Doc-To-Help Standard Template - Bio-Tek

Doc-To-Help Standard Template - Bio-Tek
BIO-TEK
®
KC4
User’s
Guide
Signature
for KC4
and KC4 v3.4
.
User’s Guide to
KC4™ v3.4 and
KC4™ Signature
March 2005
Copyright  2005
PN 5291000
Revision E
Bio-Tek Instruments, Inc.
.
Notices
BIO-TEK INSTRUMENTS, INC.
P.O. Box 998, Highland Park
Winooski, Vermont
05404-0998 USA
Customer Service & Sales:
Phone:
Fax:
E-Mail:
Internet:
(888) 451-5171 (toll free in the U.S.)
(802) 655-4740 (outside the U.S.)
(802) 655-7941
[email protected]
http://www.biotek.com
Service/Technical Assistance Center (TAC):
Phone:
Fax:
E-Mail:
(800) 242-4685 (toll free in the U.S.)
(802) 655-4740 (outside the U.S.)
(802) 655-3399
[email protected]
European Coordination Center
Bio-Tek® Instruments GmbH
Kocherwaldstrasse 34
D-74177 Bad Friedrichshall
Germany
Phone:
Fax:
E-Mail:
All Rights
Reserved
+49 (0) 7136 9680
+49 (0) 7136 968 111
[email protected]
Copyright © 2005, Bio-Tek® Instruments, Incorporated. This
publication is protected by copyright and all rights are reserved. No part
of this manual may be reproduced or transmitted in any form, or by any
means electronic or mechanical, including photocopying and recording,
for any purpose other than the purchaser’s personal use without written
permission of Bio-Tek® Instruments, Inc.
Printed in the United States of America.
Bio-Tek Part Number 5291000.
Trademarks
Bio-Tek® is a registered trademark, and KC4, KC4 v3.4, and KC4
Signature are trademarks of Bio-Tek Instruments, Inc. Bio-Cell is a
trademark of Bio-Tek Instruments and Bio-Cell™ is patented under U.S.
patent number 5,963,318.
Microsoft is a registered trademark and Windows, Windows 95/98/
NT/2000/XP, Word, and Excel are trademarks of Microsoft Corporation.
All other trademarks are the property of their respective holders.
Restrictions and
Liabilities
Information in this document is subject to change, and does not represent
a commitment by Bio-Tek Instruments, Inc. Changes made to the
information in this document will be incorporated in new editions of the
publication. No responsibility is assumed by Bio-Tek for the use or
reliability of software or equipment that is not supplied by Bio-Tek, or its
affiliated dealers.
Limited
Software
Warranty
Bio-Tek Instruments, Inc. (“Bio-Tek”) warrants, for a period of ninety
(90) days after the date of delivery, that all software manufactured and
sold by Bio-Tek (the “Software”) will conform, as to all substantial
operational features, to Bio-Tek’s then current published specifications
(meaning those specifications in effect as of the date of purchase) and
will be free of defects which, in Bio-Tek’s sole judgment, substantially
affect system performance. This Warranty is limited to the original
purchaser (the “Purchaser”) and cannot be assigned or transferred. All
claims under this Limited Warranty must be made in writing or fax to
Bio-Tek attention: Service Department.
The Purchaser must notify Bio-Tek in writing within ninety (90) days
after the date of delivery of the Software to the Purchaser of any claim
under this Limited Warranty. This 90 day period shall not be extended by
the delivery or receipt of any subsequent modifications to the Software.
Modifications to Software within a version (“Updates”) are expressly
excluded from the terms of this Warranty. If the Software is found to be
defective by Bio-Tek, Bio-Tek’s sole obligation under this Warranty is to
use efforts consistent with Bio-Tek’s regular business practices to
attempt to remedy such defect. In no event shall Bio-Tek’s liability under
this warranty exceed the original purchase price of the Software.
This is a limited warranty. This warranty contains the entire
obligation of Bio-Tek. No other warranties, express, implied, or
statutory, are given. The implied warranties of merchantability and
of fitness for any particular purpose are expressly excluded and do
not apply to the software.
Under no circumstances shall Bio-Tek be liable to the purchaser or
any other person for any direct, incidental, or consequential
damages, whether arising out of breach of warranty or otherwise.
This warranty shall not be changed or modified in any way without
the express written permission of an officer of Bio-Tek Instruments,
Inc.
This Warranty shall be NULL AND VOID if any modifications are made
to the Software by the Purchaser. Correction of difficulties or defects
traceable to Purchaser’s errors or system changes shall be billed to
Purchaser at Bio- Tek’s standard time and materials charge.
Licensing
Agreement
LICENSE: Single Use: You have the non-exclusive right to use the
enclosed KC4 program. This program can only be used on a single
computer, unless a site license or additional network passwords have
been purchased through Bio-Tek Instruments. You may transfer the
program from one computer to another provided that the program is used
with only one computer at a time. You may not electronically transfer the
program from one computer to another. You may not distribute copies of
the program(s) or documentation without the prior written consent of
Bio-Tek Instruments, Inc., or as stipulated in a sub-licensing agreement.
Network Use: You have the non-exclusive right to use the enclosed
program. This program can only be used on a single file server. The
number of concurrent users on the network at any time may not exceed
the number of licensed copies of this software. You may transfer the
program from one server to another provided that the program is used
with only one server at a time. You may not distribute copies of the
program(s) or documentation without the prior written consent of
Bio-Tek Instruments, Inc., or as stipulated in a sub-licensing agreement.
You may not use, copy, modify, loan, lease or transfer the program(s) or
documentation, or any copy except as expressly provided in this
Agreement.
COPYRIGHT: The KC4 program and its documentation are copyrighted.
You may not copy any program or the documentation except as for
loading the program into the computer/server as part of executing that
program and for back-up purposes, respectively. You must reproduce and
include the copyright notice on the back-up copy
TERMS: This license is effective until terminated. You may terminate it
by destroying the programs and documentation and all copies thereof.
This license will also terminate if you fail to comply with any term or
condition of this Agreement. You agree upon such termination to destroy
all copies of the programs and documentation.
Document
Revision
Rev / Date
Changes
A / March, 2001
First Issue.
B / December, 2002
Updated entire manual to correlate with new
licensing options, KC4 v3.1 and KC4 Signature,
and their related new and modified features.
C / September, 2003
Changed KC4 “v3.1” to “v3.3”
Added instructions for creating Read & Dispense
protocols for SynergyHTTR w/Injectors.
Added instructions for configuring and
controlling the Dispense Module.
Added instructions for using the Database
Upgrade Utility.
D / July, 2004
Changed KC4 “v3.3” to “v3.4”
Added instructions for using KC4 to control the
new Clarity™ Microplate Luminometer.
Added instructions for using the new Multi-Mode
detection method for the Synergy™ HT line of
instruments.
Added information for handling an unexpected
termination of a kinetic read.
Updated the system requirements for using
PowerReports to include Microsoft® Office XP
and Office 2003.
Updated information on COM port selection
(now supports up to nine ports).
Additional cosmetic changes throughout.
E / March, 2005
Revision D1 one-page insert removed; revision
letter changed as a result.
Contents
INTRODUCTION................................................................................................................. 1
KC4 Software ..................................................................................................................... 1
Technical Support ............................................................................................................... 4
Phone Support .............................................................................................................. 4
Facsimile Support......................................................................................................... 4
Written Communication ............................................................................................... 4
Electronic Communication........................................................................................... 4
INSTALLATION AND CONFIGURATION ......................................................................... 5
Important! ........................................................................................................................... 5
Appointing a System Administrator ................................................................................... 8
Administrator Requirements ........................................................................................ 8
Verifying Minimum System Requirements ........................................................................ 9
Upgrading from an earlier version of KC4 ....................................................................... 10
Installing KC4................................................................................................................... 11
Starting KC4 ..................................................................................................................... 18
Changing the System Administrator’s Password.............................................................. 21
Copying KCShared.mdb to a Secure Network Location .................................................. 22
Creating New User Accounts............................................................................................ 24
Connecting Reader(s) to the Computer............................................................................. 25
Where to go from here ...................................................................................................... 26
GETTING STARTED ........................................................................................................ 27
Overview........................................................................................................................... 27
Essential Information ........................................................................................................ 28
File Types ................................................................................................................... 28
File Storage ................................................................................................................ 28
Record Retention and Archival .................................................................................. 29
Plate Data Acquisition................................................................................................ 29
User’s Guide to KC4
Contents • i
Data Reduction and Analysis......................................................................................29
Biograph......................................................................................................................30
File Signatures ............................................................................................................30
Audit Trails .................................................................................................................30
Main Screen.......................................................................................................................30
Title Bar ......................................................................................................................30
Menu Bar ....................................................................................................................31
Toolbar........................................................................................................................31
Status Bar....................................................................................................................31
Quick Reference Guides....................................................................................................32
Data Menu...................................................................................................................32
Edit Menu ...................................................................................................................33
Protocol Menu.............................................................................................................33
Display Menu..............................................................................................................35
System Menu ..............................................................................................................35
About Menu ................................................................................................................36
Navigating the Help System..............................................................................................37
Quick Start.........................................................................................................................38
Quick Start for Clarity™ Microplate Luminometer Users................................................39
Sample Files ......................................................................................................................40
DATA MENU .....................................................................................................................41
Overview ...........................................................................................................................41
Working with Data Files ...................................................................................................42
Where and How is Plate Data Stored? ........................................................................42
Creating a New Data File............................................................................................43
Opening Existing Data Files .......................................................................................44
Modifying Data Files ..................................................................................................45
Saving Data Files ........................................................................................................46
Closing Data Files.......................................................................................................47
Reading Plates ...................................................................................................................48
Unintentional Termination of a Kinetic Read.............................................................49
Entering Plate Data Manually .....................................................................................50
Reading Plate Data from a File ...................................................................................51
Signing Data Files .............................................................................................................54
Append to Kinetic File ......................................................................................................56
Exporting Plate Data .........................................................................................................57
Printing Reports.................................................................................................................59
Print.............................................................................................................................59
Print Preview...............................................................................................................59
Print Setup...................................................................................................................59
ii • Contents
User’s Guide to KC4
Plate Description............................................................................................................... 60
Samples............................................................................................................................. 61
Auto Numbering......................................................................................................... 62
Import Sample Names ................................................................................................ 62
Export Sample Names ................................................................................................ 62
Printing Sample IDs ................................................................................................... 62
Data Audit Trail ................................................................................................................ 63
EDIT MENU ...................................................................................................................... 65
Editing Tools..................................................................................................................... 65
PROTOCOL MENU .......................................................................................................... 67
Overview........................................................................................................................... 67
Working With Protocols ................................................................................................... 68
Where and How Are Protocols Stored?...................................................................... 68
Opening Existing Protocol Files................................................................................. 68
Creating New Protocols.............................................................................................. 69
Modifying Protocol Parameters.................................................................................. 71
Saving Protocol Files.................................................................................................. 71
Closing Protocol Files ................................................................................................ 72
Signing Protocol Files ................................................................................................ 73
Plate Description............................................................................................................... 75
Defining Reading Parameters ........................................................................................... 76
Clarity™..................................................................................................................... 77
ELx800™ ................................................................................................................... 78
ELx808™ ................................................................................................................... 79
FL600™ ..................................................................................................................... 80
FLx800™ ................................................................................................................... 81
PowerWave™/PowerWave HT.................................................................................. 82
PowerWaveX™.......................................................................................................... 83
PowerWaveX™ Select............................................................................................... 84
PowerWave™ XS ...................................................................................................... 85
Synergy™ HT ............................................................................................................ 86
Synergy™ HT / Time-Resolved................................................................................. 87
Synergy™ HT / Time-Resolved / Injectors................................................................ 88
µQuant™ .................................................................................................................... 89
Reading Parameters Reference ......................................................................................... 90
Automatic Sensitivity Adjustment ............................................................................. 90
Bandwidth Verification .............................................................................................. 92
Detection Method ....................................................................................................... 94
Dispense Option ......................................................................................................... 94
Eject Between Filter Set Read.................................................................................... 95
Excitation/Emission Filter Sets (FL600™) ................................................................ 95
User’s Guide to KC4
Contents • iii
Excitation/Emission Filter Sets (FLx800, Synergy HT) .............................................97
Filters ..........................................................................................................................99
First Well / Last Well..................................................................................................99
General Options (FL600™) ........................................................................................99
General Options (Synergy™ HT) .............................................................................101
Kinetic Parameters ....................................................................................................102
Kinetic Parameters (FL600)......................................................................................103
Measurement Options ...............................................................................................104
Measurement Mode (FL600) ....................................................................................104
Measurement Mode (FLx800, Synergy HT).............................................................105
Measurement Mode (Synergy™ HT / Time-Resolved)............................................106
Monitor Wells ...........................................................................................................107
Multi-Mode...............................................................................................................108
Pathlength Correction ...............................................................................................109
Plate Type .................................................................................................................111
Pre-Read Blank Plate ................................................................................................111
Read Barcode ............................................................................................................111
Read & Dispense ......................................................................................................112
Read Mode................................................................................................................112
Reader/Read Method ................................................................................................112
Reading Direction .....................................................................................................113
Reading Type ............................................................................................................113
Scales for Kinetic Reads ...........................................................................................114
Scales for Spectrum Reads........................................................................................114
Scanning....................................................................................................................115
Shaking .....................................................................................................................116
Temperature Control.................................................................................................117
Time-Resolved Fluorescence (Synergy™ HT).........................................................117
Top Probe Vertical Offset.........................................................................................117
Wavelengths..............................................................................................................118
Creating a Read & Dispense Protocol .............................................................................119
Plate Mode Options...................................................................................................120
Well Mode Options...................................................................................................124
Raw Data Correction .......................................................................................................128
Blank Plate Subtraction.............................................................................................129
Blank Wells Subtraction ...........................................................................................130
Pathlength Correction ...............................................................................................130
Kinetic Analysis ..............................................................................................................131
Data to Process..........................................................................................................131
Mean V .....................................................................................................................132
Max V .......................................................................................................................132
Mean Min/Max .........................................................................................................134
Onset OD / Onset OD %...........................................................................................135
Formula.....................................................................................................................136
No Calculation ..........................................................................................................136
Integral ......................................................................................................................136
iv • Contents
User’s Guide to KC4
Spectrum Analysis .......................................................................................................... 137
Data to Process ......................................................................................................... 137
Min/Max................................................................................................................... 137
Formula .................................................................................................................... 138
Scanning Analysis........................................................................................................... 139
Mean, Std Dev, CV .................................................................................................. 139
Mean Min/Max......................................................................................................... 140
Integral ..................................................................................................................... 141
Plate Layout .................................................................................................................... 142
Well Types and IDentifiers ...................................................................................... 143
Selecting and Customizing Well Types & IDs......................................................... 144
Assigning Well IDs .................................................................................................. 149
Defining and Assigning Dilutions ............................................................................ 151
Defining and Assigning Concentrations................................................................... 155
Single Plate Transformations.......................................................................................... 159
Formula Syntax ........................................................................................................ 160
Formula Examples.................................................................................................... 161
Multi Plate Transformations ........................................................................................... 162
Syntax....................................................................................................................... 163
Automatic Formula Generation................................................................................ 163
Curves ............................................................................................................................. 164
Curve Description..................................................................................................... 164
Curve Axes ............................................................................................................... 165
Curve Fit................................................................................................................... 166
Curve Interpolation................................................................................................... 169
Validation........................................................................................................................ 171
Cutoffs ............................................................................................................................ 173
Selecting the Data to Interpret.................................................................................. 173
Defining Cutoffs....................................................................................................... 174
Defining Symbols..................................................................................................... 175
Cutoffs Evaluation.................................................................................................... 176
Reports ............................................................................................................................ 177
Audit Trail Reports................................................................................................... 178
Calculation Log Report ............................................................................................ 178
Kinetic Curves Report .............................................................................................. 179
Well Zoom Report.................................................................................................... 179
Validation Report ..................................................................................................... 180
Cutoffs Report .......................................................................................................... 180
Sample Name List Report ........................................................................................ 180
Plate vs. Table Format.............................................................................................. 181
Statistics Report........................................................................................................ 181
Export.............................................................................................................................. 182
User’s Guide to KC4
Contents • v
Protocol Audit Trail.........................................................................................................183
Security............................................................................................................................184
Protocol Options..............................................................................................................185
Raw Data...................................................................................................................185
Report........................................................................................................................186
Export........................................................................................................................187
Calculation ................................................................................................................188
DISPLAY MENU..............................................................................................................189
Overview .........................................................................................................................189
Curve(s) ...........................................................................................................................189
Calculation Warnings ......................................................................................................190
Validation Results ...........................................................................................................192
SYSTEM MENU ..............................................................................................................193
Overview .........................................................................................................................193
Reader Selection..............................................................................................................194
Port Selection ............................................................................................................195
Filters/Wavelengths ..................................................................................................196
Reader Configuration................................................................................................201
Dispenser Configuration ...........................................................................................202
Controlling Multiple Instruments Simultaneously....................................................204
Diagnostics ......................................................................................................................205
Optics Test ................................................................................................................205
System Test (Clarity) ................................................................................................206
Optics Test (FL600)..................................................................................................206
Universal Plate Test ..................................................................................................207
Security............................................................................................................................211
User Accounts...........................................................................................................212
Login and Password Parameters ...............................................................................216
Protected Functions...................................................................................................218
Audit Trail Notification Options...............................................................................220
Signature Reasons.....................................................................................................221
File Storage and Format Options ..............................................................................222
LogIn/LogOut..................................................................................................................223
Audit Trail .......................................................................................................................224
Exporting Audit Trail Events....................................................................................225
Maintain Files..................................................................................................................226
Edit Options ..............................................................................................................227
Exporting Files..........................................................................................................227
vi • Contents
User’s Guide to KC4
Importing Files ......................................................................................................... 228
Registration/Upgrade ...................................................................................................... 229
Setup ............................................................................................................................... 230
Protocol Defaults...................................................................................................... 230
Formats..................................................................................................................... 231
Start-Up .................................................................................................................... 232
Database ................................................................................................................... 233
Toolbar............................................................................................................................ 237
Plate Format .................................................................................................................... 240
Plate Description ...................................................................................................... 241
Microplate Measurement.......................................................................................... 242
Exporting/Importing Plate Formats.......................................................................... 242
Reader Control................................................................................................................ 245
Information............................................................................................................... 245
Door/Carrier ............................................................................................................. 245
Pre-Heating............................................................................................................... 246
Dispenser .................................................................................................................. 247
Clarity Control.......................................................................................................... 249
RESULTS ....................................................................................................................... 251
Overview......................................................................................................................... 251
Data Sets ......................................................................................................................... 253
Concentrations.......................................................................................................... 254
Symbols.................................................................................................................... 256
Pathlengths ............................................................................................................... 257
Kinetic Data.............................................................................................................. 258
Spectral Scan Data.................................................................................................... 260
Area Scan Data......................................................................................................... 262
Linear Scan Data ...................................................................................................... 263
Statistics .......................................................................................................................... 264
BIOGRAPH..................................................................................................................... 265
Overview......................................................................................................................... 265
File Menu........................................................................................................................ 266
Creating a New Curve .............................................................................................. 266
Closing a Curve ........................................................................................................ 266
Saving a Curve (Graph File) .................................................................................... 266
Opening a Graph File ............................................................................................... 267
Printing Curves......................................................................................................... 267
Exiting Biograph ...................................................................................................... 268
View Menu ..................................................................................................................... 268
User’s Guide to KC4
Contents • vii
The Toolbar...............................................................................................................268
The Status Bar...........................................................................................................268
Graph Menu.....................................................................................................................269
Standards...................................................................................................................269
Axes, Fit, Interpolations............................................................................................269
Overlaying Graphs ....................................................................................................270
Curve Window ................................................................................................................271
POWERREPORTS..........................................................................................................273
High-Powered Reporting.................................................................................................273
How it Works............................................................................................................274
System Requirements................................................................................................276
Preparing KC4 for PowerReporting ................................................................................276
Setting Global Options..............................................................................................276
Setting Protocol-Specific Options.............................................................................277
Defining the Protocol Parameters .............................................................................278
Creating/Editing Documents.....................................................................................278
Working with Results Objects.........................................................................................279
The Results Objects Toolbar.....................................................................................279
Object Components...................................................................................................280
Object Fulfillment.....................................................................................................281
Designing the Document .................................................................................................282
Inserting Field Objects..............................................................................................282
Inserting Plate Objects ..............................................................................................283
Inserting Table Objects .............................................................................................286
Inserting Curve Objects ............................................................................................302
Previewing and Printing the Report ................................................................................306
Previewing Options...................................................................................................306
Special Notations ......................................................................................................307
Performing the Data Export.............................................................................................308
Direct Export to Excel ..............................................................................................308
APPENDIX A: RECORDS SUBMISSION .....................................................................309
Records Submission ........................................................................................................309
APPENDIX B: WINDOWS FILE CACHING ..................................................................311
Windows File Caching ....................................................................................................311
INDEX..............................................................................................................................313
viii • Contents
User’s Guide to KC4
Introduction
KC4 Software
Bio-Tek’s KC4™ software provides rapid, flexible, and powerful assay analysis with a multitude
of applications. The user interface is simple and uncluttered, and provides a quick, step-by-step
setup and read process for even the most complex assays.
Applications include:
•
Research
•
DNA quantitation
•
Fluorescence assays
•
Bio-Industrial
•
Pharmaceutical
•
Development
Interfaces with most Bio-Tek autoreaders:
Clarity™
FLx800™
PowerWaveX 340
EL800™
PowerWave™
PowerWaveX Select
ELx800™
PowerWave 340
PowerWave XS
EL808™
PowerWave HT
Synergy™ HT
ELx808™
PowerWave HT 340
µQuant™
FL600™
PowerWaveX™
Extensive and complete analysis of your laboratory applications:
•
Fluorescence and absorbance ELISA
•
Spectral scanning from 200-999 nm with the µQuant and all PowerWave, PowerWaveX,
and Synergy HT models
•
Endpoint assays
•
Kinetic assays
•
Endotoxin analysis
•
Cytotoxicity and titer assays
User’s Guide to KC4™
Introduction • 1
Defines and controls all reading protocols:
•
Selection of filters/wavelengths
•
Shaking parameters
•
Incubation
•
Flexible kinetic options
•
Read/Dispense options
•
Optional manual entry of data
•
Reader diagnostics testing and archiving
Customized assay development:
•
Graphical input for template creation with auto-replication and fill, and customized well
ID text and colors
•
Quick entry of standard concentrations and sample dilutions with auto-increment and
auto-factor
•
Control and assay validations may be created for each assay
•
Multiple sample curves displayed and analyzed simultaneously, including interpolations
•
Powerful multi-level transformation formulas allow manipulation of raw data,
transformed data, curve-fit results, or statistical calculations at any point in the analysis
•
KC4’s zoom feature allows detailed information observed for each well, including the
ability to highlight specific kinetic reaction wells (absorbance and fluorescence)
•
Sample IDs may be entered manually or imported from a file
•
Extensive curve-fitting capabilities, including Linear or Log axis definition, Point-toPoint, Linear, Polynomial, Cubic Spline, 4-P, and interpolation and graphic editing
features
Customized display and printed reports:
•
Quick access to all available results data including raw measurement values,
transformed values, kinetic calculations, concentrations, plate layout, and statistical data
on controls, standards, and samples
•
Masking and recalculation capability for authorized users
•
Kinetic options: Real-time display of kinetic reactions, individual well zoom, Mean V,
Max V, time at Max V, Min/Max OD, Integral (area under the curve), and Onset
calculations
•
Screening options: Definition of cutoffs by algebraic formulae, and positive, negative,
etc., range calculations
•
Validation options: Formula to validate the Mean, Standard Deviation, or CV of
controls
•
Printed reports are completely customizable and can include results data sets in matrix
or column format; standard, kinetic, or dilution curves; sample list; audit trail; protocol
description; custom header; and page breaks
2 • Introduction
User’s Guide to KC4™
•
For users with Microsoft® Word/Excel 2000 (or greater) and Windows 98/NT/2000/XP,
PowerReports links KC4 with Excel and Word, providing a powerful format for creating
custom publication-quality report and export documents
•
Reports can be previewed before printing
Data exporting:
•
Data may be exported automatically for use with other programs including Excel™,
Lotus™, and Paradox™ for further manipulation or report merging
•
Exporting is not limited to just raw data; all calculated results may be exported
KC4 Signature complies with FDA’s Electronic Signatures rule, 21 CFR Part 11:
•
System Administrator. A site-designated System Administrator creates and maintains
Standard User (low level) and Power User (high level) accounts, and specifies which
KC4 functions shall be protected from use by Standard Users. The System Administrator
sets special password and login characteristics including minimum password length,
password aging, and idle session time-out.
•
Support for multiple users. Each user is identified by a unique combination of User ID
and encrypted password. Users must log into KC4 with these identifiers to gain access.
•
Time-stamped audit trails. Activities such as user login/logout, protocol and data file
creation and modification, and plate reading are permanently logged in a secure database.
•
Embedded signatures. Authorized users can electronically sign protocol and data files.
Electronic signatures are permanent and remain a part of the overall data record for the
life of that record.
•
Secure record storage. KC4 proprietary files (.prt, .pla, and .glb) are stored in a secure
shared-access database. Activities performed on files within this database (such as
rename, move, copy, overwrite, and delete) are performed within the KC4 environment,
and every change is tracked in an audit trail. In addition, the System Administrator can
configure the system so that these activities may only be performed by Power Users.
•
Protected functions. The System Administrator can protect a variety of functions from
use by Standard Users. These functions include the deletion, rename, modification, and
overwrite of various record types.
User’s Guide to KC4™
Introduction • 3
Technical Support
KC4 is backed by a superior support staff. If the software fails to function perfectly, please
contact the Technical Assistance Center (TAC) and provide the following information:
•
The KC4 software version and revision numbers (such as Version # 3.4, Rev # x) found
under the About menu.
•
The license type (‘v3.4’ or ‘Signature’).
•
The product serial number.
•
The specific steps which reproduce your problem.
•
Any error messages displayed on the screen.
•
A daytime phone number and fax number (if available).
•
Your name and company information.
Phone Support
Contact the Technical Assistance Center between 8:30 AM and 5:30 PM Eastern Standard Time
(EST), and/or Customer Service between 8:00 AM and 5:30 PM EST, Monday through Friday,
excluding holidays.
Bio-Tek Instruments’ Main Number:
(802) 655-4040
Technical Assistance Center:
(800) 242-4685 (toll free in the U.S.)
(802) 655-4740 (outside the U.S.)
European Coordination Center:
+49 (0) 7136 9680
Facsimile Support
Send a fax with your questions or requests for help 24 hours a day to:
Technical Assistance Center:
(802) 655-3399
European Coordination Center:
+49 (0) 7136 968 111
Written Communication
Send your written comments to:
Bio-Tek Instruments, Inc.
Technical Assistance Center
P.O. Box 998, Highland Park
Winooski, Vermont USA 05404-0998
Bio-Tek Instruments, GmbH
Kocherwaldstrasse 34
D-74177 Bad Friedrichshall
Germany
Electronic Communication
Send E-Mail or find us on the Web:
E-Mail: [email protected]
Web: http://www.biotek.com
To visit the our website from within KC4, select About|Bio-Tek Home Page.
4 • Introduction
User’s Guide to KC4™
Installation and Configuration
Important!
There is a certain sequence of events that must be followed to ensure that KC4 is properly installed
and configured. This chapter describes the essential installation and configuration tasks.
Review the installation options provided on the following pages and choose the one required by
your site to determine which tasks must be performed. Turn to the appropriate page number for
detailed instructions on each task.
User’s Guide to KC4™
Installation and Configuration • 5
Special note for Clarity™ Microplate Luminometer users: The Clarity PC software must also
be installed on the PC(s). Install Clarity before performing the step “Connect reader(s) to the
computer and establish communication.”
KC4 v3.4 – Single PC with one or more users
Step
Task
Page
1
Ensure that the computer meets the minimum system requirements
9
2
Install KC4 and restart the computer
11
3
Start KC4 and log in as the System Administrator
18
4
(Optional) Change the System Administrator’s password
21
5
(Optional) Copy KCShared.mdb to a secure network location
22
6
(Required if step 5 was performed) Configure KC4 to use the
KCShared.mdb file on the network
23
7
Connect reader(s) to the computer and establish communication
25
KC4 v3.4 – Multiple PCs in a network environment
Step
Task
Page
1
Ensure that all computers meet the minimum system requirements
9
2
Install KC4 on one computer and restart the computer
11
3
Start KC4 and log in as the System Administrator
18
4
Change the System Administrator’s password
21
5
Copy KCShared.mdb to a secure network location
22
6
Configure KC4 to use the KCShared.mdb file on the network
23
7
Connect reader(s) to the computer and establish communication
25
8
Repeat steps 2, 3, 6, and 7 for the remaining computers
6 • Installation and Configuration
User’s Guide to KC4™
Special note for Clarity™ Microplate Luminometer users: The Clarity PC software must also
be installed on the PC(s). Install Clarity before performing the step “Connect reader(s) to the
computer and establish communication.”
KC4 Signature – Single PC with one or more users
Step
Task
Page
1
Appoint a System Administrator
8
2
Ensure that the computer meets the minimum system requirements
9
3
Install KC4 and restart the computer
11
4
Start KC4 and log in as the System Administrator
18
5
Change the System Administrator’s password
21
6
Copy KCShared.mdb to a secure network location
22
7
Configure KC4 to use the KCShared.mdb file on the network
23
8
Create one or more new user accounts
24
9
Connect reader(s) to the computer and establish communication
25
KC4 Signature – Multiple PCs in a network environment
Step
Task
Page
1
Appoint a System Administrator
8
2
Ensure that all computers meet the minimum system requirements
9
3
Install KC4 on one computer and restart the computer
11
4
Start KC4 and log in as the System Administrator
18
5
Change the System Administrator’s password
21
6
Copy KCShared.mdb to a secure network location
22
7
Configure KC4 to use the KCShared.mdb file on the network
23
8
Create new user accounts
24
9
Connect reader(s) to the computer and establish communication
25
10
Repeat steps 3, 4, 7, and 9 for the remaining computers
User’s Guide to KC4™
Installation and Configuration • 7
Appointing a System Administrator
KC4 Signature
Before installing the software, one individual at the site must be appointed as the KC4 System
Administrator. The System Administrator is typically responsible for the following:
•
Determining which computers will have KC4 installed on them
•
Determining which users will have access to certain KC4 functions
•
Installing KC4
•
Creating and maintaining user accounts
•
Customizing security settings
•
Reviewing records
•
Establishing a procedure and schedule for record retention and archival
Administrator Requirements
The FDA’s Electronic Signatures Rule (21 CFR Part 11) contains requirements that sites must
meet in order to be in compliance. The KC4 Signature System Administrator should be cognizant
of the following:
•
Administrator: The site shall select an “Administrator” whom will be the person
responsible for all high-level administration of the program. This person will control
access to the program by adding new users, structuring the individual users authority
levels, and reporting to management, as appropriate, on any unauthorized use of the
program.
•
Personnel Qualifications: Personnel that develop, maintain, or use electronic
record/electronic signatures shall have the education, training and experience necessary
to perform their assigned tasks.
•
Written Policies: There shall be written policies which hold individuals accountable and
responsible for action initiated under their electronic signatures, in order to deter record
and signature falsification. There shall be written revision and change control procedures
to ensure that the program is administered in compliance with the FDA’s requirements.
•
Record Archiving and Deletion: The site is responsible to ensure that archiving or other
file management techniques are suitable such that electronic records generated by the
system shall be accurate and readily retrievable. Records may be removed by deleting
the entire shared database or individual files within the database. This action may be
conducted by the Administrator or by any Power User.
•
User Identity: Any person that will be authorized to use an electronic signature will have
their identity confirmed by the Administrator prior to granting them program access.
Only the genuine owner of the electronic signature is allowed to access the program
through their ID/password combination. System users should be informed that accessing
the program using someone else’s logon is a violation of the FDA rule.
8 • Installation and Configuration
User’s Guide to KC4™
•
Password Expiration/Recall: All passwords must be checked, recalled, or revised at an
interval appropriate with the security needs of the organization. Personnel whom no
longer work at the establishment shall have their program access capabilities deactivated
in a timely manner.
•
Certification to FDA: The site must certify to FDA that the electronic signatures utilized
on records are intended to be as legally binding as handwritten signatures, prior to or at
the time of record submission to FDA. The FDA rule should be consulted for details and
method to be used.
•
Notification of Attempted Security Breaches: The software utilizes an error log system
to notify the Administrator of log-in failure incidents that exceed the limits they have
established. The administrator is responsible to “immediately and urgently” notify the
appropriate personnel at the site if the activity appears to be an attempt to breach security.
•
Signature Representations: The administrator should be made aware that while
electronic signatures representations cannot be excised or added they can be copied by
screen copy techniques and pasted into other documents. These modified documents
cannot be resaved in the secure program but may be printed out as is.
Verifying Minimum System Requirements
KC4 v3.4
KC4 Signature
Before installing KC4, verify that the computer(s) meet or exceed the following requirements:
•
Pentium processor / 100 MHz minimum *
•
Microsoft Windows 95/98/NT/2000/XP operating system *
•
64 MB of RAM minimum *
•
30 MB of available hard drive space for the KC4 program files
•
Additional available hard drive space for storing protocol and plate data files
•
CD ROM drive
•
Super VGA color monitor
•
Keyboard and mouse ports
•
Serial port (for autoreader connection)
•
USB port (with USB-to-serial adapter, if required)
* For users with Microsoft® Word/Excel 2000 (or greater) and Windows 98/NT/2000/XP,
PowerReports links KC4 with Excel and Word, providing a powerful format for creating custom
publication-quality report and export documents. For the PowerReports feature to run
successfully, the computer(s) must meet or exceed the following requirements:
•
Pentium II processor / 300 MHz minimum
•
Microsoft Windows 98/NT/2000/XP operating system
•
128 MB RAM recommended
•
Microsoft Office 2000 (9.0.3821 SR-1 or newer), Office XP, Office 2003
User’s Guide to KC4™
Installation and Configuration • 9
Upgrading from an earlier version of KC4
KC4 v3.4
KC4 Signature
Upgrade Notes:
•
If you are upgrading from KC4 version 3.1, 3.2, or 3.3 and you plan to uninstall your
existing version, we recommend you do so after successfully installing version 3.4. This
will allow the 3.4 installation program to recognize your current registration information.
•
You are not required to uninstall an earlier KC4 version. If you choose to uninstall a
pre-3.1 version of KC4, however, be sure to preserve the KC4_32.pld file found in the
root KC4 folder. This file may contain custom plate formats that must be imported into
the new plate database to support any protocols that reference custom plate formats. See
“Plate Format” in System Menu to learn more.
•
If you are upgrading from KC4 version 3.1, 3.2, or 3.3 and you plan to reuse existing
Local, Shared, and/or Diagnostic databases, make a note of the names and current
locations of these databases. They will have to be upgraded for use with version 3.4.
During the version 3.4 installation, KC4 will automatically run a Database Upgrade
Utility to do this. KC4 will search all fixed, non-removable drives (e.g., diskette, ZIP,
JAZ, and network drives are not automatically searched), for the following files:
KCLocal.mdb, KC*.mdb, and Calib.mdb. You will have an opportunity to specify
additional databases for upgrade during the installation. You can also run the utility after
KC4 is installed via the System|Setup, Database menu option.
Note: When KC4 prepares to upgrade a database, it makes and saves a copy of the
original. The original database will remain unchanged and it can still be used with the
earlier KC4 version. The upgraded databases are not backwards-compatible. That is, a
KC4 version 3.4 database will not be recognized by KC4 version 3.1.
10 • Installation and Configuration
User’s Guide to KC4™
Installing KC4
KC4 v3.4
KC4 Signature
Follow these steps to install KC4:
1.
Start Windows.
Note: If you are prompted with a Windows password entry dialog, enter a valid
password; this helps to ensure a complete installation. If you were not prompted
with the Windows password entry, disregard this note.
2.
Close any unnecessary Windows programs.
3.
Place the CD in the CD ROM drive.
4.
Click the Windows Start button and select Run.
5.
When the Run dialog appears, type <drive>:\Setup.exe, where <drive>
represents the CD rom drive.
Alternatively, use the Browse function to access the drive and then click on
setup.exe.
6.
Click OK. After a brief pause, the Welcome dialog appears.
7.
Click Next to continue. An informational dialog appears:
User’s Guide to KC4™
Installation and Configuration • 11
8.
Click Next to continue. The Software License Agreement dialog appears:
9.
If you accept these terms, click Yes to continue. Otherwise, click No to discontinue the
software installation.
If you click Yes, the Choose Destination Location dialog appears:
12 • Installation and Configuration
User’s Guide to KC4™
10. The default location for the software is displayed. Click Next to accept the default
location, or click Browse to specify a different location.
After a location is specified, the Select Components dialog appears:
11. Check the components you want to install and click Next.
The Select Program Folder dialog appears:
12. Accept the default Program Folder or enter a new one. Click Next to continue with the
installation process.
User’s Guide to KC4™
Installation and Configuration • 13
After a few moments, the Registration dialog appears:
13. Enter the License Number provided with your KC4 package.
14. Click the Registration Card button to verify the number and open a registration form:
14 • Installation and Configuration
User’s Guide to KC4™
15. Enter the required information and click Send by Fax to print a copy of this form for
later faxing, or Send by E-mail to send the information to Bio-Tek via email.
Bio-Tek will respond within two business days with your password.
If you are upgrading from a previous KC4 version or from KCjunior, enter the
requested KC4 hardlock serial number, KC4 software license number, or KCjunior
serial number, as appropriate. Click Check to verify the number.
If you are reinstalling KC4, you must submit a separate Reinstallation Form to
obtain another password. Reinstallation is defined as (1) re-installing a KC4 version
on the same PC (for example, after a hard disk replacement), or (2) un-installing a
KC4 version from one PC and installing the same version on another PC.
A Reinstallation Form (.doc and .txt formats) complete with instructions can be
found in the root KC4 folder when the installation process is complete.
16. Click Close to return to the Registration window and then click OK to continue.
KC4 will automatically run the Database Upgrade Utility. This utility is used to update
Shared, Local, and Calibration databases created with earlier versions of KC4 so that they
will be compatible with the current version of KC4. For example, if you are upgrading
from KC4 version 3.1 to 3.4, your version 3.1 databases must be converted before they
can be used by KC4 version 3.4.
KC4 automatically begins a search of the fixed, non-removable drives (e.g., diskette,
ZIP, JAZ, and network drives are not automatically searched). KC4 searches for the
following files: KCLocal.mdb, KC*.mdb, and Calib.mdb. Note: This automatic
search will not find Shared database files if their names do not begin with “KC”.
17. If you know the names and locations of the databases you wish to upgrade, and/or if the
databases reside on a drive that is not automatically searched, click the Stop button to
advance to the Database Upgrade Utility dialog (see next step).
When the search is complete, if KC4 has detected database files created by former KC4
versions, an upgrade option message box appears:
18. Click Yes to upgrade the database(s) now, or No to skip the upgrade at this time. The
Database Upgrade Utility can be run manually through KC4, via System|Setup, Database.
User’s Guide to KC4™
Installation and Configuration • 15
If you click Yes, the Database Upgrade Utility dialog appears:
Click the drop-down arrows to see which databases KC4 found. Click on the desired
database name to select it for upgrading.
Click the Browse button to search for a database. When the database is located,
highlight it and click Open. KC4 will insert the name in the appropriate field
(Shared, Local, or Diagnostics).
19. Click Start Upgrading to begin the process. When prompted, enter name(s) for the
converted databases. (The original databases will remain unchanged). Default names
contains a date and time stamp, as shown below.
20. Click Save to complete the upgrade.
When the process is complete, KC4 will ask if you would like to read the Readme file:
16 • Installation and Configuration
User’s Guide to KC4™
21. Click Yes or No as desired.
If you click No, or when you exit the text editor, the installation process finishes and then
the Setup Complete dialog appears:
22. Remove the CD from the drive and click Finish to restart the computer.
User’s Guide to KC4™
Installation and Configuration • 17
Starting KC4
KC4 v3.4
KC4 Signature
To start KC4:
1.
Double-click on the KC4 icon on the desktop, or open the Start menu and select
Programs > KC4 3.4 Rev # > KC4.
KC4’s opening dialog appears (KC4 Signature users will see a different picture):
2.
Click Run KC4 in Trial Mode.
18 • Installation and Configuration
User’s Guide to KC4™
KC4’s main dialog appears:
Note: KC4 may attempt to communicate with an attached microplate reader. If you
have not yet attached a reader, do not be concerned at this time if messages resembling
“NO READER RESPONSE” and “Error when getting wavelength table from reader”
appear. These messages will disappear when a reader is properly connected and
configured.
KC4 Signature users will be presented with a Login dialog:
Enter “admin” in the Password field and click OK. The dialog will disappear and
“System Administrator” will appear in KC4’s status bar at the bottom of the dialog.
User’s Guide to KC4™
Installation and Configuration • 19
TplRep_1 Error Message
Under certain circumstances, an error message resembling ‘TplRep_1.xla could not be found’ may
appear in KC4. It is most likely to appear if an earlier version of KC4 was uninstalled and the
remaining KC4 root folder was moved, renamed, or deleted.
This error message occurs because one of the files used for building PowerReports cannot be
found. The error may occur:
•
When starting KC4, if Excel is the default reporting or exporting engine and the Preferred
file format is GLB.
•
When creating a new protocol or GLB file, if Excel is the default reporting or exporting
engine.
Follow these steps to solve the problem:
1.
Close KC4.
2.
Launch Microsoft Excel. The ‘TplRep_1.xla could not be found’ message will appear.
3.
Click OK.
4.
In Excel, select Tools/Add-Ins. The ‘TplRep_1.xla’ Add-In will be selected.
5.
Unselect the ‘TplRep_1.xla’ Add-In. Excel will display an error message indicating that
the file could not be found and prompts you to delete this Add-In.
6.
Click Yes to delete the Add-In.
7.
Quit Excel.
8.
Restart KC4. The ‘TplRep_1.xla could not be found’ message should no longer appear.
20 • Installation and Configuration
User’s Guide to KC4™
Changing the System Administrator’s Password
KC4 v3.4
KC4 Signature
We recommend that you change the System Administrator’s password immediately following
software installation to ensure a secure operating environment.
To change the System Administrator’s password:
1.
Log in as the System Administrator if you have not already done so.
KC4 v3.4: Select System|Administrator LogIn.
KC4 Signature: Select System|LogIn/LogOut.
2.
When the Login dialog appears, enter the password “admin” and click OK.
3.
Select System|Security and click the Edit button.
4.
Enter a new password, confirm the password, and then click OK.
Important! Do not forget the Administrator’s password. If you do, you will have to
reinstall KC4.
User’s Guide to KC4™
Installation and Configuration • 21
Copying KCShared.mdb to a Secure Network Location
KC4 v3.4 (optional)
KC4 Signature
KCShared.mdb is a database file installed by KC4.
•
With KC4 v3.4, KCShared contains security and plate format information.
•
With KC4 Signature, KCShared contains security and plate format information; user
accounts; protocol, plate data, and global data files and their associated audit trails; and a
system audit trail for shared events.
Following installation, KCShared.mdb is stored on the computer’s hard drive but a copy of it can
be stored in a network location for secure record storage and for multi-user access.
Note: It is important to keep either the original KCShared.mdb file or a copy of the network file
on each user’s hard drive in case the user needs to revert to it following an extended network
connection failure.
To locate the installed KCShared.mdb file and copy it to the network:
1.
In KC4, select System|Setup and click the Database tab.
2.
The Path field in the Shared Database section contains the current location of KCShared.
(To see the full pathname, click once in the Path field and press the right arrow key.)
3.
Launch Microsoft Explorer and go to this location.
Tip: With some operating systems, KCShared.mdb is installed in the Application
Data folder under \Documents and Settings\All Users. If you are looking for the
Application Data folder on your computer and can’t find it, it is probably hidden. To
reveal this folder, open Microsoft Explorer and select Tools|Folder Options|View.
Make sure that ‘Show hidden files and folders’ is enabled.
4.
Copy KCShared.mdb to the clipboard and paste it in a secure, shared-access location on
the network.
5.
Return to KC4.
22 • Installation and Configuration
User’s Guide to KC4™
To configure KC4 to use the KCShared.mdb file on the network:
1.
Within the System|Setup, Database dialog, click the Browse button. A standard
Windows-style Open dialog will appear.
2.
Use the file tree to locate the KCShared.mdb file on the network. Highlight the file and
click Open. The Database dialog will reappear and the Path field will update to show the
new location.
3.
Click OK to close the Database dialog.
Note: With KC4 Signature, an Audit Trail dialog will appear to log this event.
Enter an appropriate comment and click Close.
4.
KC4 will display the following message. Click Yes to quit KC4.
5.
Restart KC4.
User’s Guide to KC4™
Installation and Configuration • 23
Creating New User Accounts
KC4 Signature
KC4 Signature supports the creation of individual user accounts to ensure that only authorized
users can gain access to the system and to any protected functions. To create a new user account:
1.
Select System|Security|Users.
2.
Click New. The KC4 User Settings dialog will appear.
3.
Enter a unique ID using 1 to 16 alphanumeric characters. The user will enter or select this
ID when logging into KC4 and when signing files.
4.
Enter the user’s Full Name. This name will be associated with events logged by this
user’s actions and with signatures applied by this user.
5.
Choose a Group Membership.
Standard Users are denied access to protected functions. (The System Administrator
determines which functions shall be protected from use by Standard Users.)
Power Users are permitted access to protected functions. Power Users cannot
administer user accounts or modify Security settings.
6.
Enter a temporary password for the user in the New and Confirm fields.
Share the password with the user with instructions to change it as soon as possible.
24 • Installation and Configuration
User’s Guide to KC4™
7.
(Optional) Specify a folder location from which the user will most often access protocol
files. When the user selects Data|New Plate to initiate a plate read, the protocol selection
dialog will default to this Protocols Folder.
The user is permitted to modify his or her Protocols Folder.
8.
Click OK to save the changes and return to the Security dialog.
Connecting Reader(s) to the Computer
KC4 v3.4
KC4 Signature
Before KC4 can be used to communicate with a microplate reader, the reader must be properly
connected to the computer on which KC4 is installed.
The following briefly describes how to connect the reader to the computer. For more detailed
information on reader setup, refer to the reader’s user manual.
1.
Attach the appropriate cable or adapter to an available serial or USB port on the back of
the computer.
2.
Plug the other end of the cable to the serial or USB port on the reader.
3.
Connect the power cable to the back or side of the reader.
4.
Plug all power cords into grounded electrical outlets. Use of a surge protector is strongly
recommended.
5.
Use KC4 to set and test the communication parameters, as described under “Reader
Selection” in the System Menu chapter.
User’s Guide to KC4™
Installation and Configuration • 25
Where to go from here
KC4 v3.4
KC4 Signature
KC4 is a powerful software program with many features and customizable options. Learn more
now by following these recommendations:
•
Go to the Getting Started chapter and:
Review essential information on file types, plate data acquisition, data reduction, and
more.
Learn about KC4’s menu structure, and how to navigate the Help system.
Get started with the Quick Start.
Examine sample protocol and plate data files, and read the corresponding assay
descriptions.
•
Go to the Data Menu chapter and read “Working with Data Files” to learn how to access,
generate, modify, and save plate and global data files.
•
Go to the Protocol Menu chapter and read “Working with Protocols” to learn how to
access, create, modify, and save protocol files.
•
Go to the System Menu chapter and read:
“Security” to understand all of the security options available to the System
Administrator.
“Protected Functions” to learn how to protect certain KC4 functions from use by
unauthorized users.
“Database” to learn about the different databases used by KC4.
“Exporting/Importing Plate Formats” to update the plate format database to support
pre-version 3.1 protocols that reference custom plate formats.
“Importing Files” to transfer your pre-version 3.4 protocol and data files into the
shared-access database (KC4 Signature users only).
26 • Installation and Configuration
User’s Guide to KC4™
Getting Started
Overview
Welcome to KC4! Getting Started introduces some important concepts for KC4 users, including:
•
A description of KC4’s key features, under Essential Information.
•
A tour of KC4’s Main Screen.
•
An overview of each menu’s contents, under Quick Reference Guides.
•
Tips on Navigating the Help System.
•
A typical sequence of events under Quick Start.
•
A Quick Start just for Clarity Microplate Luminometer users.
•
Instructions for locating and working with Sample Files.
User’s Guide to KC4™
Getting Started • 27
Essential Information
KC4 allows you to define instructions for controlling a microplate reader, acquiring measurement
data from that reader, for reducing and analyzing the retrieved data, and for printing, exporting,
and saving the results.
The minimum instructions required for reader control and plate data acquisition are called reading
parameters. Reading parameters varying according to reader model but generally include reading
type (endpoint, kinetic, spectrum), reading method (absorbance, fluorescence, luminescence),
filters/wavelengths, shaking and incubation instructions, well monitoring instructions, plate
geometry, and various pre-reading options including pathlength correction.
Reading parameters can be combined with data reduction instructions in a protocol. In addition to
storing the reading parameters, a protocol can store plate layout information, single plate
transformations, multi plate transformations, curve-fitting parameters, cut-off and validation
criteria, report and export templates, security settings, and more.
The KC4 software package also contains the Biograph module for viewing and manipulating
curves. Biograph can be used either alone or in conjunction with KC4.
File Types
KC4 uses several proprietary file formats to store information:
•
Protocol (*.PRT) files contain instructions for controlling the attached microplate reader.
Protocols also contain information required by KC4 to analyze plate measurement data.
•
Plate data (*.PLA) files contain raw measurement values, plate description information
(prompts, comments, date and time of reading), and sample names.
•
Global data (*.GLB) files contain plate data and protocol information. The GLB file
format is offered as an alternative to maintaining two separate protocol and plate data
files, and is the required data file format for electronic signature support.
In addition, Clarity™ Microplate Luminometer users will work with Clarity protocol files, which
use a .BPF extension. These files are referenced by KC4’s PRT and GLB files and they contain
the reading parameters required to control the luminometer.
File Storage
File storage options vary depending on site requirements and the current KC4 configuration.
•
Files may be stored individually on the computer’s hard drive, on a CD or diskette, or on
a network to which the computer is attached. This is the storage method that users of
KC4 version 3.0x and earlier are familiar with. Windows Explorer or a similar
application can be used to view the file names and their locations, and to move, copy,
rename, and delete files.
•
With KC4 Signature, PRT, PLA, and GLB files may also be stored inside a secure,
shared-access database. This database, named KCShared.mdb, can be stored on a user’s
computer or on a shared-access computer. KC4 provides a special file maintenance
utility for viewing the file names and their locations, and for moving, copying, renaming,
deleting, importing, and exporting files.
28 • Getting Started
User’s Guide to KC4™
Record Retention and Archival
With KC4, users create and save protocol, plate data, and global data files containing valuable
assay information and test results. KC4 does not automatically delete or archive any of these
records. It is therefore the site’s responsibility to establish a procedure and schedule for record
retention and archival.
Important: Regardless of license type, KC4 stores information using three different databases:
KCShared.mdb, KCLocal.mdb, and CALIB.mdb. See “Database” in System Menu to learn more
about their functions and locations. Because file or network problems can potentially create
unrecoverable database errors, these databases should be backed up on a regular basis.
•
KC4 v3.4 Users:
Protocol, plate data, and global data files are stored individually on a user’s computer or
on a network computer to which the user has access. Windows Explorer or a similar
application can be used to backup/archive files by copying them to the desired medium.
•
KC4 Signature Users:
Protocol (PRT), plate data, and global data files are typically stored inside
KCShared.mdb (they can also be stored outside this database, see “File Storage and
Format Options” in System Menu for more information). To backup/archive files you can
either copy the entire database to the desired medium, or you can export files to a disk
and then copy them to the desired medium.
See “Maintain Files” in System Menu to learn more about exporting and optionally
deleting files from the shared database.
See also “Audit Trail” in System Menu to learn about exporting and optionally
deleting system audit trail events to reduce the size of KCShared.mdb.
Plate Data Acquisition
KC4 provides three methods for acquiring plate data: retrieving results from a microplate reader,
entering plate data manually, and reading data in from an existing text file. Refer to the following
for more information:
•
“Reading Plates” on page 48
•
“Entering Plate Data Manually” on page 50
•
“Reading Plate Data from a File” on page 51
Data Reduction and Analysis
When the data has been retrieved and data reduction is complete, KC4 displays the raw data
associated with the first defined filter/wavelength. The availability of additional data sets
depends on the current protocol parameters and can include raw data at other filters/wavelengths,
blanked data, corrected data, concentrations, symbols, kinetic curves, and more. Refer to the
following for more information:
•
“Results Overview” on page 251
•
“Data Sets” on page 253
User’s Guide to KC4™
Getting Started • 29
Biograph
The KC4 software package includes the Biograph module for curve viewing and manipulation.
Biograph can be used either as a stand-alone product or in conjunction with KC4. See “Biograph”
for more information.
File Signatures
KC4 Signature users can electronically sign protocol files and global data files. Signing a file is
easy; select Protocol|Sign or Data|Sign and KC4 will guide you through the process. Electronic
signatures are permanent and cannot be modified, and they remain a part of the overall protocol or
global data file for the life of that file. Signatures can be viewed by selecting Data|Audit Trail or
Protocol|Audit Trail.
Audit Trails
KC4 automatically logs events for user actions and stores these events inside audit trails:
•
KC4 v3.4 maintains a Data Audit Trail for each plate and global data file. These audit
trails contain events for Change and Mask actions applied to the measurement data.
•
KC4 Signature maintains three types of audit trails: Protocol, Data, and System. These
audit trails contain events for many user activities including login/logout, protocol and
global data file creation and modification, plate reading, and more.
Main Screen
KC4’s functions are controlled through the main screen, which contains a Title Bar, Menu Bar,
Toolbar and Status Bar.
Title Bar
The title bar shows the current open file(s). Its appearance depends on the current Preferred Data
File Format setting (selected via System|Security|Files).
When the Preferred Data File Format is set to .GLB:
•
The set of brackets [ ] embraces the Global Data File name (extension GLB).
•
If a previously-saved protocol was used to generate the .GLB file, the title bar includes
the text “created from <protocol name>.prt”
When the Preferred Data File Format is set to .PLA:
•
The first set of brackets [ ] embraces the Plate Data File name (extension PLA).
•
The second set of brackets [ ] embraces the Protocol File name (extension PRT).
•
An asterisk (*) next to any file name indicates that its contents have changed but the file
has not yet been saved.
30 • Getting Started
User’s Guide to KC4™
Menu Bar
•
The Data menu contains functions for acquiring and processing measurement data.
•
The Edit menu provides tools for copying data from the screen in KC4 and pasting it into
a spreadsheet, word processor, database manager, or text editor. (Data cannot be pasted
back into KC4.)
•
The Protocol menu contains functions for creating, modifying, and saving assay
protocols.
•
The Display menu provides access to the Biograph module and also displays the results
of the Validation criteria defined in the protocol.
•
The System menu contains special functions for configuring readers, maintaining user
accounts, configuring security parameters, setting global parameters, performing reader
diagnostics, and more.
•
On the far right of the menu bar, the About menu provides access to the Help system,
Bio-Tek’s home page, and the About KC4 screen.
Toolbar
The Toolbar contains buttons that, when clicked, provide quick access to KC4 functions.
Accessibility of a particular toolbar button depends on whether or not its function applies to the
current environment. For example, the Biograph button is accessible only if results are displayed
and a curve has been generated. When KC4 is installed, the toolbar contains a set of default
buttons. These buttons can be customized by selecting System|Toolbar.
Status Bar
The Status Bar at the bottom of the main screen displays the current function, reader and COM
port; the name of the person currently logged in (if applicable); and any enabled keyboard
functions.
User’s Guide to KC4™
Getting Started • 31
Quick Reference Guides
This section briefly describes the contents of each menu in KC4.
Data Menu
The Data Menu contains functions for acquiring and processing plate data.
New Plate
Clear memory to prepare to read a new plate.
Open...
Open an existing plate (.PLA) or global (.GLB) data file.
Close
Close the open data file. If the file has not yet been saved, KC4
provides the option to save before closing.
Save
Save the current plate data (.PLA) or global data (.GLB) file.
Save As
Save the open file under a new name.
Read Plate...
Read a plate using the attached reader, enter plate data manually, or
read plate data in from a file.
Sign
Electronically sign the current .GLB data file.
Export
Export data to a text file, to an Excel spreadsheet, or to a Word
document.
Append to
Kinetic File...
Append the current set of plate data to an existing file.
Print...
Print a report according to the Protocol|Report settings.
Print Preview
Preview the report before printing.
Print Setup
Define the print setup before printing.
Plate Description... Enter information about the current plate using up to six free-text fields
plus a large comments field.
Samples...
Define the names of samples.
Audit Trail
View the contents of the Data Audit Trail.
Exit
Exit KC4.
32 • Getting Started
User’s Guide to KC4™
Edit Menu
The Edit Menu contains editing functions commonly found in Windows.
Cut
Deletes selected data from the document and copies it to the Clipboard.
Copy
Copies selected data to the Clipboard.
Paste
Pastes data from the Clipboard into the document.
Protocol Menu
The Protocol Menu contains functions for creating, modifying, and saving assay protocols.
New
Create a new protocol.
Wizard
Launch the Wizard for help with creating protocols.
Open...
Open an existing protocol (.PRT) file.
Close
Close the current protocol file. If the file has not yet been saved, KC4
provides the option to save before closing.
Save
Save the current protocol (.PRT) file.
Save As...
Save the open file under a new name.
Sign
Electronically sign the protocol file.
Plate Description
Define the names of up to six free-text fields to appear at read time to
allow the operator to enter relevant information, such as the site name
and lot number.
User’s Guide to KC4™
Getting Started • 33
Reading
Define the reading parameters, such as reading type,
filters/wavelengths, shaking and incubation instructions, and plate type.
Raw Data
Correction
Enable/Disable corrections to raw data. Options can include Blank
Plate Subtraction, Blank Wells Subtraction, and Pathlength Correction.
Kinetic Analysis
Select the primary (and optional secondary) set of data for kinetic
analysis, as well as the calculation type.
Spectrum
Analysis
Select the data set for spectrum analysis, as well as the calculation type.
Scanning
Analysis
Select the calculation type and related options for linear scanning
analysis.
Plate Layout
Configure the layout, names, and colors of blanks, standards, controls,
and samples. Define expected concentrations or dilutions.
Single Plate
Transformations
Define and apply formulas to one or more wells within the plate.
Multi-Plate
Transformation
Define calculations between different sets of plate data.
Curves
Define curve-fitting parameters.
Validation
Define the criteria used to assess the validity of the results or the
suitability of calibrators for inclusion in subsequent calculations.
Cutoffs
Define the conditions used to screen selected data into defined
categories based on fixed or variable threshold limits.
Report
Customize the contents and appearance of the report.
Export
Customize the contents and format of the data export file.
Audit Trail
View the contents of the Protocol Audit Trail.
Security
(Administrator and Power Users) Specify which protocol- and platespecific functions, if any, shall be protected from use by Standard
Users.
Options
Select automated functions. Choose the reporting and exporting
engines. Enable the protocol audit trail function.
Advanced
Select Advanced to access special protocol menu options: Plate
Description, Raw Data Correction, Multi Plate Transformations,
Validation, and Cutoffs.
34 • Getting Started
User’s Guide to KC4™
Display Menu
The Display Menu provides access to the Biograph module and also displays the results of the
Validation criteria defined in the protocol.
Curve(s)
Launch Biograph and view the curve(s).
Calculation
Log Report
View calculation warnings associated with the current data.
Validation
Results
Check the status of every Validation condition defined in the protocol.
Toolbar
Show or hide the toolbar.
System Menu
The System Menu contains special functions for configuring readers, configuring system security,
setting global parameters, performing reader diagnostics, and more.
Readers
Change or configure the current reader.
Diagnostics
Run an Optics Test or Universal Plate Test on the attached reader.
Define characteristics for a Universal Test Plate. View historical test
results.
User’s Guide to KC4™
Getting Started • 35
Security
Configure system security parameters and maintain user accounts.
Administrator
LogIn/LogOut
Log into KC4 as the System Administrator (single-user system).
LogIn/LogOut
Log into KC4 using a unique ID and password combination (multi-user
system).
Audit Trail
View the contents of the System Audit Trail.
Registration/
Upgrade
Enter a license number and password to ensure continuous use of KC4
after the trial period is over.
Setup
Set up global parameters including the import/export text separator,
time format for kinetics, weighting of standard deviation, and
Word/Excel options. Tell KC4 whether or not to use out-of-range
values in statistics.
Toolbar
Customize the appearance of the toolbar by adding/removing icons.
Plate Formats
Create and modify plate formats, and specify a default format for new
protocols.
Reader Control
Send a command to the reader to open or close the door, insert or eject
the plate, and to control the temperature.
About Menu
The About Menu provides access to the Help system, Bio-Tek, and the About KC4 screen.
Help Topics
Open the Help window with its Contents, Index, and Find functions.
Using Help
Tips on using the Help system.
Bio-Tek
Home Page
Automatically launch the browser and go to Bio-Tek’s home page
(www.biotek.com).
About KC4
Check the software version and revision numbers, view current system
performance parameters, and find telephone and fax numbers for
technical assistance.
36 • Getting Started
User’s Guide to KC4™
Navigating the Help System
Most KC4 dialogs contain a Help button that, when clicked, opens an informational Help dialog.
In addition to this context-sensitive help, KC4 provides access to the entire Help system.
Select Help Index from the About menu to open the Help Topics dialog.
•
Double-click on a book
to see a topic’s sub-topics.
•
Double-click on a page
to view the text for a topic.
•
Highlight a topic then click Open to view the text for the topic.
•
Click the Index tab to perform a keyword search.
•
Click the Find tab to search the entire help system for a specific word or phrase.
•
Click Print to print the text for a topic.
•
Click Cancel to close the Help Topics dialog and return to KC4.
User’s Guide to KC4™
Getting Started • 37
Quick Start
When using KC4, you will typically follow some version of the steps shown below:
1.
Connect a microplate reader to the computer (if not already connected).
2.
Start KC4 and log in (if required).
3.
Select System|Readers to define reader setup parameters and establish communication.
4.
Select System|Setup to define global parameters, including special Word/Excel
reporting options.
5.
Select Data|New Plate to prepare to read a plate.
6.
Open an existing protocol or begin with an empty protocol.
7.
Select Protocol|Reading to define reading parameters such as wavelengths, plate type,
and temperature.
8.
Return to the main view and define the data reduction parameters:
create the plate layout by defining and assigning well identifiers
select curve parameters
specify cut-off and validation criteria
create single- and multi-plate transformations
define reporting and exporting options
9.
Select Data|Read Plate to read a microplate.
10. Review the results.
11. Select Data|Print to print reports.
12. Select Data|Save to save the information, or with KC4 Signature select Data|Sign to
electronically sign and then save the information.
13. Select Display|Curves to view the curve.
38 • Getting Started
User’s Guide to KC4™
Quick Start for Clarity™ Microplate Luminometer Users
Clarity users will follow a slightly different sequence of events:
1.
Connect the luminometer to the computer (if not already connected).
2.
Start the Clarity software and set up the communications port.
3.
Important: Close the Clarity software.
4.
Start KC4 and log in (if required).
5.
Select System|Readers and set Clarity as the current reader.
6.
Select System|Setup to define global parameters, including special Word/Excel
reporting options.
7.
Select Data|New Plate to prepare to read a plate.
8.
Open an existing KC4 protocol or begin with an empty one.
9.
Select Protocol|Reading to open or create a Clarity Protocol File and define its reading
parameters. (See “Parameter Input” in Chapter 4 of the Clarity Operator’s Manual to
learn about the reading parameters.)
10. Return to KC4’s main view and define the data reduction parameters:
create the plate layout by defining and assigning well identifiers
select curve parameters
specify cut-off and validation criteria
create single- and multi-plate transformations
define reporting and exporting options
11. Select Data|Read Plate to read a microplate.
12. Review the results.
13. Select Data|Print to print reports.
14. Select Data|Save to save the information, or with KC4 Signature select Data|Sign to
electronically sign and then save the information.
15. Select Display|Curves to view the curve.
User’s Guide to KC4™
Getting Started • 39
Sample Files
KC4’s installation program copies many sample protocol (PRT) and plate data files into a special
SAMPLES folder inside the root KC4 folder and, with KC4 Signature, inside the KCShared
database. Examine these files to learn how to create KC4 protocols to support your assays. The
sample files cover a range of protocol options, including single plate transformations, multi plate
transformations, curve fitting parameters, cutoff criteria, and more.
Important! The sample protocols must be considered as examples provided for demonstration and
guidance purposes only. If you plan to use these protocols or similar ones in a real application, it
is your responsibility to validate the protocol parameters, including the Excel sheet content (if
PowerReports is used), before using them.
•
The SAMPLES folder in the KC4 root folder also contains a Sample Protocol
Descriptions.doc file describing the protocols.
•
Files with the extension .PRT represent protocol files.
•
Files with the extension .PLA represent plate data files.
•
Files with the extension .GLB represent global data files (containing plate data and
protocol parameters).
•
Open protocol and plate data files with similar names, such as Quantitative.prt and
Quantitative.pla. Note: Not every .PRT file has a corresponding .PLA file.
40 • Getting Started
User’s Guide to KC4™
Data Menu
Overview
The Data Menu contains functions for acquiring, processing, and storing plate data.
This chapter covers the following topics:
•
Creating, opening, saving, and closing data files
•
Reading plates
•
Entering plate data manually
•
Retrieving plate data from an external file
•
Applying electronic signatures to global data files
•
Appending a new set of plate data to an existing kinetic file
•
Exporting plate data
•
Printing and previewing reports
•
Entering descriptive information for the plate
•
Entering sample names
•
Viewing the data file’s audit trail
User’s Guide to KC4™
Data Menu • 41
Working with Data Files
This section describes accessing, generating, modifying, and saving files that contain plate
measurement data.
Where and How is Plate Data Stored?
In KC4, there are two types of files used for storing plate data:
•
Plate data files contain raw measurement values, plate description information (prompts,
comments, date and time of reading), and sample names.
Plate data files are identified by the extension .PLA (DNA-Quant.pla)
•
Global data files contain plate data and protocol information. The GLB file format is
offered as an alternative to maintaining two separate protocol and plate files, and is the
required data file format for electronic signature support.
Global data files are identified by the extension .GLB (DNA-Quant.glb)
File storage options vary depending on site requirements and the current KC4 configuration.
•
Files may be stored individually on the computer’s hard drive, on a CD or diskette, or on
a network to which the computer is attached. This is the storage method that users of
KC4 version 3.0x and earlier are familiar with. Windows Explorer or a similar
application can be used to view the file names and their locations, and to move, copy,
rename, and delete files.
•
(KC4 Signature) Files may also be stored inside a secure, shared-access database. This
database, named KCShared.mdb, can be stored on a user’s computer or on a sharedaccess computer. KC4 provides a special file maintenance utility for viewing the file
names and their locations, and for moving, copying, renaming, deleting, importing, and
exporting files.
42 • Data Menu
User’s Guide to KC4™
Creating a New Data File
Before a plate can be read, and before plate data can be entered manually or read in from a file, a
new data file must be created.
To create a new data file:
1.
Select System|Readers and set the Current Reader to the appropriate target reader
model.
2.
Select Data|New Plate (or click the toolbar icon shown above) to clear memory to
prepare to read a new plate.
3.
If KC4 is currently configured* to create plate data (PLA) files by default, do one of the
following:
Select Protocol|New to create a new protocol.
Select Protocol|Open to open an existing protocol.
Select Data|Read Plate to initiate the plate read.
If KC4 is currently configured* to create global data (GLB) files by default, selecting
Data|New Plate opens the New Data File dialog:
This dialog allows you to choose which protocol to open and use for the plate reading.
Click the Browse button to search through the folder structure.
Highlight the desired protocol and click OK.
To start with an empty protocol and customize its parameters, highlight Empty
Protocol and click OK.
* The “Preferred Data File Format” is selected via System|Security|Files.
User’s Guide to KC4™
Data Menu • 43
Opening Existing Data Files
Select Data|Open to open a previously saved plate or global data file.
Note: The layout of the Open dialog varies slightly depending on whether or not the Secured
Protocol and Data File Storage option is enabled, but the functionality is the same. (In the
example below, this option is enabled.)
•
The Current Folder field or the Look In field (you will see one or the other) shows your
current location within the overall folder structure.
If the drive is represented as DB:, this means that files are currently being stored in
KC4’s secure, shared-access database. If the drive is represented by a single letter,
such as C: or D:, this means that files are stored individually on the computer’s hard
drive or on a network to which the computer is attached. See “Where and How is
Plate Data Stored?” for more information.
•
Click
•
Click on a folder
•
Click the Type drop-down arrow to change the type of files being viewed. Options
include:
to move up one folder level.
to open it and view its contents.
KC4 Data (*.PLA)
KC4 Data+Protocol (*.GLB)
All Files (*.*)
•
Click on a file
•
Click to update the view to show any folders/files that may have been added by another
user after you opened this dialog.
44 • Data Menu
and click OK to open it.
User’s Guide to KC4™
Modifying Data Files
After data has been retrieved from the reader (or entered manually, or read in from a file), KC4
permits authorized users to change or mask raw measurement values. KC4 automatically logs an
event to the file’s Data Audit Trail any time a change or mask action occurs.
Note: Data Audit Trail behavior differs between KC4 v3.4 and KC4 Signature. Turn to page 63
to learn more.
Important! Changing or masking wells may significantly alter or even invalidate results.
•
Only raw measurement values can be changed. Raw data set names resemble M 405,
M#3 490, and M 485/528.
•
When a well is masked, KC4 excludes its raw measurement value from further
calculations.
To change a raw measurement value within any data file:
1.
Open the desired file.
2.
Within the Results window, click the Data drop-down arrow and select a data set that
contains raw measurement values (e.g. M 405 or M#2 485/528).
3.
Click the Change box.
The System Administrator can protect the use of the Change function. If you are
trying without success to use this function, you may not be authorized to use it.
4.
Click on a well in the grid and change its value.
5.
When the changes have been made, click OK to recalculate results.
KC4 automatically updates the Data Audit Trail to reflect the action:
To mask a well within any data file:
1.
Open the desired file.
2.
Within the Results window, click the Data drop-down arrow and select the desired data
set. Note: When a well is masked, KC4 excludes its raw measurement value from
further calculations.
3.
Click the Mask box.
The System Administrator can protect the use of the Mask function. If you are
trying without success to use this function, you may not be authorized to use it.
4.
Click on a well in the grid to mask it (click again to unmask).
5.
When the well(s) have been masked, click OK to recalculate results.
User’s Guide to KC4™
Data Menu • 45
KC4 automatically updates the Data Audit Trail to reflect the action:
To modify protocol parameters within a GLB file:
1.
Open the desired file.
2.
Use the Protocol menu to select a parameter category to modify, such as Plate Layout,
Single Plate Transformations, Report, and so on. Select Protocol|Advanced to see
additional options including Cutoffs and Validation.
Note: Reading parameters (such as filters/wavelengths and plate geometry) cannot be
modified after a plate has been read.
3.
Within the appropriate protocol parameter dialog, make the desired change and click OK
to recalculate results.
The System Administrator can protect some or all protocol parameters from being
modified. If you are trying without success to modify these parameters, you may not
be authorized to do so.
KC4 Signature automatically updates the Data Audit Trail to reflect the action:
Saving Data Files
After creating or modifying a data file, save it by selecting Data|Save or Data|Save As. Save
stores the data file under its current name. (If the file does not have a name, KC4 will ask you to
enter one.) Save As copies the contents of an existing data file to a new data file with a different
name.
Note: The layout of the Save or Save As dialog varies slightly depending on whether or not the
Secured Protocol and Data File Storage option is enabled, but the functionality is the same. (In the
following example, this option is enabled.)
46 • Data Menu
User’s Guide to KC4™
•
The Current Folder field or the Save In field (you will see one or the other) shows your
current location within the overall folder structure.
If the drive is represented as DB:, this means that files are currently being stored in
KC4’s secure, shared-access database. If the drive is represented by a single letter,
such as C: or D:, this means that files are stored individually on the computer’s hard
drive or on a network to which the computer is attached. See “Where and How is
Plate Data Stored?” for more information.
•
The default file name is NewPlate.
•
Enter a new File Name and click OK to save it.
•
Click
to move up one folder level.
•
Click
to create a new folder.
•
Click on a folder
•
Click the Type drop-down arrow to change the type of files being viewed. Options
include:
to open it and view its contents.
KC4 Data (*.PLA)
KC4 Data+Protocol (*.GLB)
All Files (*.*)
•
If an existing file name is entered, KC4 will provide a warning to prevent an accidental
overwrite. Click Yes to continue with the overwrite, or No to rename the file before
saving.
Closing Data Files
Select Data|Close to close the current data file. If the file has not already been named, the Save
As dialog will appear requesting entry of a file name.
Note: Clicking
User’s Guide to KC4™
in the upper right corner of the Results window does not close the data file.
Data Menu • 47
Reading Plates
Once the new data file has been created (see “Creating a New Data File” on page 43), select
Data|Read Plate to initiate a plate read, or to enter data manually or read data from a file.
If no protocol is open, a Reading parameters dialog appears. Parameters available within this
dialog are reader-specific:
•
Define the required Reading parameters and then click OK to continue.
The Plate Reading dialog appears:
48 • Data Menu
User’s Guide to KC4™
The Plate Reading dialog allows optional information and comments to be added to the
plate. This information is filed along with the data for future reference.
(Optional) Enter data in the Plate Description fields, and enter Comments.
Tip: The data entry field names can be customized via Protocol|Plate Description.
Click Start Reading to initiate communication with the attached microplate reader.
Following a successful plate read, the measurement values will be transferred to KC4
and displayed in the Results window.
Click Enter Manually to enter plate data using the keyboard. See “Entering Plate
Data Manually” on page 50 for more information.
Click Read From File to read plate data from a stored file. See “Reading Plate Data
From a File” on page 51 for more information.
Unintentional Termination of a Kinetic Read
If a kinetic read is unintentionally terminated, or if at the end of the read KC4 receives an error
status from the reader, KC4 presents the following message:
As the message states, the read was interrupted and KC4 is providing the opportunity to save or
discard the kinetic data received up to this point. The data is unvalidated, which means the reader
did not (or could not) provide KC4 with the expected valid status at the end of the plate read.
The term “unintentional termination” applies to the following situations:
•
The reader was turned off or the serial cable was disconnected during the read.
•
KC4 received an error status string at the end of the kinetic read. Note: If the unvalidated
data is saved after this error, the last kinetic point for each well is deleted due to its
proximity to the error status.
•
KC4 received an unexpected response from the reader or the reader stopped operating
during the read (possibly due to a hardware error or an internal software error).
•
MS® Windows™ experienced a communication error in the low level COM processes.
If the message shown above appears in KC4, take a moment to try to determine its cause. If the
reader was inadvertently turned off or the serial cable was disconnected, the reader may have
performed normally up to the point of termination, in which case the data would be valid. If the
termination appears to have been induced by the reader, the data may not be valid. If a 4-digit
error code is also supplied, consult the reader’s Operator’s Manual.
Within the message box:
•
If NO is selected, all data collected up to this point is permanently deleted and cannot be
retrieved.
•
If YES is selected, all data collected up to this point will be saved. It is strongly advised
that the data be analyzed to determine its validity.
User’s Guide to KC4™
Data Menu • 49
KC4 Signature users: If Yes is selected an event is logged to the Data Audit Trail, as
demonstrated below:
Entering Plate Data Manually
KC4 provides the option to enter Endpoint and Kinetic plate data manually via the keyboard.
Typically this information comes from other instruments or from previously acquired data.
•
Use the up and down arrow keys to move from well to well in the matrix. When the
down arrow is pressed from a well in the bottom row, the cursor automatically jumps to
the well in the top row of the next column.
•
Place the cursor over the desired well and enter the OD (or RFU/RLU if fluorescence /
luminescence) value.
•
Click OK to end the editing process and calculate results.
•
For multi-wavelength reads, enter data for the first wavelength and click OK. For the
remaining wavelengths, select the appropriate data set and click Change to enter values.
•
For kinetic reads, enter data for Reading #1 and click OK. For the remaining readings,
update the Reading # field and click Change to enter values.
Note: With kinetic plate data, each reading number set must be incremented to mimic
the kinetic plate reading cycle. The time base will be taken from the kinetic reading
parameters defined in the protocol.
50 • Data Menu
User’s Guide to KC4™
Reading Plate Data from a File
KC4 can read endpoint or kinetic data from an ASCII Text file. The file must be formatted
properly, and the data must be delimited with acceptable separators.
To import Endpoint or Kinetic data:
•
Examine the text file to see what character is used to delimit the data. Typical delimiters
include semi-colon, comma, and tab.
•
In KC4, select System|Setup. Set the Import/Export Separator so that it matches the
character used within the text file.
•
If desired, create or open a protocol.
•
Select Data|Read Plate|Read from File, then select the desired file. If the file is
formatted properly, KC4 will read the data then calculate results according to the protocol
parameters.
Import File Format for an Endpoint Read
The file block size must correspond with the plate layout defined in the protocol. For example, a
96-well plate has 8 rows and 12 columns and a 24-well plate has 4 rows and 6 columns. The
following example represents the 96-well plate format:
0.003;1.409;0.483;2.022;1.463;1.200;0.876;2.240;2.057;1.867;0.435;2.147
1.776;1.284;0.760;0.037;0.229;0.911;0.368;0.415;2.471;1.114;0.298;0.012
0.022;0.945;1.329;1.428;1.504;1.518;0.416;1.658;1.127;0.880;0.143;1.519
1.958;2.007;1.300;0.755;2.190;1.817;2.390;2.314;1.348;0.356;1.155;0.588
2.156;0.524;1.949;2.109;2.492;2.499;1.529;0.981;0.666;0.743;2.100;0.059
0.940;0.232;1.693;0.141;0.022;2.297;0.690;0.682;1.470;1.728;2.094;1.816
1.212;0.513;1.859;1.171;1.145;2.373;1.861;0.271;1.498;0.963;1.838;1.522
1.431;0.903;0.379;0.563;1.063;2.007;1.293;2.475;1.879;0.864;0.422;1.643
User’s Guide to KC4™
Data Menu • 51
Import File Format for a Kinetic Read
Before importing a kinetic text file, create a protocol that specifies the exact number of kinetic
reads that will be imported.
The first line in the text file can optionally contain a time indicator. This value will be stored in
the protocol (overwriting protocol settings, if they are different), and used for any kinetic plotting.
If the file contains no time indicators then the time parameters defined via Protocol|Reading
parameters will be used as defaults.
The following example represents the kinetic data set generated from 5 readings, in the 24-well
format (to ensure a successful import of all five reads shown in this example, create a protocol and
set the kinetic parameters so that the Reading Number is 5.):
Time=00:00:00
0.000;0.007;0.013;0.020;0.026;0.033
0.004;0.011;0.017;0.024;0.030;0.037
0.008;0.014;0.021;0.028;0.034;0.041
0.012;0.018;0.025;0.032;0.038;0.045
Time=00:00:30
0.000;0.026;0.053;0.079;0.105;0.132
0.016;0.042;0.068;0.095;0.121;0.147
0.032;0.058;0.084;0.110;0.137;0.163
0.047;0.074;0.100;0.126;0.153;0.179
Time=00:01:00
0.000;0.059;0.118;0.178;0.237;0.296
0.036;0.095;0.154;0.213;0.272;0.331
0.071;0.130;0.189;0.249;0.308;0.367
0.107;0.166;0.225;0.284;0.343;0.402
Time=00:01:30
0.000;0.099;0.197;0.248;0.284;0.356
0.056;0.195;0.194;0.295;0.322;0.381
0.171;0.188;0.231;0.318;0.390;0.401
0.167;0.216;0.275;0.337;0.403;0.499
Time=00:02:00
0.000;0.176;0.251;0.299;0.378;0.446
0.117;0.281;0.295;0.375;0.397;0.441
0.257;0.275;0.331;0.388;0.438;0.482
0.239;0.296;0.369;0.402;0.492;0.557
52 • Data Menu
User’s Guide to KC4™
Importing Clarity Luminescence Data Files
Applies to Clarity™ Microplate Luminometer users only.
KC4 supports the importing of Clarity results files (*.bti) through its ‘Read from File’ feature
when Clarity is the currently selected reader. You can, for example, import a data file containing
raw luminescence data and then create a KC4 protocol to apply transformations, calculate curves,
generate reports, and so on.
The following requirements must be met to ensure a successful import:
•
The Clarity file must have a .bti extension.
•
Before the .bti file is saved in the Clarity PC software, the Displayed digits after period
field under Options|Visual Settings must be set to 0.
•
If you wish to import a .bdf file, open it in the Clarity PC software, select File|Save As,
and select the .bti file type.
•
Batch Protocols must contain a maximum of 300 reads and have a total measurement
time of less than 48 hours.
•
If the file has a measurement comment associated with it (Measurement|Measurement
Comment), the comment cannot contain any empty lines. For example, the following
comment would cause the file import to fail because the second line is empty:
(1st line) Measurement performed by Brian Smith
(2nd line) <empty>
(3rd line) Results approved by Sharon Jones
Notes on background measurement values:
If the Clarity file specified a background measurement and the ‘Subtract instrument background’
option was enabled, KC4 imports the subtracted values as well as the background value for
informational purposes. This value can be viewed in KC4 by selecting Protocol|Reading when the
data file is open.
If the Clarity file specified a background measurement and the ‘Subtract instrument background’
option was not enabled, KC4 imports the raw data values and the background value and then
subtracts the background value from all wells.
User’s Guide to KC4™
Data Menu • 53
Signing Data Files
KC4 Signature provides an option for users to permanently attach, or sign, their names to global
data (GLB) files.
Notes:
•
While KC4 does not permit users to sign plate data (PLA) files, if a plate data file and a
protocol file are open at the same time, selecting Data|Sign will combine the two
individual files into one signed GLB file.
•
KC4 only supports file signatures when the multi-user identification feature is in use.
See “Security” in System Menu for more information.
To sign a global data file:
1.
Open the desired file (if it is not already open).
2.
Select Data|Sign or click the Sign toolbar icon.
The New Data Audit Trail Signature dialog will appear:
3.
Click the Reason drop-down arrow and select the reason to be associated with this
signature.
Signature Reasons are site-defined by the System Administrator under
System|Security|Signature Reasons.
54 • Data Menu
User’s Guide to KC4™
4.
(Optional) If two users are signing on behalf of a third KC4 user, check the
Representative of box and then choose a name from the drop-down list.
5.
Enter or select your User ID.
If ‘Representative Of’ is checked, two users must enter or select their User IDs.
6.
7.
Enter your Password. The characters will not be visible on the screen.
Click Sign & Save.
If the file has not yet been saved, a Save As dialog will appear:
8.
Using the folder location tools provided, select the storage location.
9.
Enter the desired File Name and click OK. The signature is applied immediately and
KC4 logs the event in the Data Audit Trail (select Data|Audit Trail to view):
User’s Guide to KC4™
Data Menu • 55
Append to Kinetic File
KC4 provides the option to build a kinetic file using individual endpoint read plates. The
currently-open plate can be appended to the previously read plate. This feature allows plates to
be read on a discontinuous time base, unlike pure kinetic readings.
•
Alternatively, it is possible to append several kinetic readings to one file. If a kinetic
reading sequence is halted (ex. to inject reagents) then it is possible to resume the kinetic
reading and append the resumed data onto an existing file.
•
The Append feature can be set to perform automatically after a plate read. See “Protocol
Options” in Protocol Menu for more information.
To append a set of plate data to a kinetic file:
•
When the plate read is complete, select Data|Append to Kinetic File.
The Append to Kinetic File dialog appears:
The last opened plate file name appears as the default. Change the path and/or file
name if desired.
Click New if this set of data represents the starting kinetic read (i.e., if subsequent
reads will be appended to this one). If no file name has been entered, KC4 will
produce the standard Save As dialog.
Click Open to search through the directory structure to locate the target file.
Click OK to continue. A dialog will appear requesting entry of a Reading Time
relative to the first reading.
The default value represents the elapsed time since the last plate read; enter a new
value if required. Note: The time format is defined under System|Setup. For an
interval greater than 99:59:59, set the time format to HHH:MM.
56 • Data Menu
User’s Guide to KC4™
Exporting Plate Data
KC4 can export plate measurement data to a text file, a Microsoft® Excel spreadsheet, or a
Microsoft® Word document. This section contains instructions for exporting to a text file only
(using the KC4 export engine).
•
See “Export” in Protocol Menu for more information on the available export engines.
•
See PowerReports for more information on the Excel and Word exporting engines.
The export file format can be defined within a protocol (under Protocol| Export), or after the plate
is read (by selecting Data|Export). The text delimiter used during the export is selected under
System|Setup.
To export data:
•
When the plate read is complete, select Data|Export.
•
If export parameters are defined in the protocol, KC4 requests the entry of a file name.
Enter the file name and then click Save to export the data. Data exported to a text file
can be used by any other program with the ability to read an ASCII file.
•
If export parameters are not already defined in the current Protocol, an Export template
appears. The example below shows the template used to export data to a text file:
•
Select the desired data sets and then click OK to begin the export. KC4 requests the entry
of a file name. Enter the file name then click Save to export the data. Data exported to a
text file can be used by any other program with the ability to read an ASCII file.
Regardless of the Secured Protocol and Data File Storage setting, data is exported as
an individual .txt file outside the shared-access database.
See “Export” in Protocol Menu for information on customizing the export template.
User’s Guide to KC4™
Data Menu • 57
Sample Export Text File
[ProtocolDescr]
Name=C:\KC4 3.4\Sample Export.prt
Modified=No
[ReadingParameters]
PlateSize=4,6
PlateType=24 WELL PLATE
Wells=A1-D6
ReadBlankPlate=No
PathlengthCorrection=No
ReadBioCell=No
MonitorWell=No
Filter1=405
Calibrate=No
[DataDescription]
Name=C:\KC4 3.4\Sample Export.pla
Barcode=123-ABC-7890
ReadingDate=03/14/01 13:19:16
ReadingType=Reader
Temperature=
PromptTitles=Prompt #1:;Prompt #2:;Prompt #3:;Prompt #4:;
Prompt #5:;Prompt #6:
Prompts=;;;;;
Comment=This is a sample.
ReportTime=03/14/01 13:21:41
[Plate: Template]
;1;2;3;4;5;6
A;STD1:10.000;SPL1;SPL3;SPL5;SPL7;SPL9
B;STD2:20.000;SPL1;SPL3;SPL5;SPL7;SPL9
C;STD3:30.000;SPL2;SPL4;SPL6;SPL8;SPL10
D;STD4:40.000;SPL2;SPL4;SPL6;SPL8;SPL10
[Plate: M 405]
;1;2;3;4;5;6
A;0.006;2.818;0.967;4.044;2.925;2.399
B;0.751;4.480;4.114;3.733;0.871;4.295
C;1.532;2.568;1.520;0.075;0.457;1.822
D;2.737;0.829;4.943;2.228;0.595;0.023
[Plate: Concentrations]
;1;2;3;4;5;6
A;11.065;>41.500;21.774;>41.500;>41.500;37.731
B;19.367;>41.500;>41.500;>41.500;20.704;>41.500
C;28.070;39.614;27.936;11.834;16.091;31.302
D;41.498;20.236;>41.500;35.826;17.629;11.255
[Plate: Symbols]
;1;2;3;4;5;6
A;NEG;OUT+;EQV;OUT+;OUT+;POS
B;EQV;OUT+;OUT+;OUT+;EQV;OUT+
C;POS;POS;EQV;NEG;NEG;POS
D;POS;EQV;OUT+;POS;NEG;NEG
58 • Data Menu
User’s Guide to KC4™
Printing Reports
After plate measurement data has been acquired and calculations are complete, KC4 can generate
a user-customizable set of results reports. Reporting options are selected and customized within
each protocol by selecting Protocol|Reports.
Print
Select Data|Print or click the appropriate toolbar icon to print the reports selected in the protocol.
Print Preview
Select Data|Print Preview to see what the report will look like before it is printed.
Print Setup
To change the printer, paper size or source, or page orientation, select Data|Print Setup to access
a standard Print Setup dialog.
User’s Guide to KC4™
Data Menu • 59
Plate Description
The Plate Description dialog appears just prior to each plate read, to provide the optional entry of
plate-specific information.
•
The Reading Date and Time are automatically logged when the plate is read. Enter
additional data as desired and then click OK to continue with the plate read.
Data entry field names can be customized by selecting Protocol|Plate Description.
•
The information contained within this dialog is automatically stored in the data file when
Data|Save is selected.
60 • Data Menu
User’s Guide to KC4™
Samples
Select Data|Samples to assign names to the sample groups previously defined under
Protocol|Plate Layout.
Each sample ID can be entered manually, or the Auto Numbering feature can be used to
automatically increment ID suffix numbers.
•
The ID entered in cell #1 will be assigned to every replicate in Sample group one, the ID
in cell #2 will be assigned to every replicate in Sample group two, and so on.
•
Up to 20 alphanumeric characters can be used for each ID.
Note: If a sample name cannot be printed in its entirety, it is truncated and the character
‘\’ is added at the beginning of the name to indicate that the name has been truncated.
For example: ‘\LE001’, ‘\LE002’, etc.
•
Sample IDs can be entered using a barcode scanner attached to the computer. To do
this, place the cursor in cell #1 (or any empty cell). Scan the item with the barcode
scanner. The first 12 characters of the ID will appear in KC4. Move the cursor down to
the next empty cell, then scan the next item.
User’s Guide to KC4™
Data Menu • 61
Auto Numbering
To automatically number the sample IDs sequentially:
•
Check the Auto Numbering box.
•
In cell #1, type the identifying name and starting number (e.g., Smp1) for Sample group
#1. Leave the cursor in cell #1.
•
Press the down arrow through as many cells as there are sample groups on the plate.
KC4 will automatically increment the suffix number (Smp2, Smp3, and so on).
Import Sample Names
The Import option allows you to import a sample name list from a text file. Click Import and then
locate and select the file to import.
•
•
The import file must contain one name label per line.
The number of IDs in the file should equal the number of samples as defined under
Protocol|Plate Layout.
If there are more IDs in the text file than samples defined on the plate, the extra IDs at the
end of the file will remain unused.
•
If a Sample ID in the text file exceeds 12 characters, only the first 12 characters will be
imported.
Export Sample Names
The Export option allows you to export the generated identification labels to a text file for future
use. Enter the Sample IDs as required and then click Export to specify the export file name. The
export file will contain one name label per line.
Printing Sample IDs
The Print options allow you to print the Sample IDs in a plate or list format.
•
Click Print As Plate to print the Sample IDs in a matrix format corresponding with the
plate format selected in the protocol. The plate must be configured via Protocol|Plate
Layout prior to using this feature.
•
Click Print As List to print the Sample IDs in a list format. One Sample ID will be
printed on each line.
62 • Data Menu
User’s Guide to KC4™
Data Audit Trail
Every data file (plate and global) has a Data Audit Trail associated with it. This audit trail
automatically records events performed on the file since the file was created. Each event records
an action such as "Sample Plate read" or "Mask Value - <well ID>" and includes a date and time
stamp, the ID of the user logged in at the time (if applicable), and any comments that were entered
by the user when the action was performed. The audit trail cannot be edited and it remains a
permanent part of the file for the life of that file (that is, if the file is deleted, the audit trail is also
deleted).
•
With KC4 v3.4, only Change, Mask, Read, and Re-Read (delete initial values) actions are
logged in the audit trail. A file’s audit trail cannot be viewed from within KC4 but it can
be added to report and export templates.
•
With KC4 Signature, Change, Mask, and many other actions may be logged to the audit
trail. A file’s audit trail can be added to report and export templates, and can also be
viewed by selecting Data|Audit Trail:
Events are sorted by date/time stamp and include the ID of the user logged in at the
time, the action taken, and any comments that were entered by the user when the
action was performed.
The contents of the data audit trail can be copied to the Clipboard by highlighting the
desired cells and pressing Ctrl+C.
The contents of the data audit trail cannot be edited or deleted.
User’s Guide to KC4™
Data Menu • 63
64 • Data Menu
User’s Guide to KC4™
Edit Menu
Editing Tools
KC4 provides some editing tools for working with data in KC4. The two most commonly used
tools are Copy to copy selected data to the clipboard, and Undo to remove the most recent
unsaved change. Availability of editing tools depends on the current working environment; for
example, the Cut and Paste options are not available within any measurement data grids or audit
trail tables, but they are available within data entry fields (such as formulas).
Editing tools are available through the Edit menu, by clicking the right mouse button, or by
pressing the shortcut key combination, and they can include:
•
Cut (Ctrl+X): Deletes the selected data from KC4 and moves it to the clipboard. This
function is not available within measurement data grids or audit trail tables.
•
Copy (Ctrl+C): Copies the selected data from KC4 to the clipboard without deleting it.
This function is available within measurement data grids, audit trail tables, and data entry
fields. Note: The values copied to the clipboard are separated by the character selected
under System|Setup|Formats.
•
Paste (Ctrl+V): Pastes data from the clipboard into the current KC4 location (wherever
the cursor is placed). This function is not available within measurement data grids or
audit trail tables.
•
Delete (Delete key): Deletes the selected data from KC4 without moving it to the
clipboard. This function is not available within measurement data grids or audit trail
tables.
•
Select All: Selects (highlights) all text within the current field or cell. For example,
place the cursor in a formula cell within the Single Plate Transformation dialog, click the
right mouse button and click Select All to select the entire formula.
•
Undo (Ctrl+Backspace): Erase the last (unsaved) change made and restore any items
that had been modified by that last change. For example, if within the Plate Layout
dialog you mistakenly applied Sample well identifiers atop existing Standard well
identifiers, choosing Undo will restore the Standard wells.
User’s Guide to KC4™
Edit Menu • 65
66 • Edit Menu
User’s Guide to KC4™
Protocol Menu
Overview
A Protocol contains instructions for controlling a microplate reader and for analyzing and
reducing plate measurement data. Protocol parameters can be stored in a protocol file (which has a
.PRT extension) or in a global data file (which has a .GLB extension).
The topics in this chapter describe how to create and customize protocols. Customizable protocol
parameters include the following:
•
Detection method (Absorbance, Fluorescence, or Luminescence)
•
Reading type (Endpoint, Kinetic, or Spectrum)
•
Read mode (Normal, Rapid, or Sweep)
•
Read/Dispense parameters (for instruments with fluid injection)
•
Wavelength selection
•
Linear and area scanning
•
Plate type and reading zone selections
•
Blank plate / well subtraction
•
Pathlength correction
•
Well monitoring
•
Kinetic, spectrum, and scanning analysis
•
Plate layout
•
Single plate transformations
•
Multi plate transformations
•
Curve-fitting parameters
•
Validation criteria
•
Cutoff criteria
•
Reporting and exporting options
User’s Guide to KC4™
Protocol Menu • 67
Working With Protocols
This section provides instructions for creating, modifying, saving, and accessing protocols.
Where and How Are Protocols Stored?
KC4 stores protocols using a proprietary file format and the .PRT extension. File storage options
vary depending on site requirements and the current KC4 configuration.
•
Files may be stored individually on the computer’s hard drive, on a CD or diskette, or on
a network to which the computer is attached. This is the storage method that users of
KC4 version 3.0x and earlier are familiar with. Windows Explorer or a similar
application can be used to view the file names and their locations, and to move, copy,
rename, and delete files.
•
(KC4 Signature) Files may also be stored inside a secure, shared-access database. This
database, named KCShared.mdb, can be stored on a user’s computer or on a sharedaccess computer. KC4 provides a special file maintenance window for viewing the file
names and their locations, and to move, copy, rename, and delete files.
Opening Existing Protocol Files
Select Protocol|Open to open a previously saved protocol file. (If the Open option is not
available in the Protocol menu, close the plate data file.)
Note: The layout of the Open dialog varies slightly depending on whether or not the Secured
Protocol and Data File Storage option is enabled, but the functionality is the same. (In the
example below, this option is enabled.)
•
The Current Folder field or the Look In field (you will see one or the other) shows your
current location within the overall folder structure.
If the drive is represented as DB:, this means that files are currently being stored in
KC4’s secure, shared-access database. If the drive is represented by a single letter,
such as C: or D: this means that files are stored individually on the computer’s hard
drive or on a network to which the computer is attached. See “Where and How Are
Protocols Stored?” for more information.
68 • Protocol Menu
User’s Guide to KC4™
•
Click
•
Click on a folder
•
Click on a file
•
Click to update the view to show any folders/files that may have been added by another
user after you opened this dialog.
to move up one folder level.
to open it and view its contents.
and click OK to open it.
Upon opening a protocol, KC4 evaluates it to determine (1) if it was created with an earlier
version of KC4, and (2) which reader model it was created for.
•
If the protocol was created using an earlier version of KC4, the following message
appears: “This file has been created with a former version of KC4. It is going to be
converted. As a precaution, please verify after conversion that all of its parameters have
been properly recovered. Then save the converted file.” KC4 will not automatically save
the converted file; it is your responsibility to verify and then save the file either under its
existing name or a new one.
•
If the protocol’s reader model does not match the one currently selected under
System|Readers, the following message appears: “Protocol file is not compatible with the
current reader. The current reader will be changed.” Click OK to open the protocol and
allow KC4 to update the current reader selection under System|Readers, or click Cancel
to return to the main view without opening the protocol.
If you click OK, KC4 will set the ‘current reader’ to the reader that was selected when the
protocol was created. If a different reader is then selected from the System menu, KC4
will delete the reading parameters of the open protocol, but will retain the data reduction
steps of the protocol. It will also set the protocol name to “New Protocol.prt”. The
reading parameters must be re-entered and the data reduction steps (i.e. data in for curve
fits, transformations, etc.) checked for correct input. The protocol can then be saved
under a similar or new name.
Note: If the attached reader does not match the one KC4 is expecting, the messages
INCORRECT READER MODEL CONNECTED or NO READER RESPONSE
(TIMEOUT) and then Error when getting wavelength table from the reader may appear.
KC4 will proceed and change the reader under System|Readers but you will have to
attach the correct reader before running the protocol.
When the protocol is open, KC4’s title bar changes to show the file name. Use the Protocol menu
or the toolbar icons to modify or view the protocol’s parameters.
Creating New Protocols
Important: Before creating a new protocol, select System|Readers and set the Current Reader to
the appropriate target reader model. The reader does not have to be connected to the computer
when you are creating or modifying protocols.
Clarity™ Microplate Luminometer users: See page 77 for information on creating protocols.
To create a new protocol, begin by doing one of the following to enable the options under the
Protocol menu:
•
•
Close any open plate data files and then select Protocol|New.
If the Preferred Data File Format is set to .GLB, select Data|New and then choose Empty
Protocol.
User’s Guide to KC4™
Protocol Menu • 69
The next step is to define the protocol parameters required by your assay to control the reader and
perform data reduction. Do this by opening the Protocol menu and selecting each option in turn:
Reading, Kinetic Analysis, Plate Layout, and so on. Select ‘Advanced’ to see additional options
including Cutoffs and Validation. The major headings in the remainder of this chapter are aligned
with the Protocol menu options; refer to these sections for further instruction.
You may not need to use all of the options, but you must at a minimum define the Reading
parameters. For more information see “Defining Reading Parameters” later in this chapter.
Protocol Wizard
As an alternative to using the Protocol Menu to create a new protocol, KC4 provides a Wizard to
guide you through the process. Protocol parameters are conveniently categorized for easy
selection and intuitive setup. Click the Wizard toolbar button or select Protocol|Wizard to begin.
•
Click on a button (e.g. Settings) to define related parameters. KC4 displays a check
mark ( ) next to each category after it is accessed.
You do not have to define the parameters associated with every button. At a
minimum, however, you should define the Reading Parameters under the Plate
Settings tab.
•
Click on a tab (e.g. Basic Analysis) to see more buttons.
•
Click < Back or > Next to switch between tabs.
•
Click OK to close the Wizard.
70 • Protocol Menu
User’s Guide to KC4™
Modifying Protocol Parameters
To modify protocol parameters within an existing protocol (.prt) or global data (.glb) file, begin by
opening the desired file. Once the file is open, use the Protocol menu or the toolbar buttons to
select a parameter category to modify, such as Reading, Plate Layout, Single Plate
Transformations, and so on.
Notes:
Select Protocol|Advanced to see additional menu options, including Multi Plate
Transformations, Cutoffs, and Validation.
When a file is being modified, an asterisk appears next to its name in the title bar as a
reminder to save the changes: [Oligo-Quant.prt*]
With KC4 Signature, KC4 automatically logs an event to the file’s audit trail any
time a change is made.
Saving Protocol Files
After creating or modifying a protocol file, save the information by selecting Protocol|Save or
Protocol|Save As. Save stores the protocol under its current name. (If it does not have a name,
KC4 will ask you to enter one.) Save As copies the contents of an existing protocol to a new
protocol with a different name.
Note: The layout of the Save or Save As dialog varies slightly depending on whether or not the
Secured Protocol and Data File Storage option is enabled, but the functionality is the same. (In the
following example, this option is enabled.)
•
The Current Folder field or the Save In field (you will see one or the other) shows your
current location within the overall folder structure.
If the drive is represented as DB:, this means that files are currently being stored in
KC4’s secure, shared-access database. If the drive is represented by a single letter,
such as C: or D: this means that files are stored individually on the computer’s hard
drive or on a network to which the computer is attached. See “Where and How Are
Protocols Stored?” for more information.
User’s Guide to KC4™
Protocol Menu • 71
•
The default file name is NewProtocol.prt
•
Enter a new File Name and click OK to save it.
•
Click
to move up one folder level.
•
Click
to create a new folder.
•
Click on a folder
•
If an existing file name is entered, KC4 will provide a warning to avoid an accidental
overwrite. Click Yes to continue with the overwrite, or No to rename the file before
saving.
to open it and view its contents.
Closing Protocol Files
To close a protocol file, select Protocol|Close. If the protocol is newly created, or if
modifications were made to an existing protocol, KC4 provides an option to save:
•
Click Yes to save the new or modified information and close the protocol.
•
Click No to close the protocol without saving changes.
•
Click Cancel to close this dialog and return to the open protocol. The new or modified
information is retained but not yet saved.
72 • Protocol Menu
User’s Guide to KC4™
Signing Protocol Files
KC4 Signature provides an option for users to permanently attach, or sign, their names to protocol
files.
Note: KC4 only supports file signatures when the multi-user identification feature is in use. See
“Security” in System Menu for more information.
To sign a protocol file:
1.
Open the desired file (if it is not already open).
2.
Select Protocol|Sign. The New Protocol Signature dialog will appear:
3.
Click the Reason drop-down arrow and select the reason to be associated with this
signature.
Signature Reasons are site-defined by the System Administrator under
System|Security|Signature Reasons.
4.
(Optional) If two users are signing on behalf of a third KC4 user, check the
Representative of box and then choose a name from the drop-down list.
5.
Enter or select your User ID.
If ‘Representative Of’ is checked, two users must enter or select their User IDs.
6.
Enter your Password. The characters will not be visible on the screen.
7.
Click Sign & Save.
User’s Guide to KC4™
Protocol Menu • 73
If the file has not yet been saved, a Save As dialog will appear:
8.
Using the folder location tools provided, select the storage location.
9.
Enter the desired File Name and click OK. The signature is applied immediately and
KC4 logs the event in the Protocol Audit Trail (select Protocol|Audit Trail to view):
74 • Protocol Menu
User’s Guide to KC4™
Plate Description
The Plate Description dialog appears just prior to each plate read, to provide the optional entry of
plate-specific information.
This dialog gathers information to be stored in the plate file along with the measurement data.
KC4 allows you to define up to six free-text fields to appear in this dialog (they are shown above
as Name, Title, Site, Lot Code, Water Lot, and Test). Select Protocol|Plate Description to
customize the names of these fields.
•
Text entered here substitutes the Prompt # text seen in the Data|Plate Description and
Data|Plate Read dialogs.
•
To customize a prompt, simply overwrite the default text in the Prompt # field.
•
Enter up to 15 alphanumeric characters in each field.
•
If a field is left empty, the prompt will be kept out of the Plate Description dialog.
User’s Guide to KC4™
Protocol Menu • 75
Defining Reading Parameters
The configuration of the Reading parameters controls plate reading and measurement data
collection. The availability of Reading parameters varies depending on the reader type currently
selected under System|Readers. To define Reading parameters, select Protocol|Reading or click
the Settings toolbar button to open the Reading dialog.
The following pages are organized by reader model; locate your model to find the basic
instructions for defining Reading parameters. To learn more about a particular parameter, search
through the Reading Parameters Reference, which is organized alphabetically.
76 • Protocol Menu
User’s Guide to KC4™
Clarity™
When KC4 is used to control the Clarity Microplate Luminometer, two protocols are required: a
Clarity protocol file to specify the reading parameters and a KC4 protocol to specify the data
reduction parameters. The KC4 protocol references the Clarity protocol file.
Clarity protocol files have a BPF extension and they are typically (but not necessarily) stored on
the PC in the C:\Program Files\BioTek\Clarity\protocols folder. BPF files cannot be stored in
KC4’s shared database.
Clarity protocol files can be created and edited through either the Clarity PC Software or KC4. In
either case, information on Clarity’s reading parameters can be found in Chapter 4 of the Clarity
Operator’s Manual under the “Parameter Input” heading.
To create/edit/reference a Clarity protocol file within a KC4 protocol, make sure that Clarity is set
as the current reader (System|Readers) and then select Protocol|Reading. The Reading dialog
will appear, as shown below:
•
To create a new Clarity protocol file, click Create New. The Create Protocols dialog
will appear. Select a protocol type and click Create to advance to the parameter input
series of dialogs.
•
To search for and open an existing Clarity protocol file, click Browse. The file’s
extension must be .BPF. Highlight the file and click Open. The file’s name and location
will appear in the Path field.
•
To edit reading parameters for the currently selected file, click Edit. The Protocol
Settings dialog will appear. Make the necessary changes and then click Save to store the
settings using the current file name, or Save As to store the settings using a new file
name.
•
(Optional) For Fast Kinetic protocols, click the Kinetic Scales button to adjust the
min/max t and min/max y scales for curve displays.
•
When finished, click OK to save changes or Cancel to discard them. You can now define
data reduction parameters via the Protocol Menu (Plate Layout, Transformations,
Cutoffs, Reports, etc.) or run the protocol by selecting Data|Read Plate.
User’s Guide to KC4™
Protocol Menu • 77
ELx800™
1.
Select a Reading Type of Endpoint or Kinetic.
2.
Select the filter(s) to be used.
3.
Select a Plate Type and define the First and Last Wells.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
6.
(Optional) Select Pre-Read Blank Plate.
7.
(Optional) Select Monitor Wells and then click Options… to define related parameters.
8.
Click OK to verify the parameters and close this dialog.
78 • Protocol Menu
User’s Guide to KC4™
ELx808™
1.
Select a Reading Type of Endpoint or Kinetic.
2.
Select the filter(s) to be used.
3.
Define a rectangle of wells to be read if reading a partial plate.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint linear scanning protocol, click the Scanning… button to define the
horizontal reading points.
6.
(Optional) Define Shaking parameters.
7.
(Optional) Define Temperature Control parameters.
8.
(Optional) Select Pre-Read Blank Plate.
9.
(Optional) Select Read Barcode to instruct the instrument to scan the barcode as the
plate is drawn in to the reading chamber.
10. (Optional) Select Monitor Wells and then click Options… to define related parameters.
11. Click OK to verify the parameters and close this dialog.
User’s Guide to KC4™
Protocol Menu • 79
FL600™
1.
Select a Detection Method of Absorbance, Fluorescence, or Luminescence.
2.
Select a Reading Type of Endpoint or Kinetic.
3.
For a fluorescence or luminescence protocol, define the Filter Sets to be used.
4.
For an absorbance protocol, define the Wavelengths and Optics Position to be used.
5.
Select a Plate Type and choose a Reading Direction.
6.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
7.
For an endpoint or kinetic scanning protocol, enable the Scanning checkbox and then
click the Scanning… button to define the read matrix size.
8.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
9.
(Optional) Define General Options parameters.
10. (Optional) Define Shaking parameters.
11. (Optional) Define Temperature Control parameters.
12. (Optional) Select Pre-Read Blank Plate.
13. (Optional) Select Monitor Wells and then click Options… to define related parameters.
14. Click OK to verify the parameters and close this dialog.
80 • Protocol Menu
User’s Guide to KC4™
FLx800™
1.
Select a Detection Method of Fluorescence or Luminescence.
2.
Select a Reading Type of Endpoint or Kinetic.
3.
(Optional) Click Update Filters to retrieve current filter values from the instrument.
4.
Define the Filter Sets to be used.
5.
Select a Plate Type and define the First and Last Wells.
6.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
7.
For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
8.
(Optional) Define Shaking parameters.
9.
(Optional) Define Temperature Control parameters.
10. (Optional) Select Pre-Read Blank Plate.
11. (Optional) Select Monitor Wells and then click Options… to define related parameters.
12. (Optional) Select Eject Between Filter Set Read.
13. Click OK to verify the parameters and close this dialog.
User’s Guide to KC4™
Protocol Menu • 81
PowerWave™/PowerWave HT
1.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
2.
Define the Wavelengths and Read Mode to be used. Calibrate the wavelengths if
necessary.
3.
Select a Plate Type and define the First and Last Wells.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint linear scanning protocol, click the Scanning… button to define the
horizontal reading points.
6.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
7.
(Optional) Define Shaking parameters.
8.
(Optional) Define Temperature Control parameters.
9.
(Optional) Select and define Pre-Reading options.
10. (Optional) Select Read Barcode to instruct the instrument to scan the barcode as the
plate is drawn in to the reading chamber.
11. (Optional) Select Monitor Wells and then click Options… to define related parameters.
12. Click OK to verify the parameters and close this dialog.
82 • Protocol Menu
User’s Guide to KC4™
PowerWaveX™
1.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
2.
Define the Wavelengths to be used. Calibrate the wavelengths if necessary.
3.
Define a rectangle of wells to be read if reading a partial plate.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint linear scanning protocol, click the Scanning… button to define the
horizontal reading points.
6.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
7.
(Optional) Define Shaking parameters.
8.
(Optional) Define Temperature Control parameters.
9.
(Optional) Select and define Pre-Reading options.
10. (Optional) Select Read Barcode to instruct the instrument to scan the barcode as the
plate is drawn in to the reading chamber.
11. (Optional) Select Monitor Wells and then click Options… to define related parameters.
12. Click OK to verify the parameters and close this dialog.
User’s Guide to KC4™
Protocol Menu • 83
PowerWaveX™ Select
1.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
2.
Define the Wavelengths to be used. Calibrate the wavelengths if necessary.
3.
Select a Plate Type and define the First and Last Wells.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint linear scanning protocol, click the Scanning… button to define the
horizontal reading points.
6.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
7.
(Optional) Define Shaking parameters.
8.
(Optional) Define Temperature Control parameters.
9.
(Optional) Select and define Pre-Reading options.
10. (Optional) Select Read Barcode to instruct the instrument to scan the barcode as the
plate is drawn in to the reading chamber.
11. (Optional) Select Monitor Wells and then click Options… to define related parameters.
12. Click OK to verify the parameters and close this dialog.
84 • Protocol Menu
User’s Guide to KC4™
PowerWave™ XS
1.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
2.
Define the Wavelengths and Read Mode to be used. Calibrate the wavelengths if
necessary.
3.
Select a Plate Type and define the First and Last Wells.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
6.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
7.
(Optional) Define Shaking parameters.
8.
(Optional) Define Temperature Control parameters.
9.
(Optional) Select and define Pre-Reading options.
10. (Optional) Select Monitor Wells and then click Options… to define related parameters.
11. Click OK to verify the parameters and close this dialog.
User’s Guide to KC4™
Protocol Menu • 85
Synergy™ HT
1.
Select a Detection Method of Absorbance, Fluorescence, Luminescence, or Multi-Mode.
2.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
3.
For an absorbance protocol, define the Wavelengths and Read Mode to be used.
Calibrate the wavelengths if necessary.
4.
For a fluorescence, luminescence, or multi-mode protocol, define the Filter Sets to be
used.
5.
Select a Plate Type and define the First and Last Wells.
6.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
7.
For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
8.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
9.
(Optional) Define Shaking parameters.
10. (Optional) Define General Options parameters.
11. (Optional) Define Temperature Control parameters.
12. (Optional) Select and define Pre-Reading options.
13. Click OK to verify the parameters and close this dialog.
86 • Protocol Menu
User’s Guide to KC4™
Synergy™ HT / Time-Resolved
1.
Select a Detection Method of Absorbance, Fluorescence, Luminescence, or Multi-Mode.
2.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
3.
(Optional) For a fluorescence protocol, enable the Time-Resolved option.
4.
For an absorbance protocol, define the Wavelengths and Read Mode to be used.
Calibrate the wavelengths if necessary.
5.
For a fluorescence, luminescence, or multi-mode protocol, define the Filter Sets to be
used.
6.
Select a Plate Type and define the First and Last Wells.
7.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
8.
For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
9.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
10. (Optional) Define Shaking parameters.
11. (Optional) Define General Options parameters.
12. (Optional) Define Temperature Control parameters.
13. (Optional) Select and define Pre-Reading options.
14. Click OK to verify the parameters and close this dialog.
User’s Guide to KC4™
Protocol Menu • 87
Synergy™ HT / Time-Resolved / Injectors
1.
Select a Detection Method of Absorbance, Fluorescence, Luminescence, or Multi-Mode.
2.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
3.
(Optional) For a fluorescence or luminescence protocol, check Dispense.
4.
(Optional) For a fluorescence protocol, enable the Time-Resolved option.
5.
For a fluorescence, luminescence, or multi-mode protocol, define the Filter Sets.
6.
For a Dispense protocol, click Read & Dispense to define additional parameters.
7.
For an absorbance protocol, define the Wavelengths and Read Mode to be used.
Calibrate the wavelengths if necessary.
8.
Select a Plate Type and define the First and Last Wells.
9.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
10. For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
11. (Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
12. (Optional) Define Shaking parameters.
13. (Optional) Define General Options parameters.
14. (Optional) Define Temperature Control parameters.
15. (Optional) Select and define Pre-Reading options.
16. Click OK to verify the parameters and close this dialog.
88 • Protocol Menu
User’s Guide to KC4™
µQuant™
1.
Select a Reading Type of Endpoint, Kinetic, or Spectrum.
2.
Define the Wavelengths to be used. Calibrate the wavelengths if necessary.
3.
Select a Plate Type and define the First and Last Wells.
4.
For a kinetic protocol, click the Kinetic… button to define the read time and interval.
5.
For an endpoint area scanning protocol, enable the Scanning checkbox and then click
the Scanning… button to define the read matrix size.
6.
(Optional) For a spectrum protocol, click the Scales… button to customize the graphical
data display.
7.
(Optional) Select and define Pre-Reading options.
8.
(Optional) Select Monitor Wells and then click Options… to define related parameters.
9.
Click OK to verify the parameters and close this dialog.
User’s Guide to KC4™
Protocol Menu • 89
Reading Parameters Reference
All possible reading parameters and certain related topics are discussed on the following pages.
Information is organized alphabetically by parameter name or topic.
Clarity™ Microplate Luminometer users: Refer to Chapter 4 of the Clarity Operator’s Manual
for information on reading parameters.
Automatic Sensitivity Adjustment
Enable Automatic Sensitivity Adjustment to let KC4 automatically select the most appropriate
sensitivity value for a particular filter set. There are three methods to choose from: Scale to High
Wells, Scale to Low Wells, and Best Signal to Noise Ratio. The best method depends on your
application (some experimentation may be necessary).
Note for Synergy™ HT w/Injector models. With Dispense protocols, if two filter sets are
defined and Automatic Sensitivity Adjustment is enabled for the first filter set, ‘Use first filter set
sensitivity’ is the required selection for the second filter set.
Scale to High Wells
Scale to High Wells searches for two consecutive gain values where the measured values are
lower and higher than the defined high well values.
•
The High Wells represent those wells on which the calibration measurements will be
taken. They are typically wells with the highest expected signal (measurement value) on
the plate, such as the high standard wells.
•
The High Value represents the upper limit of the expected range of values for the entire
microplate. The default value for the FL600™ is 90000 RFU/RLU with a valid range of
0 to 99999. The default value for the FLx800™ and the Synergy™ HT is 80000
RFU/LFU with a valid range of 0 to 99998. The recommended high value for all of these
readers is 50,000 to 70,000 RFU/RLU.
•
The default Starting Sensitivity is 100. The valid range is from 25 to 255. The
recommended starting sensitivity is 25.
Scale to Low Wells
Scale to Low Wells searches for two consecutive gain values where the measured values are lower
and higher than the defined low value wells. Scale to Low Wells is recommended for weakly
luminescent reactions.
90 • Protocol Menu
User’s Guide to KC4™
•
The Low Wells represent those wells on which the calibration measurements will be
taken. They are typically wells with the lowest expected signal (measurement value) on
the plate, such as blank wells, negative controls, or the zero standard wells.
•
The Low Value represents the lower limit of the expected range of values for the entire
microplate. The default value is 25 RFU/RLU, and the valid range is from 0 to 99999 for
the FL600™, 0 to 99998 for the FLx800™ and the Synergy™ HT. The recommended
low value for all of these readers is 100 to 200 RFU/RLU.
•
The default Starting Sensitivity is 100. The valid range is from 25 to 255. The
recommended starting sensitivity is 25.
Best Signal to Noise Ratio
With the Best Signal to Noise Ratio method, KC4 instructs the reader to perform several prereadings on the plate to determine the optimal sensitivity. The optimum sensitivity is based on the
best signal-to-noise ratio obtained during these pre-readings.
•
Designate certain wells on the plate as Low, Experimental, and High Wells. Enter a
single well location (ex. A1) or a ‘rectangle’ (ex. B1-C2) of well locations.
Low Wells are usually blank wells, buffer wells, or negative controls, and are expected to
yield a low RFU or RLU value.
Experimental Wells are usually low standards or low positive controls, and are expected
to yield a mid-range RFU or RLU value.
High Wells are usually the highest standards or high positive controls, and are expected
to yield a high (but not over range) RFU or RLU value.
User’s Guide to KC4™
Protocol Menu • 91
•
Specify the Base and High Values.
The Base Value is the fluorescence or luminescence value that the Low and
Experimental Wells must exceed during sensitivity determination. This ensures that the
plate produces values that are large enough to be meaningful.
The High Value is the fluorescence or luminescence value that the High Well(s) should
approach but not exceed. The default setting is 90000 RFU/RLU, and the maximum
value is 99999 for the FL600™ and 99998 for the FLx800™ and the Synergy™ HT.
Practically, this value could be anywhere from 10000 to 90000.
Note: If the Best signal to Noise Ratio cannot be calculated, use Scale to High Wells.
Use First Filter Set Sensitivity
If multiple filter sets are defined, you can opt to use the sensitivity determined with filter set #1 for
other filter sets, by choosing Use First Filter Set Sensitivity.
Bandwidth Verification
(Only applies to fluorescence/luminescence readers.)
KC4 evaluates each filter set against two criteria:
1) Overlap Evaluation
By definition, the bandwidth rating on the filters used for standard fluorescence reads represents
the bandwidth at the 50% transmission level. The transmission profile of these filters is
essentially rectangular and therefore the bandwidth does not vary very much when the
transmission levels change. When KC4 checks for overlap it ensures that the excitation and
emission filter center wavelengths are far enough apart that there is no overlap of their
bandwidths. The formula used to evaluate each pair of filter sets for overlap is as follows:
|CW excitation - CW emission| > (BW excitation + BW emission)/2, where:
CW excitation is the center wavelength of the excitation filter
CW emission is the center wavelength of the emission filter
BW excitation is the bandwidth of the excitation filter*
BW emission is the bandwidth of the emission filter
*
If the Time-Resolved option is enabled, the value of BW excitation must be multiplied by
2 prior to substituting it into the formula to account for the triangular shape of the
transmission profile when using the monochromator. Specifically, although the
monochromator has a bandwidth of 10 nm at 50% transmission levels, it approximately
doubles by the time the transmission levels approach zero.
92 • Protocol Menu
User’s Guide to KC4™
If the absolute value of the difference in the excitation and emission center wavelengths is greater
than half the sum of the excitation and emission bandwidths, then no overlap condition exists. If it
is less than or equal to, then an overlap condition does exist.
•
When all of the reading parameters have been defined and the user attempts to validate
them (by clicking OK), to KC4 evaluates each filter set pair against the overlap formula.
If the result is such that there is no overlap condition for any of the defined filter set
pairs, and there are no other parameter definition violations, KC4 will allow you to
"OK" out of the Reading Parameters dialog.
If the result is such that there is an overlap condition for any filter set pair, KC4 will
display a message resembling the following: "Please select alternative filters for
filter set #1. The bandpasses overlap." KC4 will not allow you to "OK" out of the
Reading Parameters dialog until there are no overlapping filter set pairs.
2) Proximity Evaluation
Although the transmission profile is quite vertical, it does "flair out" at very low transmission
levels indicating an increase in the bandwidth. In order to account for the bandwidth at these
lower transmission levels, KC4 performs a proximity check on each filter set pair. The formula
used to evaluate each pair of filter sets to determine if they are "too close" to each other is as
follows:
|CW excitation - CW emission| > BW maximum + (BW minimum/2), where:
CW excitation is the center wavelength of the excitation filter
CW emission is the center wavelength of the emission filter
BW maximum is the larger of the two bandwidths*
BW minimum is the smaller of the two bandwidths*
*
If the Time-Resolved option is enabled, the determination of BW maximum and BW
minimum must be made by comparing the emission bandwidth to twice the excitation
bandwidth. Once again, this accounts for the triangular shape of the transmission profile
when using the monochromator.
If the absolute value of the difference in the excitation and emission center wavelengths is greater
than the sum of the maximum bandwidth and half the minimum bandwidth, then no proximity
condition exists. If it is less than or equal to, then a proximity condition does exist.
•
When all of the reading parameters have been defined and the user attempts to validate
them (by clicking OK), to KC4 evaluates each filter set pair against the proximity
formula.
If the result is such that there is no proximity condition for any of the defined filter
set pairs, and there are no other parameter definition violations, KC4 will allow you
to "OK" out of the Reading Parameters dialog.
If the result is such that there is proximity condition for any filter set pair, KC4 will
display a message resembling the following: "Warning: the bandpasses for filter set
#2 are too close." However, KC4 does not prohibit you from saving and running
protocols that produce proximity warning messages.
User’s Guide to KC4™
Protocol Menu • 93
Detection Method
Certain readers, such as the FL600™, FLx800™, and Synergy™ HT, have multi-detection read
capability. Before defining additional Reading parameters, click the radio button associated with
the detection method required for the assay.
•
Multi-Mode is only available for the Synergy HT line of instruments. When Multi-Mode
is selected, more than one detection method can be defined within a single protocol.
When a Detection Method is selected, KC4 customizes the Reading Parameters dialog so that only
the options supported by the Detection Method are available for selection.
The Detection Method selection also instructs KC4 to display measurement values as OD (optical
density), RFU (relative fluorescence unit), or RLU (relative luminescence unit).
Dispense Option
The Synergy™ HT with Injectors provides the ability to dispense fluid into microwells as part of a
Read & Dispense protocol.
When the Dispense option is enabled, the following limitations are applied:
•
Area scanning is not supported.
•
1 or 2 filter sets may be defined.
•
The Shaking options are disabled within the main Reading Parameters dialog. A Shake
step may be added to Plate Mode readings in the Read & Dispense dialog.
•
The Lag Time option is disabled.
•
The Eject Between Filter Sets option is disabled.
•
The Top Probe Vertical Offset option is disabled. The top probe height will be
automatically set to 1 mm above the top of the plate.
Note: The Dispense option is not available when Multi-Mode is selected.
94 • Protocol Menu
User’s Guide to KC4™
Eject Between Filter Set Read
This feature is available for fluorescence and luminescence protocols if you are defining an
endpoint read with two or more filter sets. Enable Eject between Filter Set Read to:
•
Perform an initial reading of a blank plate, followed by the assay plate(s) at some later
time.
•
Provide the ability to add reagents to the same plate between readings.
•
Enable reading up to 6 different plates with one protocol and plate file.
If this feature is enabled, KC4 ejects the plate after one filter set read, then displays a message to
click OK to start the next filter set read.
See also “General Options (FL600)” or “General Options (Synergy HT).”
Excitation/Emission Filter Sets (FL600™)
The FL600 can take measurements with up to six uniquely defined Filter Sets. There are many
applications for multiple filter sets, including:
•
Assays that have more than one fluorophore in the well.
•
Comparison of results between the top and bottom optics positions.
•
Comparison of results between rapid and static mode.
•
Comparison of results with varying static mode settings.
•
Comparison of results with varying sensitivity settings.
To define Filter Sets:
•
Ensure the protocol is appropriately set for Absorbance, Fluorescence, or
Luminescence detection method.
•
Select the Filter Set radio button corresponding with the number of sets required.
•
Define the wavelengths:
Absorbance detection method:
For each filter set, use the drop-down lists to select the desired wavelength.
Fluorescence detection method:
For each filter set, use the drop-down lists to select the Excitation and Emission
wavelengths.
User’s Guide to KC4™
Protocol Menu • 95
Luminescence detection method:
For maximum sensitivity, select Lum/E to indicate an empty position in the filter
wheel and a plug in position E in the excitation wheel.
For each filter set, use the drop-down lists to select the Emission wavelength.
•
Note: To prevent damage to the reader, KC4 performs bandwidth verification as part of
the validation within the Reading Parameters dialog.
•
The instrument may be equipped with one or two optical probes, positioned above (Top)
and/or below (Bottom) the assay plate. Specify the Optics Position to be used for each
filter set.
Tips:
Evaluate the proper reading position by specifying two (or more) filter sets, one each
for the Top and Bottom optics positions.
The top position is advantageous for homogeneous solutions using solid black plates.
The bottom position is advantageous for cellular assays and assays requiring a
covered plate to maintain sterility.
The recommended Optics Position for Absorbance readings is Bottom. KC4
provides a warning if Top is selected, although this selection will not be prohibited.
The recommended Optics Position for Luminescence readings is Top.
•
Fluorescence or Luminescence method only: Specify the Sensitivity setting for each
individual filter set. Valid settings are 0 and integers from 25 to 255. ‘Auto’ indicates
that the Auto Sensitivity Adjustment feature has been enabled.
The signal from the range of fluorescence and luminescence assays can be very weak,
very strong, or anywhere in between. For each assay, the sensitivity of the
photomultiplier (PMT) should be adjusted to ensure that the signals from all wells fall
within the appropriate dynamic range of 0 to 99999 relative fluorescence or luminescence
units (RFU or RLU).
Tips:
Typical assays using 96- (or fewer) well plates require sensitivities between 75 and
190. Assays using 384-well or other higher-density plates will likely require much
higher sensitivities due to narrower optical probes. Luminescent reactions may
require sensitivities up to 255.
To determine the optimum sensitivity for an assay, use Scale to High Wells or Scale
to Low Wells. Scale to High Wells is used for fluorescence and bright luminescence
assays. Scale to Low Wells is used for weakly luminescent reactions. See
Automatic Sensitivity Adjustment for more information.
As the sensitivity setting increases, so will the fluorescence values. If many wells
result in “OVRFLW” (indicating RFU or RLU values greater than 99999), the
sensitivity setting is probably too high.
If each well contains more than one fluorophore, one of which might give brighter or
weaker results, assign each filter set its own sensitivity.
If the field shows ‘Auto’, the Automatic Sensitivity Adjustment feature is enabled.
•
Click the Options… button to specify the Measurement Options for each individual filter
set (Endpoint Readings), or once for all filter sets (Kinetic Readings).
96 • Protocol Menu
User’s Guide to KC4™
Excitation/Emission Filter Sets (FLx800, Synergy HT)
The FLx800™ and Synergy™ HT can take measurements with up to six uniquely defined Filter
Sets for endpoint reads and up to two for kinetic reads. There are several applications for multiple
filter sets, including:
•
Assays that have more than one fluorophore in the well.
•
Comparison of results between the top and bottom optics positions.
•
Comparison of results with varying sensitivity settings.
To define Filter Sets:
•
Select either the Fluorescence or Luminescence detection method.
•
Select Endpoint or Kinetic reading type.
•
Click the radio button corresponding with the number of Filter Sets required.
Endpoint reads without area scanning: 1 to 6
Endpoint reads with area scanning: 1 or 2
Kinetic reads: 1 or 2
Dispense feature enabled: 1 or 2
•
Define the wavelengths:
Fluorescence detection method:
For each filter set, use the drop-down lists to select the Excitation and Emission
wavelengths.
Fluorescence detection method with Time-Resolved enabled (Synergy HTTR):
For each Excitation filter set, enter a wavelength value between 200 and 999 nm.
The bandpass is not variable; it is pre-defined to be 10 nm.
For each Emission filter set, use the drop-down lists to select the wavelengths.
Luminescence detection method:
For maximum sensitivity, select Lum/E to indicate an empty position in the filter
wheel and a plug in position E in the excitation wheel.
For each filter set, use the drop-down lists to select the Emission wavelength.
•
Note: To prevent damage to the reader, KC4 performs bandwidth verification as part of
the validation within the Reading Parameters dialog.
User’s Guide to KC4™
Protocol Menu • 97
•
The instrument may be equipped with one or two optical probes, positioned above (Top)
and/or below (Bottom) the assay plate. Specify the Optics Position to be used for each
filter set.
Tips:
Evaluate the proper reading position by specifying two (or more) filter sets, one each
for the Top and Bottom optics positions.
The top position is advantageous for homogeneous solutions using solid black plates.
The bottom position is advantageous for cellular assays and assays requiring a
covered plate to maintain sterility.
The recommended Optics Position for Luminescence readings is Top.
•
Specify the Sensitivity setting for each individual filter set. Valid settings are 0 and
integers from 25 to 255. ‘Auto’ indicates that the Auto Sensitivity Adjustment feature
has been enabled.
Note: With Dispense protocols, if two filter sets are defined the field for the second
filter set always shows Auto and will use the first filter set’s sensitivity.
The signal from the range of fluorescence and luminescence assays can be very weak,
very strong, or anywhere in between. For each assay, the sensitivity of the
photomultiplier (PMT) should be adjusted to ensure that the signals from all wells fall
within the appropriate dynamic range of 0 to 99998 relative fluorescence or luminescence
units (RFU or RLU).
Tips:
Typical assays using 96- (or fewer) well plates require sensitivities between 35 and
150. Assays using 384-well or other higher-density plates will likely require much
higher sensitivities due to narrower optical probes. Luminescent reactions may
require sensitivities up to 255.
To determine the optimum sensitivity for an assay, use Scale to High Wells or Scale
to Low Wells. Scale to High Wells is used for fluorescence and bright luminescence
assays. Scale to Low Wells is used for weakly luminescent reactions. See
Automatic Sensitivity Adjustment for more information.
As the sensitivity setting increases, so will the fluorescence values. If many wells
result in “OVRFLW” (indicating RFU or RLU values greater than 99998), the
sensitivity setting is probably too high.
If each well contains more than one fluorophore, one of which might give brighter or
weaker results, assign each filter set its own sensitivity.
If the field shows ‘Auto’, the Automatic Sensitivity Adjustment feature is enabled.
•
Click the Options… button to specify the Measurement Options for each individual filter
set.
98 • Protocol Menu
User’s Guide to KC4™
Filters
The Filters parameters include:
•
Meas. Filter: select the measurement filter for a single- or dual- wavelength read.
•
Refr. Filter: select the reference filter for a dual-wavelength read.
First Well / Last Well
Most readers support reading a subset of the entire plate, based on contiguous microplate wells.
To specify the range of wells to be read:
•
If applicable, select the appropriate Plate Type. The default Last Well field value will
automatically update to match the selected plate type.
•
Set the First Well and Last Well fields to indicate the range of wells to be read. For
example, if First Well is set to A1 and Last Well is set to B3, KC4 reads a ‘rectangle’ of
wells: A1, A2, A3, B1, B2, and B3.
General Options (FL600™)
KC4 provides options for controlling certain reader functions preceding and during plate reads,
such as lamp control and plate ejection.
Click the Options button from within the Reading Parameters dialog to open the General Options
dialog. The available/recommended settings vary, depending on the reading mode.
User’s Guide to KC4™
Protocol Menu • 99
Absorbance mode:
•
Enable Eject between Filter Set Read to perform an initial reading of a blank plate
followed by the assay plate(s) at some later time; provide the ability to add reagents to the
same plate between readings; or enable reading up to 6 different plates with one protocol
and plate file.
This feature is available for Endpoint reads only.
•
Skip reference wells is selected for absorbance readings, and cannot be changed.
•
Enable Check Lamp before measurement to check lamp output prior to measurement.
This feature is selected by default. Deselecting this feature speeds up the reading
process, however, a lamp output failure may not be detected.
•
Enable Set Lamp Off during measurement to allow for the reading of an unexcited
signal. This feature is deselected by default.
Fluorescence mode:
•
Enable Eject between Filter Set Read to perform an initial reading of a blank plate
followed by the assay plate(s) at some later time; provide the ability to add reagents to the
same plate between readings; or enable reading up to 6 different plates with one protocol
and plate file.
This feature is available for Endpoint reads only.
•
Skip reference wells is deselected by default.
The FL600 is equipped with fluorescent plastic compounds call reference wells which
help provide the best results when using Rapid Mode to read the assay plate. Skip
reference wells should be deselected for use with Rapid Mode reads.
•
Enable Check Lamp before measurement to check lamp output prior to measurement.
This feature is selected by default. Deselecting this feature speeds up the reading
process, however, a lamp output failure may not be detected.
•
Enable Set Lamp Off during measurement to allow for the reading of an unexcited
signal. This feature is deselected by default.
Luminescence mode:
•
Enable Eject between Filter Set Read to perform an initial reading of a blank plate,
followed by the assay plate(s) at some later time; provide the ability to add reagents to the
same plate between readings; or enable reading up to 6 different plates with one protocol
and plate file.
This feature is available for Endpoint reads only.
•
Skip reference wells is deselected for luminescence readings, and cannot be changed.
•
Check Lamp before measurement is deselected by default. The lamp does not need to
be checked before measurement.
•
Set Lamp Off during measurement is selected for luminescence readings, and cannot be
changed. The lamp is turned off during measurement to avoid damage to the
photomultiplier.
100 • Protocol Menu
User’s Guide to KC4™
Reading the Reference Wells
It is strongly recommended that you read the reference wells if Rapid Mode is enabled. If,
however, reading the Reference Wells must be skipped, consider the following:
•
During an Endpoint read, when prompted to "Insert plate and press OK", do so within 60
seconds.
•
If a Lag Time is specified for an Endpoint or Kinetic read, the maximum recommended
time is 60 seconds. If a longer lag time is required, read the reference wells first or use
Static Mode to read the plate.
•
During a Kinetic read, the maximum time interval is 60 seconds. If a longer time interval
is required, read the reference wells first or use Static Mode to read the plate.
If the Reference Wells are skipped and the above time limits are exceeded, the FL600 can provide
unexpectedly high fluorescence values in the first well that is read on the assay plate. Usually the
first well is A1 but since the software provides the ability to change the starting well, the first well
can be anywhere on the plate.
General Options (Synergy™ HT)
KC4 provides options for controlling certain reader functions preceding and during plate reads,
such plate ejection and top probe positioning.
If you are defining an endpoint read with two or more filter sets and are utilizing either the
fluorescence or luminescence detection method, enable Eject between Filter Set Read to:
•
Perform an initial reading of a blank plate, followed by the assay plate(s) at some later
time.
•
Provide the ability to add reagents to the same plate between readings.
•
Enable reading up to 6 different plates with one protocol and plate file.
If this feature is enabled, KC4 ejects the plate after one filter set read, then displays a
message to click OK to start the next filter set read.
The Top Probe Vertical Offset parameter is available for fluorescence or luminescence reads
when at least one of the filter sets uses the "top" optics position. It allows you to define how far
the top probe is positioned from the top surface of the plate during the read. The valid range for
the offset parameter is determined on a plate by plate basis. The calculation is based on the plate
height and the maximum travel of the top probe:
User’s Guide to KC4™
Protocol Menu • 101
In order to be considered valid, the vertical offset value must satisfy the following conditions:
•
1.0 mm < = offset < = 32.5 - plate height*
•
offset > = 15.0 mm - plate height*
* Plate heights are defined via System|Plate Formats.
Kinetic Parameters
If the Kinetic reading type is selected in the Reading dialog, click the Kinetic… button to define
related parameters.
Notes:
•
If the FL600™ is the current reader, the Kinetic dialog contains some additional
parameters. See the next section for more information.
•
The default time format for kinetics is HH:MM:SS. To change the time format, select
System|Setup.
Specify the total kinetic Run Time and the Kinetic Interval. KC4 automatically generates the
Reading Number based on these settings.
•
Run Time: Defines the duration of the complete kinetic run. The maximum run time is
48 hours.
102 • Protocol Menu
User’s Guide to KC4™
•
Interval: Defines the time interval between individual kinetic reads. The maximum valid
Interval is equal to the current Run Time. The minimum valid Interval depends on the
current reader type and certain protocol parameters, such as the number of wavelengths
specified, the plate format, the number of wells to be read, and whether or not shaking is
selected.
For certain readers, a Minimum button is available under the Interval box. When this
button is clicked, KC4 communicates with the reader to determine the minimum kinetic
interval based on the currently selected Reading parameters.
For all readers, when the OK button is clicked, KC4 automatically checks the validity of
the Interval value. If it is insufficient for the currently defined Reading parameters, a
theoretical minimum value is displayed.
Note: For readers that support more than one read mode (speed), the returned value is
based on the current reading mode. For some instruments, the read mode is set through
the instrument’s front panel. For others, it is defined as part of the protocol.
•
Reading Number: Represents the total number of kinetic reads. KC4 calculates this
automatically, based on the currently defined Run Time and Interval. The maximum
number of kinetic reads is 300.
•
Allow Well Zoom During Read: If this feature is enabled, you can click on one or more
wells in the Results dialog to view the kinetic curve(s) as the plate is being read. KC4
updates the graph and kinetic calculations as each measurement value is retrieved from
the reader. Important! When the Allow Well Zoom During Read box is checked, a
warning message appears. If the computer speed is less than 100 MHz, or if there is
insufficient memory, this feature should not be used.
Kinetic Parameters (FL600)
The Kinetic dialog contains special Mode parameters for the FL600™.
•
In Plate Mode, the FL600 repeatedly reads a whole or partial plate for a specified number
of times and for up to 6 filter sets.
User’s Guide to KC4™
Protocol Menu • 103
•
In Well Mode, the FL600™ repeatedly reads a well for a specified number of times for up
to 2 filter sets. This mode is typically used for fast kinetic reactions.
Check Eject between well to eject the assay plate between well readings. This feature is
useful, for example, for accommodating reagent addition.
Measurement Options
To define Measurement Options such as Sampling or Automatic Sensitivity Adjustment for a
particular filter set, start by clicking its Options button.
Note: If the current reader is the FL600 and the reading type is Kinetic, click the Options button
associated with Filter Set 1. Options selected here are applied to all defined filter sets.
Measurement Mode (FL600)
To define the measurement mode parameters for the FL600:
•
Choose between Rapid and Static mode:
Rapid Mode reads each well while the assay plate is in motion. This provides for
faster reading than with Static mode, but the CVs might be lower.
The FL600 is equipped with fluorescent plastic compounds called reference wells
which help provide the best results when using Rapid Mode to read the assay plate.
See “General Options (FL600)” for more information.
Static Mode performs a specified number of reads in each well, with a specified
delay between each read. The reading performs more slowly than with Rapid Mode,
but the CVs are usually better. Nb Samples, Delay before sampling, and Delay
between samples pertain specifically to Static Mode.
104 • Protocol Menu
User’s Guide to KC4™
If Static Mode was selected, specify the Sampling options:
•
Nb Samples is the number of measurements taken per well. Valid options are 1, 2, 3, 4,
10, 20, 50, and 100 measurements. The value reported for each well represents the
average of its measurements.
Usually, the more samples per well the better the CVs, although selecting a large number
of samples per well typically results in only marginal improvement. Consider a setting
that represents the optimal combination of sensitivity and speed. The recommended
setting to achieve a balance between speed and sensitivity is 10 measurements per well.
Note: The reader takes approximately 10 milliseconds to perform each measurement.
•
Delay before sampling is the pause time of the optical probe above or below the
microwell before the measurement is taken. Valid options are 0.10, 0.35, 0.50, and 2.00
seconds. The recommended setting for fluorescence assays to ensure the probe is stopped
for measurement, but for the minimum amount of time is 0.35 seconds. For
luminescence reads that allow long reading times, a delay of 2.0 seconds will provide
lower CVs. This delay of 2.0 seconds is not recommended for fluorescence reads.
•
Delay between samples is the time delay between measurements taken in each well.
The valid range is from 0 to 2550 milliseconds. The recommended setting is 10
milliseconds. Longer delays between samples may result in better CVs between
replicates.
Measurement Mode (FLx800, Synergy HT)
To define the measurement mode parameters for the FLx800™ and Synergy™ HT:
•
Nb Samples is the number of measurements taken per well. The valid range is from 1 to
255 measurements. The value reported for each well represents the average of its
measurements.
Usually, the more samples per well the better the CVs, although selecting a large number
of samples per well typically results in only marginal improvement. Consider a setting
that represents the optimal combination of sensitivity and speed. The recommended
setting to achieve a balance between speed and sensitivity is 10 measurements per well.
Note: The reader takes approximately 10 milliseconds to perform each measurement.
•
Delay before sampling is the pause time of the optical probe above or below the
microwell before the measurement is taken. The valid range is from 10 to 2550
milliseconds. The recommended setting to ensure the probe is stopped for measurement,
but for the minimum amount of time, is 350 milliseconds.
•
Delay between samples is the time delay between measurements taken in each well.
The valid range is from 0 to 255 milliseconds. The recommended setting is 1
millisecond. Longer delays between samples may result in better CVs between
replicates.
User’s Guide to KC4™
Protocol Menu • 105
Measurement Mode (Synergy™ HT / Time-Resolved)
Specify the Sampling options:
•
Nb Samples is the number of measurements taken per well. The valid range is from 1 to
255 measurements. The value reported for each well represents the average of its
measurements.
Usually, the more samples per well the better the CVs, although selecting a large number
of samples per well typically results in only marginal improvement. Consider a setting
that represents the optimal combination of sensitivity and speed. The recommended
setting to achieve a balance between speed and sensitivity is 10 measurements per well.
•
Delay before sampling is the pause time of the optical probe above or below the
microwell before the first flash. The valid range is from 10 to 2550 milliseconds. The
recommended setting to ensure the probe is stopped for measurement, but for the
minimum amount of time is 350 milliseconds.
•
Delay between samples is the time delay between the end of data collection and the
beginning of the next flash in each well. The valid range is from 0 to 255 milliseconds.
The recommended setting is 5 milliseconds. Longer delays between samples may result
in better CVs between replicates.
•
Note for Injector models. The following values are “fixed” for Dispense protocols:
Plate Mode:
Well Mode:
Nb Samples per well: 10
Nb Samples per well: 1
Delay before sampling: 350
Delay before sampling: 10
Delay between samples: 24
Delay between samples: 0
If the Time-Resolved option in the Reading Parameters dialog is checked, specify the TimeResolved Fluorescence options:
•
Delay is time delay between the flash and the beginning of data collection. It is also
referred to as the delay before integration. Valid values are 0 µsec and 20 to 16000 µsec.
The default value is 20 µsec.
Note: A delay of 0 µsec is not the same as a fluorescence read with the time-resolved
option turned off. They differ in two ways: (1) the excitation wavelength is generated by
the monochromator; and (2) the user can specify the data collection time which is not an
available parameter for normal fluorescence reads.
106 • Protocol Menu
User’s Guide to KC4™
•
Data Collection Time is the amount of time for which readings are collected after the
delay before integration time has expired. The valid range is from 20 to 16000 µsec. The
default value is 100 µsec.
The following is a simplified schematic of the measurement mode parameters:
1
= Nb Samples per well
2
= Delay before sampling
3
= Delay between samples
4
= Delay
5
= Data Collection Time
Monitor Wells
If the currently selected reader supports well monitoring, the Monitor Wells option is available.
The feature enables the instrument to repeatedly read a single well or a group of wells and
evaluate the measurements against pre-defined criteria. Once the criteria are met, the plate read
begins.
Click the Options button to define the criteria:
•
Select the Data to Monitor representing the filter or wavelength value to be used during
well monitoring. The available options depend on the current selections in the Reading
dialog. Only measured raw absorbance, fluorescence, or luminescence values are
presented for selection.
User’s Guide to KC4™
Protocol Menu • 107
•
Enter the well or Wells to Monitor. Multiple wells must be contiguous and entered as a
range, e.g. A1-A2, or G10-H12.
•
Define the Stop Monitoring When criteria, to tell the instrument when to conclude the
monitoring process:
Choose a conditional option: ‘At least one well’ in the range must meet the criteria;
the ‘Average’ of the monitored wells must meet the criteria; or ‘All wells’ in the
range must meet the criteria.
Choose an operator: >, >=, <, or <=.
Enter the measurement value to be met. For Absorbance protocols, the valid range
is -1.000 to 5.000. For Fluorescence and Luminescence protocols, the valid range is
0 to 99998 (FLx800™ and Synergy™ HT) or 99999 (FL600™).
•
If desired, enter a Delay Between Readings of the monitored well(s), in HH:MM:SS
format. The default setting of 00:00:00 allows the well(s) to be read as quickly as
possible.
Multi-Mode
(Only applies to the Synergy™ HT line of instruments.)
Synergy HT instruments with basecode versions of 2.10 or higher support KC4’s Multi-Mode
detection method option. When Multi-Mode is selected, more than one detection method can be
defined within a single protocol.
When Multi-Mode is enabled, the following Reading parameters are supported:
•
End Point reading type with up to 6 wavelengths or filter sets
•
Kinetic reading type with up to 6 wavelengths or filter sets
•
Time-Resolved Fluorescence (HTTR)
•
Pre-Read Blank Plate
•
Monitor Wells
•
Automatic Sensitivity Adjustment
The following Reading parameters are not supported:
•
Rapid and Sweep read modes (absorbance reads are always performed in Normal mode)
•
Spectrum reading type
•
Area scanning
•
Pathlength correction
•
Absorbance wavelength calibration (via the Reading Parameters dialog and before
performing the read)
•
Eject between filter set reads
•
Read & Dispense
108 • Protocol Menu
User’s Guide to KC4™
If dispensing is required during a kinetic read, here are two options:
Manual Dispense:
Create a protocol with a long kinetic interval. During an interval, when the reader is idle:
Push the carrier eject button on the front of the reader to eject the plate
Perform the dispense using a pipette
Push the carrier eject button to draw the plate back in
The read will continue as scheduled
Reader Dispense:
(Injector model only)
Create and run a total of three protocols:
A Kinetic protocol to start the read. Save results in a .PLA file.
A “Dispense-only” protocol to dispense the reagent.
A second Kinetic protocol to complete the kinetics after dispense. Note: Reading
parameters in the second protocol must be identical to the first, although kinetic
settings can differ.
Gather the results in a single .PLA file using KC4’s Append to Kinetic File feature.
Pathlength Correction
Check Pathlength Correction to normalize absorbances to 1 cm. This option is available for
readers that support multiple wavelength reads up to 999 nm.
If Pathlength Correction is enabled, KC4:
1.
Calculates the pathlength for each well.
2.
Calculates the corrected absorbance for each well.
3.
Reports the Corrected Data after Pathlength Correction is applied.
Pathlength Calculation
KC4 calculates each well’s pathlength according to the following formula:
(A977 nm - A900 nm)well
(A977 nm - A900 nm)1.0 cm water
•
The well is measured at 977 nm and again at 900 nm. KC4 calculates the difference in
measurements, then divides that result by the absorbance of water at 1 cm. Note: 977
nm and 900 nm represent default Test and Reference Wavelengths, respectively. These
wavelengths can be changed by clicking the Options button.
User’s Guide to KC4™
Protocol Menu • 109
•
KC4 uses a default value of 0.18 for the absorbance of water at 1 cm. This value can be
changed by clicking Options and defining a different Absorbance of Solution at 1 cm.
Alternatively, click Bio-Cell to calculate the absorbance of water at 1 cm value using
Bio-Tek’s Bio-Cell plate. Use the drop-down menu to select the location of the
cuvette in the plate.
Corrected Absorbance Calculation
KC4 calculates the corrected absorbance in each well according to the following formula:
(Awavelength)well
pathlength of well
•
If a Blank Plate is pre-read, and/or if blank subtraction is performed, select the
advanced protocol option Raw Data Correction to control the data used in this formula.
Pathlength Correction Options
Click the Options button to change the Test or Reference Wavelength values, or to change the
pathlength constant.
•
The valid range for the Test and Reference Wavelength fields is 200 to 999 nm.
•
The default Absorbance of solution at 1 cm value is 0.18. To use a different constant
value when calculating pathlengths, simply enter the desired value.
Reporting the Pathlength Values
KC4 automatically reports the Corrected Data after Pathlength Correction is applied. You can
easily create a Multi Plate Transformation to report the Pathlength for each well on the plate.
Look in the Help system contents under Results|Data Sets|Pathlengths for more information.
110 • Protocol Menu
User’s Guide to KC4™
Plate Type
Important! Be sure to select the appropriate Plate Type. Just prior to plate reading, KC4 sends
the plate’s dimensions to the instrument. This information guides the optical probe(s) to the
correct reading positions relative to the microplate. Choosing an inappropriate plate type could
adversely affect results.
•
Select a Plate Type from the drop-down menu. The items presented for selection come
from the plate database, which is maintained under System|Plate Formats.
•
The First Well and Last Well fields default to include the entire plate. You can change
one or both settings to read a partial plate.
Pre-Read Blank Plate
Check Pre-Read Blank Plate to perform two reads at each defined wavelength. The first read is
performed on an empty (or blank) plate, and the second on the sample plate. The "Blank Plate"
can represent a dry empty plate or a plate filled with solution.
•
For Endpoint and Kinetic reads, the blank plate is read at each wavelength or filter set
defined in the protocol.
•
For Spectrum reads, the blank plate is scanned using the wavelength range defined in the
protocol.
•
This option is not available if Scanning is enabled.
The Blank Plate data is subtracted from the actual microplate data if the Blank Plate Subtraction
option is selected in the Raw Data Corrections dialog (an advanced protocol option).
Read Barcode
If the attached reader is equipped with an internal plate barcode scanner, use the Read Barcode
feature to read the code when the plate is read. If for any reason the barcode cannot be read, you
will be prompted to manually enter the plate barcode.
See Protocol Options at the end of this chapter to find out how to use the scanned or entered
barcode as the data file name.
User’s Guide to KC4™
Protocol Menu • 111
Read & Dispense
The Synergy™ HT with Injectors provides the ability to dispense fluid into microwells as part of a
“Read & Dispense” protocol. To enable this feature, click the Dispense checkbox. To define the
Dispense, Shake, and/or Read steps, click Read & Dispense.
See “Creating a Read & Dispense Protocol” starting on page 119 for detailed instructions.
Read Mode
Some reader models support multiple Read Modes for absorbance protocols. The selection of one
read mode over another generally depends on the desired outcome of the assay (better precision
versus faster plate reading). The available read modes are:
•
Normal mode is the slowest of the three available modes. After positioning the well over
the probe, the instrument waits 100 milliseconds before taking the measurement. This
delay is to allow for more complete fluid settling.
•
Rapid mode is faster than Normal mode because the instrument does not wait before
taking the measurement.
•
Sweep mode is the fastest of the three available modes; the instrument does not stop
before taking measurements.
Reader/Read Method
The Reading parameters dialog displays the name of the currently selected Reader and the
computer port to which it is attached. (This information is configured under System|Readers.)
The available Read (or Detection) Methods are reader-dependent. If more than one option is
presented, select the desired method by clicking its radio button. This selection instructs KC4 to
represent measured values as optical densities (OD), relative fluorescence units (RFU), or relative
luminescence units (RLU). It also customizes the Reading parameters dialog to present further
options specific to the read method.
112 • Protocol Menu
User’s Guide to KC4™
Reading Direction
Some readers also support a Reading Direction setting. Choose to read in a horizontal direction
(A1-A12, B1-B12, … H1-H12), or in a vertical direction (A1-H1, A2-H2, … A12-H12).
•
The faster method for reading a full plate is Horizontal.
•
The faster method for reading a single column is Vertical.
Reading Type
Select a Reading Type before defining additional Reading parameters. The Reading Type options
Endpoint and Kinetic are available for all readers supported by KC4. For certain readers, the
Spectrum reading type is also available.
•
During an Endpoint Read, every well on the plate is read with one, two, or multiple
wavelengths.
If supported by the current reader, the Scanning feature is available for endpoint
protocols. Click the Scanning… button to define linear or area scanning parameters.
•
During a Kinetic Read, every well on the plate is read a user-specified number of times
with one, two, or multiple wavelengths. Click the Kinetic… button to define kinetic
reading parameters.
•
During a Spectrum Read, multiple readings are taken across a wavelength range. The
objective is to plot a graph with absorbance or transmittance versus wavelength. Click
the Scales… button to customize the graphical data displays (optional).
•
The Reading Type options for the Synergy™ HT with injectors contains an additional
Dispense checkbox, to enable the fluid dispense option.
User’s Guide to KC4™
Protocol Menu • 113
Scales for Kinetic Reads
By default, the Scales parameters automatically calculate an appropriate box size to display the
kinetic reactions. The default values can be manually overridden by deselecting one or both Auto
checkboxes.
•
Absorbance reads: min t is automatically set to a zero time value, min y is set to
-1.0000 OD, max t is set to the Run Time value, and max y is set to 5.0000 OD.
•
Fluorescence and luminescence reads: min t and min y are automatically set to zero
values, max t is set to the Run Time value, and max y is set to 99999 RFU/RLU (FL600),
or to 99998 RFU/RLU (FLx800™ and Synergy™ HT).
•
Individual Well Auto Scaling individually adjusts the min/max t and min/max y settings
for each well to best display its curve. It overrides the settings of the min/max t and
min/max y.
Note: This setting only affects the scale of an individual zoomed well. The kinetic
curves display, showing all wells on the plate, is not affected.
Scales for Spectrum Reads
By default, the Scales parameters automatically calculate an appropriate box size to display the
spectrum curves. The default values can be overridden manually to change the scaling of the x
and y axes.
If the Spectrum reading type is selected in the Reading dialog, click the Scales… button to open
the Scales dialog:
•
The Scales parameter defaults are set as follows:
min w is set to the Start Wavelength
max w is set to the Stop Wavelength
min y is set to -1.0000 OD
max y is set to 5.0000 OD
The default values can be manually overridden by deselecting the Auto checkboxes.
•
Individual Well Auto Scaling individually adjusts the min/max w and min/max y
settings for each well to best display each well’s curve. It overrides the settings of the
min/max w and min/max y.
114 • Protocol Menu
User’s Guide to KC4™
Scanning
If supported by the current reader, the Scanning feature is available for Endpoint reads. Some
readers support Linear scanning, others support Area scanning:
•
When performing a Linear Scan, the reader takes multiple measurements in a line across
the center of each well. Linear scanning allows you to observe a pattern that may be
present in the well bottom, such as an agglutination pattern. Readers that support Linear
scanning include the ELx808™ and all PowerWave™ models. Note for PowerWaveX™
Select: Linear scanning is supported for the 96-well plate type only.
•
When performing an Area Scan, the reader takes multiple measurements down and
across each well, in a “matrix” format. Area scanning more accurately assesses cellular
assays than reading once in the center of the well. Readers that support area scanning
include the ELx800™, µQuant™, FLx800™, and Synergy™ HT.
•
Note: If the Scanning button is dimmed and inaccessible, well scanning cannot be
performed with the currently defined plate type. This may be due to a hardware
limitation or an unacceptable combination of optic probe size and well diameter.
•
Click the Scanning… button to define the measurement points:
With Linear scanning:
•
The Horizontal Reading Points setting represents the total number of points to be read
across the center of each well. Valid entries are odd integers from 1 to 31.
•
Individual Well Auto Scaling individually adjusts the min/max x and min/max y settings
for each well to best display its scanning curve. If enabled, this feature overrides the
settings of the min/max x and min/max y.
Note: This feature only affects the scale of an individual zoomed well. The scanning
curves display, showing all wells on the plate, is not affected.
User’s Guide to KC4™
Protocol Menu • 115
With Area scanning:
•
Read Matrix Size represents the number of measurements taken across and down each
well. If, for example, the Read Matrix Size is 5 x 5 a total of 25 measurements are taken.
•
Individual Well Color Auto Scaling determines how the color palette is built when the
measurement points are graphically displayed. On color monitors and printers, blue
shades represent the lowest measurement values, purple shades represents mid-range
measurement values, and red shades represent highest measurement values. On blackand-white monitors and printers, white represents the lowest measurement values,
increasing shades of gray represent increasing measurement values, and black represents
the highest measurement values. Tip: If the plate is likely to yield values ranging from
very low to very high, enable Individual Well (Color) Auto Scaling to help with the
visual differentiation between points within each well.
Shaking
If plate shaking is available with the currently selected reader, the Shaking parameters are
accessible.
•
Endpoint / Spectrum protocols: Shaking occurs immediately before the plate is read.
•
Kinetic protocols: Shaking can occur before the first reading only, or before every
reading.
•
Intensity: Acceptable values include:
0
1
2
3
4
No Shaking
Low
Medium
High
Variable (not supported by all readers that support shaking)
•
Duration: Defines the absolute time period for shaking, in seconds.
•
Before First / Before Every Reading: Defines when the shaking will occur. For non-
kinetic reads, shaking occurs before the first reading. For kinetic reads, shaking can
occur before the first read or before every read.
116 • Protocol Menu
User’s Guide to KC4™
Temperature Control
For readers that support computer-controlled incubation, the Temperature Control parameters are
accessible:
•
Select Yes to indicate plate incubation.
•
Select No to indicate no incubation (default).
•
Check Pre-Heating to pre-heat the incubator before the plate carrier is drawn in.
•
Enter the desired set point Temperature in °C.
•
Define the Lag Time to elapse before the reader initiates the first reading cycle. The
reader draws the plate carrier into the reading chamber, waits for the Lag Time to elapse,
then reads the plate.
Lag Time is available for all reader models, including those that do not support
incubation.
The valid entry range for most readers is 00:00:00 to 00:59:59 (HH:MM:SS). For
the FL600™, the range is 00:00:00 to 08:00:00.
Time-Resolved Fluorescence (Synergy™ HT)
If supported by the reader, the Time-Resolved option is available when the selected detection
method is Fluorescence. If this option is enabled, additional measurement mode parameters
become available in the Measurement Options dialog (accessed by clicking the Option button
located in the Filters box), to specify the length of time to delay before collecting readings and the
length of time during which readings will be taken.
Top Probe Vertical Offset
See “General Options (Synergy HT).”
User’s Guide to KC4™
Protocol Menu • 117
Wavelengths
For readers equipped with a monochromator (e.g., PowerWave™, PowerWaveX™, µQuant™,
Synergy™ in Absorbance mode), the Wavelengths set of parameters is available within the
Reading parameters dialog.
For Endpoint and Kinetic reads:
•
Click the radio button associated with the desired number of wavelengths.
•
Click
to open a list of available wavelengths, then click on a wavelength to select it.
If the desired wavelength is missing from the list, type the value directly into the
wavelength field.
•
(Optional) If the reader is connected, clicking Calibrate will update KC4’s wavelength
table and the reader’s internal wavelength table.
If KC4’s wavelength table already matches the reader’s wavelength table, calibration is
not necessary.
If calibration is necessary but the reader is not connected right now, calibration will
perform automatically when a plate read begins.
For Spectrum reads:
•
Define the wavelength range by entering the Start and Stop values, in nanometers.
•
Define the reading interval by entering the Step value, in nanometers. For example, if
the Start, Stop, and Step values are 300, 600, and 50 nm, respectively, the plate will be
read at 300, 350, 400, 450, 500, 550, and 600 nm.
•
(Optional) Select Calibrate:
If Calibrate is selected, the reader will always perform calibration at the wavelengths
specified in the protocol, just prior to plate reading.
If Calibrate is not selected, the reader will calibrate at only those wavelengths specified in
the protocol that have not yet been calibrated since the reader was turned on.
118 • Protocol Menu
User’s Guide to KC4™
Creating a Read & Dispense Protocol
The Synergy™ HT with injectors provides the ability to dispense fluid into microwells as part of a
“Read & Dispense” protocol. To enable this feature, begin by clicking the Dispense checkbox
within the Reading parameters dialog.
•
Note: The Dispense option is not available if Multi-Mode is selected.
•
Define or select additional Reading parameters within this dialog, including Reading
Type, Filter Set(s), Plate Type, and Temperature Control.
•
Click the Read & Dispense button to open the Read & Dispense dialog. Use this dialog
to define the Dispense, Shake, and/or Read steps for this protocol.
•
Select a Read Mode:
If Plate mode is selected, each step in the sequence is performed on all wells of the
plate, step after step. For example, if the step sequence is Dispense / Read, fluid is
dispensed into all wells of the plate and then the wells are read. Timings are
generally long.
User’s Guide to KC4™
Protocol Menu • 119
If Well mode is selected, the full sequence of steps is performed on each well, well
after well. For example, if the step sequence is Dispense / Read, fluid is dispensed
into well A1 and the well is read, fluid is dispensed into well A2 and the well is read,
and so on. Timings are generally short (fast).
•
The Time is approximate indication at the bottom of this dialog means that Start and
End times displayed with an asterisk (*) were calculated using appromixation logic. KC4
uses approximation logic when:
A step is added with “Shortest, after…” selected.
The reader is not communicating with KC4.
The reader is unable to validate the Read & Dispense settings.
In addition, the asterisk will appear if the reader provided a time that can vary based on
other reader settings that may change before the protocol is executed (e.g., injector
position).
Plate Mode Options
The Dispense, Shake, and Read steps are all available with Plate Mode. In addition, the Timing
Control feature is available.
•
Timing Control is enabled by default. To disable Timing Control, check the box.
When Timing Control is enabled, all steps are processed in the same way with regard to
timing:
Incubation time is exactly the same for each well.
Dispense and Read steps are performed at the same pace.
When Timing Control is disabled, all steps are performed as quickly as possible:
Incubation time may vary across the microplate.
Dispense and Read steps may be performed at different speeds.
120 • Protocol Menu
User’s Guide to KC4™
•
Click the Dispense, Shake, and Read buttons to create new steps.
Up to nine (9) steps may be defined.
Steps cannot overlap. They must be performed one after another.
With Endpoint protocols, only one Read step may be defined.
Endpoint protocols do not require a Read step (the protocol can be “Dispense-only”).
Kinetic protocols require at least one Read step. Note: If the protocol contains two
or more Read steps, the Kinetic Interval is represented as ‘Uneven’ on reports.
A Kinetic Read step with Shake During Read enabled counts as two steps.
•
The steps are listed in the order in which they will be executed.
The Description column provides key information about the step, such as volume,
rate, or intensity.
The Start column indicates the time the step will begin.
The End column indicates the time the step will complete.
The Comment column contains additional information about the step, such as tip
prime settings.
To edit a step, highlight it and double-click or click the right mouse button and select
Edit. To delete a step, highlight it and press the Delete key or click the right mouse
button and select Delete.
•
The steps are also displayed graphically to show how they will be executed on the plate
over time.
Dispense step: The solid area represents the action on the first well. Cross hatches
represent the time necessary to process other wells on the plate.
Shake step: The solid area represents the action on the entire plate.
Read step: The solid area represents the action on the first well (and for each kinetic
interval, if this is a kinetic protocol). Cross hatches represent the time necessary to
process other wells on the plate.
Time indicators:
A Process time indicates the time at which a particular step will begin, relative to
the start time of the very first step in the operation. Process times are displayed in
grey (light) text.
(Kinetic protocols only.) A Kinetic time indicates the time at which a reading
begins, relative to the start time of the very first read in the operation (which is
always 00:00:00). Kinetic times are displayed underneath the Process times in black
(dark) text. Note that in a kinetic protocol, each read step will have a Process time
and a Kinetic time associated with it.
User’s Guide to KC4™
Protocol Menu • 121
Dispense Step Parameters
Use this dialog to create or modify a Dispense step:
•
Select the number of the Dispenser to be used for this step. The numbers available for
selection correspond to the numbers on the dispense box.
•
Select a Tip Prime option. Tip priming is performed in a small, removable priming
trough located in the left rear. The purpose of tip priming is to compensate for any fluid
loss at the dispense tip due to evaporation since the last dispense. The trough holds up to
1 ml of liquid and must be periodically emptied and cleaned by the user. KC4 must then
be “told” that the trough has been emptied (via the System > Reader Control dialog). If
the trough overflows, refer to the Synergy™ HT Operator’s Manual for decontamination
instructions.
None: No prime.
Before the process: Prime the tip with fluid once before the entire process begins.
Before the dispense: Prime the tip with fluid before each dispense to a well.
•
If tip priming will be performed, enter the Volume in microliters. The valid range is 5 to
20. The dispense rate will be the same as that selected for the well dispense.
•
Set the Start Time for the step. Enter the time manually (HH:MM:SS), or check
Shortest and select the step immediately after which this Dispense step shall be
performed.
•
Enter the dispense Volume, in microliters. The valid range is 5 to 1000.
•
Select the dispense Rate in microliters per second. The default rate is the maximum
available for the attached instrument.
•
Click OK to save the settings and add the Dispense step to the list.
122 • Protocol Menu
User’s Guide to KC4™
Shake Step Parameters
Use this dialog to create or modify a Shake step:
•
Set the Start Time for the step. Enter the time manually (HH:MM:SS), or check
Shortest and select the step immediately after which this Shake step shall be performed.
•
Specify the shake Intensity. Acceptable values include:
1
2
3
4
Low
Medium
High
Variable
•
Specify the shake Duration in seconds.
•
Click OK to save the settings and add the Shake step to the list.
•
Note: Shaking can also be defined as a part of the Read step if repeated shaking is
required between each kinetic reading interval.
Read Step Parameters
Use this dialog to create or modify a Read step:
•
Set the Start Time for the step. Enter the time manually (HH:MM:SS), or check
Shortest and select the step immediately after which this Read step shall be performed.
•
Kinetic protocols only:
Specify the Run Time. This is the length of time from the start of the first kinetic
read to the start of the last kinetic read.
User’s Guide to KC4™
Protocol Menu • 123
Specify the time Interval for repeat reading of the plate, section of wells, or
individual well. The maximum valid Interval is equal to the current Run Time.
KC4 automatically calculates the Reading Number by dividing the Run Time by the
Interval and adding 1.
If Shaking before each kinetic read is required, specify the shake Intensity and then
specify the shake Duration in seconds (a Duration of 0 means no shaking will
occur).
Note: Shaking can also be defined as an individual step outside of the Read step,
using the Shake button in the Read & Dispense dialog.
Acceptable Intensity values include:
1
2
3
4
•
Low
Medium
High
Variable
Click OK to save the settings and add the Read step to the list.
Well Mode Options
The Dispense and Read steps are available with Well Mode. The Timing Control feature is not
available.
•
Click the Dispense and Read buttons to create new steps.
Up to nine (9) steps may be defined.
Shaking is not permitted in Well Mode.
Endpoint protocols require one Read step.
124 • Protocol Menu
User’s Guide to KC4™
Kinetic protocols require at least one Read step. Note: If the protocol contains two
or more Read steps, the Kinetic Interval is represented as ‘Uneven’ on reports.
Dispense steps cannot overlap. (Only one dispenser can be in use at a time.)
A Read step can overlap a Dispense step. For example, the dispense may occur
during a kinetic read, to show the rate of change immediately following the injection
of fluid. (This can also be accomplished by creating two Read steps, one
immediately before and one immediately after the Dispense step).
•
The steps are listed in the order in which they will be executed.
The Description column provides key information about the step, such as volume,
rate, or intensity.
The Start column indicates the time the step will begin.
The End column indicates the time the step will complete.
The Comment column contains additional information about the step if needed.
To edit a step, highlight it and double-click or click the right mouse button and select
Edit. To delete a step, highlight it and press the Delete key or click the right mouse
button and select Delete.
•
The steps and are also displayed graphically to show how they will be executed on each
well over time. Dispenser #1, Dispenser #2, and Read steps are shown in parallel.
Dispense step: The solid area represents the execution of the Dispense for one well.
Read step: The solid area represents the execution of the Read step for one well.
Time indicators:
A Process time indicates the time at which a particular step will begin, relative to
the start time of the very first step in the operation. Process times are displayed in
grey (light) text.
(Kinetic protocols only.) A Kinetic time indicates the time at which a reading
begins, relative to the start time of the very first read in the operation (which is
always 00:00:00). Kinetic times are displayed underneath the Process times in black
(dark) text. Note that in a kinetic protocol, each read step will have a Process time
and a Kinetic time associated with it.
User’s Guide to KC4™
Protocol Menu • 125
Dispense Step Parameters
Use this dialog to create or modify a Dispense step:
•
Select the number of the Dispenser to be used for this step. The numbers available for
selection correspond to the numbers on the dispense box.
•
Select a Tip Prime option. Tip priming is performed in a small, removable priming
trough located in the left rear of the carrier. The purpose of tip priming is to compensate
for any fluid loss at the dispense tip due to evaporation since the last dispense. The
trough holds up to 1 ml of liquid and must be periodically emptied and cleaned by the
user. KC4 must then be “told” that the trough has been emptied (via the System > Reader
Control dialog). If the trough overflows, refer to the Synergy™ HT Operator’s Manual
for decontamination instructions.
None: No prime.
Before the process: Prime the tip with fluid once before the entire process begins.
Before each well: Prime the tip with fluid before dispensing to each well.
•
If tip priming will be performed, enter the Volume in microliters. The valid range is 5 to
20.
•
Set the Start Time for the step. Enter the time manually (SSSSS.sss), or check Shortest
and select the step immediately after which this Dispense step shall be performed.
•
Enter the dispense Volume, in microliters. The valid range is 5 to 1000.
•
Select the dispense Rate in microliters per second. The default rate is the maximum
available for the attached instrument.
•
Click OK to save the settings and add the Dispense step to the list.
126 • Protocol Menu
User’s Guide to KC4™
Read Step Parameters
Use this dialog to create or edit a Read step:
•
Enter the Start Time in seconds (sssss.sss).
•
Kinetic protocols only:
Specify the Run Time. This is the length of time from the start of the first kinetic
read to the start of the last kinetic read.
Specify the time Interval for repeat reading of the plate, section of wells, or
individual well. The maximum valid Interval is equal to the current Run Time.
KC4 automatically calculates the Reading Number by dividing the Run Time by the
Interval and adding 1.
•
(Optional) For non-TR Fluorescence reads, check Block light between reads to block
the light between measurements to inhibit photo bleaching.
Single filter set protocols: If light blocking is enabled, a blocking filter (Plug)
must be placed in the excitation filter wheel in one of the two positions next to the
excitation filter that is specified in the protocol.
Dual filter set protocols: If light blocking is enabled, two blocking filters (Plugs)
must be placed next to each other in the excitation filter wheel.
•
Click OK to save the settings and add the Read step to the list.
User’s Guide to KC4™
Protocol Menu • 127
Raw Data Correction
Raw data, or the optical density or fluorescence unit values retrieved from a microplate reader, can
be corrected using Blank Plate Subtraction, Blank Wells Subtraction, and/or Pathlength
Correction. The advanced protocol option Raw Data Correction controls the use of these
correction options.
•
Blank Plate Subtraction is accessible if "Pre-Read Blank Plate" is selected in the
Reading parameters dialog.
•
Blank Wells Subtraction is accessible if one or more blank wells are defined under Plate
Layout.
•
Pathlength Correction is accessible if it is selected in the Reading parameters dialog.
•
Raw Data Correction calculations are performed in the order Blank Plate Subtraction,
Blank Wells Subtraction, then Pathlength Correction.
The following example describes the plate processing and calculation steps performed when a
protocol specifies Pre-Read Blank Plate, Pathlength Correction with Bio-Cell, and the plate is
configured with blank wells. This example is for explanation only, the typical application does
not use all three raw data correction options.
Processing steps:
1.
The Blank Plate is read at every wavelength defined in the Reading parameters dialog.
2.
The Bio-Cell is read at 977 nm and 900 nm.
3.
The Sample Plate is read at 977 nm and 900 nm.
4.
The Sample Plate is read at every wavelength defined in the Reading parameters dialog.
Calculation steps performed for each wavelength defined in the Reading parameters dialog:
1.
The basis of calculations begins with the raw Sample Plate readings.
2.
The Blank Plate readings are subtracted from the Sample Plate readings. The result of
this calculation becomes the basis for step 3.
3.
The mean of the blank wells is calculated and subtracted from every well on the plate.
The result of this calculation becomes the basis for step 4.
4.
The value in each well is multiplied by the difference in readings of the Bio-Cell at 977
nm and 900 nm. The result of this calculation becomes the basis for step 5.
5.
The value in each well is divided by the difference in readings of the Sample Plate at 977
nm and 900 nm. The result of this calculation is the final corrected value.
128 • Protocol Menu
User’s Guide to KC4™
The image below shows the data sets available following a three-wavelength read with Blank
Plate Subtraction, Blank Wells Subtraction, and Pathlength Correction enabled:
•
M 977 (SP) and M 900 (SP) contain the values of the Sample Plate read at 977 nm and
900 nm, respectively (necessary for pathlength correction calculation).
•
•
Measurement numbers (M#1, M#2, M#3) correspond with the wavelength selection order
in the Reading parameters dialog.
M#1 405, M#2 450, and M#3 630 contain the raw Sample Plate values at 405 nm, 450 nm,
and 630 nm.
•
M#1 405 Blank, M#2 450 Blank, and M#3 630 Blank contain the values calculated by
subtracting the Blank Plate readings at the specified wavelength from the raw Sample
Plate readings at the specified wavelength. These values do not represent Blank Well
Subtraction.
•
M#1 405 Corr., M#2 450 Corr., and M#3 630 Corr. contain the final values after all Raw
Data Correction options have been applied.
Blank Plate Subtraction
With most readers, the Pre-Read Blank Plate option is available within the Reading parameters
dialog. If this option is selected, a blank plate is read before the sample plate, at the wavelengths
defined in the protocol. KC4 then calculates the difference in measurements between the two sets
of data (sample - blank). Note: The blank plate can represent an empty dry plate or a plate filled
with solution for blanking.
If Blank Plate Subtraction is enabled:
•
Endpoint read: For each wavelength defined in the protocol, KC4 subtracts the value of
each well of the blank plate from the corresponding well of the sample plate.
•
Kinetic read: For each wavelength defined in the protocol, within each kinetic read, KC4
subtracts the value of each well of the blank plate from the corresponding wells of the
sample plate.
•
Spectrum read: At each wavelength read within the defined spectrum range, KC4
subtracts the value of each well of the blank plate from the corresponding well and
wavelength of the sample plate.
User’s Guide to KC4™
Protocol Menu • 129
Blank Wells Subtraction
Within any protocol, if one or more wells are defined as ‘Blank’ under Plate Layout, the Blank
Wells Subtraction Raw Data Correction option automatically becomes enabled. When this option
is enabled:
•
Endpoint read: For each wavelength defined in the protocol, the average of the blank
well(s) is subtracted from every well on the plate.
Note: For readers that do not accept multiple (> 2) wavelengths, if a dual-wavelength
read is defined, KC4 automatically calculates the Delta OD first. The average of the
Delta OD values in the blank wells is then subtracted from every well on the plate.
•
Kinetic read: For each wavelength defined in the protocol, within each kinetic read, the
average of the blank wells is subtracted from every well on the plate.
Note: For readers that do not accept multiple (> 2) wavelengths, if a dual-wavelength
read is defined, KC4 automatically calculates the Delta OD first. Within each kinetic
read, the average of the Delta OD values in the blank wells is then subtracted from every
well on the plate.
•
Spectrum read: A blank average is calculated for each wavelength in the spectrum
reading range. The blank average for each wavelength is then subtracted from the
absorbance read at the corresponding wavelength in each well.
Pathlength Correction
With readers that support multiple-wavelength reads up to 999 nm, the Pathlength Correction
option is available within the Reading parameters dialog. If this option is selected, absorbances
are normalized to 1 cm.
If Pathlength Correction is enabled, KC4 first calculates the pathlength for each well. The
absorbance in each well is then divided by the pathlength, resulting in the corrected absorbance.
See “Pathlength Correction” in the Reading Parameters Reference section to learn more.
130 • Protocol Menu
User’s Guide to KC4™
Kinetic Analysis
Use Kinetic Analysis to select pre-data reduction parameters for kinetic reads.
This option becomes available within the Protocol menu when the Kinetic read method is selected.
Data to Process
This option allows the selection of the type of data to be analyzed, i.e., the measured response
value for kinetic analysis. The drop-down list displays the available data types.
Typical uses of this feature:
•
To blank data prior to the kinetic slope calculation. First, define one or more blank wells
in the Plate Layout, and make sure Blank Wells Subtraction is enabled in the Raw Data
Correction dialog. Then, select ‘<wavelength> Corr.’ from the Kinetic Analysis Data to
Process pull-down list.
•
With some readers it is possible to perform kinetic reading with multiple filters or
wavelengths. In this case, two levels of Data to Process options can be selected (e.g.,
‘M#1 405’ and ‘M#2 550’) to perform and compare kinetic calculations.
Note: When two data sets are selected, the kinetic curves are automatically overlaid in
the kinetic graphs and in the kinetic zooms.
User’s Guide to KC4™
Protocol Menu • 131
Mean V
Mean V is the calculated value of the mean slope. KC4 determines the mean slope by calculating
a linear regression on points that are in the calculation zone. To set the calculation zone, select
Mean V, then click on Calculation Options.
Calculation Zone
Typically, the Calculation Zone is adjusted to ignore erroneous data point(s) generated at the
beginning of a kinetic assay due to assay "noise".
•
From t / To t : Defines a calculation zone based on time. The default values span the
defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of reads. The default values
include all reading points.
Units
•
mOD or OD dependent on kinetic dynamic range.
•
Per sec or Per sec2 dependent on rate order.
•
Per min or Per min2 dependent on rate order.
•
Per hour or Per hour2 dependent on rate order.
Max V
With Max V, the calculated value is the maximum slope. KC4 calculates the maximum slope as
follows:
•
Starting at the first point in the calculation zone, KC4 evaluates n points and calculates
the slope among these n points.
•
Next, KC4 repeats the operation, starting at the second point in the calculation zone, and
repeats again, starting at the third point, and so on.
•
KC4 then compares all calculated slopes to determine the maximum slope.
•
KC4 will register the Delta t time in the middle of the point where the V max is
calculated.
132 • Protocol Menu
User’s Guide to KC4™
KC4 also calculates the kinetic Lag Time, which is the time intercept between the line of
maximum slope of the propagation phase and the absorbance baseline at time = 0, as illustrated
below:
By default, the calculation zone starts at time zero. To change the calculation zone, select Max V,
then click Calculation Options.
Calculation Zone
Typically, the Calculation Zone is adjusted to ignore erroneous data point(s) generated at the
beginning of a kinetic assay due to assay “noise”.
•
From t / To t : Defines a calculation zone based on time. The default values span the
defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of reads. The default values
include all reading points.
User’s Guide to KC4™
Protocol Menu • 133
V calculation on n points
•
This option defines the number of successive points to be included in the calculation of
the maximum slope.
•
The minimum number of points is 2. The maximum number of points is the total number
of points within the calculation zone.
•
Only the points defined within the calculation zone are used.
Units
•
mOD or OD dependent on kinetic dynamic range.
•
Per sec or Per sec2 dependent on rate order.
•
Per min or Per min2 dependent on rate order.
•
Per hour or Per hour2 dependent on rate order.
Mean Min/Max
The Mean Min OD is the mean minimum OD based on n points. The Mean Max OD is the mean
maximum OD based on n points. KC4 calculates the Mean Min and Mean Max ODs as follows:
•
Starting at the first point in the calculation zone, KC4 evaluates n points and calculates
the mean among these n points.
•
Next, KC4 repeats the operation, starting at the second point in the calculation zone, and
repeats again, starting at the third point, and so on.
•
KC4 then compares all of the calculated means to determine the minimum and maximum
of these values.
•
KC4 also calculates the Delta t at Min/Max OD.
•
Note: For fluorescence and luminescence reads, KC4 performs calculations based on
fluorescence units (Mean Min/Max RFU) and luminescence units (Mean Min/Max RLU),
respectively.
To set the calculation zone, select Mean Min/Max OD, then click Calculation Options.
134 • Protocol Menu
User’s Guide to KC4™
Calculation Zone
Typically, the Calculation Zone is adjusted to ignore erroneous data point(s) generated at the
beginning of a kinetic assay due to assay "noise".
•
From t / To t : Defines a calculation zone based on time. The default values span the
defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of reads. The default values
include all reading points.
Mean Min/Max on n points
•
Selects the number of points to be included in the average calculation.
Onset OD / Onset OD %
The calculated value is the time when the OD reaches a set decrease/increase (Onset OD), that can
be defined by an absolute value or a relative value (%), compared with a reference OD (the Basis
Value). You can either manually enter this value, or have KC4 calculate it (mean of the n first
points). By default n=1, thus the Basis Value is the OD of the first point.
•
KC4 returns the time for each well to pass the Onset OD value.
•
Note: For fluorescence and luminescence reads, KC4 performs calculations based on
fluorescence units (Onset RFU) and luminescence units (Onset RLU), respectively.
To set the calculation zone, select Onset OD or Onset OD %, then click Calculation Options.
Basis Value Calculation
•
Mean of the n first points: For relative determination of the Basis Value.
•
OD = : For absolute determination of the Basis Value.
Examples:
Onset OD=0.5, Basis Value=Mean of first 2 points:
KC4 calculates the time that is required for the OD to increase by 0.5 OD, compared with
the mean of the 2 first points of the kinetics.
Onset OD=15%, Basis Value=Mean of the first point:
KC4 calculates the time that is required for the OD to increase by 15%, compared with
the first point of the kinetics.
Onset OD=1.6, Basis Value=0:
KC4 calculates the time that is required for the OD to reach the value of 1.6 (Basis Value
+ Onset OD).
User’s Guide to KC4™
Protocol Menu • 135
Formula
Select Formula to define a formula to calculate a value using data from individual reading points
from the same well. Designate an individual reading point using the identifier Rn, where n
represents the reading number, then enter a Result Name in which to store the formula results.
This name can be used as a Data-In setting in Transformations. Formula examples:
•
The formula R10-R5 calculates the difference in readings between data points 10 and 5.
•
The formula R# (e.g., R1) extracts a single reading from each well for endpoint data
reduction.
No Calculation
This option, if selected, overrides any calculations on the kinetic data, and allows the display of
kinetic graphics without data reduction.
•
No Calculation is typically used to display overlaid curves.
Integral
Select Integral to calculate the area under the curve according to the trapezoidal method, shown
below:
i=n–1
Integral =
∑
(( yi + yi+1 ) / 2 ) × ( xi+1 – xi )
i=1
Where y = measurement value and x = read point value.
By default, the calculation zone spans the full Run Time and includes all of the reading points
defined in the protocol. Click Calculation Options to change the calculation zone:
•
From t / To t : Defines a calculation zone based on time. The default values span the
defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of reads. The default values
include all reading points.
136 • Protocol Menu
User’s Guide to KC4™
Spectrum Analysis
KC4 provides the option for performing additional calculations on spectral data. This is available
for Spectrum reading types only.
Open the Spectrum Analysis dialog by selecting Protocol|Spectrum Analysis:
Data to Process
Select up to two levels of data to be used in calculations.
•
When two data sets are selected, the spectrum curves are automatically overlaid in the
spectrum graphs and in the spectrum zooms.
Min/Max
Select Min/Max to determine the minimum and maximum values for the points defined within the
calculation zone. This is the default setting. Upon completion of a read, the following is reported
for each well:
•
Minimum OD and Wavelength at Minimum OD
•
Maximum OD and Wavelength at Maximum OD
If the Calculation Type is set for Min/Max, certain calculation parameters may be customized.
Access the Spectrum Analysis - Min/Max dialog by clicking the Calculation Options button.
User’s Guide to KC4™
Protocol Menu • 137
•
From w and To w allow the specification of a range of wavelengths upon which to
perform calculations. The default values correspond with the Start and Stop values in
the Protocol|Reading Parameters dialog.
•
From # and To # allow the specification of a range of read numbers upon which to
perform calculations. The default values correspond with the total number of
wavelengths to be read, based on the Start, Stop, and Step values in the
Protocol|Reading Parameters dialog.
Example: Start/Stop/Step values set to 400/600/10, respectively. The default From # and
To # values are 1 and 21, indicating a calculation zone from 400-600 nm. Setting From #
to 2, and To # to 18 would change the calculation zone to 410-570 nm.
•
Mean Min/Max specifies the number of successive wavelength readings to be averaged
prior to the determination of the Minimum and Maximum OD values.
Formula
Check the Formula radio button to define a formula to structure calculations by a formula. Enter
the formula into the long field.
Example: The formula W1+W2+W3 calculates the sum of the ODs for the readings at
wavelengths 1, 2, and 3. If the plate is read from 400 to 410 nm in 2 nm increments, W1 will
correspond with the OD at 400 nm, W2 with the OD at 402 nm, and W3 with the OD at 404 nm.
•
Enter a Result Name in which to store the formula results. This name can be used as a
Data-In variable in Transformations.
•
Mean Min/Max specifies the number of successive wavelength readings to be averaged
prior to the determination of the Minimum and Maximum OD values.
138 • Protocol Menu
User’s Guide to KC4™
Scanning Analysis
If linear scanning is selected in an endpoint protocol, select Protocol|Scanning Analysis to
specify data reduction options:
•
Select the Calculation Type to be performed on each scanned well.
•
Click the Calculation Options button to define the calculation zone. By default, the
calculation zone includes all of the Horizontal Reading Points defined in the protocol.
Mean, Std Dev, CV
Select Mean, Std Dev, CV to calculate and report the Mean, Standard Deviation, and Coefficient
of Variation for all points in the calculation zone.
By default, the calculation zone includes all of the Horizontal Reading Points defined in the
protocol. To change the calculation zone, click Calculation Options.
•
Currently, only the "From x: / To x: " method for determining the calculation zone is
supported.
By default, From x: represents the outermost scanned point to the left of center (0), and
To x: represents the outermost scanned point to the right of center. These values can be
changed to base calculations on a subset of all scanned points.
User’s Guide to KC4™
Protocol Menu • 139
In the example below, the number of Horizontal Reading Points in the protocol is 13.
The default From x: and To x: settings are - 6 and 6, respectively. To change the
calculation zone to include only the three points to the left and right of center (0), set
From x: to -3 and To x: to 3.
-6
-3
0
3
6
Points in calculation zone
All scanned points (13 total)
Mean Min/Max
Select Mean Min/Max to determine and report the Mean Minimum and Mean Maximum
measurement values based on n points.
By default, the calculation zone includes all of the Horizontal Reading Points defined in the
protocol, and the n value is 1. To change the calculation zone and/or the n value, click
Calculation Options.
•
Currently, only the "From x: / To x: " method for determining the calculation zone is
supported. See "Mean, Std Dev, CV" for more information.
•
The default number of points (n) used to calculate the Mean Minimum and Mean
Maximum values is 1. KC4 calculates the Mean Minimum and Mean Maximum values
as follows:
Starting at the first point in the calculation zone, KC4 evaluates n points and
calculates the mean.
KC4 repeats the operation, starting at the second point in the calculation zone, then
repeats again starting at the third point, and so on.
KC4 then compares all mean values to determine the minimum and maximum
values.
140 • Protocol Menu
User’s Guide to KC4™
Integral
Use the Integral calculation option to provide the area under the curve according to the trapezoidal
method, shown below:
i=n–1
Integral =
∑
(( yi + yi+1 ) / 2 ) × ( xi+1 – xi )
i=1
Where y = absorbance and x = scan point value.
Note that in linear scanning, ( xi+1 - xi ) is equal to 1, and thus the calculation formula is reduced
to:
i=n–1
Integral =
∑
yi – (( y1 + yn ) / 2 )
i=1
By default, the calculation zone includes all of the Horizontal Reading Points defined in the
protocol. To change the calculation zone, click the Calculation Options button in the Scanning
Analysis dialog.
•
Currently, only the "From x: / To x: " method for determining the calculation zone is
supported. See Mean, Std Dev, CV for more information.
User’s Guide to KC4™
Protocol Menu • 141
Plate Layout
KC4 allows you to describe the layout of the microplate(s) that will be associated with a protocol.
You are not required to do this. To perform blank well subtraction, generate curves, assign
dilution values, or define formulas that reference well identifiers, however, you must create a
Plate Layout. The process involves:
•
Selecting a well type (Blank, Assay Control, Sample, Sample Control, or Standard).
•
Defining well identifier(s) to be associated with the well type.
•
Defining dilution or concentration values (if required).
•
Assigning the well identifiers to the appropriate locations on a row x column grid.
To perform these tasks, select Protocol|Plate Layout, or click the Layout toolbar icon.
The Plate Layout dialog appears, in the row x column format of the plate type selected under
Protocol|Reading:
142 • Protocol Menu
User’s Guide to KC4™
•
For readers that support multiple plate formats, make sure that the correct plate type is
selected in the Reading parameters dialog before creating the plate layout.
•
Use the Well Settings options to select a well type, customize the text and color of its
IDentifier(s), and define dilutions or concentrations (if required).
•
Enter the number of Replicates then select the replicate mapping direction, Horizontal or
Vertical.
•
Check an Auto Selection option to automatically increment well ID group numbers
and/or concentration or dilution values.
•
Select the group mapping direction, Horizontal or Vertical.
•
Highlight one or more or wells in the grid to assign the identifier(s).
Tips:
•
Click the Undo button to undo the last action.
•
Click the Print button to print the current plate layout. If the plate contains 384 wells, the
printout will cover four pages, one per plate quadrant.
•
The Plate Layout dialog can be maximized to fill your entire screen. Click the maximize
icon in the upper right corner of the Plate Layout dialog.
•
Within the grid, rows and columns can be resized (to see all wells of a 384-well plate,
for example). Place the cursor over a row or column separator until it changes shape.
Click and hold the mouse, then drag the cursor up or to the left to make rows or columns
smaller. Drag the cursor down or to the right to make rows or columns larger.
Well Types and IDentifiers
Select and customize Well Types and Well IDentifiers within the Well Settings area of the Plate
Layout dialog, then assign them to one or more locations within the grid area of the dialog.
•
The Well Type tells KC4 which function to perform using the measurement value in the
well. KC4 supports five different Well Types:
Blank, used during full plate blank subtraction.
Assay Control, used during assay validation or as calibrators.
Sample, representing the unknown samples to be processed.
Sample Control, representing the control well(s) that are associated with a specific
sample.
Standard, used to generate a standard curve on which data will be interpolated.
•
The Well ID is the descriptive link between the measurement value retrieved for the well
and the Well Type assigned to it.
For example: The average of the values in the wells of type Blank and labeled with the
Well ID "BLK" will be subtracted from every well on the plate, as a part of KC4’s blank
subtraction function.
User’s Guide to KC4™
Protocol Menu • 143
Selecting and Customizing Well Types & IDs
The customization features vary among the different Well Types. Each Well Type is addressed
individually starting below and continuing on the following pages.
Blank Wells
During data reduction, the average of the raw data values in the blank wells is subtracted from the
raw data values in all wells on the plate (or from the corrected data values if a Blank Plate was
read).
KC4 requires that the same identifier be used for all blank wells. The maximum number of blank
wells that can be defined is limited only by the number of available wells on the plate. You
cannot define concentrations or dilutions for blank wells.
The default blank well ID is BLK, and its default color is white (it will print in black on reports).
To change the ID text and/or color, or to define a descriptive name for the blank wells for use on
reports, click the "…" box to the right of the ID field.
•
Enter an ID using 1 to 10 characters.
The ID can contain letters, numbers, and underscores. It cannot contain any spaces.
‘Blank_Well’ is valid, ‘Blank Well’ is not.
The ID cannot be identical to a formula operand (X, MIN, MAX, LOG, POW, SQRT,
CV, SD, and MEAN are not valid IDs).
If an ID is currently used in the grid, its text can be changed here. The change will
automatically be reflected in the grid.
•
(Optional) Enter a descriptive Name using up to 20 alphanumeric characters. This name
can be included on reports.
•
Click on the Color box to open a drop-down list showing all of the available colors.
Click on the desired color.
144 • Protocol Menu
User’s Guide to KC4™
Assay Control Wells
Assay control wells are typically used to validate the assay, or they can be used as calibrators.
Unique IDs must be used to distinguish between the different control groups (e.g. “NC” for
negative controls, “PC” for the positive controls). The maximum number of unique assay control
well IDs that can be defined is limited only by the number of available wells on the plate.
Concentrations and dilutions can be optionally assigned.
The default IDs are CTL1, CTL2, CTL3, etc., each with a different color. To change the IDs
and/or colors, or to define descriptive names for use on reports, click the “…” box to the right of
the ID field.
•
Enter an ID using 1 to 10 characters.
The ID can contain letters, numbers, and underscores. It cannot contain any spaces.
‘High_POS’ is valid, ‘High POS’ is not.
The ID cannot be identical to a formula operand (X, MIN, MAX, LOG, POW, SQRT,
CV, SD, and MEAN are not valid IDs).
If an ID is currently used in the grid, its text can be changed here. The change will
automatically be reflected in the grid.
If an ID is not currently used in the grid, it can be deleted from this list by deleting the
contents of the ID field.
•
(Optional) Enter a descriptive Name using up to 20 alphanumeric characters. This name
can be included on reports.
•
Click on the Color box to open a drop-down list showing all of the available colors.
Click on the desired color.
User’s Guide to KC4™
Protocol Menu • 145
Sample Wells
Sample wells contain the unknown sample solutions to be processed. Dilutions or concentrations
can be optionally assigned.
Unique IDs should be used to distinguish between the different sample groups. The maximum
number of unique sample well IDs that can be defined is limited only by the number of available
wells on the plate.
The default ID prefix is SPL, and the default IDs are SPL1, SPL2, SPL3, etc. The default color
for all IDs is black. To change the ID prefix and/or colors, click the "…" box to the right of the ID
field.
•
Enter an ID Prefix using 1 to 7 characters.
The prefix can contain letters, numbers, and underscores.
It cannot contain any spaces, and it cannot end in a number.
If the prefix is currently used in the grid, its text can be changed here. The change will
automatically be reflected in the grid.
•
For each group number, click on the Color Sequence box to open a drop-down list
showing all of the available colors. Click on the desired color.
KC4 uses the first color for SPL1, then the second for SPL2, and so on. It restarts with
the first color when the whole list of colors has been used.
To remove the last color of the current sequence, set it to End of Sequence (the last item
in the list).
146 • Protocol Menu
User’s Guide to KC4™
Sample Control Wells
Sample Controls are the controls that are associated with specific samples. For example, SPLC1
is associated with SPL1 (assuming the default names are used). This well type can be used to
represent sample blanks, or spikes.
Unique IDs should be used to distinguish between the different sample control groups. The
maximum number of unique sample control well IDs that can be defined is limited only by the
number of available wells on the plate.
The default ID prefix is SPLC, and the default IDs are SPLC1, SPLC2, SPLC3, etc. The default
color for all IDs is dark purple. To change the ID prefix and/or colors, click the “…” box to the
right of the ID field.
•
Enter an ID Prefix using 1 to 7 characters.
The prefix can contain letters, numbers, and underscores.
It cannot contain any spaces, and it cannot end in a number.
If the prefix is currently used in the grid, its text can be changed here. The change will
automatically be reflected in the grid.
•
For each group number, click on the Color Sequence box to open a drop-down list
showing all of the available colors. Click on the desired color.
KC4 uses the first color for SPLC1, then the second for SPLC2, and so on. It restarts
with the first color when the whole list of colors has been used.
To remove the last color of the current sequence, set it to End of Sequence (the last item
in the list).
User’s Guide to KC4™
Protocol Menu • 147
Standard Wells
Standard wells are used to generate a standard curve on which data will be interpolated. Expected
concentrations must be defined for each standard group.
Unique IDs must be used to distinguish between the different standard groups. The minimum
number of standard groups that can be defined is 2, and the maximum number is 20. Note: Each
curve fit type has its own required minimum number of standards.
The default ID prefix is STD, and the default IDs are STD1, STD2, STD3, etc. The default color
for all standard wells is dark blue. To change the ID prefix and/or color, click the “…” box to the
right of the ID field.
•
Enter an ID Prefix using 1 to 7 characters.
The prefix can contain letters, numbers, and underscores.
It cannot contain any spaces, and it cannot end in a number.
If the prefix is currently used in the grid, its text can be changed here. The change will
automatically be reflected in the grid.
•
(Optional) Enter a descriptive Name using up to 20 alphanumeric characters. This name
can be included on reports.
•
Click on the Color box to open a drop-down list showing all of the available colors.
Click on the desired color.
148 • Protocol Menu
User’s Guide to KC4™
Assigning Well IDs
After defining the Well Settings, assign the Well ID(s) in the grid area of the Plate Layout dialog.
Here is a very simple example for assigning blank wells to the plate:
•
Select the Blank well type from the Type drop-down menu:
•
The ID field shows the default identifier, BLK:
•
Assuming you wish to use the "BLK" identifier, highlight the well(s) in the template that
should contain blanks, then release the mouse button:
•
The average of the measurement values in wells A1 and A2 will be subtracted from every
well on the plate.
KC4 provides options for defining replicates and automatically updating the Well ID numbers.
The following example shows you how to define Replicates and use the Auto Select and Filling
features to assign samples in triplicate.
•
Select the Sample well type from the Type drop-down menu:
•
The ID field shows the first ID in the sequence, SPL1:
User’s Guide to KC4™
Protocol Menu • 149
•
Set the number of Replicates to 3, then select the Horizontal filling option. Sample
replicates will be assigned horizontally, A1/A2/A3, B1/B2/B3, and so on.
•
Choose the Auto Select option Next ID. This automatically increments sample groups,
SPL1, SPL2, SPL3, and so on.
•
Select the Filling option Vertical to assign groups vertically.
•
Finally, go to the grid and click the mouse once in the box above the A and to the left of
the 1. This fills all wells with samples.
•
During data reduction, KC4 will automatically calculate and display the Mean, Standard
Deviation, and CV % for each sample group (click the Statistics button from within the
Data display to view this information):
150 • Protocol Menu
User’s Guide to KC4™
Defining and Assigning Dilutions
KC4 provides the ability to define dilutions for Assay Controls, Samples, and Sample Controls.
Defining Dilutions
Dilution values can be assigned to wells when the well IDs are assigned, or they can be assigned
to existing well IDs by clicking the Dilutions Only button. This section discusses assigning
dilutions and well IDs at the same time.
When defining dilutions, start by selecting the desired well type. The dilution values will remain
associated with this well type. Customize the well IDs if necessary.
When the Type and ID fields are set, click the "…" button to the right of the Dil. Field. The
Concentrations\Dilutions dialog appears:
Use this dialog to enter the dilution values required for the assay. Dilutions are must be entered in
ascending or descending order, starting with Dilution #1 (10.000 in the example above):
•
Choose the Conc./Dil. Type option Dilution.
•
Enter the starting dilution in cell #1. Subsequent dilutions can be entered manually or
automatically:
Enter dilutions manually in cells #2, #3, and so on. Use the down arrow key to advance
to the next cell, or click the mouse in the desired cell.
To increase dilutions automatically, choose an Auto option of Incr or Fact. Enter the
required Increment or Factor value, then click on cell #2 to assign the next dilution. Use
the down arrow key to advance to the next cell, or click the mouse in the desired cell.
User’s Guide to KC4™
Protocol Menu • 151
Incr: Increments dilutions by the entered value. In the example above, the starting
dilution is 10, and subsequent dilutions increase by 10. Enter a negative number (e.g. 10) to decrement dilution values.
Fact: Increase dilutions by an entered factor value. If the starting dilution is 2,
increasing by a factor of 2, subsequent dilutions become 4, 8, 16, 32, 64, and so on.
•
Click the Clear button to delete all values from all cells.
•
When all of the dilution values have been entered, click OK to return to the Plate Layout
dialog.
Assigning Dilutions
There are different techniques for assigning dilutions. You may wish to experiment with the
software, to get comfortable with assigning Replicates, choosing neither, one, or both Auto Select
options, and toggling the Horizontal/Vertical filling options. It might also be helpful to sketch
your plate map on a piece of paper to use as a reference.
To assign dilutions (with Well IDs):
•
Select the necessary Well Settings (Type, #, and ID), then choose the first dilution value
to be assigned.
•
Enter the number of dilution Replicates per control or sample.
•
Choose the dilution replicate filling direction, Horizontal or Vertical.
•
To advance well IDs and/or dilution values automatically, choose Next ID (if available),
and/or Next Dil.
•
Choose the dilution group filling direction, Horizontal or Vertical.
•
Highlight the well(s) to assign dilutions and well IDs.
Examples are provided on the following pages.
152 • Protocol Menu
User’s Guide to KC4™
•
Dilution values are defined as 2.000, 4.000, 8.000, 16.000, 32.000, and 64.000.
•
The Auto Select option Next Dilution is selected. The Next ID option is not available for
Assay Control groups; they must be incremented manually.
•
Wells A1 through A6 are highlighted to assign CTRL1 and its dilutions. CTRL2 is then
selected and assigned to wells B1 through B6.
User’s Guide to KC4™
Protocol Menu • 153
•
Dilution values are defined as 10.000, 20.000, 30.000, 40.000, 50.000, and 60.000.
•
Replicates is set to 3, and both Auto Select options are enabled.
•
Wells A1 through F12 are highlighted to assign IDs and dilutions.
•
Dilution values are defined as 1.000, 10.000, 100.000, and 1000.000.
•
Replicates is set to 1, and both Auto Select options are selected.
•
Wells A2 through D2 are highlighted to assign the ID and dilutions, then A4 through D4,
and so on to A12 through D12.
154 • Protocol Menu
User’s Guide to KC4™
Defining and Assigning Concentrations
If the plate layout contains Standard wells, or if you want to generate curves based on control or
sample wells (e.g. for a titer assay), the location and the expected concentrations of these wells
must be defined and assigned in the plate layout. KC4 uses the expected concentration values as
the basis for calculating the curve; they represent the x-axis values. The measurement values
retrieved from the reader (or the results of transformations applied to them), represent the y-axis
values.
KC4 provides the ability to define concentrations for Assay Controls, Samples, Sample Controls,
and Standards.
Defining Concentrations
When defining concentrations, start by selecting the desired well type. The concentration values
will remain associated with this well type. Customize the well IDs if necessary.
When the Type and ID fields are set, click the "…" button to the right of the Conc. field (for well
types other than Standard, this field will initially be labeled “Dil.”). The Concentrations or
Concentrations\Dilutions dialog will appear, depending on the well type.
Use this dialog to enter the expected concentration values required for the assay. Values must be
entered in ascending or descending order, starting with concentration #1 (1.000 and 5.000E-3 in
the examples above):
•
Enter the starting concentration in cell #1. Subsequent concentrations can be entered
manually or automatically:
When entering concentrations manually, use the down arrow key to advance to the next
cell, or click the mouse in the desired cell.
User’s Guide to KC4™
Protocol Menu • 155
To increase concentrations automatically after entering the starting concentration, choose
an Auto option of Incr or Fact then define the required Increment or Factor value. Click
on cell #2 to assign the next concentration. Use the down arrow key to advance to the
next cell, or click the mouse in the desired cell.
Incr: Increments concentrations by the defined value. If the starting concentration is
10.000 with an Increment of 10, subsequent concentrations become 20.000, 30.000, and
so on. Enter a negative number (e.g. -5) to decrement concentration values.
Fact: Increases concentrations by the defined value. If the starting concentration is
1.000 with a Factor of 2, subsequent concentrations become 2.000, 4.000, 8.000, 16.000,
and so on.
•
(Optional) Enter the Unit of measure for the X-axis values on the curve, using up to five
alphanumeric characters.
•
Click the Clear button to delete all values from all cells.
•
For Sample Controls, KC4 provides the Calculate Recovery % option. If this feature is
enabled, KC4 automatically generates a “Recovery %” results data set, containing the
results of the following formula:
[Conc(SPLCn) - Conc(SPLn) ÷ ExpectedConc(SPLCn)] x 100
•
When all of the concentration values have been entered, click OK to return to the Plate
Layout dialog.
156 • Protocol Menu
User’s Guide to KC4™
Assigning Concentrations
To assign concentrations (with well IDs):
•
Select the necessary Well Settings (Type, #, and ID), then choose the first concentration
value to be assigned.
•
Enter the number of standard, sample, or control Replicates.
•
Choose the replicate filling direction, Horizontal or Vertical.
•
If you want well IDs and/or concentration values to advance automatically, choose Next
ID (if available), and/or Next Dil.
•
Choose the group filling direction, Horizontal or Vertical.
•
Highlight the well(s) to assign the concentrations and well IDs.
Examples are provided below and on the next page.
•
Concentrations are defined as 10, 20, 30, 40, 50, and 60.
•
The Auto Select option Next Concentration is selected. The Next ID option is not
available for Standard groups; they must be incremented manually.
•
Wells A1 through F2 are highlighted to assign the concentrations and IDs.
User’s Guide to KC4™
Protocol Menu • 157
•
Concentrations are defined as 1, 2.5, 5, 10, 20, 40, 80, and 160.
•
Replicates is set to 2, and both Auto Select options are selected.
•
Wells A1 through H12 are highlighted to assign the concentrations and IDs.
158 • Protocol Menu
User’s Guide to KC4™
Single Plate Transformations
KC4 provides for up to three levels of Single Plate Transformations. The three different levels
of transformations may be interdependent, or completely independent of each other. Typical uses
include:
•
Using specific wells as local blanks for other wells.
•
Calculating ratios between wells.
The Single Plate Transformation dialog functions similarly to a spreadsheet. The data entry
template resembles a microplate in row x column format. Formulas are entered in "cells"
representing the wells within the microplate.
To define Single Plate Transformations:
•
For readers that support multiple plate formats, ensure the correct format is selected
under Protocol|Reading.
•
Ensure that identifiers have been assigned under Protocol|Plate Layout.
•
Select Protocol|Single Plate Transformations:
Place the cursor in a well and enter the desired formula. The formula is visible in the
Current Formula box at the bottom of the dialog.
The Transf. # field indicates the current transformation level. Level 1
transformations can be passed for further manipulation into Level 2 and Level 3
transforms for successive manipulation.
Data In represents the data set upon which the formulas will be applied. The Data In
choices will vary depending on the defined protocol parameters.
User’s Guide to KC4™
Protocol Menu • 159
For kinetic protocols, a Kinetic checkbox is provided. If this box is checked, the
Data-In options represent plate data, such as “M#1 405” or “M#2 630”. If this box is
not checked, the Data-In options represent the results of kinetic calculations, such as
“Max V” and “Coeff Reg”.
The Data Out field assigns a name to the transformed data. The transformed data
can then be used for further calculations, including Multi Plate Transformations.
Select Copy Current Formula to copy the current formula into one or more wells.
Clicking on the title row or column auto-fills the wells selected with the current
formula. Clicking on the top left corner auto-fills the whole plate.
Select Edit Well to edit the formula in a particular well.
Select Diff. Between Lines to perform subtraction between rows, then select A-B or
B-A.
Select Diff. Between Cols to perform subtraction between columns, then select 1-2
or 2-1.
Click Init. All Transf. to reinitialize all Single Plate Transformations to their most
recently saved settings.
to expand the width of the columns, to view and access long formulas.
Click
The maximum expansion shows a single column of wells in full screen.
Click
to contract the width of the columns.
Formula Syntax
The following tables contain symbols and functions that can be used in Single-Plate
Transformations:
Type of Symbol
Description/Example
Algebraic symbols
+,-, /,*,()
Figures (scientific notation)
2.45E-08
x or X
Represents the current well value.
coordinates of the well
Represents the value of a particular well, such as A2 or H12.
160 • Protocol Menu
User’s Guide to KC4™
Functions Applied
to the Identifier
Description/Example
MEAN[POS1]
Represents the mean of identifier POS1. ‘POS1’ by itself
also represents the mean of all POS1 wells on the plate.
SD[NEG1]
Represents the standard deviation of all valid wells in group
NEG1.
CV[BLK]
Represents the coefficient of variation of all valid wells in
group BLK.
Advanced Mathematical
Functions
Description/Example
LOG(x)
Represents the log10 function.
POW(x;y)
Raises x to the power of y.
POW(10;x)
Calculates the Anti-Log of the current well.
SQRT(x)
Square root of x.
MIN(x1;x2;x3...)
Minimum value of defined variables.
Example: MIN(A1;B1;C1).
MAX(x1;x2;x3...)
Maximum value of defined variables.
Example: MAX(CV[CTRL1];CV[CTRL2]).
Formula Examples
See also the installed sample protocol files for both Single and Multi Plate Transformation
examples; these can be found in the SAMPLES folder in your main KC4 folder.
CTRL1-3*SD[CTRL1]
The result of this formula is the mean of the wells containing the identifier CTRL1 minus
three Standard Deviations of these wells. It is the equivalent of MEAN[CTRL1]3*SD[CTRL1].
X+(A5+A6)/2
The result of this formula is the sum of the current well value and the mean of the A5 and
A6 wells.
LOG(X/MEAN[POS1])
The result of this formula will log the ratio of the current well over the mean of POS1.
MAX(A1;A2;A3)-MIN(B1;B2;B3)
This formula calculates the difference between the maximum value of A1, A2, A3 and
minimum value of B1, B2, B3.
% = (SPLn-BLKn) / (CTRLn-BLKn)
This formula can be used for toxicology assays, to calculate toxicity percentages.
User’s Guide to KC4™
Protocol Menu • 161
Multi Plate Transformations
KC4 provides for up to three levels of Multi Plate Transformations. The three different levels of
transformations may be interdependent, or completely independent of each other. In addition, the
results of any Single Plate Transformations can be incorporated into a Multi Plate
Transformation.
To define Multi Plate Transformations:
•
Ensure that all protocol parameters that have an effect on data reduction have been
defined.
•
Select Protocol|Multi Plate Transformations:
The Transf. # field indicates the current transformation level. Level 1
transformations can be passed for further manipulation into Level 2 and Level 3
transforms for successive manipulation.
Data In represents the data set upon which the formulas will be applied. The Data In
choices will vary depending on the protocol type and reading parameters.
Within one Multi Plate Transformation, you can select up to three discrete assay
data sets for combined calculations. You must select and position each data set in
the appropriate location holder (i.e., X1, X2 and X3). The data sets must be of
similar type.
162 • Protocol Menu
User’s Guide to KC4™
You can select previously stored data files (these files must be correctly formatted
text files and only contain end-point readings).
For kinetic protocols, Reading # can be set to All, or from 1 to the total number of
readings.
Use the location variables X1, X2 and X3 to construct the Formula.
The Name field assigns a name to the transformed data and saves the settings and
formula. The transformed data can then be used as the Data-In for further
calculations, including Kinetic Analysis and other Multi Plate Transformations.
Syntax
The following table contains symbols that can be used in Multi Plate Transformations:
Type of Symbol
Description/Example
Algebraic symbols
+,-, /,*,()
Figures (scientific notation)
2.45E-08
Automatic Formula Generation
For readers with multiple wavelength (> 2) reading capability, a default Multi Plate
Transformation is automatically created when exactly 2 wavelengths or filter sets are specified.
These default parameters can be changed.
•
The Protocol menu switches to Advanced Mode.
•
The Multi Plate Transformation dialog contains:
X1 = <Meas. OD#1>
X2 = <Meas. OD#2>
Formula = X1 - X2
Data Out = Delta OD
•
In addition, if the protocol is Kinetic:
The ‘Kinetic’ checkbox becomes enabled.
The Reading # fields for both X1 and X2 are set to All.
Note: Once the Reading Parameters dialog is saved and closed with 2 filter sets/wavelengths
specified, the Delta OD Multi-Plate Transformation will not automatically ‘clear’ if the protocol is
then changed to 1 or 3-6 filter sets/wavelengths. The user must open the Multi Plate
Transformation dialog and manually empty/modify the fields.
User’s Guide to KC4™
Protocol Menu • 163
Curves
Select Protocol|Curves to define various curve fitting parameters, including the data set to
interpolate and the fitting method. Interpolations are defined here as well.
Curve Description
Select Protocol|Curves to select the data set to be used for curve calculations, and to specify the
origin of the curve.
•
The Data to Interpolate setting represents the type of data to be used for curve fitting.
•
Select Current Plate if the standard curve will be generated using data from the current
plate.
•
Select File if the standard curve will be based on a previously stored curve. The stored
curve will be used as the basis for calculating concentrations for the current plate.
Click Browse... to search for the desired file. The file must have a *.GPH extension.
Refer to the Biograph module for information on storing curves.
If a stored curve is used and the plate layout contains standard identifiers, then any use of
STDn in formulas will refer to the standard well(s) on the current plate and not to the
standards in the stored curve.
164 • Protocol Menu
User’s Guide to KC4™
Curve Axes
By default, the x- and y-Axis data is transformed linearly, and the Extrapolation Factor is set to
1.1. These options can be modified by selecting Protocol|Curves|Axes.
•
Select either Lin or Log for the transformation of x axis or y axis data.
•
If Display Grid is checked, the graph will be displayed and printed with gridlines.
•
The text in the x Axis and y Axis Title fields appears on standard curve displays and
reports. To modify a title (e.g.,
"% toxicity" or "Delta OD"), overwrite the existing text.
•
The Extrapolation Factor enables you to extend the abscissa range of the curve fitting
routine beyond the limits of the standard data points generated from the assay. See
Defining Extrapolation below for more information.
Exercise caution when using extrapolation, as the implied assumption is that the
relationship of the x and y variables is valid outside the range defined by the standards.
This type of extrapolation should only be applied when prior knowledge of the
relationship is known. Typically this feature is used in assays where linear relationships
are known. Inappropriate use of the Extrapolation Factor feature may invalidate results
generated from data falling into the extrapolation zone.
•
By default, the Min and Max y axis values (for display and reporting purposes) are
determined automatically by KC4. These values can be overridden by deselecting Auto
and manually entering the Min and Max values.
User’s Guide to KC4™
Protocol Menu • 165
Defining Extrapolation
The extrapolation factor range must be between 1 and 3.
Using a factor of 1 implies that the defined upper and lower standards are the limits for
concentration interpretation. However, given some assays, the upper and lower limits may be
extended, in this case tentative extension of the range may be imposed.
•
Example: Assuming a linear x axis we wish to extend the upper and lower limit by 20%.
The lower limit denoted by the first standard is 50 units. The upper limit denoted by the
last standard is 150 units. The dynamic range therefore is 150-50 = 100 units. Imposing
the 20% extension means that the new range will be: 100 x 1.2 equals 120 units.
This represents an increase in the dynamic of 10 units at the lower limit and 10 units at
the upper limit. Therefore the new lower limit will be 50-10 equals 40 units. The new
upper limit will be 150 + 10 equals 160 units.
•
Notes: Sample values that fall into the new limits will be calculated. Those that fall
outside these limits will be assigned > than or < than, respectively. Extrapolation cannot
be used for the Spline routine nor the Point-to-Point fit. These algorithms cannot
calculate values outside the upper and lower defined standard points.
Curve Fit
Use the Protocol|Curves|Curve Fit dialog to select the method by which the standard curve will
be generated. The default setting is Linear Regression.
166 • Protocol Menu
User’s Guide to KC4™
It is important that the appropriate curve fit is applied to the data otherwise the results may be
invalid. Whichever curve fitting routine is used, it is essential to check the shape of the curve
prior to evaluating data.
The curve fit methods are described below.
Point to Point
Linear interpolation between standard points. The minimum number of standards is 2.
Linear Regression
Linear regression y = ax + b.
y represents unknown response
x represents concentration
a represents the slope of the linear regression
b represents the intercept to the y axis
•
The linear regression fit uses the least squares technique. The better the quality of the fit
the more the absolute value of R tends to 1. This type of curve fitting technique can be
used when you think the data will fall in a predictable linear pattern. It may be necessary
to transform the x and/or y components using Log axes.
•
Click the Options button to enable or disable the averaging of standard replicates before
curve calculation. By default, average replicates is enabled.
Polynomial Regression
This type of regression fitting is an extension of the linear regression equations.
y = an Xn + an-1 Xn-1 + … a1 X + a0
n represents the degree of the polynomial regression.
Note: The degree must be less than or equal to the number of standards minus 2. The unknown
concentrations are calculated by using an approximate calculation method, linear interpolation
between 200 points evenly spaced on the x axis.
•
Certain assays require forcing the curve through the origin. KC4 provides a feature that
allows you to specify the point at which the curve should meet the Y axis.
Click the Options button to enable this feature and then enter the Y axis intercept value.
Enter 0 (the default value) to force the curve through the origin.
User’s Guide to KC4™
Protocol Menu • 167
The value entered here determines the coefficient ‘a’ of the polynomial equation:
y = g*x6 + f*x5 + e*x4 + d*x3 + c*x2 + b*x + a
Note: By forcing the curve through the origin, the R-squared value may be decreased
because the equation may not describe the data of the standards as well. It is the user’s
responsibility to check the validity of the data when using this feature.
Spline with Smoothing
Spline fitting with a smoothing factor. The curve will appear smoother as the smoothing factor is
increased. The zero factor will force the curve fit through all data points. The minimal number of
standards for the spline fit is 4.
Note: The unknown concentrations are calculated using an approximate calculation method,
linear interpolation between 200 points evenly spaced on the x axis.
4 Parameter logistic fit
a = (theoretical) response at concentration = 0
b = measure of slope of curve at its inflection point
c = value of x at inflection point
d = (theoretical) response at infinite concentration
x = concentration
y = response (OD)
The minimum number of standards is 4.
It is recommended that:
•
at least one standard is not far from each asymptote.
•
at least 2 standards fall within the linear area of the curve, one either side of the inflection
point.
168 • Protocol Menu
User’s Guide to KC4™
Curve Interpolation
Select Protocol|Curves|Curve Interpolation to define Y values and/or formulas for which a
concentration will be calculated.
KC4 supports the definition of up to 25 Interpolation values and formulas.
•
Before defining interpolations that reference well identifiers, ensure that all identifiers
have been assigned under Protocol|Plate Layout.
•
Each interpolation can contain any of the following:
Numeric values, such as 1.500, 85000, or 2.0E-8.
Well identifiers, such as NC, BLK, or STD1.
Well coordinates, such as A1 , H12, or P24.
A formula combining any or all of the above, such as CTL1/CTL2*100.
•
Click the Biograph button after a plate is read to view the Interpolation Results.
•
Interpolation results can be included on reports and in export files.
•
Interpolation formula syntax is described on the next page.
User’s Guide to KC4™
Protocol Menu • 169
Interpolation Formulas
The following table contains valid symbols and functions for use within Interpolation formulas:
Symbol or Function
Description
Example
+
Addition
CTL1+0.100
-
Subtraction or negation
BLK-0.010
/
Division
CTL1/CTL2
*
Multiplication
STD1*0.10
Represents inclusion
(CTL1/CTL2)*100
Well coordinates
The value of a specific well
A1/B1
MEAN[<ID>]
The mean of the specified well
identifier
MEAN[POS1]
SD[<ID>]
The standard deviation of the specified
well identifier
SD[BLK]
CV[<ID>]
The coefficient of variation of the
specified well identifier
CV[STD1]
LOG(<x>)
Represents the LOG10 function
LOG(SPL10)
POW(<x>;<y>)
The value of <x> raised to the power of
<y>
POW(STD1;3)
SQRT(<x>)
The square root of <x>
SQRT(A1*B1)
MIN(<x>;<y>;<z>;…)
The minimum of the defined variables
MIN(CV[CTL1];
CV[CTL2])
MAX(<x>;<y>;<z>;…)
The maximum of the defined variables
MAX(A1;B1;C1)
( )
170 • Protocol Menu
User’s Guide to KC4™
Validation
Validation is a set of determinant criteria used to assess the suitability of calibrators for the
inclusion in subsequent calculations. Validation criteria, or Conditions, are expressions based on
threshold limits previously determined from known standardized conditions.
Select Protocol|Validation (with the Protocol Menu in ‘Advanced’ mode) to define Validation
Sets and Conditions.
•
Before defining validation criteria, ensure that all identifiers have been selected and
defined under Protocol|Plate Layout.
•
Define up to 5 different Validation Sets, each containing up to 10 Conditions.
•
The Data Type setting represents the set of data to which the Conditions equations will
be applied.
•
The Conditions fields allow fixed thresholds and/or algebraic formulas to be used in
validating data to satisfy known conditions of the defined assay. A Condition typically
combines well identifiers (see Plate Layout) with the following symbols:
<
less than
<=
less than or equal to
>
greater than
>=
greater than or equal to
User’s Guide to KC4™
Protocol Menu • 171
•
Example 1:
Conditions to be met:
1) During the first of several kinetic readings, the mean value of STD1 must fall
between 0.200 and 0.300 OD, and the mean value of STD2 must fall between, 0.900
and 1.300 OD.
2) For each kinetic reading point, the mean value of POS1 must be less than the mean
value of POS2.
•
Example 2:
Condition to be met:
The mean of every sample replicate group must be greater than 0:
The # (pound) sign is a generic symbol that can be used to represent all indices of a
particular identifier.
172 • Protocol Menu
User’s Guide to KC4™
Cutoffs
Cutoffs screen selected data into defined categories based on fixed or variable threshold limits.
During data reduction, KC4 evaluates results against the cutoff criteria assigns each well a
Symbol.
Select Protocol|Cutoffs (with the Protocol Menu in ‘Advanced’ mode) to define Cutoffs and
Symbols.
•
After plate reading, open the Symbols data set to see the assigned symbols and the
calculated cutoff values.
Selecting the Data to Interpret
The Data to Interpret setting represents the set of data to which the cutoffs will be applied. For
example, if Data to Interpret is set to ‘Concentrations’, the cutoffs will be applied to the calculated
concentrations.
KC4 presents for selection the applicable data sets that are made available by the current protocol
definition.
User’s Guide to KC4™
Protocol Menu • 173
Defining Cutoffs
KC4 supports the definition of up to 40 cutoffs. These are the criteria against which the plate data
will be evaluated.
•
Enter cutoff values or formulas in the C.O.#. = fields. Click
previous 8 fields.
•
Before defining cutoffs that reference well identifiers, ensure that all identifiers have
been assigned under Protocol|Plate Layout.
•
Cutoffs must be entered in ascending order. If entering formulas, the calculated results
of the formulas must be in ascending order to be considered valid.
or
to see the next or
For example, if Data to Interpret is set to ‘Concentrations’ and the cutoffs are defined as
‘STD1’, ‘STD2’, and ‘STD3’, then the calculated concentrations must be in ascending
order, such as 10.000, 20.000, and 30.000.
•
Each cutoff can contain any of the following:
Numeric values (1.500, 85000, 2.0E-8)
Well identifiers (NC, BLK, STD1)
Well coordinates (A1 , H12, P24)
A formula combining any of the above (CTL1/CTL2*100)
Cutoff Formulas
The following table contains valid symbols and functions for use within cutoff formulas:
Symbol or Function
Description
Example
+
Addition
CTL1+0.100
-
Subtraction or negation
BLK-0.010
/
Division
CTL1/CTL2
*
Multiplication
STD1*0.10
Represents inclusion
(CTL1/CTL2)*100
Well coordinates
The value of a specific well
A1/B1
MEAN[<ID>]
The mean of the specified well identifier
MEAN[POS1]
SD[<ID>]
The standard deviation of the specified well
identifier
SD[BLK]
CV[<ID>]
The coefficient of variation of the specified well
identifier
CV[STD1]
LOG(<x>)
Represents the LOG10 function
LOG(SPL10)
POW(<x>;<y>)
The value of <x> raised to the power of <y>
POW(STD1;3)
SQRT(<x>)
The square root of <x>
SQRT(A1*B1)
MIN(<x>;<y>;<z>;…)
The minimum of the defined variables
MIN(CV[CTL1];
CV[CTL2])
MAX(<x>;<y>;<z>;…)
The maximum of the defined variables
MAX(A1;B1;C1)
( )
174 • Protocol Menu
User’s Guide to KC4™
Defining Symbols
Symbols are assigned to wells after cutoff evaluation. After plate reading, open the ‘Symbols’
results data set to see the assigned symbols as well as the calculated cutoff values.
The number of available Symbols fields exceeds the number of defined cutoffs by one. During
data reduction, if the value of a well is less than the first cutoff, the first symbol is assigned to the
well. If the value of a well is greater than or equal to the first cutoff, but less than the second
cutoff, the second symbol is assigned to the well, and so on. See Results, Symbols later in this
manual for more information.
Each Symbol can contain up to 8 alphanumeric characters. Common symbols include NEG, EQV,
POS, -, +. The appearance of each symbol can be customized by clicking its corresponding Font
button:
•
The default Font is MS Sans Serif Regular 10 point (or similar), in Black. KC4 presents
for selection the fonts that are already installed on your PC (typically in your
\Windows\Fonts folder.
•
Change the Font, Font Style, Size, and color, as desired. Add Effects such as a Strikeout
or an Underline. Symbols will appear on the results display and on printed reports as
they are defined within this dialog.
User’s Guide to KC4™
Protocol Menu • 175
Out-of-Range Symbols
When ‘Data to Interpret’ is set to ‘Concentrations’, the Out of Range Symbols fields are
available at the bottom of the Cutoffs dialog.
Following data reduction:
•
Wells with concentrations less than the minimum standard concentration are assigned the
‘Concentration below minimum’ symbol.
•
Wells with concentrations greater than the maximum standard concentration are assigned
the ‘Concentration above maximum’ symbol.
•
Note: Use of the Extrapolation Factor may cause the upper and lower limits for
concentration interpretation to be extended.
The default out of range symbols are OUT- and OUT+. Each Symbol field can contain 0 to 8
alphanumeric characters. The appearance of each symbol can be customized by clicking its
corresponding Font button, as described in the previous section.
Cutoffs Evaluation
Cutoffs are evaluated during data reduction. If the cutoffs are in increasing order, symbol
assignment is determined as follows:
•
Each well is evaluated against the first cutoff. If the value of the well is less than the
criteria, then the first symbol is assigned to the well.
•
If the value of the well is greater than or equal to the first cutoff, then it is evaluated
against the next cutoff. If the value of the well is less than the next criteria, then the
symbol immediately preceding the cutoff criteria is assigned to the well.
•
This process continues until the last cutoff criteria is encountered. If the value of the well
is less than the last criteria, then the symbol immediately preceding the last cutoff criteria
is assigned to the well. If the value of the well is greater than or equal to the last criteria,
then the symbol immediately following the last cutoff criteria is assigned to the well.
176 • Protocol Menu
User’s Guide to KC4™
Reports
With KC4’s standard reporting function you can customize the contents, and to some extent the
appearance, of printed reports. All KC4 users have access to this standard function. Reporting
through Microsoft® Excel 2000 and Word 2000 is also supported with KC4’s PowerReports
feature.
This section only discusses the standard reporting function (the KC4 report engine). The report
engine is selected under Protocol|Options.
Select Protocol|Reports to customize the report for the open protocol:
•
Check Header and enter the text to appear at the top of every report.
•
Check Protocol Description and Assay Description to include this information on
reports.
•
The Available Data window displays the data and information available for reporting.
Available Data selectable items are dependent upon the type of protocol selected, as well
as the defined Reading Parameters.
Click the left mouse button to select an individual item.
To select groups of like items, hold the Ctrl key down while highlighting them.
Click the Add>> button to append the item(s) to the Selected Data list.
User’s Guide to KC4™
Protocol Menu • 177
Click the Insert>> button to insert item(s) into the Selected Data list. Highlight the
item in the Selected Data list above which the inserted item should appear.
You can drag and drop selected items between the two lists.
The Form Feed option inserts a page break.
Audit Trail Reports
If Protocol Audit Trail is selected, a list of all changes made to the protocol is included on the
report.
If Data Audit Trail is selected, a list of all changes made to the data file is included on the report.
Calculation Log Report
If Calculation Log Report is selected, warning(s) appear on reports if a special event occurs
during the calculations. Events can include a failure during the calculation of a transformation, the
standard curve, or cut-offs:
178 • Protocol Menu
User’s Guide to KC4™
Kinetic Curves Report
The Kinetic Curves report shows the kinetic curve in every well, in a plate format. For example:
Well Zoom Report
Each Well Zoom report contains the kinetic curve for one well.
•
To specify a Well Zoom report, highlight Well Zoom in the Available Data box, enter the
desired Wells for kinetic zoom, then click the Add button.
•
Up to 8 kinetic wells can be overlaid in a single graph. Select the Wells for kinetic
zoom, then click Overlay Wells.
User’s Guide to KC4™
Protocol Menu • 179
Validation Report
The Validation report contains every validation condition defined for the protocol and its resulting
status.
•
The possible status values are Verified, Not Verified, and Could Not Be Evaluated.
Cutoffs Report
The Cutoffs report lists the defined cutoffs and their calculated results.
•
In the example above, cutoffs were based on the Concentrations.
Sample Name List Report
The Sample Name List contains all sample names defined for the plate.
180 • Protocol Menu
User’s Guide to KC4™
Plate vs. Table Format
Depending on the nature of the item chosen under Available Data, information can be reported
either in Plate format or in Table format.
•
With the Plate format selected, data will print in a matrix format corresponding with the
plate layout.
•
Data for individual kinetic reads can be printed in the Plate format. Select the type of
data (e.g., Meas. OD, Delta OD), then set the range of reads to include in the report using
the From # and To # fields.
•
The Table format is available for kinetic and scanned data. All kinetic or scanned
readings for one or multiple wells. Set the range of wells to include in the report using
the From and To fields.
Statistics Report
Statistical expansion of the data can be reported. This information is specifically related to the
Identifiers, e.g. controls, standards or samples.
•
To specify a Statistics report, select an item from the Available Data box, click the
Controls, Standards, or Samples radio button in the Statistics box, then click the Add
button.
User’s Guide to KC4™
Protocol Menu • 181
Export
With KC4’s standard exporting function you can customize the contents of export files. All KC4
users have access to this standard function. Exporting through Microsoft® Excel 2000 and Word
2000 is also supported with KC4’s PowerReports function.
This section only discusses the standard exporting function (the KC4 report engine). The export
engine is selected under Protocol|Options.
Select Protocol|Export to customize the export file for the open protocol:
•
Check Protocol Description and Assay Description to include this information in the
file.
•
The Available Data window displays the data and information available for exporting.
Available Data selectable items are dependent upon the type of protocol selected, as well
as the defined Reading Parameters.
Select an individual item by clicking it with the left mouse button. To select groups
of like items, hold the Ctrl key down while highlighting them.
Click the Add>> button to transfer the item(s) to the bottom of the Selected Data
list.
Click the Insert>> button to insert item(s) into the Selected Data list. Highlight the
item in the Report field above which the inserted item should appear.
•
See “Formats” in System Menu, Setup for information on choosing the text delimiter.
182 • Protocol Menu
User’s Guide to KC4™
Protocol Audit Trail
This feature only applies to KC4 Signature
Every protocol file has a Protocol Audit Trail associated with it. This audit trail automatically
records events performed on the file since the file was created (first saved). Each event records
an action such as "Plate Layout changed" or "Reading parameters changed" and includes a date
and time stamp, the ID of the user logged in at the time, and any comments that were entered by
the user when the action was performed. The audit trail cannot be edited and it remains a
permanent part of the file for the life of that file (that is, if the file is deleted, the audit trail is also
deleted).
To view a protocol’s audit trail, select Protocol|Audit Trail:
•
Events are sorted by date/time stamp and include the ID of the user logged in at the time,
the action taken, and any comments that were entered by the user when the action was
performed.
•
The contents of the protocol audit trail can be copied to the Clipboard by highlighting the
desired cells and pressing Ctrl+C.
•
The contents of the protocol audit trail cannot be edited or deleted.
User’s Guide to KC4™
Protocol Menu • 183
Security
KC4 provides the ability for the Administrator to protect the use of certain protocol/data functions
by unauthorized users. To view or modify the protected functions associated with a particular
protocol, select Protocol|Security (advanced function).
Checked
functions may not be performed by unauthorized users.
•
With KC4 v3.4, an unauthorized user is any user other than the System Administrator.
•
With KC4 Signature, an unauthorized user is one who is designated as a Standard User.
The following list includes all protocol-level protectable functions and describes the behavior the
unauthorized user can expect from KC4 for each function when it is protected:
•
Re-read plate. The user cannot re-read a plate if one is already open, because re-reading
a plate deletes the initial values. The user must first select Data|New Plate and then
Data|Read Plate.
•
Mask a value. Within the Results window, the user cannot click the Mask box to
exclude wells from calculations.
•
Change a value. Within the Results window, the user cannot click the Change box to
change any measurement values.
•
Edit Protocol Parameters. If this box is checked, the user cannot edit any protocol
parameters.
•
Edit <individual protocol parameter>. The user cannot modify the particular protocol
parameter. For example, if ‘Edit Plate Layout’ is protected, the user cannot define or
assign new well IDs, expected concentration values, or dilution values. The user can,
however, select Protocol|Plate Layout to view the layout.
184 • Protocol Menu
User’s Guide to KC4™
Protocol Options
Select Protocol|Options to customize various options for this protocol, including automatic file
saving, report printing, and data exporting. Reporting, exporting, and calculation options can also
be modified here if the desired options are different than the default ones specified under
System|Setup.
Raw Data
Select Protocol|Options|Raw Data to specify automatic save and append options for raw data.
•
Enter the Save Directory in which to store plate data files (*.pla and *.glb) associated
with this protocol. Click Browse to search for and select the desired location.
Note: If files will not be saved automatically (see below), this location can be
changed manually by selecting Plate|Save or Plate|Save As.
•
Check Automatic Save after reading to automatically initiate the save plate data file
function when the read is complete.
Select Prompt for Name to prompt the operator to manually enter the file name after
the plate read, or select Auto Name to let KC4 automatically name the file, using the
system date and a sequence number. The use of ‘Auto Name’ ensures a unique file
name each time.
•
If the reader is equipped with a barcode scanner and "Read Barcode" is selected under
Protocol|Reading, the Use barcode as filename option is available. Select this option to
automatically save the data file using the barcode name.
User’s Guide to KC4™
Protocol Menu • 185
•
Select Automatic Append to Kinetic File after reading to append the current data to an
existing data file. This feature allows you to perform several endpoint reads on one plate.
You can temporarily remove the plate to incubate it externally, or perhaps to add
reagents.
Enter the target directory and file name in the Kinetic File Name field. Click the
Browse button to search for and select the desired file.
Note: ‘Automatic Append to Kinetic File’ is not available if the protocol specifies
pathlength correction or the pre-reading of a blank plate.
Report
Select Protocol|Options|Report to select the reporting engine and to set printing options.
•
Select the Engine for designing report templates:
KC4: This is the standard method for designing reports that KC4 has always
provided. The contents are customizable but the appearance is completely controlled
by KC4 (you cannot change the font style or size, for example).
Word: If your computer has Microsoft® Word 2000 installed on it, you can design
the report using Word and take advantage of all its formatting features.
Excel: If your computer has Microsoft® Excel 2000 installed on it, you can design
the report using Excel and take advantage of all of its formatting features. In
addition, you can create custom Excel formulas that reference KC4 data.
•
If you chose Word as the engine, choose a Document Formatting option to tell KC4
how to prepare documents for printing and print previewing.
If Standard is selected, document formatting is faster because KC4 does not apply
any custom font characteristics from the Plate Layout or Cutoffs dialogs. Well IDs
and cutoff symbols are printed using just the default font characteristics.
If Enhanced is selected, document formatting is slower because KC4 does apply
custom font characteristics to Well IDs and cutoff symbols.
•
Select Automatic Print after reading to automatically print reports when the plate read is
complete. This is available for all reporting engines.
•
Select Keep Open After Printing to keep Word or Excel open after the print preview or
after printing, to allow for temporary formatting adjustments. Note: To make permanent
changes, modify the report via Protocol|Report.
If Keep Open After Printing is enabled, the Protect option can be enabled to prevent
unauthorized changes to the document.
186 • Protocol Menu
User’s Guide to KC4™
Export
Select Protocol|Options|Export to select the export engine and define related parameters.
•
Select the Engine for designing export templates:
KC4: This is the standard method for designing export files that KC4 has always
provided. The contents are customizable but the appearance is completely controlled
by KC4 (you cannot change the font style or size, for example).
Word: If your computer has Microsoft® Word 2000 installed on it, you can design
export file templates using Word and take advantage of all of its formatting features.
Excel: If your computer has Microsoft® Excel 2000 installed on it, you can design
export file templates using Excel and take advantage of all of its formatting features.
In addition, you can create custom Excel formulas that reference KC4 data.
•
If you chose Word as the engine, choose a Document Formatting option of Standard or
Enhanced.
If Standard is selected, document formatting is faster because KC4 does not apply
any custom font characteristics from the Plate Layout or Cutoffs dialogs. Well IDs
and cutoff symbols are printed using just the default font characteristics.
If Enhanced is selected, document formatting is slower because KC4 does apply
custom font characteristics to Well IDs and cutoff symbols.
•
Enter the Export Directory in which to store exported files associated with this protocol.
Click Browse to search for and select the desired location. Note: Select the export text
file delimiter under System|Setup.
•
Select Automatic Export after reading to automatically export plate data to a file when
the read is complete.
If ‘Automatic Save after reading’ is selected, the export file name will match the
manually entered file name, the automatically generated file name, or the barcode,
depending on the automatic save parameters, and will have a .txt extension. If
‘Automatic Save after reading’ is not selected, the export file name will be
NONAME.TXT.
•
If the export engine is Excel, choose a File Format of Excel, Excel CSV, or Excel Text.
User’s Guide to KC4™
Protocol Menu • 187
•
Select Keep Open After Exporting to keep the Word or Excel application open after the
preview or after exporting, to allow for temporary formula and/or formatting
adjustments. (To make permanent changes, modify the export file via Protocol|Export.)
If Keep Open After Exporting is enabled, the Protect option can be enabled to
prevent unauthorized changes to the document.
Calculation
Select Protocol|Options|Calculation to select a weighting of standard deviation and tell KC4
whether or not to include out-of-range values in statistics calculations.
•
•
Select an option for the Weighting of Standard Deviation:
N-1
Used in calculations where the data is assumed to represent a sample from
a larger population.
N
Used in calculations where the data is assumed to represent the total
population from which the sample was taken.
Choose whether or not to use out of range values in statistics. Out of range values are
those values reported as ‘<xxxx’ or as ‘>xxxx’.
Example: SPL1 has three replicates reported with the following concentrations:
well A1 : 10.230
well A2 : <10 (out of range value)
well A3 : 10.450
If the option is not selected, the software will not use the concentration of well A2 in the
statistic calculations. Therefore the mean, standard deviation and CV of SPL1 will be
10.34, 0.155, and 1.5%, respectively. If the option is selected, concentration of well A2
is taken into account (the value of 10 is used in the statistic calculations). Therefore the
mean, standard deviation and CV of SPL1 will be reported as <10.22, 0.225, and 2.2%,
respectively.
Important! Exercise caution when enabling this option, as it can lead to biased results
particularly for standard deviation and CV. As a warning, statistic results arising out of
the use of this option are displayed in italic in the report.
188 • Protocol Menu
User’s Guide to KC4™
Display Menu
Overview
The Display menu allows you to check the verification status of your data and/or to activate the
Biograph module to view, adjust, and otherwise interact with test results as displayed on a curve
or set of curves.
Curve(s)
Select Display|Curves to load Biograph and view the curve or curves generated using the current
plate data. This option is available only if the protocol defines a standard curve or dilution curves.
•
When Biograph is loaded, every curve calculated for the current plate appears in a
window. See “Biograph” for information on changing the curve fitting method, deleting
standard points, printing the graph, and more.
•
To quit Biograph and return to KC4, select File|Exit.
User’s Guide to KC4™
Display Menu • 189
Calculation Warnings
Select Display|Calculation Warnings to view a list of data reduction calculation warnings issued
by KC4 against the current set of data. (If this list is empty, KC4 found no errors with the
calculations.)
Warnings and their probable causes include:
•
At least one validation condition failed.
The protocol defines validation conditions and one or more conditions has failed. Select
Display|Validation Results to see the current status of each condition.
•
Cutoffs are not in increasing order. Symbols can not be determined.
The protocol defines cutoff criteria, the results of which are not in increasing order and
therefore Symbols (+, -, Pos, Neg, etc.) cannot be determined. For example, if criteria is
defined like this:
and if CTL1=2.050 and CTL2=1.432, cutoffs are not in increasing order. Select
Protocol|Cutoffs to view/modify the criteria.
190 • Display Menu
User’s Guide to KC4™
•
Data In not available for Multi Plate Transformation #<1, 2, or 3>
A multi plate transformation references a data set that no longer exists. Select
Protocol|Multi Plate Transformation to choose another data set and then recalculate
results.
•
Data In not available for Single Plate Transformation #<1, 2, or 3>
A single plate transformation references a data set that no longer exists. Select
Protocol|Single Plate Transformation to choose another data set and then recalculate
results.
•
Data to interpolate are not available. Standards could not be calculated.
The data set specified for curve fit calculation may no longer exist. Select
Protocol|Curves, Curve Description. If the Data to Interpolate field contains asterisks,
this is the case. Choose another data set and recalculate results.
•
Data to process <#> not available for kinetic calculations.
The data set specified for kinetic analysis may no longer exist. Select Protocol|Kinetic
Analysis. If the Data to Process (1 or 2) field is empty, this is the case. Choose another
data set and recalculate results.
•
Log Axis cannot be used with a negative or null value.
KC4 was unable to calculate the curve because one or both Axes is set to calculate
logarithmically and an input value (such as a standard OD) is negative or null. Select
Protocol|Curves, Curve Fit to view the current curve parameters.
•
Not enough valid standards.
Each curve fit requires a minimum number of valid standards; either not enough
standards are defined in the plate layout, or following results calculation too many
standards were determined to be invalid.
•
Some cutoffs could not be calculated. Symbols could not be determined.
The cutoff criteria may reference a data set or well ID that no longer exists, or a
referenced well ID may contain an invalid value. Select Protocol|Cutoffs to examine the
current criteria.
•
The archived curve could not be loaded. Path is probably wrong.
This message was likely preceded by a "<pathname><filename>.gph was not found"
message. If this is the case, the protocol references a stored curve in the location
specified in that message. Select Protocol|Curves, Curve Description. Under Curve
Origin, if File is selected click Browse to locate the appropriate .gph stored curve file.
•
The curve fit method is not compatible with the standards. The curve could not be
calculated.
Every curve fit requires a minimum number of valid standards; there may not be enough
valid standards to support the current curve fit. Select Protocol|Plate Layout to add more
standard wells or select Protocol|Curves to choose another curve fit and then recalculate
results.
•
The curve is not strictly increasing nor strictly decreasing.
The curve is ambiguous; more than one X value was calculated for a particular Y value
(ex. more than one concentration was calculated for at least one OD/FU/LU).
User’s Guide to KC4™
Display Menu • 191
•
There must be at least two valid standards.
The plate currently contains only one valid standard and at least two are required to
calculate the curve. Check to see if any of the standard wells is currently masked or
contains an out-of-range or another non-numeric notation.
•
Values out of range. Log Axis cannot be calculated.
KC4 was unable to calculate the curve because one or both Axes is set to calculate
logarithmically and an input value (such as a standard OD) is out of range. Select
Protocol|Curves, Curve Fit to view the current curve parameters.
Validation Results
Selecting Display|Validation Results shows the current status of each of the Validation
conditions defined in the protocol:
•
•
The Condition column lists the Validation criteria defined in the protocol.
The Status column tells you whether or not each Condition has been met:
VERIFIED: The condition has been met.
NOT VERIFIED: The condition has not been met and results are therefore suspect.
COULD NOT BE EVALUATED: The condition could not be evaluated. This may occur
if, for example, a condition refers to an identifier with masked replicate values.
•
Important! If a validation condition receives the status NOT VERIFIED then the results
are suspect. The responsibility for any masking or editing of the data lies with the user.
192 • Display Menu
User’s Guide to KC4™
System Menu
Overview
The options available under the System menu allow you to:
•
Select the current reader and then set and test communication parameters.
•
Run an optics test or a universal plate test on the attached microplate reader and
view/print/store results.
•
Configure security settings, set up user accounts, and define protected functions.
•
Log into and out of KC4.
•
Set protocol default settings, including report and export engine, and the weighting
standard deviation.
•
Set global parameters, including the import/export text separator and the time format for
kinetics.
•
Customize the arrangement of the toolbar buttons.
•
Create and maintain microplate measurement information.
User’s Guide to KC4™
System Menu • 193
Reader Selection
Select System|Readers to select or change the current reader.
Important! Be sure to set and test communication parameters before attempting to read a
microplate.
•
Choose a reader from the Available Readers list, then define the Configuration, Port, and
Filters/Wavelengths parameters.
•
Click Port to define communication parameters including Port number, Transmission
Speed, Data Bits, Stop Bits, and Parity.
•
Click Filters/Wavelengths to align KC4’s wavelengths table with the reader’s internal
table.
•
If applicable, click Configuration to define the optical probe size so that it matches the
current hardware configuration.
•
When all parameters are set, click the Current Reader button. KC4 will attempt to
communicate with the attached reader. If communication fails, KC4 provides
information for solving the problem. Follow the instructions and then try again.
Note: If the current reader is set to “Clarity”, the Configuration and Port buttons are dimmed and
inaccessible because configuration parameters and port settings can only be accessed through the
Clarity™ PC software. The Filters/Wavelengths button is dimmed and inaccessible because it
does not apply to the luminometer.
Note: If the computer has more than one COM port, you can control more than one instrument at a
time by running multiple instances of KC4. See “Controlling Multiple Instruments
Simultaneously” at the end of this section for more information.
194 • System Menu
User’s Guide to KC4™
Port Selection
Use the Port Selection dialog to select the COM port number (1 to 9), and to access additional
communication parameters.
Clarity™ Microplate Luminometer users: Port settings can only be accessed through the Clarity
PC software. See the Clarity Operator’s Manual for more information.
•
Click the drop-down arrow and select the number that represents the COM Port KC4
should use when communicating with the reader.
•
Click Setup to define additional communication parameters.
Important! It is the user’s responsibility to define the correct parameters, otherwise
communication errors will likely occur. Ensure the communication settings match
those defined on the reader.
Transmission speed: Values commonly accepted by readers are between 2400 and
9600 bps.
Data Bits: Options range from 4-8 bits.
Parity: Options include No, Odd, Even and Mark.
Stop bits: Options include 1, 1.5 and 2 bits.
User’s Guide to KC4™
System Menu • 195
Filters/Wavelengths
Use the Wavelengths dialog to ensure that KC4’s wavelengths table is aligned with the reader’s
internal table.
Note: It is essential that you conform to the specific Bio-Tek reader procedures for altering the
configuration. When using filter-based readers, it is your responsibility to ensure that correct
filters are allocated to their respective positions otherwise incorrect data will result.
Clarity™ Microplate Luminometer users: KC4’s Filters/Wavelengths options do not apply to the
luminometer.
ELx-Series / PowerWave Series / µQuant
Note: To exchange wavelength information between KC4 and the instrument, the two must be
communicating. The currently selected reader and COM port are displayed at the top of this
dialog. If you need to change the COM port, click Cancel now to exit this dialog and then click
the Port button.
KC4’s currently-defined Wavelength values are displayed in nanometers in fields 1 through 6.
The settings here will be made available as default selections within the Protocol|Reading
Parameters dialog.
•
To change, download, and calibrate wavelength values, entered the desired values in
these fields and then click Send Wavelengths. The values will be downloaded to the
instrument, overwriting its existing wavelength table.
•
To retrieve wavelength values from the instrument, click Get Wavelengths. The values
will be uploaded to KC4, overwriting the values in this dialog.
196 • System Menu
User’s Guide to KC4™
FL600™
KC4 cannot automatically retrieve the wavelength information from the FL600, therefore this
information must be entered manually. Consult the Order Entry Form that was shipped with the
unit to determine which filters were originally installed. If this information cannot be found, note
the reader serial number then contact Bio-Tek Instruments.
Tip: If the exact filter setup is unknown, you can remove the FL600’s shroud to examine the
filters in the Excitation and Emission filter wheels. The filters should be imprinted with the
wavelength and bandwidth. Refer to the FL600 Hardware Guide for instructions.
•
A - F represent the 6 filter positions on the Excitation and Emission filter wheels.
Excitation refers to the light directed at the sample in the well. Emission refers to the
light emitted by the sample in the well.
•
Center is the center wavelength, in nanometers, of the installed filter.
•
Bandwidth is the filter bandpass width, in nanometers (nm), of the installed filter (does
not apply to absorbance filters).
Example: The Excitation/Center setting of 485/20 refers to a center wavelength of 485
nm with a the total bandpass of 20 nm. This combination will transmit light from 475 to
495 nm (10 nm on either side of the center).
•
A check in the Absorb. column informs the software that the corresponding filter is an
absorbance filter. If at least one Absorb. box is checked, the ‘HOLE’ notation appears in
the Center field for Excitation position F.
Note: It is not advised to check Absorb. for fluorescence filters because the bandpass is
not fixed at 8 to 10 nm but can be as wide as 50 nm or more. Some fluorescence filters
with narrower bandpasses, however, might be able to function as absorbance filters.
•
A check in the Lum. column informs the software that the corresponding filter will be
used for luminescence reading. If at least one Lum. box is checked, the ‘PLUG’ notation
appears in the Center field for the Excitation position E.
Note: When a Lum. box is checked, its corresponding Bandwidth field is not disabled,
although the bandwidth is not used during luminescence plate reading.
User’s Guide to KC4™
System Menu • 197
FLx800™
The FLx800 has one four-position excitation wheel and one four-position emission filter wheel.
Each filter wheel can contain any combination of filters, plugs, and empty locations, depending on
assay requirements. Use this dialog to specify the exact contents of each filter wheel. The settings
here will be made available as default selections within the Protocol|Reading Parameters dialog.
Important! It is essential that you conform to the specific Bio-Tek instrument procedures for
altering the filter wheel configuration. The FLx800 does not automatically detect which filters (if
any) are installed. Therefore, it is your responsibility to ensure that KC4’s wavelength table
exactly matches the contents of each filter wheel. For instructions for physically changing filters,
see the FLx800 operator’s manual.
Note: To exchange wavelength information between KC4 and the reader, the two must be
communicating. The currently selected reader and COM port are displayed at the top of this
dialog. If you need to change the COM port, click Cancel to exit this dialog now and then click
the Port button.
To change the current settings and download them to the instrument:
•
Enter wavelength values in the Center fields, or use the drop-down boxes to select PLUG
or HOLE. ‘PLUG’ indicates a solid plug in the filter wheel. A plug in the Excitation
filter wheel is typical for luminescence assays, to prevent ambient light from entering the
measurement chamber. ‘HOLE’ indicates an empty location in the filter wheel. An
empty location in the Emission filter wheel is typical for luminescence assays.
•
For each Center wavelength value, enter its accompanying Bandwidth. Note: This
information is printed on the side of each filter. See the FLx800 operator’s manual for
details. ‘Bandwidth’ is the filter bandpass width, in nanometers, of the installed filter.
For example, the Center/Bandwidth combination of 485/20 will transmit light from 475
to 495 nm (10 nm on either side of the center).
•
When all values have been entered, click Send Filters. The values will be downloaded
to the instrument, overwriting its existing wavelength table.
To retrieve filter wheel settings from the instrument:
•
Click Get Filters. The values will be uploaded to KC4, overwriting the values in this
dialog.
198 • System Menu
User’s Guide to KC4™
Synergy™ HT (Absorbance)
The Synergy HT performs absorbance reads in the range of 200 to 999 nm. Use this dialog to
specify and calibrate six wavelengths to be made available as default selections within the Reading
Parameters dialog.
Note: To exchange wavelength information between KC4 and the instrument, the two must be
communicating. The currently selected reader and COM port are displayed at the top of this
dialog. If you need to change the COM port, click Cancel to exit this dialog now and then click
the Port button.
KC4’s currently defined wavelength values are displayed in fields 1 through 6.
•
To change, download, and calibrate wavelength values, entered the desired values in
these fields and then click Send Wavelengths. The values will be downloaded to the
instrument, overwriting its existing wavelength table.
•
To retrieve wavelength values from the instrument, click Get Wavelengths. The values
will be uploaded to KC4, overwriting the values in this dialog.
User’s Guide to KC4™
System Menu • 199
Synergy™ HT (Fluorescence/Luminescence)
The Synergy HT instrument has one four-position excitation wheel and one four-position emission
filter wheel. Each filter wheel can contain any combination of filters, plugs, and empty locations,
depending on assay requirements. Use this dialog to specify the contents of each filter wheel. The
settings here will be made available as default selections within the Reading Parameters dialog.
Important! It is essential that you conform to the specific Bio-Tek instrument procedures for
altering the filter wheel configuration. The Synergy HT does not automatically detect which
filters (if any) are installed. Therefore, it is your responsibility to ensure that KC4’s wavelength
table exactly matches the contents of each filter wheel. For specific instructions on changing
filters, see the Synergy HT Operator’s Manual.
Note: To exchange wavelength information between KC4 and the instrument, the two must be
communicating. The currently selected reader and COM port are displayed at the top of this
dialog. If you need to change the COM port, click Cancel to exit this dialog now and then click
the Port button.
To change the current settings and download them to the instrument:
•
Enter wavelength values in the Center fields, or use the drop-down boxes to select PLUG
or HOLE. ‘PLUG’ indicates a solid plug in the filter wheel. A plug in the Excitation
filter wheel is typical for luminescence assays, to prevent ambient light from entering the
measurement chamber. ‘HOLE’ indicates an empty location in the filter wheel. An
empty location in the Emission filter wheel is typical for luminescence assays.
•
For each Center wavelength value, enter its accompanying Bandwidth. This is the filter
bandpass width, in nanometers, of the installed filter. For example, the
Center/Bandwidth combination of 485/20 will transmit light from 475 to 495 nm (10 nm
on either side of the center). The bandwidth is printed on the side of each filter. See the
SynergyHT Operator’s Manual for details.
•
When all values have been entered, click Send Filters. The values will be downloaded
to the instrument, overwriting its existing wavelength table.
200 • System Menu
User’s Guide to KC4™
To retrieve filter wheel settings from the instrument:
•
Click Get Filters. The values will be uploaded to KC4, overwriting the values in this
dialog.
Special filter position requirements for the Synergy™ HT with Injectors model:
•
For Read & Dispense protocols, KC4 offers an option to block the light between
measurements to inhibit photo bleaching.
Single filter set protocols: If light blocking is enabled, a blocking filter (Plug) must be
placed in the excitation filter wheel in one of the two positions next to the excitation filter
that is specified in the protocol.
Dual filter set protocols: If light blocking is enabled, two blocking filters (Plugs) must
be placed next to each other in the excitation filter wheel.
Reader Configuration
KC4 provides probe size information for some readers via the Reader Configuration dialog.
Clarity™ Microplate Luminometer users: KC4’s Reader Configuration parameters do not apply
to the luminometer.
The contents of this dialog vary depending on the currently selected reader. The following
parameters/options may be available:
•
The Absorbance Probe size field displays the diameter of the absorbance probe.
•
The Top Probe size field displays the diameter of the top probe. If the reader is an
FL600™, use the drop-down list to choose the size.
•
The Bottom Probe size field displays the diameter of the bottom probe. If the reader is
an FL600, use the drop-down list to choose the size.
•
If supported by the reader, click the Get Probe Sizes button to retrieve the size(s) of the
probe(s) currently installed in the attached reader.
User’s Guide to KC4™
System Menu • 201
Dispenser Configuration
For the Synergy™ HT with injectors, KC4 provides access to special configuration functions and
settings.
Initializing the Dispenser
If the dispenser module was connected before the reader was turned on, or if a system test was run,
the dispenser should initialize automatically. If for any reason the dispenser does not initialize
automatically, you can initialize it from KC4. This may be necessary if the module was connected
to the reader after the reader was turned on, or following the installation of a new syringe.
To initialize the dispenser:
•
In KC4, select System|Readers, click the Configuration button, and then click the
appropriate Dispenser tab (#1 or #2).
•
Click the Initialize button. The syringe drive will move to its home position and its
sensors will be verified.
Upon successful completion, the Status should show “Initialized.”
202 • System Menu
User’s Guide to KC4™
Setting the Injector Position
The instrument dispenses fluid using two injectors. The injector heads can be positioned to
dispense fluid from three possible locations: two adjacent to the top probe and one above the
bottom probe. The default positioning is one adjacent to the top and one above the bottom.
Moving the injector head from one position to another must be done manually (see the Synergy™
HT Operator’s Manual for instructions). After making an adjustment, the new position(s) must be
configured in KC4 for recognition by the reader.
To configure the injector positions in KC4:
•
In KC4, select System|Readers, click the Configuration button, and then click the
appropriate Dispenser tab (#1 or #2).
•
Click the radio button corresponding with the current physical injector head position,
Next to Top Probe or Above Bottom Probe.
•
Click OK to save the change and close the dialog.
Syringe Setup
KC4 provides access to special syringe setup functions for maintenance and calibration purposes.
Important! Do not change the syringe positions or calibrate the dispensers unless instructed to do
so as part of installation, upgrade, or maintenance.
Moving a Syringe to its Maintenance Position
If a syringe needs to be installed or replaced, it must first be moved to its Maintenance Position.
To do this using KC4:
•
In KC4, select System|Readers, click the Configuration button, and then click the
appropriate Dispenser tab (#1 or #2).
•
Click Move Syringe to its Maintenance Position.
•
The syringe plunger will move to its furthest-from-home position. The syringe can then
be disconnected form the drive bracket and unscrewed from the valve (see the Synergy
HT Operator’s Manual for detailed instructions.)
•
Click OK to close the dialog.
User’s Guide to KC4™
System Menu • 203
Controlling Multiple Instruments Simultaneously
If the computer has more than one COM port, more than one instrument can be controlled at a
time by running multiple instances of KC4.
Important! Please review the items under “Technical Issues” before attempting to control
multiple instruments simultaneously.
To control multiple instruments simultaneously:
•
Connect the instruments to the computer, one per COM port.
•
Launch a single instance of KC4.
•
Open System|Readers.
•
Select a reader model, define and test its Port settings, then set up its Filter/Wavelengths
table. Repeat for each attached reader.
•
Once communication is established for all attached readers, launch additional instances of
KC4 (one per reader).
•
Create, open, and run protocols within each KC4 instance.
Technical Issues
The use of certain functions is restricted when running multiple instances of KC4. These
restrictions are in place to maintain integrity with respect to communication, data retrieval, data
reduction, and reporting.
When running multiple instances of KC4:
•
Individual KC4 instances must control unique reader models. For example, KC4 cannot
control two ELx808 instruments at the same time. KC4 can control an ELx808
instrument and an ELx808-I instrument at the same time.
•
Under System|Readers a new Current Reader can be set but its communication and
filter/wavelength parameters cannot be changed.
•
Software filter/wavelengths tables cannot be updated at any time. For example, clicking
the Calibrate button from within the Protocol|Reading dialog generates an error message.
•
System|Setup parameters cannot be modified.
•
System|Users parameters cannot be modified.
•
System|Plate Formats parameters cannot be modified.
•
Only one PowerReports document can be edited/printed at a time.
204 • System Menu
User’s Guide to KC4™
Diagnostics
The Optics Test and Universal Plate Test should be performed routinely to validate and
document the consistency of a reader’s optical and electronic performance, alignment, accuracy,
reproducibility, and linearity.
Optics Test
The Optics Test (also referred to as the “System Test”) validates and documents the consistency
of reader optical and electronic performance. For most readers, this test can be performed from
within KC4.
To perform an Optics Test for all readers except the Clarity™ and the FL600™:
•
Ensure that the reader is properly connected to the computer, and that all communication
parameters are set correctly.
•
Select System|Diagnostics|Run Optics Test. The Run Optics Test dialog appears.
•
Enter the reader serial number, then click Start to run the test. When the test is complete,
the Identification dialog appears:
Enter the From, Company, Telephone, Fax Number, and Comments as
appropriate.
Click OK to continue. KC4 displays the results and then saves them in a database in
the KC4 folder.
User’s Guide to KC4™
System Menu • 205
Optics Test History
Following each Optics Test, KC4 saves the test results in a database (CALIB.MDB), located in the
KC4 folder.
To view historical Optics Test results:
•
Select Setup|Diagnostics|Optics Test History. The Optic Test History dialog appears:
To view a list of tests for a specific reader, click the Reader drop-down arrow.
To view a list of tests for a specific operator, click the Operator drop-down arrow.
To view test results, highlight the desired date/time from the list then click View.
To permanently delete test results from the database, highlight the desired date/time
from the list, then click Delete.
System Test (Clarity)
The System Test for the Clarity™ Microplate Luminometer must be performed using the Clarity
PC software. Refer to the Clarity Operator’s Manual for instructions.
Optics Test (FL600)
To perform an Optics Test with the FL600™:
•
Ensure that the reader is properly connected to the computer, and that all communication
parameters are set correctly.
•
Select System|Diagnostics|Optics Test.
•
The FL600 Reader Diagnostics dialog will appear. Click Start to run the test.
•
As each individual test completes, its pass/fail status appears on the display.
•
When the tests are complete, the results will resemble the following:
206 • System Menu
User’s Guide to KC4™
Click View Report to see a complete listing of commands exchanged between KC4
and the FL600 during the diagnostic procedures. This report can be printed and
faxed to Bio-Tek for help with technical support.
Click Close to close the dialog and return to the main display. Test results are
automatically saved in a database in the KC4 folder.
Universal Plate Test
The Universal Plate Test uses Bio-Tek’s Universal Calibration Test Plate to validate and
document the consistency of an absorbance reader’s alignment, accuracy, reproducibility, and
linearity.
Every Bio-Tek Universal Test Plate comes with a Calibration Test Plate data sheet. The data
sheet lists the expected absorbance reading for each location in the test plate at several different
wavelengths. This data must be entered into KC4 before the Universal Test Plate can be used.
To define Universal Test Plates:
•
Select Setup|Diagnostics|Define Universal Plates. The Define Universal Test Plates
dialog appears, listing the currently defined test plates by serial number.
To modify an existing plate, highlight the desired serial number then click Modify, or
to add a new plate, click Add.
User’s Guide to KC4™
System Menu • 207
The Edit Universal Plate dialog appears:
If this is a new plate, select the Plate Type then enter its Serial Number.
The top row of the wavelength grid shows eight unique wavelengths for use with
the Universal Plate Test. The default wavelengths correspond with the calibration
test plate data sheet. To modify one or more of these wavelengths click Wavelength
List.
Refer to the calibration data sheet that came with the test plate to determine the
expected OD for each test well at each wavelength. For each Location (C1, D4, E2,
F5, G3, H6), enter the OD associated with that well at each wavelength.
For readers that support spectral scanning, a Peak Wavelength value is required.
Enter the value in nanometers (nm) .
Click OK to save the changes and close the dialog, or Cancel to close the dialog
without saving changes.
To run the Universal Plate Test:
•
Ensure that the reader is properly connected to the computer, and that all communication
parameters are set correctly.
•
The reader should be equipped with the necessary filters to perform the test. Refer to the
calibration data table that came with the test plate to determine the required wavelengths.
•
Select System|Diagnostics|Run Universal Plate Test.
208 • System Menu
User’s Guide to KC4™
The Run Universal Plate Test dialog will appear.
Enter the Serial Number of the reader under test, or click the drop-down arrow to
choose an existing number.
Click the Universal Plate drop-down arrow to select the test plate to be used. If the
desired test plate is not in the list, select Setup|Diagnostics|Define Universal Plate
to define a new test plate.
If supported by the reader, the Peak Absorbance Search option is available. If the
box is checked, the Peak Absorbance Search is performed in addition to the standard
Universal Plate Test.
Choose the wavelength(s) to be included in the test by clicking the required cells in
the Test (Yes/No) column. Click in a cell to toggle Yes/No. Note: If ‘N/A’ appears
for a wavelength that you want to test, update the reader’s filter/wavelength table to
include the wavelength.
Enter the Operator name, or click the drop-down arrow to choose an existing name.
Enter Comments pertaining to this plate test (optional).
Click Run Test to begin. Follow the directions provided for loading and turning the
test plate. When the test is complete, KC4 displays the results and then saves them
in the CALIB.MDB database in the KC4 folder.
User’s Guide to KC4™
System Menu • 209
Universal Plate Test History
Following each Universal Plate Test, KC4 saves the test results in a database (CALIB.MDB),
located in the KC4 folder.
To view historical Universal Plate Test results:
•
Select Setup|Diagnostics|Universal Plate Test History. The Universal Plate Test
History dialog will appear.
To view a list of tests for a specific reader, click the Reader drop-down arrow.
To view a list of tests for a specific operator, click the Operator drop-down arrow.
To view test results, highlight the desired date/time from the list then click View.
To permanently delete test results from the database, highlight the desired date/time
from the list, then click Delete.
210 • System Menu
User’s Guide to KC4™
Security
KC4 provides several security options to help protect the system and its data from intentional or
inadvertent harm or loss. Select System|Security to view or edit these options:
Up to six feature tabs are available within the Security dialog. Availability depends on the current
KC4 license and whether or not the System Administrator is logged in.
KC4 v3.4
KC4 Signature
Users
Edit the Administrator’s password.
Edit the Administrator’s password. Establish and
edit individual user accounts and enable or disable
Multi-User Identification.
Login
(does not apply)
Enable and define various Login and Password
parameters for application in a multi-user
environment.
Protected
Functions
Determine which KC4 functions
shall be protected from use when
the Administrator is not logged in.
Determine which KC4 functions shall be protected
from use by Standard Users.
Audit Trail
(does not apply)
Enable or disable the appearance of an Audit Trail
Notification dialog each time an event is logged to a
Data or Protocol Audit Trail, or to the System Audit
Trail.
Signature
Reasons
(does not apply)
Define a list of terms for users to choose from when
signing files, to explain why they are signing.
Files
Select a Preferred Data File format.
Enable or disable the Secured Protocol and Data
File Storage feature and select a Preferred Data File
format.
User’s Guide to KC4™
System Menu • 211
User Accounts
This feature only applies to KC4 Signature
A significant component of a secure software environment is the ability to create individual user
accounts to ensure that only authorized users can gain access to the system and to any restricted
functions. KC4 Signature’s Multi-User Identification feature allows a site-designated System
Administrator to create and maintain user accounts.
Multi-User Identification is automatically enabled upon KC4 Signature software installation, and
it must remain enabled to ensure 21 CFR Part 11 compliance. If, however, you wish to disable
this feature, select System|Security and uncheck the Multi-User Identification box. This action
will cause all user accounts, except the Administrator’s, to be permanently deleted.
When Multi-User Identification is disabled, users are not required to login to KC4 but the
Administrator can still log in and perform the following tasks:
•
Change the Administrator’s password
•
Define system-level protected functions
•
Set audit trail notification options
•
Enable or disable the Secured Protocol and Data File Storage feature
•
Select a Preferred Data File Format
212 • System Menu
User’s Guide to KC4™
Creating and Modifying User Accounts (Administrator)
To create or modify a user account:
1.
Login as the System Administrator.
2.
Select System|Security and click New, or highlight the desired account name and click
Edit. The KC4 User Settings dialog will appear:
3.
Enter a unique ID using 1 to 16 alphanumeric characters. The user will enter or select this
ID when logging into KC4 and when signing files.
4.
Enter the user’s Full Name. This name will be associated with events logged by this
user’s actions and with signatures applied by this user.
5.
Choose a Group Membership.
Standard Users are denied access to protected functions. (The System Administrator
determines which functions shall be protected from use by Standard Users.)
Power Users are permitted access to protected functions. Power Users cannot
administer user accounts or modify Security settings.
6.
Enter a temporary password for the user in the New and Confirm fields.
Share the password with the user with instructions to change it as soon as possible.
7.
(Optional) Specify a folder location from which the user will most often access protocol
files. When the user selects Data|New Plate to initiate a plate read, the protocol selection
dialog will default to this Protocols Folder.
8.
Click OK to save the changes and return to the Security dialog.
User’s Guide to KC4™
System Menu • 213
Modifying User Accounts (Standard and Power Users)
Standard Users and Power Users are authorized to modify the Password and Protocols Folder
fields within their own accounts. (The ID, Full Name, Group Membership, and Status parameters
can only be modified by the System Administrator.)
To modify your user account, begin by logging into KC4 with your user ID and Password. Once
logged in, select System|Security, highlight your name, and click Edit. The KC4 User Settings
dialog will appear.
To set a new password:
1.
Enter your Current password (if one exists).
2.
Enter a New password using up to ten alphanumeric characters (no spaces). Note: If the
New password field is inaccessible, the correct Current password must first be entered.
3.
Enter the new password again to Confirm it.
4.
Click OK to save the password and close the dialog.
If the message "Password minimum length is not respected" appears, this means that
the System Administrator has set a minimum password length between 2 and 10
characters and the password you entered is too short.
Important! The System Administrator cannot retrieve your password if you forget it.
If you do forget your password, contact the System Administrator to request a
temporary one and then change it immediately.
214 • System Menu
User’s Guide to KC4™
To specify a Protocols Folder:
1.
Using the Browse button, select the folder location from which you will most often
access protocol files. When you select Data|New Plate to initiate a plate read, the
protocol selection dialog will default to the folder defined here. If no folder is specified,
KC4 will default to the most recently-accessed folder.
2.
Click OK to save the change and close the dialog.
Specifying a Protocols Folder is optional, and is only available if the ‘Secured
Protocol and Data File Storage’ feature is in use.
Deleting a User Account (Administrator)
To permanently delete a user account:
1.
Login as the System Administrator.
2.
Select System|Audit Trail to see if the user is currently logged into KC4. You can still
delete the account but it will not take effect until the user logs out and tries to log back in,
or tries to sign a file.
3.
Select System|Security.
4.
Highlight the desired account name and then click Delete. A confirmatory message will
appear; click Yes to confirm or No to cancel the process.
Note: The System Administrator’s account cannot be deleted.
Changing an Account's Status (Administrator)
The Status box within the KC4 User Settings dialog shows whether or not that user’s account is
currently locked, and allows the System Administrator to lock or unlock the account. When a
user’s account is locked, the user cannot log into KC4 and cannot sign files. A user’s account may
become locked through one of three ways:
•
Intentional lock by the Administrator through this dialog
•
Automatic lock if the user has exceeded the defined number of successive failed login
attempts
•
Automatic lock if the user’s password has expired
To unlock an account, uncheck the Status box and click OK to return to the Security dialog.
Click OK again to return to the main view. The change is effective immediately; the unlocked
user is now permitted log into KC4 and to sign files.
To lock an account, check the Status box and click OK to return to the Security dialog. Click OK
again to return to the main view. The change is effective immediately; the locked user is no
longer permitted to log into KC4 or to sign files.
•
Note: If the user is presently logged into another KC4 session, he or she is prevented
from signing files but is not automatically logged out.
User’s Guide to KC4™
System Menu • 215
Login and Password Parameters
This feature only applies to KC4 Signature
Select System|Security, Login to access Login and Password parameters. Only the System
Administrator is authorized to modify these parameters.
Note: The options shown as enabled above must be enabled to ensure 21 CFR Part 11
compliance. The options’ values, however, are site-definable.
To set Login parameters:
1.
Enable the desired Login parameters by checking the appropriate boxes.
2.
Specify the number of successive failed login attempts a user may make before
becoming locked out of KC4. This feature only applies to Standard and Power User
accounts; the System Administrator’s account cannot become locked. Only the System
Administrator is permitted to reinstate locked user accounts. Valid entry range: 2 to 10
attempts.
3.
Specify the number of minutes that the KC4 session may remain idle before it becomes
locked and users are required to re-login. A session is considered idle if the user is not
operating the keyboard or mouse and if the software is not currently controlling an
instrument. Valid entry range: 1 to 1440 minutes.
Note: For security purposes, KC4 prevents lower-level users from logging in after a
higher-level user has been automatically logged out of a session (if a user
intentionally logs out, this restriction does not apply).
For example, if the Administrator is automatically logged out, only the Administrator
is permitted to re-login. If a Power User is automatically logged out, only Power
Users and the Administrator are permitted to re-login. If a Standard User is
automatically logged out, any valid user may re-login.
216 • System Menu
User’s Guide to KC4™
4.
Enable Force user to type ID to require the user to manually type in his or her User ID in
the Login and Signature dialogs. Disable this option to permit the user to select his or her
User ID from a drop-down list.
5.
Click OK to save the changes and close this dialog.
To set Password parameters:
1.
Enable the desired Password parameters by checking the appropriate boxes.
2.
Specify the minimum number of alphanumeric characters required to define a valid
password. Valid entry range: 2 to 10 characters.
3.
Specify the number of days that a password may be used before it KC4 requires the user
to change it. If a user allows a password to expire, his or her account will become
locked. Only the System Administrator is permitted to reinstate locked user accounts.
Valid entry range: 1 to 10000 days.
4.
Specify the number of days in advance of the password expiration date that KC4 should
alert the user to the upcoming expiration. Valid entry range: 1 to the Password
Expiration value or 30, whichever is smaller.
5.
Specify the number of unique passwords that KC4 shall “remember” for each user’s
account to prevent that user from defining a recently used password. Valid entry range:
2 to 20 passwords.
6.
Click OK to save the changes and close this dialog.
User’s Guide to KC4™
System Menu • 217
Protected Functions
Select System|Security, Protected Functions to access a list of functions which the System
Administrator can protect from use by unauthorized users. Note: The picture below shows all
system-level protectable functions; it may or may not represent your present KC4 configuration.
KC4 Signature users: The functions shown as protected below must be protected to ensure 21
CFR Part 11 compliance.
Checked
functions may not be performed by unauthorized users.
•
With KC4 v3.4, an unauthorized user is anyone other than the System Administrator.
•
With KC4 Signature, an unauthorized user is one who is designated as a Standard User.
218 • System Menu
User’s Guide to KC4™
The following list includes all system-level protectable functions and describes the behavior the
unauthorized user can expect from KC4 for each function when it is protected:
•
Modify Reader Settings: Under System|Readers, the user can change the current reader
but cannot alter the Configuration, Port, or Filter/Wavelengths settings. The user will be
prevented from changing the current reader or from reading a plate if the action requires
an update to KC4’s filters/wavelengths table.
•
Modify Plate Formats: Under System|Plate Formats, the user can view the dimensions
of any plate. The user cannot create, modify, or delete plates.
•
Modify System Setup: Under System|Setup, the user can view the current Protocol
Defaults, Formats, Start-Up, and Database settings. The user cannot modify any of these
settings.
•
Repair/Compact Databases: Under System|Setup, Database tab, the user cannot access
the Repair/Compact buttons for the KCShared and KCLocal databases.
•
Define Universal Plates: Under System|Diagnostics|Define Universal Test Plates the
user can view the test plate settings but cannot add, modify, or delete a test plate. The
user can run a Universal Plate Test.
•
Delete Optics tests/Universal Plate tests from history: Under
System|Diagnostics|Optics Test History and Universal Plate Test History, the user can
view historical test results but cannot delete any of them.
The remaining functions are only found in KC4 Signature:
•
Delete System Audit Trail events after export: Via the Export button within the
System|Audit Trail dialog the user can export audit trail events to a text file but cannot
select the ‘Delete events after export’ option.
•
Create Folder (in secured file storage): When ‘Secured Protocol and Data File
Storage’ is enabled, the user cannot create new storage folders within the
System|Maintain Files, Protocol|Save As, and Data|Save As dialogs.
•
Rename Folder/File (in secured file storage): When ‘Secured Protocol and Data File
Storage’ is enabled, the user cannot rename folders or files within the System|Maintain
Files, Protocol|Save As, and Data|Save As dialogs.
•
Delete/Overwrite Folder/File (from secured file storage): When ‘Secured Protocol and
Data File Storage’ is enabled, the user cannot delete or overwrite existing folders or files
within the System|Maintain Files dialog.
•
Move Folder/File (in secured file storage): When ‘Secured Protocol and Data File
Storage’ is enabled, the user cannot move folders or files within the System|Maintain
Files dialog.
•
Import File (to secure file storage): When ‘Secured Protocol and Data File Storage’ is
enabled, the user cannot import (paste from clipboard or drag-and-drop from another
location) folders or files into the System|Maintain Files dialog.
•
Export File (from secured storage): When ‘Secured Protocol and Data File Storage’ is
enabled, the user cannot export folders or files out of the System|Maintain Files dialog.
User’s Guide to KC4™
System Menu • 219
Audit Trail Notification Options
This feature only applies to KC4 Signature
Select System|Security, Audit Trail to enable or disable the appearance of an Audit Trail
Notification dialog each time an event is logged to the Data, Protocol, or System Audit Trail.
Only the System Administrator is authorized to modify these options.
Enabling the User Notification options allows users to enter comments for an event. For example,
if a user modifies a protocol’s Plate Layout, the logged event is simply ‘Plate Layout changed.’ If
the Protocol Audit Trail notification option is enabled, however, the user can provide additional
text that will be logged with the event, such as "Added blank wells A1 and A2".
Here is a sample Audit Trail Notification dialog:
Here is a sample Protocol Audit Trail showing the comments entered above:
220 • System Menu
User’s Guide to KC4™
Signature Reasons
This feature only applies to KC4 Signature
Select System|Security, Signature Reasons to define a list of terms for users to select from when
signing protocols and data files to explain why they are signing. Only the System Administrator is
authorized to modify these terms.
Here is a sample ‘New Data Signature’ dialog showing the Reason drop-down list:
User’s Guide to KC4™
System Menu • 221
File Storage and Format Options
Select System|Security, Files to enable or disable the Secured Protocol and Data File Storage
feature and to select a Preferred Data File Format. Only the System Administrator is authorized to
modify these parameters.
Secured Protocol and Data File Storage
(KC4 Signature only) If this option is enabled, all protocol, plate data, and global data files are
stored inside a secure, shared-access database (named KCShared.mdb by default).
•
These files can only be accessed from within KC4; they cannot be viewed or otherwise
manipulated using separate file management software such as Windows Explorer.
•
Important: To ensure 21 CFR Part 11 compliance, Secured Protocol and Data File
Storage must be enabled.
•
Clarity™ Microplate Luminometer users: Clarity protocol files (with a BPF extension)
cannot be stored in KC4’s Shared Database. See page 77 for more information.
Preferred Data File Format
When a user chooses to read a plate, the default data file format will be PLA (raw data) or GLB
(raw data with protocol parameters), whichever is selected as the Preferred Data File Format in
this dialog.
•
For sites unconcerned with 21 CFR Part 11 requirements, the selection made here is
merely a matter of preference (pre-version 3.1 KC4 users may be more comfortable with
setting this parameter to .pla).
•
For 21 CFR Part 11-compliant sites, setting this parameter to .glb will help to streamline
the file signing process.
222 • System Menu
User’s Guide to KC4™
LogIn/LogOut
During typical use with KC4 v3.4, individual users do not log into the software. A System
Administrator can optionally log into change the Administrator password, specify protected
functions, and specify a preferred data file format.
During typical use with KC4 Signature, users must log into KC4 to gain access. Each user is
identified by a short User ID, a longer Full Name, and a 2 to 10 character Password (see “User
Accounts” on page 212 for more information). In addition, the System Administrator logs in to
set up and maintain user accounts, and to customize security options.
To log into KC4:
1.
Select System|LogIn or System|Administrator LogIn. The Login dialog will appear:
With KC4 Signature, the Login dialog also appears each time KC4 is launched.
2.
Click the User drop-down arrow and select your User ID.
If the User field does not have a drop-down arrow, type the ID directly into the field.
3.
Type your Password. For security purposes it will appear as asterisks on the screen.
4.
Click OK. KC4 will determine whether or not the User ID / Password combination is
valid, and will check to see if your account is "locked" for any reason. If the combination
is valid and the account is not locked, KC4’s main view will appear.
The system may be configured to automatically lock user accounts after a certain
number of successive failed login attempts. If your account is locked, contact the
System Administrator.
To log out of KC4 v3.4:
1.
Select System|Administrator LogOut. Control will return to KC4’s main view and all
unprotected functions will become accessible.
To log out of KC4 Signature:
1.
Select System|LogOut. The Login dialog will appear. KC4’s functions will remain
inaccessible until the Administrator or another user logs back in.
Recommendation: To maintain a secure environment, log out any time you are
away from the computer. The Login dialog will remain on the screen and you (or
another user) can log in at any time. The System Administrator may also set KC4 to
automatically log out after a set number of minutes if the system is idle.
User’s Guide to KC4™
System Menu • 223
Audit Trail
This feature only applies to KC4 Signature
Select System|Audit Trail to view a list of all system-level events stored in the shared and local
databases currently in use (to see which databases are in use, select System|Setup|Database):
The System Audit Trail displays Shared and Local events recorded for the following activities:
•
User login and logout
•
System security updates (user accounts, login and password parameters, protected
functions, audit trail notifications, and file location and format)
•
Plate format modifications
•
Universal test plate modifications
•
Reader settings changes (reader type, probe selection, communication parameters, and
filters/wavelengths)
•
System setup changes (protocol defaults, format settings, start-up options, and database
settings)
•
User-customizable application settings (such as toolbar and window characteristics)
A note on event sorting under the Shared tab:
The Shared tab displays events recorded by all computers on the network. The date of an event is
supplied by the computer on which the event occurs. Because the clocks on different computers
are not synchronized, consecutive events on different computers may not be recorded with
consecutive dates. Therefore, events under the Shared tab are displayed in the order of their
recording in the shared database, not in the order of the event dates.
224 • System Menu
User’s Guide to KC4™
Exporting Audit Trail Events
System audit trail events can be exported to a text file and optionally deleted from the System
Audit Trail. This feature may be used as part of a site’s procedure for archiving past events.
Deleting events after export helps to reduce the size of the KCShared and/or KCLocal databases.
Important: When audit trail events are exported to a text file, the text file does not reside within
KC4’s secure database; it resides as a single file in a user-designated location. It is the site’s
responsibility to ensure the security of exported system audit trail event files.
To export audit trail events:
1.
Select System|Audit Trail.
2.
Click the Shared or Local tab.
3.
Click Export. The Export Audit Trail Events dialog will appear:
4.
Enter a valid date range, in mm/dd/yyyy format.
5.
Select a destination folder for the text file.
6.
Enter an identifying file name.
7.
(Optional) Check Delete events after exportation to permanently remove the events
from the audit trail.
8.
Click OK to export the events.
If events were deleted after export, the System Administrator should do the following to compact
the database:
1.
Ensure that no KC4 users are currently logged into the shared database.
2.
Select System|Setup, Database.
3.
In the Shared Database or Local Database area as appropriate, click Repair/Compact,
and then click OK. When the process is complete, the database should be smaller in size.
User’s Guide to KC4™
System Menu • 225
Maintain Files
This feature only applies to KC4 Signature
KC4 Signature provides a Windows™ Explorer-style file maintenance utility for working with
files stored in the shared database. Authorized users can create folders, move files, delete files,
and so on, all within the secure KC4 environment. Events associated with all actions are
automatically logged in the System Audit Trail.
To access the file maintenance utility, select System|Maintain Files. All files and folders
currently stored in the shared database will be displayed:
•
The Maintain Files dialog contains two frames. The left frame displays the folder/file
tree. The right frame displays the contents of the current folder. All changes are made
within the right frame.
•
Click
to move up one folder level.
•
Click
to create a new folder.
•
Click on a folder
•
Highlight a folder or file and click
•
Click to update the view to show any folders/files that may have been added by another
user after you opened this dialog.
•
Click the right mouse button to open an editing menu (see “Edit Options” on the next
page).
226 • System Menu
to open it and view its contents.
to delete it.
User’s Guide to KC4™
Edit Options
Click the right mouse button to open an editing menu:
With the options available through this menu, authorized users can:
•
Export the highlighted item(s) to a user-designated location outside the shared database.
•
Delete (Cut) the highlighted item(s) and move them to the clipboard.
•
Copy the highlighted item(s) to the clipboard without deleting them.
•
Paste (or import) folders and files from the clipboard into the current folder.
•
Rename the highlighted folder or file.
•
Delete the highlighted item(s) without first moving them to the clipboard.
Exporting Files
Protocol, plate data, and global data files residing inside the shared database can be exported out
of the database through the Maintain Files dialog.
Important: When files are exported out of KC4’s secured database, they will reside on a disk or
the network in a user-designated location. It is the site’s responsibility to ensure the security of
exported files.
To export a file:
1.
Select System|Maintain Files. Locate the desired file.
2.
Highlight the file, click the right mouse button, and select Export to Disk.
The System Administrator can protect the use of the ‘Export’ and ‘Delete Files
After Extraction’ functions. If you are trying without success to export and/or delete
files, you may not be authorized to do so.
User’s Guide to KC4™
System Menu • 227
The Select a Folder dialog will appear.
3.
Select the target Drive and Folder (either the local hard drive or a network drive).
4.
(Optional) Check Delete files after extraction to permanently remove files from the
database after they are exported.
5.
Click OK to continue.
Files are exported in their proprietary formats. Files can be opened by a KC4
program that does not use the shared database, or they can be imported (pasted from
the clipboard) into another shared database.
If files were deleted after export, the System Administrator should do the following to compact
the database:
1.
Ensure that no KC4 users are currently logged into the shared database.
2.
Select System|Setup, Database.
3.
In the Shared Database area, click Repair/Compact, and then click OK. When the
process is complete, the database should be smaller in size.
Importing Files
Protocol, plate data, and global data files residing outside the shared database can be imported into
the database through the Maintain Files dialog. This includes any unsaved files that were
automatically saved to a temporary location following a database connection failure.
To import a file:
1.
Select System|Maintain Files. Locate and make current the folder in which the imported
file will reside.
2.
Launch Windows Explorer and locate the desired file.
3.
Highlight the file, click the right mouse button, and select Copy.
4.
Switch back to KC4. Place the cursor inside the Maintain Files dialog, click the right
mouse button, and select Paste.
The System Administrator can protect the use of the Import function. If you are
trying without success to import files, you may not be authorized to do so.
228 • System Menu
User’s Guide to KC4™
Registration/Upgrade
Twenty one (21) days after installation (when the Trial mode is over), your KC4 software will
switch to Demonstration mode unless you enter a valid password. To apply for and enter a
password:
1.
Select System|Registration. The Registration dialog will appear:
2.
If the License Number field is empty, enter the license number provided with your KC4
software package.
3.
Click the Registration Card to verify the license number and then open a form. Enter
the required information and send the form to Bio-Tek via fax or email.
4.
Bio-Tek will respond with a password via fax or email. When you receive your password
from Bio-Tek, enter it in the Password field and click OK.
KC4 Reinstallation
If you are reinstalling KC4, you must fill out and submit a Reinstallation Form to obtain another
password. Reinstallation is defined as:
•
Re-installing a KC4 version on the same PC (for example, after a hard disk replacement).
•
Un-installing a KC4 version from one PC and installing the same version on another PC.
A Reinstallation Form (.doc and .txt formats) complete with instructions can be found in the root
KC4 folder.
User’s Guide to KC4™
System Menu • 229
Setup
Select System|Setup to set various global parameters, including reporting and exporting engines,
import/export separator, and startup options.
Protocol Defaults
Select System|Setup|Protocol Defaults to specify several default settings for new protocols:
•
Select the default Reporting and Exporting Engines.
With KC4’s standard reporting and exporting engines you can customize the contents
(and to some extent the appearance) of printed reports and exported plate data. All KC4
users have access to these standard functions. Reporting and exporting with userdesigned Microsoft® Excel 2000 and Word 2000 documents is supported with KC4’s
PowerReports feature. For more information on the Word/Excel reporting options, see
PowerReports near the end of this manual.
If Word was chosen as the reporting and/or exporting engine, choose a Document
Formatting option of Standard or Enhanced to tell KC4 how to prepare documents for
printing and print previewing. The default selection is Standard.
If Standard is selected, document formatting is faster because KC4 does not apply
any custom font characteristics from the Plate Layout or Cutoffs dialogs. Well IDs
and cutoff symbols are printed using just the default font characteristics.
If Enhanced is selected, document formatting is slower because KC4 does apply
custom font characteristics to Well IDs and cutoff symbols.
230 • System Menu
User’s Guide to KC4™
•
•
Select the Weighting of Standard Deviation:
N-1
Used in calculations where the data is assumed to represent a sample
from a larger population.
N
Used in calculations where the data is assumed to represent the total
population from which the sample was taken.
Choose whether or not to use out of range values in statistics. Out of range values are
those values reported as ‘<xxxx’ or as ‘>xxxx’.
Example: SPL1 has three replicates reported with the following concentrations:
well A1 : 10.230
well A2 : <10 (out of range value)
well A3 : 10.450
If the option is not selected, the software will not use the concentration of well A2 in
the statistic calculations. Therefore the mean, standard deviation and CV of SPL1
will be 10.34, 0.155, and 1.5%, respectively. If the option is selected, concentration
of well A2 is taken into account (the value of 10 is used in the statistic calculations).
Therefore the mean, standard deviation and CV of SPL1 will be reported as <10.22,
0.225, and 2.2%, respectively.
Important! Exercise caution when enabling this option, as it can lead to biased
results particularly for standard deviation and CV. As a warning, statistic results
arising out of the use of this option are displayed in italic in the report.
Formats
Select System|Setup|Formats to choose an import/export separator and a time format for
kinetics.
User’s Guide to KC4™
System Menu • 231
•
Select an Import/Export Separator (delimiter) for use in export file formats. Choose
semi-colon, comma, or tab, or specify a custom separator (such as | ) in the Other field.
Before importing a plate data file, ensure that the selected Import/Export Separator
matches what is used in the file.
•
KC4 offers the following Time Formats for Kinetics:
HH:MM:SS
Average duration kinetics
HHH.MM
Long duration kinetics
SSSS.sss
Average duration kinetics, reported in seconds and milliseconds
SSSSSSSS
Average duration kinetics, reported in seconds
sssssssss
Fast kinetics, reported in milliseconds
When involved in calculations such as Transformations, Cutoffs and Validation
conditions, time values are always expressed in milliseconds, regardless of the time
formats chosen in the System|Setup menu. If you need to use other units, define a
single-plate transformation to make the conversion.
When time values are used on the Y axis of a Standard Curve, they are always
expressed in seconds.
Start-Up
Select System|Setup|Start-Up specify behavior each time KC4 is launched.
•
Specify whether or not to start Word or Excel when launching KC4. Keeping these
applications open saves time when creating new report and export templates; you won’t
have to wait for them to load each time you make a change.
•
If you are running KC4 with an external robotic application (such as Bio-Tek’s Bio-Stack
PC Control software), click Minimize KC4 when running in OLE mode. When KC4
starts it will automatically minimize, permitting access to the external application.
232 • System Menu
User’s Guide to KC4™
Database
KC4 uses three different databases for storing information. These databases are secure, meaning
that they can only be opened and updated through KC4.
•
KCShared.mdb contains security and plate format information. With KC4 Signature,
this database also contains user accounts; protocol, plate data, and global data files and
their associated audit trails; and a system audit trail for shared events. Following
installation, KCShared.mdb is stored on the computer’s hard drive but it can be moved to
a network location for secure storage and multi-user access. This database may also be
copied and renamed (if, for example, your site requires a separate shared database for
each project).
•
KCLocal.mdb contains all of the settings defined by the user under System|Readers and
System|Setup. With KC4 Signature, this database also contains an audit trail for local
events. KCLocal.mdb is stored on the computer’s hard drive, and it cannot be moved or
renamed.
•
CALIB.mdb contains Optics and Universal Plate Test setup values and saved test results.
CALIB.mdb is stored in the root KC4 folder. It cannot be moved or renamed, and there
are no special functions associated with it.
Select System|Setup|Database to access information and functions related to KC4’s shared and
local databases.
Shared Database
The Shared Database area contains the current location and name of the shared database, a
Restore Connection button, and a Repair/Compact button.
The Path setting must be changed if the location and/or the name of the KCShared.mdb file
changes. For example, following KC4 installation the System Administrator is advised to make a
copy of the installed KCShared.mdb file and store the copy on a shared location on the network.
To gain access to this shared file, the Path setting must change to reference the network location.
•
Before changing the Path, a KCShared.mdb file must already exist in the new location;
this feature does not create a new KCShared.mdb file.
•
The shared database name is not required to be KCShared.mdb. If your site requires the
use of multiple shared databases (one per project, for example), the file can be renamed
via Windows Explorer. The .mdb extension IS required.
User’s Guide to KC4™
System Menu • 233
•
Tip: With some operating systems, KCShared.mdb is installed in the Application Data
folder under \Documents and Settings\All Users. If you are looking for the Application
Data folder on your computer and can’t find it, it is probably hidden. To reveal this
folder, open Microsoft Explorer and select Tools|Folder Options|View. Make sure that
‘Show hidden files and folders’ is enabled.
To change the shared database Path:
1.
Click Browse. A standard file open dialog will appear.
2.
Locate the desired shared database file and click Open. Control will return to the
Database dialog and the Path field will fill in with the new name.
3.
Click OK. A confirmatory message will appear, stating that the database change will not
take effect until KC4 is closed down and relaunched.
“Modify system setup” is a protectable function. If you have tried without success to
change the Path, you may not be authorized to do so. Contact your KC4 System
Administrator.
4.
Click Yes to close KC4.
5.
Multi-user sites: Repeat these steps for all computers that use the shared database.
The Restore Connection button becomes active after a critical database error is detected (if the
database path did not change). This button must be clicked after a critical database error occurs.
For example, if the network connection drops while KC4 is open, a critical database error can
occur because the shared database is no longer accessible. When the network connection is
restored, click Restore Connection to reconnect KC4 with the database.
It may be necessary to click the Restore Connection button several times to successfully restore
the database connection. If this message appears:
•
Click Retry to try to restore the database connection again.
•
Click Cancel to abort the process and regain control of KC4.
•
Click Quit KC4. Unsaved files will be saved as temporary files on the computer’s hard
drive; the pathname will be provided. These files can be renamed and moved using
Windows Explorer and optionally imported into the shared database.
The Repair/Compact button should be used following these events:
•
After reconnecting to the shared database following a database error, in case the database
has been corrupted.
•
(KC4 Signature) After System Audit Trail events are exported and deleted, to reduce the
size of the database.
234 • System Menu
User’s Guide to KC4™
•
(KC4 Signature) After files are exported and deleted from the Maintain Files dialog, to
reduce the size of the database.
•
Note: Repair/Compact may only be performed if no other KC4 session is using this file.
When Repair/Compact is clicked, KC4 performs a safety backup of the database in its present
location (KCShared_yymmdd_hhmmss.mdb) before beginning the repair/compact procedure.
Local Database
The Local Database area of the Setup|Database dialog contains the current location of
KCLocal.mdb and a Repair/Compact button.
The Path setting for KCLocal.mdb cannot be changed. If the pathname is larger than the width of
the field, click once in the field and press the right arrow button on your keyboard to scroll to the
right. Knowing where KCLocal.mdb is stored is useful if your site requires a periodic back up of
this file.
•
Tip: With some operating systems, KCLocal.mdb is installed in the Application Data
folder under \Documents and Settings\All Users. If you are looking for the Application
Data folder on your computer and can’t find it, it is probably hidden. To reveal this
folder, open Microsoft Explorer and select Tools|Folder Options|View. Make sure that
‘Show hidden files and folders’ is enabled.
The Repair/Compact button should be used following a KCLocal.mdb database error. When the
button is clicked, KC4 performs a safety backup of the database in its present location (KCLocal_
yymmdd_hhmmss.mdb) before beginning the repair/compact procedure.
User’s Guide to KC4™
System Menu • 235
Database Upgrade Utility
Use the Database Upgrade Utility to update Shared, Local, and Calibration databases created
with earlier versions of KC4 so that they will be compatible with the current version of KC4. For
example, if you are upgrading from KC4 version 3.1 to 3.4, your version 3.1 databases must be
converted before they can be used by KC4 version 3.4. This utility runs automatically during
installation. It can also be run manually from the System|Setup, Database dialog.
To run the Database Upgrade Utility:
1.
If possible, identify the locations and names of all databases to be upgraded.
2.
Close all other instances of KC4 running on the PC.
3.
If upgrading a database located on a network drive, make sure that no other KC4 users
are currently accessing that database.
4.
Close any open, unsaved KC4 files.
5.
Select System|Setup and click the Database tab.
6.
Click the Database Upgrade Utility button.
KC4 automatically begins a search of the fixed, non-removable drives (e.g., diskette,
ZIP, JAZ, and network drives are not automatically searched). KC4 searches for the
following files: KCLocal.mdb, KC*.mdb, and Calib.mdb. Note: This automatic
search will not find Shared database files if their names do not begin with “KC”.
If you know the names and locations of the databases you wish to upgrade, and/or if
the databases reside on a drive that is not automatically searched, click the Stop
button.
When the search is complete, or when the Stop button is clicked, the Database Upgrade Utility
dialog appears:
•
Click the drop-down arrows to see which databases KC4 found. Click on the desired
database name to select it for upgrading.
•
Click the Browse button to search for a database. When the database is located,
highlight it and click Open. KC4 will insert the name in the appropriate field (Shared,
Local, or Diagnostics).
•
Click Start Upgrading to begin the process. When prompted, enter the name(s) for the
converted databases. (The original databases will remain unchanged).
•
When the process is complete, restart KC4. The upgraded database(s) will be in use.
236 • System Menu
User’s Guide to KC4™
Toolbar
The Toolbar contains buttons that, when clicked, allow quick access to KC4 functions.
Accessibility of a particular toolbar button depends on whether or not its function may be used in
the current environment. For example, the Biograph button is accessible only if results are
displayed and a curve has been generated.
When KC4 is installed, the toolbar contains a set of default buttons. These buttons can be
customized by selecting System|Toolbar.
•
To add a button to the toolbar, highlight the desired button under Available Toolbar
Buttons, then click Add.
•
To remove a button from the toolbar, highlight the button under Current Toolbar Buttons,
then click Remove.
•
To change the position of a button in the Current toolbar list, highlight it then click Move
Up or Move Down.
•
To restore the toolbar to its default buttons, click Reset.
•
To save the changes and update the toolbar, click Close.
On most displays, the toolbar can easily display up to 16 icons. All possible icons are shown and
described below and on the following pages:
Create a protocol using the Protocol Wizard. This is offered as an alternative to
using the Protocol Menu. (Protocol|Wizard)
Initialize memory for a New data file. Click this button before reading a plate.
(Data|New)
User’s Guide to KC4™
System Menu • 237
Read a plate (or enter data manually, or read data from a file). Before clicking this
button, ensure that the reader is connected to the computer, and that the
communication parameters are properly configured. (Data|Read Plate)
Save a data file. Plate data files (raw data) use the extension *.PLA. Global data
files (raw data + protocol parameters) use the extension .GLB. Selection of the
target directory is possible using the Browse facility. (Data|Save)
Sign a data file. In a multi-user system, global data files (raw data + protocol
parameters) can be electronically signed. (Data|Sign)
Print the results report. The appearance of the button depends on the Report
Engine setting under Protocol|Options (Excel, Word, or KC4). Report templates
are created under Protocol|Report. (Data|Print)
Preview the results report before printing. The appearance of the button depends
on the Report Engine setting under Protocol|Options (Excel, Word, or KC4).
Report templates are created under Protocol|Report. (Data|Print Preview)
Export the plate data to a file. The appearance of the button depends on the Export
Engine setting under Protocol|Options (Excel, Word, or KC4). Export templates
are created under Protocol|Export. (Data|Export)
Enter Sample IDs associated with the plate. This can be done before a plate is
read, or after a plate read when results are displayed in KC4. (Data|Samples)
Define the text to replace the prompts in the Data|Plate Description and Data|Read
Plate dialogs. (Protocol|Plate Description)
Define or modify reading parameter settings. If this button is inaccessible, select
Protocol|New Protocol to select a protocol type, then click Settings to define the
reading parameters. (Protocol|Reading)
Configure the plate Layout by assigning a meaningful identifier to each well.
Typical identifiers include “BLK”, “STD”, and “SPL”. Standard concentrations
and dilutions are also defined here. (Protocol|Plate Layout)
For kinetic, spectrum, or scanning protocols, define related Analysis parameters
for data reduction. (Protocol|<type> Analysis)
Define or modify Single Plate Transformations to perform calculations within the
same plate/data set. Typical uses include using specific wells as local blanks for
other wells and calculating ratios between wells. (Protocol|Single Plate
Transformations)
Define or modify Multi Plate Transformations to perform calculations utilizing
more than one plate/data set. For example, create a multi plate transformation to
subtract the 630 nm measurement values from the 405 nm measurement values in
all wells. (Protocol|Multi Plate Transformations)
238 • System Menu
User’s Guide to KC4™
Define or modify Curve fitting parameters. Configurable parameters include Data
to Interpolate, Curve Fit Method, and Interpolation values. This button is available
if well identifiers with concentrations or dilutions are defined in the Plate Layout.
(Protocol|Curve)
Define up to five sets of Validation criteria to assess the suitability of select wells.
If the criteria are not met, KC4 generates a warning to indicate that results are
suspect. (Protocol|Validation)
Define Cutoffs to screen selected data into defined categories based on fixed or
variable threshold limits. During data reduction, results are evaluated against the
cutoff criteria and each well is assigned a user-defined symbol. (Protocol|Cutoffs)
Create a report template. The appearance of the button depends on the Report
Engine setting under Protocol|Options (Excel, Word, or KC4). The template is
stored with the protocol when it is saved. (Protocol|Report)
Create an export template. The appearance of the button depends on the Export
Engine setting under Protocol|Options (Excel, Word, or KC4). The template is
stored with the protocol when it is saved. (Protocol|Export)
Define various protocol options including the report and export engines, and
automatic save, append, report, and export procedures. (Protocol|Options)
Specify which functions in the currently open protocol shall be protected from use
by unauthorized users. (Protocol|Security)
Open the Maintain Files dialog to create folders, move files, delete files, and so on,
within the shared database. (System|Maintain Files)
Launch the Biograph application to view the calculated curve(s). This button is
accessible only if results are displayed and a curve has been successfully generated.
(Display|Curve)
Control the attached reader’s door/carrier movement or incubation. This button is
accessible only if these functions are supported by the current reader.
(System|Reader Control)
User’s Guide to KC4™
System Menu • 239
Plate Format
This dialog lists all of the plate types currently stored in the shared database. For each plate type,
the database contains formatting information such as width, length, height, and the number of
rows and columns. When a plate read is initiated, KC4 checks to see which plate type is selected
in the Reading Parameters dialog and then sends the appropriate formatting information to the
reader. The reader uses this information to precisely position the plate when taking measurements.
Not all readers support all of the plate formats in this list. Consult your reader’s operator’s manual
for a list of supported plate sizes.
KC4 comes with format information for more than forty industry-standard microplates. You can
add your own microplates by selecting System|Plate Formats:
The Plate Formats dialog lists the currently defined plate types.
•
To add a new plate, click New.
•
To modify a plate, highlight the plate name and click Modify.
Warning! Modifying the dimensions of the installed plate types is not recommended;
consider adding a new plate type instead. Please contact Bio-Tek with any questions
regarding the current dimensions.
•
To view the format information for any plate, highlight the plate name and click View.
•
To delete a plate, highlight the plate name and click Delete.
•
To export plate format information to a text file, click Export. The export function is
useful if you wish to add custom plate formats from earlier KC4 versions. See
“Exporting/Importing Plate Formats” for more information.
•
To import the contents of an external plate database file (either a .txt or a .pld file), click
Import. The import function is useful if you wish to work with custom plate formats
created with an earlier KC4 version. See “Exporting/Importing Plate Formats” for more
information.
240 • System Menu
User’s Guide to KC4™
Plate Description
When creating or modifying a plate, information is entered in the Plate Description dialog:
•
Name stores descriptive, unique text appropriate for the microplate. This name appears
in the Plate Type selection list within the Reading Parameters dialog.
•
Manufacturer stores the name of the microplate manufacturer.
•
Catalogue # stores the microplate identification number.
•
Length is the longest dimension of the plasticware (the x axis).
•
Width is the shorter dimension of the plasticware (the y axis).
•
Top Left X is the distance from the left side of the plate to the center of well A1.
•
Top Left Y is the distance from the top of the plate to the center of well A1.
•
Bottom Right X is the distance from the left side of the plate to the center of the last well
on the plate. In the case of a 96-well plate, this would be H12.
•
Bottom Right Y is the distance from the top of the plate to the center of the last well on
the plate. In the case of a 96-well plate, this would be H12.
•
Nb Columns is the number of vertical columns of wells when viewing the plate in its
normal orientation. In the case of a 96-well plate, this would be 12.
•
Nb Rows is the number of horizontal rows of wells when viewing the plate in its normal
orientation. In the case of a 96-well plate, this would be 8.
•
Well Diameter is the diameter of any well.
•
Height is the distance from the bottom mounting surface of the plate to the top face of the
plate.
User’s Guide to KC4™
System Menu • 241
Microplate Measurement
When creating or updating plate dimensions, it is essential that precise measurements be taken.
Use calipers with precision to 0.01 microns to gather the following measurements. A standard
ruler is not precise enough.
Important! Dimension values are used by the software to calculate reading positions. Any
inaccuracies in these dimensions could significantly affect your results.
The following diagram demonstrates the proper method for taking measurements for a standard
96-well plate.
Top Left X
Diameter
Top Left Y
Width
Bottom
Right Y
Bottom Right X
Length
Exporting/Importing Plate Formats
If the following message appears when opening a protocol created with a 3.0x/2.x version of KC4,
the protocol references a custom plate format not currently found in KC4:
If you wish to work with 3.0x/2.x protocols that reference custom plate formats, you have two
options:
#1 Export the current plate format information into a text file, manually add the custom plate
format(s) to this file, and then re-import the file back into the shared database. This will
both preserve your current plate format information and add the custom plate formats.
#2 Import the plate database (KC4_32.pld) from the earlier KC4 version. Warning! This
will overwrite the current plate format information in the shared database.
242 • System Menu
User’s Guide to KC4™
To manually update KC4 with the custom plate formats from the earlier KC4 version (option #1):
1.
Locate the KC4_32.pld file containing the desired custom plate formats (this file can be
found in the root KC4 3.0x/2.x folder). Open this file using a text editor such as
Notepad. Keep this file open in the background.
2.
Launch KC4 v3.4 / KC4 Signature.
3.
Select System|Plate Formats and click Export. The Export plate database as dialog
will appear. Tip: If the Plate Formats option is not available in the System Menu, this
means that the currently selected reader does not support customizable plate formats.
Select System|Readers and choose a more appropriate reader.
4.
Using the file tree, choose a target location. Enter an identifying file name and click
Save.
5.
Open this file using a text editor such as Notepad.
6.
Switch to the open KC4_32.pld file. Locate the custom plate format; it will resemble the
following:
7.
Highlight the entire line, click the right mouse button, and select Copy.
8.
Switch to the open <plate database>.txt file. Scroll to the end and place the cursor on the
first available blank line. Click the right mouse button and select Paste. The custom
plate format should appear.
9.
Save the <plate database>.txt file and close it. Return to KC4.
1034789489=Custom Format 1;126570,84830;25150,22860,102310,61740;2,3;34990;Bio-Tek;12345
10. Within the System|Plate Formats dialog, click Import. Locate the modified <plate
database>.txt file and click Open. The Plate Formats list will update to show the
contents of this file.
Important: KC4 does not verify that the imported file has a valid structure or contains
valid values. It is the user’s responsibility to verify the structure and content of the plate
formats after they have been imported.
11. Click OK to return to the main view.
Note: KC4 version 3.02 introduced a new Plate Height dimension for plate formats.
This is the distance from the bottom mounting surface of the plate to the top face of the
plate. If you are importing a plate database from an earlier KC4 version, be sure to
measure the plate height and add it via the Modify button in the Plate Formats dialog.
User’s Guide to KC4™
System Menu • 243
To import the plate database from the earlier KC4 version (option #2):
1.
Launch KC4 v3.4 / KC4 Signature.
2.
Select System|Plate Formats and click Import. The Select plate database file to import
dialog will appear. Tip: If the Plate Formats option is not available in the System Menu,
this means that the currently selected reader does not support customizable plate formats.
Select System|Readers and choose a more appropriate reader.
3.
Set Files of Type to ‘KC4 3.0x/2.x Plate Database (*.pld)’.
4.
Use the file tree to locate the KC4_32.pld file from the previous KC4 version, and then
click Open. The Plate Formats list will update to show the contents of this file.
Important: KC4 does not verify that the imported file has a valid structure or contains
valid values. It is the user’s responsibility to verify the structure and content of the plate
formats after they have been imported.
5.
Click OK to return to the main view.
Note: KC4 version 3.02 introduced a new Plate Height dimension for plate formats.
This is the distance from the bottom mounting surface of the plate to the top face of the
plate. If you are importing a plate database from an earlier KC4 version, be sure to
measure the plate height and add it via the Modify button in the Plate Formats dialog.
244 • System Menu
User’s Guide to KC4™
Reader Control
If supported by the current reader, Reader Control is available under the System menu. This
feature can be used to view information about the attached reader, control the reader door or
carrier, and to control incubation.
Information
Before contacting Bio-Tek for technical assistance, use the Information dialog to retrieve the
serial number, part number, software version, and basecode checksum from the attached reader.
Door/Carrier
The Door/Carrier functions are reader-dependent. The attached reader must have robotic
capability (such as the ELx808™ or FL600™).
•
Select Plate In to move the plate carrier into the plate reading chamber and close the door
(if applicable).
•
Select Plate Out to move the plate carrier out of the plate reading chamber and open the
door (if applicable).
User’s Guide to KC4™
System Menu • 245
Pre-Heating
Use the Pre-Heating dialog to set the reading chamber temperature, turn the incubation unit on or
off, or to set a timer to beep after a specified duration or when the requested temperature has been
reached.
•
To set the reading chamber temperature, enter the temperature in the Requested field
and then check On to begin pre-heating. On enables the incubation unit; the temperature
will rise or fall to the Requested temperature, as appropriate. Off disables the incubation
unit; the temperature will fall to ambient.
•
The Actual field reports the current temperature (Celsius) of the incubation unit.
•
For the FL600™, the Ambient field reports the current temperature of the outside
environment, via a sensor located within the reader (refer to the FL600 user manual for
sensor location information).
•
The Total Time field reports the total time elapsed since the On box was checked.
Associated with the Total Time field, the Beep after checkbox and field can be enabled
to request that KC4 "beep" continuously after a specified duration. The duration begins
when the incubation unit is turned on.
•
The Time since reached field reports the total time elapsed since the incubation
temperature has reached the Requested temperature. Associated with the Time since
reached field, the Beep after checkbox and field can be enabled to request that KC4
"beep" continuously after a specified duration. The duration begins when the incubation
temperature reaches the Requested temperature.
•
The REACHED indication appears when the incubation temperature has reached the
Requested temperature.
Note: As the temperature of the incubation unit approaches the Requested temperature, it may
take a few minutes to settle within an acceptable tolerance (+/- 0.5° C). During this settling
period, the REACHED indication may appear for a few seconds, disappear, then reappear moments
later. The Time since reached field will automatically reset to 00:00:00 each time the
REACHED indication appears. If the Beep after checkbox associated with Time since reached
field is enabled and set to, for example, 10 minutes, KC4 will "beep" 10 minutes after the
incubation temperature has consistently settled within +/- 0.5° C of the Requested temperature.
246 • System Menu
User’s Guide to KC4™
Dispenser
Synergy™ HT with injectors
Use the Dispenser dialog to perform dispenser-specific functions such as prime and purge.
Priming the Dispenser
Before running a Read & Dispense protocol, the syringe(s) and tubing must be primed with the
fluid to be used.
To prime the dispenser:
•
Fill the supply bottle with a sufficient volume of the fluid to be used for the prime.
•
Important! Place the priming plate on the carrier (Bio-Tek PN 7142002).
•
In KC4, select System|Reader Control and click the Dispenser tab.
•
Select the Dispenser (1 or 2) associated with the supply bottle.
•
Enter the volume to be used for the prime, from 5 to 5000 µl.
•
Select a prime Rate in µl/second.
•
Click Prime to start the process.
•
When the process is complete, carefully remove the priming plate from the carrier and
dispose of the fluid.
Purging the Dispenser
KC4 provides a special utility to purge fluid from the dispense tubing and syringes by pumping
the fluid in reverse, back into the supply bottle.
To purge the dispenser:
•
In KC4, select System|Reader Control and click the Dispenser tab.
•
Select the Dispenser (1 or 2) associated with the supply bottle.
•
Enter the desired purge Volume from 5 to 5000 µl.
•
Select a prime Rate in µl/second.
•
Click Purge to start the process.
User’s Guide to KC4™
System Menu • 247
Emptying the Tip Prime Trough
Read & Dispense protocols can specify a tip prime to compensate for any fluid loss at the dispense
tip due to evaporation since the last dispense. Tip priming is performed in a small, removable
priming trough located in the left rear of the carrier. The prime trough holds up to 1 ml of liquid
and must be periodically emptied and cleaned by the user. The trough must be emptied
periodically, and KC4 must be “told” that the trough has been emptied.
To empty the tip prime trough:
•
Carefully remove the trough from the carrier and empty it.
•
Clean the trough as required by your site and then put it back on the carrier. Refer to the
Synergy HT Operator’s Manual for decontamination instructions.
•
In KC4, select System|Reader Control and click the Dispenser tab. The Tip Prime
Vessel value shows KC4’s estimate of how much fluid is in the trough.
•
Click Dump all Tip Prime Troughs. The Tip Prime Vessel value will reset to 0.
Note: If the tip priming trough overflows, refer to the Synergy HT Operator’s Manual for
decontamination instructions.
Note: When running a Read & Dispense protocol, KC4 may prompt you to empty the tip prime
trough. In this case, KC4 will automatically open the System|Reader Control, Dispenser dialog.
248 • System Menu
User’s Guide to KC4™
Clarity Control
Clarity™ Microplate Luminometer
Use the Clarity Control dialog to control the microplate holder, prime the reagent lines and
injectors, and control plate heating.
Microplate Holder Control
The Clarity’s microplate holder can be opened and closed only through software control.
•
To extend the microplate holder, click Plate Out.
•
To retract the microplate holder into the reading and heating chamber, click Plate In.
Note: Do not forcibly push in or pull out the microplate holder! If for any reason you cannot
control the holder using the software, use the Allan key supplied with the instrument to extend or
retract the holder. Refer to the Clarity™ Operator’s Manual for more information.
Prime
The reagent lines and injectors should be primed with the dispensing fluid before running the
protocol. Click the Prime button to open the Priming Parameters dialog.
Refer to the Clarity Operator’s Manual for complete priming instructions.
To prime the injectors:
1.
Fill the reagent bottle(s) with the fluid to be dispensed.
2.
Access the Priming Parameters dialog. All installed injectors are displayed. Injectors
marked by an asterisk (*) have yet to be initialized. Initialization takes place
automatically when the injector is used.
3.
Click on the injector’s check box. The drop-down list boxes Direction, Volume [µl] and
Strokes are enabled.
User’s Guide to KC4™
System Menu • 249
4.
In the Direction list box select To Mpl. In the Volume [µl] box specify the injection
volume, and in the Strokes box specify the number of cycles. The total liquid volume is
displayed in the Total Volume column.
5.
Click <Prime> to prime the injector(s).
Heating
The Clarity’s optional incubator is controlled via software. Click the Heating button to access the
Temperature Control dialog.
Refer to the Clarity Operator’s Manual for complete heating instructions.
250 • System Menu
User’s Guide to KC4™
Results
Overview
When plate reading is complete, or after data has been entered manually or read from a file, KC4
displays the Results as demonstrated below:
•
Click the Data drop-down arrow to view a different data set.
•
Click Statistics to view statistical information for the identifiers assigned to this plate.
•
Check Change to modify one or more raw data values, then click OK to recalculate and
revalidate results. Note: The System Administrator can protect the use of the Change
function.
User’s Guide to KC4™
Results • 251
•
Check Mask to mask one or more wells to temporarily exclude them from calculations.
Click on the desired well(s) then click OK to recalculate and revalidate results. To
unmask a well, click Mask, click on the well, then click OK. Results will automatically
recalculate.
Important! Masking wells may significantly change or even invalidate the results. The
System Administrator can protect the use of this function.
•
To copy data to another application, highlight the desired wells then select Copy from
the Edit menu. Switch to the target application and select a Paste option.
•
For all instruments except the FL600, a well will contain '?????' if any of the following
conditions occurs:
The plate layout specifies the contents of a well that is not read.
The absorbance value for the well cannot be calculated (absorbance reads only).
The RFU/RLU value exceeds 99998 (fluorescence and luminescence reads only). Tip: If
many or most of the wells contain '?????' the sensitivity setting in the protocol may be
too high.
Note: Wells containing ????? are excluded from transformation equations and
statistical calculations.
•
For the FL600™, a well will contain '?????' if the plate layout specifies the contents of a
well that is not read, or if the absorbance value for the well cannot be calculated
(absorbance reads only).
Note: Wells containing ????? are excluded from transformation equations and
statistical calculations.
•
For the FL600™ a well will contain 'OVRFLW' if the absorbance value for a well can be
calculated, but it is greater than 4.0 O.D. (absorbance reads only), or if the RFU/RLU
value exceeds 99999 (fluorescence and luminescence reads only). Tip: If many or most
of the wells contain 'OVRFLW', the sensitivity setting in the protocol may be too high.
Note: Wells containing OVRFLW are excluded from transformation equations and
statistical calculations.
252 • Results
User’s Guide to KC4™
Data Sets
The Results data sets presented for viewing depend on the current protocol parameters. Click the
Data menu drop-down arrow to choose from a list of available data sets.
The following describes just a subset of the many different Results data sets:
•
Template displays the identifier and any concentration or dilution value assigned to each
well under the Protocol|Plate Layout dialog.
•
M 977 (SP) and M 900 (SP) contain the measurement values of the Sample Plate at 977
nm and 900 nm, respectively (necessary for pathlength correction calculation).
•
Measurement numbers (M#1, M#2, … M#6) correspond with the wavelength selection
order in the Reading parameters dialog.
•
A data set resembling M#1 405 represents the raw measurement values for the Sample
Plate at this wavelength.
•
A data set resembling M#1 405 Blank represents the measurement values for the Blank
Plate at this wavelength.
•
A data set resembling M#1 405 Corr. represents the corrected values after Blank Plate
Subtraction, Blank Wells Subtraction, and/or Pathlength Correction.
•
The Delta OD data set represents the difference in measurement values between two
wavelengths.
•
The Concentrations data set represents the calculated concentration values. (Launch
Biograph to see the curve.)
•
The Conc./Dil. data set contains values calculated by multiplying the interpolated
concentrations by any dilution factor imposed on the concentration.
•
The Symbols data set contains the cut-off symbols assigned in accordance with the cutoff criteria defined in the protocol.
•
The Kinetic Curves and Scanning Curves data sets contain the graphical representation
of the measurement values in each well.
•
The results of any single- or multi-plate transformation can be identified by the name
defined within the Protocol| Single Plate Transformation dialog or the Protocol|Multi
Plate Transformation dialog. For example, if the name of a single plate transformation is
'% toxicity' select the '% toxicity' data set to view the results.
User’s Guide to KC4™
Results • 253
Concentrations
Concentrations are reported if standards are defined in the Plate Layout and the curve generated
successfully.
•
Values outside the range of the standard curve are reported as '<lowest conc.' or as
'>highest conc.' where the 'lowest conc.' and 'highest conc.' represent the minimum and
maximum concentrations plotted on the standard curve, respectively. Note: These two
values may be different than the min/max standard concentrations if an extrapolation
factor is applied.
See System Menu, Setup, Format for information on using 'out of range' values in
statistical calculations.
•
To calculate values outside the normal limits of the curve, select Protocol|Curves|Axes
to increase the Extrapolation Factor.
•
If a concentration could not be calculated for a well, the ????? symbol is reported. One
reason this may occur is if the value exceeds the limits of the standard curve.
254 • Results
User’s Guide to KC4™
•
Select Display|Curve(s) or click the Biograph toolbar button to view curves:
User’s Guide to KC4™
Results • 255
Symbols
If Cutoffs are defined in the protocol, the specified set of data is evaluated against the cutoff
criteria and each well is assigned a user-defined Symbol.
To view the assigned symbols, select the Symbols data set:
•
If the cutoffs are in increasing order, symbol assignment is determined as follows:
Each well is evaluated against the first cutoff. If the value of the well is less than the
criteria, then the first symbol is assigned to the well. If the value of the well is greater
than or equal to the first cutoff, then it is evaluated against the next cutoff. If the value
of the well is less than the next criteria, then the symbol immediately preceding the cutoff
criteria is assigned to the well.
This process continues until the last cutoff criteria is encountered. If the value of the well
is less than the last criteria, then the symbol immediately preceding the last cutoff criteria
is assigned to the well. If the value of the well is greater than or equal to the last
criteria, then the symbol immediately following the last cutoff criteria is assigned to the
well.
•
If the cutoffs are not in increasing order, the message "Cutoffs are not in increasing order.
Symbols can not be determined" appears, and ????? is assigned to every well in the
Symbols data set.
•
????? may also appear in one or more wells if a cutoff formula references a well ID that
is not defined in the Plate Layout, a well that is masked, or a well that is out-of-range or
has been failed by the attached microplate reader.
•
256 • Results
If a well contains OUT- or OUT+ cutoffs are based on concentrations and the well fell
below or above the lowest or greatest X value of the calculated standard curve.
User’s Guide to KC4™
•
Click the Cutoffs button to see the cutoff criteria and the calculated cutoff value(s).
Cutoffs Values
The contents of the Formulae in this dialog correspond with the Cutoffs defined within the
protocol.
The calculated value of each cutoff is displayed in the Value column.
If a Value is ????? the cutoff could not be calculated. Possible causes include:
•
A cutoff formula references a well ID that is not defined in the Plate Layout.
•
A cutoff formula references a well that is masked.
•
A cutoff formula references a well that is out-of-range or has been failed by the attached
microplate reader.
Pathlengths
Pathlength Correction can be enabled to normalize absorbances to 1 cm. Its use is controlled
within the Protocol|Reading parameters dialog.
If Pathlength Correction is enabled, KC4 first calculates the pathlength for each well. The
absorbance in each well is then divided by the pathlength, resulting in the corrected absorbance.
When calculations are complete, KC4 automatically reports the corrected values, available by
selecting M 405 Corr., M 630 Corr., etc. from the Data drop down list.
You can easily create a Multi Plate Transformation to report the pathlength of each well. You
can give the transformation a descriptive name such as "Pathlengths" and the resulting data set
will be available from the Data drop down list. To learn how to do this, access KC4’s help system
contents and select Results > Data Sets > Pathlengths. Click on “Click here for more information
on reporting the pathlength for each well.”
User’s Guide to KC4™
Results • 257
Kinetic Data
KC4 provides several options for viewing kinetic data. The default view is Kinetic Curves.
•
Each well displays a graph of the measurement values taken over time.
•
Hold down the Ctrl key and click on up to 8 wells to overlay the kinetic curves in one
display.
•
Click the Scales button to adjust the data points displayed for each well. The current
scaling parameters are shown in the lower left corner of the Kinetic Curves display.
•
Click the Calculations button to alter the kinetic calculation zone for all wells.
•
Click on a well to enlarge the view of its kinetic curve:
258 • Results
User’s Guide to KC4™
This dialog can display up to 8 overlaid kinetic curves.
Kinetic calculations such as Max V, Coeff Reg, Delta t at Max V, and Lag Time
associated with the currently selected plot are displayed. Click on the down arrow
associated with Selected Curve to choose a different curve.
Moving the mouse cursor over a kinetic data point in the currently selected curve
will display coordinates for that point. Data displayed can include Read #, OD, and
Reading Time.
Click on an individual data point to temporarily exclude it from further calculations,
then click the Recalculate button to exclude the point from kinetic calculations.
Important! Modifying data may adversely affect the final results.
To restore an excluded data point, click on the point and then click Recalculate.
Click the Options button to adjust the Kinetic Analysis parameters for the curves
shown in this dialog. Refer to Kinetic Analysis under “Protocol Reading
Parameters” in the Protocol Menu section of this guide for more information.
Dispense During Kinetic Read
The Synergy™ HT with injectors model provides the ability to dispense fluid into microwells as
part of a fluorescence or luminescence Read & Dispense protocol.
When the protocol specifies a dispense operation during a kinetic read, the Kinetic Curves display
will represent the dispense operation in a box with cross hatches on the Time axis, as shown
below. No readings are taken during a dispense operation.
User’s Guide to KC4™
Results • 259
Spectral Scan Data
The default view for spectral scan data is Spectrum Curves:
•
Each well displays a graph of the measurement values taken across a wavelength range.
•
Hold down the Ctrl key and click on up to 8 wells to overlay the spectrum curves in one
display.
•
Click the Scales button to adjust the data points displayed for each well. The current
scaling parameters are shown in the lower left corner of the Spectrum Curves display.
•
Click the Calculations button to alter the spectrum calculation zone for all wells.
260 • Results
User’s Guide to KC4™
•
Click on a well to enlarge the view of its spectrum curve.
The spectrum curve initially contains all values measured at all wavelengths as
defined in the protocol. Note: If a data point is "missing" for a particular
wavelength, this most likely means that the measurement value over ranged.
Spectrum calculations such as Min OD/Max OD and W at Min OD/Max OD,
associated with the currently selected plot are displayed. Click on the down arrow
associated with Selected Curve to choose a different curve.
Moving the mouse cursor over a data point in the currently selected curve will
display coordinates for that point. Data displayed can include Wavelength #, OD,
and Wavelength value in nanometers.
Click on an individual data point to temporarily exclude it from further calculations,
then click the Recalculate button to exclude the point from calculations.
Important! Modifying data may adversely affect the final results.
To restore an excluded data point, click on the point then click Recalculate.
Click the Options button to adjust the Spectrum Analysis parameters for the curves
shown in this dialog.
User’s Guide to KC4™
Results • 261
Area Scan Data
The default view for area scan results contains the graphical representation of the measurements
taken down and across each well:
•
To view a different set of data, click the Data drop-down arrow and select Mean
OD/RFU/RLU, Std Dev OD/RFU/RLU, or CV OD/RFU/RLU.
•
Click on a well to zoom in on it:
Picture Mode (the default view) is a graphical representation of all measurements
taken across the well. Place the cursor over any point in the well to see its value and
its X/Y location on the plate. Click Data Mode to view the measurement values for
all points.
To exclude/restore a point from the scanning calculations, click on the desired point
and then click Recalculate.
262 • Results
User’s Guide to KC4™
Linear Scan Data
The default view for linear scan results contains the graphical representation of the measurements
taken across each well:
•
To view a different set of data, click the Data drop-down arrow and select Mean OD, Std
Dev OD, or CV OD.
•
Click on a well to zoom in on it:
Place the cursor over any point in the well to see its measurement value and its X
location in the well.
If the graph contains more than one curve, use the Selected Curve menu to choose a
different curve and view its Mean OD, Std Dev OD, and CV OD values.
Click Options to adjust the Calculation Zone, then click Recalculate to update the
calculations.
User’s Guide to KC4™
Results • 263
Statistics
From within any Results window, click the Statistics button to view the statistical data associated
with the results.
•
Tip: To view statistics for a different data set, go back to the main Results window,
choose the desired data set and then click Statistics.
•
To view statistics for a different set of identifiers, click Samples, Controls, or
Standards.
•
The available columns are protocol-dependent:
Name: The name of the sample associated with the current well.
Conc.\Dil.: Concentration or dilution of the current well.
Well: Coordinates of the current well.
Nb: Represents the number of replicates that were used to calculate statistics.
Mean: Mean of the replicate group.
Std Dev: Standard deviation of the replicate group.
CV (%): Coefficient of variation of the replicate group, i.e. the standard deviation
expressed as a percentage of the mean.
264 • Results
User’s Guide to KC4™
Biograph
Overview
The KC4 software package includes the Biograph module for curve viewing and manipulation.
Biograph can be used either as a stand-alone product or in conjunction with KC4.
The Biograph functions are described in the following sections.
•
To start Biograph from within KC4 and view the current curve(s), select Display|Curves
or click the Biograph toolbar button.
•
To launch Biograph in stand-alone mode, select Biograph.exe from within the KC4
folder.
Both KC4 and Biograph allow you to select the mathematical curve that best fits the standards,
and this curve is used for calculating unknowns. Each curve is displayed with its formula and the
calculated coefficients and correlation parameters.
User’s Guide to KC4™
Biograph • 265
File Menu
Use the File Menu to work with curves and graph (.gph) files.
Creating a New Curve
Select File|New to define a new curve by manually entering the coordinates of the standard points.
•
Enter the abscissa values in the x column. These are typically the standard concentration
values.
•
Enter the ordinate values in the y columns. These are typically the measurement values
(OD, RFU, RLU). You can enter up to 12 ‘y’ values (replicates) per standard.
The M y column calculates the mean for each row of y values. Note: The curve is
calculated based on the mean y values, not the individual replicate values.
The SD y column calculates the standard deviation for each row of y values.
•
To mask a y value, check the Mask box then click on the desired y value. It will
temporarily change to *****.
•
To unmask a y value, keep the Mask box checked and click on the desired *****. It will
change back to its original value.
•
Click OK to display the new curve.
Closing a Curve
Select File|Close to close the current curve.
•
If the current curve is one generated dynamically using data from KC4, clicking Close
will close both the curve and the Biograph application.
•
If the current curve is a stored .gph file, clicking Close will close the file but leave
Biograph open.
Saving a Curve (Graph File)
Select File|Save or File|Save As to save the current curve as a graph file.
•
Graph files of the type *.GPH contain calculated curve-fitting values. A stored .GPH file
can be used as the basis for calculating concentrations in a protocol that does not contain
standard wells.
•
Tip: You can use refer to this curve in KC4 as the basis for calculating concentrations in
a protocol that contains no standards. To do this, select Protocol|Curves in KC4, then
enter the name of this .GPH file in the Curve Origin, File field.
266 • Biograph
User’s Guide to KC4™
Opening a Graph File
Select File|Open to retrieve a stored graphic file with the extension .GPH and manipulate it in the
same manner as newly created graphs.
•
To open a file, highlight its name then click Open.
•
To look for a file in a different folder, click the drop-down arrow next to the Look In
field, or click the previous folder button.
Printing Curves
Select File|Print to print the current curve, File|Print Preview to preview the printout, or File|Print
Setup to adjust the printer settings.
The report contains a picture of the curve, the name of the curve fit method, the equation, and the
calculated coefficients.
User’s Guide to KC4™
Biograph • 267
Exiting Biograph
Select File|Exit to close Biograph and return to KC4 or Windows, wherever Biograph was
originally launched from.
•
Upon returning to KC4, all changes are lost and the following message appears:
View Menu
Use the View Menu to customize Biograph by hiding/displaying the status bar and the tool bar.
The Toolbar
By default, the Toolbar is displayed below the menu options:
•
To hide the Toolbar, select View|Toolbar to remove the checkmark.
•
To redisplay the Toolbar, select View|Toolbar to reinstate the checkmark.
The Status Bar
By default, the Status Bar is displayed at the bottom of the screen:
•
To hide the Status Bar, select View|Status Bar to remove the checkmark.
•
To redisplay the Status Bar, select View|Status Bar to reinstate the checkmark.
268 • Biograph
User’s Guide to KC4™
Graph Menu
Use the Graph Menu to view and/or modify certain characteristics of the current curve.
Standards
Use the Standards dialog to view, enter, or modify the ‘x’ and ‘y’ values of standards. Note:
When Biograph is launched from KC4, these values cannot be modified.
•
The abscissa values are listed in the x column. These are typically the standard
concentration values.
•
The ordinate values are listed in the y columns. These are typically the measurement
values (OD, RFU, RLU). You can enter up to 12 ‘y’ values (replicates) per standard.
The M y column calculates the mean for each row of y values. Note: The curve is
calculated based on the mean y values, not on the individual replicate values.
The SD y column calculates the standard deviation for each row of y values.
•
To mask a y value, check the Mask box then click on the desired y value. It will
temporarily change to *****.
•
To unmask a y value, keep the Mask box checked and click on the desired *****. It will
change back to its original value.
•
Click OK to display the curve.
Axes, Fit, Interpolations
These features function similarly to functions of the same name in KC4.
See “Curves” under Protocol Menu for more information.
User’s Guide to KC4™
Biograph • 269
Overlaying Graphs
Biograph allows you to view multiple graphs in the same session.
•
The graphs must be open before they can be overlaid.
•
Up to 8 graphs can be overlaid, regardless of curve fit type, but all must have the same
axes (either all linear or all logarithmic). The curve overlay will display in a new
window labeled OVERLAYED CURVES.
The curves in this window cannot be edited. New interpolations can, however, be
imposed.
Interpolations cannot be adjusted within this window (make adjustments in the
individual curve windows).
However, any changes which you make to an individual curve or curves are reflected
immediately in the "shadow curve" in the OVERLAYED CURVES window once
you have calculated the change using Graph|Recalculate.
270 • Biograph
User’s Guide to KC4™
Curve Window
The replicate of each data point is displayed with a green square and the mean replicate value is
displayed in a red circle.
•
Click on an individual data point to temporarily exclude it from further calculations, then
select Graph|Recalculate button to recalculate the curve without the point.
Important! Modified data may adversely affect the final results.
Changes made here are not automatically transferred over to KC4. To make permanent
curve-fitting changes, return to KC4 and select Protocol|Curves.
•
To restore an excluded data point, click on the point then select Graph|Recalculate.
•
Select File|Print to print the graph.
User’s Guide to KC4™
Biograph • 271
272 • Biograph
User’s Guide to KC4™
PowerReports
High-Powered Reporting
Since its inception, KC4 has helped users with designing professional, high-quality reports
tailored to meet their specific needs. KC4 now takes information presentation to an even higher
level with PowerReports.
PowerReports links KC4 with Microsoft® Excel/Word 2000, providing you with a new format for
creating custom report and export documents.
With PowerReports you can:
•
Take full advantage of Excel/Word’s document customization features to generate
publication-quality reports.
•
Include pictures, drawings, and company logos in reports.
•
Create custom formulas in Excel to perform additional calculations on KC4 data.
•
Take advantage of Excel’s charting capabilities to create bar graphs, pie charts, and
more.
•
Perform a direct export of plate data to Excel without first having to create an export
template.
User’s Guide to KC4™
PowerReports • 273
How it Works
PowerReports uses Object Linking and Embedding (OLE) technology to interact with Microsoft
Excel and Word, providing you with total control over all report formatting using results objects
created by KC4.
KC4 sends information about the current protocol parameters to an Excel or Word document.
You design the document by selecting the pieces of information to include on the report or in the
export file, defining the locations for the different pieces of information, and customizing the
appearance of the document. The document then becomes a part of the protocol, and is available
for editing any time the protocol is open.
274 • PowerReports
User’s Guide to KC4™
User’s Guide to KC4™
PowerReports • 275
System Requirements
To put the power in PowerReports, your computer must meet or exceed the following
requirements:
•
Pentium II processor (or equivalent) / 300 MHz or faster
•
64 MB RAM (128 recommended)
•
Microsoft Office 2000 (9.0.3821 SR-1 or newer), Office XP, Office 2003
To check your Office 2000 version, launch Word or Excel and select Help|About
Microsoft Word (or Excel).
•
Windows 98/2000/NT/XP
Important! If your computer does not currently meet all of these requirements, you can still print
reports and export data. With KC4’s standard reporting and exporting functions you can
customize the contents, and to some extent the appearance, of printed reports and exported plate
data. All KC4 users have access to these standard functions.
Preparing KC4 for PowerReporting
KC4 offers three reporting/exporting drivers, or engines: KC4, Microsoft Excel, and Microsoft
Word. KC4 needs to know which engine(s) to you want to engage for editing, previewing, and
printing reports and for editing and performing exports. The following topics explain how to
select default global reporting and exporting engines and how to override them when necessary.
Setting Global Options
Under System|Setup|Protocol Defaults, KC4 allows you to select a default Reporting Engine
and a default Exporting Engine. When you create a new protocol, KC4 refers to these settings to
know which engine(s) to engage. These default settings can be overridden within a protocol
(explained in the next section).
•
Select the default Reporting Engine and Exporting Engine (the ones you plan to use
most frequently).
276 • PowerReports
User’s Guide to KC4™
•
If you have selected Microsoft Word for either or both engines, choose a Word
Document Formatting option of Standard or Enhanced.
With Standard formatting, the print preview and print functions perform quickly but
any custom Plate Layout or Cut-Offs fonts are not applied.
With Enhanced formatting, the print preview and print functions may take longer
but all of your custom Plate Layout and Cut-Offs fonts will be applied.
•
Click the Start-up tab to customize additional options:
(Optional) Check Start when launching KC4 if you want Word and/or Excel to
open automatically when KC4 is launched. With the program open in the
background, document opening and formatting will perform more quickly.
Setting Protocol-Specific Options
If you have opened a protocol and want to change the reporting or exporting engine but do not
want to change the global default settings, select Protocol|Options or click the Options toolbar
button. Click the Report or Export tab as appropriate.
•
Select the desired Report and/or Export Engines, and modify other parameters as
necessary.
User’s Guide to KC4™
PowerReports • 277
Important! If you change the engine after a document has been created for the protocol,
the current document will be discarded and a new one must be created:
Click OK to approve the selection of a new report or export engine. The current
document (if one exists) will be discarded.
Click Cancel to keep the previous engine and retain the current document (if one
exists).
•
See “Protocol Options” at the end of Protocol Menu for more information on the different
Report and Result Export options.
Defining the Protocol Parameters
It is a good idea to define all or at least the majority of the protocol parameters before creating a
report or export document. KC4 customizes its lists of options that can be inserted into the
document based on the current protocol parameters.
For example:
•
Define Standard wells in the Plate Layout dialog and set the curve-fitting parameters if
the report will contain information about the standard curve (such as a picture of the
curve, the curve equation, and the R/R2 values).
•
Define Sample wells in the Plate Layout dialog and specify cutoff criteria if the report
will contain custom formulas to determine the frequency of symbols assigned to
samples and then generate a pie chart showing the percentages.
•
Define the wavelengths in the Reading parameters dialog and assign blank wells in the
Plate Layout dialog if the export file will contain raw and blanked measurement data
at 405 nm and 490 nm.
Creating/Editing Documents
When you are ready to create or edit a document, select Protocol|Report or Protocol|Export, or
click the appropriate toolbar button:
Create/edit an Excel
Create/edit an Excel
report document.
export document.
Create/edit a Word
Create/edit a Word
report document.
export document.
Tip: If you don’t see these buttons in KC4’s toolbar, you can add them by selecting
System|Toolbar.
278 • PowerReports
User’s Guide to KC4™
Working with Results Objects
A Results Object is a placeholder of information in an Excel or Word document that is filled in
with results data during the print preview, print, or export function.
The Results Objects Toolbar
When KC4 launches Excel or Word, it automatically adds a Result Objects toolbar to the
standard Excel/Word toolbar:
Remember that a results object is a placeholder of information which will be filled in by KC4
after the plate is read. Each object specifies which item of information to use, where the
information will be located in the document, and how it will be presented (font style, color, size,
etc.).
Insert a Field Object. Each object contains a single piece of information, such as a
cut-off value, a file name, or a curve coefficient. Clicking the Fields button opens
a dialog with lists of available categories and related items to choose from.
Categories include Curve Fit, Cut-Offs, File Information, Plate Description, and
Reading Parameters.
Insert a Plate Object to present data in a row x column format matching the plate
type defined in the protocol. Clicking the Plates button opens a list of available
items to include in the plate, such as Well ID, 405, 405 Corr., and Concentrations.
Insert a Table Object to present data in a columnar format. Clicking the Tables
button opens a list of available items to present in this format, including Statistics,
Well Data Analysis, and Fitting Results.
Insert a Curve Object, such as a standard curve, a set of dose response curves, or a
kinetic, scanning, or spectrum well zoom.
Delete an object. The cursor must be placed inside the object when this button is
clicked.
Close (minimize) Excel or Word and return to KC4. The document is
automatically saved as a part of the protocol.
Get Help on PowerReports.
User’s Guide to KC4™
PowerReports • 279
Object Components
Objects are inserted into the document using the Result Objects toolbar.
See Field Objects, Plate Objects, Table Objects, or Curve Objects for specific instructions for
inserting the different types of objects.
The example below shows a Field Object in an Excel document:
In the report or export document, any object contains 3 unique items:
•
An identifying title, as shown in cell B2.
The text and appearance of this title can be modified. To change the text, simply
overwrite the existing title. To change the appearance, use the application’s font
customization tools.
•
A dollar sign ($) character, as shown in cell C2.
The dollar sign will be replaced by actual information during the print, print preview,
or export process. In the example above, the dollar sign will be replaced by the
system date recorded when the plate was read.
By default, the font color of this character is set to ‘Auto’. The color will be
whatever the color is in KC4. The color (and font type and size) can be overridden
in the template using the application’s font customization tools.
Do not delete or move a dollar sign without deleting or moving the entire object.
•
A gray editing button, as shown in cell D2.
To change the contents of an existing object, double-click the gray editing button.
The subsequent dialog depends on the object type.
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Object Fulfillment
After a plate is read and the operator initiates the print preview, print, or export function KC4
begins the formatting process and transfers data into the prepared document. A Formatting Office
document… message lets you know that something is happening:
When formatting is complete, the document can be printed, viewed, or exported depending on the
current process.
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Designing the Document
This section explains how to work with Field, Plate, Table, and Curve Objects.
Inserting Field Objects
A Field Object represents a single item of information, such as the name of the protocol file, the
calculated value of a cut-off, or the r-squared value of a curve.
To insert a Field Object, click the Fields button in the RSO toolbar. The Insert Field Object
dialog appears:
•
Select the Category of information that represents this Field Object. Available options
include Curve Fit, Cut-Offs, File Information, Plate Description, Reading Parameters,
and System Information.
•
Select the Name of the field within that category.
If the only available item in the list is ‘*****’ the current protocol parameters do not
generate any fields for this category. For example, if the protocol does not specify
cut-off criteria there will be no items available under the Cut-Offs category.
Tip: To learn more about the different fields and to see some examples, click on the
Insert Field Object Help button then click on a Category link.
•
If the field will contain a numeric value, set the Notation to Decimal (ex. 0.003) or
Scientific (ex. 3E-03).
•
If Decimal notation is selected, specify the number of decimal places (Nb Dec) to be
displayed, 0 to 5.
•
Click OK to close this dialog and insert the Field Object into the document.
The document shown on the next page contains six Field Objects. Each object is represented by a
1 row x 3 column table. Notice that Field objects do not have to be separated by a blank line or
row. See “Object Components” for more information.
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Inserting Plate Objects
A Plate Object presents information in a matrix corresponding with the plate type selected in the
protocol. For example, if the protocol specifies a 96-well plate, the plate object contains 8 rows
and 12 columns of data.
To insert a Plate Object, click the Plate button in the RSO toolbar. A drop-down list appears,
containing all of the data sets available for inclusion in the Plate Object:
•
Data set availability is based on the current protocol settings.
•
Each object can contain from 1 to 20 data sets. For example, an object can contain the
Well ID, the corrected data value, and the concentration value for every well. Or, it can
contain just the concentrations.
•
To proceed, select one data set (even if you want to include multiple data sets in the same
object).
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The Plate Properties dialog appears:
The Top-Left Well and Bottom-Right Well set the boundaries for the plate object.
The default settings coordinate with the plate type defined in the protocol.
The Reading # field is available for kinetic protocols to specify which kinetic read to
include in the object. Each plate object can display information for one kinetic read.
•
Click the Data tab to select the data sets to include in the plate object. KC4 presents one
or more default Data sets for inclusion in the object, although any available data set(s)
can be selected.
To add a data set, highlight the first empty Data field and click the left mouse button
once. A list of available data sets will appear; click on the desired set to select it.
To change an existing data set, highlight it and click the left mouse button once. A
list of available data sets will appear; click on the desired set to select it.
To delete an existing data set, highlight its entire row then click the right mouse
button and select Delete.
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To insert a new data set between two existing sets, highlight the row above which
the new data set will be inserted. Click the right mouse button and select Insert.
For data sets that represent numerical information, click the corresponding Format
field then select Decimal or Scientific. For the Decimal format, click the
corresponding Precision field to set the number of decimal places (0 to 5).
Click OK to save the settings and insert the new Plate object into the document, as
demonstrated below:
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Inserting Table Objects
A Table Object presents results data in a columnar format.
To insert a Table object, click the Table button in the RSO toolbar. A drop-down list of available
data categories appears:
•
This list contains all available categories of data, based on the current protocol settings.
•
Available categories can include Statistics, Well IDs, Well Data, Well Analysis Results,
Validation Results, Fitting Results, Protocol/Data Audit Trail, and Calculation Log
Report.
Statistics
A Statistics table calculates the Mean, Standard Deviation, and Coefficient of Variation (CV%)
for the Standard, Sample, Control, and/or Blank groups defined in the protocol. To create a
Statistics table, click the Tables button in the RSO toolbar, then highlight Statistics. A list of
available data sets appears:
•
The list contains those data sets made available by the current protocol parameters.
•
Select the data set to be represented in the table.
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The Statistic Table Properties dialog appears:
Select the Identifier(s) to be represented in the table.
If Sample and/or Assay Control is selected, Index is by default set to All, meaning all
sample and/or assay control wells will be included in the table.
To include a subset of all sample and/or assay control wells, use the Index From and
To fields to enter the limiting group numbers. In the example above, Sample groups
1 through 10 (SPL1 to SPL10) will be represented in the table.
Click the Data tab to customize the contents of the table.
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KC4 presents some default Data sets for inclusion in the table, although any
available data set(s) can be selected.
To add a data set, highlight the first empty Data field and click the left mouse button
once. A list of available data sets will appear; click on the desired set to select it.
To change an existing data set, highlight it and click the left mouse button once. A
list of available data sets will appear; click on the desired data set to select it.
To delete an existing data set, highlight the entire row then click the right mouse
button and select Delete.
To insert a new data set between two existing sets, highlight the row above which
the new data set will be inserted. Click the right mouse button and select Insert.
For data sets that represent numerical information, an Operator can be applied to
perform a specific calculation.
Nb displays the number of wells in the group.
Mean calculates the average measurement for each well group.
Std Dev calculates the Standard Deviation for each well group.
CV (%) calculates the Coefficient of Variation for each well group as (Std Dev /
Mean) x 100.
Click OK to close this dialog and insert the object into the document.
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Well IDs
A Well IDs Table lists well identifiers and their names. To create a Well IDs table, click the
Tables button in the RSO toolbar and then click on Well IDs. The Statistic Table Properties
dialog appears.
•
Select the Identifier(s) to be represented in the table.
•
If Sample and/or Assay Control is selected, Index is by default set to All, meaning all
sample and/or assay control wells will be included in the table.
To include a subset of all sample and/or assay control wells, use the Index From and
To fields to enter the limiting group numbers. In the example above, Sample groups
1 through 10 (SPL1 to SPL10) will be included in the table.
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•
Click the Data tab to customize the contents of the table.
For the Well IDs table, KC4 automatically inserts the Well ID and Name data sets.
Any available data set can be included in the list. See “Statistics” for information on
working with data sets.
Click OK to close this dialog and insert the object into the document.
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Well Data
A Well Data table presents measurement values for kinetic, spectrum, or scanning protocols.
•
For Kinetic protocols, the table lists measurement values by time of kinetic reading
•
For Spectrum protocols, the table lists measurement values by wavelength
•
For Linear Scanning protocols, the table lists measurement values by reading position
•
For Area Scanning protocols, each table presents one well’s data in a matrix format
To create a Well Data table, click the Tables button in the RSO toolbar and then highlight Well
Data to open a list of available data sets. Click on the data set to be represented in the table; the
Well Data Table Properties dialog appears.
Kinetic, Spectrum, and Linear Scanning protocols:
•
Define a rectangle of wells to be included in this table. Note: This table reports
information in columns, one column per well. You may wish to experiment with the
maximum number of columns that can display legibly per table. If you want to display
information for many wells (i.e. more than 10-12), consider inserting multiple Well Data
Table objects.
Area Scanning protocols:
•
Each table reports the area scanning data for one well. Specify the Well to be represented
in the table.
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•
Area scanning data is reported in a matrix format, representing the measurement points
taken across and down each well. Specify the First and Last Matrix Column to include
in this table object.
Note: If Word is the reporting/exporting engine, each table object can contain a
maximum of 15 columns, so you may need to create multiple table objects to print all of
the information for the well. For example, if the Read Matrix Size is 25 x 25, create one
table object with First and Last Matrix Columns set to 1 and 15, then create another table
object with First and Last Matrix Columns set to 16 and 25.
All protocols:
•
Click the Data tab to change and/or customize the data set.
Each Well Data table reports information for one data set. If the current protocol
parameters generate multiple data sets, click on the Data field to open a drop-down
list of all available data sets.
Click the Format field then select Decimal or Scientific. Click the Precision field to
set the number of decimal places (0 to 5).
Click OK to close this dialog and insert the object into the document.
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Well Analysis Results
A Well Analysis Results table lists various statistics for kinetic, spectrum, or scanning protocols.
•
For Kinetic protocols, the table lists the kinetic analysis calculations (e.g. Max V, Delta t
at Max V, Lag Time, etc.), determined for each well.
•
For Spectrum protocols, the table lists the Minimum and Maximum measurements and
their corresponding wavelength values for each well.
•
For Area and Linear Scanning protocols, the table lists the Mean OD, Standard
Deviation, and Coefficient of Variation calculated for each well.
To create a Well Analysis Results table, click the Tables button in the RSO toolbar and then click
on Well Analysis Results. The Well Analysis Table Properties dialog appears:
•
The Selected Wells list contains all wells that will be included in the table.
To append a well to the Selected Wells list, enter the well’s Coordinate in a format
resembling A1 or F11, then click the Add>> button.
To remove a well from the Selected Wells list, highlight the well then click
<<Remove.
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•
Click the Data tab to customize the information to be included in the table for the
selected wells.
KC4 presents a default list of Data sets to be included in the table, although any
available data set can be selected. See “Statistics” for information on working with
data sets.
Click OK to close this dialog and insert the object into the document.
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Validation Results
A Validation Results table lists the validation conditions and their corresponding statuses.
To create a Validation Results table, click the Tables button in the RSO toolbar, then click on
Validation Results. The table object is automatically inserted into the document.
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Fitting Results
A Fitting Results table lists coefficient values for all curves in the assay.
To create a Fitting Results table, click the Tables button in the RSO toolbar, and then click on
Fitting Results. The Curve Parameter Table Properties dialog appears.
•
Select the Identifier(s) to be represented in the table.
If Sample and/or Assay Control is selected, Index is by default set to All, meaning all
sample and/or assay control wells will be included in the table.
To include a subset of all sample and/or assay control wells, use the Index From and
To fields to enter the limiting group numbers. In the example above, Sample groups
1 and 2 (SPL1 and SPL2), will be included in the table.
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•
Click the Data tab to customize the contents of the table.
KC4 presents a default set of Data items that will be included in the table, although any
available item can be included in the list.
To add an item to the list, highlight the first empty Data field and click the left
mouse button once. A list of available data items will appear. Click on the desired
item.
To modify an item in the list, highlight it and click the left mouse button once. A list
of available data items will appear. Click on the desired item.
To delete an item from the list, highlight the row then click the right mouse button
and select Delete.
To insert a new item between two existing items, highlight the row above which the
new item will be inserted. Click the right mouse button and select Insert.
For data sets that represent numerical information:
Click the corresponding Format field then select Decimal or Scientific.
For the Decimal format, click the corresponding Precision field to set the number of
decimal places (0 to 5).
Click OK to close this dialog and insert the object into the document.
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Interpolation Results
An Interpolation Results table lists the calculated X-values for the
Y-values and formulas entered under Protocol|Curves|Interpolations.
To create an Interpolation Results table, click the Tables button in the RSO toolbar, and then click
on Interpolation Results. The Curve Parameter Table Properties dialog appears:
•
Standard is the only Identifier that can be included in an Interpolation Results report.
•
Click the Data tab to customize the contents of the Interpolation Results table.
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KC4 presents a default set of Data items that will be included in the table, although any
available item can be included in the list. See the previous topic, “Fitting Results” for
information on working with Data items.
Click OK to close this dialog and insert the object into the document.
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Protocol/Data Audit Trail
Protocol and Data Audit Trail information can be included on reports and in export files.
Note: The Protocol Audit Trail is only available if the Preferred Data File Format setting is PLA.
To create a Protocol or Data Audit Trail table, click the Tables button in the RSO toolbar then
click on Protocol Audit Trail or Data Audit Trail. The table is automatically inserted into the
document (the example below shows two separate tables).
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Calculation Log Report
A Calculation Log Report can be included on reports and in export files.
To create a Calculation Log Report table, click the Tables button in the RSO toolbar and then
click on Calculation Log Report. The table is automatically inserted into the document:
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Inserting Curve Objects
A Curve Object inserts a graph showing one or multiple overlaid curves. To insert a Curve
Object, click the Curves button in the RSO toolbar. A drop-down list showing the available curve
type will appear:
•
This list contains all available curve types, based on the current protocol settings.
Available curve types can include Standard, Dose Response, and Well Zoom.
Standard Curve
A standard curve is generated by a protocol that has Standard wells defined in the Plate Layout.
To insert a Standard Curve, click on the Curves button in the RSO toolbar, then click on
Standard Curve to insert the object.
•
KC4 generates a temporary curve using fictitious data. After the plate is read, KC4
rebuilds the curve with real plate data.
•
Certain characteristics of this curve can be modified, including colors and labels.
Double-click anywhere on the graph to enter the editing mode; a shaded box should
appear around the graph. When this box appears, click the right mouse button once to
open a menu of items that can be edited.
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Dose-Response Curve(s)
Dose response curves are generated by a protocol with assay control, sample, or sample control
wells with concentrations or increasing dilutions defined. Up to 5 curves can be overlaid in one
curve object. To insert a Dose Response curve, click on the Curves button in the RSO toolbar,
then click on Dose Response. The Graph Properties dialog appears, listing all available curves
by Well ID:
•
The Available Curves box lists all curves made available by the current protocol
parameters.
•
The Selected Curves box lists all curves that will be overlaid in a single curve object.
•
To add a curve to the list, highlight an available curve, then click Add >>.
•
To remove a curve from the list, highlight the curve, then click << Remove.
•
Click OK to close the dialog and insert the object into the document.
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KC4 generates temporary curve(s) using fictitious data. After the plate is read, KC4
rebuilds the curve(s) with real plate data.
Certain characteristics of this curve can be modified, including colors and labels.
Double-click anywhere on the graph to enter the editing mode; a shaded box should
appear around the graph. When this box appears, click the right mouse button once
to open a menu of items that can be edited.
Well Zoom Curve(s)
Well zoom curves are available for kinetic, spectrum, and scanning protocols. Up to five curves
can be overlaid in one object. To insert a Well Zoom curve object, click on the Curves button in
the RSO toolbar, then click on Well Zoom. The Well Zoom Properties dialog appears:
The Selected Curves box lists all wells that will be overlaid in the curve object.
•
To add an item to the Selected Curves list:
Choose a Data Type from the drop-down list. The contents of this list depend on the
current protocol parameters. Data types can represent raw, blank plate, and corrected
data.
Enter the Well coordinates. The corresponding Well ID and Conc./Dil. value will
appear, if applicable.
Click Add >>.
•
To remove an item from the list, highlight the item then click << Remove.
•
Click OK to close the dialog and insert the object into the document.
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KC4 generates temporary curve(s) using fictitious data. After the plate is read, KC4
rebuilds the curve(s) with real plate data.
Certain characteristics of this curve can be modified, including colors and labels.
Double-click anywhere on the graph to enter the editing mode; a shaded box should
appear around the graph. When this box appears, click the right mouse button once
to open a menu of items that can be edited.
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Previewing and Printing the Report
When plate reading and data reduction are complete, the report is ready for formatting and then
previewing and/or printing.
To format the report (that is, to fill all of the objects in with current plate data), select Data|Print
Preview or Data|Print, or click the appropriate toolbar button.
During the formatting process, KC4 displays a progress bar, as shown below:
Previewing Options
When preparing to print a newly created or recently edited report, it’s a good idea to preview it
first to check column widths, page breaks, fonts, colors, and so on.
If a change is required before printing, two options are available:
•
Modify the report document and go through the print preview process again. This is the
recommended option because changes will be saved with the protocol.
•
Make a temporary change in the preview document. The Protocol|Options parameter
Keep Open After Printing must be enabled.
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Special Notations
When displaying results or when reporting results using the standard (KC4) reporting engine, KC4
uses various font characteristics to denote masked wells and biased values.
When exporting results or when reporting results using the Excel and Word reporting engines,
KC4 uses some special characters to denote masked wells and biased values. This is to avoid
conflict with any special font characteristics that may be assigned by the report/export file
designer.
•
To denote a masked well, KC4 surrounds the value with asterisks: *0.234*, *?????*,
*98999*.
•
To denote biased values, KC4 surrounds the value with exclamation points: !0.068!,
!23.456!.
Note: Biased values may appear in a Statistics table if Use out of range values in statistics is
enabled under System|Setup. If, for example, the concentration for one of three standard
replicates is reported as ‘>40’, the Standard Deviation and CV% values are considered suspect.
KC4 surrounds the values with exclamation points to indicate that it is the user’s responsibility to
determine whether or not the results are valid.
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Performing the Data Export
When plate reading and data reduction are complete, the export document is ready for formatting
and saving.
To format the export document (that is, to fill all of the objects in with current plate data), select
Data|Export or click the appropriate toolbar button. When formatting completes, KC4 displays
the Save As dialog:
•
Change the current folder and file name if desired, then click Save to complete the
export.
•
If the Protocol|Options parameter Keep Open After Exporting is enabled, the document
stays open after the export is complete. This allows you to, for example, print the
contents of the file.
•
See “Special Notations” on the previous page for information
on how KC4 denotes masked wells and biased values in export files.
Direct Export to Excel
KC4 provides a way to directly export data to Excel without first having to create an export
template. To export plate data directly to Excel:
1.
Under Protocol|Options (or under System|Setup if no protocol is open) set the export
engine to Microsoft® Excel.
2.
Read the plate.
3.
Select Data|Export. KC4 will launch Excel and insert the RSO toolbar.
4.
Insert objects (Field, Plate, Table, and/or Curve) as explained throughout this chapter.
5.
Click Save As to export the data.
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Appendix A: Records Submission
Records Submission
When preparing electronic records for submission to the FDA, contact Bio-Tek Instruments’
Marketing & Sales Department for guidance, and to obtain a demonstration copy of KC4 to
include with your package. The FDA will need KC4 to view and print your records.
Contact information:
Phone: (888) 451-5171 (toll free in the U.S.)
(802) 655-4740 (outside the U.S.)
Fax:
(802) 655-7941
E-mail: [email protected]
User’s Guide to KC4™
Appendix A: Records Submission • 309
310 • Appendix A: Records Submission
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Appendix B: Windows File Caching
Windows File Caching
This topic applies to sites accessing a shared database stored on a network/server.
By design, most write and read database operations are intended to be performed synchronously
by KC4. For example, when a system audit trail event or a file is created by a KC4 instance, it
should be immediately visible in other instances using the same database. However, because of
other factors beyond the control of KC4, like network and operating system delayed-write
optimizations, there is no way to guarantee such behavior. To improve synchronization, it is
recommended, when KC4 is used on several networked PCs connected to the same shared
database, to disable the network redirector file caching.
In addition, while file caching is enabled there is a potential for data loss following a network
connection failure. According to Microsoft, when the Windows redirector opens a file for read or
read/write access, the redirector utilizes the Windows system cache. Therefore, when data is
written to the file, it is written to the cache and not immediately flushed to the redirector. The
cache manager flushes the data at a later time. If an unrecoverable network error occurs while the
data is being transferred to the remote server, it may cause the write request to fail and an
application error to occur.
Here is an example scenario: The network connection failed because of a problem on the server.
The KC4 user, unaware of the failure, chose to save a GLB file to the shared database just seconds
after the failure. Because file caching was enabled, the file actually went into temporary storage
in the Windows system cache, to await redirection to the shared database on the network. KC4
gave the appearance that the file was saved successfully (no error message appeared, and the
asterisk disappeared from the title bar). After a moment, Windows detected the failure and
displayed an appropriate error message, and the user decided to quit KC4. Upon closing, KC4 did
not present an option to save the file because KC4 “thought” that the file was already saved.
When the network connection was re-established, the user launched KC4 but could not locate the
GLB file because it never reached the shared database.
For more information, visit http://support.microsoft.com and search for current articles on “file
caching” or “write caching”.
User’s Guide to KC4™
Appendix B: Windows File Caching • 311
312 • Appendix B: Windows File Caching
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Index
!#! indication in statistics,
307
*#* indication in statistics,
307
????? indication in Symbols,
256
4 Parameter logistic fit, 164,
168
A
Absorb. checkbox, 197
Absorbance read method,
112
Administrator
Appointing a System
Administrator, 8
Initial password, 19
System Administrator
requirements, 8
Administrator:, 8
Allow well zoom during
read, 103
Anti-Log, 160
Append to kinetic file, 56,
185
Application Data folder, not
visible in Explorer, 22,
234, 235
Archiving records, 29
Area scanning, 115, 262
Area under the curve, 136
Assay control wells, 143,
145
Assay description, 177, 182
Assigning names to sample
groups, 61
Audit trail, 63
Data audit trail, 63
Notification dialog, 220
User’s Guide to KC4™
PowerReports, 300
Protocol audit trail, 183
System audit trail, 224
Automatic append to kinetic
file after reading, 185
Automatic export after
reading, 187
Automatic print after
reading, 186
Automatic save after
reading, 185
Automatic sensitivity
adjustment
Best signal to noise ratio,
91
Scale to high wells, 90
Scale to low wells, 90
Selecting in a protocol, 96,
98
Available readers, 194
Averaging in formulas, 160
Axes, 165
B
Bandwidth, 197, 200
Bandwidth verification, 92
Barcode
Enabling the option to
read, 111
Using as file name, 185
Base value, 92
Basis value calculation, 135
BDF file, 53
Importing, 53
Best signal to noise ratio, 91
Bias value, 96, 98
Biograph, 189, 265
Index • 313
Bio-Stack
Minimizing KC4 when
running in OLE mode,
232
Sample IDs, 61
Bio-Tek, contacting for
technical assistance, 4
Blank plate subtraction, 111,
128
Blank wells, 143, 144
Blank wells subtraction, 128
Block light between reads
Filter position
requirements, 201
Read step option, 127
BPF file, 77
Clarity protocol files, 28
Creating new, 77
Opening existing, 77
BTI file, 53
Importing, 53
Buttons in toolbar, 237
C
Calculate recovery percent,
155
Calculation log report, 178,
301
Calculation options, 188
Calculation warnings, 190
CALIB.mdb database, 233
Calibrate
Endpoint reads, 118
Kinetic reads, 118
Spectrum reads, 118
Calibration plate test, 205,
207
Calibrators, 143
Changing measurement
values
How to, 45
Results window, 251
Check Lamp before
measurement, 99, 100
Clarity, 77
Background measurement
value, 53
File importation failures,
53
Heating, 250
314 • Index
Importing BTI files, 53
Priming, 249
Protocol definition, 77
Protocol files, 28
Reader control, 249
Reading parameters, 77
Coefficient of variation, 160
Coefficient of Variation, 264
Communication port, 195
Compact database
After exporting and
deleting audit trail
events, 225
After exporting and
deleting files, 227
Local database, 235
Shared database, 233
Computer requirements, 9
Concentrations
Defining in plate layout,
155
Contacting Bio-Tek
Instruments, Inc., 4
Control wells, 143
Controlling multiple
instruments
simultaneously, 194
Converting databases, 15,
236
Copy Current Formula, 160
Corrected data, 128
Critical database error
Restoring connection to
KCShared, 234
Current folder, 44, 47, 68, 71
Current plate, 164
Current reader, 194
Curve axes, 165
Curve description, 164
Curve fitting, 164, 166
Curve interpolation, 169
Curve objects in
PowerReports, 279,
302
Curve through origin, 168
Curves
Printing, 189
Stored, 266
Viewing, 189
Custom plate formats, 240,
242
User’s Guide to KC4™
Cutoffs
Defining, 173
Evaluation method, 176
Evaluation of results, 256
Examples, 174
Formula examples, 174
Report, 180
Symbols, 173
Viewing calculated values,
257
Viewing results, 256
CV function, 160, 264
D
Data audit trail
Data menu, 63
PowerReports, 300
Data bits, 195
Data files, 42
Changing measurement
values, 45
Contents, 28
Exporting, 57
Masking wells, 45
Modifying protocol
parameters, 45
Plate description, 60, 75
Signing electronically, 54
Storage options, 28
Data mode, 262
Data to interpolate, 164
Data to interpret, 173
Data to monitor, 107
Data to process, 131, 137
Database connection error
Restoring connection to
KCShared, 234
Database upgrade utility, 15,
236
Databases
KCLocal.mdb, 233, 235
KCShared.mdb, 233
DB (database) drive, 44, 47,
68, 71
Default reporting and
exporting engines, 230
Delay before sampling, 105
Delay between readings, 108
Delay between samples, 105
Delimiters, 231
User’s Guide to KC4™
Delta OD, 130, 163, 181
Detection method, 94, 108,
112
Diagnostics
CALIB database, 233
Optics test, 205, 206
Universal plate test, 205,
207
Dilutions, 151
Direct export to Excel, 308
Dispense protocol, 94, 119
Dispenser
Configuration, 202
Control, 247
Display grid, 165
Document formatting
options for Word, 277
Dose response curves, 303
Duration, plate shaking, 116
E
Eject between filter set read
FLx800, 95
Eject between Filter Set
Read
FL600, 100
SynergyHT/HTTR, 101
Eject between well, FL600,
104
Electronic signatures
Data audit trail, 63
Global data files, 54
Protocol audit trail, 183
Protocol files, 73
ELx800, 78, 196
ELx808, 79, 196
Emission filter wheel, 197,
200
Emission wavelength, 95, 97
Endpoint read
Selecting in a protocol,
113
Engine
Export, 187
Report, 186
Error when getting
wavelength table from
the reader, 69
Index • 315
Excel
Reporting or exporting
engine, 276
Requirement for
PowerReports, 276
Using to design reports
and export files, 273
Excitation filter wheel, 197,
200
Excitation wavelength, 95,
97
Experimental wells, 91
Export engine, 187
Exporting a plate database,
242
Exporting events, 225
Exporting files from the
shared database, 227
Exporting plate data
Automatic export after
reading, 187
Direct export to Excel, 308
Excel, 187, 308
Exporting sample names,
61, 62
KC4, 182, 187
Keep open after exporting,
187
Protecting the document,
187
Selecting a text separator,
231
Selecting data sets for
export, 57
Selecting the exporting
engine, 187, 230
Word, 187, 308
Extrapolation factor, 165,
254
F
Failed login attempts, 216
Failed to open document,
242
Fax numbers, Bio-Tek, 4
Field objects in
PowerReports, 279,
282
File format options, 222
316 • Index
File importation failure,
possible causes, 53
Files
File types, 28
Maintenance, 226
Signing, 30
Storage options, 222
Filter Sets, 95, 97, 103
Filter wheels, 197, 200
Filters/Wavelengths, 194,
196
Filter sets for
SynergyHT/HTTR, 97
Filter sets for the FLx800,
97
KC4 wavelength table
setup, 196
First well, 99
Fitting results, 296
FL600, 80
Absorbance mode, 112
Eject between well, 104
Filter Sets, 95, 103
Fluorescence mode, 112
General Options, 99
Kinetic reading
parameters, 103
Luminescence read
method mode, 112
Measurement Options, 104
Optics Position, 96
Plate Mode, 103
Rapid/Static Mode, 100,
104
Reader Configuration, 201
Sensitivity, 96
Well Mode, 104
Fluorescence
Read method, 112
Time resolved, 117
Fluorophore, 95, 97
FLx800, 81
Fluorescence mode, 112
Luminescence read
method, 112
Measurement Options, 104
Sensitivity, 98
Wavelengths table, 198
Force user to type ID, 217
Formats, 231
User’s Guide to KC4™
Formula examples, 40, 159,
162, 170, 174
Formula for kinetic analysis,
136
Formula for spectrum
analysis, 137, 138
Formula syntax, 160
Multi plate
transformations, 163
Single plate
transformations, 160
G
General Options, FL600, 99
General Options,
SynergyHT, 101
Get filters from reader, 201
Get wavelengths from
reader, 196, 199
GLB files
Global data files, 28, 42
Signing electronically, 54
Global data files, 28, 42
GPH files, 266
Graph files, 266
Group membership, 213
H
High value, 92
High wells, 90, 91
HOLE notation, 197, 200
Horizontal Reading Points,
linear scanning, 115
I
Identifiers, 181
Importing a plate database,
242
Importing files into the
shared database, 228
Importing plate data
Clarity BTI files, 53
Importing plate data, 51
Expected file formats, 51
Importing sample names,
61
Selecting a text separator,
231
Importing sample IDs, 61
User’s Guide to KC4™
INCORRECT READER
MODEL
CONNECTED
message, 69
Incubation
Pre-heating, 117, 246
Temperature control, 117
Individual Well Auto
Scaling, 114, 115
Individual Well Color Auto
Scaling, 116
Init. All Transf., 160
Installing the software, 11
Instruments, controlling
more than one at a
time, 194
Integral, 136
Kinetics, 136
Scanning, 141
Intensity, plate shaking, 116
Interpolation, 169
Examples, 169, 170
Formula syntax, 170
Interpolation results, 298
K
KC4_32.pld file, 10, 244
KCLocal database
Compacting, 235
Explained, 233
Location, 235
Repairing, 235
KCShared database
Compacting, 233
Configuring KC4, 22
Explained, 233
Location, 233
Renaming, 233
Repairing, 233
Restoring connection to,
233
Used for file storage, 222
Keep open after exporting,
187
Keep open after printing,
186
Kinetic analysis, 131, 163,
259
Data to process, 131
Formula, 136
Index • 317
Integral, 136
Lag time, 133
Max V, 132
Mean Min/Max, 134
Mean V, 132
No calculation, 136
Onset OD, 135
Kinetic curves
Overlaying, 258
Report, 179
Viewing results, 258
Kinetic interval, 103
Uneven indication on
reports, 121, 125
Kinetic read, 131
Choosing the time format,
231
Read was interrupted, 49
Real time kinetic zoom,
103
Selecting in a protocol,
113
Kinetic reading parameters,
102, 103
Allow well zoom during
read, 103
Eject between well, 104
FL600, 103
Interval, 103
Minimum kinetic interval,
103
Plate Mode, 103
Reading Number, 103, 136
Run Time, 102
Scales, 114
Well Mode, 104
L
Lag time
Delay before reading, 101
Kinetics, 133, 259
Lamp control, 99
Last well, 99
Lin axis, 165
Linear regression, 166
Linear Regression, 164, 167
Linear scanning, 115, 139
Local database
Compacting, 235
Explained, 233
318 • Index
Location, 235
Repairing, 235
Log axis, 165
LOG function, 160
Log report, 301
Logging in to and out of
KC4, 223
Login parameters, 216
Low wells, 90, 91
Lum. checkbox, 197
Luminescence, 197
Emission filter wheel, 200
Read method, 112
M
Maintain files, 226
Manual data entry, 50
Masking wells
How to, 45
Results window, 252
MAX function, 161
Max V, 132
MEAN function, 160
Mean Min/Max, 134, 138
Mean V, 132
Meas. Filter, 99
Measurement Options, 104
Microplates, measuring, 242
MIN function, 161
Min/Max, spectrum analysis,
137
Minimize KC4, 232
Minimum kinetic interval,
103
Minimum password length,
217
Monitor wells, 107
Multi plate transformations,
162
Multi Plate Transformations
Formula syntax, 163
Overview, 162
Multi user identification,
211, 212
Multi-mode, 94, 108
Multiple instruments,
controlling
simultaneously, 194
Multiple shared databases,
using, 233
User’s Guide to KC4™
Multiple wavelength reads,
109, 118
N
Naming samples, 61
No Calculation, 136
NO READER RESPONSE
(TIMEOUT) message,
69
Normal Mode, 112
Normalizing absorbances to
1 cm, 109
O
OLE mode, 232
OLE mode, minimizing
KC4, 232
Onset OD/FU/LU, 135
Optics Position, 96, 98
Optics test, 205, 206
OUT indication, 256
Out of range wells, 256
OVRFLW, 252
P
Parity, 195
Password parameters, 216
Passwords, 213
Changing, 214
Expiration, 217
Minimum length, 217
Unique, 217
Pathlength Correction, 130
Enabling, 109
Raw data correction, 128
Pathlengths
Calculating, 109
Reporting the pathlength
for each well, 257
Peak OD test, 208
Picture mode, 262
PLA files
Plate data files, 28, 42
Plate data files, 28, 42
Plate database
Exporting, 242
Importing, 242
Plate definition not found,
242
User’s Guide to KC4™
Viewing contents of, 240
Plate definition not found in
plate database, 242
Plate description, 60, 75
Plate format, reports, 181
Plate formats
Custom, 242
Exporting, 242
Importing, 242
List of, 240
Plate height, 241
Plate In/Plate Out, 245
Plate layout
Assay control wells, 145
Blank wells, 144
Dilutions, 151
Sample control wells, 147
Sample wells, 146
Standard wells, 148
Undo the last action, 143
Well IDs, 143, 144
Well settings, 143
Plate Layout, 142, 181
Plate mode
FL600, 103
SynergyHTTR with
Injectors, 119
Plate objects in
PowerReports, 279,
283
Plate shaking, 116
Plate Shaking
Before First / Every
Reading, 116
Duration, 116
Intensity, 116
Plates
Signing electronically, 54
PLD file, 10, 244
PLUG notation, 197, 200
Point to Point curve fit, 164,
167
Polynomial Regression, 164,
167
Port, 195
POW function, 161
Power users
Group membership, 213
Protocol-level protected
functions, 184
Index • 319
System-level protected
functions, 218
PowerReports
Curve objects, 279, 302
Dollar sign character, 280
Editing an object, 280
Field objects, 279, 282
How it works, 274
Overview, 273
Plate objects, 279, 283
Results objects, 279
RSO toolbar, 279
System requirements, 276
Table objects, 279, 286
PowerWave, 82, 196
PowerWaveHT, 82, 196
PowerWaveX, 83, 196
PowerWaveX Select, 84,
196
PowerWaveXS, 85
Preferred data file format,
222
Pre-Heating, 117, 246
Pre-read blank plate, 111
Print a list of sample names,
61
Printing data automatically,
186
Printing reports, 177, 306
Probe orientation, FL600, 96
Probe orientation,
FLx800/SynergyHT/H
TTR, 98
Probe size, 194, 201
Prompt #, 75
Prompt for name, 185
Protected functions
Protocol level, 184
System level, 218
Protocol
Creating a new protocol,
69
Defaults, 230
Defined, 28, 67
Modify, 71
Options, 185
Protected functions, 184
Save, Save As, 71
Security, 184
Protocol audit trail
PowerReports, 300
320 • Index
Protocol menu, 183
Protocol defaults, 230
Protocol description, 177,
182
Protocol file not compatible
with current reader, 69
Protocol files
Contents, 28
Creating, modifying, etc.,
68
Signing electronically, 73
Storage options, 28
Protocol options
Calculation, 188
Export, 187
Raw data, 185
Report, 186
Protocol reading parameters
Automatic sensitivity
adjustment, 96, 98
Endpoint read, 113
Filter Sets, 95, 97
Kinetic read, 113, 131
Measurement Options, 104
Optics Position, 96, 98
Plate shaking, 116
Read Mode, 112
Reading Type, 113
Sensitivity, 96, 98
Spectrum read, 113
Temperature Control, 117
Protocols folder in user
account, 213
PRT files
Protocol files, 68
Protocol Files, 28
Signing electronically, 73
Q
Quick Start, 38
For Clarity luminometer
users, 39
For other instrument users,
38
R
Rapid Mode, 100, 104, 112
Raw data
Options, 185
Raw data correction, 128
User’s Guide to KC4™
Read and dispense protocol,
94, 119
Read from file, 51
Read Matrix Size, area
scanning, 116
Read method, 94, 108, 112
Read Mode
Defining in protocol, 112
Reader configuration, 194
Reader Configuration, 201
Reader control, 245
Reader model, 112
Readers, controlling more
than one at a time, 194
Reading direction, 113
Reading Number, 103, 136
Reading parameters
Clarity, 77
ELx800, 78
ELx808, 79
FL600, 80
FLx800, 81
PowerWave, 82
PowerWaveHT, 82
PowerWaveX, 83
PowerWaveX Select, 84
PowerWaveXS, 85
SynergyHT, 86
SynergyHTTR, 87
SynergyHTTR with
Injectors, 88
uQuant, 89
Reading parameters
reference, 90
Reading plate data from a
file, 51
Reading Type, 113
Real time kinetic zoom, 103
Records, 29
Archival, 29
Retention, 29
Recovery percent
calculation, 155
Reference wells, FL600,
100, 104
Refr. Filter, 99
Registering KC4, 229
Reinstalling KC4, 229
Report
Engine, 186
User’s Guide to KC4™
Reports
Excel, 186
KC4, 177, 186
Keep open after printing,
186
Preview before printing,
59
Printing, 59
Protecting the document,
186
Selecting the reporting
engine, 186, 230
Word, 186
Restoring connection to
KCShared, 234
Retaining records, 29
Run KC4 in trial mode, 18
Run Time, 102
S
Sample control wells, 143,
147
Sample name list report, 180
Sample names, 61
Sample protocol and plate
data files, 40
Sample wells, 143, 146
Saving data automatically,
185
Scale to high wells, 90
Scale to low wells, 90
Scales
Individual Well Auto
Scaling, 114
Kinetic reads, 114
Spectrum reads, 114
Scanning, 262
Analysis, 139
Area, 115
Integral, 141
Linear, 115, 139
SD function, 160
Secured protocol and data
file storage, 222
Security, 184, 211
Send filters to reader, 200
Send wavelengths to reader,
196, 199
Sensitivity, 96, 98
Session idle timer, 216
Index • 321
Session timeout, 216
Set Lamp Off during
measurement, 100
Setup, 230
Formats, 231
Protocol defaults, 230
Shaking, 116
Shared database
Explained, 233
File maintenance, 226
Location, 233
Renaming, 233
System audit trail, 224
Used for file storage, 222
Signature reasons
Applying, 54, 73
Defining, 221
Signing files
Global data files, 54
Protocol files, 73
Simulation data, 50
Single Plate
Transformations, 159
Formula syntax, 160
Overview, 159
Skip reference wells, 100
Software
Trial mode, 18
Software installation, 11
Software version and
revision numbers, 4
SP data, 129
Spectral scanning, 118
Spectrum analysis, 137, 261
Data to process, 137
Formula, 137, 138
Min/Max, 137
Spectrum read
Selecting in a protocol,
113
Spectrum reading
parameters
Scales, 114
Wavelengths, 118
Spline curve fit, 164, 168
SQRT function, 161
Standard curve, printing, 302
Standard deviation, 160
Standard deviation,
weighting of, 188, 230
Standard users
322 • Index
Group membership, 213
Protocol-level protected
functions, 184
System-level protected
functions, 218
Standard wells, 143, 148
Standards, defining, 155
Static Mode, 104
Statistics, 181, 230, 251, 264
Statistics report, 181, 286
Status bar, 31
Stop bits, 195
Stop monitoring when, 108
Stored curves, 266
Sweep Mode, 112
Symbols, 160, 163, 171
????? indication, 256
Defining for cutoffs, 175
How they are assigned,
176
Method of assignment, 256
Out of range, 176
OUT+/- indication, 256
Viewing results, 256
SynergyHT, 86, 199, 200
SynergyHT/HTTR
Detection method, 112
Measurement Options, 104
Reader Configuration, 201
Sensitivity, 98
Top Probe Vertical Offset,
101
SynergyHTTR, 87
SynergyHTTR with
Injectors
Dispenser configuration,
202
Dispenser control, 247
Emptying the tip prime
troughs, 247
Initializing the dispenser,
202
Priming the dispenser, 247
Purging the dispenser, 247
Read and dispense
protocol, 119
Reading parameters, 88
Setting the injector
position, 202
Syringe setup, 202
User’s Guide to KC4™
System Administrator
Administrator
requirements, 8
Appointing, 8
Initial password, 19
System audit trail
Exporting events, 225
Local events, 224
Shared events, 224
System menu
Diagnostics, 205
Overview, 193
Plate formats, 240
Reader control, 245
Readers, 194
Setup, 230
System requirements, 9
System security, 211
System test, 205
T
Table format, reports, 181
Table objects in
PowerReports, 279,
286
Technical assistance center
(TAC), 4
Technical support, 4
Temperature Control, 117
Temporary files
Creation following
database connection
failure, 233
Importing into shared
database, 228
Text separators for
importing and
exporting, 231
Time formats for kinetics,
231
Time is approximate
indication, 120
Time resolved fluorescence,
117
Timeout, session, 216
Timing control, 120
Tip prime troughs, 247
Tip prime troughs, 122
Toolbar, 31, 237
Buttons, 237
User’s Guide to KC4™
Customizing, 237
Top Probe Vertical Offset,
101
TplRep_1 could not be
found, 20
Transformations
Formula syntax, 160, 163
Multi Plate, 162
Single Plate, 159
Transmission speed, 195
Trial mode, 18
U
Unauthorized users
Protected functions, 218
Protocol security options,
184
Uneven kinetic interval, 121,
125
Uninstall notes, 10
Universal plate test, 205,
207
Unmasking wells, 252
Upgrade notes, 10
Upgrading databases, 15,
236
uQuant, 89, 196
Use barcode as filename,
185
Use first filter set sensitivity,
92
Use out of range values in
statistics, 188, 230
User accounts, 211
Creating, 24, 212
Deleting, 215
Locking or unlocking, 215
Modifying, 214
Status, 215
User ID, 213
User notification options,
220
V
Validation, 171
Validation report, 180
Validation results, 192, 295
Index • 323
W
Wavelength Selection, 118
Filter Sets, 95
Filters, 99
Wavelengths (using
monochromators), 118
Wavelengths
Defining in protocol, 118
Spectrum reads, 118
Weighting of standard
deviation, 188, 230
Well analysis results, 293
Well IDs, 143, 144
Well mode
324 • Index
FL600, 104
SynergyHTTR with
Injectors, 120
Well settings, 143
Well zoom curves, 304
Well zoom report, 179
Wells to monitor, 108
WL#, 95
Word
Reporting or exporting
engine, 276
Requirement for
PowerReports, 276
Using to design reports
and export files, 273
User’s Guide to KC4™
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