Beckman Coulter ACT Service manual

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Page 1

BECKMAN COULTER™ AC•T™ 5diff Hematology Analyzer

Service Manual

PN 4237616B (September 2000) COULTER CORPORATION

A Beckman Coulter Company Miami, Florida 33196-2500 USA

Page 2

LEGAL NOTICES

Beckman Coulter, Inc. makes no representation that, upon furnishing this service manual, the holder of the manual will have the necessary technical capabilities and know-how to properly troubleshoot and repair any of the equipment specified in the manual. Beckman Coulter, Inc. assumes no liability whatsoever, including consequential and incidental damages, resulting from improper operation of Beckman Coulter instruments after maintenance of Beckman Coulter instruments has been performed by persons not employed by Beckman Coulter, Inc. Furthermore, Beckman Coulter, Inc. assumes no liability whatsoever for any personal injury or property damage resulting from maintenance and/or repair of Beckman Coulter instruments performed by persons not employed by Beckman Coulter, Inc.

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.

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.

"This Service Manual contains confidential information of Beckman Coulter, Inc. and its receipt or possession does not convey any rights to reproduce, disclose its contents, or to manufacture, use, or sell anything it may describe. Reproduction, disclosure, or use without specific written authorization of Beckman Coulter, Inc. is strictly forbidden."

Page 3

REVISION STATUS

Initial Issue, 3/2000

Released by CN 040130-0003 Software Version 0.11

Issue B, 07/00

Released by CN 040150-0025 Software version 1.03

The material in the revision B change pages was updated for software version 1.03 and for any hardware changes since revision A. The changes include updating the adjustment procedures for the bath assembly, HGB blank, RBC/PLT gain, WBC/BASO, motor current, thresholds, and the optical bench; updating the replacement procedures for the heater assembly, power supply, start switch, reagent syringes, count syringe, sample prove, waste syringe, 5diff syringe, flow-cell coax, optical bench lamp, diluent reservoir, and sample syringe; updating the procedure for testing and configuring the bar-code reader; updating the parts lists; updating the tubing lists and associated circuit connections; adding procedures for balancing

the WBC count, setting the diff information on the LX300 + printer.

Changes were made on the following pages:

1.1-1, 1.1-2, 1.1-4

2.1-4, 2.6-1, 2.6-2, 2.6-3, 2.6-4, 2.6-5, 2.8-3, 2.8-11, 2.8-12, 2.8-13, 2.8-15, 2.10-2, 2.10-3

3.1-1, 3.2-1, 3.2-2, 3.2-3, 3.2-6, 3.2-7, 3.2-9, 3.2-10, 3.3-1, 3.3-7, 3.3-8 added 3.3-9 and 3.3-10

4.1-1, 4.1-2, 4.1-3, 4.1-4, 4.2-1, 4.2-2, 4.2-4, 4.2-5, 4.2-6 through 4.2-8, 4.4-1, 4.5-1 through 4.5-4

4.6-1 through 4.6-8, 4.7-2

4.14-1, 4.14-2, 4.17-2, 4.17-3, 4.18-1 4.19-1 through 4.19-8, 4.20-1, 4.20-2, 4.20-3, 4.20-4 deleted 4.20-5 and 4.20-6

4.21-1 through 4.21-3, 4.23-1 through 4.23-6, 4.24-1 through 4.24-6, 4.25-1 through 4.25-8 deleted 4.25-9 through 4.25-12

4.26-1, 4.26-3, 4.26-4, 4.27-1 through 4.27-8 deleted 4.27-9 through 4.27-12

4.29-1, 4.29-3 through 4.29-6, 4.30-1, 4.30-3 through 4.30-5, 4.31-1, 4.31-3, 4.31-4

4.32-1 through 4.32-3, 4.33-1, 4.33-2, 4.35-1, 4.35-2, 4.35-3, 4.35-10, 4.36-1, 4.36-2, 4.36-5, 4.36-6 added 4.37-1 and 4.37-2, 4.38-1 and 4.38-2, 4.39-1 through 4.39-10

5.2-3, 6.3-2 through 6.3-6, 7.3-2

8.1-1 through 8.1-12, 8.2-7, 8.2-8, 8.2-9, 8.2-16, 8.2-17, 8.2-42, 8.2-46, 8.2-48 A.2-9, A.3-3, A.4-1, C.1-1, C.1-2 and C.1-7.

/diff- thresholds, and replacing the new Main card; and adding

4.8-1, 4.9-1, 4.9-2, 4.10-1, 4.10-2, 4.11-1, 4.12-1, 4.12-2, 4.13-1; 4.13-2

The change page packet also includes the latest revision of the Pneumatic/Hyraulic Schematic, 7616069B.

Changes that are part of the most recent revision are indicated in the printed copy by a bar in the margin of the amended page.

This document applies to the latest software listed and higher versions. When a subsequent software version affects the information in this document, the changes will be included on minor revision change pages or summarized on a Notice of Information Update form and will be released by service memo.

PN 4237616B

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Page 4

REVISION STATUS

PN 4237616B

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LEGAL NOTICES, 2

REVISION STATUS, iii

CONTENTS, v

1 INTRODUCTION, 1.1-1

1.1 MANUAL DESCRIPTION, 1.1-1 Scope, 1.1-1

Notification of Updates, 1.1-1 Intended Audience, 1.1-1 Organization, 1.1-1 Numbering Format, 1.1-2 Special Headings, 1.1-3

WARNING, 1.1-3

CAUTION, 1.1-3

IMPORTANT, 1.1-3

ATTENTION, 1.1-3

Note, 1.1-3 Conventions, 1.1-3 Graphics, 1.1-4

1.2 SAFETY PRECAUTIONS, 1.2-1 Electronic, 1.2-1 Biological, 1.2-1 Troubleshooting, 1.2-1

2 INSTRUMENT DESCRIPTION, 2.1-1

2.1 INTRODUCTION TO THE A

Purpose, 2.1-1 Function, 2.1-1 Description, 2.1-1

Components, 2.1-1

Interaction with the A

•T 5diff Hematology Analyzer, 2.1-1

Modes of Operation, 2.1-3

CBC Mode, 2.1-3 CBC/DIFF Mode, 2.1-3

Reagent Consumption, 2.1-4

2.2 OPERATION PRINCIPLES, 2.2-1 Overview, 2.2-1 Measurement Principles, 2.2-1

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

V Technology, 2.2-3

Dual Focused Flow (DFF), 2.2-3 Flow Cell, 2.2-3 Focused Flow Impedance, 2.2-4 Absorbance Cytochemistry, 2.2-4

•T 5diff HEMATOLOGY ANALYZER, 2.1-1

PN 4237616B

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CONTENTS

Signal Processing, 2.2-4 Thresholds, 2.2-4

WBC/BASO Methodology,2.2-5 Sample Analysis Overview,2.2-5

Aspiration, 2.2-5 Dilution, 2.2-6 Delivery, 2.2-6 Sample Partitioning, 2.2-7

2.3 CYCLE DESCRIPTION, 2.3-1 Cycle Start Conditions, 2.3-1 Sample Flow, 2.3-2

2.4 SAMPLE ANALYSIS, 2.4-1 RBC and Platelet Analysis, 2.4-1

Parameter Results Obtained from the RBC/Plt Dilution, 2.4-2 Hgb Measurement, 2.4-3 WBC Count and Differential, 2.4-4

Parameter Results Obtained from the WBC/BASO Dilution, 2.4-5

Differential, 2.4-5

Parameter Results Obtained from the DIFF Dilution, 2.4-6 Dilution Summary, 2.4-7

2.5 RBC PARAMETER DEVELOPMENT, 2.5-1 RBC/Plt Dilution, 2.5-1 RBC Count, 2.5-1 RBC Histogram, 2.5-1 Parameter Results Obtained Using the RBC Histogram, 2.5-2

Hct Measurement, 2.5-2 MCV Calculation, 2.5-2 RDW Calculation, 2.5-2

RBC Distribution Flags, 2.5-2

RBC1 and RBC2 Thresholds, 2.5-3 Flags, 2.5-3

Hgb Determination, 2.5-3

Hgb Blank Reading, 2.5-3 Sample Reading, 2.5-4 Hgb Specific Flags, 2.5-4

MCH and MCHC Calculations, 2.5-4

MCH Calculation, 2.5-4 MCHC Calculation, 2.5-4

2.6 PLATELET PARAMETER DEVELOPMENT, 2.6-1 RBC/Plt Dilution, 2.6-1 Plt Count, 2.6-1 Platelet Distribution Curve, 2.6-1 Parameter Results Obtained Using the Plt Histogram, 2.6-2

MPV Measurement, 2.6-2 Pct Calculation, 2.6-2 PDW Calculation, 2.6-2

PN 4237616B

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Detecting Abnormal Platelet Distributions, 2.6-3

Identifying a Normal Distribution, 2.6-3 Interference on the Lower End of the Platelet Distribution Curve, 2.6-3 Microcytic Interferences on the Upper End of the Platelet Distribution Curve, 2.6-3 Microcytic Interference with a Distinct Valley between 18 fL and 25 fL, 2.6-4 Microcytic Interference with a Valley below 18 fL, 2.6-4 Interference with No Distinct Valley, 2.6-5

2.7 WBC PARAMETER DEVELOPMENT, 2.7-1 Overview, 2.7-1 WBC/BASO Dilution, 2.7-1 WBC Count, 2.7-1 BASO Count, 2.7-1 DIFF Dilution, 2.7-2 DiffPlot Development, 2.7-2 DiffPlot Regions Defined, 2.7-3

Neutrophil (Neut), 2.7-3 Lymphocyte (Lymph), 2.7-3 Monocyte (Mono), 2.7-3 Eosinophil (Eos), 2.7-3 Debris, 2.7-3

Immature White Blood Cells, 2.7-4

Immature Granulocytes, 2.7-4 Band Cells, 2.7-4 Blast Cells, 2.7-4

DiffPlot Thresholds, 2.7-4

CONTENTS

2.8 PNEUMATIC/HYDRAULIC SYSTEM, 2.8-1 Functions of Valves, 2.8-1 Pneumatic Diagrams, 2.8-4 Diluter System, 2.8-11

Diluent Input (Figure 2.8-10), 2.8-11 5diff Syringe and Flow Cell, 2.8-12 Probe and Probe Rinse, 2.8-13 Diluent to Baths, 2.8-13

Waste System, 2.8-15

2.9 ELECTRONIC SYSTEM, 2.9-1 Plug/Jack Labels, 2.9-1 Optical Preamplifier Card, 2.9-1 LCD and Keypad Card, 2.9-1 LED Card, 2.9-1 Motor Interconnect Card, 2.9-1 Traverse Interconnect Card, 2.9-1

2.10 SOFTWARE STRUCTURE, 2.10-1 Overview, 2.10-1 Menu Trees, 2.10-1 How to Select a Menu Item, 2.10-1

PN 4237616B

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CONTENTS

3 INSTALLATION PROCEDURES, 3.1-1

3.1 PREINSTALLATION CHECKS, 3.1-1 Environment, 3.1-1

Altitude Range, 3.1-1

Ambient Temperature, 3.1-1 Space and Accessibility Requirements, 3.1-1 Electrical Input, 3.1-1

Power Requirements, 3.1-2

Grounding, 3.1-2

Installation Category, 3.1-2 Electromagnetic Environment Check, 3.1-2 Inspection Report, 3.1-2

3.2 INITIAL SETUP, 3.2-1 Preinstallation Checks, 3.2-1 Supplies, 3.2-1 Unpacking, 3.2-1

Inspection, 3.2-1 Unpack the Analyzer, 3.2-1 Unpack the Installation Kit, PN XEA484A, 3.2-1

Unpack the Waste Alarm Kit, PN 6912680, 3.2-1 Verify All Caution and Compliance Labels are in Place, 3.2-1 Connect the Waste System, 3.2-3

Connect the Waste Tubing, 3.2-3

Install the Waste Alarm, 3.2-3 Connect the Reagents, 3.2-5

Connect the Diluent Tubing, 3.2-5

Install the Reagent Bottles, 3.2-6 Install the Printer, 3.2-7 Power On the Instrument, 3.2-7 Enter Reagent Lot Numbers, 3.2-8 Prime the Instrument, 3.2-8 Configure the Instrument Printer Settings, 3.2-9 Set the User Mode, 3.2-9 Verification, 3.2-10

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3.3 PRINTER INSTALLATION, 3.3-1

EPSON

LX™- 300 and LX™- 300+ Printer Connection, 3.3-1 Unpack the Printer, 3.3-1 Install the Knob, 3.3-1 Install the Ribbon Cartridge, 3.3-2 Connect the Printer, 3.3-3 Paper Feed Options, 3.3-4

Single Sheet Paper Feed Setup, 3.3-4 Loading Continuous Feed Paper Feed, 3.3-5

Configure the Printer, 3.3-7

LX300 Printer, 3.3-7 LX300+ Printer, 3.3-7

Complete the Instrument Installation, 3.3-9

PN 4237616B

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4 SERVICE AND REPAIR PROCEDURES, 4.1-1

4.1 GUIDELINES FOR SERVICING THE A ANALYZER, 4.1-1 General Guidelines, 4.1-1

Safety Precautions, 4.1-1 Accessibility, 4.1-1 Electronic Precautions, 4.1-1 Environment Protection, 4.1-1

Procedures, 4.1-1

Tools and Supplies, 4.1-2

Instrument Performance Verification, 4.1-2 Service Password, 4.1-2 User Mode, 4.1-2

How to Disable the Right Side Door Interlock, 4.1-2

How to Reactivate the Right Side Door Interlock, 4.1-3 Power Down / Power Up the Instrument, 4.1-3

Purpose, 4.1-3

Power Down, 4.1-3

Power Up, 4.1-4 Reset the Instrument, 4.1-4

System Reset Cycle, 4.1-4

Hardware Reset, 4.1-4

•T 5diff HEMATOLOGY

CONTENTS

4.2 OPENING OR REMOVING INSTRUMENT DOORS, PANELS, AND COVERS, 4.2-1 Purpose, 4.2-1 Tools/Supplies Needed, 4.2-1 Opening the Right Side Door, 4.2-1

Bypassing the Right Side Door interlock, 4.2-1

Removing the Left Side Panel, 4.2-2

Removal, 4.2-2 Opening the Main Card Door, 4.2-3 Installation, 4.2-3

Removing the Rear Access Panel, 4.2-3

Removal, 4.2-4 Installation, 4.2-4

Removing the Top Cover, 4.2-4

Removal, 4.2-4 Installation, 4.2-5

Removing the Front Cover, 4.2-5

Removal, 4.2-5 Installation, 4.2-7

4.3 PREPARATION TO SHIP THE INSTRUMENT, 4.3-1 Purpose, 4.3-1 Tools/Supplies Needed, 4.3-1 Bleach the Baths (20 minutes), 4.3-1 Clean the External Surfaces (20 minutes), 4.3-2 Clean the Tubing and Chambers (60 minutes), 4.3-2

PN 4237616B

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CONTENTS

Preparation, 4.3-2 Cycle Routine, 4.3-3 Drain and Rinse, 4.3-3

4.4 FLOW CELL CHECKS AND ADJUSTMENTS, 4.4-1 Purpose, 4.4-1 Tools/Supplies Needed, 4.4-1 Preparation, 4.4-1 Flow Cell Checks, 4.4-1 DIFF Lamp Voltage Adjustment, 4.4-3

Preparation, 4.4-3 Adjustment, 4.4-3 Interim Verification, 4.4-4

Resistive Channel Adjustment, 4.4-5

Preparation, 4.4-5 Adjustment, 4.4-6 Interim Verification, 4.4-6

Absorbance Channel Adjustment, 4.4-7

Preparation, 4.4-7 Adjustment, 4.4-7 Interim Verification, 4.4-8

Final Verification, 4.4-9

4.5 BATHS ASSEMBLY ALIGNMENT CHECK AND ADJUSTMENT, 4.5-1 Purpose, 4.5-1 Tools/Supplies Needed, 4.5-1 Preparation, 4.5-1 Alignment Check, 4.5-1 Alignment Adjustment, 4.5-3

Verification, 4.5-4

4.6 SAMPLE PROBE CHECKS AND ADJUSTMENTS, 4.6-1 Purpose, 4.6-1 Tools/Supplies Needed, 4.6-1 Sample Probe Checks, 4.6-2

Home Position Check, 4.6-2 Inside Bath Position Check, 4.6-3

Sample Probe Adjustments, 4.6-4

Home Position Adjustment, 4.6-4 Inside Bath Position Adjustment, 4.6-5

4.7 HGB BLANK ADJUSTMENT, 4.7-1 Purpose, 4.7-1 Tools/Supplies Needed, 4.7-1 Preparation, 4.7-1 Adjustment, 4.7-2 Verification, 4.7-2

4.8 APERTURE CURRENT CHECK, 4.8-1 Purpose, 4.8-1 Tools/Supplies Needed, 4.8-1

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Procedure, 4.8-1

4.9 RBC/PLT GAIN ADJUSTMENT, 4.9-1 Purpose, 4.9-1 Tools/Supplies Needed, 4.9-1 Procedure, 4.9-1

Preparation, 4.9-1 Adjustments, 4.9-2

4.10 WBC/BASO GAIN ADJUSTMENT, 4.10-1 Purpose, 4.10-1 Tools/Supplies Needed, 4.10-1 Procedure, 4.10-1

Preparation, 4.10-1 Adjustment, 4.10-2

4.11 DRAIN SENSOR ADJUSTMENT, 4.11-1 Purpose, 4.11-1 Tools/Supplies Needed, 4.11-1 Preparation, 4.11-1 Adjustment, 4.11-2 Wrap Up, 4.11-2

CONTENTS

4.12 TRANSFER SENSOR ADJUSTMENT, 4.12-1 Purpose, 4.12-1 Tools/Supplies Needed, 4.12-1 Preparation, 4.12-1 Adjustment, 4.12-2 Wrap Up, 4.12-2

4.13 MOTOR CURRENT ADJUSTMENT, 4.13-1 Purpose, 4.13-1 Tools/Supplies Needed, 4.13-1 Procedure, 4.13-1

4.14 THRESHOLD ADJUSTMENTS, 4.14-1 Purpose, 4.14-1 Tools/Supplies Needed, 4.14-1 Procedure, 4.14-1

4.15 REAGENT TEMPERATURE CHECK AND ADJUSTMENT, 4.15-1 Purpose, 4.15-1 Tools/Supplies Needed, 4.15-1 Reagent Temperature Check, 4.15-1 Reagent Temperature Adjustment, 4.15-3

Preparation, 4.15-3 Adjustment, 4.15-4

PN 4237616B

4.16 BATH ENCLOSURE TEMPERATURE CHECK AND ADJUSTMENT, 4.16-1 Purpose, 4.16-1 Tools/Supplies Needed, 4.16-1

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CONTENTS

Bath Enclosure Temperature Check, 4.16-1 Bath Enclosure Temperature Adjustment, 4.16-2

Preparation, 4.16-2 Adjustment, 4.16-3

4.17 VACUUM CHECKS AND ADJUSTMENTS, 4.17-1 Purpose, 4.17-1 Tools/Supplies Needed, 4.17-1 Waste Syringe Vacuum Check, 4.17-1 Count Syringe Vacuum Check and Adjustment, 4.17-2

Count Syringe Vacuum Check, 4.17-2 Count Syringe Vacuum Adjustment, 4.17-3

4.18 MIX BUBBLE ADJUSTMENT, 4.18-1 Purpose, 4.18-1 Tools/Supplies Required, 4.18-1 Procedure, 4.18-1

4.19 HEATER ASSEMBLY REPLACEMENT, 4.19-1 Purpose, 4.19-1 Tools/Supplies Needed, 4.19-1 Removal, 4.19-1 Installation, 4.19-5

4.20 POWER SUPPLY REPLACEMENT, 4.20-1 Purpose, 4.20-1 Tools/Supplies Needed, 4.20-1 Removal, 4.20-1 Installation, 4.20-3 Verification, 4.20-4

4.21 START SWITCH REPLACEMENT, 4.21-1 Purpose, 4.21-1 Tools/Supplies Needed, 4.21-1 Removal, 4.21-1 Installation, 4.21-3

4.22 OPTICAL BENCH DISASSEMBLY AND REPLACEMENT, 4.22-1 Purpose, 4.22-1 Tools/Supplies Needed, 4.22-1 Removal, 4.22-1 Installation, 4.22-3

4.23 BAR-CODE READER TESTING AND CONFIGURATION, 4.23-1 Purpose, 4.23-1 Read Test, 4.23-1 Default Settings, 4.23-2

Codabar - Start/Stop Equality Check/Output, 4.23-4 Interleaved 2-of-5 Options, 4.23-5

xii

4.24 REAGENT SYRINGES ASSEMBLY REPLACEMENTS, 4.24-1

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Purpose, 4.24-1 Tools/Supplies Needed, 4.24-1 Preparation, 4.24-1 Removal, 4.24-2 O-Ring, Washer, and Piston Replacement, 4.24-2 Installation, 4.24-5 Verification, 4.24-5

4.25 COUNT SYRINGE COMPONENT REPLACEMENTS, 4.25-1 Purpose, 4.25-1 Tools/Supplies Needed, 4.25-1 Preparation, 4.25-1 Removal, 4.25-2 O-ring, Washer, and Piston Replacement, 4.25-5

Piston Replacement, 4.25-5

O-ring and Washer Replacement Only, 4.25-6 Installation, 4.25-7 Verification, 4.25-8

CONTENTS

4.26 SAMPLE PROBE AND RINSE BLOCK ASSEMBLY COMPONENT REPLACEMENTS, 4.26-1 Purpose, 4.26-1 Tools/Supplies Needed, 4.26-1 Preparation, 4.26-1 Removal, 4.26-1 Sample Probe Replacement, 4.26-2 Rinse Block Assembly Component Replacement, 4.26-2 Installation, 4.26-4

Verification, 4.26-4

4.27 WASTE SYRINGE COMPONENT REPLACEMENTS, 4.27-1 Purpose, 4.27-1 Tools/Supplies Needed, 4.27-1 Preparation, 4.27-1 Removal, 4.27-2

Piston Replacement, 4.27-5

O-ring and Washer Replacement Only, 4.27-6 Installation, 4.27-7 Verification, 4.27-8

4.28 CLEANING THE BATH ENCLOSURE, 4.28-1 Purpose, 4.28-1 Tools/Supplies Needed, 4.28-1 Procedure, 4.28-1

PN 4237616B

4.29 5diff SYRINGE ASSEMBLY REPLACEMENTS, 4.29-1 Purpose, 4.29-1 Tools/Supplies Needed, 4.29-1 Preparation, 4.29-1 Removal, 4.29-1 O-ring Replacements, 4.29-3

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CONTENTS

Installation, 4.29-6 Verification, 4.29-6

4.30 FLOW CELL COAXIAL CABLE REPLACEMENT, 4.30-1 Purpose, 4.30-1 Tools/Supplies Needed, 4.30-1 Preparation, 4.30-1 Removal, 4.30-1 Replacement, 4.30-4 Verification, 4.30-5

4.31 OPTICAL BENCH LAMP REPLACEMENT, 4.31-1 Purpose, 4.31-1 Tools/Supplies Needed, 4.31-1 Preparation, 4.31-1 Removal, 4.31-3 Lamp Replacement, 4.31-4 Verification, 4.31-5

4.32 DILUENT RESERVOIR REPLACEMENTS, 4.32-1 Purpose, 4.32-1 Tools/Supplies Needed, 4.32-1 Preparation, 4.32-1 Removal, 4.32-1 O-ring and Washer Replacement, 4.32-2 Installation, 4.32-3 Verification, 4.32-3

4.33 SAMPLE SYRINGE ASSEMBLY REPLACEMENTS, 4.33-1 Purpose, 4.33-1 Tools/Supplies Needed, 4.33-1 Preparation, 4.33-1 Removal, 4.33-1 O-ring Replacements, 4.33-3 Installation, 4.33-4 Verification, 4.33-5

4.34 DRAINING BATH REPLACEMENTS, 4.34-1 Purpose, 4.34-1 Tools/Supplies Needed, 4.34-1 Preparation, 4.34-1 O-ring Replacements, 4.34-1 Verification, 4.34-2

xiv

4.35 O-RING REPLACEMENTS IN THE COUNTING BATHS (RBC and WBC/BASO Baths), 4.35-1 Purpose, 4.35-1 Tools/Supplies Needed, 4.35-1 Preparation, 4.35-1 Removal, 4.35-2 O-ring Replacements, 4.35-3

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Replacing the Coaxial Cable O-ring on the Bath Electrode, 4.35-3

Replacing the Aperture O-rings in the Counting Head, 4.35-5 Installation, 4.35-8 Align the Bath Assembly, 4.35-8 Verification, 4.35-9

4.36 OPTICAL BENCH PRELIMINARY ADJUSTMENTS, 4.36-1 Purpose, 4.36-1 Tools/Supplies Needed, 4.36-1 Preparation, 4.36-1

Verify the Flow Cell is Free of Bubbles, 4.36-2

Course Adjustments, 4.36-2

Y-Axis Adjustment, 4.36-2 X-Axis Adjustment, 4.36-3

Lamp Alignment, 4.36-5

4.37 FLOW CELL WBC BALANCE, 4.37-1 Purpose, 4.37-1 Tools/Supplies Needed, 4.37-1 Procedure, 4.37-1

CONTENTS

4.38 SETTING diff+/diff- THRESHOLDS, 4.38-1 Purpose, 4.38-1 Tools/Supplies Needed, 4.38-1 Procedure, 4.38-1

4.39 MAIN CARD REPLACEMENT AND SOFTWARE TRANSFER, 4.39-1 Purpose, 4.39-1 Tools/Supplies Needed, 4.39-1 Preliminary Setup, 4.39-1 Removing Main Card, 4.39-1 Transferring EPROMs to Replacement Card, 4.39-3 Installing Main Card, 4.39-4 Verifying System Configuration, 4.39-5

Patient Ranges, Action Ranges and Thresholds, 4.39-6

Verifying Main Card Settings, 4.39-7

5 MAINTENANCE PROCEDURES, 5.1-1

5.1 RECOMMENDED MAINTENANCE SCHEDULE, 5.1-1

5.2 MAINTENANCE WORKLIST, 5.2-1 Purpose, 5.2-1 Tools/Supplies Needed, 5.2-1

Additional Tools or Supplies Needed for 1-Year Maintenance, 5.2-1

Additional Tools or Supplies Needed for the Every 2-Years Maintenance, 5.2-1 User Mode, 5.2-1 Worklist Instructions, 5.2-2

Overview, 5.2-2

Maintenance Category Identifier, 5.2-2

Basic Instructions, 5.2-2

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CONTENTS

Preparation, 5.2-3 Reagent Syringes Assembly, 5.2-4

Replacement Parts, 5.2-4 Procedure, 5.2-4

5diff Syringe Assembly, 5.2-5

Replacement Parts, 5.2-5 Procedure, 5.2-5

Count Syringe Assembly, 5.2-5

Replacement Parts, 5.2-5

Procedure, 5.2-5 Interim Verification Check, 5.2-6 Replace the Optical Bench Lamp, 5.2-6

Replacement Part, 5.2-6

Procedure, 5.2-6 Replace the Flow Cell Coaxial Cable, 5.2-7

Replacement Part, 5.2-7

Procedure, 5.2-7 Preparation, 5.2-7 Sample Probe and Rinse Block Assembly, 5.2-8

Replacement Parts, 5.2-8

Procedure, 5.2-8 Sample Syringe Assembly, 5.2-8

Replacement Part, 5.2-8

Procedure, 5.2-8 Waste Syringe Assembly, 5.2-9

Replacement Parts, 5.2-9

Procedure, 5.2-9 Diluent Reservoir, 5.2-9

Replacement Parts, 5.2-9

Procedure, 5.2-9 Interim Verification Check, 5.2-10 Preparation, 5.2-10 Draining Baths, 5.2-11

Replacement Parts, 5.2-11

Procedure, 5.2-11 Counting Heads, 5.2-11

Replacement Parts, 5.2-11

Procedure, 5.2-11 Clean the Bath Enclosure, 5.2-11 Wrap Up, 5.2-12

xvi

5.3 SYSTEM VERIFICATION PROCEDURES, 5.3-1 Purpose, 5.3-1 Tools/Supplies Needed, 5.3-1 Preparation, 5.3-1 Startup, 5.3-1 Reproducibility, 5.3-1 Calibration, 5.3-2

Autocalibration, 5.3-2 Run Calibration, 5.3-2

PN 4237616B

Page 17

Calibration Passed, 5.3-2

Step-By-Step Cycle Check, 5.3-3

Preparation, 5.3-3 Instrument At Rest, 5.3-3 Sample Preparation (Making the Dilutions), 5.3-3 Count and Measurement of the WBC Group, 5.3-5 RBC/PLT Group Count, 5.3-6 Permanent Rinse Flow (PRF), 5.3-6 Filling of Diluent Reservoir, 5.3-6 Completing the Cycle, 5.3-6 Wrap Up, 5.3-6

6 SCHEMATICS, 6.1-1

6.1 ENGINEERING SCHEMATIC DRAWINGS, 6.1-1 Engineering Schematics, 6.1-1 Additional Pneumatic/Hydraulic Information, 6.1-1

Tubings and Connectors, 6.1-1 Pneumatic/Hydraulic Circuit Connections, 6.1-1

Additional Interconnect Information, 6.1-1

CONTENTS

6.2 PNEUMATIC / HYDRAULIC SCHEMATIC, 6.2-1 Layout, 6.2-1 Color Coding, 6.2-1 Tubing Designations, 6.2-1 Solenoid Valves, 6.2-1

6.3 PNEUMATIC/HYDRAULIC TUBINGS AND CONNECTIONS, 6.3-1 Tubings and Connectors List, 6.3-1 Pneumatic/Hydraulic Circuit Connections, 6.3-3

6.4 INTERCONNECT DIAGRAM, 6.4-1

6.5 MOTORS AND CABLES, 6.5-1

7 TROUBLESHOOTING, 7.1-1

7.1 ERROR MESSAGES, 7.1-1

7.2 CHECKING THE MOTORS, 7.2-1 Purpose, 7.2-1 Tools/Supplies Needed, 7.2-1 Preparation, 7.2-2

To Check All Motors, 7.2-2 To Only Check Motors in the Right Side Compartment, 7.2-2

To Only Check Motors in the Left Side Compartment, 7.2-2 Motors Check, 7.2-3 Wrap Up, 7.2-3

PN 4237616B

7.3 CHECKING THE VALVES, 7.3-1 Purpose, 7.3-1 Tools/Supplies Needed, 7.3-1

xvii

Page 18

CONTENTS

Preparation, 7.3-1

To Only Check Valves in the Left Side Compartment, 7.3-1

To Only Check Valves in the Right Side Compartment, 7.3-1 Valves Check, 7.3-2 Wrap Up, 7.3-3

8 PARTS LISTS, 8.1-1

8.1 MASTER PARTS LISTS, 8.1-1

8.2 ILLUSTRATED PARTS, 8.2-1

A QUICK REFERENCE INFORMATION, A.1-1

A.1 TOLERANCE AND LIMITS, A.1-1

A.2 CIRCUIT CARD LAYOUTS WITH KEY COMPONENT DESCRIPTIONS, A.2-1

Main Card, A.2-1

Component Locations, A.2-1

Tes t P oi nt s, A. 2- 2

Potentiometers, A.2-4

Jumper Settings, A.2-5 Optical Preamplifier Card, A.2-6

Component Locations, A.2-6

Connectors, A.2-6 LCD and Keypad Card, A.2-7

Component Locations, A.2-7 LED Card, A.2-8

Connectors, A.2-8 Motor Interconnect Card, A.2-9

Connectors, A.2-9 Traverse Card, A.2-10

Connectors, A.2-10

xviii

A.3 A

•T 5diff MODULE LOCATIONS AND FUNCTIONS, A.3-1 Overview, A.3-1 Analyzer Modules, A.3-1

Mechanical and Hydraulic Modules Locations, A.3-2 Rear Panel, A.3-4

A.4 SOFTWARE MENU TREE, A.4-1

B SOFTWARE INTERFACE, B.1-1

B.1 FORMAT, B.1-1

B.2 PACKET TYPE PIN ASSIGNMENTS, B.2-1

C FLAG SENSITIVITY AND THRESHOLDS, C.1-1

C.1 OVERVIEW, C.1-1

Flag Sensitivity, C.1-1

PN 4237616B

Page 19

Thresholds, C.1-2

Plt Threshold, C.1-2 WBC and BASO Thresholds, C.1-2 DiffPlot Thresholds, C.1-3 DiffPlot - Volume Thresholds, C.1-4 DiffPlot - Absorbance Thresholds, C.1-6 NL, NE, and MN Alarms, C.1-7 RBC Histogram, C.1-7

C.2 SETTING FLAG SENSITIVITY, C.2-1

Purpose, C.2-1 Tools/Supplies Needed, C.2-1 Procedure, C.2-1 Verification, C.2-1

C.3 SETTING THRESHOLDS, C.3-1

Purpose, C.3-1 Tools/Supplies Needed, C.3-1 Procedure, C.3-1 Verification, C.3-2

CONTENTS

ABBREVIATIONS, ABBREVIATIONS-1

INDEX, 1

TRADEMARKS, 1

PN 4237616B

xix

Page 20

CONTENTS

ILLUSTRATIONS

1.2-1 Warning and Information Label, 1.2-2

2.1-1 User Interfaces on the A

2.2-1 Coulter Principle, 2.2-2

2.2-2 Dual Focused Flow Process, 2.2-3

2.2-3 Signal Processing, 2.2-4

2.2-4 Basophil Thresholds, 2.2-5

2.2-5 Bath Assembly, 2.2-6

2.2-6 Sample Delivery Using Tangential Flow, 2.2-6

2.2-7 CBC/DIFF Mode Sample Partitions inside the Probe, 2.2-7

2.2-8 CBC Mode Sample Partitions inside the Probe, 2.2-7

2.3-1 Sample Probe and LED at Start of a Cycle, 2.3-1

2.3-2 Baths Assembly at Start of a Cycle, 2.3-1

2.3-3 Rinsing Probe Exterior After Aspiration, 2.3-2

2.3-4 Making the RBC/PLT First Dilution, 2.3-2

2.3-5 Making the WBC/BASO Dilution, 2.3-3

2.3-6 Making the DIFF Bath Dilution, 2.3-3

2.3-7 Double Rinse of the Sample Probe, 2.3-4

2.3-8 Aspirating from the First Dilution, 2.3-4

2.3-9 Rinsing the Outside of the Probe, 2.3-5

2.3-10 Making the RBC/Plt Dilution, 2.3-5

2.4-1 Bath Assembly, 2.4-1

2.4-2 Bath Assembly, 2.4-4

2.4-3 Flow Cell Operation, 2.4-5

2.4-4 DiffPlot Regions, 2.4-6

2.5-1 Typical RBC Histogram, 2.5-1

2.5-2 RBC1 and RBC2 Positions - RBC Histogram, 2.5-2

2.6-1 Typical Plt Histogram, 2.6-1

2.6-2 Area of the Plt Histogram Used to Determine the PDW Parameter Result, 2.6-2

2.6-3 Typical Platelet Distribution Curve, 2.6-3

2.6-4 Microcytic Interference with a Valley between 18 fL and 25 fL, 2.6-4

2.6-5 Microcytic Interference with a Valley below 18 fL, 2.6-4

2.6-6 Interference with no Distinct Valley, 2.6-5

2.7-1 Areas Used to Determine WBC and BASO Parameter Results, 2.7-1

2.7-2 DiffPlot Regions, 2.7-2

2.7-3 Volume Thresholds, 2.7-5

2.7-4 Absorbance Thresholds / NL, NE and MN Alarms, 2.7-6

2.8-1 Valve 1 through Valve 16 Locations, 2.8-1

2.8-2 Valve 17 and 18 Location, 2.8-1

2.8-3 Valve 20 to Valve 31 Locations, 2.8-2

2.8-4 Hgb Lyse Reagent Circuit, 2.8-5

2.8-5 Fix Reagent Circuit, 2.8-6

2.8-6 WBC Lyse Reagent Circuit, 2.8-7

2.8-7 Rinse Reagent Supply Circuit, 2.8-8

2.8-8 Probe Rinse Reagent Circuit, 2.8-9

2.8-9 WBC/BASO Rinse Reagent Circuit, 2.8-10

2.8-10 Diluent Reagent Circuit, 2.8-11

•T 5diff Hematology Analyzer, 2.1-2

PN 4237616B

Page 21

CONTENTS

2.8-11 Probe Diluent Reagent Circuit, 2.8-13

2.8-12 Bath Diluent Reagent Circuit, 2.8-14

2.8-13 Waste Circuit, 2.8-15

2.10-1 User Menu Tree, 2.10-2

2.10-2 Service Menu Tree, 2.10-3

3.2-1 Warning and Caution Label Locations on the Instrument, 3.2-2

3.2-2 Plastic Blocker Locations, View with Right Door Open, 3.2-2

3.2-3 Rear Panel Connections, 3.2-3

3.2-4 Loop-Side Velcro Strip Attachment, 3.2-4

3.2-5 Waste Alarm and Float Sensor Setup, 3.2-4

3.2-6 Position the Waste Alarm on the Rear Access Panel, 3.2-5

3.2-7 Reagent Bottle Locations, 3.2-6

3.2-8 Printer Configuration Menu, 3.2-9

3.3-1 Carefully Remove All Packing Materials, 3.3-1

3.3-2 Paper-Feed Knob Installation, 3.3-1

3.3-3 Insert the Ribbon Cartridge, 3.3-2

3.3-4 Threading the Ribbon, 3.3-2

3.3-5 Proper Ribbon Placement, 3.3-3

3.3-6 Cable Connection at the Instrument, 3.3-3

3.3-7 Cable Connections, 3.3-4

3.3-8 Paper Support for Printing Single Sheets of Paper, 3.3-5

3.3-9 Printer Sprockets, 3.3-5

3.3-10 Replace Paper Guide, 3.3-6

3.3-11 Printer Ready for Continuous Feed Printing, 3.3-6

3.3-12 LX300 Printer Control Panel, 3.3-7

3.3-13 LX300+ Printer Control Panel, 3.3-7

4.1-1 Location of the Power On/Off Rocker Switch, 4.1-3

4.2-1 Opening the Right Side Door, 4.2-1

4.2-2 Removing the Left Side Panel, 4.2-3

4.2-3 Rear Access Panel Screw Locations, 4.2-4

4.2-4 Top Cover - Side Screw Locations, 4.2-5

4.2-5 Reagent Compartment Screw Locations, 4.2-6

4.2-6 Torx Screw Location Inside Front Panel, 4.2-6

4.2-7 Torx Screw Location on Right Frame, 4.2-6

4.2-8 Front Panel Screw Locations, 4.2-7

4.2-9 Connector for the Keypad and LCD Card, 4.2-7

4.3-1 Rear Panel Connections, 4.3-3

4.4-1 Diff Adjustment Screen, 4.4-2

4.4-2 Potentiometer R11 - Location on the Optical Bench Assembly, 4.4-3

4.4-3 Potentiometer R11 Location - Top View from the Front of the Instrument, 4.4-3

4.4-4 Main Card Flow Cell Adjustments, 4.4-5

4.4-5 Front Adjustment Knob - Optical Bench, 4.4-7

4.4-6 Side Adjustment Screw - Optical Bench, 4.4-7

4.4-7 DiffPlot Regions, 4.4-9

4.5-1 Sample Probe Position at the Rinse Bath, 4.5-2

4.5-2 Close-up of Probe Position, 4.5-2

4.5-3 Sample Probe at the WBC/BASO Bath, 4.5-2

4.5-4 Close-up of Probe at the WBC/BASO Bath, 4.5-2

4.5-5 Location of Screws Securing the Baths Support Panel, 4.5-3

4.5-6 Sample Probe Position at the Rinse Bath, 4.5-3

PN 4237616B

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Page 22

CONTENTS

4.5-7 Close-up of Acceptable Probe Position, 4.5-3

4.5-8 Sample Probe Position, Right Side, 4.5-4

4.5-9 Close-up of Acceptable Probe Position, 4.5-4

4.6-1 Dilution Screen, 4.6-1

4.6-2 Acceptable Distance between the Sample Probe Tip and the Traverse, 4.6-2

4.6-3 Measuring the Distance between the Sample Probe Tip and the Traverse, 4.6-2

4.6-4 Correct View through the Port, 4.6-3

4.6-5 Incorrect View through the Port, 4.6-3

4.6-6 Properly Adjusted Sample Probe Tip, 4.6-4

4.6-7 Ideal Probe Position, 4.6-5

4.6-8 Improper Probe Tip Position - Too Forward, 4.6-5

4.6-9 Improper Probe Tip Position - Too Backward, 4.6-5

4.6-10 Improper Probe Tip Position - Too High, 4.6-6

4.6-11 Improper Probe Tip Position - Too Low, 4.6-6

4.7-1 Main Card Hgb Blank Adjustment, 4.7-1

4.8-1 LMNE CIS, GR (RBC), and GB (WBC) Coax Locations, 4.8-1

4.9-1 Main Card RBC/PLT Gain Adjustments, 4.9-1

4.10-1 Main Card WBC/BASO Gain Adjustment, 4.10-1

4.11-1 Main Card Drain Sensor Adjustment, 4.11-1

4.12-1 Main Card Transfer Sensor Adjustment, 4.12-1

4.13-1 Main Card Motor Current Adjustments, 4.13-2

4.14-1 Main Card Threshold Adjustments, 4.14-2

4.15-1 Thermometer Probe inside the DIFF Bath, 4.15-1

4.15-2 Main Card Heating Status LED Location, 4.15-2

4.15-3 Location of the Label Containing the Temperature Value for the Heater Assembly, 4.15-3

4.16-1 Temperature Sensor Location - View with the Right Side Door Open, 4.16-1

4.16-2 Location of the Label Containing the Temperature Value for the Temperature Sensor, 4.16-2

4.17-1 Attach the Vacuum Meter to the Waste Syringe, 4.17-1

4.17-2 Attach the Vacuum Meter to the Count Syringe, 4.17-2

4.19-1 Captive Hex Screw Locations - Reagent Syringes and 5diff Syringe, 4.19-1

4.19-2 CHC M3 x 6 Screw Locations, 4.19-2

4.19-3 Heater Assembly, 4.19-3

4.19-4 Heater Assembly - Tubing Port Locations, 4.19-3

4.19-5 Main Card Heater Assembly Replacement, 4.19-4

4.19-6 Heater Assembly After Removal, 4.19-5

4.19-7 Heater Assembly - Tubing Port Locations, 4.19-5

4.19-8 Heater Assembly - Orientation Inside the Instrument, 4.19-6

4.19-9 Heater Assembly - Port Locations, 4.19-6

4.19-10Baths Assembly Support Panel Nut Locations, 4.19-7

4.19-11Location of Openings in the Baths Assembly Support Panel, 4.19-7

4.19-12Heater Assembly Screw Locations, 4.19-8

4.20-1 Main Card - J2 Location, 4.20-1

4.20-2 Main Card - J37 Location, 4.20-2

4.20-3 Optical Module - Lamp Supply Cable Location, 4.20-2

4.20-4 Location of Screws Securing the Power Supply to the Rear Panel, 4.20-3

4.20-5 Screws Securing the Power Supply to the Instrument Frame, 4.20-3

4.20-6 Main Card Heater Assembly Replacement Adjustment, 4.20-4

xxii

PN 4237616B

Page 23

CONTENTS

4.21-1 Fan Removal - Right Side Compartment, 4.21-1

4.21-2 Start Switch Screw Locations - With Fan Removed, 4.21-2

4.21-3 Disconnected Start Switch - Front View with Front Panel Removed, 4.21-2

4.21-4 Start Switch Orientation, 4.21-3

4.22-1 5diff Syringe Port Locations, 4.22-1

4.22-2 Optical Bench - Ground Fitting Location, 4.22-2

4.22-3 Disconnection Sites for Named Components, 4.22-2

4.22-4 Captive Screw Locations - Optical Bench Assembly, 4.22-3

4.22-5 Connection Sites for Named Components, 4.22-4

4.22-6 Optical Bench - Ground Fitting Location, 4.22-4

4.22-7 5diff Syringe Port Locations, 4.22-5

4.24-1 Valve and Screw Locations - Left Side View, 4.24-2

4.24-2 O-rings and Washers - Reagent Syringes Assembly, 4.24-3

4.24-3 Bottom Plate Screw Locations and Tightening Patterns, 4.24-4

4.24-4 Valve and Screw Locations - Left Side View, 4.24-5

4.25-1 Rear Access Panel Screw Locations, 4.25-1

4.25-2 Motor Interconnect Card - Count Syringe Connector Locations, 4.25-2

4.25-3 Count Syringe - Ground Wire Location, 4.25-3

4.25-4 Count Syringe Housing - Tubing Locations, 4.25-3

4.25-5 Count Syringe - Captive Screw Locations, 4.25-4

4.25-6 Count Syringe - Piston, O-ring, and Washer Replacement, 4.25-5

4.25-7 Count Syringe - O-ring and Washer Replacement, 4.25-6

4.25-8 Count Syringe Housing - Tubing Locations, 4.25-8

4.26-1 Probe Rinse Block Screw Locations, 4.26-1

4.26-2 Lift the Sample Probe Lock-Lever, 4.26-2

4.26-3 Remove Probe and Rinse Block Assembly, 4.26-2

4.26-4 Reassemble the Rinse Block Assembly, 4.26-3

4.27-1 Rear Access Panel Screw Locations, 4.27-1

4.27-2 Motor Interconnect Card - Waste Syringe Connector Locations, 4.27-2

4.27-3 Waste Syringe - Tie Wrap and Ground Wire Location, 4.27-3

4.27-4 Waste Syringe - Captive Screw Locations, 4.27-4

4.27-5 Waste Syringe - Piston, O-ring, and Washer Replacement, 4.27-5

4.27-6 Waste Syringe - O-ring and Washer Replacement, 4.27-6

4.29-1 5diff Syringe Port Locations, 4.29-2

4.29-2 Valve and Tubing Locations - Left Side View, 4.29-2

4.29-3 Screw Locations - 5diff Syringe, 4.29-3

4.29-4 Bottom Plate - 5diff Syringe, 4.29-3

4.29-5 Bottom Plate and Attachments - Housing Removed, 4.29-4

4.29-6 Illustrated Parts - 5diff Syringe, 4.29-5

4.30-1 Optical Bench Cover Screw Locations, 4.30-1

4.30-2 T-Connector Location, 4.30-2

4.30-3 Disconnecting the Isolator Chamber from the T-connector, 4.30-2

4.30-4 Coaxial Cable Connector - Top View, 4.30-3

4.30-5 Flow Cell Screw Locations - Top View, 4.30-3

4.30-6 Ground Screw Removal, 4.30-4

4.30-7 Connector Location - Top View, 4.30-5

4.31-1 Optical Bench Lamp Assembly, 4.31-2

4.31-2 Power Connector Location - Top View, 4.31-3

4.31-3 Screw Locations - Lamp Housing, 4.31-3

4.31-4 Winged Metal Bracket - Top View, 4.31-4

PN 4237616B

xxiii

Page 24

CONTENTS

4.32-1 Diluent Reservoir Screw Locations, 4.32-1

4.32-2 Diluent Reservoir O-ring and Washer Positioning, 4.32-2

4.33-1 Disconnect Tubing at Valve 18, Port 2, 4.33-1

4.33-2 Screw Locations - Sample Syringe Housing, 4.33-2

4.33-3 Sample Syringe Output Port, 4.33-2

4.33-4 Screw Locations on the Bottom of the Sample Syringe Housing, 4.33-3

4.33-5 Illustrated Parts - Sample Syringe, 4.33-4

4.34-1 Removing a Draining Bath, 4.34-1

4.34-2 Draining Bath O-ring Placement, 4.34-2

4.34-3 Draining Bath O-Ring Locations, 4.34-2

4.35-1 Sample Probe Position at the Rinse Bath, 4.35-2

4.35-2 Close-up of Probe Positioning, 4.35-2

4.35-3 Baths Assembly Screw Locations, 4.35-3

4.35-4 Electrode Screw Locations, 4.35-3

4.35-5 Removing the Coaxial Cable O-ring from the Bath Electrode, 4.35-4

4.35-6 Making a Protective Cover from a Micropipette Tip, 4.35-4

4.35-7 Bath Electrode Locations, 4.35-5

4.35-8 Location of the Aperture and Its Two O-Rings, 4.35-6

4.35-9 Sample Probe Position at the Rinse Bath, 4.35-8

4.35-10Close-up of Acceptable Probe Position, 4.35-8

4.35-11Sample Probe Position, Right Side, 4.35-9

4.35-12Close-up of Acceptable Probe Position, 4.35-9

4.36-1 Optical Bench Cover Screw Locations, 4.36-1

4.36-2 Checking the Lens to Flow Cell Gap, 4.36-2

4.36-3 Front Knob for Y-Axis Adjustment, 4.36-3

4.36-4 Side Screw - X-Axis Adjustment, 4.36-3

4.36-5 How to Position the White Paper, 4.36-4

4.36-6 Ideal Lamp Filament Projection Image, 4.36-4

4.36-7 Side Screw - X-Axis Adjustment, 4.36-5

4.36-8 Lamp Adjustment Screw, 4.36-5

4.36-9 Ideal Lamp Filament Projection Image, 4.36-6

4.36-10Side Screw - X-Axis Adjustment, 4.36-6

4.37-1 WBC/Flow Cell Balance Screen, 4.37-1

4.38-1 diff Flag Sensitivity Screen, 4.38-1

4.39-1 Main Card - Connections and EPROMs, 4.39-2

4.39-2 Main Card - Potentiometers /Test Points, 4.39-9

6.5-1 Horizontal Traverse Motor (PN - XBA391A), 6.5-1

6.5-2 Upper Fan (PN - XBA393A), 6.5-2

6.5-3 Horizontal Traverse Sensor (IR Sensor, PN - XBA394AS), 6.5-3

6.5-4 Bath Drain and DIFF Transfer Sensor (IR Sensor, PN - XBA395AS), 6.5-4

6.5-5 Vertical Traverse Sensor (IR Sensor, PN - XBA396AS), 6.5-5

6.5-6 IR Sensor (PN - XBA397AS), 6.5-6

6.5-7 RBC/WBC Coaxial Cable (PN - XBA398A), 6.5-7

6.5-8 DIFF Flow Cell Coaxial Cable (PN - XBA399AS), 6.5-8

6.5-9 Bar-Code Reader Cable (PN - XBA402AS), 6.5-9

6.5-10 Diluent Level Sensor (PN - XDA605AS), 6.5-10

7.3-1 Valve 1 through 16 Locations, 7.3-2

7.3-2 Valves 17 and 18 Location, 7.3-2

7.3-3 Valves 20 through 31 Locations, 7.3-2

xxiv

PN 4237616B

Page 25

CONTENTS

8.2-1 11-Valves Assembly (See Table 8.2-1), 8.2-1

8.2-2 5-Valves Assembly (See Table 8.2-2), 8.2-2

8.2-3 2-Valves Assembly (See Table 8.2-3), 8.2-3

8.2-4 7-Valves Assembly (See Table 8.2-4), 8.2-4

8.2-5 5-Valves Assembly (See Table 8.2-5), 8.2-5

8.2-6 Right Side Compartment, Lower Rear Area (See Table 8.2-6), 8.2-6

8.2-7 Diluent Reservoir Assembly (See Table 8.2-7), 8.2-7

8.2-8 Count Syringe and Motor Assembly (See Table 8.2-8), 8.2-8

8.2-9 Count Syringe Assembly (See Table 8.2-9), 8.2-9

8.2-10 Count Syringe Motor Assembly (See Table 8.2-10), 8.2-10

8.2-11 Count Syringe Piston Assembly (See Table 8.2-11), 8.2-11

8.2-12 Reagent Syringes and Motor Assembly (See Table 8.2-12), 8.2-12

8.2-13 Reagent Syringes Assembly (See Table 8.2-13), 8.2-13

8.2-14 5diff Syringe and Motor Assembly (See Table 8.2-14), 8.2-14

8.2-15 5diff Syringe Assembly (See Table 8.2-15), 8.2-15

8.2-16 Waste Syringe and Motor Assembly (See Table 8.2-16), 8.2-16

8.2-17 Waste Syringe Assembly (See Table 8.2-17), 8.2-17

8.2-18 Syringe Motor (See Table 8.2-18), 8.2-18

8.2-19 Sample Motor (See Table 8.2-19), 8.2-19

8.2-20 Sample Syringe and Motor Assembly (See Table 8.2-20), 8.2-20

8.2-21 Sample Assembly Syringe (See Table 8.2-21), 8.2-21

8.2-22 Sample Syringe Motor Assembly (See Table 8.2-22), 8.2-22

8.2-23 Syringe Motor Housing Assembly (See Table 8.2-23), 8.2-23

8.2-24 Syringe Motor Guide Block Assembly (See Table 8.2-24), 8.2-24

8.2-25 Sample Probe Retainer and Guide Assembly (See Table 8.2-25), 8.2-25

8.2-26 Sample Probe Retainer (See Table 8.2-26), 8.2-26

8.2-27 Rinse Block Assembly (See Table 8.2-27), 8.2-27

8.2-28 Sample Probe (See Table 8.2-28), 8.2-28

8.2-29 Traverse Vertical Movement Components - Belt Retainer (See Table 8.2-29), 8.2-29

8.2-30 Vertical Traverse Vertical Movement Components - Belt (See Table 8.2-30), 8.2-30

8.2-31 Traverse Vertical Movement Components - Motor and Pulley (See Table 8.2-31), 8.2-31

8.2-32 Traverse Vertical Movement Components - Motor (See Table 8.2-32), 8.2-32

8.2-33 Traverse Vertical Movement Components - Home Sensor (See Table 8.2-33), 8.2-33

8.2-34 Traverse Horizontal Movement Components - Motor (See Table 8.2-34), 8.2-34

8.2-35 Traverse Horizontal Movement Components - Belt (See Table 8.2-35)t, 8.2-35

8.2-36 Traverse Horizontal Movement Components - Free Wheel and Home Sensor (See Table 8.2-36), 8.2-36

8.2-37 Optical Bench Assembly (See Table 8.2-37), 8.2-37

8.2-38 Optical Bench Lamp (See Table 8.2-38), 8.2-38

8.2-39 DIFF Flow Cell Assembly (See Table 8.2-39), 8.2-39

8.2-40 Optics Preamplifier (See Table 8.2-40), 8.2-40

8.2-41 LED Card (See Table 8.2-41), 8.2-41

8.2-42 Bath Enclosure Compartment (See Table 8.2-42), 8.2-42

8.2-43 Bath Enclosure Fan Assembly (See Table 8.2-43), 8.2-43

8.2-44 Bath Enclosure Door Interlock (See Table 8.2-44), 8.2-44

8.2-45 Reagent Heating Coil Assembly (See Table 8.2-45), 8.2-45

PN 4237616B

xxv

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CONTENTS

8.2-46 Baths Assembly (See Table 8.2-46), 8.2-46

8.2-47 Hgb Photometer Assembly (See Table 8.2-47), 8.2-47

8.2-48 WBC/BASO Bath Assembly (See Table 8.2-48), 8.2-48

8.2-49 Rear Frame Assembly (See Table 8.2-49), 8.2-49 A.2-1 Main Card Components, A.2-1 A.2-2 Main Card Jumper Settings, A.2-5 A.2-3 Optical Preamplifier Card Components, A.2-6 A.2-4 Keypad and LCD Card Components, A.2-7 A.2-5 LED Card Components, A.2-8 A.2-6 Motor Interconnect Card Components, A.2-9 A.2-7 Traverse Card Components, A.2-10 A.3-1 View of an AC•T 5diff Hematology Analyzer with the Right Side Door

Open, A.3-2

A.3-2 View of an A

•T 5diff Hematology Analyzer with the Left Side Panel

Removed, A.3-2

A.3-3 Rear Panel - A

•T 5diff Hematology Analyzer, A.3-4 A.4-1 Software Menu Tree , A.4-1 C.1-1 PLT Threshold, C.1-2 C.1-2 WBC and BASO Threshold, C.1-2 C.1-3 DiffPlot, C.1-3 C.1-4 DiffPlot - Volume Thresholds (Y-axis), C.1-4 C.1-5 DiffPlot - Absorbance Thresholds (X-Axis), C.1-6 C.2-1 Flags Sensitivity Screen, C.2-1 C.3-1 Thresholds Screen, C.3-1

xxvi

PN 4237616B

Page 27

TABLES

2.1-1 AC•T 5diff Hematology Analyzer Reagent Consumption, Software Version

1.03, 2.1-4

2.2-1 A

2.4-1 Technical Characteristics for Obtaining RBC and Platelet Counts, 2.4-2

2.4-2 Technical Characteristics for the Measurement of the Hemoglobin, 2.4-3

2.4-3 Characteristics Required to Obtain WBC and BASO Results, 2.4-4

2.4-4 Technical Characteristics for Acquisition of the DiffPlot, 2.4-6

2.4-5 Summary of Dilutions, 2.4-7

2.8-1 Valves and their Functions, 2.8-3

3.1-1 Space Requirements, 3.1-1

3.2-1 Whole-Blood Reproducibility CV Limits for 20 Cycles, 3.2-10

3.2-2 Calibration Factors - Acceptable Range, 3.2-11

3.3-1 LX300+ Printer Controls and Indicators, 3.3-7

3.3-2 LX300+ Printer Default Settings, 3.3-8

4.13-1 Motor Voltage Limits, 4.13-1

4.14-1 Threshold Voltage Limits, 4.14-1

4.18-1 Mixing Bubble Limits, 4.18-1

4.20-1 Power Supply Voltages, 4.20-4

4.23-1 Test Labels With Check Digit (Checksum), 4.23-1

4.23-2 Test Labels Without Check Digit, 4.23-2

4.23-3 Bar-code Labels for Default Configuration, 4.23-3

4.39-1 Main Card - Plug/Jack Connections, 4.39-3

4.39-2 A

4.39-3 AcT 5diff - Main Card Settings, 4.39-7

4.39-4 Whole-Blood Reproducibility CV Limits for 20 Cycles, 4.39-8

5.1-1 Maintenance Schedule, 5.1-1

6.3-1 Instrument Tubing and Connectors , 6.3-1

6.3-2 Circuit Connections , 6.3-3

7.1-1 Error Messages, 7.1-1

8.1-1 Part Categories, 8.1-1

8.1-2 Return Parts, 8.1-1

8.1-3 Nonreturn Parts, 8.1-2

8.1-4 Peripherals, Accessories and Consumables, 8.1-6

8.1-5 Tools, 8.1-7

8.1-6 Fitting Kit Parts PN - XEA311AS, 8.1-8

8.1-7 Screws Kit Parts PN - XEA293AS, 8.1-8

8.1-8 Installation Kit, PN - XEA484AS, 8.1-10

8.1-9 Waste Alarm Kit, PN - 6912680, 8.1-11

8.1-10 6 Month Maintenance Kit, PN - XEA485AS, 8.1-11

8.1-11 1 Year Maintenance Kit, PN - XEA486AS, 8.1-11

8.1-12 Every 2 Years Maintenance Kit, PN - XEA581AS, 8.1-11

8.1-13 100 mN-m Torque Driver Kit, - PN 6915456, 8.1-12

8.1-14 400 mN-m Torque Driver Kit, - PN 6915457, 8.1-12

8.1-15 Assorted Tools Kit, - PN 6915458, 8.1-12

8.2-1 11-Valves Assembly (See Figure 8.2-1), 8.2-1

8.2-2 5-Valves Assembly (See Figure 8.2-2), 8.2-2

8.2-3 2-Valves Assembly (See Figure 8.2-3), 8.2-3

8.2-4 7-Valves Assembly (See Figure 8.2-4), 8.2-4

•T 5diff Analyzer Measurement Technologies, 2.2-1

•T 5diff Menu Paths - System Settings, 4.39-5

CONTENTS

PN 4237616B

xxvii

Page 28

CONTENTS

8.2-5 5-Valves Assembly (See Figure 8.2-5), 8.2-5

8.2-6 Right Side Compartment, Lower Rear Area (See Figure 8.2-6), 8.2-6

8.2-7 Diluent Reservoir Assembly (See Figure 8.2-7), 8.2-7

8.2-8 Count Syringe and Motor Assembly (See Figure 8.2-8), 8.2-8

8.2-9 Count Syringe Assembly (See Figure 8.2-9), 8.2-9

8.2-10 Count Syringe Motor Assembly (See Figure 8.2-10), 8.2-10

8.2-11 Count Syringe Piston Assembly (See Figure 8.2-11), 8.2-11

8.2-12 Reagent Syringes and Motor Assembly (See Figure 8.2-12), 8.2-12

8.2-13 Reagent Syringes Assembly (See Figure 8.2-13), 8.2-13

8.2-14 5diff Syringe and Motor Assembly (See Figure 8.2-14), 8.2-14

8.2-15 5diff Syringe Assembly (See Figure 8.2-15), 8.2-15

8.2-16 Waste Syringe and Motor Assembly (See Figure 8.2-16), 8.2-16

8.2-17 Waste Syringe Assembly (See Figure 8.2-17), 8.2-17

8.2-18 Syringe Motor (See Figure 8.2-18), 8.2-18

8.2-19 Sample Motor (See Figure 8.2-19), 8.2-19

8.2-20 Sample Syringe and Motor Assembly (See Figure 8.2-20), 8.2-20

8.2-21 Sample Assembly Syringe (See Figure 8.2-21), 8.2-21

8.2-22 Sample Syringe Motor Assembly (See Figure 8.2-22), 8.2-22

8.2-23 Syringe Motor Housing Assembly (See Figure 8.2-23), 8.2-23

8.2-24 Syringe Motor Guide Block Assembly (See Figure 8.2-24), 8.2-24

8.2-25 Sample Probe Retainer and Guide Assembly (See Figure 8.2-25), 8.2-25

8.2-26 Sample Probe Retainer (See Figure 8.2-26), 8.2-26

8.2-27 Rinse Block Assembly (See Figure 8.2-27), 8.2-27

8.2-28 Sample Probe (See Figure 8.2-28), 8.2-28

8.2-29 Traverse Vertical Movement Components - Belt Retainer (See

Figure 8.2-29), 8.2-29

8.2-30 Vertical Traverse Vertical Movement Components - Belt (See

Figure 8.2-30), 8.2-30

8.2-31 Traverse Vertical Movement Components - Motor and Pulley (See

Figure 8.2-31), 8.2-31

8.2-32 Traverse Vertical Movement Components - Motor (See Figure 8.2-32), 8.2-32

8.2-33 Traverse Vertical Movement Components - Home Sensor (See

Figure 8.2-33), 8.2-33

8.2-34 Traverse Horizontal Movement Components - Motor (See Figure 8.2-34), 8.2-34

8.2-35 Traverse Horizontal Movement Components - Belt (See Figure 8.2-35), 8.2-35

8.2-36 Traverse Horizontal Movement Components - Free Wheel and Home Sensor

(See Figure 8.2-36), 8.2-36

8.2-37 Optical Bench Assembly (See Figure 8.2-37), 8.2-37

8.2-38 Optical Bench Lamp (See Figure 8.2-38), 8.2-38

8.2-39 DIFF Flow Cell Assembly (See Figure 8.2-39), 8.2-39

8.2-40 Optics Preamplifier (See Figure 8.2-40), 8.2-40

8.2-41 LED Card (See Figure 8.2-41), 8.2-41

8.2-42 Bath Enclosure Compartment (See Figure 8.2-42), 8.2-42

8.2-43 Bath Enclosure Fan Assembly (See Figure 8.2-43), 8.2-43

8.2-44 Bath Enclosure Door Interlock (See Figure 8.2-44), 8.2-44

8.2-45 Reagent Heating Coil Assembly (See Figure 8.2-45), 8.2-45

8.2-46 Baths Assembly (See Figure 8.2-46), 8.2-46

8.2-47 Hgb Photometer Assembly (See Figure 8.2-47), 8.2-47

8.2-48 WBC/BASO Bath Assembly (See Figure 8.2-48), 8.2-48

xxviii

PN 4237616B

Page 29

8.2-49 Rear Frame Assembly (Figure 8.2-49), 8.2-49 A.1-1 Flow Cell Adjustment Limits, A.1-1 A.1-2 Motor Voltage Limits, A.1-1 A.1-3 Thresholds Voltage Limits, A.1-1 A.1-4 Mixing Bubble Limits, A.1-1 A.1-5 Power Supply Voltages, A.1-2 A.1-6 Whole-Blood Reproducibility CV Limits for 20 Cycles, A.1-2 A.1-7 Calibration Factor Limits, A.1-2 A.2-1 Main Card Test Points , A.2-2 A.2-2 Main Card Potentiometers, A.2-4 A.2-3 Connectors on the Optical Preamplifier Card, A.2-6 A.2-4 Connectors on the Keypad and LCD Card, A.2-7 A.2-5 Connector on the LED Card, A.2-8 A.2-6 Connectors on the Motor Interconnect Card, A.2-9 A.2-7 Connectors on the Traverse Card, A.2-10 A.3-1 Mechanical and Hydraulic Modules, A.3-3 C.1-1 Flag Sensitivity Default Values, C.1-1 C.1-2 WBC/BASO Factory-Set Threshold Values, C.1-3 C.1-3 DiffPlot - Volume Thresholds (Y-Axis), C.1-5 C.1-4 DiffPlot - Absorbance Thresholds (X-Axis), C.1-6 C.1-5 DiffPlot - FNL, FNE, and FMN Thresholds, C.1-7

CONTENTS

PN 4237616B

xxix

Page 30

CONTENTS

xxx

PN 4237616B

Page 31

1 INTRODUCTION, 1.1-1

1.1 MANUAL DESCRIPTION, 1.1-1 Scope, 1.1-1

Notification of Updates, 1.1-1 Intended Audience, 1.1-1 Organization, 1.1-1 Numbering Format, 1.1-2 Special Headings, 1.1-3

WARNING, 1.1-3

CAUTION, 1.1-3

IMPORTANT, 1.1-3

ATTENTION, 1.1-3

Note, 1.1-3 Conventions, 1.1-3 Graphics, 1.1-4

1.2 SAFETY PRECAUTIONS, 1.2-1 Electronic, 1.2-1 Biological, 1.2-1 Troubleshooting, 1.2-1

ILLUSTRATIONS

1.2-1 Warning and Information Label, 1.2-2

PN 4237616B

1-i

Page 32

CONTENTS

1-ii

PN 4237616B

Page 33

1.1 MANUAL DESCRIPTION

Scope

This manual provides the reference information and procedures needed for servicing and maintaining the BECKMAN COULTER™ A

referred to as the A online and in hard copy. The online manual is released on the Service Resource Kit CD-ROM, PN 6417471.

This manual is to be used in conjunction with the following customer documents and does not contain information and procedures already covered in these documents:

Document Language Part Number

•T 5diff hematology analyzer or the instrument). It is available both

INTRODUCTION

•T™ 5diff hematology analyzer (hereafter

Operator’s Guide English

French Italian German Spanish Chinese

Host Transmission Specification English 4277065

Notification of Updates

Any service memo that affects the information in this manual will include either minor revision change pages or a Notice of Information Update form for this manual. A Notice of Information Update form will summarize the changes and will list the specific headings, figures, and tables affected.

4237615 4237630 4237631 4237632 4237633 4237634

Intended Audience

To use this manual effectively, you need the following:

r An operator’s knowledge of the A r A thorough understanding of -

t Basic electronic and pneumatic principles and devices t Reagent systems

•T 5diff hematology analyzer

PN 4237616B

t Quality control t Troubleshooting concepts

r The ability to -

t Use basic mechanical tools and understand related terminology t Use a digital voltmeter (DVM) t Read pneumatic/hydraulic schematics and understand related terminology t Read electronic schematics and understand related terminology

Organization

The material in this manual is organized into eight chapters and two appendices. To make it easier to access the information:

1.1-1

Page 34

INTRODUCTION

MANUAL DESCRIPTION

r In the online manual, each page has a Contents button linked to a master table of

contents and an Index button linked to an alphabetic index.

r In the printed manual, there is a master table of contents at the beginning of the manual,

a chapter-specific table of contents at the beginning of each chapter, and an alphabetic index at the end of the manual.

The chapters / appendices contain:

Chapter 1, INTRODUCTION - A brief description of this manual and essential safety information.

Chapter 2, INSTRUMENT DESCRIPTION - An introduction to the A analyzer and a description of how it functions.

Chapter 3, INSTALLATION PROCEDURES - Installation and verification procedures.

Chapter 4, SERVICE AND REPAIR PROCEDURES - The procedures for servicing/repairing

the A

•T 5diff hematology analyzer.

•T5 diff hematology

Chapter 5, MAINTENANCE PROCEDURES - The procedures for maintaining the

•T 5diff hematology analyzer.

Chapter 6, SCHEMATICS - The schematic diagrams and tubing lists.

Chapter 7, TROUBLESHOOTING - An error message table.

Chapter 8, PARTS LISTS - The master parts list followed by the illustrated parts list.

Appendix A, QUICK REFERENCE INFORMATION - Quick reference information:

tolerances and limits; connectors, test points and jumpers for the circuit cards; the software menu trees; location diagrams and summarized functions for main analyzer components.

Appendix B, SOFTWARE INTERFACE - Tables of fatal and non-fatal error messages.

Appendix C, FLAG SENSITIVITY AND THRESHOLDS - An overview of the theory including default values; also includes the setup procedures.

ABBREVIATIONS - A list of abbreviations, acronyms, and reference designators used in this manual.

Numbering Format

Each chapter of this manual is further divided into topics that are numbered sequentially, beginning at one. The numbering format for the topic heading, which is called the primary heading, is chapter number, decimal point, topic number. For example, the primary heading number for the third topic covered in Chapter 2 is 2.3.

1.1-2

The page, figure, and table numbers are tied directly to the primary heading number. For example, Heading 2.3 begins on page 2.3-1, the first figure under Heading 2.3 is Figure 2.3-1 and the first table under Heading 2.3 is Table 2.3-1.

PN 4237616B

Page 35

INTRODUCTION

MANUAL DESCRIPTION

Primary headings always begin at the top of a right-hand page.

Note:

Special Headings

Throughout this manual, WARNING, CAUTION, IMPORTANT, ATTENTION, and Note headings are used to indicate potentially hazardous situations and important or helpful information.

WARNING

A WARNING indicates a situation or procedure that, if ignored, can cause serious personal injury. The word WARNING is in bold-faced text in the printed manual and is red in the online manual.

CAUTION

A CAUTION indicates a situation or procedure that, if ignored, can cause damage to the instrument.The word CAUTION is in bold-faced text in the printed manual and is red in the online manual.

IMPORTANT

An IMPORTANT indicates a situation or procedure that, if ignored, can result in erroneous test results.The word IMPORTANT is in bold-faced text in the printed manual and is red in the online manual.

ATTENTION

An ATTENTION contains information that is critical for the successful completion of a procedure and/or operation of the instrument.The word ATTENTION is in bold-faced text in the printed manual and is red in the online manual.

Note

A Note contains information that is important to remember or helpful in performing a procedure.

Conventions

This manual uses the following conventions.

r Instrument or analyzer refers to the A r Main card refers to the motherboard in the instrument. r Main Menu refers to the initial menu displayed on the instrument after Startup. r Each menu option consists of an item number followed by bold, uppercase text. For

example,

Note:

number next to the menu item indicates the numeric pushbutton on the front of the analyzer that can be pressed to select the menu option.

r Keys on the analyzer keypad are in bold, uppercase letters. For example, press

indicates the operator should press the

3. REAGENTS

Both the menu item number and text are displayed on the LCD screen. The item

is the third option on the Main Menu.

•T 5diff hematology analyzer.

pushbutton on the instrument keypad.

ENTER

PN 4237616B

1.1-3

Page 36

INTRODUCTION

MANUAL DESCRIPTION

r To select a menu item,

t Use the arrow keys to highlight the desired menu item then press the

t Simply press the numeric pushbutton (on the front of the analyzer) that correlates

ENTER

pushbutton on the front of the analyzer to select the highlighted option. or

with the desired option. This is the faster way to select a menu item.

For example, to select the

3. REAGENTS

t Use the down arrow to highlight the 3. REAGENTS

menu item from the Main Menu, you may:

option and then press the

ENTER

pushbutton on the front of the analyzer. or

t Press the pushbutton labeled

on the front of the analyzer.

r Select menu item tt sub-menu item indicates the software options you have to select, as

well as the order in which you should select them. For example, to prime the diluent reagent:

From the Main Menu, select

3. REAGENTS

3. PRIME

1. DILUENT

r Italics us used to indicate screen messages. For example:

The message CYCLE IN PROGRESS. PLEASE WAIT . . . appears on the screen.

r A r A r A r A r A

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

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

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

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

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

r In the electronic version of the manual:

t Links to additional information are in blue and are underlined. To access the linked

information, select the blue underlined text.

1.1-4

t The material is divided into many small sections (electronic files) to enhance the

loading and accessibility features.

t Every primary heading is a separate file and whenever possible the amount of

material contained within one primary heading is limited to four to ten pages.

t If a primary heading must be large, such as an illustrated parts list (IPL), invisible

breaks are added to the electronic file to further divide it. Note: Unless you are scrolling, these divisions are invisible. If you choose to scroll

through the IPL, you will encounter stop points. When you scroll to the end of a section and encounter a stop point, use the navigation bar to access the next section.

t To move from one section (electronic file) to the next in the HTML version of the

manual, use the right and left arrows on the navigation bars displayed at the top and bottom of each section.

Graphics

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

PN 4237616B

Page 37

1.2 SAFETY PRECAUTIONS

Electronic

INTRODUCTION

SAFETY PRECAUTIONS

WARNING

attached to power can cause personal injury from electric shock. Power down completely before removing covers to access electronic components.

WARNING

service on the instrument, rings and other metal jewelry can become caught in the instrument. To avoid personal injury or damage to the instrument, remove rings and other metal jewelry before performing maintenance or service on the electronic components of the instrument.

CAUTION

circuit cards and components, the instrument could be damaged. To prevent damage to electronic components, always be sure power is OFF before removing or replacing printed circuit cards and components.

CAUTION

circuit cards and other electronic components. If there is a possibility of ESD damage with a procedure, then perform that procedure at an ESD workstation, or wear an antistatic wrist strap attached to a metal part of the chassis connected to an earth ground.

Risk of personal injury. Contacting exposed electronic components while the instrument is

Risk of personal injury or damage to electronic components. While performing maintenance or

Risk of damage to electronic components. If the power is ON while removing or replacing printed

Risk of damage to electronic components. Electrostatic discharge (ESD) can damage add-in

Biological

WARNING

the instrument with the doors open, you may become injured or contaminated. To prevent possible injury or biological contamination, you must wear appropriate safety glasses, a lab coat, and gloves when servicing the instrument with the doors open.

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

PN 4237616B

Use care when working with pathogenic materials. Means must be available to decontaminate the instrument, provide ventilation, and to dispose of waste liquid. 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

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

Troubleshooting

Bring the following Warning to the customer’s attention before advising that customer to perform any service, maintenance or troubleshooting procedures on the A hematology analyzer. Also, make sure customers are aware of the Warning and information labels shown in Figure 1.2-1.

•T 5diff

1.2-1

Page 38

INTRODUCTION

SAFETY PRECAUTIONS

WARNING

Risk of personal injury or contamination. If you do not properly shield yourself while performing service, maintenance, and troubleshooting procedures, residual fluids in the instrument could injure or contaminate you. Beckman Coulter recommends that you wear barrier protection, such as appropriate safety glasses, a lab coat, and gloves throughout the performance of service, maintenance, and troubleshooting procedures to avoid contact with cleaners and residual fluids in the instrument.

Figure 1.2-1 Warning and Information Label

MOD

T 5diff

MOD NO.

ASSY

xxxxxx

NO.

50/60100-240

VOLTS

HZ AMPS

MANUFACTURED BY COULTER CORPORATION A BECKMAN COULTER COMPANY

ECKMAN

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

OULTER

PATTENTS ISSUED AND/OR PENDING

AUTOMATED DIFFERENTIAL CELL COUNTER

FOR IN V IT R O D IAG N OS T IC U S 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 T O S E R VICING . 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.

S/N

xxxxxx

WATTS

•

NO.

ASSY NO.

VO LTS

xxxxxx

ECKMAN

OULTER

50-60100-240

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

S/N

xxxxxx

0.9-2.0 200

WATTS

AUTOMATED DIFFERENTIAL CELL COUNTER

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

CAUTION:

TO REDUCE THE RISK OF ELECTRICAL SHOCK DO NOT REMO VE THE CO VER OR BACK. REFER SERVICING TO QUALIFIED SERVICE PERSONNEL. ELECTRIC SHOCK HAZARD. DISCONNECT UNIT FROM PO W ER SOURCE PRIOR TO SERVIC IN G. F O R C O N T IN U E D P R O T E C T IO N A G A IN S T R F IR E H A Z A R D , R E P L A C E O N L Y W ITH SAM E TYPE AN D RATIN G OF FUSE. F O R S A F E T Y R E A S O N S , E Q U IP M E N T R E Q U IR E S C O N N E C T IO N T O PROTECTIVE EARTH GROUND.

T H IS A R E A M A Y C O N T A IN B I O 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/PAN ELS M UST BE SECUR ED IN PLACE PRIOR TO IN STRUM ENT OPER ATIO N. REFER TO PRO DUCT REFERENCE M ANUAL FOR PROPER INSTALLATION.

2429555

CAUTION

1.2-2

PN 4237616B

Page 39

2 INSTRUMENT DESCRIPTION, 2.1-1

2.1 INTRODUCTION TO THE A

Purpose, 2.1-1 Function, 2.1-1 Description, 2.1-1

Components, 2.1-1

Interaction with the A

•T 5diff Hematology Analyzer, 2.1-1

Modes of Operation, 2.1-3

CBC Mode, 2.1-3 CBC/DIFF Mode, 2.1-3

Reagent Consumption, 2.1-4

2.2 OPERATION PRINCIPLES, 2.2-1 Overview, 2.2-1 Measurement Principles, 2.2-1

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

V Technology, 2.2-3

Dual Focused Flow (DFF), 2.2-3 Flow Cell, 2.2-3 Focused Flow Impedance, 2.2-4 Absorbance Cytochemistry, 2.2-4 Signal Processing, 2.2-4 Thresholds, 2.2-4

WBC/BASO Methodology,2.2-5 Sample Analysis Overview,2.2-5

Aspiration, 2.2-5 Dilution, 2.2-6 Delivery, 2.2-6 Sample Partitioning, 2.2-7

•T 5diff HEMATOLOGY ANALYZER, 2.1-1

PN 4237616B

2.3 CYCLE DESCRIPTION, 2.3-1 Cycle Start Conditions, 2.3-1 Sample Flow, 2.3-2

2.4 SAMPLE ANALYSIS, 2.4-1 RBC and Platelet Analysis, 2.4-1

Parameter Results Obtained from the RBC/Plt Dilution, 2.4-2 Hgb Measurement, 2.4-3 WBC Count and Differential, 2.4-4

Parameter Results Obtained from the WBC/BASO Dilution, 2.4-5

Differential, 2.4-5

Parameter Results Obtained from the DIFF Dilution, 2.4-6 Dilution Summary, 2.4-7

2-i

Page 40

CONTENTS

2.5 RBC PARAMETER DEVELOPMENT, 2.5-1 RBC/Plt Dilution, 2.5-1 RBC Count, 2.5-1 RBC Histogram, 2.5-1 Parameter Results Obtained Using the RBC Histogram, 2.5-2

Hct Measurement, 2.5-2 MCV Calculation, 2.5-2 RDW Calculation, 2.5-2

RBC Distribution Flags, 2.5-2

RBC1 and RBC2 Thresholds, 2.5-3 Flags, 2.5-3

Hgb Determination, 2.5-3

Hgb Blank Reading, 2.5-3 Sample Reading, 2.5-4 Hgb Specific Flags, 2.5-4

MCH and MCHC Calculations, 2.5-4

MCH Calculation, 2.5-4 MCHC Calculation, 2.5-4

2.6 PLATELET PARAMETER DEVELOPMENT, 2.6-1 RBC/Plt Dilution, 2.6-1 Plt Count, 2.6-1 Platelet Distribution Curve, 2.6-1 Parameter Results Obtained Using the Plt Histogram, 2.6-2

MPV Measurement, 2.6-2 Pct Calculation, 2.6-2 PDW Calculation, 2.6-2

Detecting Abnormal Platelet Distributions, 2.6-3

2.7 WBC PARAMETER DEVELOPMENT, 2.7-1 Overview, 2.7-1 WBC/BASO Dilution, 2.7-1 WBC Count, 2.7-1 BASO Count, 2.7-1 DIFF Dilution, 2.7-2 DiffPlot Development, 2.7-2 DiffPlot Regions Defined, 2.7-3

Neutrophil (Neut), 2.7-3 Lymphocyte (Lymph), 2.7-3 Monocyte (Mono), 2.7-3 Eosinophil (Eos), 2.7-3 Debris, 2.7-3

2-ii

PN 4237616B

Page 41

Immature White Blood Cells, 2.7-4

Immature Granulocytes, 2.7-4 Band Cells, 2.7-4 Blast Cells, 2.7-4

DiffPlot Thresholds, 2.7-4

2.8 PNEUMATIC/HYDRAULIC SYSTEM, 2.8-1 Functions of Valves, 2.8-1 Pneumatic Diagrams, 2.8-4 Diluter System, 2.8-11

Diluent Input (Figure 2.8-10), 2.8-11 5diff Syringe and Flow Cell, 2.8-12 Probe and Probe Rinse, 2.8-13 Diluent to Baths, 2.8-13

Waste System, 2.8-15

2.9 ELECTRONIC SYSTEM, 2.9-1 Plug/Jack Labels, 2.9-1 Optical Preamplifier Card, 2.9-1 LCD and Keypad Card, 2.9-1 LED Card, 2.9-1 Motor Interconnect Card, 2.9-1 Traverse Interconnect Card, 2.9-1

CONTENTS

2.10 SOFTWARE STRUCTURE, 2.10-1 Overview, 2.10-1 Menu Trees, 2.10-1 How to Select a Menu Item, 2.10-1

ILLUSTRATIONS

2.1-1 User Interfaces on the AC•T 5diff Hematology Analyzer, 2.1-2

2.2-1 Coulter Principle, 2.2-2

2.2-2 Dual Focused Flow Process, 2.2-3

2.2-3 Signal Processing, 2.2-4

2.2-4 Basophil Thresholds, 2.2-5

2.2-5 Bath Assembly, 2.2-6

2.2-6 Sample Delivery Using Tangential Flow, 2.2-6

2.2-7 CBC/DIFF Mode -

Sample Partitions inside the Probe, 2.2-7

2.2-8 CBC Mode -

Sample Partitions inside the Probe, 2.2-7

2.3-1 Sample Probe and LED at Start of a Cycle, 2.3-1

2.3-2 Baths Assembly at Start of a Cycle, 2.3-1

2.3-3 Rinsing Probe Exterior After Aspiration, 2.3-2

2.3-4 Making the RBC/PLT First Dilution, 2.3-2

2.3-5 Making the WBC/BASO Dilution, 2.3-3

2.3-6 Making the DIFF Bath Dilution, 2.3-3

2.3-7 Double Rinse of the Sample Probe, 2.3-4

2.3-8 Aspirating from the First Dilution, 2.3-4

2.3-9 Rinsing the Outside of the Probe, 2.3-5

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CONTENTS

2.3-10 Making the RBC/Plt Dilution, 2.3-5

2.4-1 Bath Assembly, 2.4-1

2.4-2 Bath Assembly, 2.4-4

2.4-3 Flow Cell Operation, 2.4-5

2.4-4 DiffPlot Regions, 2.4-6

2.5-1 Typical RBC Histogram, 2.5-1

2.5-2 RBC1 and RBC2 Positions - RBC Histogram, 2.5-2

2.6-1 Typical Plt Histogram, 2.6-1

2.6-2 Area of the Plt Histogram Used to Determine the PDW Parameter Result, 2.6-2

2.6-3 Typical Platelet Distribution Curve, 2.6-3

2.6-4 Microcytic Interference with a Valley between 18 fL and 25 fL, 2.6-4

2.6-5 Microcytic Interference with a Valley below 18 fL, 2.6-4

2.6-6 Interference with no Distinct Valley, 2.6-5

2.7-1 Areas Used to Determine WBC and BASO Parameter Results, 2.7-1

2.7-2 DiffPlot Regions, 2.7-2

2.7-3 Volume Thresholds, 2.7-5

2.7-4 Absorbance Thresholds / NL, NE and MN Alarms, 2.7-6

2.8-1 Valve 1 through Valve 16 Locations, 2.8-1

2.8-2 Valve 17 and 18 Location, 2.8-1

2.8-3 Valve 20 to Valve 31 Locations, 2.8-2

2.8-4 Hgb Lyse Reagent Circuit, 2.8-5

2.8-5 Fix Reagent Circuit, 2.8-6

2.8-6 WBC Lyse Reagent Circuit, 2.8-7

2.8-7 Rinse Reagent Supply Circuit, 2.8-8

2.8-8 Probe Rinse Reagent Circuit, 2.8-9

2.8-9 WBC/BASO Rinse Reagent Circuit, 2.8-10

2.8-10 Diluent Reagent Circuit, 2.8-11

2.8-11 Probe Diluent Reagent Circuit, 2.8-13

2.8-12 Bath Diluent Reagent Circuit, 2.8-14

2.8-13 Waste Circuit, 2.8-15

2.10-1 User Menu Tree, 2.10-2

2.10-2 Service Menu Tree, 2.10-3

2-iv

TABLES

2.1-1 AC•T 5diff Hematology Analyzer Reagent Consumption, Software Version

1.03, 2.1-4

2.2-1 A

2.4-1 Technical Characteristics for Obtaining RBC and Platelet Counts, 2.4-2

2.4-2 Technical Characteristics for the Measurement of the Hemoglobin, 2.4-3

2.4-3 Characteristics Required to Obtain WBC and BASO Results, 2.4-4

2.4-4 Technical Characteristics for Acquisition of the DiffPlot, 2.4-6

2.4-5 Summary of Dilutions, 2.4-7

2.8-1 Valves and their Functions, 2.8-3

•T 5diff Analyzer Measurement Technologies, 2.2-1

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INSTRUMENT DESCRIPTION

2.1 INTRODUCTION TO THE AC•T 5diff HEMATOLOGY 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.

Function

The AC•T 5diff analyzer is a quantitative, fully automated (microprocessor controlled) hematology analyzer and leukocyte differential counter

clinical laboratories. The A (CBC) and white blood cell differential (DIFF) on open-vial, whole-blood specimens.

The CBC consists of white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (Plt), and mean platelet volume (MPV).

The DIFF (a 5-part leukocyte differential) consists of the percentage (%) and absolute number (#) of the following WBC populations: neutrophils (NE% and NE#), lymphocytes (LY% and LY#), monocytes (MO% and MO#), eosinophils (EO% and EO#), and basophils (BA% and BA#).

•T 5diff hematology analyzer reports a complete blood count

For In Vitro Diagnostic Use

Six parameters are qualitative and are

Procedures

(PDW), percentage and absolute number of immature cells (IMM% and IMM#), and percentage and absolute number of atypical lymphocytes (ATL% and ATL#).

. These parameters include the plateletcrit (Pct), platelet distribution width

For Research Use Only. Not For In Vitro Diagnostic

Description

Components

The A only for the external printer, the diluent reagent container, and a waste container (if used).

Interaction with the A

The A (Figure 2.1-1) initiates a cycle. When the sample probe is submerged in a whole-blood specimen and the aspirate switch is pressed, sample is pulled from the specimen tube into the sample probe. As the cycle continues, the instrument then dilutes and analyzes this sample. When the analysis is complete, results appear on a LCD (Figure 2.1-1) and are available to the printer.

•T 5diff hematology analyzer is contained in one unit, with additional space needed

•T 5diff Hematology Analyzer

•T 5diff analyzer uses an Open-Vial mode of operation. Pressing the aspirate switch

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2.1-1

Page 44

INSTRUMENT DESCRIPTION

INTRODUCTION TO THE A

Figure 2.1-1 User Interfaces on the AC•T 5diff Hematology Analyzer

LC D

Keypad

Aspirate

sw itch

•T 5diff HEMATOLOGY ANALYZER

7616094A

You also interact with the instrument through the use of a menu system displayed on a 128 by 240 pixels LCD and a control panel keypad with buttons that are used to setup and operate the instrument (Figure 2.1-1).

Since most input/output functions of the operating system software are controlled by the user, the pushbutton keypad and LCD screen are particularly important because they provide the physical user interface with the software.

See Heading 2.10, SOFTWARE STRUCTURE for more specific information as well as a graphic representation of the available menus and menu items (or options).

2.1-2

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INSTRUMENT DESCRIPTION

INTRODUCTION TO THE A

•T 5diff HEMATOLOGY ANALYZER

Modes of Operation

The AC•T 5diff hematology analyzer has two operating modes: CBC and CBC/DIFF.

CBC Mode

Twelve parameters are generated in the CBC mode of operation - 10 parameters

Diagnostic Use Vitro Diagnostic Procedures

and two qualitative parameters that are

For Research Use Only. Not For In

For In Vitro

Parameters

(For In Vitro Diagnostic Use)

WBC Pct

RBC PDW Hgb

Hct MCV MCH

MCHC

RDW

Plt

MPV

Parameters

(For Research Use Only)

CBC/DIFF Mode

26 parameters are generated in the CBC/DIFF mode of operation - 20 parameters

Diagnostic Use Vitro Diagnostic Procedures

(For In Vitro Diagnostic Use)

and six qualitative parameters that are

Parameters

(For Research Use Only)

For Research Use Only. Not For In

For In Vitro

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WBC Pct

RBC PDW Hgb IMM% and IMM#

Hct ATL% and ATL# MCV MCH

MCHC

RDW

Plt

MPV

NE% and NE#

LY % and LY #

MO% and MO#

EO% and EO# BA% and BA#

2.1-3

Page 46

INSTRUMENT DESCRIPTION

INTRODUCTION TO THE A

Reagent Consumption

Table 2.1-1 shows the instrument reagent consumption by cycle.

Table 2.1-1 AC•T 5diff Hematology Analyzer Reagent Consumption, Software Version 1.03

•T 5diff HEMATOLOGY ANALYZER

Cycle

•T 5diff reagents with usage per cycle

Approximate Duration

Diluent WBC Lyse Rinse Fix Hgb Lyse

CBC 20.5 mL 2.1 mL 0.9 mL Not used 0.4 mL 1 minute CBC/DIFF 25.6 mL 2.1 mL 0.9 mL 1.0 mL 0.4 mL 1 minute

Startup

†

62.0 mL 2.1 mL 3.7 mL 1.0 mL 1.4 mL 3 minutes 40 seconds

Shutdown 25.5 mL Not used 14.0 mL Not used 1.0 mL 2 minutes 45 seconds Prime diluent 35.5 mL Not used Not used Not used Not used 2 minutes 30 seconds Prime rinse Not used Not used 25.8 mL Not used Not used 1 minute 20 seconds Prime fix Not used Not used Not used 25.8 mL Not used 1 minute 30 seconds Prime WBC Lyse Not used 25.8 mL Not used Not used Not used 1 minute 20 seconds Prime Hgb Lyse 2.5 mL Not used Not used Not used 4.2 mL 1 minute Prime All Reagents 23.7 mL 16.0 mL 16.0 mL 16.0 mL 4.2 mL 3 minutes 20 seconds Extended Cleaning 12.5 mL Not used 6.0 mL Not used Not used 1 minute 35 seconds System Reset Cycle 24.0 mL Not used 1.4 mL Not used 1.0 mL 1 minute 25 seconds

†

For one background count only. The maximum is three.

2.1-4

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2.2 OPERATION PRINCIPLES

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

(Absorbance Cytochemistry and Volume Technology) and the white blood cell / basophil (WBC/BASO) methodologies.

The A WBC/BASO methodology uses differential lysis, impedance technology, and differential thresholds. See Table 2.2-1.

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

INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

V Technology

Table 2.2-1 AC•T 5diff Analyzer Measurement Technologies

Fluid Dynamics Technology Measurements Output

Dual Focused Flow

Volume aperture Differential lysis using the

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

V Technology

Coulter Principle

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

Measurement Principles

Coulter Principle

In the A platelet (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 nonconductive 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.

•T 5diff analyzer, the Coulter Principle is used to analyze the final red blood cell and

PN 4237616B

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

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

WBC/BASO bath.

2.2-1

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INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

To sense particles using the Coulter Principle (Figure 2.2-1), 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 (Figure 2.2-1). 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.

Figure 2.2-1 Coulter Principle

Solution to be analyzed

Vacuum constant

Constant current

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 2.2-1).

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 A

•T 5diff analyzer makes several dilutions of an aspirated whole-blood sample. The RBC/Plt dilution begins in the DIL1/HGB (first dilution/hemoglobin) 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.

2.2-2

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

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INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

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.

ACV Technology

In the DIFF (differential) bath, 25 µL of whole blood is mixed with 1,000 µL of AC•T5diff

Fix reagent for 12 seconds, then stabilized with 1,000 µL of A additional three seconds. 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).

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

Dual Focused Flow (DFF)

DFF fluid dynamics uses a hydrodynamic focusing process to focus individual cells or particles in a stream of diluent (Figure 2.2-2). The focused sample stream of the A

analyzer is about 40 µm in diameter.

•T 5diff Diluent for an

•T5diff

Figure 2.2-2 Dual Focused Flow Process

DFF uses sheath fluid to surround and force cells suspended in diluent to pass one at a time through the center of the flow cell. 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 flow cell. Hydrodynamic focusing in the flow cell enables accurate and rapid cell-by-cell measurements on a large number of individual cells.

Flow Cell

Sequential analyses for cell volume (impedance) and light absorbance are performed in the flow cell. A total of 72 µL of sample is injected through the flow cell for 15 seconds. The flow cell incorporates a 60 µm aperture for cellular volume analysis and about a 40 µm measurement area for light absorbance.

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INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

Focused Flow Impedance

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

Absorbance Cytochemistry

As a cell passes through the optical portion of the flow cell, 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 flow cell 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 2.2-3.

Figure 2.2-3 Signal Processing

Thresholds

2.2-4

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INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

WBC/BASO Methodology

In the WBC/BASO bath, 10 µL of whole blood is mixed with 2,000 µL of AC•T5diff WBC Lyse reagent. This reaction lyses the red blood cells and specifically differentiates between 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 80 µm 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 2.2-4).

Figure 2.2-4 Basophil Thresholds

Sample Analysis Overview

Aspiration

When the sample probe is immersed in a whole-blood specimen 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.

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

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2.2-5

Page 52

INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

Dilution

Using the Sequential Dilution System (SDS) technique, the aspirated sample is partitioned as it is distributed to make a series of dilutions in a series of baths (Figure 2.2-5).

Figure 2.2-5 Bath Assembly

Delivery

In the CBC and the CBC/DIFF modes, each aliquotted sample is delivered to its appropriate bath using a tangential flow (Figure 2.2-6) of reagent, which mixes the diluted sample and minimizes viscosity problems.

Figure 2.2-6 Sample Delivery Using Tangential Flow

Probe

Reagent

input

Bath

Tangential flow

7616002

2.2-6

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INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

Sample Partitioning

Figure 2.2-7 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 2.2-7 CBC/DIFF Mode Sample Partitions inside the Probe

CBC/DIFF Mode

After aspiration in the CBC/DIFF mode, aliquots of the whole-blood sample are distributed to the

Diluent

various baths as follows (Figure 2.2-5):

r The 3 µL sample aliquot at the tip of the probe

Air bubble

is discarded into the rinse chamber as the exterior of the sample probe is rinsed, ensuring sample integrity.

Not used

DIFF dilution

r 10 µL of sample is delivered to the DIL1/HGB

bath for use in preparing the primary RBC/Plt dilution and for measuring the Hgb value.

r 10 µL of sample is delivered to the WBC/BASO

WBC/BASO dilution

RBC/PLT/HGB first dilution

Not used

7616001A

bath for the WBC/BASO count.

r 25 µL of sample is delivered to the DIFF bath

for development of the DiffPlot.

r 5 µL of remaining sample is discarded into the

rinse chamber.

Figure 2.2-8 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.)

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Figure 2.2-8 CBC Mode Sample Partitions inside the Probe

Diluent

Air bubble

Not used

WBC/BASO dilution

RBC/PLT/HGB first dilution

Not used

CBC Mode

After aspiration in the CBC mode, aliquots of the whole-blood sample are distributed to the various baths as follows (Figure 2.2-5):

r The 3 µL sample aliquot at the tip of the probe

is discarded into the rinse chamber as the exterior of the sample probe is rinsed, ensuring sample integrity.

r 10 µL of sample is delivered to the DIL1/HGB

bath for use in preparing the primary RBC/Plt dilution and for measuring the Hgb value.

r 10 µL of sample is delivered to the WBC/BASO

bath for the WBC/BASO count.

r 7 µL of remaining sample is discarded into the

rinse chamber.

7616056A

2.2-7

Page 54

INSTRUMENT DESCRIPTION

OPERATION PRINCIPLES

2.2-8

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Page 55

2.3 CYCLE DESCRIPTION

This cycle description focuses on the sequence of the sample probe movement among the baths. It also focuses on the volume of sample and reagents being delivered to make the dilutions needed for sample analysis.

Cycle Start Conditions

INSTRUMENT DESCRIPTION

CYCLE DESCRIPTION

Figure 2.3-1 Sample Probe and LED at Start of a Cycle

Figure 2.3-2 Baths Assembly at Start of a Cycle

r The sample probe is in its home position. r The green LED is glowing indicating the

instrument is ready.

r All the baths (except the rinse chamber) are

filled with clean diluent.

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2.3-1

Page 56

INSTRUMENT DESCRIPTION

CYCLE DESCRIPTION

Sample Flow

r To initiate a cycle, submerge the sample probe in

a well-mixed whole-blood specimen and press the aspirate switch to start the cycle.

r All the baths drain. r A sample of the whole-blood specimen is

aspirated.

t 53 µL in the CBC/DIFF mode. t 30 µL in the CBC mode.

Figure 2.3-3 Rinsing Probe Exterior After Aspiration

Figure 2.3-4 Making the RBC/PLT First Dilution

r The horizontal traverse assembly positions the

sample probe over the rinse chamber.

r 3 µL sample aliquot at the tip of the sample

probe is discarded into the rinse chamber as the exterior of the sample probe is rinsed. Discarding this aliquot helps ensure sample integrity.

r The horizontal traverse assembly positions the

sample probe over the DIL1/HGB (first dilution/Hgb) bath.

r The vertical traverse assembly moves the probe

downward into the bath. The probe tip is positioned to produce a tangential flow when the sample and diluent are simultaneously dispensed into the bath. For a more detailed description of tangential flow, see Delivery under Heading 2.2, OPERATION PRINCIPLES.

2.3-2

r 10 µL of the whole-blood partitioned for making

the first dilution is delivered to the DIL1/HGB bath using a tangential flow of 1.7 mL of diluent.

r The tangential flow of reagent mixes the sample

and the diluent. Mixing bubbles enter the bath to make a uniform suspension of cells. This 1:170 dilution is commonly referred to as the first dilution.

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INSTRUMENT DESCRIPTION

CYCLE DESCRIPTION

Figure 2.3-5 Making the WBC/BASO Dilution

Figure 2.3-6 Making the DIFF Bath Dilution

r The horizontal traverse assembly positions the

sample probe over the WBC/BASO bath

r The vertical traverse moves the probe downward

into the bath. The tip of the probe is positioned so that a tangential flow occurs as the 10 µL of the whole-blood sample and 2.0 mL of WBC Lyse are simultaneously dispensed into the bath.

r The tangential flow of reagent mixes the sample

and reagent. Mixing bubbles enter the bath to make a uniform suspension of cells. The WBC Lyse destroys the red blood cells and the specific lytic action on the white blood cells differentiates the basophils from other WBCs.

WBC/BASO Bath Dilution

Whole-blood volume 10 µL

Volume of A Dilution ratio 1:200

•T 5diff WBC Lyse reagent

2000 µL

r The horizontal traverse assembly moves the

sample probe over the DIFF bath.

r The vertical traverse assembly moves the probe

downward into the bath.

r The tip of the probe is positioned so that a

tangential flow occurs as 25 µL of the whole-blood sample and 1.0 mL of Fix reagent are simultaneously dispensed into the bath.

r The tangential flow of reagent mixes the sample

and the Fix reagent. Mixing bubbles enter the bath to make a uniform suspension of cells. The Fix reagent lyses the red blood cells, stabilizes the WBCs in their native form, and differentially stains the lymphocytes, monocytes, neutrophils, and eosinophils, with the eosinophils staining most intensely.

r After 12 seconds of incubation, the staining

process inside the DIFF bath is completed by adding another 1.0 mL of diluent which stops the cytochemical reaction.

DIFF Bath Dilution

Whole-blood volume 25 µL

Volume of A Volume of A

Final dilution ratio 1:80

•T 5diff Fix reagent

•T 5diff Diluent

1000 µL 1000 µL

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2.3-3

Page 58

INSTRUMENT DESCRIPTION

CYCLE DESCRIPTION

Figure 2.3-7 Double Rinse of the Sample Probe

Figure 2.3-8 Aspirating from the First Dilution

r The horizontal traverse assembly moves the

sample probe over the rinse chamber.

r A double rinsing (interior and exterior) of the

probe removes residual whole-blood sample from inside the probe.

t In the CBC/DIFF mode, 5 µL is discarded in

the rinse chamber.

t In the CBC mode, 7 µL is discarded in the

rinse chamber.

r The horizontal traverse assembly moves the

sample probe over the DIL1/HGB bath.

r The vertical traverse assembly moves the probe

downward into the bath.

r 42.5 µL of the 1:170 first dilution is aspirated

into the sample probe.

2.3-4

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Page 59

INSTRUMENT DESCRIPTION

CYCLE DESCRIPTION

Figure 2.3-9 Rinsing the Outside of the Probe

r While still inside the DIL1/HGB bath, the

exterior of the sample probe is rinsed with

0.4 mL of diluent.

r The vertical traverse assembly moves the probe

up out of the bath.

r 0.4 mL of Hgb Lyse is added to the bath. The

Hgb Lyse reagent rapidly destroys the red blood cells and converts a substantial proportion of the hemoglobin to a stable pigment so a hemoglobin value can be determined.

r Mixing bubbles enter the bath to ensure a

uniform dilution.

DIL1/HGB Bath Dilution

First dilution 1:170 Volume of first dilution removed 42.5 µL

Volume of A Volume of A

Final dilution ratio 1:250

•T 5diff Hgb Lyse reagent

•T 5diff Diluent reagent

400 µL 400 µL

Figure 2.3-10 Making the RBC/Plt Dilution

r The horizontal traverse assembly moves the

sample probe over the RBC bath.

r The vertical traverse assembly moves the probe

downward into the bath.

r The tip of the probe is positioned so that a

tangential flow occurs as the 42.5 µL of 1:170 dilution obtained from the first dilution in the DIL1/HGB bath and 2.0 mL of diluent are simultaneously dispensed into the bath

r An additional 0.5 mL of diluent is dispensed

through the probe at the end of the second dilution.

RBC Bath Dilution

Volume 1:170 dilution from

Volume of A Final dilution ratio 1:10,000

•T 5diff Diluent reagent

DIL1/HGB

bath 42.5 µL

2500 µL

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2.3-5

Page 60

INSTRUMENT DESCRIPTION

CYCLE DESCRIPTION

2.3-6

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2.4 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 DIL1/HGB bath, and the secondary dilution is made in the RBC bath (Figure 2.4-1).

Figure 2.4-1 Bath Assembly

INSTRUMENT DESCRIPTION

SAMPLE ANALYSIS

PN 4237616B

Table 2.4-1 summarizes the technical characteristics required to obtain RBC and Platelet results.

2.4-1

Page 62

INSTRUMENT DESCRIPTION

SAMPLE ANALYSIS

Table 2.4-1 Technical Characteristics for Obtaining RBC and Platelet Counts

Dilution Characteristics

Primary Dilution for RBC and Plt:

Initial volume of whole-blood 10 µL Volume A

Primary dilution ratio 1:170

Secondary Dilution for RBC and Plt

Volume of primary dilution 42.5 µL 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)

Measurement Characteristics

•T 5diff diluent

1700 µL

2500 µL

Method of analysis Coulter Principle Aperture diameter 50 µm Count vacuum 200 mb (5.9 in. Hg) Count period 2 x 5 seconds

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 the Plt count. r Develop the Plt histogram, which is needed to obtain the MPV, Pct, and PDW results.

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INSTRUMENT DESCRIPTION

SAMPLE ANALYSIS

Hgb Measurement

Hemoglobin is determined from the dilution in the DIL1/HGB bath (Figure 2.4-1). This dilution is prepared in two stages – the primary (first) dilution and the secondary (last) dilution.

The primary dilution is made and 42.5 µL 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 DIL1/HGB bath using a spectrophotometric technique at a wavelength of 550 nm. 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.

Table 2.4-2 summarizes the technical characteristics required for measuring hemoglobin.

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

Table 2.4-2 Technical Characteristics for the Measurement of the Hemoglobin

Dilution Characteristics

Volume of whole-blood 10 µL

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 550 nm

•T 5diff diluent

•T 5diff Hgb Lyse

•T 5diff diluent

1700 µL

42.5 µL

400 µL 400 µL

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INSTRUMENT DESCRIPTION

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 2.4-2).

The WBC count and the BASO count are determined simultaneously.

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

The dilution analyzed in the flow cell is prepared in the DIFF bath (Figure 2.4-2).

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

Figure 2.4-2 Bath Assembly

2.4-4

Table 2.4-3 summarizes the technical characteristics required to obtain WBC and BASO results.

Table 2.4-3 Characteristics Required to Obtain WBC and BASO Results

Dilution Characteristics

Volume of whole-blood 10 µL Volume A

Dilution ratio 1:200 Reaction temperature 35°C (95°F)

Measurement Characteristics

Method of analysis Coulter Principle Aperture diameter 80 µm Count vacuum 200 mb (5.9 in. Hg) Count period 2 x 6 seconds

•T 5diff WBC Lyse

2,000 µL

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INSTRUMENT DESCRIPTION

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

•T 5diff Fix reagent, which lyses the red blood cells, stabilizes the WBC in their native forms, and differentially stains the lymphocytes, monocytes, neutrophils, and eosinophils, with the eosinophils staining most intensely.

The solution is then stabilized with diluent for three seconds and transferred to the measuring bath. See Figure 2.4-3. Each cell is measured in absorbance (cytochemistry) and resistivity (volume).

Figure 2.4-3 Flow Cell Operation

2) Second focused flow for optical detection

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1) Primary focused flow for impedance

Table 2.4-4 summarizes the technical characteristics required for acquisition of the DiffPlot.

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INSTRUMENT DESCRIPTION

SAMPLE ANALYSIS

Table 2.4-4 Technical Characteristics for Acquisition of the DiffPlot

Dilution Characteristics

Volume of whole-blood 25 µL 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 60 µm Diameter of the flow 42 µm Volume injected 72 µL

•T 5diff Fix

•T 5diff Diluent

1000 µL 1000 µL

Injection duration 15 seconds Data accumulation 12 seconds

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 2.4-4 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 2.4-4 DiffPlot Regions

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INSTRUMENT DESCRIPTION

SAMPLE ANALYSIS

Dilution Summary

Table 2.4-5 summarizes the dilution characteristics required to obtain CBC and CBC/DIFF parameter results.

Table 2.4-5 Summary of Dilutions

Technical Characteristics for

WBC Count and BASO count

(in the WBC/BASO bath)

Differential Acquisition with

Differential WBC Count (in the DIFF bath)

Hemoglobin Measurement

(in the DIL1/HGB bath)

RBC and PLT Count

(in the RBC bath)

Note:

The primary dilution (1:170) is made in the DIL1/HGB bath.

Whole-Blood Volume Reagent(s)

10 µL

25 µL

10 µL

42.5 µL of the 1:170 dilution (from the DIL1/HGB bath)

•T 5diff WBC Lyse

•T 5diff Fix

•T 5diff Diluent

After removing 42.5 µL of the 1:170 dilution:

•T 5diff Diluent

•T 5diff Hgb Lyse

•T 5diff Diluent

Reagent Volume Dilution Ratio

2000 µL Final

1:200 1000 µL 1000 µL

1700 µL

400 µL 400 µL

2500 µL Secondary

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)

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INSTRUMENT DESCRIPTION

SAMPLE ANALYSIS

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2.5 RBC PARAMETER DEVELOPMENT

RBC/Plt Dilution

The final 1:10,000 dilution in the RBC bath contains red blood cells, white blood cells, and platelets. Thresholds are used to separate the platelet pulses, which are much smaller, from the red and white blood cell pulses. Since white blood cells fall in the red blood cell size range, they are counted and sized as RBCs. The WBCs are not sorted out because any interference is usually insignificant; there are normally very few WBCs (thousands) in comparison to the number of RBCs (millions). Only when the white count is markedly elevated is the red cell count or histogram influenced.

RBC Count

The AC•T 5diff hematology analyzer uses duplicate counting criteria, voting criteria, and 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 volume unit x Calibration factor

INSTRUMENT DESCRIPTION

RBC PARAMETER DEVELOPMENT

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

Cells per microliter (cells/µL) is the US reporting unit format. See Heading A.7 in the

Note:

Operator’s Guide for the other available formats.

cells /µL.

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 2.5-1 is an example of an RBC histogram with a normal RBC size distribution.

Figure 2.5-1 Typical RBC Histogram

30 300

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INSTRUMENT DESCRIPTION

RBC PARAMETER DEVELOPMENT

Parameter Results Obtained Using the RBC Histogram

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. The Hct is displayed and printed as a percentage (%).

Note: Percentage (%) is the US reporting unit format. See Heading A.7 in the Operator’s Guide for the other available formats.

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: Femtoliters (fL) is the US reporting unit format. See Heading A.7 in the Operator’s Guide for the other available formats.

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.

K SD

---------------

RDW (%)=

MCV

where:

K = System constant SD = Calculated standard deviation based on the red cell distribution MCV = Mean Cell Volume of the red cells

RBC Distribution Flags

Once the RBC distribution curve is developed, two positions on the distribution curve are located (Figure 2.5-2):

Figure 2.5-2 RBC1 and RBC2 Positions - RBC Histogram

RBC1 RBC2

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INSTRUMENT DESCRIPTION

RBC PARAMETER DEVELOPMENT

RBC1 and RBC2 Thresholds

Thresholds RBC1 and RBC2 define the

MICRO and MACRO regions and are calculated based

on standard deviation (SD) of the RBC population.

The RBC1 threshold (monitoring area for microcytes) and the RBC2 threshold (monitoring area for macrocytes) identify the points on the curve that are ±2 SD from the mean (Figure 2.5-2).

Flags

Note:

MICRO

and

MACRO

flags will be activated in software version 1.0 and higher.

MICRO

The to the total number of RBCs exceeds the preset default limit of 5%. The

flag is generated when the percentage of cells in the microcytic region compared

MACRO

flag is generated when the percentage of cells in the macrocytic region compared to the total number of RBCs exceeds the preset default limit of 7.5%. A laboratory may establish its own limits to replace the preset default values.

Note:

The

MICRO

and

MACRO

flags are independent of the

Microcytosis

Macrocytosis

and

flags that are generated from the Low and High patient limits.

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 DIL1/HGB bath using a spectrophotometric technique at a wavelength of 550 nm. 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 in g/dL represents: absorbance value obtained x Calibration factor.

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

Grams per deciliter (g/dL) is the US reporting unit format. See Heading A.7 in the

Note:

Operator’s Guide for the other available formats.

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Hgb Blank Reading

The Hgb blank value measured during the first patient cycle after a Startup cycle is stored as a reference blank. This blank must be greater than 2.5 Vdc. During each analysis cycle, the instrument checks the measured Hgb blank against the stored Hgb blank reference value using the following formula:

(Blank

Ref

x 1/3) + (BlankS x 2/3) = Blank

where:

Ref

Blank Blank Blank

Note:

= Hgb blank reference value

= Hgb blank value from the current cycle

= New Hgb blank reference value for comparison

If the new Hgb blank reference value is within 3% of the old reference value,

the Hgb blank reference value is changed to this new value.

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INSTRUMENT DESCRIPTION

RBC PARAMETER DEVELOPMENT

Sample Reading

This value is based on the sample, diluent, and Hgb Lyse reagent mixture in the DIL1/HGB bath during sample measurement.

Hgb Specific Flags

If the Hgb blank value is less than 2.5 Vdc, a reject (

If the difference between the new Hgb blank reference value and the original Hgb blank reference value is greater than 3%, a review (

(

) flags occur on the Hgb blank reference value, the (

For each Hgb sample read value, the instrument takes three readings. If the difference between these readings exceeds the predefined limits (default setting is 60 A to D units), a

voteout (

) flag is generated.

MCH and MCHC Calculations

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:

) flag occurs on the Hgb value.

) flag is generated. If three consecutive review

. . . . .

) code replaces the Hgb result.

Hgb

-----------RBC

Picograms (pg) is the US reporting unit format. See Heading A.7 in the Operator’s

Note:

10 × MCH (pg)=

Guide for the other available formats.

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:

Hgb

----------

Note:

100 × MCHC (g/dL)=

Hct

Grams per deciliter (g/dL) is the US reporting unit format. See Heading A.7 in the

Operator’s Guide for the other available formats.

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2.6 PLATELET PARAMETER DEVELOPMENT

RBC/Plt Dilution

Platelet counting and sizing is also done in the RBC bath. Thresholds separate the platelet pulses, which are much smaller, from the red and white blood cell pulses.

Plt Count

The AC•T 5diff hematology analyzer uses duplicate counting criteria, voting 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 volume unit x Calibration factor.

INSTRUMENT DESCRIPTION

PLATELET PARAMETER DEVELOPMENT

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

Cells per microliter (cells/µL) is the US reporting unit format. See Heading A.7 in the

Note:

Operator’s Guide for the other available formats.

cells /µL.

Platelet Distribution Curve

Platelets are 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 between 2 fL and 30 fL. The Plt distribution curve shows cells in their native size. Figure 2.6-1 is an example of a Plt histogram with a normal Plt size distribution.

Figure 2.6-1 Typical Plt Histogram

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INSTRUMENT DESCRIPTION

PLATELET PARAMETER DEVELOPMENT

Parameter Results Obtained Using the Plt Histogram

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).

Note: Femtoliters (fL) is the US reporting unit format. See Heading A.7 in the Operator’s Guide for the other available formats

Pct Calculation

The Pct (plateletcrit or thrombocrit) is calculated according to the formula:

Plt 10

------------------------------------------ ---------------------

The Pct parameter result is displayed and printed as a percentage (%).

PDW Calculation

PDW (Platelet Distribution Width) is calculated from the Plt histogram as the width of the curve between S1 and S2.

/µL()MPV (fL)×

10 000,

Pct%=

As shown in Figure 2.6-2, S1 and S2 are placed so that:

r 15% of the platelets occur between 2 fL and S1. r 15% of the platelets occur between S2 and the variable upper threshold.

Note: This threshold is explained under the Detecting Abnormal Platelet Distributions heading that follows.

r The PDW result is determined on the platelets between S1 and S2.

Figure 2.6-2 Area of the Plt Histogram Used to Determine the PDW Parameter Result

15%

PDW

S1 S2

15%

7615002A

2.6-2

The PDW parameter result is displayed and printed as a percentage (%).

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INSTRUMENT DESCRIPTION

PLATELET PARAMETER DEVELOPMENT

Detecting Abnormal Platelet Distributions

Particles of approximately platelet size can interfere with the platelet histogram and count. Small particles, such as microbubbles or dust, can overlap the low end. Microcytic red cells can intrude at the upper end.

Identifying a Normal Distribution

When a platelet histogram is being evaluated, a mobile threshold can move from its starting position at 25 fL to 18 fL (Figure 2.6-3). The computer searches for a valley between the platelet and red cell populations. If no valley is detected between 18 fL and 25 fL, the threshold remains at 25 fL and no flag is generated.

Figure 2.6-3 Typical Platelet Distribution Curve

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 microbubbles 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 appears to alert the operator that a significant number of small cells or interference, such as microbubbles, are present.

Microcytic Interferences on the Upper End of the Platelet Distribution Curve

Microcytic red cells can intrude at the upper end of the platelet distribution curve. If the specimen contains microcytes, the A

the influence of this interference by repositioning the variable threshold (25 fL threshold) and excluding the microcytes.

25fL

•T 5diff analyzer may be able to successfully eliminate

SCL

flag

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INSTRUMENT DESCRIPTION

PLATELET PARAMETER DEVELOPMENT

Microcytic Interference with a Distinct Valley between 18 fL and 25 fL

If the intrusion of microcytes creates a valley between the 25 fL and the 18 fL thresholds (Figure 2.6-4). The 25 fL threshold is repositioned at the valley to minimize interference to the platelet parameter results. Therefore, the reported platelet results are acceptable. The (microcytes) flag appears to alert the operator that microcytes are present.

Figure 2.6-4 Microcytic Interference with a Valley between 18 fL and 25 fL

MIC

25fL

Microcytic Interference with a Valley below 18 fL

If the microcytes are extremely small so that the valley between the platelet population and the microcyte population falls below the 18 fL limit, the threshold is placed at the 18 fL limit

MIC

(Figure 2.6-5). The

flag appears and the platelet count is flagged to alert the operator that the extremely small microcytes present in this sample could not be eliminated. The platelet count and associated parameters are not reliable and should be verified by an alternative method. To effectively eliminate the microcytes, the Operator’s Guide suggests the customer use platelet rich plasma (PRP) or a manual count to verify the results.

Figure 2.6-5 Microcytic Interference with a Valley below 18 fL

2.6-4

25fL

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INSTRUMENT DESCRIPTION

PLATELET PARAMETER DEVELOPMENT

Interference with No Distinct Valley

Interference present in the upper area of the platelet distribution curve that blends with the platelet population so that there is no clear distinction between the platelets and the interference suggest the presence of schistocytes (fragmented red cells) or platelet aggregates (platelet clumps).

If the threshold cannot be positioned in the 25 fL to 18 fL region, the threshold defaults to the

SCH

18 fL position (Figure 2.6-6). The flagged to alert the operator that the interference (which is most likely either schistocytes or platelet clumps) could not be eliminated. The platelet count and associated parameters are not reliable and must be verified using an alternative method.

Figure 2.6-6 Interference with no Distinct Valley

(schistocytes) flag appears and the platelet count is

25fL18

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INSTRUMENT DESCRIPTION

PLATELET PARAMETER DEVELOPMENT

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2.7 WBC PARAMETER DEVELOPMENT

Overview

WBC parameter results are generated from two different dilutions: the 1:200 WBC/BASO dilution which is made and analyzed in the WBC/BASO bath and the 1:80 DIFF dilution which is made in the DIFF bath but analyzed in the flow cell.

WBC/BASO Dilution

The WBC and basophil counts are determined from the 1:200 dilution made in the WBC/BASO bath. To make this dilution, 10 µL of whole blood is mixed with 2,000 µL of

•T 5diff WBC Lyse reagent. The reaction that occurs lyses the red blood cells and

A specifically differentiates between basophils and other leukocytes by volume.

WBC Count

The AC•T 5diff hematology analyzer uses duplicate counting criteria, voting criteria, and proprietary flagging information to confirm the parameter result prior to reporting it. To obtain an 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 also the count reported.

INSTRUMENT DESCRIPTION

WBC PARAMETER DEVELOPMENT

A second WBC count is determined in the flow cell during acquisition of the DiffPlot. The two counts are compared and if they differ more than the predefined limit, a flag occurs.

WBC count: Number of cells per volume x calibration factor.

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

Cells per microliter (cells/µL) is the US reporting unit format. See Heading A.7 in the

Note:

Operator’s Guide for the other available formats.

cells /µL.

BASO Count

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

•T 5diff WBC Lyse-specific lytic action. See Figure 2.7-1.

Figure 2.7-1 Areas Used to Determine WBC and BASO Parameter Results

bc d

basophilsWBC

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In Figure 2.7-1, basophils are located in the area between the thresholds labeled candd. One 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

andd.

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INSTRUMENT DESCRIPTION

WBC PARAMETER DEVELOPMENT

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

BASO count = Number of cells per volume x calibration factor in a percentage relative to the number of counted cells (basophils plus other WBC nuclei).

BASO count

DIFF Dilution

The data for the DiffPlot is accumulated as the dilution made in the DIFF bath is injected into the flow cell. To make the 1:80 DIFF dilution, 25 µL of the whole-blood sample is mixed with

1,000 µL of A the white blood cells, and differentially stains the lymphocytes, monocytes, neutrophils, and eosinophils, with the eosinophils staining most intensely. After 12 seconds of incubation,

1,000 µL of A dilution is injected through the flow cell 15 seconds. For 12 of these 15 seconds, data for developing the DiffPlot is accumulated.

•T 5diff Fix reagent. The Fix reagent lyses the red blood cells, stabilizes the

•T 5diff Diluent reagent is added to stop the cytochemical reaction. This

BASO%

---------------------WBC%

andd (Figure 2.7-1)

WBC count×=

DiffPlot Development

The DiffPlot analysis on the AC•T 5diff hematology analyzer is based on three essential 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. Figure 2.7-2 shows the DiffPlot regions.

Figure 2.7-2 DiffPlot Regions

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INSTRUMENT DESCRIPTION

WBC PARAMETER DEVELOPMENT

DiffPlot Regions Defined

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.

Neutrophil (Neut)

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 2.7-2).

Lymphocyte (Lymph)

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 2.7-2). 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 2.7-2).

The lower area of the lymphocyte zone is normally empty; however, when small lymphocytes are present, a population may exist in this area (Figure 2.7-2).

The presence of platelet aggregates is indicated by a distribution pattern that moves from the DiffPlot origin into the lymphocyte region (Figure 2.7-2).

NRBC cytoplasmic membranes lyse like those of mature erythrocytes. The small nuclei that remain appear in the debris and small lymphocyte regions (Figure 2.7-2).

Monocyte (Mono)

Monocytes are typically large cells with a kidney-shaped nucleus and agranular (granule-free) 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 2.7-2).

Very large monocytes may be found in the IMM (immature cell) region.

Eosinophil (Eos)

With the reagent action, eosinophils are the most intensely stained cells 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 2.7-2).

Debris

Platelets and debris from erythrocyte lysis represent the background debris population located in the lower region of the DiffPlot.

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INSTRUMENT DESCRIPTION

WBC PARAMETER DEVELOPMENT

Immature White Blood Cells

Immature Granulocytes

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

Band Cells

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

Blast Cells

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

DiffPlot 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. Volume thresholds and definitions are shown in Figure 2.7-3. Absorbance thresholds and definitions are shown in Figure 2.7-4. The NL, NE and MN alarms are also included in Figure 2.7-4.

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Figure 2.7-3

Volume Thresholds

INSTRUMENT DESCRIPTION

WBC PARAMETER DEVELOPMENT

DL Threshold

lymphocytes.

DN Threshold

neutrophils.

SL Threshold

lymphocytes.

LN Threshold

neutrophils.

DE Threshold LMN Threshold

lymphocyte, monocyte, and neutrophil thresholds.

AL Threshold

atypical lymphocytes.

LMU Threshold

slope between atypical lymphocytes and monocytes.

LMD Threshold

slope between atypical lymphocytes and monocytes.

- Separates debris and small

- Separates debris and lower

- Separates small lymphocytes and

- Separates neutrophils and lower

- Separates debris and eosinophils.

- Intersection point between the

- Separates lymphocytes and

- Lower point on the separation

- Upper point on the separation

MN Threshold

slope between monocytes and neutrophils.

UM Threshold

monocytes.

UN Threshold

neutrophils.

- Upper point on the separation

- Separates monocytes and upper

- Separates neutrophils and upper

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INSTRUMENT DESCRIPTION

WBC PARAMETER DEVELOPMENT

Figure 2.7-4

Absorbance Thresholds / NL, NE and MN Alarms

NL Threshold

- Separates lymphocytes and

neutrophils.

RMN Threshold

- Separates upper monocytes and

upper neutrophils.

NE Threshold

- Separates neutrophils and

eosinophils.

NL, NE and MN Alarms

FNL

- # of channels for NL alarm area.

FNE

- # of channels for NE alarm area.

FMN

- # of channels for MN alarm area.

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2.8 PNEUMATIC/HYDRAULIC SYSTEM

Functions of Valves

Valve blocks are located close to the elements concerned. Five different blocks:

r Valves 1 to 11 (Figure 2.8-1):

t In the left side compartment behind the Main card, t Horizontal block, t Above the 5diff syringe and reagent syringes assembly.

r Valves 12 to 16 (Figure 2.8-1):

t In the left side compartment behind the Main card, t Vertical block, t Beside the count syringe.

Figure 2.8-1 Valve 1 through Valve 16 Locations

INSTRUMENT DESCRIPTION

PNEUMATIC/HYDRAULIC SYSTEM

PN 4237616B

r Valves 17 to 18 (Figure 2.8-2):

t In the right side compartment, t Horizontal block, t At the top of the vertical traverse (above the sample syringe assembly).

Figure 2.8-2 Valve 17 and 18 Location

2.8-1

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r Valve 19 is not used on the A r Valves 20 to 26 (Figure 2.8-3):

t In the right side compartment, t Vertical block, t Beside the waste syringe.

r Valves 27 to 31 (Figure 2.8-3):

t In the right side compartment (bath enclosure area), t Horizontal block, t Below the baths assembly.

Figure 2.8-3 Valve 20 to Valve 31 Locations

•T 5diff hematology analyzer.

See Table 2.8-1 for a description of the functions for each valve in the AC•T 5diff hematology analyzer.

2.8-2

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Table 2.8-1 Valves and their Functions

Valve Function Action

1 Differential diluent Select flow cell sheath 2 / DIFF bath 2 Differential diluent Select flow cell sheath 1 / sheath 2 3 Differential diluent Select input/output for flow cell Diluent syringe 4 Flow cell sample supply Opens pathway from the DIFF bath to the flow cell 5 Flow cell sample injector Opens waste path for sample injector syringe 6 Hgb Lyse syringe flow Selects input/output of Hgb Lyse syringe 7 Rinse syringe flow Selects input/output of Rinse syringe 8 Fix syringe flow Selects input/output of Fix syringe 9 Diluent syringe flow Selects input/output of Diluent syringe 10 Diluent output control Routes diluent to probe rinse block or heating coil 11 WBC Lyse syringe flow Selects input/output of WBC Lyse syringe 12 Rinse output control Selects rinse to probe rinse block or WBC/BASO bath

13 Count syringe vent Opens vent line of count syringe 14 RBC/PLT count valve Opens vacuum count line for RBC bath 15 Diluent reservoir vent Selects between vacuum and vent for diluent reservoir 16 Count syringe drain Opens count syringe drain path 17 Probe rinse drain Opens drain line for probe rinse block 18 Probe diluent Routes diluent/rinse to sample syringe or rinse block 19 Spare Not used 20 Waste syringe vent Opens waste vent (through the rinse chamber) 21 Sweep flow diluent Routes diluent to heating coil or sweep flow 22 Diluent bath select Route diluent (via heating coil) to Hgb or RBC bath 23 WBC/BASO count vacuum Routes vacuum direct or through RBC/PLT count head 24 Flow cell drain Opens path from flow cell output to DIFF bath for drain 25 Diluent reservoir input Opens diluent source to diluent reservoir 26 Waste syringe control Selects waste to syringe / syringe waste out 27 Rinse chamber drain Opens drain path from rinse chamber 28 HGB bath drain Opens drain path from Hgb bath 29 DIFF bath drain Opens drain path from the DIFF bath 30 RBC bath drain Opens drain path from the RBC bath

PN 4237616B

31 WBC/BASO bath drain Opens drain path from WBC/BASO bath drain

2.8-3

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Pneumatic Diagrams

To locate the pneumatic diagram for a desired reagent or the waste circuit, see the designated figure:

r For the Hgb Lyse reagent circuit, see Figure 2.8-4. r For the Fix reagent circuit, see Figure 2.8-5. r For the WBC Lyse reagent circuit, see Figure 2.8-6. r For the Rinse reagent supply circuit, see Figure 2.8-7. r For the Probe Rinse reagent circuit, see Figure 2.8-8. r For the WBC/BASO Rinse reagent circuit, see Figure 2.8-9. r For the Diluent reagent circuit, see Figure 2.8-10. r For the Probe Diluent reagent circuit, see Figure 2.8-11. r For the Bath Diluent reagent circuit, see Figure 2.8-12. r For the waste circuit, see Figure 2.8-13.

2.8-4

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Figure 2.8-4 Hgb Lyse Reagent Circuit

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2.8-5

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Figure 2.8-5 Fix Reagent Circuit

2.8-6

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Figure 2.8-6 WBC Lyse Reagent Circuit

INSTRUMENT DESCRIPTION

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2.8-7

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Figure 2.8-7 Rinse Reagent Supply Circuit

2.8-8

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Figure 2.8-8 Probe Rinse Reagent Circuit

INSTRUMENT DESCRIPTION

PNEUMATIC/HYDRAULIC SYSTEM

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2.8-9

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Figure 2.8-9 WBC/BASO Rinse Reagent Circuit

2.8-10

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Diluter System

Figure 2.8-10)

Diluent Input

Diluent for the A from a diluent container and is stored in the diluent reservoir. The input tubing from the diluent container should be no longer than 2 meters (78.7 in.) and the top of the container cannot be lower than 80 cm (31.5 in.) from the input fitting on the instrument. Vacuum, produced by the count syringe, is used to draw diluent into the diluent reservoir from the diluent container. This vacuum is applied to port 2 of the diluent reservoir. Solenoid valve 15, Diluent Reservoir Vent, is activated to connect the diluent reservoir to the count syringe. When in the normal inactive state, this valve vents the diluent reservoir to atmosphere. The vent tubing from port 1 of valve 15 is notched to ensure it does not seal against any surface and is routed to the left side drip tray. A float sensor located in the reservoir is used to determine when the reservoir is full, or needs more diluent.

Figure 2.8-10 Diluent Reagent Circuit

(

•T 5diff hemtology analyzer enters a fitting at the rear of the instrument

PN 4237616B

2.8-11

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Two ports distribute diluent from the diluent reservoir. Port 1 supplies diluent to be used by the flow cell while port 3 supplies diluent for the baths and aspirate probe. Solenoid valve 3 normally connects port 1 of the diluent reservoir to the center syringe piston in the 5diff syringe assembly, allowing the syringe to fill with diluent. For diluent output from the 5diff syringe see 5diff Syringe and Flow Cell.

Solenoid valve 9 connects the diluent reagent syringe to port 3 of the diluent reservoir, and when it is in a normal inactive state, this path is open, allowing the syringe to be filled. For output from the main diluent syringe, see Diluent to Baths and Probe and Probe Rinse.

5diff Syringe and Flow Cell

Diluent for several flow cell requirements originates at the center syringe piston of the 5diff syringe assembly. When solenoid valve 3 is active, valves 1 and 2 have control of the diluent path. If valve 2 is energized, diluent is sent from the large center syringe piston to (and through) the left injector piston. A short upward stroke is used to fill the small left injector piston with clean diluent. Since the center syringe piston is much larger, even a short stroke will produce excess diluent, which exits out the top of the left injector piston and through the flow cell.

When valve 2 is in the normal state, valve 1 determines where diluent is routed. An energized valve 1 allows diluent to be sent through the heater assembly to port 2 of the DIFF bath. This is used for the second part of the DIFF dilution when 1 mL of diluent is added to the sample 12 seconds after the original dilution with Fix. This stops the staining action of the Fix.

Solenoid valve 1 in its normal inactive state creates a diluent path for the flow cell outer sheath. During normal flow cell operation, the center syringe piston (solenoid valve 3 energized and solenoid valves 1 and 2 in a normal inactive state) sends diluent through an electrical isolator and T-fitting to ports 2 and 4 of the flow cell. This creates an outer sheath or fluid pipe around the stream of fluid exiting the flow cell aperture. This sheath is approximately 140 µm in diameter.

During the upward stroke of normal flow cell operation, the 5diff syringe assembly creates the inner sheath flow. Diluent exits from the top of the left injector piston and enters the flow cell at port 5. It then forms the inner sheath, creating a fluid pipe around the injected sample. The sample injector creates sample flow of 40 µm in diameter, while the sample and inner sheath is forced through the aperture, which is 60 µm in diameter. On exiting the aperture, an outer sheath is created and this double sheath around the sample is called Dual Focused Flow, or DFF.

Sample is sent through the flow cell with the right injector piston (see Figure 2.8-13), but it first must be positioned. The sample dilution, created in the DIFF bath, is drained with the waste syringe. The vacuum path to the waste syringe, starting at the bath, is through fluid sensor M1, an energized solenoid valve 4, through the right injector piston (note that the piston is not being used at this time), through an energized solenoid valve 5, T-fitting T6, waste fluid sensor M2, a fluidic/electrical isolator, and solenoid valve 26 in its normal inactive state. Fluid is drained from the bath until sensor M1 detects air. The length and size of the tubing ensures that the sample does not actually reach or enter the right injector piston when sensor M1 detects air and stops flow. This is critical. When the injector piston pushes up, sample in the tubing between T-fitting T2 and port 5 of the right injector piston is sent through the flow cell.

2.8-12

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Probe and Probe Rinse

When valves 9 and 10 are both energized (Figure 2.8-11), diluent output from the diluent syringe is sent to the probe and probe rinse block by way of solenoid valve 18. An energized solenoid 18 routes diluent through the sample syringe and out the probe for backwash and sometimes dilution. Note that the sample syringe is not used at this time. Since the piston seal is an O-ring at the base of the piston, rather than a seal at the top of the piston, the bottom fitting and top fitting on the syringe assembly have an open fluid path. Diluent is sent through an inactive sample syringe by the diluent syringe. The sample syringe is only used when aspirating or dispensing sample.

Figure 2.8-11 Probe Diluent Reagent Circuit

PN 4237616B

With solenoid valve 18 in its normal inactive state, the diluent is sent to fitting 1 on the probe rinse block. This fluid exits the rinse block from fitting 2 and passes through solenoid valve 17 to the waste system. Usually, this occurs while the probe is moving up through the rinse block to clean blood off the exterior of the probe. However, this is not always the case. On occasion, solenoid 17 is not opened, and the probe does not move. Diluent is forced to flow down the outside of the probe. This is done once to clean the exterior of the probe without moving it and another time to add a small amount of diluent to a dilution.

Diluent to Baths

Energizing solenoid valve 9 routes the diluent syringe output through solenoid valve 10 (Figure 2.8-12). In the normal state of valve 10, diluent is routed for use at the sample baths. There are 3 uses of diluent at the baths, diluent for dilution in the Hgb bath, diluent for dilution in the RBC bath, and rinse for the WBC/BASO counting head.

2.8-13

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Solenoid valve 21 routes diluent for dilutions through valve 22 when in a normal inactive state. Valve 22 selects the RBC bath (normal, inactive state) or the Hgb bath (energized state). The fluid paths to the baths both go through a heater block. Each path has a 1 mL coil of fluid in the heater block which allows 1 mL of reagent to be heated to 35°C prior to being delivered to the bath.

Figure 2.8-12 Bath Diluent Reagent Circuit

2.8-14

When solenoid valve 21 is energized, fluid is sent to the WBC/BASO counting head to rinse the pathway and counting head. This is necessary since fluid in this area is used to flush cells from the rear of the RBC/Plt aperture, a technique called the Rinse Flow System (RFS). During the RBC/Plt count, the WBC/BASO count head is connected to the RBC/Plt count head through an inactive (normal state) valve 23. The vacuum applied to the RBC/Plt aperture actually draws fluid (rinse solution, not diluent) from the WBC/BASO bath, through the WBC/BASO aperture, and past the rear of the RBC/Plt aperture, sweeping away any RBC cells from the rear of the aperture. When solenoid valve 23 is energized, the WBC/BASO counting head is connected directly to the counting syringe, providing vacuum for the WBC/BASO count.

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Waste System

The waste and drain system (Figure 2.8-13) comprises many components. The waste syringe itself is used to drain the baths, and to expel waste from the baths. The count syringe expels any waste that it accumulates during count directly into the waste system through normally closed solenoid valve 16. Waste from the probe rinse block is pushed out by the diluent syringe as well as being evacuated with the waste syringe.

The bath drain system connects each bath, including the rinse chamber, through a normally closed solenoid valve to the waste system with a series of T-fittings. There is a fluid isolator chamber between the baths and the waste syringe and container. This electrically isolates the baths (and flow cell and aspirate probe) from any interference that can be picked up by the external waste system. Draining waste from any bath involves opening the associated solenoid valve while the drain syringe is filling. Solenoid valve 26 is then energized, which connects the waste syringe to the external waste system, and waste is expelled.

There is also a fluid sensor, M2, in the waste system, just before the isolator. It is used to detect whether the waste system has fluid or air at the appropriate times. It is not used to stop a drain action, like M1 does when detecting that sample has been drained from the DIFF bath.

Figure 2.8-13 Waste Circuit

PN 4237616B

2.8-15

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2.8-16

PN 4237616B

Beckman Coulter ACT Service manual

Specifications and Main Features

Frequently Asked Questions

User Manual

BECKMAN COULTER™ AC•T™ 5diff Hematology Analyzer

REVISION STATUS

CONTENTS

CONTENTS

1.1 MANUAL DESCRIPTION

Scope

INTRODUCTION

Notification of Updates

Intended Audience

Organization

Numbering Format

Special Headings

WARNING

CAUTION

IMPORTANT

ATTENTION

Note

Conventions

Graphics

1.2 SAFETY PRECAUTIONS

Electronic

Biological

Troubleshooting

CONTENTS

INSTRUMENT DESCRIPTION

2.1 INTRODUCTION TO THE AC•T 5diff HEMATOLOGY ANALYZER

Purpose

Function

Description

Components

Modes of Operation

CBC Mode

CBC/DIFF Mode

Reagent Consumption

2.2 OPERATION PRINCIPLES

Overview

Coulter Principle

Measurement Principles

Aperture Sensor System

Applying the Coulter Principle

ACV Technology

Dual Focused Flow (DFF)

Flow Cell

Focused Flow Impedance

Absorbance Cytochemistry

Signal Processing

Thresholds

WBC/BASO Methodology

Sample Analysis Overview

Aspiration

Dilution

Delivery

Sample Partitioning

2.3 CYCLE DESCRIPTION

Cycle Start Conditions

Sample Flow

2.4 SAMPLE ANALYSIS

RBC and Platelet Analysis

Parameter Results Obtained from the RBC/Plt Dilution

Hgb Measurement

WBC Count and Differential

Parameter Results Obtained from the WBC/BASO Dilution

Differential

Parameter Results Obtained from the DIFF Dilution

Dilution Summary

2.5 RBC PARAMETER DEVELOPMENT

RBC/Plt Dilution

RBC Count

RBC Histogram

Parameter Results Obtained Using the RBC Histogram

Hct Measurement

MCV Calculation

RDW Calculation

RBC Distribution Flags

RBC1 and RBC2 Thresholds

Flags