that occurs in the CBC/DIFF mode. Notice there are
three aliquots of the aspirated whole-blood sample that will be used to make dilutions.

Figure 2.6 shows the sample partitioning that occurs in the CBC mode. Notice there are only
two aliquots of the aspirated whole-blood sample that will be used to make dilutions in this
mode of operation. (The DIFF aliquot is not needed in the CBC mode.)

To ensure sample integrity, the sample aliquot at the tip of the probe is never used to make a
dilution; it is discarded into the Rinse bath.

C

•T 5diff analyzer aspirates either 30µL (CBC mode) or 53µL

PN 4237615B

2-5

OPERATION PRINCIPLES

SAMPLE ANALYSIS OVERVIEW

Figure 2.5 Sample Partitions Inside the Probe ­CBC/DIFF Mode

Diluent

Air bubble

Not used

DIFF dilution

WBC/BASO dilution

RBC/PLT/HGB first dilution

Not used

7616001A

Figure 2.6 Sample Partitions Inside the Probe ­CBC Mode

Diluent

Air bubble

Not used

WBC/BASO dilution

RBC/PLT/HGB first dilution

Not used

7616056A

Dilution

Using the Sequential Dilution System (SDS) technique, the instrument makes a series of
dilutions in a series of baths (Figure 2.7).

Figure 2.7 Bath Assembly

Rinse bath

b

First Dilution/Hgb bath

c

DIFF bath

d

RBC bath

e

WBC/BASO bath

f

2-6

PN 4237615B

OPERATION PRINCIPLES

A

SAMPLE ANALYSIS OVERVIEW

CBC Mode

After aspiration in the CBC mode, aliquots of the whole-blood sample are distributed as
follows (Figure 2.5):

r The 3µL sample aliquot at the tip of the probe is discarded into the Rinse bath as the

exterior of the sample probe is rinsed, ensuring sample integrity.

r 10µL of sample is delivered to the First Dilution/Hgb bath for use in preparing the

primary RBC/Plt dilution and for measuring the Hgb value.

r 10µL of sample is delivered to the WBC/BASO bath for the WBC/BASO count.
r 7µL of remaining sample is discarded into the Rinse bath.

CBC/DIFF Mode

After aspiration in the CBC/DIFF mode, aliquots of the whole-blood sample are distributed as
follows (Figure 2.6):

r The 3µL sample aliquot at the tip of the probe is discarded into the Rinse bath as the

exterior of the sample probe is rinsed, ensuring sample integrity.

r 10µL of sample is delivered to the First Dilution/Hgb bath for use in preparing the

primary RBC/Plt dilution and for measuring the Hgb value.

2

r 10µL of sample is delivered to the WBC/BASO bath for the WBC/BASO count.
r 25µL of sample is delivered to the DIFF bath for development of the DiffPlot.
r 5µL of remaining sample is discarded into the Rinse bath.

Delivery

In the CBC and the CBC/DIFF modes, each aliquotted sample is delivered to its appropriate
bath using a tangential flow (Figure 2.8) of reagent, which mixes the diluted sample and
minimizes viscosity problems.

Figure 2.8 Sample Delivery Using Tangential Flow

Probe

Reagent

input

Tangential flow

PN 4237615B

Mixing bath

7616002

2-7

OPERATION PRINCIPLES

SAMPLE ANALYSIS

2.6 SAMPLE ANALYSIS

RBC and Platelet Analysis

The RBC/Plt dilution analyzes red blood cells and platelets. This dilution is prepared in two
stages – the primary (first) dilution and the secondary (last) dilution.

The primary dilution is made in the First Dilution/Hgb bath, and the secondary dilution is
made in the RBC bath (Figure 2.9). Table 2.2 summarizes the technical characteristics
required to obtain RBC and Platelet results.

Figure 2.9 Bath Assembly

Rinse bath

b

First Dilution/Hgb bath

c

DIFF bath

d

RBC bath

e

WBC/BASO bath

f

Table 2.2 Technical Characteristics for Obtaining RBC and Platelet Counts

Dilution Characteristics

Primary Dilution for RBC and Plt:

Initial volume of whole-blood 10µL
Volume A
Primary dilution ratio 1:170

Secondary Dilution for RBC and Plt

Volume of primary dilution 42.5µL
Volume A
Secondary dilution ratio 1:58.8
Final dilution for RBC and Plt results 1:170 x 1:58.8 = 1:10,000
Reaction temperature 35°C (95°F)

C

•T 5diff diluent

C

•T 5diff diluent

:

1700µL

2500µL

2-8

Measurement Characteristics

Method of analysis Coulter Principle
Aperture diameter 50µm
Count vacuum 200mb (5.9in. Hg)
Count period 2x5 seconds

PN 4237615B

OPERATION PRINCIPLES

SAMPLE ANALYSIS

Parameter Results Obtained from the RBC/Plt Dilution

This final 1:10,000 RBC/Plt dilution is used to:

r Determine the RBC count,
r Develop the RBC histogram, which is needed to obtain the Hct, MCV, and RDW results,
r Determine Plt count,
r Develop the Plt histogram, which is needed to obtain MPV, Pct, and PDW results.

Hgb Measurement

Hemoglobin is determined from the dilution in the First Dilution/Hgb bath (Figure 2.9). This
dilution is prepared in two stages – the primary (first) dilution and the secondary (last)
dilution.

The primary dilution is made and 42.5µL of that dilution is removed for making the RBC/Plt
dilution. A
dilution.

The Hgb concentration is based on the transmittance of light through the optical part of the
First Dilution/Hgb bath using a spectrophotometric technique at a wavelength of 550nm. The
transmittance of the sample dilution is compared to the transmittance of a reagent blank. The
system calculates the Hgb using the blank and sample readings.

C

•T 5diff Hgb Lyse and additional Diluent are added to make the final 1:250

2

Table 2.3 summarizes the technical characteristics required for measuring hemoglobin.

Table 2.3 Technical Characteristics for the Measurement of the Hemoglobin

Dilution Characteristics

Volume of whole-blood 10µL
Volume A
Preliminary dilution ratio 1:170
Volume of the 1:170 dilution removed

(for making the RBC/Plt dilution)
Additional volume of A
Volume of A
Final dilution for Hgb determination 1:250
Reaction temperature 35°C (95°F)

Measurement Characteristics

Method of analysis Spectrophotometry
Wavelength 550nm

C

•T 5diff diluent

C

•T 5diff Hgb Lyse

C

•T 5diff diluent

1700µL

42.5µL

400µL
400µL

PN 4237615B

2-9

OPERATION PRINCIPLES

SAMPLE ANALYSIS

WBC Count and Differential

The WBC count is determined twice using two different methodologies:

r The reference WBC count is the count obtained in the WBC/BASO bath (Figure 2.10).

The WBC count and the BASO count are determined simultaneously.

r A second WBC count is determined in the flowcell during acquisition of the DiffPlot.

The dilution analyzed in the flowcell is prepared in the DIFF bath (Figure 2.10).

The WBC counts from the two methodologies are compared, and, if they exceed the defined
limits, will be flagged.

Figure 2.10 Bath Assembly

Rinse bath

b

First Dilution/Hgb bath

c

DIFF bath

d

RBC bath

e

WBC/BASO bath

f

Table 2.4 summarizes the technical characteristics required to obtain WBC and BASO results.

Table 2.4 Characteristics Required to Obtain WBC/BASO Results

Dilution Characteristics

Volume of whole-blood 10µL
Volume A
Dilution ratio 1:200
Reaction temperature 35°C (95°F)

Measurement Characteristics

Method of analysis Coulter Principle
Aperture diameter 80µm

C

•T 5diff WBC Lyse

2,000µL

2-10

Count vacuum 200mb (5.9in. Hg)
Count period 2x6 seconds

PN 4237615B

OPERATION PRINCIPLES

SAMPLE ANALYSIS

Parameter Results Obtained from the WBC/BASO Dilution

The final 1:200 dilution is used to:

r Determine the WBC count, and
r Develop the WBC/BASO histogram, which is needed to obtain the BASO count.

Differential

Twenty-five microliters (25µL) of whole blood is delivered to the DIFF bath in a flow of

C

•T 5diff Fix reagent, which lyses the red blood cells, stabilizes the WBC in their native

A
forms, and differentially stains the lymphocytes, monocytes, neutrophils, and eosinophils,
with eosinophils staining most intensely.

The solution is then stabilized with Diluent for three seconds and transferred to the
measuring bath. See Figure 2.11. Each cell is measured in absorbance (cytochemistry) and
resistivity (volume).

Figure 2.11 Flowcell Operation

2) Second focused flow for optical detection

2

PN 4237615B

1) Primary focused flow for impedance

2-11

OPERATION PRINCIPLES

SAMPLE ANALYSIS

Table 2.5 summarizes the technical characteristics required for acquisition of the DiffPlot.

Table 2.5 Technical Characteristics for Acquisition of the DiffPlot

Dilution Characteristics

Volume of whole-blood 25µL
Volume A
Volume A
Final dilution ratio 1:80
Reaction temperature 35°C (95°F)
Incubation duration 12 seconds

Measurement Characteristics

Method of analysis Impedance with hydrofocus
Aperture diameter 60µm
Diameter of the flow 42µm

C

•T 5diff Fix

C

•T 5diff Diluent

1000µL
1000µL

Injection duration 15 seconds
Data accumulated 12 seconds
Volume injected 72µL

Parameter Results Obtained from the DIFF Dilution

From these measurements, a DiffPlot is developed with optical transmission (absorbance) on
the X-axis and volume on the Y-axis. Figure 2.12 shows the DiffPlot regions.

From the DiffPlot, four out of five leukocyte (white blood cell) populations are determined:
lymphocytes, monocytes, neutrophils, and eosinophils.

In a typical whole-blood sample, the basophil population (determined in the WBC/BASO
bath) is very small compared to the other four white blood cell populations.

Figure 2.12 DiffPlot Regions

2-12

PN 4237615B

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

Dilution Summary

Table 2.6 summarizes the dilution characteristics required to obtain CBC and CBC/DIFF
parameter results.

Table 2.6 Summary of Dilutions

2

Technical
Characteristics

WBC Count and BASO

Count
(in the WBC/BASO
bath)

Differential Acquisition

with Differential WBC
Count
(in the DIFF bath)

Hgb Measurement

(in the First
Dilution/Hgb bath)

RBC and Plt Count

(in the RBC bath)

The primary

Note:

dilution (1:170) is made
in the First Dilution/Hgb
bath.

Whole-Blood
Volume Reagent(s)

10µL

25µL

10µL

42.5µL of the
1:170 dilution
(from the
First
Dilution/Hgb
bath)

C

A

•T 5diff WBC Lyse

C

A

•T 5diff Fix

C

•T 5diff Diluent

A

C

A

•T 5diff Diluent

After removing

42.5 µL of the 1:170
dilution:

C

A

•T 5diff Diluent

C

•T 5diff Hgb Lyse

A

C

A

•T 5diff Diluent

Reagent
Volume

2,000µL Final

1,000µL
1,000µL

1700µL

400µL
400µL

2,500µL Secondary

Dilution
Ratio

1:200

Final
1:80

Preliminary
1:170

Final
1:250

1:58.8
1:170 x

1:58.8 =
Final

1:10,000

Reaction
Temperature

35°C (95°F)

35°C (95°F)

35°C (95°F)

35°C (95°F)

2.7 PARAMETER DEVELOPMENT

RBC Parameters

RBC Count

C

The A
proprietary flagging information to confirm the parameter result prior to reporting it. To
obtain an RBC count result, the instrument compares the data from the two 5-second count
periods then votes and rejects any questionable data.

RBC count = Number of cells counted per unit volume x Calibration coefficient

The RBC count is displayed and printed as: RBC = N x 10
(

UNIT SELECTION

PN 4237615B

•T 5diff hematology analyzer uses duplicate counting criteria, voting criteria, and

: cells/µL is the US unit format. Other formats are available. See

Note

.)

6

cells/µL.

Heading A.7, REPORTING

2-13

OPERATION PRINCIPLES

A

PARAMETER DEVELOPMENT

RBC Histogram

In addition to being counted, red blood cells are categorized according to size (from 30 fL to
300 fL) by a 256-channel pulse-height analyzer. The pulse-height analyzer uses a number of
thresholds to sort the particles into several size (volume) categories and to develop a size
distribution curve of the particles. The RBC distribution curve shows cells in their native size.
Figure 2.13 is an example of an RBC histogram with a normal RBC size distribution.

Figure 2.13 Typical RBC Histogram

30 300

7616036

Parameter Results Obtained Using the RBC Histogram

r Hct measurement: The height of the pulse generated by the passage of a cell through the

aperture is directly proportional to the volume of the analyzed red blood cell. The
hematocrit (Hct) is the sum of all the digitized pulses. Hct is displayed and printed as %
(percentage). (Note: % is the US unit format. Other formats are available. See

A.7, REPORTING UNIT SELECTION

.)

Heading

r MCV calculation: The MCV (Mean Cell Volume) is calculated using the Hct and the RBC

count. The MCV is displayed and printed in femtoliters (fL). (Note: fL is the US unit
format. Other formats are available. See

Heading A.7, REPORTING UNIT SELECTION

.)

r RDW calculation: The RDW (Red cell Distribution Width) is an index of the variation or

spread in the size of the red blood cells. The study of the RBC distribution detects
erythrocyte anomalies linked to anisocytosis and enables the clinician to follow the
evolution of the width of the curve relative to the cell number and average volume.
Displayed and printed as a percentage, RDW is calculated using the standard deviation
(SD) of the RBC population and the MCV.

RDW(%)

K SD

--------------=
MCV

where:

K = System constant

2-14

SD = Calculated standard deviation based on the red cell distribution

MCV = Mean Cell Volume of the red cells

PN 4237615B

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

MCH and MCHC Calculations

r MCH calculation: The MCH (Mean Cell Hemoglobin) is calculated from the Hgb value

and the RBC count and describes the average weight of hemoglobin in a red cell. The
calculation for MCH is:

Hgb

MCH (pg)

(

: pg is the US unit format. Other formats are available. See

Note

UNIT SELECTION

r MCHC calculation: The MCHC (Mean Cell Hemoglobin Concentration) is calculated

using the Hgb and Hct values and describes the average concentration of hemoglobin in
the red blood cells. The calculation for MCHC is:

MCHC (g/dL)

(

: g/dL is the US unit format. Other formats are available. See

Note

REPORTING UNIT SELECTION

-----------­RBC

.)

Hgb

---------­Hct

10×=

Heading A.7, REPORTING

100×=

Heading A.7,

.)

Plt Parameters

Platelet counting and sizing is also done in the RBC bath. Thresholds separate the platelet
pulses, which are much smaller, from the red blood cell pulses. Platelets are also categorized
according to size by a 256-channel pulse-height analyzer. A pulse-height analyzer uses a
number of thresholds to sort the particles into several size (volume) categories and to develop
a size distribution curve of the particles. The Plt distribution curve shows cells in their native
size. Figure 2.14 is an example of a Plt histogram with a normal Plt size distribution.

2

Figure 2.14 Typical Plt Histogram

Interference on the Lower End of the Platelet Distribution Curve

Particles that are approximately platelet size can interfere with the platelet histogram and
count. Small particles, such as micro-bubbles or dust, can interfere at the low end. If the
number of pulses in the 2 to 3 fL region is higher than the predefined limits, an SCL flag
appears to alert the operator that a significant number of small cells or interference, such as
micro-bubbles, are present.

PN 4237615B

2-15

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

Microcytic Interferences on the Upper End of the Platelet Distribution Curve

Microcytic red blood cells can intrude at the upper end of the platelet distribution curve. If
the sample contains microcytes, the A
the influence of this interference by repositioning the variable threshold and excluding the
microcytes.

Parameter Results Obtained Using the Plt Histogram

r Plt Count: The A

criteria, and proprietary flagging information to confirm the parameter result prior to
reporting it. To obtain a Plt count result, the instrument compares the data from the two
5-second count periods then votes and rejects any questionable data.

Plt count = Number of cells counted per unit volume x Calibration coefficient.
Plt count is displayed and printed as Plt = Nx10

(Note: µL is the US unit format. Other formats are available. See

UNIT SELECTION

r MPV Measurement: The MPV (Mean Platelet Volume) is measured directly from analysis

of the platelet distribution curve. The MPV is displayed and printed in femtoliters (fL).

C

•T 5diff analyzer may be able to successfully eliminate

C

•T 5diff hematology analyzer uses duplicate counting criteria, voting

3

cells/µL.

Heading A.7, REPORTING

.)

r Pct Calculation: The Pct (plateletcrit) is calculated according to the formula:

3

Pct%

Plt 10

------------------------- ----------------------------- ---------=

/µL()MPV (fL)×

10 000,

r PDW Calculation: PDW (Platelet Distribution Width) is calculated from the Plt

histogram as the width of the curve between S1 and S2.
As shown in Figure 2.15, S1 and S2 are placed so that:

t 15% of the platelets occur between 2fL and S1.
t 15% of the platelets occur between S2 and the variable upper threshold.
t The PDW result is determined on the platelets between S1 and S2.

Figure 2.15 Area of the Plt Histogram Used to Determine the PDW Parameter Result

2-16

15%

PDW

S1 S2

15%

7615002A

PN 4237615B

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

Hgb Determination

The hemoglobin (Hgb) released by the lysis of the red blood cells combines with the
potassium cyanide to form a stable cyanmethemoglobin compound.

This compound is measured through the optical part of the First Dilution/Hgb bath using a
spectrophotometric technique at a wavelength of 550nm. Transmittance of the sample
dilution is compared with the transmittance of a reagent blank. The system calculates the Hgb
using both the blank and sample readings.

The final Hgb result represents: absorbance value obtained x coefficient of calibration.

Hgb is displayed and printed as Hgb = N g/dL.
(

: g/dL is the US unit format. Other formats are available. See

Note

UNIT SELECTION

.)

Heading A.7, REPORTING

WBC Count, BASO Count, and DiffPlot Development

WBC Count

C

The A
proprietary flagging information to confirm the parameter result prior to reporting it. To
obtain a WBC count result, the instrument compares the data from the two 5-second count
periods then votes and rejects any questionable data. This is the reference WBC count, which
is reported.

•T 5diff hematology analyzer uses duplicate counting criteria, voting criteria, and

2

A second WBC count is determined in the flowcell during acquisition of the DiffPlot.

WBC count: number of cells per unit volume x coefficient of calibration.

BASO Count

Differentiation between basophils and other leukocytes is obtained by means of the

C

A

•T 5diff WBC Lyse-specific lytic action. See Figure 2.16.

In Figure 2.16, basophils are located in the area between the thresholds labeled
hundred percent (100%) of the leukocytes is represented by the total number of nucleated
particles plus the basophils within the area between the thresholds labeled

The basophil percentage is calculated from the number of particles existing in the area
between the thresholds labeled

Figure 2.16 Areas Used to Determine WBC and BASO Parameter Results

c

and d (Figure 2.16).

c

b

and d.

and d. One

bc d

basophilsWBC

PN 4237615B

2-17

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

BASO count: number of cells per unit volume x coefficient of calibration in percentage
relative to the number of counted cells (BASO plus WBC nuclei).

BASO count

DiffPlot Development

The DiffPlot analysis on the A
principles:

r Dual Focused Flow (DFF) fluid dynamics, which is a process by which individual cells

or particles are focused in a stream of diluent (hydrodynamic focusing).

r The volume measurement (Coulter Principle).
r The measurement of transmitted light with zero degree (0°) angle, which permits a

response proportional to the internal structure of each cell and its absorbance.

From these measurements, a DiffPlot is developed with optical transmission (absorbance) on
the X-axis and volume on the Y-axis. See Figure 2.17.

BASO%

---------------------­WBC%

WBC count×=

C

•T 5diff hematology analyzer is based on three essential

Figure 2.17 DiffPlot Regions

The study of the DiffPlot permits the clear differentiation of four out of five leukocyte
populations. In a typical whole-blood sample, the basophil population is very small when
compared with the other four white cell populations.

2-18

PN 4237615B

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

Table 2.7 defines the DiffPlot regions. Table 2.8 defines immature white blood cells.

Table 2.7 DiffPlot Regions Defined

Region Definition

2

Neutrophil

(Neut)

Lymphocyte

(Lymph)

Monocyte

(Mono)

Eosinophil

(Eos)

Neutrophils, with their cytoplasmic granules and segmented nuclei, scatter light
according to their morphological complexity. A hypersegmented neutrophil
gives an increased optical response when compared to a young neutrophil
population. The higher the complexity of the cell, the further to the right they
appear in the DiffPlot (Figure 2.17).

Lymphocytes, typically being small with regular shape are smaller in volume
and lower in absorbance than the other cells, and are positioned in the lower
region of the DiffPlot (Figure 2.17). Normal lymphocyte populations typically
have a homogeneous volume with a Gaussian (bell-shaped) distribution.

Large lymphocytes, reactive lymphoid forms, stimulated lymphocytes and
plasma cells are found in the upper portion of the lymphocyte region
(Figure 2.17).

The lower area of the lymphocyte zone is normally empty; however, when small
lymphocytes are present, a population may exist in this area (Figure 2.17).

The presence of platelet aggregates is indicated by a distribution pattern that
moves from the DiffPlot origin into the lymphocyte region (Figure 2.17).

NRBC cytoplasmic membranes lyse like those of mature erythrocytes. The
small nuclei that remain appear in the debris and small lymphocyte regions
(Figure 2.17).

Monocytes are typically large cells with a kidney-shaped nucleus and agranular
cytoplasm. These cells neither scatter nor absorb large amounts of light and,
therefore, are positioned in the lower end of the absorbance axis. Due to their
size, the monocytes are clearly positioned high on the volume axis
(Figure 2.17).

Very large monocytes may be found in the IMM (immature cell) region.
With the reagent action, eosinophils are the most intensely stained for optical

separation. Due to the staining and their size, the eosinophils will show higher
absorbance than the neutrophils, but will be of similar volume (Figure 2.17).

PN 4237615B

Debris Platelets and debris from erythrocyte lysis represent the background debris

population located in the lower region of the DiffPlot.

2-19

OPERATION PRINCIPLES

PARAMETER DEVELOPMENT

Table 2.8 Immature White Blood Cells

Immature Cell Type Definition

Immature Granulocytes Immature granulocytes are detected by their larger volume and by the presence

Band Cells Band cells are typically larger or of similar size to the neutrophils; however, due

Blast Cells Blast cells are generally larger than monocytes and have similar absorbance.

of granules that increase the intensity of the scattered light.
Due to their increased volume and similar absorbance, promyelocytes,

myelocytes, and metamyelocytes are located above the neutrophil population
and are typically counted as IMM cells. IMM cells are included in the reported
neutrophil value.

to their low level of cellular complexity, they absorb less light. As a result, band
cells tend to appear in the region between the neutrophils and the monocytes.

When blast cells are present, they are generally located above the monocytes,
which means they will be included in the IMM cell count.

Small blasts will be located between the normal lymphocyte and monocyte
populations.

2-20

PN 4237615B

3.1 INSTRUMENT SPECIFICATIONS

Dimensions and Weight

See Figure 3.1.

SPECIFICATIONS/CHARACTERISTICS

3

3

WARNING

handles, and it weighs more than one person should lift. Therefore, to prevent injury, at least two people
following necessary safety precautions should lift the instrument together.

Figure 3.1 Instrument Dimensions and Weight

23 in.
58 cm

Risk of operator injury if only one person lifts the instrument. The instrument has no lifting

81.0 lb.

37.0 Kg

17.5 in.

44.4 cm

19.75 in.

50.10 cm

Power

Supply

From 100 Vac to 240 Vac. From 50 Hz to 60 Hz.

PN 4237615B

Consumption

200 watts maximum.

Installation Category

The instrument is designed to be safe for transient voltages according to Installation
Category II and Pollution Degree 2.

Grounding Requirements

To protect against electrical shock, the wall ground (earth) plug must be correctly connected
to the laboratory grounding electricity installation.

Temperature, Ambient Operating

The ambient operating temperature is 16°C to 34°C (61°F to 93°F). If you keep the
instrument at a temperature less than 10°C (50°F), allow the instrument to remain at the
ambient operating temperature for one hour before use.

3-1

SPECIFICATIONS/CHARACTERISTICS

INSTRUMENT SPECIFICATIONS

Altitude Range

The instrument can be operated at any altitude up to 3,000 meters (9,800 feet).

Recommended Location

Place the instrument on a clean, level bench, allowing at least 20cm (8 in.) of space behind
the instrument for ventilation.

Electromagnetic Environment Check

The instrument produces less than the acceptable level of electromagnetic interference when
properly placed. Electromagnetic interferences are limited to levels that allow the correct
operation of other instruments conforming to their placement.

If there is a problem, ensure that the instrument is not placed near electromagnetic fields or
short wave emissions (such as radar, X-ray machines, scanners, and so forth).

Recommended Reagents

Beckman Coulter recommends these reagents:

C

r A
r A
r A
r A
r A

•T 5diff Diluent,

C

•T 5diff Fix,

C

•T 5diff WBC Lyse,

C

•T 5diff Hgb Lyse, and

C

•T 5diff Rinse.

Heading 1.3, REAGENTS

See

for additional information about these reagents.

Recommended Controls

AC•T 5diff Control is the recommended control. See
for additional information.

Heading 1.2, CONTROLS AND CALIBRATORS

Recommended Calibrator

AC•T 5diff Cal Calibrator is the recommended calibrator. See

CALIBRATORS

for additional information.

Heading 1.2, CONTROLS AND

Recommended Anticoagulant

The recommended anticoagulant is K3EDTA with the proper proportion of blood to
anticoagulant as specified by the tube manufacturer.

Sample Volume Aspirated

r 30 µL of whole blood is aspirated in the CBC mode.
r 53 µL of whole blood is aspirated in the CBC/DIFF mode.

Dilution Ratios

WBC/BASO: 1/200
DIFF: 1/80
RBC/Plt: 1/10,000
Hgb: 1/250

3-2

PN 4237615B

SPECIFICATIONS/CHARACTERISTICS

INSTRUMENT SPECIFICATIONS

Throughput

The instrument can process up to 60 samples per hour in either mode – CBC or CBC/DIFF.

The instrument achieves nominal throughput when used in a routine laboratory environment
with samples having normal hematology parameters. Depending on sample mix and
workflow conditions, slightly higher or lower throughput might be observed.

Sample Stability

Sample stability is based on an average of 20 clinical normal and abnormal whole-blood
samples.

r CBC parameters are stable up to 48 hours at room temperature.
r DIFF parameters are stable up to 24 hours at room temperature.

Sample Identification

You can enter a sample ID using the instrument’s control panel, setup the instrument to
autonumber the IDs, or scan the tube’s barcode label with the optional hand-held barcode
reader.

3

Output

The instrument can transmit startup, sample, and control data to a host computer. The
Sample Results screen shows the sample identification number, sample results, and any result
flags.

The instrument prints a report (Figure 3.2).

Figure 3.2 Sample Report

PN 4237615B

3-3

SPECIFICATIONS/CHARACTERISTICS

INSTRUMENT SPECIFICATIONS

Measurements and Computation

r Impedance for WBC, Plt, RBC, and BA.
r Photometry for Hgb using cyanmethemoglobin method with 550nm diode light source.
r Impedance and light absorbance for NE, LY, MO, EO, ATL, and IMM.
r Computation from stored data that was directly measured for Hct, MCV, MCH, MCHC,

RDW, MPV, Pct, and PDW.

Counting Aperture Diameters

WBC/BASO: 80 µm
DIFF: 60 µm
RBC/Plt: 50 µm

Reagent Consumption

Table 3.1 shows the instrument’s reagent consumption by cycle.

Table 3.1 Reagent Consumption by Cycle in mL

Approximate

Cycle Reagent

Diluent WBC Lyse Rinse Fix Hgb Lyse

CBC 20.5 2.1 0.9 – 0.4 60 sec
CBC/DIFF 25.6 2.1 0.9 1.0 0.4 60 sec

†

Startup
Shutdown 25.5 – 14 – 1.0 2 min 45 sec
Prime Diluent 35.5 ––––2 min 30 sec
Prime Rinse ––25.8 –– 1 min 20 sec
Prime Fix –––25.8 – 1 min 30 sec
Prime WBC Lyse – 25.8 ––– 1 min 20 sec
Prime Hgb Lyse 2.5 –––4.2 1 min
Prime All Reagents 23.7 16.0 16.0 16.0 4.2 3 min 20 sec
Autoclean 12.5 – 6.0 –– 1 min 35 sec
System Reset Cycle 24.0 – 1.4 – 1.0 1 min 25 sec

62.0 2.1 3.7 1.0 1.4 3 min 40 sec

†

For one background count only. The maximum is three.

– indicates not applicable.

Duration

3-4

Environmental Protection

Removal and recycling of this instrument must be done by a properly qualified laboratory in
accordance with local legislation.

PN 4237615B

3.2 PERFORMANCE SPECIFICATIONS

The stated performance specifications apply to an instrument that has been properly
maintained as indicated in
reagents listed in

Recommended Reagents

Reproducibility

Reproducibility (Table 3.2) is based on 20 consecutive replicate runs from one normal, fresh
whole-blood sample without flags.

Table 3.2 Reproducibility Specifications

Parameter CV% Test Level

Chapter 8, DIAGNOSTICS

SPECIFICATIONS/CHARACTERISTICS

PERFORMANCE SPECIFICATIONS

3

, and one that uses only the recommended

.

WBC <2.0%
RBC <2.0%
Hgb <1.0% 15.0 g/dL
Hct <2.0% 45.0%
Plt <5.0%

10.0x10

5.00x10

300.0x10

3

6

/µL
/µL

3

/µL

Linearity

Linearity (Table 3.3) is assessed on serially diluted material. Each dilution is analyzed four
times.

Table 3.3 Linearity Specifications

Difference

Parameter Units Linearity Range

WBC
RBC
Plt
Hgb g/dL 0 to 22.9 ±0.3 or ±2%
Hct % 1.8 to 55.9

10
10
10

3

/µL

6

/µL

3

/µL

0.4 to 90.0 ±0.2 or ±3%

0.23 to 7.70 ±0.05 or ±2%
4 to 1,000 ±10 or ±6%

56.0 to 63.8

(Whichever is Greater)

±2 or ±3%
±5 or ±5%

PN 4237615B

Accuracy

Accuracy (Table 3.4) is assessed by duplicate analysis of clinical specimens when compared
to an automated hematology analyzer that has been properly calibrated and maintained
according to the manufacturer’s recommendation.

Table 3.4 Accuracy Specifications

Parameter Correlation r

WBC >0.95
RBC >0.95
Hgb >0.95

3-5

SPECIFICATIONS/CHARACTERISTICS

PERFORMANCE SPECIFICATIONS

Table 3.4 Accuracy Specifications (Continued)

Parameter Correlation r

Hct >0.95
Plt >0.95

Carryover

Carryover (Table 3.5) is assessed by analyzing whole blood with high values followed by a
whole blood sample with low values. Each sample is run consecutively in triplicate.

Carryover is calculated as follows:

Table 3.5 Carryover Specifications

Parameter Carryover

Carryover

Low 1 - Low 3

----------------------- --------------­High 1 - Low 3

100×=

WBC <2.0%
RBC <2.0%
Plt <2.0%
Hgb <2.0%

Reportable Range

The reportable range (Table 3.6) is the range of results that the instrument displays, prints,
and transmits. Results between the linear range and the reportable range will be flagged.

Table 3.6 Reportable Range

Parameter Units Reportable Range

WBC
RBC
Plt
Hct % 0.0 – 80.0
Hgb g/dL 0.0 - 30.0

10
10
10

3

6

3

/µL
/µL
/µL

0.0 – 100.0

0.00 – 10.00

0.0 – 1500.0

3-6

PN 4237615B

3.3 PERFORMANCE CHARACTERISTICS

Reproducibility

Reproducibility was measured twice to show precision at two levels of WBC counts – one
relatively lower than the other:

r Table 3.7 shows the precision values based on 20 replicate samples that were analyzed

consecutively on the same instrument from one normal, fresh, whole-blood sample with
a low, normal WBC and without flags.

r Table 3.8 shows the precision values based on 20 replicate samples analyzed

consecutively on the same instrument from one normal, fresh, whole-blood sample with
a high, normal WBC and without flags.

Table 3.7 Reproducibility Characteristics From a Normal Sample with a Low Normal WBC Count

Parameter Mean Standard Deviation (SD) CV%

WBC 6.40 0.09 1.42
RBC 4.39 0.03 0.59

SPECIFICATIONS/CHARACTERISTICS

PERFORMANCE CHARACTERISTICS

3

Hgb 13.80 0.06 0.44
Hct 38.60 0.25 0.66
Plt 288.40 7.86 2.73
NE% 60.8 0.73 1.21
LY% 27.3 0.55 2.02
MO% 8.10 0.49 6.09
EO% 3.30 0.32 9.64
BA% 0.60 0.12 20.21

Table 3.8 Reproducibility Characteristics From a Normal Sample with a High Normal WBC Count

Parameter Mean Standard Deviation (SD) CV%

WBC 12.50 0.18 1.41
RBC 5.17 0.03 0.61
Hgb 16.80 0.06 0.33
Hct 48.90 0.32 0.64
Plt 173.60 4.05 2.33
NE% 85.20 0.31 0.36
LY% 7.70 0.37 4.80
MO% 5.30 0.17 3.30

PN 4237615B

EO% 0.90 0.14 15.10
BA% 0.90 0.10 10.59

3-7

SPECIFICATIONS/CHARACTERISTICS

LIMITATIONS

Accuracy

Accuracy (Table 3.9) for the CBC and DIFF parameters was defined as agreement between
the comparator instrument and the A
expected range of performance.

Table 3.9 Accuracy Characteristics

Parameter Correlation r

WBC 0.99
RBC 0.99
Hgb 0.99
Hct 0.99
Plt 0.98
NE% 0.99
LY% 0 .9 9
MO% 0.88

C

•T 5diff analyzer using clinical specimens covering the

EO% 0.98

Carryover

Carryover (Table 3.10) was assessed by analyzing whole blood with high values followed by a
whole-blood sample with low values. Each sample was run consecutively in triplicate.

Carryover is calculated as follows:

Table 3.10 Carryover Characteristics

Parameter Units Low Level High Level Carryover %

WBC
RBC
Plt
Hgb g/dL 6.3 25.4 0.26%

3.4 LIMITATIONS

Maintenance

Failure to properly execute the maintenance procedures in
compromise the instrument’s reliability.

10
10
10

3

6

3

/µL
/µL
/µL

Low 1 - Low 3

Carryover

1.5 54.6 0.00%

2.0 8.1 0.16%

35.5 818.0 0.45%

----------------------- --------------­High 1 - Low 3

100×=

Chapter 8, DIAGNOSTICS

may

3-8

Blood Specimens

If any abnormal test result (including flagged results or results outside the normal range)
occur, use reference methods or other standard laboratory procedures to verify the results.
For additional information, see

Heading 3.5, INTERFERING SUBSTANCES

.

PN 4237615B

3.5 INTERFERING SUBSTANCES

Table 3.11 shows a list of known limitations of automated blood cell counters that use
impedance and light absorbance as measurement principles.

Table 3.11 Interfering Substances

Parameter Interfering Substance

WBC Unlysed RBCs: In rare instances, the erythrocytes in the blood sample may not completely

lyse and are detected on the WBC histogram with an *WBC flag or as an elevated baseline on
the lymphocytes. Non-lysed RBCs will cause a falsely elevated WBC count.

Multiple myeloma: The precipitation of proteins in multiple myeloma patients may cause
elevated WBC counts.

Leukemia: A very low WBC count may result in this disease state due to the possible fragility
of the leukocytes; some of these cells may be destroyed during counting. WBC fragments
will also interfere with the WBC DIFF parameters.

Chemotherapy: Cytotoxic and immunosuppressive drugs may increase the fragility of the
leukocytes, which may cause low WBC counts.

Cryoglobulins: Increased levels of cryoglobulin that may be associated with myeloma,
carcinoma, leukemia, macroglobulinemia, lymphoproliferative disorders, metastic tumors,
autoimmune disorders, infections, aneurysm, pregnancy, thromboembolic phenomena,
diabetes, and so forth, which can elevate the WBC, RBC, or Plt counts and the Hgb
concentration. The specimen must be warmed to 37°C (99°F) in a water bath for 30 minutes
and reanalyzed immediately (analyzer or manual method).

Agglutinated WBCs: Leukoagglutination.

†

RBC

Hgb Turbidity of the blood sample: Any number of physiologic and/or therapeutic factors may

Agglutinated RBCs: May cause a falsely low RBC count. Blood samples containing the
agglutinated RBCs may be suspected by elevated MCH and MCHC values and shown by
examination of the stained blood film.

Cold agglutinins: IgM immunoglobulins elevated in cold agglutinin disease may lower RBC
and Plt counts and increase MCV.

produce falsely elevated Hgb results. To obtain accurate Hgb results when increased
turbidity of the blood sample occurs, determine the cause of the turbidity and follow the
appropriate method below:

Elevated WBC: An extremely elevated WBC will cause excessive light scatter. If this

r

occurs:

1. Use the reference (manual) methods.

2. Centrifuge the diluted sample.

3. Measure the supernatant fluid with a spectrophotometer.
Elevated lipids: Elevated lipids in the blood sample will give the plasma a milky

r

appearance. This condition can occur with hyperlipidemia, hyperproteinemia (as in
gammapathies), and hyperbilirubinemia. Accurate hemoglobin determinations can be
achieved by using reference (manual) methods and a plasma bank.

Increased turbidity: This may be seen in cases where the RBCs are resistant to lysing.

r

This condition will cause a falsely elevated Hgb result but may be detected by
observing the abnormal MCH, MCHC values, and the increased baseline on the leading
edge of the WBC histogram. Erroneous Hgb results will cause the results of MCH and
MCHC to also be erroneous.

Fetal bloods: The mixing of fetal and maternal blood may produce a falsely elevated

r

Hgb value.

SPECIFICATIONS/CHARACTERISTICS

INTERFERING SUBSTANCES

3

PN 4237615B

3-9

SPECIFICATIONS/CHARACTERISTICS

INTERFERING SUBSTANCES

Table 3.11 Interfering Substances (Continued)

Parameter Interfering Substance

Hct RBC agglutination: May produce erroneous Hct and MCV values. RBC agglutination may be

detected by observing abnormal MCH and MCHC values, and by examining the stained blood
film. Use the manual method to obtain an accurate Hct value.

MCV RBC agglutination: May produce an erroneous MCV value. RBC agglutination may be

detected by observing abnormal MCH and MCHC values, and by examining the stained blood
film. Use the manual method to obtain an accurate MCV value.

Excessive numbers of large platelets: This condition and/or the presence of an excessively
high WBC count may interfere with the accurate determination of the MCV value. Carefully
examine the stained blood film to detect the problem.

MCH MCH is determined according to the Hgb value and the RBC count, which means that

anything listed as an interfering substance for Hgb and/or RBC will impact MCH and may
cause erroneous MCH values.

MCHC MCHC is determined according to the Hgb and Hct values, which means that anything listed

as an interfering substance for Hgb and/or Hct will impact MCHC and may cause erroneous
MCHC values.

RDW RDW is determined according to the RBC count and may be impacted by the following

conditions:

Agglutinated RBCs: May cause a falsely low RBC count and erroneous RDWs. Blood

r

samples containing the agglutinated RBC may be detected by observing abnormal MCH
and MCHC values and by examining the stained blood film.

Nutritional deficiency or blood transfusion: May cause elevated RDW results due to

r

iron, cobalamin, and/or folate deficiencies.

Plt Very small RBCs (microcytes), RBC fragments (schizocytes), and WBC fragments: May

interfere with the proper counting of platelets and cause elevated Plt counts.
Agglutinated RBCs: May trap platelets, causing an erroneously low Plt count. The presence

of agglutinated RBCs may be detected by observing abnormal MCH and MCHC values and by
examining the stained blood film.

Excessive numbers of large platelets: May cause an erroneously low Plt count since these
large platelets may exceed the upper threshold for the Plt parameter are not counted.

Chemotherapy: Cytotoxic and immunosuppressive drugs may increase the fragility of these
cells, which may cause low Plt counts. Use the manual (reference) method to obtain an
accurate Plt count.

Hemolysis: Hemolysed specimens contain RBC stroma which may elevate Plt count.
ACD (acid-citrate-dextrose) blood: Blood anticoagulated with ACD may contain clumped Plt

which could depress the Plt count.
Plt Agglutination: Clumped platelets may cause a decreased Plt count and/or elevated WBC

count; *WBC, SL, and SL1 flags may be generated. Reanalyze the specimen as follows:

3-10

1. Recollect the specimen in sodium citrate anticoagulant to prevent platelet
agglutination.

2. Reanalyze the specimen for only the Plt count.

3. Correct the final Plt result for the effect of the sodium citrate dilution.

PN 4237615B

Table 3.11 Interfering Substances (Continued)

Parameter Interfering Substance

SPECIFICATIONS/CHARACTERISTICS

INTERFERING SUBSTANCES

3

‡

MPV

NE#, NE% The neutrophil count is derived from the WBC count. The presence of excessive eosinophils,

LY#, LY% The lymphocyte count is derived from the WBC count. The presence of erythroblasts, certain

MO#, MO% The mononuclear cell count absolute is derived from the WBC count. The presence of large

EO#, EO% The eosinophil cell count is derived from the WBC count. The presence of abnormal granules

Giant platelets: May exceed the upper threshold of the Plt parameter and may not be counted
as platelets. Consequently, these larger platelets will not be included in the instrument’s
calculation of MPV.

Very small RBCs (microcytes), RBC fragments (schizocytes), and WBC fragments: May
interfere with the proper counting of platelets.

Agglutinated RBCs: May trap platelets, causing an erroneous MPV result. You may be able to
detect the presence of agglutinated RBCs by observing abnormal MCH and MCHC values and
by examining the stained blood film.

Chemotherapy: May also affect the sizing of platelets.

metamyelocytes, myelocytes, promyelocytes, blasts, and plasma cells may interfere with an
accurate neutrophil count.

parasites, and RBCs that are resistant to lysis may interfere with an accurate LY count.
Interfering substances pertaining to WBC also pertain to the LY# and LY%.

lymphocytes, atypical lymphocytes, blasts, and an excessive number of basophils may
interfere with an accurate monocyte count. Interfering substances pertaining to WBC also
pertain to the MO# and MO%.

(degranulated areas, toxic granules, and so forth) may interfere with the eosinophil count.
Interfering substances pertaining to WBC also pertain to the EO# and EO%.

BA#, BA% The basophil cell count is derived from the WBC count. Interfering substances pertaining to

WBC also pertain to the BA# and BA%.

†

The RBC dilution contains all formed elements in the blood, erythrocytes, leukocytes, and platelets. During the counting of the RBCs,

‡

Blood samples collected in EDTA will not maintain a stable MPV because platelets swell depending on the time post-collection and

platelets are not counted if their size falls below the RBC minimum threshold.

storage temperature.

PN 4237615B

3-11

SPECIFICATIONS/CHARACTERISTICS

INTERFERING SUBSTANCES

3-12

PN 4237615B

4.1 DEFINITIONS

Warnings

Anything that can cause user injury is considered a hazard and is noted in the text as
WARNING. Warnings appear where needed throughout this manual.

Cautions

Anything that can cause instrument damage is considered a caution and is noted in the text as
CAUTION. Cautions appear where needed throughout this manual.

Importants

Anything that can cause misleading results or data corruption is considered an important and
is noted in the text as IMPORTANT. Importants appear where needed throughout this
manual.

Attention

An ATTENTION provides additional information to be considered when performing a
procedure.

PRECAUTIONS/HAZARDS

4

4

4.2 SAFETY PRECAUTIONS

Electronic

WARNING

To prevent possible injury or shock, do not tamper with the instrument and do not remove any components
(covers, doors, panels, and so on) unless otherwise instructed within this document.

Biological

WARNING

servicing the instrument, you may become injured or contaminated. To prevent possible injury or biological
contamination, you must wear proper laboratory attire, including gloves, a laboratory coat, and eye
protection.

Use care when working with pathogenic materials. Be sure that you have a procedure
available to decontaminate the instrument, provide ventilation, and dispose of waste liquid
and sharps. Refer to the following publications for further guidance on decontamination.

r Biohazards Safety Guide, 1974, National Institute of Health.
r Classifications of Etiological Agents on the Basis of Hazards, 3d ed., June 1974, Center

Risk of personal injury from electronic shock. Electronic components can shock and injure you.

Risk of personal injury or contamination. If you do not properly shield yourself while using or

for Disease Control, U.S. Public Health Service.

PN 4237615B

Moving Parts

WARNING

personal injury. When you operate the instrument, be sure all covers and doors are closed.

Risk of personal injury. Operating the instrument with doors and/or covers open can cause

4-1

PRECAUTIONS/HAZARDS

OPERATIONAL HAZARDS

4.3 OPERATIONAL HAZARDS

Safety symbols alert you to potentially dangerous conditions. These symbols, together with
text, apply to specific procedures and appear as needed throughout this manual.

Symbol Warning Condition Action

Biohazard

!

(specimens, reagents, controls,
calibrators, and so forth)

.

Consider all materials

and areas

Wear standard laboratory attire and follow safe
laboratory procedures when handling any
material in the laboratory.

these materials come into
contact with

infectious.

Probe hazard.

!

!

and may contain biohazardous
materials, such as controls and
calibrators.

Electrical shock hazard

of electrical shock when
instrument is plugged into the
power source.

as being potentially

The probe is sharp

.

Possibility

Avoid any unnecessary contact with the probe
and probe area.

Before continuing, unplug the A
analyzer from the electrical outlet.

C

•T 5diff

4-2

PN 4237615B

5.1 BEFORE ANALYSIS

Do the following procedures:

Waste Container Level Check

r

Printer Check

r

Startup

r

Specimen Collection and Mixing

r

Running Cell Controls to Verify Calibration

r

Waste Container Level Check

At the beginning of each day, check the waste container to determine if it needs to be
replaced. If so, do

Printer Check

At the beginning of each day, be sure the printer is ready to print.

,

,

,

, and

.

Replacing the Waste Container

RUNNING SAMPLES

5

.

5

1

Be sure there is an adequate paper
supply in the printer.

r If so, go to step 2.
r If not, add paper according to the

printer’s user manual.

2

Press the printer’s ON/OFF switch until
the control LEDs are on.

3

Be sure the printer is properly
configured. See

CONFIGURATION

Heading A.10, PRINTER

for details.

PN 4237615B

5-1

RUNNING SAMPLES

BEFORE ANALYSIS

Startup

Startup During Power Up

When you turn on the instrument, Startup is automatically done if the unit has been put in
Shutdown. If you want to do Startup again, do

1

Turn the instrument on.
The instrument performs the Startup

routine (a rinse cycle followed by a
background count, which is an analysis
cycle on reagent without any blood
specimen).

Upon completion of the Startup cycle,
the instrument displays and prints the
results.

Startup After Power Up





.

16:05

IMPORTANT

Risk of erroneous results if the
instrument’s heating devices have not reached
35°C (95°F). Allow the instrument to warm to
35°C (95°F). This may take several minutes to
do. Keep the right door closed.

Review the Startup results.

2

r If Startup passed, go to

Collection and Mixing

Specimen

.

r If Startup failed, go to step 3.



BACKGROUND COUNT IN PROGRESS





10%

5-2

PN 4237615B

If the background counts are not

3

within acceptable limits after the first
Startup cycle, the instrument
automatically performs Startup up to
two more times. If Startup fails after the
third attempt, a STARTUP FAILED
message appears on the screen and on
the report for every cycle.

: The background count limits are:

Note

3

/µL

3

/µL

3

WBC = 0.3 x 10
RBC = 0.03 x 106/µL
Hgb = 0.3 g/dL

Plt = 7.0 x 10
a. Do
b. If Startup continues to fail, contact

Startup After Power Up

a Beckman Coulter representative.

RUNNING SAMPLES

BEFORE ANALYSIS

3

3

.

5

If the system determines that there is
insufficient reagent to complete the
day’s work, a REAGENT LOW LEVEL
message appears.

r Identify the low reagent and

change it according to the
procedures in

OR

r Continue and change the reagent

when the specific reagent low
message is displayed.

Startup After Power Up

Do this procedure if you want to run Startup after the instrument has already gone through
the initial Startup routine at power up.

Press .

1

Replacing Reagents

.

PN 4237615B

5-3

RUNNING SAMPLES

BEFORE ANALYSIS

2

Review the Startup results.
r If Startup passed, go to

Collection and Mixing

.

r If Startup failed, go to step 3.

If the background counts are not

3

within acceptable limits after the first
Startup cycle, the instrument
automatically performs Startup up to
two more times. If Startup fails after the
third attempt, a STARTUP FAILED
message appears on the screen and on
the report for every cycle.

Note: The background count limits are:

WBC = 0.3 x 10

3

/µL
RBC = 0.03 x 106/µL
Hgb = 0.3 g/dL

3

/µL

3

a. Do

Plt = 7.0 x 10

Startup After Power Up

b. If Startup continues to fail, contact

a Beckman Coulter representative.

If the system determines that there is
insufficient reagent to complete the
day’s work, a REAGENT LOW LEVEL
message appears.

Specimen

3

3

.

5-4

r Identify the low reagent and

change it according to

Reagents

.

Replacing

OR

r Continue and change the reagent

when the specific reagent low
message is displayed.

PN 4237615B

Specimen Collection and Mixing

!

RUNNING SAMPLES

BEFORE ANALYSIS

5

IMPORTANT

specimen collection tube is not filled to the
quantity required by the tube manufacturer. Fill
the specimen collection tube as required.

Using K3EDTA as the anticoagulant,

1

collect the required amount of venous
specimen according to the tube
manufacturer’s requirements.

Note:

microcontainer with a minimum
volume of 100µL for analysis on this
instrument.

Mix the blood specimen gently and

2

thoroughly before analysis.

Risk of erroneous results if the

You can collect blood into a

PN 4237615B

Running Cell Controls to Verify Calibration

Before analyzing patient samples, ensure that the system is within acceptable operating limits
by analyzing three levels (low, normal, and high) of cell control material.

The cell control for the A

C

•T 5diff hematology analyzer is AC•T 5diff Control.

!

CBC

Press to select the desired analysis

1

mode (CBC or CBC/DIFF). The mode
selected appears on the screen.

DIFF

5-5

RUNNING SAMPLES

BEFORE ANALYSIS

2

Enter the cell control number as the
sample ID.

!

Mix each control according to the

3

instructions in the cell control package
insert.

Inspect the vial’s contents to ensure
that all cells are uniformly distributed;
if not, repeat this step.

!

Present the cell control vial to the

4

sample probe, and press the aspirate
switch.

The LEDs flash during sample
aspiration.

b

c

5-6

When the red LED illuminates, remove

5

the cell control tube from the probe.
When the green LED remains

illuminated, the instrument is ready for
the next analysis.

PN 4237615B

Repeat steps 1 through 5 until you have

6

run all three levels of cell control.

Review the control results to ensure

7

they are within the acceptable ranges.
r If so, then you are ready to analyze

patient samples. See

ANALYSIS

r If not, go to step 8.

When control results are not within the

8

acceptable ranges:

.

Heading 5.2,

RUNNING SAMPLES

BEFORE ANALYSIS

5

a. Rerun the control. If results are

still outside the acceptable ranges,
do step b.

b. Clean the system. See

System

c. Rerun the control. If the results are

still outside the acceptable ranges,
do step d.

d. Analyze a new cell control vial. If

the results are still outside the
acceptable ranges, do step e.

e. Recalibrate the system. See

Heading 7.3, AUTO-CALIBRATION

rerun the control.

r If the results are still outside

r If your cell control results are

.

the acceptable ranges, contact
a Beckman Coulter
representative.

within the acceptable ranges,
you are ready to analyze
patient samples in

Heading 5.2, ANALYSIS

Diluter

and

.

PN 4237615B

5-7

RUNNING SAMPLES

ANALYSIS

5.2 ANALYSIS

Running Whole-Blood Samples

!

Verify that the sample ID in the Next

1

ID field is correct.

r If so, go to step 2.
r If not, enter the sample ID as

instructed in

Heading 5.5, ENTERING

THE SAMPLE IDENTIFICATION (ID)

.

If you want to change the sample

2

analysis mode from what is currently

CBC

selected, press .

DIFF

The current analysis mode and range
are displayed on the bottom right of the
screen.

If you want to change the Range from

3

what is currently selected, press
until the desired flagging range
appears.

Mix the sample according to your

4

laboratory’s protocol.

RANGE

ID 1007
RBC

4.62

HGB

13.4

HCT

40.5

MCV

88

MCH

29.1

MCHC

33.1

RDW

13.

PLT

280

ANALYZING
NEXT ID 1008

WBC
NE
LY
MO
EO
BA

12 / 07 / 99

12.3 H

64.1

27.8

5.6

1.8

0.7

16:05

CBC/DIFF 1

5-8

PN 4237615B

Remove the cap from the sample tube

5

according to your laboratory’s protocol.

!

Present the sample to the probe and

6

press the aspirate switch.
The LEDs flash during sample

aspiration.

b

RUNNING SAMPLES

ANALYSIS

c

5

When the red LED remains

7

illuminated, remove the tube from the
probe.

When the green LED remains
illuminated, the instrument is ready for
the next analysis.

The sample results appear on the

8

screen and print according to
instrument setup.

ID 1007
RBC

4.62

HGB

13.4

HCT

40.5

MCV

88

MCH

29.1

MCHC

33.1

RDW

13.

PLT

280

ANALYZING
NEXT ID 1008

WBC
NE
LY
MO
EO
BA

12 / 07 / 99

12.3 H

64.1

27.8

5.6

1.8

0.7

16:05

CBC/DIFF 1

PN 4237615B

5-9

RUNNING SAMPLES

AFTER ANALYSIS

If Autonumbering is on, the instrument

9

is ready to run the next sample.
If Autonumbering is off, enter the next

sample ID manually or with the
optional barcode reader.

5.3 AFTER ANALYSIS

Results

When analysis is completed, the instrument displays the results and prints the report.
Figure 5.1 is an example of a sample report.

If flags appear on the results, see

Figure 5.1 Sample Report

Chapter 6, REVIEWING RESULTS

.

5-10

Printing Results for Last Sample Analyzed

There are two ways you can print results for the last sample analyzed:

r If the run screen is displayed, press .
r If the run screen is not displayed, beginning at the Main menu, select

PRINT LATEST RESULT

tt

.

SETUP

tt

PRINTER

PN 4237615B

Auto-Clean

An auto-clean (automatic cleaning) is performed by the instrument after a specified number
of samples is analyzed. You can set the frequency from 1 to 75; see

FREQUENCY SETTING

5.4 SHUTDOWN

At the end of each day, do this procedure to rinse the instrument and place it in a stand-by
mode.

Press .

1

The instrument cycles Rinse reagent for
cleaning and goes into a stand-by
mode.

RUNNING SAMPLES

SHUTDOWN

Heading A.19, AUTO-CLEAN

.

5

When Shutdown is complete:

2

r Allow the instrument to remain in

stand-by mode,
OR

r Turn the instrument off.

: After doing Shutdown, you must

Note

do a

Startup

instrument again.

before operating the

5.5 ENTERING THE SAMPLE IDENTIFICATION (ID)

Three methods are available for entering sample IDs on this instrument: auto-numbering,
manual, and barcode (optional). For details on selecting the sample ID mode, see

Heading A.14, SELECTING THE SAMPLE IDENTIFICATION (ID) MODE

ATTENTION:

analysis cycle will not start.

Auto-Numbering

If your instrument is configured to the Autonumbering ID mode, the instrument
automatically assigns a sample ID (from 1 to 99999) and increments the number before each
analysis.

If the system is set up for a manual ID and no sample ID has been entered, the

.

PN 4237615B

If you want to override the current autonumber and enter another sample ID, do this
procedure.

5-11

RUNNING SAMPLES

ENTERING THE SAMPLE IDENTIFICATION (ID)

!

Enter the sequence number in the

1

Next ID field.

Press when the ID is complete.

2

Running Whole-Blood Samples

Do

3

.

The ID of the current sample appears in
the upper left corner of the screen, and
the ID of the next sample appears in
the Next ID field.

Manual Sample ID

If your instrument is configured for Manual ID entry, do this procedure to enter a sample
identification. You can enter up to 16 alphanumeric characters in the sample ID.

!

Press or to scroll through the

1

available alpha characters, or enter the
number at the numeric keypad.

5-12

Move the cursor by pressing or

2

.

PN 4237615B

Repeat steps 1 and 2 until you have

3

entered the sample ID.

Press when the ID is complete.

4

The ID of the current sample appears in
the Analyzing field, and the ID of the
next sample appears in the Next ID
field.

Running Whole-Blood Samples

Do

5

RUNNING SAMPLES

ENTERING THE SAMPLE IDENTIFICATION (ID)

.

5

Scanning the Sample ID with the Barcode Reader

The barcode reader is optional. If your system is equipped with a barcode reader, you can scan
the sample ID into the system.

ATTENTION:

correct sample identification.

Beckman Coulter recommends that you verify each barcode reading to ensure

!

Locate the barcode on the sample tube

1

label.

PN 4237615B

5-13

RUNNING SAMPLES

ENTERING THE SAMPLE IDENTIFICATION (ID)

IMPORTANT

the entire barcode is not captured with the barcode
reader, especially with Interleaved 2-of-5 barcode
format. Position the barcode reader over the label
to capture the entire barcoded sample ID.
Otherwise, part of the sample ID may not be
scanned, resulting in mis-identification. Pass the
barcode reader over the barcode label on the
sample tube.

Pass the barcode reader over the

2

Risk of sample mis-identification if

barcode label on the sample tube.
The ID of the current sample appears in

the Analyzing field, and the ID of the
next sample appears in the Next ID
field.

Verify each barcode reading to ensure

3

correct sample identification.

5-14

PN 4237615B

6.1 GENERAL

Patient sample results are generated from sample analysis. There may be instances when a
patient sample result is flagged or a parameter number is replaced by a flag.

Carefully review all patient sample results, especially results with flags and/or messages. For
details, see

MESSAGES

Heading 6.3, FLAGS GENERATED BY THE INSTRUMENT

.

REVIEWING RESULTS

6

Heading 6.4, INTERPRETIVE

and

6

IMPORTANT

rare instances, especially for samples where fibrin or other debris is likely to occur (such as pediatric or
oncology samples), a transient or partial blockage may not be detected by the instrument. Therefore, verify
flagged results for accuracy and review any result that exceeds your laboratory’s limits.

Risk of result inaccuracy if a transient or partial blockage is not detected by the instrument. In

6.2 FLAGS AND INTERPRETIVE MESSAGES

Flags

Definition

A flag is a symbol, set of symbols, or letters generated by the instrument to signal that a
certain parameter requires additional attention. Flags can be:

r Linked to a result when it exceeds the normal limits.
r Linked to a problem in the morphology of the blood cell population.
r Linked to instrument operation.

For details, see

Types of Flags

This instrument uses two types of flags – replacement and non-replacement flags.

Heading 6.3, FLAGS GENERATED BY THE INSTRUMENT

.

PN 4237615B

r Replacement flags, also called codes, replace a parameter’s numeric results.
r Non-replacement flags appear next to the parameter results. Up to two of these flags can

be displayed for a parameter.

Types of Flag Printout Formats

The system provides two printout formats for reporting the DiffPlot and histogram flags on
the patient report –

r If the DIFFPLOT AND HISTROGRAM FLAGS print option is

flagged using the

r If the DIFFPLOT AND HISTOGRAM FLAGS print option is selected, samples are flagged

using the

For additional information on print options, see
additional information on flag printout formats, see

Suspect

Detailed

and

Suspect

format.

Detailed

format.

.

: This is the default setting.

Note

Configuring the Instrument’s Printer Settings

Suspect or Detailed Flag Format

selected, samples are

not

.

. For

6-1

REVIEWING RESULTS

FLAGS GENERATED BY THE INSTRUMENT

Interpretive Messages

Definition

Interpretive messages are triggered from the flagging limits established by your laboratory.
These messages indicate possible pathological disorders. For details, see

INTERPRETIVE MESSAGES

.

6.3 FLAGS GENERATED BY THE INSTRUMENT

The following sections define these general instrument-generated flags:

r Results Exceeding Instrument Capacity,
r Hemoglobin Errors,
r Voteout Flag,
r WBC Count Flag,
r DiffPlot Flags,
r CBC Flags, and
r Patient Ranges and Action Ranges.

Heading 6.4,

Results Exceeding Instrument Capacity

If a result exceeds instrument capacity, the result will be indicated as follows:

r If the result is below the lower limits of the instrument, the result will be reported as 0.

For example, if the WBC is less than 0.1x10

r If the result is outside the limits at which the parameter can be calculated, the result is

replaced by

r If the result is above the instrument’s linear range, the result is flagged with

result is above the instrument’s reportable range, the result is replaced by
In addition, related parameters may also be flagged or replaced.

. . . .

.

3

/µL, WBC is reported as 0.0.

+

, or if the

++++

Hemoglobin Errors

Hgb Blank Error

The instrument establishes a reference blank reading and compares each sample blank to the
reference result. If the blank differs from the reference by more than an allowable amount, the
Hgb, MCH, and MCHC results are flagged with a review “ R” flag.

If three consecutive samples produce a Hgb blank error, the Hgb, MCH, and MCHC results
are replaced by

. . . .

on the third sample.

.

6-2

Hgb Read Error

The instrument reads each sample three times. If the difference among the three readings
exceeds a predefined limit, the Hgb, MCH, and MCHC results are flagged with a voteout “V”
flag.

PN 4237615B

REVIEWING RESULTS

FLAGS GENERATED BY THE INSTRUMENT

Voteout Flag

The instrument performs two counts on the WBC, RBC, Hct, and Plt. If the results for the two
counts differ by more than a predefined limit, the WBC, RBC, Hct, and Plt results are flagged
with a voteout “V” flag.

r If the WBC result is flagged with a V, then the DIFF number results are also flagged with

a V.

r If the RBC result is flagged with a V, then the MCV, MCH, MCHC, and RDW results are

replaced by

r If the Hct result is flagged with a V, then the MCV and MCHC results are replaced by

.

. . . .

r If the Plt counts votes out, then the Plt result is flagged with a V.

. . . .

.

WBC Count Flag

During the data collection for the DiffPlot, the instrument also determines the WBC count
from the flowcell.

The WBC flag DIFF- or DIFF+ is reported:

6

r If the WBC count from the flowcell exceeds the WBC count from the WBC/BASO bath

by more than a predefined amount, DIFF+ is displayed.

r If the WBC count from the flowcell is less than the WBC count from the WBC/BASO

bath by more than a predefined amount, DIFF- is displayed.

r When a DIFF- or a DIFF+ flag occurs, the WBC count and all DIFF parameters are

flagged with an

: The comparison between the WBC count from the WBC/BASO bath and the WBC

Note

count from the flowcell will not be performed when the sample is analyzed in the CBC mode
or when this option is disabled in setup.

.

*

DiffPlot Flags

When populations in the DiffPlot exceed the limits set for that region, a review (R) flag will
occur on the DIFF parameter related to that region.

If the R flag occurs on a DIFF parameter, further investigate the result.

Twelve different flags may occur related to the position of the populations within the DiffPlot:

Reject r UM (upper monocyte)

r

r DB (debris) r LN (lower neutrophil)
r SL (small lymphocytes) r UN (upper neutrophil)

PN 4237615B

r SL1 (small lymphocytes 1) r NE (neutrophil/eosinophil)
r NL (neutrophil/lymphocyte) r ATL (atypical lymphocytes)
r MN (monocyte/neutrophil) r IMM (immature cells)

See Table 6.1 for additional information.

6-3

REVIEWING RESULTS

FLAGS GENERATED BY THE INSTRUMENT

Table 6.1 Definition of DIFF Flags

DiffPlot
Region
Flag DiffPlot Region Affected Description Flags

Suspected
Abnormalities

DB

The system
detects a problem
with volume and
absorbance
measurements in
the flowcell.

More than 50% of
the pulses were
rejected.

Occurs when the
number of pulses
in the DB region
exceeds the DB#
limit.

Default values:
100% or 120
particles.

next to all

R

DIFF
parameters.

is displayed

DB

and printed in
WBC flag area.

Plt aggregates
Increased Plt count
RBCs resistant to

lysis (stroma)
NRBCs
Reagent

contamination

SL

Occurs when the
number of
particles that are
counted in the SL
region is higher
than the SL# limit.

Default values:
100% or 50
particles.

next to:

R

NE%, NE#,
LY%, LY#,
MO%, MO#,
EO%, EO#,
ATL%, ATL#,
IMM%, IMM#.

displayed

SL

and printed in
WBC flag area.

Small lymphocytes
Plt aggregates
NRBCs
RBCs resistant to

lysis (stroma)

6-4

PN 4237615B

FLAGS GENERATED BY THE INSTRUMENT

Table 6.1 Definition of DIFF Flags (Continued)

DiffPlot
Region
Flag DiffPlot Region Affected Description Flags

REVIEWING RESULTS

6

Suspected
Abnormalities

SL1

NL

Occurs when the
number of
particles in the SL
region is higher
than the SL1
number limit
when the
percentage of
particles in the SL
region, relative to
the lymphocyte
region, exceeds
the SL1
percentage limit.

Default values: 5%
or 45 particles.

Occurs when the
number of
particles in the NL
separation region
is above the limits
set.

Default values: 3%
or 120 particles.

and

May trigger
interpretive
messages.
NRBCs, Plt
aggregates, and
NRBCs plus Plt
aggregates

is displayed

SL1

and printed in
the WBC flag
area.

next to:

R

NE%, NE#,
LY%, and LY#.

is displayed

NL

and printed in
WBC flag area.

Plt aggregates
NRBCs
RBCs resistant to

lysis (stroma)
Small abnormal

lymphocytes

Small Neutrophils
without granules
and/or slight
nuclear
segmentation

Lymphocytes with
segment nuclei

Neutrophils with
weak membranes
(smudge/smear
cells)

PN 4237615B

MN

Occurs when the
number of
particles in the MN
separation region
is above the limits
set.

Default values:
100% or 120
particles.

next to:

R

ATL%, ATL#,
IMM%, and
IMM#.

Replaces NE%,
NE#, MO%, and
MO# with

. . . .

.

is displayed

MN

and printed in
WBC flag area.

Monocytes with
granules or
hyperbasophilic
monocytes

Immature
neutrophils with
non-segmented
nuclei (band cells)

6-5

REVIEWING RESULTS

FLAGS GENERATED BY THE INSTRUMENT

Table 6.1 Definition of DIFF Flags (Continued)

DiffPlot
Region
Flag DiffPlot Region Affected Description Flags

Suspected
Abnormalities

UM

LN

Occurs when the
number of
particles in UM
region is above the
limits set.

Default values:

1.1% or 999
particles.

Occurs when the
number of
particles in the LN
region is above the
limits set.

Default values:

2.5% or 999
particles.

next to:

R

NE%, NE#,
MO%, MO#,
IMM%, and
IMM#.

displayed

UM

and printed in
WBC flag area.

next to all

R

WBC DIFF
parameters.

is displayed

LN

and printed in
WBC flag area.

Large monocytes
Hyperbasophilic

monocytes
Myelocytes
Promyelocytes
Large blasts

Neutrophil
degradation due to
improper storage or
sample age

Plt aggregates
RBCs resistant to

lysis (stroma)
Reagent

contamination

UN

Occurs when the
number of
particles in the UN
region is above the
limits set.

Default values:

1.1% or 999
particles.

next to:

R

NE%, NE#,
IMM%, IMM#

is displayed

UN

and printed in
WBC flag area.

Large neutrophils
Immature

granulocytes:

Metamyelocytes

r

Myelocytes

r

Promyelocytes

r

6-6

PN 4237615B

FLAGS GENERATED BY THE INSTRUMENT

Table 6.1 Definition of DIFF Flags (Continued)

DiffPlot
Region
Flag DiffPlot Region Affected Description Flags

REVIEWING RESULTS

6

Suspected
Abnormalities

NE

ATL

Occurs when the
number of
particles the NE
separation region
is above the limits
set.

Default values:

1.1% or 60
particles.

Occurs when a
significantly large
population is
located in the ATL
region.

ATL flag is
triggered from the
Patient Limits, and
the interpretive
message (

Lymphocyte

triggered from the
Action Limits.

Default values: 2%
or 0.2x10

Atypical

) is

9

/L.

next to:

R

IMM% and
IMM#.

Replaces NE%,
NE#, EO%, and
EO# with

. . . .

.

is displayed

NE

and printed in
WBC flag area.

is displayed

ATL

and printed in
WBC flag area.

May be
displayed and
printed as
ATL% and
ATL#.

Young eosinophils
Giant

hypersegmented
neutrophils

Eosinophils with
low
intracytoplasmic
material (agranular
eosinophils)

Large lymphocytes
Reactive

lymphocytes
Stimulated

lymphocytes
Plasma cells

PN 4237615B

IMM

Occurs when a
significantly large
population of cells
is located in UN,
UM, and channel
127 regions.

IMM flag is
triggered from the
Patient Limits, and
the interpretive
message (

Immature Cell

triggered from the
Action Limits.

Default values: 2%
or 0.2x10

Large

9

/L.

) is

is

IMM

displayed and
printed in WBC
flag area.

May be
displayed and
printed as
IMM% and
IMM#.

Large monocytes
Hyperbasophilic

monocytes
Myelocytes,

metamyelocytes,
promyelocytes

Large blasts
Large neutrophils

6-7

REVIEWING RESULTS

FLAGS GENERATED BY THE INSTRUMENT

CBC Flags

There are three types of CBC flags:

r WBC/BASO histogram flags,
r RBC histogram flags, and
r Plt histogram flags.

See Table 6.2 for additional information.

Table 6.2 CBC Histogram Flags

Histogram Flag Illustrations of Histogram Flags Description

WBC/BASO

*WBC Figure 6.1 WBC/BASO Histogram Flags:

CBC Mode

WBC

BA1 BA2 BA3

MB

(Mono

Figure 6.2 WBC/BASO Histogram Flags:
CBC/DIFF Mode

Baso)

BA1 BA2 BA3

BASO+

BASO

Determined from the ratio of the cells
counted between the 0 channel and
BA1.

Indicates the presence of an
abnormal number of cells in
comparison to leukocytes. Plt
aggregates and NRBCs may be found
in this region.

Default value: 3.5% or 999 particles.

Generated when the percentage of
basophils found in the BA channel is
above the percentage of the
LY/MO/NE raw count found on the
DIFF channel.

If the BASO% exceeds 50%, a
BASO+ flag is generated. The
basophils are not taken away from
the DiffPlot LY/MO/NE populations.

is displayed and printed instead

. . . .

of the BA% and BA#.

6-8

PN 4237615B

FLAGS GENERATED BY THE INSTRUMENT

Table 6.2 CBC Histogram Flags (Continued)

Histogram Flag Illustrations of Histogram Flags Description

REVIEWING RESULTS

6

RBC

MICRO
and/or
MACRO

Figure 6.3 MICRO and MACRO Regions
on RBC Histogram

RBC2

RBC1

%MICRO

%MACRO

MICRO and MACRO flags are
generated when the percentage of
cells counted in the microcytic
(MICRO) and macrocytic (MACRO)
regions compared to the total
number of RBCs are above the
established limits set by your
laboratory.

Thresholds RBC1 and RBC2 define
the MICRO and MACRO regions and
are calculated based on the standard
deviation of a normal RBC
population.

Default value: 5% for MICRO and

7.5% for MACRO.
: MICRO and MACRO flags will

Note

be activated in software version 1.0
and higher.

PN 4237615B

6-9

REVIEWING RESULTS

FLAGS GENERATED BY THE INSTRUMENT

Table 6.2 CBC Histogram Flags (Continued)

Histogram Flag Illustrations of Histogram Flags Description

Plt

MIC
and
SCH

Figure 6.4 Plt Flags

3

25µ

30

Figure 6.5 Mobile Threshold Positioned
in the Standard Regions (Between 18fL
and 25fL)

2

18

25µ

30

Figure 6.6 Mobile Threshold Cannot Be
Positioned in the Standard Region

3

18

25µ

30

The Plt histogram has 256 channels
between 2fL and 30fL. A mobile
threshold (at 25fL by default)
(Figure 6.4) moves according to the
presence of microcytic RBCs present
in the Plt analysis region. Plt flags
generate when the following three
conditions occur.

1. If the mobile threshold can be
positioned in the standard region,
between 18fL and 25fL, the MIC
(Microcytes) flag will be shown in
the Plt alarm region. See
Figure 6.5.

The Plt result is reliable.

2. If a valley is not detected by the
18fL threshold, the threshold is
placed at the 18fL position and a
MIC flag is generated. If the
interference is significant, the Plt
count will also be flagged.

3. If the mobile threshold cannot be
positioned between 18fL and
25fL, the threshold is placed at
the 18fL position, an SCH
(schistocytes) flag is generated,
and the Plt count is flagged.

Suspected abnormalities include
the presence of schistocytes
and/or the presence of Plt
aggregates. See Figure 6.7

The Plt result is not reliable.
Verify the result by an alternative
method.

6-10

Figure 6.7 Mobile Threshold Cannot Be
Positioned

25µ18

2

30

PN 4237615B

FLAGS GENERATED BY THE INSTRUMENT

Table 6.2 CBC Histogram Flags (Continued)

Histogram Flag Illustrations of Histogram Flags Description

REVIEWING RESULTS

6

Plt

(continued)

SCL

23

An SCL (small cell) flag indicates the
presence of small cells in the 2fL and
3fL regions.

Rerun the sample and verify the
results.

Suspect or Detailed Flag Format

As described in
and

Detailed

Suspect Flag Format

If the DIFFPLOT AND HISTOGRAM FLAGS option is not selected (default setting) on the
instrument’s printer configuration screen, the flags are reported (displayed and printed) in the

Suspect

format as follows:

r DB prints as DB.
r The DIFF flag replaces the SL, SL1, NL, MN, UM, LN, UN, and NE flags.

Types of Flag Printout Formats

formats.

, the two types of flag printout formats are

Suspect

r IMM prints as IMM.
r ATL pr in ts as ATL .
r The WBC/BA flag replaces the DIFF+, DIFF-, *WBC, MB, and BASO+ flags.
r The HISTO flag replaces the MICRO, MACRO, SCL, MIC, and SCH flags.
r The flags will be printed on the patient report in the area labeled “SUSPECT”.

For example, when the option is not selected, flags on the patient sample report may be
shown as:

SUSPECT:
WBC: WBC/BA DB DIFF IMM ATL
RBC: HISTO
PLT: HISTO

For additional information about the DIFFPLOT AND HISTOGRAM FLAGS print option, see

Configuring the Instrument’s Printer Settings

.

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6-11

REVIEWING RESULTS

INTERPRETIVE MESSAGES

Detailed Flag Format

If the DIFFPLOT AND HISTOGRAM FLAGS option is selected on the instrument’s printer
configuration screen, the DiffPlot and histogram flags are reported (displayed and printed) in
the detailed format.

For example, when the option is selected, flags on the patient sample report may be shown as:

FLAGS
WBC: *WBC DB SL UM IMM ATL
RBC: MICRO
PLT: MIC

For additional information about the DIFFPLOT AND HISTOGRAM FLAGS print option, see

Configuring the Instrument’s Printer Settings

Patient Ranges and Action Ranges

Table 6.3 shows the four flags that can be generated based on patient ranges and action
ranges.

Table 6.3 Patient Range and Action Range Flags

.

Flag Description

H

Result is above the patient limit set by your laboratory and may generate an
interpretive message on the printout.

L

Result is below the patient limit set by your laboratory and may generate an
interpretive message on the printout.

HH

Result is above the action limit set by your laboratory and may generate an
interpretive message on the printout.

LL

Result is below the action limit set by your laboratory and may generate an
interpretive message on the printout.

6.4 INTERPRETIVE MESSAGES

ATTENTION:

for assisting with quickly and efficiently screening abnormal samples and for diagnosis. It is
recommended that your laboratory use suitable reference methods to confirm diagnoses.

The interpretive messages print in the flag area on the patient report. Tables 6.4 through 6.11
list interpretive messages and triggering conditions.

Only one DIFF interpretive message can be displayed for each DIFF parameter. The message
generated from the absolute count for that parameter takes priority. For example, if a relative
LYMPHOPENIA (LY% < LY% LL) and an absolute LYMPHOCYTOSIS (LY# > LY# HH) occur,
only the LYMPHOCYTOSIS message will be displayed.

Interpretive messages indicate a possible pathological disorder and should be used

6-12

The following sections define:

r WBC Interpretive Messages,
r RBC Interpretive Messages,
r Plt Interpretive Messages, and
r Combination WBC/RBC/Plt Interpretive Messages.

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WBC Interpretive Messages

r Table 6.4 lists WBC interpretive messages from Action Ranges.
r Table 6.5 lists WBC interpretive messages from the DiffPlot.

Table 6.4 WBC Interpretive Messages from Action Ranges

Printed Message Triggering Condition

REVIEWING RESULTS

INTERPRETIVE MESSAGES

6

LEUCOCYTOSIS
LEUCOPENIA
LYMPHOCYTOSIS
LYM PH OP EN IA
NEUTROPHILIA
NEUTROPENIA
EOSINOPHILIA
MONOCYTOSIS
BASOPHILIA
LARGE IMMATURE

CELLS
ATYPICAL

LYM PH OC YT E
MYELEMIA
BLASTS

Table 6.5 WBC Interpretive Messages from DiffPlot

WBC > WBC HH
WBC < WBC LL
LY# > LY# HH, or LY % > LY% HH
LY# < LY# LL, or LY% < LY% LL
NE# > NE# HH, or NE% > NE% HH
NE# < NE# LL, or NE% < NE% LL
EO# > EO# HH, or EO% > EO% HH
MO# > MO# HH, or MO% > MO% HH
BA# > BA# HH, or BA% > BA% HH
IMM# > IMM# HH, or IMM% > IMM% HH

ATL# > ATL# HH, or ATL% > ATL% HH

NE% > NE% HH
BA# > BA# HH

IMM# > IMM# HH

and

IMM# > IMM# HH

and

and

UM

H means above the patient range.

HH means above the action range.

LL means below the action range.

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Message Triggering Condition

LEFT SHIFT

MN or NL

and

UN

RBC Interpretive Messages

r Table 6.6 lists RBC interpretive messages from Action Ranges.
r Table 6.7 lists RBC interpretive messages from Flag Sensitivity.

Table 6.6 RBC Interpretive Messages from Action Ranges

Message Triggering Condition

ANEMIA
ANISOCYTOSIS
HYPOCHROMA
COLD AGGLUTININ
MICROCYTOSIS

Hgb < Hgb LL
RDW > RDW HH
MCHC < MCHC LL
MCHC > MCHC HH
MCV < MCV LL

6-13

REVIEWING RESULTS

INTERPRETIVE MESSAGES

Table 6.6 RBC Interpretive Messages from Action Ranges (Continued)

Message Triggering Condition

MACROCYTOSIS
ERYTHROCYTOSIS

MCV > MCV HH
RBC > RBC HH

Table 6.7 RBC Interpretive Messages from Flag Sensitivity

Message Triggering Condition

MICROCYTE
MACROCYTE

MICRO% > MICRO% Flag Sensitivity limit
MACRO% > MACRO% Flag Sensitivity limit

Plt Interpretive Messages

r Table 6.8 lists platelet interpretive messages from Action Ranges.
r Table 6.9 lists platelet interpretive messages from the Plt histogram.

Table 6.8 Plt Interpretive Messages from Action Ranges

Message Triggering Condition

THROMBOCYTOSIS
THROMBOCYTOPENIA
MACROPLATELETS

Plt > Plt HH
Plt < Plt LL
MPV > 11

HH means above the action range.

LL means below the action range.

HH means above the action range.

LL means below the action range.

6-14

Table 6.9 Plt Interpretive Messages from the Plt Histogram

MESSAGE Triggering Condition

MICROCYTOSIS
SCHISTOCYTE
SMALL CELL

Derived from Plt histogram
Derived from Plt histogram
Derived from Plt histogram

Combination WBC/RBC/Plt Interpretive Messages

r Table 6.10 lists interpretive messages from a combination of WBC/RBC/Plt Action

Ranges.

r Table 6.11 lists conditions causing NRBCS and PLATELET AGGREGATES interpretive

messages.

Table 6.10 Interpretive Messages from a Combination of WBC/RBC/Plt Action Ranges

Message Triggering Condition

PANCYTOPENIA

WBC < WBC LL

RBC < RBC LL

and

Plt < Plt LL

and

HH means above the action range.

LL means below the action range.

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Table 6.11 NRBCs and PLATELET AGGREGATES Interpretive Messages

Message Triggering Condition

REVIEWING RESULTS

INTERPRETIVE MESSAGES

6

PLT AGGREGATES

NRBCS

NRBCS & PLATELET
AGGREGATES

3

Plt < 150x10
DB and PDW > 20, or

DB and MPV > 10, or
DB and Plt < 150x10
DB and WBC Voteout

*WBC and PDW > 20, or
*WBC and MPV > 10, or
*WBC and Plt < 150x10

SL, or
SL and WBC Voteout, or
*WBC and WBC Voteout, or
SL1 and WBC Voteout

If none of the individual conditions defined for

AGGREGATES

/mm3

occur

WBC voteout

and

3

/mm3, or

3

3

/mm

*WBC or SL1 or WBC Voteout occur.

and

NRBCS

or

PLATELET

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REVIEWING RESULTS

INTERPRETIVE MESSAGES

6-16

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