Luminex 100 User Manual

Lum/nex

Luminex 100

Version 1.7

™

User Manual

© LUMINEX CORPORATION, 1998 - 2004. All rights reserved. No part of this publication may be reproduced, transmitted, transcribed, or translated into any language or computer language, in any form or by any means without prior express, written consent of:

LUMINEX CORPORATION

12212 Technology Boulevard

Austin, Texas 78727-6115

U.S.A.

Voice: (512) 219-8020

Fax: (512) 219-5195

Technical Support U.S. and Canada: toll-free 1-877-785-2323

Technical Support international: +1 512-381-4397

Technical Support fax: 512-219-5195

Technical Support website:

http://luminexcorp.custhelp.com.

Luminex® 100™ User Manual Version 1.7

CN-M027-01

PN 89-00002-00-063 Rev. A

March, 2004

Luminex reserves the right to modify its products and services at any time. This guide is subject to change without notice. Although prepared to ensure accuracy, Luminex assumes no liability for errors or omissions, or for any damages resulting from the application or use of this information.

The following are trademarks of Luminex Corporation: Luminex, Luminex 100, Luminex HTS, Luminex 100 IS, LabMAP, LumAvidin, Luminex XYP, Luminex SD, xMAP, and FlexMAP. All other

trademarks, including Windows, Cheminert, Tween, Pentium, Dell, Alexa and Titertube trademarks of their respective companies.

The contents of this manual and the associated Luminex software are the property of LUMINEX CORPORATION and are copyrighted. Any reproduction in whole or in part is strictly prohibited.

are registered

End-User License Agreement (EULA) for Luminex® Software

This Luminex End-User License Agreement (“EULA”) is a legal agreement between you (either an individual or a single entity, also referred herein as “you”) the end-user and Luminex Corporation (“Luminex”) regarding the use of the Luminex software product identified above, which includes computer software and online or electronic documentation and may include associated media and printed materials (if any) (“SOFTWARE PRODUCT” or “SOFTWARE”).

The SOFTWARE PRODUCT is protected by copyright laws and international copyright treaties, as well as other intellectual property laws and treaties. The SOFTWARE PRODUCT is licensed, not sold.

1. GRANT OF LICENSE. Subject to the terms and conditions of this EULA, Luminex hereby grants to you a nonexclusive, nontransferable, nonassignable license (without right to sublicense) under Luminex’s copyrights and trade secrets to use the SOFTWARE PRODUCT on a hardware platform purchased from Luminex pursuant to Luminex’s terms and conditions of sale. You may make one (1) copy of the SOFTWARE PRODUCT for backup or archival purposes only. Although no rights or licenses under any of Luminex's patents are granted by or shall be implied from the license of the SOFTWARE or the sale of Luminex instrumentation to you, the purchaser, you may obtain a license under Luminex’s patents, if any, to use this unit of Luminex instrumentation with fluorescently labeled microsphere beads authorized by Luminex by purchasing such microspheres from Luminex or an authorized Luminex reseller.

2. RESTRICTIONS.

• You must maintain all proprietary notices on all copies of the SOFTWARE PRODUCT.

• You may not distribute copies of the SOFTWARE PRODUCT to third parties.

• You may not reverse-engineer, decompile, disassemble, or otherwise attempt to derive source code from the SOFTWARE PRODUCT.

• You may not copy (other than one backup or archival copy), distribute, sublicense, rent, lease, transfer or grant any rights in or to all or any portion of the SOFTWARE PRODUCT.

• You must comply with all applicable laws regarding the use of the SOFTWARE PRODUCT.

• You may not modify or prepare derivative works of the SOFTWARE PRODUCT.

• You may not use the SOFTWARE PRODUCT in a computer-based service business or publicly display visual output of the SOFTWARE PRODUCT.

• You may not transmit the SOFTWARE PRODUCT over a network, by telephone, or electronically by any means.

3. TERM AND TERMINATION. Your rights under this EULA are effective until termination. You may terminate this EULA at any time by destroying the SOFTWARE PRODUCT, including all computer programs and documentation, and erasing any copies residing on your computer equipment. Luminex may terminate this EULA upon thirty (30) days written notice to you. Your rights under this EULA automatically terminate without further action on the part of Luminex if you do not comply with any of the terms or conditions of this EULA. Upon any termination of this EULA, you agree to destroy the SOFTWARE PRODUCT and erase any copies residing on your computer equipment.

4. RIGHTS IN SOFTWARE. All rights and title in and to the SOFTWARE PRODUCT and any copies thereof are owned by Luminex or its suppliers. This EULA is not a sale and does not transfer to you any title or ownership interest in or to the SOFTWARE or any patent, copyright, trade secret, trade name, trademark or other intellectual property right therein. You shall not remove, alter, or obscure any proprietary notices contained on or within the SOFTWARE and shall reproduce such notices on any back-up copy of the SOFTWARE. All title and intellectual property rights in and to the content which may

be accessed through use of the SOFTWARE PRODUCT is the property of the respective content owner and may be protected by applicable copyright or other intellectual property laws and treaties. This EULA grants you no rights to use such content.

5. EXPORT RESTRICTIONS. You agree that you will not export or re-export the SOFTWARE PRODUCT to any country, person, entity, or end-user subject to U.S.A. export restrictions. You hereby warrant no state or federal agency has suspended, revoked, or denied your export privileges.

6. NO WARRANTY. THE SOFTWARE PRODUCT IS LICENSED “AS IS.” ANY USE OF THE SOFTWARE PRODUCT IS AT YOUR OWN RISK. THE SOFTWARE PRODUCT IS PROVIDED FOR USE ONLY WITH LUMINEX PRODUCTS. TO THE MAXIMUM EXTENT PERMITTED BY APPLICABLE LAW, LUMINEX AND ITS SUPPLIERS DISCLAIM ALL WARRANTIES, EITHER EXPRESS OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, AND NONINFRINGEMENT.

7. LIMITATION OF LIABILITY. IN NO EVENT SHALL LUMINEX OR ITS SUPPLIERS BE LIABLE FOR ANY SPECIAL, INCIDENTAL, INDIRECT, OR CONSEQUENTIAL DAMAGES WHATSOEVER (INCLUDING, WITHOUT LIMITATION, DAMAGES FOR LOSS OF BUSINESS PROFITS, BUSINESS INTERRUPTION, LOSS OF BUSINESS INFORMATION, OR ANY OTHER PECUNIARY LOSS) ARISING OUT OF THE USE OF OR INABILITY TO USE THE SOFTWARE PRODUCT, EVEN IF LUMINEX HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES.

MISCELLANEOUS. This EULA is governed by the laws of the State of Texas, U.S.A., without reference to conflicts of laws principles. You shall not assign or sublicense or otherwise transfer the rights or license granted hereunder, by agreement or by operation of law, without the prior written consent of Luminex, and all assignments in violation of this prohibition shall be null and void. This EULA is the complete and exclusive agreement of Luminex and you and supersedes all other communications, oral or written, relating to the subject matter hereof. No change to this EULA shall be valid unless in writing and signed by the party against whom enforcement is sought. The waiver or failure of Luminex or you to exercise in any respect any right or rights provided for herein shall not be deemed a waiver of any further right hereunder. If any provision of this EULA is held unenforceable, the remainder of this EULA will continue in full force and effect.

EULA PN: 89-30000-00-070

Contents

Introduction 1-1

Principle of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

Basic Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2

Excitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-3

xMAP Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Reporter Fluorochromes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Fluorescence Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

Repetitive Microsphere Measurements . . . . . . . . . . . . . . . . . .1-4

About this Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-4

Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-5

Safety 2-1

Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1

Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1

Warnings and Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-2

Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2

Electrical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3

Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

Mechanical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7

Biological . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7

Heat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

Blue Indicator Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8

System Overview 3-1

Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

xMAP Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2

Luminex 100 Analyzer Specifications . . . . . . . . . . . . . . . . . . . . . .3-2

General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2

Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2

Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3

Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3

Signal Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

PC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-3

Luminex XYP Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4

General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4

Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

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Luminex 100 User Manual Version 1.7 xMAP Technology

Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Recommended Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Printer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Uninterruptible power supply (UPS) . . . . . . . . . . . . . . . . . . . . 3-4

Surge Protector. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4

Luminex 100 Analyzer Overview . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

Electronic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

Fluidic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-6

Optical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-9

Luminex XYP Instrument Overview . . . . . . . . . . . . . . . . . . . . . . 3-10

Reservoir . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

Heater Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-10

Installation 4-1

Luminex 100 System Installation. . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

Luminex 100 Software Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3

Adjusting the Sample Probe Height . . . . . . . . . . . . . . . . . . . . . . . . 4-4

Installing Software and Firmware . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

What to Do Next. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

System Startup 5-1

Luminex Main Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-2

Add New Classification Channel Calibration Lots . . . . . . . . . 5-2

Add New Reporter Channel Calibration Lots . . . . . . . . . . . . . 5-4

Calibrate the Luminex 100 Analyzer. . . . . . . . . . . . . . . . . . . . 5-5

Preview or Print Calibration Report . . . . . . . . . . . . . . . . . . . . 5-7

Analyzer menu commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-9

Data Acquisition 6-1

Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

User tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

Acquisition tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

Advanced tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

Info tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Setup XY Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Sessions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-5

Create a Session Using Default Settings. . . . . . . . . . . . . . . . . 6-6

Create a Session Using a Template . . . . . . . . . . . . . . . . . . . . . 6-6

Open and Add to an Existing Session . . . . . . . . . . . . . . . . . . . 6-8

Open an Existing Session and Save it as a New Session. . . . . 6-9

Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Create a Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Modify a Template. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-9

Delete a Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10

Multiplex Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10

Menu Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10

ii PN 89-00002-00-063 Rev. A

xMAP Technology Contents

File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-10

Edit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-11

View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-11

Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-12

Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-12

Main Control and Session Detail . . . . . . . . . . . . . . . . . . . . . . 6-12

Right-Click Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14

Session Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14

General tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-14

Bead Set tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-16

Histogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-17

Gates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18

Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-19

Bar Graph/Dot Plot Display . . . . . . . . . . . . . . . . . . . . . . . . . . 6-20

System Monitor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-22

Status Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-24

Data Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24

Run an Entire Plate of Samples . . . . . . . . . . . . . . . . . . . . . . .6-25

Run a Range of Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-26

Run a Single Sample. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-27

Interrupt a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-27

Reports. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-28

Maintenance and Cleaning 7-1

Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1

Shutdown. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-2

Check Fluid Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-2

Check for Leaks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

Calibrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

Sanitize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

Clean Accessible Surfaces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

Clean Sample Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4

Replace Air Intake Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4

Replace Syringe Seal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-6

Clean Luminex 100 Analyzer Ventilation Filter. . . . . . . . . . . . . . . 7-7

Change Sheath Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-7

Change the Sheath Filter and Quick-disconnect O-rings . . . . . . . .7-8

Change Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-8

Adjust Sample Probe Vertical Height . . . . . . . . . . . . . . . . . . . . . . .7-9

Adjusting the Sample Probe Horizontal Position . . . . . . . . . . 7-11

Decontaminating the Luminex 100 Analyzer for

Return Shipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-12

PN 89-00002-00-063 Rev. A iii

Luminex 100 User Manual Version 1.7 xMAP Technology

Troubleshooting 8-1

Power Supply Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2

Communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2

Pressurization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-3

Fluid Leaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-4

Sample Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5

Calibration Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-6

Acquisition Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-8

Bead Detail Irregularities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-10

Error Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-12

Printing Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14

Verification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-14

Protocols 9-1

xMAP Microsphere Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1

Microsphere Dispersion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1

Probe Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-2

Bath Sonicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-2

Enumeration of xMAP Microsphere Suspensions. . . . . . . . . . 9-3

xMAP Microsphere Separation Methods. . . . . . . . . . . . . . . . . 9-3

xMAP Microsphere Agitation During Assay . . . . . . . . . . . . . 9-3

xMAP Microsphere Stability and Storage . . . . . . . . . . . . . . . . 9-4

Two-Step Carbodiimide Coupling of Protein to xMAP

Carboxylated Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5

Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-5

Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6

Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6

Technical Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-7

One-Step Carbodiimide Coupling of Oligonucleotides to xMAP

Carboxylated Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8

Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8

Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8

Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-9

Technical notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10

Binding Biotin-conjugated Molecules to LumAvidin-Modified

xMAP Microspheres. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10

Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10

Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-11

Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-11

Technical notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-12

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Glossary A-1

Product Numbers B-1

Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1

Software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-2

Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3

Luminex Sheath Delivery System C-1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2

Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3

Using the Sheath Delivery System . . . . . . . . . . . . . . . . . . . . . . . . C-5

Audible Alarm and

Fault LED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-5

Maintenance and Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . C-5

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6

Contact Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-6

Labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-7

Index Index-1

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Introduction

The Luminex® 100 system is a compact analysis unit consisting of a Luminex 100 analyzer, the Luminex XY Platform (Luminex XYP™), a PC, Luminex software, and reagents. The Luminex 100 analyzer integrates optics, fluidics, and advanced signal processing in a 17 inch by 20 inch footprint. The Luminex XYP provides efficient handling of 96-well microtiter plates. The PC uses Luminex software to control the analyzer. The optional Luminex Sheath Delivery system (Luminex SD™) is used to provide sheath fluid directly from a 20 liter container.

Photodiodes and a photomultiplier tube receive fluorescent signals from xMAP

and delivers the signals to a digital signal processor (DSP). Proprietary algorithms function with the DSP to greatly increase the sensitivity.

microspheres. The analyzer digitizes the waveforms

Hardware and preloaded software simplify installation and setup, and with the help of this manual, you will be able to begin using xMAP technology.

Principle of Operation

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The Luminex 100 system analyzes immunoassays, complex genetic analyses, and enzymatic assays in one simple format. The reactants (antibodies, oligonucleotides, substrates, etc.) of these assays are anchored to the surfaces of uniquely fluorescent microscopic beads, called microspheres. The unique fluorescent emission spectra of a given microsphere identifies each of the assays performed simultaneously on a single sample.

The Luminex 100 analyzer individually analyzes xMAP microspheres in a flow stream. Every xMAP microsphere is accurately classified to its own subset, based on its fluorescent

Luminex 100 User Manual Version 1.7 xMAP Technology

signature. In addition, the Luminex 100 analyzer scans each microsphere for the presence of a reporter fluorescence that quantifies the assay at the microsphere’s surface.

The Luminex XYP works with 96-well plates that are no thicker than

0.75 inches. If you are not using the heater block, any brand of microtiter plate that meets this size requirement will work. If you are using the heater block, we recommend one of these two plates:

• Polyfiltronics black.

•Costar

The system reads the wells in column-first order. An entire column of wells is read; that is, well A1, B1, C1, D1, and so on to the end of the column. Then the XY Platform moves over to the next column and reads well A2, B2, C2, D2, and so on. When placing a plate on the plate holder, place it so well A1 is in the upper-left corner of the holder. You can run an entire plate, a consecutive range of samples from the plate, or a single sample from the plate.

model P.

96-well plate, 200 µL, catalog #7703-1902,

Thermowell® 96-well plate, thin-wall polycarbonate,

Basic Concepts Luminex has pioneered a versatile and robust technology for the

measurement of soluble analytes. The Luminex 100 system performs simultaneous, discrete measurements of multiple microsphere-based reactions from a single specimen aliquot. A clear understanding of the concepts and functionality associated with the instrument and xMAP microspheres contributes to greater success with this technology. We present a brief overview of some of the basic concepts. For more information, refer to Practical Flow Cytometry, 4th edition, by Howard M. Shapiro, M.D. (New York: Wiley-Liss Inc., 2003).

Fluidics There are two fluidic paths in the Luminex 100 analyzer. The first

path is a syringe-driven mechanism that controls the sample uptake. This mechanism permits small sample uptake volumes from small reaction volumes. The syringe-driven system transports a userspecified volume of sample from a microtiter plate to the cuvette. The sample is injected into the cuvette at a steady rate for analysis. After analysis, the sample path is purged with sheath fluid by the second fluidics path. This process expels sheath fluid into the sample container and effectively removes residual sample within the tubing, valves, and probe. The second fluidics path is driven under positive air pressure and supplies sheath fluid to the cuvette.

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xMAP Technology Introduction

Excitation The excitation system in the Luminex 100 analyzer uses two solid-

state lasers. A reporter laser excites fluorescent molecules bound to biological reactants at the microsphere surface, and a classification laser excites fluorochromes embedded in the microsphere. The lasers illuminate the xMAP microspheres as they flow single-file through the cuvette. These fluorescent signals are discriminated with selective emission filters and are converted into intensity units by a digital signal processor.

xMAP Microspheres The xMAP microspheres are highly uniform, polystyrene particles

that have been crosslinked during polymerization for great physical and thermal stability. Varying amounts of fluorochromes embedded within each microsphere give each microsphere set an unique fluorescent signal. To ensure the stability of this address, it is essential to protect the xMAP microspheres from light. Do not subject xMAP microspheres to prolonged high temperatures, and protect them from freeze/thaw manipulations.

Reporter Fluorochromes Each xMAP microsphere is dyed to emit light in a certain

classification channel. All xMAP microspheres of a given emission represent a distinct assay within a multiplex of assays. A reporter channel is used to detect fluorescence bound to the surface of each microsphere, and each reporter emission quantitates each of the distinct assays. Only one reporter emission is used for multi-analyte assays in the Luminex 100 analyzer. The Luminex 100 analyzer uses an excitation wavelength of 532 nm, and has an emission wavelength of 575 nm ± 12 nm.

Fluorochromes R-Phycoerythrin, and Alexa

Luminex 100 analyzer, but you are not limited to these two. You may have success using other fluorochromes such as Cyanine 3.

Phycoerythrin

Formula weight (Daltons) 240,000 470

Absorbance max (nm) 480, 546, 565 531

Extinction max (M

-1cm-1

)

1,960,000 83,800

532 work well with the

R-

Alexa

532

Emission max (nm) 578 554

Quantum yield 0.82 0.8

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Fluorescence Compensation

Repetitive Microsphere Measurements

Fluorescent spillover occurs when an emission spectrum overlaps other emission criteria; therefore, selective filtering cannot occur. In most analyzers, emission spillover is corrected using a technique called compensation. Compensation involves subtraction of emission percentage from another emission signal. In the Luminex 100 system, compensation is not necessary—the reporter signal does not spill over into classification emissions.

Bound to reagents at the surfaces of the xMAP microspheres, the reporter provides raw analytical data. Because a microsphere suspension provides near liquid phase reaction kinetics, each microsphere theoretically binds an equal number of reporter molecules. Equal binding results in a statistically even distribution of reporter on each microsphere in a set. When the Luminex 100 analyzer registers numerous events for each set, the system reports individual population values based on an even coating of reporter. This means that numerous replicates for each microsphere population are measured from a single reaction vessel. The confidence in a given measurement strengthens with increased replicate measurements. For adequate confidence, 100 collected events per microsphere set in each reaction vessel is usually sufficient. Measurements are reported as the median for the xMAP microspheres having a specific color signal.

About this Manual This manual introduces you to the Luminex 100 system. With step-

by-step instructions, the manual guides you through initial setup and powering-on procedures. Next, it offers a concepts section which describes some of the key topics encountered during system operation. Once you have a working knowledge of the analyzer, the manual’s text and figures lead you through the data collection process, giving detailed explanations of available options and offering examples when necessary. Glossary and troubleshooting sections assist as references. In short, this manual instructs you through each operational stage—from unpacking the Luminex 100 analyzer and XYP to collecting your data.

The conventions in this document assume a basic familiarity with

computers and a knowledge of Windows document only one method of accessing a command, although many commands are available through more than one method, such as

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software. We typically

xMAP Technology Introduction

from the main menu bar, from the toolbar, and from menus that appear when you right-click an area of the screen.

Technical Assistance

You can find answers to frequently asked questions (FAQs) on our website: http://luminexcorp.custhelp.com.

Technical assistance is available to users in the U.S. and Canada by calling toll free 1-877-785-BEAD (1-877-785-2323) between the hours of 7:00 a.m. and 7:00 p.m. Central Time, Monday through Friday. Users outside of the U.S. and Canada can call us at +1 512381-4397 between the hours of 7:00 a.m. and 7:00 p.m. Central Time, Monday through Friday.

Inquiries may also be sent by email to support@luminexcorp.com.

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Safety

Your safety is important. Please do not perform procedures on your Luminex 100 system that are not specifically contained in this manual, unless you are directed to do so by Luminex Technical Support. This chapter discusses the following safety topics

• Intended Use

• Symbols

• Safety Precautions

Intended Use The Luminex 100 analyzer is designed for a wide range of indoor

laboratory testing applications measuring biomolecular reactions on the surfaces of xMAP microspheres. This system is intended for indoor research use only and not for use in diagnostic procedures.

The Luminex XYP instrument is intended for use with the Luminex 100 analyzer. With the Luminex XYP, you can process 96-well microtiter plates without handling individual samples. The Luminex XYP works with microtiter plates that are no thicker than 0.75 in.

Symbols These symbols describe warnings, cautions, and general information

used in the operation of this analyzer.

Symbol Description

1 Alternating current

2 Protective ground

3On

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Symbol Description

4Off

5 Warning (see manual).

6 Warning (see manual).

7 Warning (see manual).

8 Warning (see manual).

9 Warning (see manual).

Warning (refer to manual)

Warnings and Notes Informational notes and warnings may appear in this manual.

Note: A note provides general helpful information. No safety or performance issues are involved.

Caution: This message is used in cases where the hazard is minor or only potential hazard is present. Failure to comply with the caution may result in potentially hazardous conditions.

Warning: This message is used in cases where danger to the operator or to the performance of the instrument is present. Failure to comply with the warning may result in incorrect performance, instrument failure, invalid results, or hazard to the operator.

Danger: This message is used in cases where significant risk of serious injury or death is present.

Safety Precautions Please read the following safety information before setting up or

using the Luminex 100 system. A user should always be present during system operation. This system contains electrical, mechanical, and laser components which, if handled improperly, are potentially harmful. In addition, biological hazards may be present during Luminex 100 analyzer operation. Therefore, Luminex Corporation recommends that all Luminex 100 analyzer users become familiar

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xMAP Technology Safety

with the specific safety advisories below, in addition to adhering to standard laboratory safety practices. The protection provided by the equipment may be impaired or the warranty voided if the equipment is used in a manner not specified by Luminex Corporation.

Electrical The Luminex 100 analyzer and Luminex XYP must be connected to

approved power sources.

Do not perform any maintenance or cleaning of the system’s electrical components (except for the fuses).

This caution label appears on the back of Luminex 100 analyzers using the Coherent laser, and on the Luminex XYP:

Figure 2-1. Fuse Replacement Warning Label

This label appears on the back of Luminex 100 analyzers using the Uniphase laser:

Figure 2-2. Fuse Replacement Warning Label - Uniphase Laser

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This label appears on the back of the Luminex 100 analyzer and the Luminex XYP.

Figure 2-3. European Safety Requirements Label

The Luminex 100 analyzer complies with European Union (EU) safety requirements and, therefore, may be marketed in the Europe Single Market.

One of the following voltage labels displays on the back of the Luminex 100 analyzer, depending if the instrument uses a Coherent or Uniphase laser:

Figure 2-4. Luminex 100 Voltage Label (Coherent)

Figure 2-5. Luminex 100 Voltage Label (Uniphase)

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xMAP Technology Safety

This voltage label displays on the back of the Luminex XYP:

Figure 2-6. Luminex XYP Voltage Label

Fluidics The Luminex 100 analyzer contains fluidics. In the event of a fluid

leak, turn off all power to the system and disconnect all power cords.

Warning: If the system is used

to test biological samples, use your standard laboratory safety practices when handling system waste.

Remember that the on/off switch is not a disconnect means - the power cord must be removed from the outlet. Contact Luminex Corporation for further information.

You must manually monitor waste levels. Do not allow the waste container to overflow. Empty the waste container each time you fill the sheath fluid container. Do not place the waste container on top of the analyzer.

United States and international regulations require the following warnings to appear on the analyzer during operation and maintenance.

This label appears on the back panel of the analyzer:

Figure 2-7. Laser Class Label

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This label appears on the back panel of the Luminex 100 analyzer:

Figure 2-8. Laser Caution Label

Do not remove the analyzer cover. When performing routine maintenance, ensure that power to the analyzer is OFF and the power cord is disconnected.

All laser apertures in the analyzer are contained within a protective housing. This label appears on the optics cover within the Luminex 100 analyzer.

Figure 2-9. Laser Caution Label on Optics Cover

This label appears above the laser apertures located inside the optics enclosure inside the Luminex 100 analyzer.

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xMAP Technology Safety

Figure 2-10. Avoid Exposure Label

Caution: Use of controls or adjustments or performance of

procedures other than those specified herein may result in hazardous radiation exposure.

Caution: To avoid exposure to hazardous radiation, only perform

procedures and adjustments as specified in this manual.

Mechanical

Warning: During operation, this system contains exposed,

moving parts. Risk of personal injury is present. Observe all warnings and cautions.

Note: Access doors must be

closed while operating the instrument. An operator must be present during operation of the instrument.

Biological

Warning: During operation, this system contains exposed,

moving parts which could result in puncture hazard. Risk of personal injury is present. Keep hands and fingers away from the sample probe. Keep hands and fingers out of the Luminex XYP slot during operation.

Warning: During operation, this system contains exposed,

moving parts which could result in pinch point hazard. Risk of personal injury is present. Keep hands and fingers away from the syringe arm.

Warning: Human/animal samples may contain biohazardous

infectious agents. Where exposure (including aerosol) to potentially biohazardous materials exists, follow appropriate biosafety procedures and use personal protective equipment, such as gloves, gowns, laboratory coats, face shields or masks, eye protection, and ventilation devices. Observe all local, state, and federal biohazard handling regulations when disposing of biohazardous waste material.

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Heat

Warning: The heater plate of the Luminex XYP may be hot and

could cause personal injury if touched. Do not touch the heater plate.

Blue Indicator Light

The blue light above the sample arm simply indicates the on/off status of the instrument, and is harmless. The blue light emitting diode (LED) does not emit light in the UV spectrum.

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System Overview

Hardware The Luminex 100 Version 1.7 system includes a Luminex 100

Version 1.7 analyzer, the Luminex XYP, and a PC.

The Luminex 100 analyzer includes the following hardware:

• Luminex 100 analyzer

• Sample tube holders (1.5 mL and 1.2 mL)

• Sheath fluid container

• Waste container

• Communications cable to connect analyzer with computer

The Luminex XYP includes the following hardware:

• Luminex XYP instrument

• Power cord

• Communication cable to connect to the Luminex XYP

• Alignment guide

• Luminex XYP sample probe (long probe)

• Reservoir

•Shield

• Luminex XYP heater block

• Probe alignment toolkit

• Allen wrench, 3/32 inch hexagonal

The PC includes the following hardware:

• Computer: a Pentium

• Monitor

• Power cords

• Keyboard

•Mouse

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Luminex 100 User Manual Version 1.7 xMAP Technology

In addition, you can purchase the optional Luminex Sheath Delivery system (Luminex SD).

xMAP Reagents • xMAP classification calibration microspheres (CAL1)

• xMAP reporter calibration microspheres (CAL2)

• Sheath fluid

Software Luminex Data Collector software provides complete control of the

Luminex 100 analyzer and performs real-time digital analysis of assays. The Luminex 100 system is preloaded with the Luminex software. However, we supply a software CD in case you need to reinstall the software.

Luminex 100 Analyzer Specifications

General • Operating temperature: 15°C to 30°C (59°F to 86°F)

• Storage temperature: 0°C to 50°C (32°F to 122°F)

• Humidity: 20% to 80%, noncondensing

• Altitude: designed to operate up to 2400 m (7874 ft.) above mean sea level

• Physical dimensions: 43 cm (17 in) W x 51 cm (20 in) D x 23 cm (9 in) H

• Weight: 23 kg (60 lbs)

• UL installation category: UL Installation Category II, as defined in Annex J of UL 61010A-1

• Pollution degree: UL Pollution Degree 2, as defined in section

3.7.3.2 of UL 61010A-1

Power • Input voltage range:

• 100-240 V~, 1.5 Amps, 47-63 Hz

• 100-120 V~ and 200-240 V~, 1.4 Amps, 47-63 Hz. See label

on rear of instrument.

• AC inlet fuse: 3A, 250V~, Fast Acting OR 2A, 250 V~, Fast Acting. See label on rear of instrument.

• Shipping and storage: allowable shipping and storage temperature and humidity ranges, 0°C to +50°C and 20%-80% noncondensing, respectively.

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xMAP Technology System Overview

Laser • Reporter laser: 532 nm, nominal output 10-16.5 mW, maximum

500 mW, frequency-doubled diode; mode of operation mW, continuous wave (CW)

• Classification laser: 635 nm, nominal output 9.1 mW ± 6%, maximum output 25 mW, diode; mode of operation, continuous wave (CW)

Fluidics • Sheath flow rate: 90 µL/second ± 5 µL/second

• Cuvette: 200 micron square flow channel

• Sample injection rates: 60 µL/minute, 45 µL/minute, or 30 µL/ minute

• Sample uptake volume: 10-200 µL

Electronics • Reporter channel detection: photomultiplier tube,

A/D resolution, 14 bits

• Classification and doublet discriminator channel detection: avalanche photo diodes (APD) with temperature compensation, A/D resolution 12 bits

• Communications interface: RS 232

Signal Processing • Signal modes: linear, with logarithmic or linear display option

PC • Computer: a Pentium III microprocessor or equivalent class PC,

minimum speed 933 MHz

• Main memory: minimum of 128 MB

• Hard disk drive: 10 GB minimum storage capacity

• Communications, serial ports: minimum of 2 RS-232 compatible serial ports

• Operating system support: MS

• CE marked and UL listed

• Two-button mouse or equivalent

• Monitor: minimum of 17-inch diagonal monitor

• Keyboard: a 104-key keyboard or equivalent

• Power cords for the US and Canada are included in the system. Power cords for other countries must be ordered separately.

• Power: 115-230 V~, Maximum 6.0 Amps, 50-60 Hz

Windows

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Luminex XYP Specifications

General • Operating temperature: 15°C to 30°C (59°F to 86°F)

• Humidity: 20% to 80%, noncondensing

• Altitude: designed to operate at 2400 m (7874 ft.) above mean sea level or below.

• Physical dimensions: 44 cm (17.25 in) W x 61 cm (24 in) D x 8 cm (3 in) H

• Weight: 14.5 kg (32 lbs)

• UL installation category: UL Installation Category II, as defined in Annex J of UL 3101-1.

• Pollution degree: UL Pollution Degree 2, as defined in section

3.7.3.2 of UL 3101-1.

• Heater operating range: 35°C to 60°C (95°F to 140°F) with tolerance 0°C to +2°C.

• Shipping and storage: allowable shipping and storage temperature and humidity ranges, 0°C to +50°C and 20%-80% noncondensing, respectively.

Power • Input voltage range: 100-240 V~, 1.8 Amps, 47-63 Hz

• AC Inlet Fuse: 3 Amp, 250 V~, Fast Acting

Electronics Communications interface: RS-232

Recommended Equipment

You may find the following items helpful to protect the electronic components of your Luminex 100 System.

Printer We recommend that you have a printer installed to your system so

that you can print reports and diagnostics. We recommend an HP LaserJet 2200 printer.

Uninterruptible power supply (UPS)

Luminex highly recommends using an uninterruptible power supply (UPS) to protect your system from a power outage. Choose a supply that can provide 1050 Watts for at least 45 minutes. The UPS should be UL listed and CSA certified. The UPS also needs to be CE marked if used internationally.

Surge Protector If you do not use a UPS, use a surge protector. Choose a protector

that meets your needs. Factors to consider include electrical environment, endurance, suppressed voltage rating, and method of protection. It should have six outlets, be rated at least 1500 Watts, and be UL listed, CSA certified, CE marked for nondomestic use when used internationally.

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xMAP Technology System Overview

Luminex 100 Analyzer Overview

The Luminex 100 analyzer consists of three subsystems: electronic, fluidic and optical. The following section describes the useraccessible components of each subsystem. Chapter 7, "Maintenance and Cleaning", describes routine maintenance for each of these components.

Electronic Power input module: Houses the analyzer on/off switch and

fuses.

P1 communications port (DB9): Connects the Luminex 100 analyzer to the computer.

Analyzer ventilation filter: Provides filtration for the analyzer ventilation system. To ensure proper ventilation, do not obstruct the area below the analyzer. Allow at least two inches of clearance around the analyzer.

Air Intake Access Door

Power Input Module

XYP Instrument Ventilation Filter

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Communications Ports (DB9)

Figure 3-1. Rear View of Luminex 100 Analyzer

XYP to PC Serial cable

Analyzer Ventilation Filter

Luminex 100 User Manual Version 1.7 xMAP Technology

Fluidic Sample Arm: Transports the sample from the sample plate to the

cuvette. Upon operation, the carriage drops automatically to the sample plate for sample retrieval.

Sample Probe: Acquires sample from the sample plate.

Warning: During operation,

this system contains exposed, moving parts which can cause puncture wounds. Risk of personal injury is present. Keep hands and fingers away from the sample probe.

Note: The probe shield is not in

place for this photo. You should always replace the probe shield before operating the system.

Cheminert

® fitting: Holds the sample probe in place. Use the

wheel to adjust the sample probe height.

Alignment Guide: Directs the sample probe into the Luminex XYP instrument.

Cheminert Fitting

Vertical Adjustment Screw

Alignment Guide

Figure 3-2. Sample Uptake Components

Access doors: There are two access doors on the face of the analyzer. The center door provides access to the syringe. The left door provides service panel access. Inside the left door, a bracket holds the heater block for the Luminex XYP. There is also a clip for the allen wrench included with the Luminex 100 analyzer.

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+ 116 hidden pages