Luminex 100 User Manual

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Luminex 100

Version 1.7

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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

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

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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xMAP Technology Contents

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

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

1 - 4 PN 89-00002-00-063 Rev. A

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

2 - 2 PN 89-00002-00-063 Rev. A

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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microprocessor class PC

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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xMAP Technology System Overview

Left Door, Access to Service Panel

Figure 3-3. Front View of Luminex 100 Analyzer Showing Access Doors

Center Door, Access to Syringe

Air filter and access door: Cleans the air used to pressurize sheath fluid. This filter is enclosed behind an access door located on the back of the Luminex 100 analyzer.

Figure 3-4. Air Filter on Rear of Instrument

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Syringe: Delivers sample from the sample plate to the cuvette. It is located in the compartment behind the center door.

Syringe Seal

Syringe

Figure 3-5. Syringe

Sheath filter: Removes particles greater than 10 microns in diameter from the sheath fluid. It is located behind the left access door.

Sheath Filter Assembly

Figure 3-6. Sheath Filter Assembly

Air, waste fluid, and sheath fluid connectors: Connect to the sheath and waste fluid containers. The air connector is green, the sheath fluid connector is blue, and the waste fluid connector is orange.

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xMAP Technology System Overview

Sheath Fluid Connector (Blue)

Figure 3-7. Left Side of Analyzer - Air and Fluid Connectors

Air Connector (Green)

Waste Connector (Orange)

Sheath fluid container (not shown): Holds sheath fluid. For proper operation, place the container at the same level as the analyzer. Do not place the container on top of the Luminex 100 analyzer.

The sheath fluid level should be below the air inlet connection and above the sheath outlet connection. Sheath fluid levels must be monitored manually. Check the sheath fluid level before starting a run or procedure.

Waste fluid container (not shown): Receives waste from the system. Monitor the waste fluid levels—do not allow the waste container to overflow! Empty the waste container each time you fill the sheath fluid container. Do not put the waste container on top of the Luminex 100 analyzer.

Optical The optical system contains the optics assembly and the excitation

lasers. The optical assemblies do not require user adjustment.

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Luminex XYP Instrument

The Luminex XYP is a self-contained system. There is one access door that contains a plate holder and a reservoir. A heater plate can be placed in the plate holder.

Overview

Reservoir You can use 70% isopropanol or 70% ethanol, deionized water, and

10%-20% bleach solution in the reservoir for Luminex 100 analyzer cleaning and maintenance procedures.

Heater Block Use the heater block as required by your assay procedures. When

you are not using the heater block you can store it in the bracket inside the left access door of the Luminex 100 analyzer.

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Installation

Luminex 100 System Installation

Note: The Luminex 100 System is

too heavy for one person to lift alone. Two people should unpack and lift the system to the workbench.

Ensure that the facility complies with all system and safety requirements.

If you are using the optional Luminex SD, install it after completely installing the Luminex 100 analyzer and the Luminex XYP. See the Luminex Sheath Delivery System appendix for instructions.

 To set up and start up the Luminex 100 system:

1. Unpack the Luminex 100 analyzer, Luminex XYP instrument, and the PC. Review the hardware list on page 3-1 and identify each system component.

2. Place the Luminex XYP on a clean, flat surface.

3. Remove the red shipping pin. Leave the silver knob in place.

Shipping Pin

Figure 4-1. XYP With Shipping Pin in Place

4. Attach the power cord and the communication cable to the Luminex XYP. Do not plug the power cord into the outlet.

5. Place the Luminex 100 on top of the Luminex XYP. Do not plug it into the power outlet.

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6. Replace the short sample probe with the long sample probe as follows: Unsnap the light housing above the probe. Loosen the Cheminert fitting that holds the sample probe in place. Gently grasp the probe and push up. Remove the sample probe out of the top of the sample arm. Push the long sample probe into the top of the sample arm, and tighten the Cheminert fitting.

7. Install the alignment guide by screwing it into the Luminex XYP as shown below. Adjust the position of the Luminex 100 analyzer with the Luminex XYP as necessary.

Alignment Guide

Figure 4-2. Front of Luminex 100 System, Alignment Guide in Place

8. Attach the waste container.

9. Fill and attach the sheath fluid container. Fill the container with sheath fluid to just below the air intake.

 To set up the PC:

1. Place the PC to the right of the Luminex 100 analyzer and Luminex XYP. Do not place the PC on top of the analyzer.

2. Place the monitor on top of the PC and connect the cable between the monitor and the PC.

3. Attach a power cable to the power input modules of the PC and monitor. Do not plug the power cable into the power outlet.

4. Connect the mouse and keyboard to the PC.

5. Connect one of the communications cables between the Luminex 100 analyzer communications port and COM Port 1 on the PC.

6. Connect the other communications cable to between the XYP instrument communications port to and COM port 2 on the PC.

7. Plug all instrument power cords into an approved power source.

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Luminex 100 Software Setup

After performing the Luminex 100 system assembly, ensure that the proper interface between the analyzer and PC is in place.

 To set up the software:

1. Turn on the Luminex 100 analyzer, then turn on the PC. The Luminex software launches automatically.

2. Click Ye s to accept the End-User License Agreement. If the system does not prompt you to accept this agreement, read the agreement shown after the title page of this manual. If you reject the agreement, you cannot use the Luminex 100 system. Contact Luminex Technical Support if you reject the agreement.

3. Verify that the analyzer status, shown in the System Monitor box on the Luminex Main window, is at “Standby.” If the status is “Bad Link,” exit the Luminex software and start it again.

4. On the Analyzer menu, click Setup. The Machine Setup dialog box opens.

Figure 4-3. Machine Setup Dialog Box

5. Ensure that the analyzer type shown is Luminex 100.

6. Make sure that COM Port is set to 1. This setting refers to the port on your computer, not on the Luminex 100 analyzer.

7. Make sure that the XY Port is set to 2. If communication between the Luminex 100 analyzer and the Luminex XYP instrument fails, switch ports so that the Luminex 100 analyzer is on COM port 2 and the XY Port is set to 1. This task enables you to check if the serial cable is the cause of the communication errors.

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Adjusting the Sample Probe Height

Note: Adjusting the sample probe

vertical height is critical.

You must adjust the Sample Probe vertical height each time you install a Sample Probe, change the type or style of microtiter plate, or move the instrument.

Thumb Wheel

ent

Loc

Figure 4-4. Sample Probe Height Adjustment Tools

 To adjust the Sample Probe’s vertical height:

1. Turn on the Luminex XYP and the Luminex 100 analyzer, and PC. Wait for the Luminex Data Collector software to start.

2. If already installed, remove the clear plastic shield covering the sample probe area.

3. In a 96-well microtiter plate where overall height is no more than

0.75 inches, place the appropriate alignment tool in the plate.

• For a standard plate with flat-bottomed wells, stack two of

the larger (5.08 mm diameter) alignment disks in well A1.

• For filter-bottom plates, stack three of the larger alignment

disks into well A1.

• For half-volume plates with flat-bottomed wells, stack two

small (3.35 mm diameter) alignment disks in well A1.

• For a plate with conical wells, place one alignment sphere

into well A1.

• For round-bottom wells, stack 2 small disks in well A1.

4. Click Eject.

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xMAP Technology Installation

5. Place the 96-well microtiter plate on the Luminex XYP with position A1 in the top left corner and click Retract to retract the plate.

6. Use the 3/32 inch hexagonal allen wrench to loosen the height adjustment locking screw. See the photo on page 4-4.

7. Click Options on the toolbar.

8. Click Setup XY, then click Setup. This dialog box appears:

9. Click Test to lower the sample probe into well A1.

10. Using the thumb wheel, raise and lower the Sample Probe until it rests on, but does not apply pressure to, the top of the alignment disk or sphere.

Note: To avoid adjusting the sample probe too low, open the

Luminex XYP door as you begin adjusting the probe height. The plate should not bounce up or plunge down as you lower or raise the sample arm. DO NOT PUT YOUR HAND INSIDE THE LUMINEX XYP!

11. Use a 3/32 inch hexagonal allen wrench to tighten the height adjustment locking screw.

12. Click Test to raise the Sample Probe, then click OK to close the dialog box.

13. Replace the clear plastic shield that covers the Sample Probe area.

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Installing Software and Firmware

The Luminex 100 system arrives preloaded with the Data Collector software. It is possible that you may need to reload the software at some time.

To reinstall the software:

1. Backup log files. Copy your template directory and the file named calibrationlog.mdb to a backup location. These files may be found in the Luminex setup directory.

2. Uninstall the Luminex Data Collector software using the Windows Add/Remove programs function. On the lower left corner of your screen, click Start > Settings > Control Panel. The Control Panel window opens. Click Add/Remove

Programs. The Add/Remove Programs window opens. Click Luminex Data Collector, then click Change/Remove. Follow

the prompts throughout the rest of the removal process.

3. Insert the Luminex CD into the CD ROM drive. The program manager beings installing the software, displaying instructions when necessary.

4. Restart your computer after installing the Luminex software.

5. Copy your template file directory and the calibrationlog.mdb file from the backup location toe the Luminex setup directory.

6. Verify that the firmware version, listed in the Help menu, is the most recent version. Contact Technical Support to check this information.

What to Do Next After you assemble and power the system, verify that your analyzer

is correctly installed and functions properly using the “Verification” section beginning on page 8-14 in the “Troubleshooting” chapter.

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

For assistance, contact Luminex Technical Support. Users in the U.S. and Canada can call us at 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 +1 512-381-4397 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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System Startup

Title B

This chapter describes how to start up your system. The topics included are:

• Luminex Main Window - a tour of the Luminex Main window

• Setup - describes how to set up the system

• Calibration - describes how to calibrate the instrument, add new lots of calibration microspheres, how to print calibration reports.

Luminex Main Window

Menu Bar

Tool Bar

Startup Display

Status Bar

Use the Luminex software to start the system. To start the software and open the main window, double-click the Luminex Data Collector icon on the Windows desktop.

System Monitor

Figure 5-1. Luminex Main Window

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The Luminex Main window has six components: Title bar, Menu bar, Tool bar, Startup display, Status bar, and System monitor.

The Title bar shows the name of the software, and changes depending on what section of the software you are using.

The Menu bar contains commands to set up the Luminex 100 analyzer and perform data acquisition. See Chapter 6 for more detail.

The Tool bar, also discussed in greater detail in Chapter 6, contains icons for commands that are frequently used when running the Luminex software.

The Startup display shows icons for many frequently-used commands. You use most of these commands when starting the system, acquiring data, and shutting the system down.

The Status bar shows the status of current software events.

The System monitor displays Device and Calibration status.

Calibration You should perform Luminex 100 analyzer calibration after system

installation, instrument service, whenever the system is moved, and monthly with routine use. For optimal performance, calibrate the instrument daily as part of your startup routine and whenever the d Cal Temp temperature shown on the system monitor panel indicates

Add New Classification Channel Calibration Lots

a change of more than 3 degrees Celsius ( xMAP microspheres with known light-scattering properties and known fluorescent intensities in the reporter (RP1), classification 1 (CL1), and classification 2 (CL2) wavelength ranges. The calibration process uses these xMAP microspheres to adjust voltage settings for optimal and consistent microsphere classification and reporter readings. Current calibrated settings automatically apply to any new session.

Before you perform an initial calibration for the analyzer, and whenever you receive a new supply of xMAP calibration microspheres, enter the product and lot numbers and the calibration target values.

°C). The calibrators are

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 To add a new lot of xMAP classification calibration

microspheres:

1. Click Calibrate. The Start Calibration dialog box opens.

Figure 5-2. Start Calibration Dialog Box

2. Click New in the Classification Channel box. The Add New Classification Calibrator dialog box opens.

Figure 5-3. Add New Classification Calibrator Dialog Box

3. Enter the information into the dialog box, using the values on the Certificate of Quality that came with your xMAP calibration microspheres. You do not need to enter a value for CL3; however, you must click within the CL3 box and press Enter to save the values.

4. Click OK. You can now use this classification channel xMAP calibration microsphere lot to calibrate your instrument.

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Add New Reporter Channel Calibration Lots

Before you perform initial calibration for the analyzer, and whenever you receive a new supply of xMAP Calibration microspheres, enter the product and lot numbers and the calibration target values.

 To add a new lot of xMAP reporter calibration microspheres:

1. Click Calibrate. The Start Calibration dialog box opens.

Figure 5-4. Start Calibration Dialog Box

2. Click New in the reporter channel area. The Add New Reporter Calibrator dialog box opens.

Figure 5-5. Add New Reporter Calibrator Dialog Box

3. Fill in the information on this dialog box using the values on the Certificate of Quality Analysis included with your xMAP calibration microspheres. Press Enter.

4. Click OK. You can now use this xMAP reporter channel calibration microsphere lot to calibrate your instrument.

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Calibrate the Luminex 100 Analyzer

Note: Do not dilute the xMAP

calibration microspheres. Limit exposure to light!

In addition to calibrating the analyzer after installation, you can enhance optimal performance by calibrating the instrument daily as part of your startup routine and whenever the d Cal Temp temperature shown on the system monitor panel indicates a change of more than 3 degrees Celsius (

°C).

 To calibrate the Luminex 100 analyzer:

1. Turn on the Luminex 100 analyzer. Click Warmup on the main screen, then click OK on the dialog box that appears. After the 30-minute warmup, go on to the next step.

2. Click Prime on the main screen. Click OK when you want to begin the prime cycle.

3. Click Options on the toolbar and ensure that the default settings for alcohol flush is set to Reservoir, and that the wells selected for CAL1 and CAL2 correspond to the wells in the plate that you want to use for calibration.

4. Vortex the xMAP calibration microspheres (CAL1 and CAL2).

5. Dispense 5 drops (~ 200 µL) of xMAP classification calibration microspheres (CAL1) and 5 drops of xMAP reporter calibration microspheres into the wells you designated in the Setup XY tab of the options menu.

Place classification calibrator in a well (i.e., A1).

(You may assign different well locations, if

Place reporter calibrator in a well (i.e., B1).

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Figure 5-6. 96-well Plate. Well A1 and B1 Filled

6. Click Eject on the toolbar and place the plate on the plate holder with well A1 in the top, left-hand corner.

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7. Fill the reservoir with a solution of 70% isopropanol or 70% ethanol. Click Retract on the toolbar to retract the plate holder.

8. Click Alcohol Flush, then click OK. The alcohol flush removes air from the fluidics. This takes about 5 minutes.

9. Click Calibrate on the main screen. The Start Calibration dialog box opens.

Figure 5-7. Start Calibration Dialog Box

10. Enter your name in the Operator text box.

11. Select the Product / Lot Number for the xMAP classification calibration and reporter microspheres.

12. Click OK. The Calibration dialog box opens.

Figure 5-8. Calibration Dialog Box

13. Make sure Classification is selected, and click Start.

The progress bar shows the calibration progress, which should take less than two minutes. If the status changes to “Sample Empty” before calibration completes, a “Calibration Failed” message appears. Click OK and restart the process. When calibration finishes for the classification microspheres, a

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“Calibration Succeeded” message appears. The settings and any comment you enter are logged into the calibration report.

14. Click OK in the confirmation dialog box. The Calibration dialog box opens again.

15. Select Reporter and click Start. The progress bar shows you the calibration progress. Calibration should take less than two minutes. If the status in the system monitor changes to “Sample Empty” before calibration completes, a “Calibration Failed” message appears. Click OK and start the process again. When calibration finishes for the reporter microspheres, a “Calibration Succeeded” message appears. The settings and any comment you enter are logged into the calibration report.

16. Click OK on the confirmation dialog box. xMAP Calibration microspheres are very concentrated. Each time you calibrate the instrument, follow it with 4 wash cycles using deionized water or sheath fluid.

Preview or Print Calibration Report

17. Click Eject from the toolbar and remove the plate.

18. Click Retract to retract the plate holder.

The calibration report is a quality control tool that shows the frequency of past calibrations, operators of past calibrations, and trends in instrument settings. The calibration report is available as a log or as a detailed report.

 To print or preview the calibration report:

1. Close all open sessions.

2. Click Print on the toolbar. The Print dialog box opens.

Figure 5-9. Print Dialog Box

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3. Check the box in front of the report type you want. You can check both boxes to see both kinds of calibration report.

4. Click OK to print the calibration report, or Preview to see the report on your screen. Figure 5-10 shows an example of a calibration log. Figure 5-11 shows an example of calibration detail report.

Figure 5-10. Calibration Log

The calibration log shows gain values associated with each calibration. A gain value is a representation of the voltage. Use this log to observe the gain values. If you see a spike in voltage (20 V or more) in the gain value for a channel from one calibration to the next, the difference may indicate a problem with the instrument. It is normal to see a value of -1 for RP1 gain for classification calibrations. You also see gain values of -1 listed for DD, CL1, and CL2 for reporter calibrations.

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Analyzer menu commands

Figure 5-11. Luminex Calibration Detail Report

The calibration detail report is often used by Luminex field service personnel when performing system maintenance. The report shows system temperature, Voltage, and pressure readings.

Use the Analyzer commands to prepare the Luminex 100 analyzer for sample acquisition, analysis, and shut down. On the Analyzer menu, choose the command you want to perform. Many of these commands also have buttons on the main screen.

Calibrate: Opens the Start Calibration dialog box. See page 5-2 for calibration procedures.

Setup: Opens the Machine Setup dialog box. System connections must be defined with initial installation of the instrument, and again if a Luminex XYP instrument is added to the system. See page 4-3 for more information.

Sanitize: Decontaminates the sample lines and cuvette after biohazard contact. Use a 10%-20% bleach solution to sanitize the system after running biohazardous samples. See page 7-3 for the sanitizing procedure.

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Prime: Removes air pockets from the fluidic pathways. Use this operation after system installation and following replacement of the sheath fluid. Sheath fluid flows freely for several seconds followed by a fill-and-purge action by the syringe. Repeat until the waste line outside the instrument appears free of all significant air pockets.

Alcohol flush: Removes air bubbles from the walls of the cuvette. Use 70% isopropanol or 70% ethanol for the alcohol flush solution. After an alcohol flush, do 2 wash cycles with distilled water.

Wash cycle: Removes residual sample from the sample acquisition lines. Place at least 200 µL of distilled water in the Luminex XYP reservoir. After calibration, run 4 wash cycles with distilled water.

Back flush: Removes obstructions from the cuvette. Use this if

Note: Always follow a Back Flush

with a 70% isopropanol OR a 70% ethanol Alcohol Flush. Follow the Alcohol Flush with two distilled water or sheath fluid wash cycles.

fluid does not flow through the waste tubing during prime cycles or during sample acquisition. Routine back flushes are not recommended; use back flush only as a troubleshooting method.

Drain: Temporarily drains the cuvette, then does a fill command. Useful for cleaning debris buildup in the cuvette, or for clearing bubbles from the cuvette. Routine drains are not recommended. Sheath fluid is expelled at the end of the drain function.

Soak: Holds a solution in the fluidics lines for an extended time. When shutting down the instrument overnight or for an extended period of time, use a distilled water soak. Salt in the sheath fluid can form salt crystals in the sample probe or instrument tubing. When restarting the instrument, follow the routine startup procedure shown on page 7-1 to re-equilibrate the system with sheath fluid. Before you use the Soak command, place 1.2 mL of soak solution in the Luminex XYP reservoir.

Warmup: Performs an initial 30-minute optics warmup. While powered, the optics remain engaged for 4 hours after each acquisition. After 4 hours of inactivity, the lasers automatically shut off, and must be warmed up again before the next acquisition. If Start is activated before warming up, an automatic 30-minute warmup phase is started. The warmup phase can be cancelled using the Cancel warmup command on the Analyzer menu. We strongly recommend that you do not cancel warmup. Your test results will be accurate only if the system is completely warmed up before you begin acquisition. A complete warmup cycle is critical for accurate calibration.

XYP Retract: Retracts the plate into the Luminex XYP slot.

Enable Heater: Warms the Luminex XYP heater block.

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Data Acquisition

The Luminex 100 analyzer uses Luminex Data Collector software to acquire data. This chapter describes the following topics:

• Options

• Sessions

• Templates

• Multiplex Acquisition

• Data Collection

•Reports

Options Before you can acquire data, you must set up some of the different

options available. Click Options on the toolbar to open the Options dialog box. The Options dialog box has these tabs: User, Acquisition, Advanced, Setup XY, and Info. These options apply to all sessions.

User tab This tab has controls to set preferences for how the system works.

Figure 6-1. Options Dialog Box - User Tab

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Sound effects: Enables sounds during system operation.

Clear recent file list: Removes the list of recent files from the File

menu. Do this each time you want to clear the list of recent files.

Show project settings dialog on new session: Opens the project settings dialog box when you start a new session. You can also click

Settings on the toolbar to open the settings dialog box.

Display full path of session on title bar: If this option is unchecked,

an abbreviated file name displays on the title bar.

Auto resize acquisition grid: Resizes the acquisition grid depending on the length of text within each column.

Default beadmap: Controls the beadmap (or MAP) used when you begin a session via the New Session button on the Main screen. Click Browse to change the setting. Each MAP refers to a certain combination of xMAP microspheres. The 100, 50, and 25 Region MAPs are standard MAPs for multiple analyte data collection. Use the Development MAP for development microspheres. The Calibrator MAP and NoRegion MAPS are for system diagnostics.

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Acquisition tab On this tab you can select the default folder for your session files. If

you want a folder other than the one shown, click Browse and select a different folder.

Figure 6-3. Options Dialog Box - Acquisition Tab

Advanced tab Use the Advanced Tab to set properties in the Luminex Data

Collector software. You can select multiple commands in this tab.

Figure 6-4. Options Dialog Box - Advanced Tab

Show Dot Plot. Enables toggling between a bar graph and dot plot.

Show Statistics. Displays all session statistics in the acquisition

window.

Show Prep Mode. Sets a gate before actually running an assay.

Show RP1 Gate. This is a Field Service option. Do not select this

option unless directed by Technical Support.

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Show APD Voltages. This is a Field Service option. Do not select this option unless directed by Technical Support

Show Project Properties. This is a Field Service option. Do not select this option unless directed by Technical Support.

View Calibration Detail. Shows the detail screen while calibrating the instrument.

Allow FileMode. Enables use to replay previously run sessions.

Enable Liquid Handler. This is a Field Service option. Do not

select this option unless directed by Technical Support.

Info tab View the serial number, version numbers and build date. This

information is for display only; you cannot change it.

Figure 6-5. Options Dialog Box - Info Tab

Setup XY Tab This tab has controls to set preferences for how the Luminex XYP

operates.

Figure 6-6. Options Dialog Box - Setup XY Tab

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To use the heater, check Enabled in the Heater area. Use the Set Point control to select a temperature between 35

°C and 60°C (95°F

to 140°F). To maintain heater temperature between sessions, check Hold Temperature between sessions checkbox. If this is not checked, the heater turns off between acquisition sessions.

Warning: The heater plate is hot

when in use and may cause personal injury. Do not touch the heater plate. Allow the heater block to cool after use before touching it.

To have the Luminex XYP automatically retract the plate holder when you click Start, check the Auto Retract checkbox.

To consistently open a session in single sample mode, check Single Sample Start Acquisition.

To control plate positions, use the controls in the Default Sample Locations area. If you change these settings, be sure to select a location that is large enough to hold the amount of sample required for the command. The reservoir holds up to 3000 µL.

Command Size of location needed

Sanitize 1025 µL

Soak 275 µL

Back Flush 0 µL

Wash 200 µL

Alcohol Flush 1025 µL

Drain 200 µL

Click Apply and OK when you finish changing the options.

Sessions You must create a session in order to collect assay data. A session is

a folder that contains individual assay data and specified settings. When you first open a session, the session uses default settings. These default values reflect calibrated and recommended settings.

There are four ways to create a session:

• use default settings

• use a template

• open an existing session and add new data

• open an existing session and save as a new session.

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Create a Session Using Default Settings

 To create a session using default settings:

1. On the Luminex main window, click New Session. The Settings dialog box opens. The values shown in the General tab are recommended settings.

Figure 6-7. Settings Dialog Box - General Tab

2. Enter a name for the session in the Description field. Enter your name in the Operator field. Modify other fields as necessary. A full description of each of the fields begins on page 6-14. Click OK.

Create a Session Using a Template

3. When the analyzer finishes acquiring data, click save to save the session under a folder heading of your choice.

 To create a session using a template:

1. Click Tem pl at e on the toolbar to open the Choose a Template dialog box.

Figure 6-8. Choose a Template Dialog Box

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2. Select the template that you want to use for this session and click OK. If the template already contains a beadmap (or MAP), the template defaults to that MAP. If you have not yet selected a beadmap, this Choose Bead Map dialog box opens:

Figure 6-9. Choose Bead Map Dialog Box

Select the MAP you want to use and click OK. The 100 Region, 50 Region, and 25 Region MAPs are the standard MAPs for multiple analyte data collection. The Calibrator MAP is available for system diagnostics. Use the Development MAP only with development microspheres. Use the NoRegion MAP for system diagnostics only.

3. In the save current settings dialog box, Type the name of the session in the New folder field. Select the folder in which you want to create the session, then click OK.

Figure 6-10. Save Current Settings Dialog Box

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Open and Add to an Existing Session

 To open an existing session and add to it:

1. Click Open on the toolbar. The Open an Existing Session dialog box opens.

Figure 6-11. Open an Existing Session Dialog Box

2. Double-click the session folder that contains the session that you want to use. The session information displays in the session group box. Click OK. The Multiplex Acquisition window opens.

Figure 6-12. Multiplex Acquisition Window

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3. Use the settings shown or modify them, then highlight the first vacant sample.

4. Click Start to collect the new data; it is added to the established session.

Open an Existing Session

 To open an existing session and save it as a new session:

and Save it as a New Session

1. Click Open on the toolbar.

2. Choose the session folder and click OK. The established session appears.

3. Click File on the main menu bar and select Save As, then rename your session. Use the settings shown or modify the settings.

4. If you want to keep the settings, but not the data, click the Clear All button.

5. Click Start to collect data for the new session.

Templates Templates contain customized session settings: numbers of events

desired, numbers of runs, panel settings, gates, and other session information. You can quickly apply these commonly-used settings to a new session. Templates do not store data, and do not store older calibration settings. Any session you create from a template contains the current calibration settings.

This section explains how to create, modify, and delete templates.

Create a Template  To create a template:

1. Open an existing session or create a new session.

2. Make desired setting changes. See “Session Settings” on page 6-

14.

3. Click Edit on the main menu bar and select Create Template. When prompted, supply a name for this template. The software saves the template in the Templates folder.

Modify a Template  To modify a template:

1. Create a new session from the template that you want to modify. Default templates cannot be changed.

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2. Make desired setting changes. See “Session Settings” on page 6-

14.

3. Click Edit on the main menu bar and select Create Template. Supply the same name as the loaded template.

4. When prompted to overwrite an existing template, select Ye s .

5. Close the session and select No when asked to save changes to the current session.

Delete a Template  To delete a template:

1. Click Too ls on the main menu bar and select Manage Te mp lat es.

2. Select the template you want to delete and click Delete. Default templates cannot be deleted.

Multiplex Acquisition

Use Multiplex Acquisition to analyze individual microsphere sets. For each microsphere set, the system supplies information regarding size and fluorescence intensity. The microspheres emit signals in the classification channels. Use a detection fluorochrome (reporter) for quantitation. The reporter emits in the RP1 channel.

The Multiplex Acquisition window opens when you click New Session on the Luminex main window. Before you start using the Luminex software, you should become familiar with the features of this screen. Check the information to make sure all your settings are correct before collecting data.

Menu Bar These bars appear at the top of all Luminex software modules. The

title bar shows the Luminex icon and name and indicates the current session. The main menu bar provides access to software commands and functions. The menus included are File, Edit, Analyzer, Sample, Tools, View, and Help.

Figure 6-13. Menu Bar

File New. Opens a new session

Template. Opens the Choose Template dialog box. After you select a template, you are prompted to begin a new session.

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Open. Opens an existing session

Close Session. Closes the current session. Prompts you to save

the session before closing.

Save. Saves the current session.

Save As. Saves the current session under a new name.

Print. Opens the print dialog box. You have the option of printing a

results table, calibration log, bar graph, histogram, and dot plot.

Print Setup. Opens the Print Setup dialog box. Use the settings in this dialog box to choose printer options.

Exit. Exits the Luminex software. Prompts you to save the current session before exiting.

Edit Project Settings. Opens the Settings dialog box.

Clear Project. Clears the table, graphs, and collected data files in the open project.

Create Template. Opens the Create Template dialog box.

Reset to Calibrated Settings - . Resets the session to the

previously calibrated settings.

Analyzer The Analyzer commands are discussed in detail in Chapter 5.

Sample Load Data. Loads data to a session.

Start/Cancel. Starts or cancels a session.

Delete Data. Deletes the data in the Multiplex Acquisition window.

Copy. Copies the current information to another file.

Tools Options. Opens the Options dialog box.

Manage Templates. Opens the Choose Template to Delete dialog box.

View Toolbar. Toggles the toolbar on or off.

System Monitor. Toggles the System Monitor on and off.

Expand/Contract. This command is available when the Multiplex

Acquisition window is open. It toggles the bead gates views on and off.

Next Window/Previous Window. These commands show the window related to sequential windows related to sample acquisition.

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Help Contents. Opens the online help.

Search for Help On. Opens a searchable index for the online help.

Luminex on the Web. Opens a web browser window and

navigates to the Luminex website.

About Luminex Data Collector. Provides information about the Data Collector software.

Toolbar The toolbar gives easy access to important commands of the main

menu. Each of the commands are listed in the previous section.

Figure 6-14. Toolbar

Main Control and Session Detail

This section includes session information such as the operator’s name, session description, and results. The results table shows you session data as the analyzer acquires the information. Controls on the Main control and Session detail section include Clear All, Autosize, Start, and Single.

Double click inside any of the settings boxes to open the settings dialog box.

Click between headings to resize

Double click a cell to name the sample. The cell turns yellow.

Sample number up to 100 (96 for XYP); single click to select the cell, or right click to see a command menu.

Figure 6-15. Multiplex Acquisition Window - Session Detail

Displays the total number of microspheres counted during sample analysis.

Double click to make a comment about the run. You may see some automatic comments here. The cell turns yellow when active.

Indicates acquisition by downward movement of microsphere.

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Clear All: Clears the screen of all data and graphs. This function deletes all associated files. A confirmation dialog displays when you

click this button to make sure that you intend to delete the table, graphs, and the data files for the session.

Autosize: Adjusts column widths to fit the data and header sizes.

Statistic: This dropdown list offers a choice of statistic types

displayed in the table. Changing the statistic type does not affect collected or recorded data.

Trimmed: Select this option to trim the top 5% and bottom 5% of the collected data.

Prep: Initiates a pre-acquisition mode that allows you to prepare the instrument for a session. In this mode, data is displayed but not saved. You can use this function to optimize instrument settings (such as gates and regions) before beginning a session. There is no event number to indicate the end of a sample. Acquisition ends when you click Cancel or when all of the sample is processed.

Note: To avoid the formation of air

bubbles in the cuvette, only click

Start when a sample is present.

Start: Initiates a sample read. The sample probe lowers, draws

sample out of the well, and retracts to its original position. The sample moves from the probe to the sample loop. If you click Start before warming up the analyzer, an automatic 30-minute warmup phase begins.

If you click Start without a sample present in the well, the analyzer draws air into the sample loop and injects it into the flow chamber. As a result, bubbles form in the cuvette and interfere with analysis. If you click Start without a sample present, complete a 70% isopropanol or 70% ethanol Alcohol flush cycle, followed by two distilled water Wash cycles before acquiring further data.

Cancel: When the sample test begins, the Start button becomes a Cancel button. The sample acquisition ends when you select Cancel

or if the entire sample is analyzed before the desired number of events has been collected. At the end of sample acquisition the sample probe lowers, then the syringe pump purges the sample loop and probe with sheath fluid, sending a fraction of sheath fluid back into the sample well.

Single: Check this box to run one sample at a time. When you check the single check box, you need to start each run individually.

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Right-Click Menu Other commands appear on the right-click menu. To open this menu,

right-click any sample cell.

Figure 6-16. Right-Click Menu

Load Data: Displays the data, including histogram and events information, of a selected (highlighted) sample.

Start/Cancel: Begins or ends data acquisition.

Delete Data: Deletes the data from a selected sample row.

Copy: Copies selected information from the results table to the

Windows clipboard.

Session Settings Click Settings on the toolbar to see the Settings dialog box. The

Settings dialog box contains two tabs, General and Bead Set. These settings apply only to the open session.

Figure 6-17. Settings Dialog Box - General Tab

General tab Use the General tab to provide information about the data collection.

Description: Describe the experiment.

Operator: Identify the experimenter.

Events: Enter a number between 1 and 100,000 if you are using total

events, or a number between 1 and 1000 if you are using events per microsphere set.

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We recommend using at least 100 events per microsphere set. This guarantees that each bead set selected acquires 100 events before completing sample acquisition. If each selected bead set does not acquire this number of events, you see a warning that not enough bead events were acquired. You can choose to keep the data or disregard it. Because of this option, when selecting bead sets on the Bead Set tab, you should choose only the bead sets present in your sample. If you select bead sets that are not present, the analyzer continues to acquire, trying to reach the number of events per bead for bead sets that are not in the sample.

If you choose total events instead of events per bead, the analyzer stops acquiring data as soon as it acquires the specified number of beads within any combination of bead sets. This means that some bead sets may not have a minimum number of events.

Override Histogram Gates: Check this box to set known gate values.

Min events: Set the minimum events needed for display on the graphs and results table. When set to a number greater than 0, the analyzer does not display data for bead sets not generating events equal to or less than this value. Enter 0 to display all events. The minimum events must be less than the number you entered in the

events field.

Samples: Enter the number of tubes or wells to be analyzed. The

maximum number of samples you can enter is 96 if you are using an Luminex XYP, and 100 if you are not using an Luminex XYP.

Sample size: Set this to the amount in µL that you want the analyzer to take up for each sample. You can enter a number between 10 µL and 200 µL. We recommend a sample size of 25 µL or larger.

Note: To avoid air uptake, make sure that your sample well contains

at least 25 µL of sample in addition to the sample size, so that at least 25 µL of sample remains in the sample well after acquisition.

Flow rate: Set the flow rate to Slow, 30 µL/minute; Medium, 45 µL/ minute; or Fast, 60 µL/minute.

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Bead Set tab On the Bead Set tab you can select and name the appropriate

microsphere sets. Select only microsphere sets that are present in your samples.

Figure 6-18. Settings Dialog Box - Bead Set Tab

Availab le: Lists available bead sets for the default beadmap (or MAP) selected in Options. See page 6-2 for more information. If the bead set you need is not on the Available list, click Options on the toolbar and select a different default MAP. If you change the default MAP for an open session, close the session and open it again to update the default MAP.

Selected: Displays and describes the bead sets to be used in a session. The arrow buttons allow you to add or remove bead sets from the selection (individually or as a group). For example, to select bead sets, simply click the arrows pointing toward the Selected box. The single arrow button selects the highlighted bead set, and the double arrow button selects all of the bead sets. To remove unwanted bead sets, click the arrows pointing toward the Available box. Select at least one bead set before running your assay.

If you double click on a bead set in the Available section, the bead set appears in the Selected section, and vice versa.

Caption: Allows you to label or rename beads to identify the assay bound to that particular bead set (for example, 909 IgG). First, highlight the bead set to be renamed. Click in the Caption edit box. Then type in the new label or name. In the Selected window, the original numeric designation remains in the first column, and the corresponding new label or name appears in the second column.

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Histogram The histogram is located in the lower-left corner of the screen. The

histogram provides a graphical display of the real-time data collection. An example of a histogram follows:

Figure 6-19. Histogram Example

The Y axis of the graph displays collected events.

 To autoscale the Y axis:

Click the up-and-down arrow button shown just above the graph.

 To manually set the Y axis scale:

1. Right-click anywhere in the histogram to open the right-click menu. Select Set scale. The Set Scale dialog box opens.

Figure 6-20. Set Scale Dialog Box

2. Enter the maximum value in the dialog box, and click OK.

 To change the X axis parameter:

1. Right-click in the histogram to open the right-click menu.

Figure 6-21. Right-Click X-Axis Menu

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2. Click X axis, then click on the desired parameter name.

If you select reporter 1 for the X axis parameter, the histogram displays reporter-generated intensity data. The central collected wavelength is 575 nm. With classification 1 or classification 2, peaks are based on the emission wavelengths of the classification dyes inside the microspheres; the central collected wavelengths are 658 nm and 712 nm, respectively. Select Doublet Discriminator to display data relating to the light-scattering properties of the microspheres.

Gates An additional histogram feature is the ability to gate a specific

parameter before data collection. The gate filters graphical displays and generated statistical data by eliminating particles smaller or larger than a microsphere. Always set the X axis parameter to doublet discriminator before setting a gate. Setting a gate on any other parameter gives inaccurate results. We recommend setting the gate on the primary peak of the doublet discriminator display, thereby removing data collected from doublets or contaminants.

Note: The gate that is in effect

when data is collected determines which values are used in the result. Applying a gate or changing a gate for existing data does not change your calculated values.

Gate Boundaries

Gate Numerical Position

Figure 6-22. Gate Histogram

 To set a gate:

1. Set the X axis to Doublet Discriminator.

2. Right-click in the histogram; a pull-down menu appears.

3. Scroll to Gate and follow the arrow to the right.

4. Click Create. Dotted vertical lines appear in the histogram.

5. Use your mouse to drag each line where you want it.

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Use the Show Bead options to set the histogram to show events for only one bead set, all gated events, or all events.

There are five buttons to the upper right of the histogram:

Auto scale: Automatically adjusts the maximum number of events shown on the Y axis. Click during acquisition to readjust the Y axis scale.

Zoom: Enlarges a specific area on the histogram. Use the cursor and left mouse button to adjust the graph’s range.

Log/linear: Toggles the X axis scale between logarithmic

and linear modes.

Maximize:

Analyzer: Opens the Analyzer dialog box, which shows

calibrated settings for the threshold and RP1 (reporter channel). You can change the doublet discriminator threshold and the reporter 1 parameters.

Enlarges the entire histogram frame.

Figure 6-23. Analyzer Dialog Box

Analyzer The threshold establishes the minimum doublet detector voltage for

the detection of an event. Proper threshold adjustment removes background noise contributed by the analyzer or the sample. Unlike a gate, a threshold value excludes any data below it. The excluded data does not appear in the table and it is not stored in the run file. A default threshold value is automatically applied to a new session. We

strongly recommend that you use the default threshold setting.

The RP1 PMT value applies to reporter 1 channel data. Calibration sets this parameter to a standard value that can be used as a reference

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for later assays or sessions. By increasing this voltage setting, you may gain sensitivity; however, you may also compromise range. Decreasing this value has the opposite effect. To return to the calibrated RP1 setting, click Edit on the main menu bar and select Reset to Calibrated Settings. We recommend that you use the calibrated RP1 value.

Bar Graph/Dot Plot Display

You can toggle between the bar graph and dot plot displays. Each bead set has a color signal that corresponds with a numeric designation called the Bead ID.

In the bar graph view, the bead sets you selected in the Settings dialog box appear on this graph along the Y axis. The X axis shows progress as the data is collected in the form of a count or a result. You can change which of these two values appears.

Figure 6-24. Bead Set Graph

The dot plot view gives a real-time, two-parameter display of the accumulating data. Each event is shown as a tiny dot. When you select the dot plot display, the Density Dot Plot button and Decaying Dot Plot button activate.

Figure 6-25. Density and Decaying Dot Plot Buttons

Use these buttons to view a density dot plot or decaying dot plot. A density dot plot shows a constant accumulation of events. Increasing

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density is indicated by contrasting colors. A decaying dot plot shows the 100 most recent events acquired by the Luminex 100 analyzer.

Figure 6-26. Dot Plot Display

In the upper right corner of the frame, four buttons let you control the display:

Count/Result: Switches the display between count and result. This is either a count of the number of events collected for the bead ID, or an RP1 intensity result. The result is the median value for the events.

Switch to Dot Plot: Switches the display between the bar graph and dot plot.

Show all: Shows the bead IDs (001, 002, 003, ...100 instead of 010, 020, 030, ...100). The Maximize button makes the display easier to read.

Zoom: Enlarges a specific area on the display.

Lin/Log: Switches the dot plot display between a linear

and logarithmic view.

Maximize/minimize: Enlarges the entire histogram frame, or returns it to its smaller size.

Bead details. You can view information about a specific bead set by clicking the bar for that bead set, or by clicking the Y axis position for that bead set. This brings up a Bead details dialog box for that specific bead ID, showing the bead ID, the bead name, and the count (number of events for that bead). A density plot shows how

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the beads are hitting with respect to their region. The beads should hit within the white region shown. The colored dots show where the beads actually hit.

Figure 6-27. Bead Details Box

This display can be useful in troubleshooting problems. See the “Troubleshooting” chapter on page 8-1 for examples. If the beads do not register properly during sample analysis, recalibrate the Luminex 100 analyzer.

System Monitor The system monitor is an information bar on the right side of the

screen. It shows the status of the Luminex 100 analyzer.

Shows current system status and warnings.

Shows temperature in degrees Celsius, pressure in pounds per square inch, and high voltage status during operation and while idle.

Shows total events, gated events, and region events.

Shows rate of bead acquisition.

Shows events per microliter based upon the selected flow rate.

Shows XYP status.

Shows XYP temperature. When the number is red, the system has not stabilized at the set point temperature. Wait for it to turn green before you start collecting data. The heater temperature does not display if the heater is off.

Figure 6-28. System Status Bar

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The message at the top reflects the current status of the Luminex 100 analyzer.

Ready: Indicates that the Luminex 100 analyzer is ready to collect data.

Pressure: Pressure normally displays during a routine pressure check at the beginning of each run. However, if the pressure message remains for more than 1.5 minutes, a lack of pressure exists in the system. Check for the following problems:

• Low sheath fluid

• Loose hose connections

• Loose cap on sheath fluid bottle.

• Leak in sheath fluid container, tubing, or bubble trap

Not Ready: Normally indicates that the analyzer is initializing.

Busy: Indicates an intermediate state between Standby and Ready.

Standby: Shows that the flow of sheath fluid stopped; the system is

idle.

Bad Link: The communication between the analyzer and the computer is not working. Check that:

• The analyzer is powered on.

• The cables between the analyzer communications port and computer are properly installed.

If the situation has been corrected, restart the Luminex software application to reinitialize this link.

Warm Up: Indicates a 30-minute warmup of the analyzer’s optics. While powered, the optics remain active for four hours after each acquisition.

Running: Indicates that the Luminex 100 analyzer is collecting data.

Sample empty: Indicates an empty sample syringe. The system automatically cancels data collection and adds a comment in the notes section.

Connect: Indicates that the communication between the analyzer and the computer was broken, then re-established. You must restart the Luminex software application to reinitialize communication links.

Sheath fluid: Indicates empty sheath fluid. Fill the sheath fluid container before continuing.

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The system monitor section also shows information about temperature, pressure, voltage, and events. Air pressure generally runs a little higher than sheath pressure. It is normal to see a drop in pressure if several minutes elapse between sample runs. Information in the calibration box of the systems monitor, which appears when no session is open, is shown for the last successful calibration. Normal readings for these parameters are shown in the table below. If poststabilization readings deviate from normal readings, call Luminex for technical support.

Parameter Normal reading

DD Temp Between ambient temperature and 5°C

above ambient temperature

d Cal Temp (delta calibration temperature, how far away the current temperature is from the temperature of the last calibration)

Air Pressure 6 - 9 psi after system stabilization

Sheath Pressure 6 - 9 psi after system stabilization

Hi Voltage 200 ± 20 during acquisition, 10 while idle

Status Bar

This section displays the acquisition time and status. It also lists the session’s file path.

Data Collection  To collect data:

1. Make sure the analyzer has been warmed up and calibrated. See the calibration section beginning on page 5-2 for more information about calibration. Your startup routine may also include priming, flushing with alcohol, and washing.

0 ± 3°C

Note: If the d Cal Temp falls outside this

range, recalibrate the analyzer.

Figure 6-29. Status Bar

2. Create a session. See page 6-5 for more information.

3. Make any changes needed to the session settings.

4. Prepare your assay samples.

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5. In the results table of the new session, highlight the sample where you want to begin collecting data.

6. Click Start. You can see the progress of data collection on the histogram and the bar graph. Make sure that the Single box remains unchecked for continued sample acquisition.

7. After the analyzer finishes collecting data, select Save on the File menu. The analyzer saves your data in the session folder you selected when you created the session.

Run an Entire Plate of Samples

Note: Ensure that the sample

needle height is adjusted to the 96-well plate in use. See page 7-9

 To run an entire plate:

1. Create the session for this run.

2. Click Eject from the toolbar to eject the plate holder.

3. Place a 96-well microtiter plate on the plate holder.

4. Click Retract from the toolbar to retract the plate holder.

5. Click Settings and make sure the number of samples is set to 96, then click OK.

Figure 6-30. Settings Dialog Box

6. On the results table in the acquisition screen, highlight the row for the first sample.

7. Click Start on the main control screen. The Luminex 100 automatically reads the entire plate of samples.

Note: If the Single box is checked, the software pauses between

samples. Click OK to continue acquiring sample.

8. After all samples have been read, click Eject from the toolbar to eject the plate holder.

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9. Remove the microtiter plate from the plate holder.

10. Click Retract from the toolbar to retract the plate holder.

Run a Range of Samples You can run a specific range of samples, as demonstrated in the

highlighted samples shown below.

1 2 3 4 5 6 7 8 9 10 11 12

Figure 6-31. Sample Plate with Row of Samples

 To run a range of samples on a plate, follow these steps:

1. Create the session for this run.

2. Click Eject from the toolbar to eject the plate holder.

3. Place a 96-well microtiter plate on the plate holder.

4. Click Retract from the toolbar to retract the plate holder.

5. Click Settings and set the number of samples to the number of the last sample that you want to read, then click OK.

To find the number of the last sample, start counting with well A1, and count down an entire column. Then move to the next column. For the example shown, you would set the number of samples to 15.

6. On the results table part of the acquisition screen, select the sample where you want to begin collecting sample. For the example shown, you would highlight well D2.

7. Click Start on the main control screen.

The Luminex 100 analyzer automatically reads the range of samples beginning with the selected sample.

8. After the samples have been read, click Eject from the toolbar to eject the plate holder.

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9. Remove the microtiter plate from the plate holder.

10. Click Retract from the toolbar to retract the plate holder.

Run a Single Sample  To run a single sample:

1. Click Eject from the toolbar to eject the plate holder.

2. Place a 96-well microtiter plate on the plate holder.

3. Click Retract from the toolbar to retract the plate holder.

4. On the results table part of the acquisition screen, select the sample that you want to read.

5. Check the Single checkbox below the start button on the main control screen.

6. Click Start on the main control screen. The Luminex 100 reads just the selected sample.

Interrupt a Run

Between Samples The Single checkbox becomes accessible for several seconds

During Sample

Acquisition

7. Click Eject from the toolbar to eject the plate holder.

8. Remove the microtiter plate from the plate holder.

9. Click Retract from the toolbar to retract the plate holder.

 To interrupt a run while the system acquires a sample:

1. Click Cancel. Sampling stops after you click Cancel, and a window pops up asking if you want to acquire the next sample. If you click Ye s, sampling proceeds with the next sample. Click No to stop the session completely.

2. To continue acquiring sample, highlight the sample with which you want to resume sampling with and click Start.

between samples. Click the Single checkbox. The software then pauses between acquiring the next sample run.

Click OK to continue acquiring the next sample.

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 To interrupt sample acquisition between sample runs:

1. Click Cancel. Sampling stops after you click Cancel.

2. Click OK to continue acquiring the next sample.

Reports You can print the results table or histogram from any session.

 To print reports:

Note: The analyzer does not

support printing with the Windows NT operating system.

1. Load the desired data.

2. Click File on the main menu bar and select Print. The Print dialog box opens.

Figure 6-32. Print Dialog Box

Selecting both histogram and bar graph activates the Single Page option.

3. Choose the type of report, and click OK.

 To print session results to a text file:

1. On the Open the Windows Start menu (located on the lower left corner of the screen). Select Settings > Printers. The Printers window opens.

2. Double click Add Printer. The Add Printer Wizard opens.

3. Click Next to begin the install.

4. In the second page of The Add Printer Wizard, select Local Printer. Deselect the Automatically detect option. Click Next.

5. In the Select the Printers Port window of the Add Printer Wizard, select LPT1.

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6. In the Manufacturers List, click Generic. In Printers list, click Generic/Text Only.

7. Click OK throughout the rest of the windows.

8. You can now open report pages in Notepad, then print them to this printer.

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Maintenance and Cleaning

Warning: When analyzing

potentially infectious biological samples on the Luminex 100 analyzer, follow proper biosafety procedures. These safety precautions should also be taken when cleaning or maintaining the Luminex 100 analyzer.

Proper maintenance and cleaning of the Luminex 100 analyzer should be performed in order to preserve the analyzer’s longevity and reliability.The maintenance log at the end of this chapter will help you to keep track of when maintenance procedures are performed. It lists and defines the frequency of each task.

This section describes the following maintenance procedures:

•Startup

• Shutdown

• Check Fluid Levels

• Check for Leaks

• Calibrate

• Sanitize

• Clean Accessible Surfaces

• Clean Sample Probe

• Replace Air Intake Filterss

• Replace Syringe Seal

• Clean Luminex 100 Analyzer Ventilation Filter

• Change Sheath Filter

• Change Fuses

• Adjust Sample Probe Vertical Height

O NOT REMOVE THE ANALYZER COVER!

Startup  Perform the entire startup procedure on a daily basis.

1. Turn on the analyzer and PC.

2. Click Warmup.

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Note: There is NO sensor to warn

you of low sheath fluid levels. Fill the sheath fluid container before the level reaches the sheath outlet line. There is NO indicator light to warn you of high waste volume. Empty the waste container each time you fill the sheath container.

warm up - about 30 minutes - check the levels of sheath fluid and waste fluid. Check that the sheath container cap is tight.

4. Prime the system.

5. Perform an Alcohol Flush with 70% isopropanol or 70% ethanol.

6. Wash twice with distilled water or sheath fluid. For optimal performance, Calibrate the system and then Was h four times with distilled water. See page 5-2 for more information about calibration.

Shutdown  Perform the shutdown procedure when you finish running

samples.

1. Dispense 1.2 mL of a fresh 10%-20% bleach solution into the Luminex XYP reservoir. Run a Sanitize command.

2. Run a Soak command with distilled water.

3. Raise the sample probe.

3. While you are waiting for the analyzer and optics system to

4. Click Eject on the toolbar to eject the plate holder.

5. Remove the microtiter plate and the reservoir.

6. Clean the plate holder and reservoir with a 10%-20% bleach solution.

7. Replace the reservoir, then click Retract on the toolbar to retract the plate holder.

8. Loosen the sheath fluid container cap to release pressure in the system.

9. Turn the analyzer and PC off.

Check Fluid Levels Check these sheath and waste fluids daily, more often if you run a lot

of samples. Replace the sheath fluid and empty the waste container as needed. You must manually monitor the sheath fluid and waste container levels. Release the Luminex 100 analyzer’s pressure by removing the lid from the sheath fluid container. Add sheath fluid, filling the sheath fluid container to just below the air intake. Discard waste by appropriate means. After you add sheath fluid and discard

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xMAP Technology Maintenance and Cleaning

the waste, remove air from the lines by clicking Prime until no more air shows in the lines.

Check for Leaks  Check for leaks on a weekly basis.

Note: In the event of a leak or spill

inside the analyzer, make sure the power to the system is off and that the power cord is disconnected from the wall source.

Open all analyzer doors and visually inspect for leaks. If you see a leak or spill, contact Luminex Corporation. Users in the U.S. and Canada can call 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.

Calibrate Calibrate at least monthly during routine use. Follow the instructions

beginning on page 5-2.

Sanitize  You should sanitize the system on a monthly basis and every

time you run biological samples.

1. Perform four Wash cycles using distilled water.

2. Dispense 1.2 mL of a fresh 10%-20% bleach solution into a sample tube. Place the tube into the sample tube holder, and begin the Sanitize cycle.

Clean Accessible Surfaces

PN 89-00002-00-063 Rev. A 7 - 3

 Clean the exterior surfaces of the Luminex 100 system on a

monthly basis, or more often if you are running biological samples.

1. Turn off the power switch on the rear of the Luminex 100 analyzer.

2. Unplug the analyzer power cord from the wall source.

3. Wipe all exterior surfaces with mild germicidal detergent, followed by a 10%-20% bleach solution.

4. Open the front doors of the analyzer and clean all accessible surfaces with detergent followed by a 10%-20% bleach solution.

5. Clean the Luminex XYP reservoir and plate holder with a 10%20% bleach solution.

6. Dry the sheet metal surfaces to prevent corrosion.

Luminex 100 User Manual Version 1.7 xMAP Technology

Clean Sample Probe  Clean the sample probe on a monthly basis, or whenever you

suspect that it may be clogged.

1. Remove the sample probe as follows: Unsnap the light housing located above the probe. Unscrew the Cheminert fitting that holds the sample probe in place. Gently grasp the probe and push up. Remove the sample probe out of the top of the sample arm.

Warning: Be sure that the

system is not operating when you remove the sample probe.

Note: The sample probe should

slide up easily while removing it from the sample arm. If you feel resistance, do not force the probe up. Contact Technical Support.

2. Use a bath sonicator or 10 mL syringe to clean the sample probe. If you are using a sonicator, place the smaller end of the sample probe in the sonicator for 2-5 minutes. If you are using a syringe, force 10%-20% bleach through the larger end of the sample probe.

3. Replace the sample probe. Adjust the vertical height if necessary.

4. Perform the following commands:

• Three Backflushes

• Three Drain commands

• Two Alcohol Flushes using 70% isopropanol or 70% ethanol.

• Three Washes - use water.

Vertical Adjustment Screw

Sample Probe

Figure 7-1. Sample Probe

Replace Air Intake Filters

You should replace the filters on the Luminex 100 analyzer and Luminex XYP every six months.

 To replace the air intake filter on the Luminex 100 analyzer:

Note: Hold on to the tubing! Do

not allow the tubing to fall inside the analyzer.

7 - 4 PN 89-00002-00-063 Rev. A

1. Turn off the power switch on the rear of the Luminex 100 analyzer, then unplug the power cord from the wall source.

xMAP Technology Maintenance and Cleaning

2. On the back of the Luminex 100 analyzer, remove the thumbscrew at the top of the air intake filter access door.

3. Pull the access door off of the Luminex 100 analyzer.

4. Pull the filter out from the unit, about three or four inches.

5. Grasp the tubing. Remove the filter with one hand, and hold the tubing with the other hand.

6. Connect a new filter to the tubing.

7. Put the filter inside the panel, then reattach the panel door.

Figure 7-2. Rear of Luminex 100 Analyzer, Air Filter Exposed

 To replace the Luminex XYP Air Intake Filter:

1. Disconnect the Luminex XYP from ac power by turning off the power switch on the rear of the instrument, then unplug the power cord from the wall source.

2. On the back of the instrument, to the left side, gently remove the screen from the filter. Do not remove the screws.

Figure 7-3. Back of Luminex XYP, Removing Air Filter

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Luminex 100 User Manual Version 1.7 xMAP Technology

3. Replace the filter and replace the screen.

4. Plug in and turn on the Luminex XYP.

Replace Syringe Seal  Replace the syringe plunger seal every 6 months.

1. Turn off and unplug the Luminex 100 analyzer.

2. Open the center door on the front of the Luminex 100 analyzer.

3. Unscrew the knob on the syringe arm at the bottom of the

Warning: The syringe arm does

not deactivate when changing the plunger; injury could result if the system is not unplugged.

syringe, and forcefully push the syringe arm down.

4. Unscrew the syringe from the top of its housing; pull the plunger out of the syringe.

5. Remove and replace the plunger seal.

6. Return the plunger to the syringe.

7. Screw the syringe back into the top of its housing.

8. Return the syringe arm to its original position, and hand-tighten the screw on the syringe arm.

9. Plug in the power cord and turn the Luminex 100 analyzer on.

10. Prime the Luminex 100 analyzer 5 times and watch for any leaks in the syringe area.

11. When finished, close the center door.

Syringe Seal

Syringe

Figure 7-4. Front of Luminex 100 Aanalyzer, Access Door Opened

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xMAP Technology Maintenance and Cleaning

Clean Luminex 100 Analyzer Ventilation Filter

Change Sheath Filter

 Check the Luminex 100 analyzer ventilation filter every 6

months. Clean the filter only when soiled.

1. Turn off the Luminex 100 analyzer at the power switch.

2. Unplug the power cord from the wall source.

3. On the bottom of the unit on the left side, push the filter out toward the right side.

4. Grasp the filter and pull it out from underneath the Luminex 100 analyzer.

5. If the filter is dirty, clean it with a vacuum cleaner or in running, distilled water. Stand it upright to air-dry.

6. Reinstall the filter with the arrows facing up.

 Change the sheath filter once a year.

1. Disconnect the sheath fluid bottle

2. Squeeze the metal quick release connections at both ends of the filter assembly, then pull the tubing away from the filter assembly.

3. Connect the new sheath filter, matching up the color-coded fittings. The arrow on the sheath filter should point up.

4. Reconnect the sheath fluid bottle.

5. Prime the instrument 5 times to remove any air introduced into the system.

Figure 7-5. Sheath Fluid Filter

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Luminex 100 User Manual Version 1.7 xMAP Technology

Change the Sheath Filter and

 Change the sheath quick-disconnect o-rings as necessary.

1. Turn off the Luminex 100 analyzer at the power switch.

Quick-disconnect

2. Remove the quick-disconnect fitting that has the faulty o-ring.

O-rings

3. Use a small flat-head screwdriver to remove the worn out o-ring.

4. Place the new o-ring over the tip of a pair of closed needle-nose pliers.

5. Gently separate the tips of the pliers to stretch the o-ring just enough to fit it over the top of the quick-disconnect.

6. Slide the o-ring over the quick disconnect, then remove the pliers.

Change Fuses  To replace fuses on the Luminex 100 analyzer or the Luminex

XYP instrument.

1. Turn off the power switch on both the Luminex 100 analyzer and the Luminex XYP.

DANGER! To avoid serious injury

or death by electric shock, you must turn off the system and unplug it from the wall outlet.

2. Unplug the power cord from the analyzer or instrument, depending on which fuses you are changing.

3. Use a small, flathead screwdriver to open the power module door. See Figure 7-6.

4. Remove the red cartridge. Check both fuses for damage.

5. Replace damaged fuses with the type specified on the sticker to the right of the power input module.

6. Push the cartridge back into the power module, then push the power module door back into place.

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xMAP Technology Maintenance and Cleaning

Power Module Door

Figure 7-6. Power Module

Adjust Sample Probe Vertical Height

Note: Adjusting the sample probe

vertical height is critical.

The vertical height determines how far the sample probe goes into the sample tube or sample well when aspirating a sample. It is set when the Luminex 100 analyzer is shipped. Readjust the vertical alignment any time the instrument is moved, the probe is removed or replaced, the type of plate you are running changes, or any time sample acquisition is slow or sporadic.

Thumb Wheel

ent

Figure 7-7. Sample Probe

 To adjust the sample probe’s vertical height:

1. Turn on the Luminex XYP, the Luminex 100 analyzer, and PC. Wait for the Luminex Data Collector software to start.

2. Remove the clear plastic shield covering the sample probe area.

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3. In a 96-well microtiter plate where overall height is no more than

0.75 inches, place the appropriate alignment tool in the plate.

• For a standard plate with flat-bottomed wells, stack two of the larger (5.08 mm diameter) alignment disks in well A1.

• For filter-bottom plates, stack three of the larger alignment disks into well A1.

• For a half-volume plate with flat-bottomed wells, stack two of the smaller (3.35 mm diameter) alignment disks in well A1.

• For a plate with conical wells, place one alignment sphere in well A1.

• For round-bottom wells, stack 2 small disks in well A1.

4. Click Eject.

5. Place the 96-well microtiter plate on the Luminex XYP with position A1 in the top left corner and click Retract to retract the plate.

6. Use the 3/32 inch hexagonal allen wrench to loosen the height adjustment locking screw. See the photo on page 7-9.

7. Click Options on the toolbar.

8. Click Setup XY, then click Setup. This dialog box appears:

Figure 7-8. XY Setup Dialog Box

9. Click Test to lower the sample probe into well A1.

10. Using the thumb wheel, raise and lower the sample probe until it rests on, but does not apply pressure to, the top of the alignment disk or sphere.

Note: To avoid adjusting the sample probe too low, open the

Luminex XYP door as you begin adjusting the probe height. The plate should not bounce up or plunge down as you lower or raise the sample arm.

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xMAP Technology Maintenance and Cleaning

11. Use a 3/32 inch hexagonal allen wrench to tighten the height adjustment locking screw.

12. Click Test to raise the sample probe, then click OK to close the dialog box.

13. Replace the clear plastic shield that covers the sample probe area.

Adjusting the Sample Probe Horizontal Position

Luminex aligns the sample probe on your Luminex 100 analyzer prior to shipping, but you may need to adjust the horizontal position after installing the Luminex XYP. Adjust the sample probe’s horizontal position if it rubs or scrapes against the side of the alignment guide. Some rubbing is acceptable, as long as the probe movement is smooth.

Note: Improper placement of the Luminex 100 analyzer on the

Luminex XYP may cause horizontal misalignment. Verify that they are stacked squarely before proceeding.

 To adjust the horizontal position of the sample probe:

1. Turn on the Luminex XYP and the Luminex 100 analyzer. Start the Luminex Data Collector software.

2. Use a 3/32 inch hexagonal allen wrench to loosen the arm pivot locking screw.

3. Move the sample arm to the left or right until the sample probe is in the center of the alignment guide.

4. Tighten the arm pivot locking screw.

The arm pivot

locking screw Thumb Wheel

Height Adjustment Locking Screw

Figure 7-9. Sample Probe

PN 89-00002-00-063 Rev. A 7 - 11

is behind the

probe arm.

Luminex 100 User Manual Version 1.7 xMAP Technology

Decontaminating the Luminex 100 Analyzer for Return Shipment

Note: If you are returning the

Luminex XYP instrument or the Luminex SD instrument, decontaminate accessible surfaces using 10% bleach.

Technical Support will supply a Return Material Authorization (RMA) number if they direct you to return the analyzer to Luminex, and instruct you in returning the analyzer according to Luminex procedures. Decontaminate the unit before shipping.

Before returning the Luminex 100 analyzer, sanitize and decontaminate the accessible surfaces and internal fluidics system. This is particularly important if you have analyzed biohazardous samples. Make a copy of this page to complete and return with the analyzer. Submit the completed checklist, both signed and dated, with the Luminex 100 analyzer.

1. Replace the sheath fluid with a 10%-20% bleach solution.

2. Remove specimens, disposables, and reagents from the analyzer.

3. Sanitize the analyzer with a 10%-20% bleach solution using the Sanitize command on the main screen of the system. Follow this by washing twice with distilled water.

4. Turn off the power switch on the back of the analyzer, then unplug the power cord from the wall source.

5. Disconnect the sheath fluid and waste containers.

6. Drain the sheath fluid and waste containers.

7. Rinse the waste container with a 10%-20% bleach solution.

8. Wash all exterior surfaces with a mild detergent, followed by a 10% bleach solution.

9. Open both front doors of the Luminex 100 analyzer. Clean all accessible surfaces with mild detergent, followed by 10% bleach.

10. Pack the system in a biohazard bag. Return the unit in its original packaging or an approved shipping container. Include a copy of this checklist in the packaging.

Was there an internal leak in the system? Yes No

Print Name: __________________________________

Signature: ___________________________________RMA

Number: __________________ Date: ___________________

7 - 12 PN 89-00002-00-063 Rev. A

PN 89-00002-00-063 Rev. A

Luminex 100 Maintenance Log Month(s): ________________________________________Year:_______________

Note: Follow your standard laboratory safety practices when cleaning or maintaining the system. Do not remove the analyzer cover under any circumstances.

Procedure: To use this log, fill in the month(s) and year above. This form is designed to record information for four weeks. Make photocopies as necessary. Fill in the dates for which this

log is used in line one of the table below. For each item listed at the left, add your initials under each date you perform the item.

Dates

Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Check waste level

Check sheath fluid

Tighten sheath cap

Laser warmup

Daily maintenance

Startup Initials: (for each item listed at the left, fill in your initials under each date that you perform the item)

sheath fluid)

Alcohol Flush (70% isopropano l)

Wash twice (distilled water or

Shut down Initials: (for each item listed at the left, fill in your initials under each date that you perform the item)

Sanitize (10%-20% bleach)

Soak (water)

Loosen sheath cap

Weekly

Turn the system off (optional )

Visual inspection Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Monthly

Calibrate Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Every six months

Replace air intake filter, analyzer Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Wipe exterior surfaces Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Clean sample probe Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Replace air intake filter, XYP

(optional)

Replace syringe plunger seal, or

Yearly

Replace sheath filter Date/Initials: Date/Initials: Date/Initials: Date/Initials:

As required

Check analyzer ventilation filter Date/Initials: Date/Initials: Date/Initials: Date/Initials:

syringe

Replace fuses Date/Initials: Date/Initials: Date/Initials: Date/Initials:

Comments:

Troubleshooting

Troubleshooting procedures help users isolate, identify, and remedy problems with the Luminex 100 analyzer and Luminex XYP instrument. This chapter does not troubleshoot problems with the PC. For help with PC problems, please call Dell Technical Support at 800-624-9896.

To troubleshoot a problem, start by selecting a general symptom Next, identify the possible problem and remedy it with one of the solutions listed.

This troubleshooting chapter supplies information for the following topics.

• Power Supply Problems

• Communication

• Pressurization

• Fluid Leaks

• Sample Probe

• Calibration Problems

• Acquisition Problems

• Bead Detail Irregularities

• Error Messages

• Printing Errors

• Verification

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

Technical assistance is available to users in the U.S. and Canada by calling 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

PN 89-00002-00-063 Rev. A 8 - 1

Luminex 100 User Manual Version 1.7 xMAP Technology

outside of the U.S. and Canada can call us at +1 512-381-4397 between the hours of 7:00 a.m. and 7:00 p.m. Central Time, Monday through Friday. You may send inquiries by email to support@luminexcorp.com. You can also read answers to common troubleshooting questions at http://luminexcorp.custhelp.com.

Power Supply Problems

Symptom Possible problem Solution

Analyzer will not turn on, or XYP will not turn on.

Fuses continue to open (blow). A component has a short circuit. Contact Technical Support.

Power supply problems often involve a blown fuse, faulty electronic component, or even something as simple as a disconnected cable. Use extreme care when you replace a fuse.

The power cord is disconnected. Verify that the power cord is

Plugged in.

No voltage is coming from the electrical outlet.

The power supply is faulty. Contact Technical Support.

A fuse has burned out. See page 7-8 for instructions on

Verify that the electrical outlet is operational.

changing fuses.

Communication The communication problems described in this section involve the

links between the data system (PC and Luminex Data Collector software) and the Luminex 100 analyzer and XYP instrument. This section does not address communication issues with other peripheral devices.

The term “Communication” refers to:

• The transfer of data between the PC and the analyzer.

• The current status of the analyzer and XYP instrument.

• Instrument readbacks.

• Instrument control, sample acquisition, session uploading, and start, stop and pause features.

Symptom Possible Problem Solution

PC cannot initialize communication with analyzer.

8 - 2 PN 89-00002-00-063 Rev. A

The communications cable is unplugged.

Check the communications cable connections.

xMAP Technology Troubleshooting

“Bad Link” error message There is a bad link between the

PC, analyzer, and/or XYP.

The Machine Setup is using the wrong COM Port.

XY Platform microtiter plate connect error. Command timed out.

There is a bad link between the XYP and PC.

Ensure that the instruments have power.

Check the power cord and communication cable connections

Shut down the PC and analyzer. Turn on the analyzer before turning on the PC.

On the Analyzer menu, click Setup. Verify that the correct ports are selected for each instrument. If you have make changes, restart the system.

Ensure that the communication cable between the PC and XYP is connected.

Pressurization Normal air and sheath pressure readings vary between 6-9 psi while

the compressor runs. If the system pressure is out of range, your sample acquisition will fail or return poor results.

Symptom Possible Problem Solution

Pressurization Fails or Pressure too low.

The sheath and waste lines are not fully connected.

An air leak is present in the sheath bottle.

The sheath or waste bottle fittings are cracked.

There is a leak in the system. Check for system leaks.

The compressor does not engage.

The Cheminert Fitting is loose. Ensure that the fitting connects

Fluid leaks in the system. See Fluid Leak on page 8-4.

Make sure the lines between the sheath and waste bottles and the analyzer are fully connected.

Remove and retighten the sheath bottle cap.

Inspect the fittings to be sure they form a tight seal.

Run a Prime command. If you do not hear the compressor turn on, call Technical Support.

tightly above the sample probe, below the blue light.

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The sheath bottle has an air leak.

Pressure too High The sheath bottle is overfilled. Ensure that the sheath bottle is

Disconnect the sheath and waste bottle connections from the analyzer. Run a Prime command. If pressure builds, remove and retighten the sheath fluid bottle cap, then reconnect fluid lines to the analyzer. If pressurization fails again, replace the sheath bottle.

not filled above the fill line.

Fluid Leaks Fluid leaks can result in poor pressurization and failed sample

acquisition.

Symptom Possible Problem Solution

Pressure too low The sample probe is clogged. Clean the sample probe. See

page 7-4.

Large amount of fluid pooled around instrument

Fluid coming from sample probe

Fluid leaking from front of analyzer

The syringe seal leaks. Replace the syringe seal. See

page 7-6.

The syringe valve leaks. Hand-tighten the syringe

connection (silver knob) on the syringe valve. Run a Prime command. If leaks continue, call Technical Support.

Fittings or fluid lines are damaged.

The sample probe is clogged. Clean the sample probe. See

The sample 3-way valve is faulty.

The syringe seal leaks. Replace the syringe seal.

The syringe valve leaks. Hand-tighten the syringe

Contact Technical Support.

page 7-4.

Contact Technical Support.

connection (silver knob) to the white syringe valve. Run a Prime. If leaks continue, contact Technical Support.

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xMAP Technology Troubleshooting

Sample Probe Problems with the sample probe can lead to fluid leaks and

pressurization problems, as well as inhibit sample acquisition.

Symptom Possible Problem Solution

Sample probe leak The sample probe is clogged. Clean the sample probe. See

page 7-4.

Sample arm stuck in the up position

Sample arm stuck in the down position

The system isn’t properly pressurized.

The sample probe height is too low, or the path to the well is blocked.

If a PRESSURIZATION error message displays, see Pressurization on page 8-3.

DO NOT turn off Luminex XYP instrument.

1. Remove the blue light housing on the Luminex 100 analyzer.

2. Unscrew the tubing connector

connecting the sample tube to the top of the sample arm. The system monitor changes from “Busy” to “Running” and sample acquisition continues. Click Cancel to make adjustments before continuing with the rest of the samples.

3. If sample arm still does not raise, save all the data that has been collected to this point. Turn off the analyzer, but LEAVE THE XYP instrument turned on.

4. Exit Data Collector.

5. Turn the analyzer back on and restart the software.

6. Replace the sample tubing

and blue light. Adjust the sample arm.

7. Run a calibration with water

to reset the sample probe optical switch.

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Sample arm does not go down smoothly.

The 96-well plate is incorrectly seated in the XYP.

The 96-well plate is warped. Inspect the 96-well plate. Replace

The sample arm is misaligned. Readjust the sample arm

The analyzer is misaligned with the XYP instrument.

The sample probe is bent. Remove the sample probe from

Adjust the 96-well plate.

it if it is warped.

horizontal alignment.

Reposition the Luminex 100 analyzer on top of the Luminex XYP instrument so that the holes match up correctly. The alignment guide should tighten and loosen fairly easily if the instruments are aligned correctly. Continue to square the instruments until the guide loosens and tightens easily.

the Luminex 100 analyzer. Roll it on a flat surface. If it does not roll smoothly, replace it with a new sample probe.

Calibration Problems

Calibration is slow or fails There is a bad link between the

PC and the analyzer.

The calibration microspheres are not fully suspended.

Wrong calibration lot chosen in the Calibration Setup window.

Verify that all instruments turned on and all communications cables are in place on back of instruments.

Vortex the calibration vials to resuspend the microspheres.

In the Main Window of the Luminex Data Collector software, click Calibrate. In the Start Calibration dialog box, make sure that the correct lot numbers are shown in both the Classification Channel and Reporter Channel menus.

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Luminex 100 User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual