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Pyros EQS v1.2 Help

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0% found this document useful (0 votes)

2K views154 pages

Pyros EQS v1.2 Help

manual

Uploaded by

Branco Yubini

We take content rights seriously. If you suspect this is your content, claim it here.

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Associates of Cape Cod, Inc.

124 Bernard E. Saint Jean Drive

East Falmouth, MA 02536-4445 USA
Tel: (888) 395-2221 or (508) 540-3444
Fax: (508) 540-8680

© Copyright 2013, Associates of Cape Cod, Inc., All rights reserved

This document has been prepared for use by the customers and authorized personnel of Associates of
Cape Cod, Inc. The information contained in this manual is proprietary. The manual may not be copied,
reproduced, translated or transmitted in any form without the express written permission of Associates
of Cape Cod, Inc.
While Associates of Cape Cod, Inc. has made every effort to produce an accurate and useful manual, no
representation or warranties of any kind, expressed or implied, are made regarding the contents of this
manual, including, but not limited to, completeness or typographical errors.

Adobe® is a registered trademark of Adobe Systems Incorporated in the United States and/or other
countries.
Crystal Reports® is a registered trademark of Business Objects SA or its affiliated companies in the
United States and other countries.
Microsoft®, Microsoft® .NET, Windows Vista®, Windows® XP, Windows Server® and Excel® are
registered trademarks of Microsoft Corporation in the United States and/or other countries.
Windows® and the Windows logo are trademarks of the Microsoft group of companies.
Oracle® is a registered trademark of Oracle Corporation and/or its affiliates.
Pentium® is a registered trademark of Intel Corporation in the U.S. and other countries.
Pyros® and Pyros Kinetix® are registered trademarks of Associates of Cape Cod, Inc.

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 2
Table of Contents
Section One: Use of Manual and Introduction ...................................................................................... 8
1.1 Using This Manual ........................................................................................................................... 8
1.2 Introduction ..................................................................................................................................... 8
1.3 Kinetic Assays .................................................................................................................................. 9
1.4 Pyros EQS v1.2 Intended Use Statement ...................................................................................... 10
Section Two: Pyros EQS and 21 CFR Part 11 Compliance .................................................................... 11
2.1 Overview of 21 CFR Part 11 ........................................................................................................... 11
2.2 Meeting the Requirements of Part 11........................................................................................... 11
2.3 Part 11 Functionality in Pyros EQS ................................................................................................ 15
2.3.1 Security Settings ............................................................................................................................ 15
2.3.1.1 Authentication ...................................................................................................................... 16
2.3.2 Audit Trail Reports ......................................................................................................................... 18
2.3.2.1 Assay Audit Trail Report ....................................................................................................... 19
2.3.2.2 System Audit Trail Report ..................................................................................................... 20
2.3.2.3 User Audit Trail Report ......................................................................................................... 21
2.3.2.4 Custom Audit Trail Report .................................................................................................... 22
Section Three: Installation ................................................................................................................. 23
3.1 Equipment Required...................................................................................................................... 23
3.1.1 Instrumentation Requirements ..................................................................................................... 23
3.1.2 Computer System Requirements .................................................................................................. 23
3.2 Installation Procedures.................................................................................................................. 24
3.2.1 Hardware ....................................................................................................................................... 24
3.2.2 Software ........................................................................................................................................ 24
3.2.2.1 Installation Procedure .......................................................................................................... 24
3.2.2.2 Application Installation Procedure ....................................................................................... 25
3.2.2.3 Database Server Installation Procedure ............................................................................... 28
3.2.2.4 Pyros EQS Application Error Conditions ............................................................................... 32
3.2.2.5 Registration........................................................................................................................... 32
3.2.3 Testing the System ........................................................................................................................ 34
Section Four: Summary of Test Procedures ........................................................................................ 36
4.1 Summary of Test Procedures ........................................................................................................ 36
Section Five: System Preparation ....................................................................................................... 42
5.1 System Preparation - Introduction ................................................................................................ 42
5.2 File Menu ....................................................................................................................................... 42
5.2.1 Delete ............................................................................................................................................ 42
5.2.2 Create Data File from Test............................................................................................................. 43
5.2.3 Logout ............................................................................................................................................ 44
5.2.4 Exit ................................................................................................................................................. 44

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 3
5.3 Options Menu ................................................................................................................................ 45
5.3.1 Select Default Settings................................................................................................................... 45
5.3.2 Store Curve for Analysis ................................................................................................................ 46
5.3.3 Well Uniformity Test (Pyros Kinetix Only) ..................................................................................... 46
5.4 Admin Menu .................................................................................................................................. 48
5.4.1 Change Admin Settings ................................................................................................................. 48
5.4.1.1 Additional Field Designation ................................................................................................. 48
5.4.1.2 Tube Reader Parameters ...................................................................................................... 49
5.4.2 Change Password .......................................................................................................................... 51
5.4.3 Change Security Settings ............................................................................................................... 51
5.4.4 Edit Default Settings ...................................................................................................................... 52
5.4.4.1 General Tab........................................................................................................................... 53
5.4.4.2 Options Tab........................................................................................................................... 55
5.4.4.3 Hardware Tab ....................................................................................................................... 56
5.4.4.4 Lots Tab ................................................................................................................................. 57
5.4.4.5 Custom Fields Tab ................................................................................................................. 58
5.4.5 Setup User ..................................................................................................................................... 59
5.4.6 Administrator Override ................................................................................................................. 62
5.4.6.1 Sign-off Override................................................................................................................... 62
5.4.6.2 Test Complete Override ........................................................................................................ 62
5.4.6.3 Unlock Assay ......................................................................................................................... 63
5.5 Help Menu ..................................................................................................................................... 64
5.5.1 Pyros EQS Help .............................................................................................................................. 64
5.5.2 About ............................................................................................................................................. 64
5.6 System Tray Icon ........................................................................................................................... 65
Section Six: Data Collection ................................................................................................................ 66
6.1 Data Collection .............................................................................................................................. 66
6.2 Test Information Screen ................................................................................................................ 67
6.2.1 General Tab ................................................................................................................................... 69
6.2.2 Options Tab ................................................................................................................................... 72
6.2.3 Hardware Tab ................................................................................................................................ 73
6.2.4 Custom Fields Tab ......................................................................................................................... 73
6.3 Instrument Communication .......................................................................................................... 73
6.4 Collect Data Main Screen .............................................................................................................. 76
6.5 Negative Control Setup ................................................................................................................. 77
6.6 Standard Curve Setup .................................................................................................................... 78
6.7 Positive Controls ............................................................................................................................ 80
6.8 Sample Setup ................................................................................................................................. 81
6.9 View Menu .................................................................................................................................... 83
6.9.1 Graphic Circle ................................................................................................................................ 83

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 4
6.9.2 Numerical Data .............................................................................................................................. 85
6.9.3 Standard Curve Regression Plot .................................................................................................... 86
6.9.4 Reagent Lots Report ...................................................................................................................... 87
6.9.5 Well Info ........................................................................................................................................ 88
6.10 Options Menu ................................................................................................................................ 89
6.10.1 Terminate Test ...................................................................................................................... 89
6.10.2 Printer Setup ......................................................................................................................... 89
6.10.3 Save Format .......................................................................................................................... 90
6.11 Edit................................................................................................................................................. 90
6.12 Test Info ......................................................................................................................................... 91
6.13 Print Test Setup ............................................................................................................................. 92
6.14 Close .............................................................................................................................................. 92
6.15 Right-Click Functionality ................................................................................................................ 92
Section Seven: Data Analysis ............................................................................................................. 94
7.1 Data Analysis ................................................................................................................................. 94
7.2 Standard Curve for Analysis .......................................................................................................... 96
7.2.1 Standard Curve Info....................................................................................................................... 96
7.2.2 Lots ................................................................................................................................................ 98
7.2.3 Analysis Tools ................................................................................................................................ 99
7.3 Graphics ....................................................................................................................................... 101
7.3.1 Data Plots .................................................................................................................................... 101
7.3.2 Standard Curve Regression Plot .................................................................................................. 104
7.4 View ............................................................................................................................................. 105
7.4.1 Test Info ....................................................................................................................................... 105
7.4.2 Test Errors ................................................................................................................................... 106
7.4.3 Well Info ...................................................................................................................................... 109
7.4.4 Reagent Lots ................................................................................................................................ 110
7.4.5 Temperatures .............................................................................................................................. 111
7.4.6 Tube Insertion Delay ................................................................................................................... 111
7.5 Reports ........................................................................................................................................ 112
7.5.1 Report Cover Sheet ..................................................................................................................... 113
7.5.2 Data Summary ............................................................................................................................. 115
7.5.3 Individual Sample ........................................................................................................................ 117
7.5.4 Individual Well Results ................................................................................................................ 119
7.5.5 Assay Audit Trail .......................................................................................................................... 120
7.5.6 Raw Data/OD ............................................................................................................................... 121
7.5.7 Pass/Fail ....................................................................................................................................... 122
7.5.8 Well Uniformity (Pyros Kinetix Only)........................................................................................... 124
7.5.9 Potency ........................................................................................................................................ 126
7.6 Options ........................................................................................................................................ 127

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 5
7.6.1 Test Comments............................................................................................................................ 127
7.6.2 Exclude/Include ........................................................................................................................... 127
7.6.3 Save Format ................................................................................................................................. 128
7.6.4 Supervisor Sign-off ...................................................................................................................... 129
7.6.5 Printer Setup ............................................................................................................................... 130
7.7 Edit............................................................................................................................................... 131
7.8 Test Info ....................................................................................................................................... 131
7.9 Print ............................................................................................................................................. 132
7.10 Close ............................................................................................................................................ 132
Section Eight: Data Trending ............................................................................................................ 133
8.1 Data Trending .............................................................................................................................. 133
Section Nine: Troubleshooting ......................................................................................................... 137
9.1 Tube Reader ................................................................................................................................ 137
9.1.1 No Power ..................................................................................................................................... 137
9.1.2 Front Panel LED Not Red on Instrument Power-up (Pyros Kinetix Only) .................................... 137
9.1.3 Tube Reader Not Detected .......................................................................................................... 137
9.1.4 Tubes Not Detected in the Software During Data Collection ..................................................... 138
9.1.5 Tube-Detect LEDs On but No Tubes Inserted .............................................................................. 138
9.1.6 Tubes Inserted; LEDs Not On ....................................................................................................... 139
9.1.7 Tubes Loose/Tight ....................................................................................................................... 139
9.1.8 Number Ring Lifted (Pyros Kinetix Only) ..................................................................................... 139
9.1.9 Inconsistent Results or Poor Replication ..................................................................................... 139
9.1.10 A/D values out of range ...................................................................................................... 140
9.1.11 Temperature Out of Range ................................................................................................. 140
9.1.12 Bad Data Blocks .................................................................................................................. 140
9.1.13 Missing Data Blocks ............................................................................................................ 141
9.1.14 Interpretation of Data Blocks ............................................................................................. 141
9.2 Pyros EQS..................................................................................................................................... 142
9.2.1 Signed-off Assay Requires Editing ............................................................................................... 142
9.2.2 Assay Will Not Close Using the Terminate Test Icon ................................................................... 142
9.2.3 Assay Closed Abnormally and Not Visible in Data Analysis ......................................................... 143
9.2.4 Service Packs ............................................................................................................................... 143
9.2.5 Login Failure ................................................................................................................................ 143
9.2.6 Enabling/Disabling Audit Trail ..................................................................................................... 144
9.2.7 Keyboard Curve ........................................................................................................................... 144
9.2.8 Report ID Generation .................................................................................................................. 144
9.2.9 Multiple Plot Printing .................................................................................................................. 144
9.2.10 Creating First User .............................................................................................................. 144
9.2.11 System Tray Icon is Red ...................................................................................................... 145
9.2.12 Oracle Errors ....................................................................................................................... 145

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 6
9.2.13 Test Started but Collect Data Screen Remains Blank ......................................................... 145
9.2.14 Test ID Does Not Appear in the Selection List .................................................................... 146
9.2.15 Axes Origin for Plots ........................................................................................................... 146
9.2.16 Field Validation Errors – Red Exclamation .......................................................................... 146
9.2.17 Spaces in Field Values ......................................................................................................... 146
9.2.18 License Registration – Serial Number Entry ....................................................................... 146
9.2.19 Data Group Creation ........................................................................................................... 147
9.2.20 Series is “Broken” After Editing .......................................................................................... 147
9.2.21 Pyros EQS Unable to Generate an Individual Wells Results Report ................................... 147
9.2.22 Backup and Restore ............................................................................................................ 148
Appendix A: Program Calculations ................................................................................................... 149
A.1 Calculation of Optical Density from Numerical Data .................................................................. 149
A.2 Baseline Adjustment.................................................................................................................... 149
A.3 Linear Regression ........................................................................................................................ 149
Appendix B: Pre-Install Checklist ...................................................................................................... 152
B. 3 Pyros EQS Registration Checklist ................................................................................................. 153

Section One: Use of Manual and Introduction

1.1 Using This Manual
Before setting up your Pyros Kinetix® or Pyros Kinetix® Flex system, read:
 Introduction (Section 1) of this manual. Pay particular attention to Section 1.3.
 Installation (Section 3) and follow the directions carefully.
 The user manual accompanying your tube reader.
Before using the system for the first time, read:
 System Preparation (Section 5).
Information related to Pyros® EQS compliance with 21 CFR Part 11 is found in Section 2.
Use the Summary of Test Procedures (Section 4) as a reference for the basic methodology to
setup and run an assay. Pyros EQS is largely self-explanatory and easy to learn. After several
tests, it should only be necessary to use the manual for reference purposes.

1.2 Introduction
Limulus Amebocyte Lysate (LAL) is an extract made from the blood cells (amebocytes) of the
horseshoe crab (Limulus polyphemus). This extract becomes turbid and clots in the presence of
bacterial endotoxin. Endotoxin is a component of the outer membrane of gram-negative
bacteria. By far, it is the most common and significant pyrogen (agents that induce fever). If
endotoxin is present as a contaminant in parenteral drugs or medical devices, it may elicit a
response in humans ranging from mild fever to septic shock, possibly leading to death. A
reliable method of detecting bacterial endotoxin is, therefore, of vital importance to the
pharmaceutical and medical device industries.
In the 1940s the USP Pyrogen Test became the standard test for pyrogens. In this test,
temperature rise (a pyrogenic response) in rabbits is monitored following injection with the
substance being tested. In 1977 the U.S. Food and Drug Administration licensed the in vitro LAL
assay as an alternative to the pyrogen test. Since then, the USP Bacterial Endotoxins Test (i.e.
the LAL Test) has steadily replaced the pyrogen test. Because the LAL reagent is more sensitive
than the rabbit test, it is possible to detect concentrations of endotoxin below the pyrogenic
level.
In all LAL tests, and in horseshoe crabs in nature, endotoxin activates the clotting cascade via
the intermediate factors, C and B. The activated enzyme then clips a peptide from the substrate
coagulogen to form the clotting protein, coagulin. As the coagulin concentration increases, the
sample increases in turbidity. There are three principal LAL methodologies: gel-clot,
chromogenic and turbidimetric.
1. The Gel-Clot Test. The gel-clot test is the simplest LAL method. It is performed by adding
reconstituted LAL to an equal volume of liquid sample in a small glass test tube. The
mixture is incubated at 37°C for an hour. If, after this time, a solid gel-clot forms that can
withstand a 180 degree inversion of the tube without breaking, the test is considered
positive for the presence of endotoxin.
2. The Turbidimetric Test. The turbidimetric test is a refinement of the gel-clot test in that the
turbidity that precedes clot formation is measured. The test is performed by adding LAL to
the liquid sample or standard endotoxin dilution. The turbidity of the reaction mixture is
measured in a spectrophotometer. At higher endotoxin concentrations, clotting enzyme is
activated more rapidly, so the sample becomes more turbid. The assay is read as a kinetic

assay, where the time for each sample to reach a particular optical density (OD) is
measured.
3. The Chromogenic Test. In the chromogenic method, the clotting enzyme acts on a colorless
synthetic substrate and cleaves a terminal chromophore that is colored when separated
from the rest of the molecule. At higher endotoxin concentrations, clotting enzyme is
activated more rapidly, so the rate of chromophore cleavage and color development is
greater. The test can either be read as an endpoint, after a fixed incubation period, or as a
kinetic test in which the time taken to reach a particular OD is determined.

1.3 Kinetic Assays

In kinetic assays, the optical density of an LAL/sample mixture is recorded at regular intervals at
37°C. Pyros EQS stores the data in a database residing on a hard drive or designated network
server. Pyros EQS tracks the data during collection and calculates the amount of time taken for
the OD to cross a fixed threshold level. The amount of time for a given reaction to cross the
threshold is referred to as its Onset Time. Higher endotoxin levels result in faster Onset Times.
Note the kinetic reactions and the threshold line in the figure below entitled Optical Density
Plotted Against Time. For kinetic reactions, the log of endotoxin concentration is inversely
proportional to the log of onset time. Utilizing this, Pyros EQS performs a log-log regression of
onset times and known endotoxin concentrations (standards) to calculate endotoxin
concentration in samples as shown in the second figure below entitled Standard Curve
Regression Plot.

Optical Density Plotted Against Time

Standard Curve Regression Plot

The Pyros Kinetix and Pyros Kinetix Flex tube readers are designed for use with 8x75 mm borosilicate
tubes. Each reaction is timed individually, starting when a tube is inserted into the tube reader. Pyros
Kinetix tube readers provide data on a 10 second read interval. This interval is pre-programmed into
the firmware of the instruments and cannot be user-modified. Pyros EQS is designed to capture the
data from these instruments on the same 10 second read interval.

1.4 Pyros EQS v1.2 Intended Use Statement

Pyros EQS is software designed for use in Limulus Amebocyte Lysate kinetic turbidimetric or
chromogenic assays for the quantitation of gram-negative bacterial endotoxin. The application
collects and processes data from the Pyros Kinetix Flex and Pyros Kinetix tube readers, stores
the information in a database, and produces reports on test results and data trends. Pyros EQS
is designed to be compliant with 21 CFR Part 11.

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 10
Section Two: Pyros EQS and 21 CFR Part 11 Compliance

Section Two: Pyros EQS and 21 CFR Part 11 Compliance

2.1 Overview of 21 CFR Part 11
21 CFR Part 11 Electronic Records and Signatures became effective in 1997. The regulation
describes the steps that must be followed to ensure electronic records, electronic signatures,
and handwritten signatures executed to electronic records, are trustworthy, reliable, and
generally equivalent to paper records and handwritten signatures executed on paper.
The scope of Part 11 includes all records in electronic form that are created, modified,
maintained, archived, retrieved, or transmitted, under any record-related requirement
expressed in FDA regulations.
Software associated with computerized systems used in GxP, (cGMP, GLP, and GCP), processes
must be Part 11 compliant. However, no software by itself is capable of being compliant;
software can only be developed with the technical controls necessary for compliance with Part
11. Pyros EQS was developed with these required technical controls.
To ensure that a software application is compliant with Part 11, procedures and systems must
be in place to establish the required environment. In fact, approximately 50% of the elements
of the Part 11 regulation deal with procedures and systems an organization must have in place
for a software application to meet the requirements of Part 11.
One of the major requirements of Part 11 is that software must be validated to ensure
accuracy, reliability, and consistent intended performance.
Another aspect of the software validation process is that end-users must validate the software
system in their operating environment. ACC has created Validation Protocol Packages specific
to the Pyros Kinetix and the Pyros Kinetix Flex tube readers to assist with this requirement. To
order a Pyros EQS Validation Protocol Package contact a local sales representative or call the
Customer Service department at (800) LAL-TEST (525-8378) or (508) 540-3444.

2.2 Meeting the Requirements of Part 11

The table below contains a listing of the elements of 21 CFR Part 11 and the method of
obtaining compliance: either through Pyros EQS, an end-user procedure, or both.

21 CFR Part 11 Sub-part Part B Electronic Records

11.10 Controls for Closed Systems Method of Compliance

Validation of systems to ensure accuracy, reliability, The end-user must validate the Pyros EQS
a. consistent intended performance, and the ability to system. To assist the user, a Validation
discern invalid or altered records. Protocol package is available from ACC.

The ability to generate accurate and complete copies of

records in both human readable and electronic form
b. Feature of Pyros EQS
suitable for inspection, review, and copying by the
agency.

Protection of records to enable their accurate and ready

c. Feature of Pyros EQS
retrieval throughout the records retention period.

d. Limiting system access to authorized individuals. Feature of Pyros EQS

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 11
Section Two: Pyros EQS and 21 CFR Part 11 Compliance

21 CFR Part 11 Sub-part Part B Electronic Records

Use of secure, computer-generated, time-stamped audit
trails to independently record the date and time of
operator entries and actions that create, modify, or
delete electronic records. Record changes will not
Feature of Pyros EQS and end-user
e. obscure previously recorded information. Such audit
procedure
trail documentation will be retained for a period at least
as long as that required for the subject electronic
records and will be available for agency review and
copying.
Use of operational system checks to enforce permitted
f. Feature of Pyros EQS
sequencing of steps and events, as appropriate.
Use of authority checks to ensure that only authorized
individuals can use the system, electronically sign a
Feature of Pyros EQS and end-user
g. record, access the operation or computer system input
procedure
or output device, alter a record, or perform the
operation at hand.
Use of device (e.g., terminal) checks to determine, as
h. appropriate, the validity of the source of data input or Feature of Pyros EQS
operational instruction.
Determination that persons who develop, maintain, or
use electronic record/electronic signature systems have
i. End-user procedure
the education, training, and experience to perform their
assigned tasks.
The establishment of, and adherence to, written policies
that hold individuals accountable and responsible for
j. End-user procedure
actions initiated under their electronic signatures, in
order to deter record and signature falsification.
Use of appropriate controls over systems
k. (See below)
documentation including:

(1) Adequate controls over the distribution of, access to,

and use of documentation for system operation and End-user procedure
maintenance.

(2) Revision and change control procedures to maintain

an audit trail that documents time-sequenced
End-user procedure
development and modification of systems
documentation.
(3) Persons who use open systems to create, modify,
maintain, or transmit electronic records will employ
procedures and controls designed to ensure the
authenticity, integrity, and, as appropriate, the
confidentiality of electronic records from the point
of their creation to the point of their receipt. Such Not applicable. Pyros EQS operates under
procedures and controls will include those identified a closed system.
in Section 11.10, as appropriate and additional
measures such as document encryption and use of
appropriate digital signature standards to ensure, as
necessary under the circumstances, record
authenticity, integrity, and confidentiality.

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 12
Section Two: Pyros EQS and 21 CFR Part 11 Compliance

11.50 Signatures and Manifestations Method of Compliance

Signed electronic records will contain information
associated with the signing that clearly indicates all of
the following:
(1) The printed name of the signer;
a. (2) The date and time when the signature was executed; Feature of Pyros EQS
and
(3) The meaning (such as review, approval,
responsibility, or authorship) associated with the
signature.

The items identified in paragraphs (a) (1), (a) (2), and (a)
(3) of this section (11.50) will be subject to the same
b. controls as for electronic records and will be included as Feature of Pyros EQS
part of any human readable form of the electronic
record (such as electronic display or printout).

11.70 Signatures Linking Method of Compliance

Electronic signatures and handwritten signatures
executed to electronic records will be linked to their
respective electronic records to ensure that the Feature of Pyros EQS and end-user
signatures cannot be excised, copied or otherwise procedure
transferred to falsify an electronic record by ordinary
means.
21 CFR Part 11 Sub-part Part C Electronic Signatures
11.100 General Requirements Method of Compliance
Each electronic signature will be unique to one
a. individual and will not be reused by, or reassigned Feature of Pyros EQS and end-user procedure
to, anyone else.
Before an organization establishes, assigns,
certifies, or otherwise sanctions an individual's
b. End-user procedure
electronic signature, the organization will verify
the identity of the individual.
Persons using electronic signatures will, prior to or
at the time of such use, certify to the agency that
the electronic signatures in their system, used on
c. End-user procedure
or after August 20, 1997, are intended to be the
legally binding equivalent of traditional
handwritten signatures.
11.200 General Requirements Method of Compliance
Electronic signatures that are not based on
a. (See below)
biometrics will:
(1) Employ at least two distinct identification
components such as an identification code and Feature of Pyros EQS
password.

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 13
Section Two: Pyros EQS and 21 CFR Part 11 Compliance

21 CFR Part 11 Sub-part Part B Electronic Records

(i) When an individual executes a series of signings
during a single, continuous period of controlled
system access, the first signing will be executed
using all electronic signature components;
Feature of Pyros EQS
subsequent signings will be executed using at
least one electronic signature component that is
only executable by, and designed to be used
only by, the individual.
(ii) When an individual executes one or more
signings not performed during a single,
continuous period of controlled system access, Feature of Pyros EQS
each signing will be executed using all of the
electronic signature components.
(2) Be used only by their genuine owners. End-user procedure
(3) Be administered and executed to ensure that
attempted use of an individual's electronic
signature by anyone other than its genuine Feature of Pyros EQS and end-user procedure
owner requires collaboration of two or more
individuals.
Electronic signatures based upon biometrics will
Not applicable. Pyros EQS does not use elec-
b. be designed to ensure that they cannot be used by
tronic signatures based on biometrics.
anyone other than their genuine owners.
11.300 Controls for identification codes/
Method of Compliance
passwords
Maintaining the uniqueness of each combined
identification code and password, such that no two
a. Feature of Pyros EQS and end-user procedure
individuals have the same combination of
identification code and password.
Ensuring that identification code and password
issuances are periodically checked, recalled, or
b. Feature of Pyros EQS and end-user procedure
revised (e.g., to cover such events as password
aging).
Following loss management procedures to
electronically de-authorize lost, stolen, missing, or
otherwise potentially compromised tokens, cards,
c. and other devices that bear or generate iden- Feature of Pyros EQS and end-user procedure
tification code or password information, and to
issue temporary or permanent replacements using
suitable, rigorous controls.
Use of transaction safeguards to prevent
unauthorized use of passwords and/or
identification codes, and to detect and report in an
d. immediate and urgent manner any attempts at Feature of Pyros EQS and end-user procedure
their unauthorized use to the system security unit,
and, as appropriate, to organizational
management.

© Copyright 2013, Associates of Cape Cod, Inc. Pyros® EQS v1.2 Page 14
Section Two: Pyros EQS and 21 CFR Part 11 Compliance

21 CFR Part 11 Sub-part Part B Electronic Records

Initial and periodic testing of devices, such as
tokens or cards, that bear or generate Not applicable. Pyros EQS does not use devices
e. identification code or password information to to bear or generate identification code or
ensure that they function properly and have not password information.
been altered in an unauthorized manner.

2.3 Part 11 Functionality in Pyros EQS

Pyros EQS was developed with flexibility in mind. One organization may desire to use Part 11
settings with some degree of customization whereas another may not be subject to Part 11 and
consequently prefer not to utilize the Part 11 features at all.

2.3.1 Security Settings

When Pyros EQS has been procured and installed in the Part 11 compliant version, an
Administrator will have the access and rights to enable Part 11 features. By default,
Part 11 features will remain deactivated. If Pyros EQS compliant version is installed and
run without activating the Part 11 features, an Administrator can subsequently activate
(or modify) Part 11 features (see Sections 5.4.2, 5.4.3 and 5.4.5 for more information
on access rights), and such events will be captured in the Pyros EQS Audit Trail. The
Part 11 settings are located on the “Security Settings and Audit Trail” screen, accessed
by selecting the following from the “Main Application” screen: Admin  Change
Security Settings.

Security Settings and Audit Trail Screen

The following is a description of each of the Part 11 features found on the “Security
Settings and Audit Trail Activation” screen.
Audit Trail Activation: In Pyros EQS there are four categories of audit trails: System,
User, Assay and Custom. Each captures different information. All four of the categories
are either activated or deactivated together through use of the Enable Audit Trail check
box. Audit trails for individual categories cannot be separately activated or deactivated.
This ensures that if the Audit Trail is activated, all pertinent information is recorded by
Pyros EQS. The decision to not activate Audit Trail tracking may compromise 21 CFR

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Part 11 compliance and should, therefore, only be considered in applications not

subject to regulation by the United States Food and Drug Administration (FDA).
The System Audit Trail records information concerning changes to the system settings
such as user setup, password expiration, authentication requirements, audit trail
tracking, and default settings. Change information is listed in the System Audit Trail
Report.
The User Audit Trail records actions performed by each active user. It records login
events, and particular actions that the user has performed. Actions that write or read
information to and from the database can be recorded and reported in the User Audit
Trail Report.
The Assay Audit Trail captures particular access or change events to a particular test ID.
This information is available in the Assay Audit Trail Report.
The Custom Audit Trail allows the user to select specific events to be reported from the
System, User and Assay Audit Trail events. These events are reported in the Custom
Audit Trail Report.
2.3.1.1 Authentication

User Authentication Dialog Box

Authentication is the application of an electronic signature to a logged event. This is

accomplished by entering the unique user name and password of the individual
performing the action. When a valid user name and password are entered, the user is
allowed to proceed with the desired operation in Pyros EQS. If an invalid attempt is
made, the user is prevented from proceeding. Both valid and invalid entries are
captured in the User Audit Trail.
To be Part 11 compliant, particular actions or events require an explanation or reason
for the occurrence being captured in the audit trail. The “Secondary Authentication”
screen provides users with a method for adding comments to edits performed during
Data Collection and Data Analysis, and when updates are made to various Pyros EQS
settings. This screen is displayed when the Secondary Authentication feature is active
(checked) in the "Security Settings and Audit Trail" screen. The user is forced to enter
comments only if the Reason for Change feature has been activated (checked) in the
“Security Settings and Audit Trail” screen.

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Secondary Authentication Dialog Box

There are several instances in Pyros EQS where secondary authentication may be
required for compliance with 21 CFR Part 11. Secondary Authentication provides an
additional verification of user credentials for the audit trail prior to recording changes
in test or system information. The following is a list of the Secondary Authentications
with a description of each.
Require Authentication when:
 Pyros EQS is opened: Authentication when the Pyros EQS application is started.
 User (Administrator or Supervisor) commits changes to settings: Authentication
when the user edits and accepts changes to Default Settings.
 User edits data within a test ID: Authentication when the user edits metadata
(user input). An example is a user’s determination that the tubes in the standard
curve have been inserted in the reverse order, and that the well descriptions
should be updated to reflect the insertion error. The original data will still be secure
in the database and will not be overwritten.
 When user deletes a Stored Curve: Authentication when the user deletes a Stored
Curve using the "Select Item to Delete” screen by selecting File  Delete from the
“Main Application” screen.
 When user deletes a Format: Authentication when the user deletes a Format using
the “Select Item to Delete” screen by selecting File  Delete from the “Main
Application” screen.
 When user deletes Default Settings Set: Authentication when the user deletes a
Default Settings Set using the “Select Item to Delete” screen by selecting File 
Delete from the “Main Application” screen.
 When user excludes/includes wells: Authentication when the user excludes or
includes a well in the “Analyze Data” screen. Individual wells may be excluded if an
authorized user determines that particular test well is invalid. Alternatively,
excluded wells may be re-included if an authorized user needs to use that data.
Security Settings: The following is a description of each of the security setting options.
 Limit User Access to Audit Trails: When checked (default), users with Technician
level access will not be able to view the System or User Audit Trail Reports. They
will only be able to view the Assay Audit Trail Report. Administrators and
Supervisors are able to view all three audit trail reports.

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 Re-authentication Timeout: This feature allows an Administrator to define a time

period that allows a user to authenticate once and then perform actions without
requiring the user to re-authenticate for each action performed. All actions during
this time period are automatically authenticated and tracked in the audit trails.
After this defined time period has ended, the next action that requires
authentication will cause the User Authentication window to reappear, forcing the
user to re-authenticate. The Administrator can choose to disable the need for re-
authentication by enabling the No Timeout option. The Timeout Authentication
dialog box appears as depicted below.

Timeout Authentication

2.3.2 Audit Trail Reports

Audit trail information can be retrieved via the "Audit Trail Report" screen, which is
accessed by clicking the “Audit Trail” icon on the “Main Application” screen.
On the "Audit Trail Report" screen there are four audit trail reports available: Assay,
System, User, and Custom. Prior to selecting a report, the user can filter the report's
contents to a desired date range, user(s), instrument(s), and system(s). For a Custom
Report, the user can select the audit trail events that the user wants reported on the
Custom Report.
Clicking and selecting the appropriate radio button displays the corresponding report:
 Assay Audit Trail Report
 System Audit Trail Report
 User Audit Trail Report
 Custom Audit Trail Report

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Audit Trail Reports Selection

The User Name field allows for the selection of the users to be included in the Audit
Trail Report. The default is "All," indicating that all users have been selected. A single
user can be selected by clicking the down arrow and then clicking on the desired user
name in the list. The selected user name will then be highlighted.
The Computer ID can be used to refine the contents of an Audit Trail Report.
The Start Date indicates the beginning date that information will be retrieved from the
database for inclusion in the Audit Trail Report. The End Date is the last date for
information to be included. The date for each field can be selected by clicking the down
arrow and then clicking the desired date on the pop-up calendar. The selected date will
be displayed in the field. All data between and including the Start and End Dates will be
displayed in the report. The default date range used to populate these data fields is the
current date.
When the appropriate filtering has been applied using the fields on the "Audit Trail
Report" screen, an Audit Trail Report can be displayed by clicking the appropriate
report button.
Clicking the "Cancel" button returns the user to the “Main Application” screen.

2.3.2.1 Assay Audit Trail Report

The Assay Audit Trail Report displays modifications associated with a Test ID, or Test
IDs within the specified date range. For each assay (Test ID), the following are
displayed: date and time the modification was made, user name, test ID, pre- and post-
modification values, and comments.

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Assay Audit Trail Report

Note that changes or edits made to well series and/or replicates will be recorded as
individual well edits in the Audit Trail Report.

2.3.2.2 System Audit Trail Report

The System Audit Trail Report tracks changes to system wide settings. The report lists
the date and time of the change, the user that authenticated the change, the
parameter that was changed and comments related to it.

System Audit Trail Report

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2.3.2.3 User Audit Trail Report

The User Audit Trail Report shows successful logins, logouts, unsuccessful login
attempts, and the creation and closure of test IDs. The date and time of the event, and
the user name of the individual who performed the event, are also included.

User Audit Trail Report

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2.3.2.4 Custom Audit Trail Report

The Custom Audit Trail Report allows the user to select specific events categorized
under the System, User and Assay Audit Trail Reports discussed above, and generate a
custom report.

Custom Audit Trail Report

Section Three: Installation

3.1 Equipment Required

3.1.1 Instrumentation Requirements

Refer to the manual accompanying the Pyros Kinetix or Pyros Kinetix Flex tube reader
for information regarding each instrument’s requirements.

3.1.2 Computer System Requirements

Note: It is strongly recommended that the Windows environment is up to date with the latest
available service packs. To check a system, run Windows Update to automatically scan
the computer and determine if any upgrades are necessary. Even new computers may
need upgrades.
The following items are required for the installation of Pyros EQS but are not supplied:
Pyros EQS Application
System Requirements for Windows 32-bit
Requirement Value
Operating System Windows 7 or Windows XP (English,
German or Japanese)
Physical Memory 2 GB minimum
Free Disk Space 2 GB minimum

System Requirements for Windows 64-bit

Requirement Value
Operating System Windows 7 (English, German or Japanese)
Physical Memory 3 GB minimum
Free Disk Space 2 GB minimum

Pyros EQS Oracle Server

For additional information please see the Oracle Database Pre-installation Requirements
section of the Oracle Database Installation Guide included on the Pyros EQS installation
media.

System Requirements for remote database

Requirement Value
Operating System Windows Server 2008 R2
Processor Architecture AMD64 or Intel EM64T
Physical Memory 2 GB minimum
Free Disk Space 10 GB minimum
15 GB or greater recommended
Screen Resolution 1024 x 768 minimum

Other Static ip address

System Requirements for local database Windows 32-bit

Requirement Value
Operating System Windows 7 or Windows XP (English,
German or Japanese)
Processor Architecture Intel (x86)
Physical Memory 1 GB minimum
Free Disk Space 10 GB minimum
15 GB or greater recommended
Screen Resolution 1024 x 768 minimum

System Requirements for local database Windows 64-bit

Requirement Value
Operating System Windows 7 (English, German or Japanese)
Processor Architecture AMD64 or Intel EM64T
Physical Memory 2 GB minimum
Free Disk Space 10 GB minimum
15 GB or greater recommended
Screen Resolution 1024 x 768 minimum

3.2 Installation Procedures

3.2.1 Hardware
Refer to the manual accompanying the Pyros Kinetix or Pyros Kinetix Flex instrument
for information regarding installation of the tube reader.

3.2.2 Software
This installation procedure and the instructions below assume that the user is familiar
with basic Windows operations. If additional assistance is required, contact the
Technical Services Department of Associates of Cape Cod, Inc. at (800) 848-3248,
(508) 540-3444 or email [email protected] for assistance.

3.2.2.1 Installation / Upgrade Procedure

Note: Please consult Appendix B: Pre-Install Checklist prior to attempting any installation of the
Oracle database and/or the Pyros EQS application.
Pyros EQS v1.2 Software has two components the Pyros EQS Application and the Pyros
EQS Oracle Server. The two components can be installed on separate computers in a
client / server configuration or they can be installed on the same computer in a
standalone configuration.

3.2.2.2 Application Installation / Upgrade Procedure

Note: A Pyros EQS Database Server must be installed prior to installing the Pyros EQS
Application. No changes are necessary to existing Pyros EQS Database installations (v1.0,
1.2 and 1.2 SP1). The database EZConnect connection string
(DB_SERVER_NAME/DATABASE_NAME) and the Oracle SYS user password will be
required to install the Pyros EQS Application.

Note: Pyros EQS uses TCP connections for inter-process communication and database access.
Some antivirus / firewall software may block this form of communication. This can cause
unexpected early test termination and other exceptions. Please review your antivirus /
firewall policies and if necessary exclude the EQS installation directory.
Step 1. Verify that a Pyros EQS Database is installed before initiating application installation.
Step 2. Remove any installed version of the Pyros EQS Application prior to v1.2 SP1 installed on
the computer using Add / Remove Programs.
Step 3. Ensure that the Pyros EQS Application and the Pyros EQS Tray Icon are closed (if
applicable).
Step 4. Insert the Pyros EQS setup disk into your computer’s DVD drive and launch the Pyros
EQS Setup program from the root of the DVD. Accept the Pyros EQS License Agreement
and click “Install” to start the setup routines.

Step 5. The Pyros EQS Setup program will now preform a system check and will install any
needed prerequisites.

Step 6. After any needed prerequisits have been installed the Pyros EQS Setup Wizard will
appear. Click “Next” to continue.

Step 7. The Configuration screen will now be displayed. Enter the Pyros EQS Oracle Database
Connection String and the Pyros EQS Oracle Server SYS Password and click “Next”.

Examples:
For a local Oracle Database with a global database name Ora10g enter:
localhost/ora10g

For a remote Oracle Database on the server oracleserver with a global database name
ora10g enter: oracleserver/ora10g

Note: Port 1521 (the Oracle SQL Net Listener default port) will be used if no port
number is specified in the connection string.

Step 8. Click “Install” to continue the installation.

Step 9. The Pyros EQS Setup program will install the Pyros EQS Application and Install / Update
the Pyros EQS Schema on the specified Pyros EQS Oracle Database.

Step 10. After the installation has completed click “Finish” and then “Close”.

3.2.2.3 Database Server Installation Procedure

Pyros EQS is supplied with a copy of Oracle Database 11g Standard Edition One for use
with Pyros EQS. The steps below outline a typical installation scenario. For additional
information please see the Oracle Database Installation Guide included on the Pyros EQS
installation media or contact ACC Technical Services Department at (800) 848-3248, (508)
540-3444 or via email at [email protected].

Step 1. Insert the Pyros EQS setup disk into your computer’s DVD drive and launch the Oracle
Universal Installer (setup.exe) from the Oracle Database folder in the root of the DVD.
Step 2. When the Configure Security Updates screen is displayed click “Next”.

Step 3. The “Download Software Updates” screen will be displayed. Select “Skip software
updates” and then click “Next”.

Step 4. At the “Select Installation Option” screen select “Create and configure a database” and
then click “Next”.

Step 5. At the System Class screen select “Desktop Class” and click “Next”.

Step 6. At the “Typical Install Configuration” screen verify the installation paths are appropriate
for your enviornment, the Database Edition is Standard Edition One and the Character
Set is AL32UTF8, WE8MSWIN1252 or WE8ISO8859P1. AL32UTF8 is recommended for
all new installs.

Note: Record the Global database name and the Administrative password (SYS user
password). These will be required to install the Pyros EQS Application.

Step 7. The Oracle Universal Installer will check your system for the minimum installation
requirements. If any checks fail please resolve these before continuing.

Step 8. The “Summary” screen will be displayed. Click “Install” to install the Oracle Database.
Step 9. The Installation will progress.

Step 10. When the Database Configuration Assistant completes click “OK” to close the window.

Step 11. Click “Close” to close the Oracle Universal Installer and complete the installation.

3.2.2.4 Pyros EQS Application Error Conditions

If any errors occur, contact ACC Technical Services Department at (800) 848-3248, (508)
540-3444 or email at [email protected] for assistance.

3.2.2.5 Registration
Prior to using Pyros EQS for the first time, the system (computer/instrument
combination) must be registered with Associates of Cape Cod, Inc. This is very simple to
do, but requires that the Pyros EQS Registration Checklist, found in Appendix B.3, be
completed. Once the necessary fields have been filled out, please fax a copy to (508)
540-8680. ACC can then email the required codes. Alternatively, call our Customer
Service department at (800) LAL-TEST (525-8378) or (508) 540-3444 and speak with a
representative in order to immediately obtain the authorization codes.
Note: It is very important that the License Registration dialog box not be closed
and/or the application exited until the appropriate authorization codes
have been received and entered. The registration codes are randomly
generated and will change if the dialog box is closed and reopened, thus
rendering the authorization codes provided by ACC for the prior
registration code(s) nonfunctional.
If a Network Version of the software has been installed, performing the registration
process on a single client machine will unlock all the Client applications installed. For
Standalone Versions, the registration process has to be performed on every single
computer on which Pyros EQS is installed.
Once registered, Pyros EQS may be used to collect data, analyze data, or simply for
demonstration purposes.
To register a copy of Pyros EQS, double-click the application icon on the computer
desktop. The Login dialog box will be displayed.

At the “Login” screen, enter "admin" in the User Name field and "password" (or
previously established password) in the Password field then click “OK”. Pyros EQS
should now display the “Main Application” screen.
Click on the “Collect Data” icon. The License Registration dialog box will be displayed
(image below). It will be necessary to select the Instrument Type, and enter its 6
character serial number (letters in capitals as it is case-sensitive) separated by a
hyphen. For Pyros Kinetix Flex instruments, use the last 6 characters of the serial
number. Check the Compliance License Purchased box if a 21 CFR Part 11 compliant
version of Pyros EQS has been purchased (optional). The software will now generate a
Registration Code (two codes if the compliance check box is checked). At this time,
please refer to Appendix B.3 and fill out the relevant information. The information can
be emailed or faxed to the Customer Service Department at ACC.
Note: The answers provided regarding company and end-user information will be
kept confidential, but are necessary to track Oracle licensing agreements
and fees. Please see Appendix B.3 for additional details.

Pyros EQS Registration Screen

Note: Do NOT close the License Registration dialog box and/or exit the
application until the appropriate authorization codes have been received
and entered. The registration codes are randomly generated and will
change if the dialog box is closed and reopened, thus rendering the
authorization codes provided by ACC for the prior registration code(s)
nonfunctional.

If ACC has been contacted by phone, the customer service representative will then read
off an Authorization Code that must be entered on this screen. Note that if the
Compliance License Purchased check box is enabled, two 9 digit serial number codes
will be provided. Enter these codes one after the other, separated by a hyphen, as
shown here: 123456789-987654321.
Authorization codes for faxed and emailed registration requests will be communicated
as indicated during the request.
Once successfully entered, click “Save” and a message box will be displayed indicating
“Note: Valid License. Save operation is complete”. Click “OK” and the “Test
Information” screen will be displayed.

3.2.3 Testing the System

After the hardware has been connected and the software installed, test the
system to check communication between the instrument and the computer.
Start the computer. Double-click on the “Pyros EQS” icon or go to Start 
Programs  Associates of Cape Cod  Pyros EQS, to start the program.
At the login screen, enter “admin” in the User Name field and “password” in the
Password field, and click “Enter”.
Note: As the above password is a "published" password, the Administrator must
change the Admin password to a new password, to prevent unauthorized
access to the system (Sections 5.4.2 and 5.4.3). For an installation of the
Pyros EQS software to be compliant with 21 CFR Part 11 guidelines,
changing this “published” password is essential.
Note: It is strongly recommended that two Administrator level users are setup
on the system after installation: a ‘system’ Administrator (based in the
laboratory) and a ‘super’ Administrator (based in the customer IT
department). These login credentials should be recorded and retained as
appropriate in order to prevent lock-outs. See Section 5.4.5: Setup User for
more information.
From the “Main Application” screen click the “Collect Data” icon.

The “Test Information” screen will appear. Enter a test ID in the Test ID field.
Select or enter a Data Group. Select the various tabs to edit the test parameters.
Use the Hardware tab to select the instrument type (Turbidimetric or
Chromogenic), instrument (Pyros Kinetix or Pyros Kinetix Flex, variable
configuration), 6 character instrument serial number, computer system ID and
serial port (COM). Specific test parameters for the Pyros Kinetix tube readers are
defined in the General tab. Make sure all fields have appropriate values entered,
using the LAL Reagent Product Insert for reference. Click “OK”. The program will
then test for communication with the instrument. Accept that the device is ready,
and click “OK”.
If communication is not established, an error box will open with a message
indicating that there is no communication with the instrument. Clicking the
“Abort” button will return the user to the”Main Application” screen. There are a
number of possible reasons for the lack of communication. These are listed below
with remedies.
1. Tube reader is not turned on - turn on the instrument.
2. Tube reader is not properly connected to the computer - make and/or check
cable connections.
3. Tube reader connected to the wrong serial port of the computer - connect to
the correct port or change the port setting by selecting the Hardware tab and
update the information in the Serial Port field to the correct port designation.
4. Instrument did not properly reset – turn off the power supply on the Pyros
Kinetix power adapter, or main power switch on Pyros Kinetix Flex tube
readers, for 1 minute, then turn on and retry.
If the cause of a communications error cannot be found or corrected, contact the
Technical Services Department of Associates of Cape Cod, Inc. at (800) 848-3248
or (508) 540-3444.

Section Four: Summary of Test Procedures

4.1 Summary of Test Procedures
This section is a quick guide to the necessary steps required to setup, run, and analyze a test
using Pyros EQS. More detailed descriptions of the various screens and fields are provided in
other sections of this manual, as referenced.
1. Follow the installation procedures to install the Oracle Database, Client and Pyros EQS
application as described in Section 3: Installation of this manual. Once the installation
procedures have been completed, a Pyros EQS shortcut icon will appear on the computer
desktop as shown below.

Pyros EQS Shortcut Icon

2. Double-click the “Pyros EQS” icon on the computer desktop to open the program. The
following screen will be displayed.

Pyros EQS Login Screen

3. The user will be prompted to supply a user name and password. As a first time user enter
"admin" as the user name and “password" as the password. These are "published" values
and should be changed by the System Administrator as soon as possible, to prevent
unauthorized access. See Sections 5.4.2 and 5.4.5 respectively for changing passwords and
setting up new users. The user will be required to re-authenticate after making changes to
various fields based on the controls setup in Section 2.3.1.
4. The “Main Application” screen of Pyros EQS will now be displayed. This screen is shown
below. The user may either setup default settings and users via the Admin  menu options
(Change Admin Settings, Change Security Settings, Edit Default Settings, or Setup Users) or
start a test. See Section Five: System Preparation for more information on default settings.

Pyros EQS Main Application Screen

5. To start a test, click the “Collect Data” icon. This will open the “Test Information” screen.
Several tabs: General, Options, and Hardware will be displayed across the top of the screen,
with General as the default tab (an additional tab, Custom Fields, may also appear). If
Default Settings have been configured (Section 5.4.4) it should only be necessary to enter a
Test ID. The Data Group listed will be dependent on the default settings applied. The user
can elect to create the test in a different Data Group, if additional Data Groups have been
created by the Administrator and “Read + Write” privileges to those data groups have been
assigned to the logged in user. The Test ID is the assay name and Data Group designates
the area of the database the test will be assigned to. For more information on Data Groups
and access controls see Section 5.4.5. Make sure all the fields in the various tabs are filled
in and the appropriate serial port is selected under the Hardware tab for connection to the
Pyros Kinetix or Pyros Kinetix Flex instrument being used.

Test Information Screen

When fully expanded by clicking on the “Show More Info” button, the “Test Information”
screen looks as depicted below.

Test Information Screen – Show More Info Expanded

6. After entering the appropriate test information click the “OK” button.
If a Pyros Kinetix Flex instrument is selected on the Hardware tab, the software will initially
display the “Setting instrument wavelength” screen. This screen will cycle through four
messages: “Setting calibration mode”, “Sending wavelength select command”, “Sending
wavelength set value command”, and “Exiting calibration mode”. At any time, if desired,
the user can click “Abort” to return to the “Test Information” screen.
Upon completion of the “Exiting calibration mode”, the “Device Status” screen will display.

Pyros Kinetix Flex – Setting Instrument Wavelength

Upon completion of the wavelength calibration, the “Device Status” screen will appear. On
assays where the Pyros Kinetix instrument is selected in the Hardware tab, the “Device
Status” screen will be displayed immediately after clicking “OK” on the “Test Information”
screen.
This screen will update to reflect the status of communication with the instrument, the
type of instrument in use, the temperature of the instrument, and whether any tubes are

present in the instrument. At any time, if desired, the user can click “Abort” to return to the
“Test Information” screen.

Device Status Screen – Awaiting Instrument Response

Device Status Screen – Instrument Communication

Device Status Screen – Instrument with Tubes Detected

Once the target temperature has been reached and no tubes are detected in the
instrument, this screen will close automatically and a confirmation screen will be displayed.

Instrument Communication Confirmation Screen

Click “OK” to continue to the “Collect Data” main screen.

At the “Collect Data” main screen, use the icon buttons at the top of the screen, to assign
wells as Negative Controls, Standards, Positive Controls (available option under Std. Curve),
or Samples. See Sections 6.5 thru 6.8 for additional information on entering values in these
fields. Well descriptors may be entered or changed before, during and after a test. Once a
test is complete (Data Collection complete), well descriptors for wells with no tubes (no
raw data) may not be assigned. Further, once a test has been signed-off by a Supervisor, no
test parameters, including well descriptors, can be modified.

Collect Data Main Screen – Test Wells Defined

7. The test should be performed as described in the LAL Reagent Product Insert.

8. When the test has run for a sufficient amount of time, such that all of the standards have
crossed the Threshold OD (Section 6.2.1) and all samples have run at least as long as the
lowest standard, the test may be terminated by clicking the “Close” button at the top of the
screen. If a maximum test time or Auto-end option (Section 6.2.1) has been applied, the

assay may be left to run, until it auto-terminates. Once the test ends, the following dialog
box will appear.

Data Collection Complete Dialog Box

Clicking “OK” on the dialog box will take the user to the “Analyze Data” main screen where
Pyros EQS will display the assay results.

9. The “Analyze Data” main screen will now be displayed. Using the icon buttons at the top of
the screen the user may look at and print various reports, standard curve regressions,
include/exclude wells, etc. See Section 7: Data Analysis for more information on the
options and reports available to the user.

Analyze Data Main Screen

Section Five: System Preparation

5.1 System Preparation - Introduction
This section is designed to help the user understand the functionality available on the “Main
Application” screen of Pyros EQS. More specifically, this section elaborates on the definition of
Default Settings and the configuration of Admin/Security Settings parameters.
Default Settings define the user's common test procedure information. Default settings also
define system information such as the test method (Turbidimetric or Chromogenic),
instrument type and configuration, serial numbers and the serial port that will be used to
communicate with the Pyros Kinetix or Pyros Kinetix Flex. Admin/Security Settings limit
individual user access rights based on their level of access: Technician, Supervisor, or
Administrator. Many of these functions have 21 CFR Part 11 implications and are referred to in
Section Two: Pyros EQS and 21 CFR Part 11 Compliance.

5.2 File Menu

5.2.1 Delete
The Delete menu item from the File menu brings up the following dialog box.

File  Delete Screen

Three types of items can be deleted from the Pyros EQS system: Default Settings,
Stored Formats, and Stored Curves. Test IDs cannot be deleted.
Depending on the Data Group selected from the list of available Data Groups in the
drop-down, and the Item Type selected, all available items belonging to the selected
type (ex: Default Settings) and the selected data group will be listed under Item to
Delete. Select the item to be deleted and click “OK”.
Note that only Administrators have delete privileges. This menu item will be disabled
for users without Administrative rights.

5.2.2 Create Data File from Test

This feature allows the user to create raw data files from tests that have been run on
the Pyros EQS system. These raw data files are created by Pyros EQS with a .tst
extension and can be used to run the same test in simulation mode. More information
about running tests in Simulation Mode can be found in Section 6.2.3.

Create Data File from Test Screen

Create Data File from Test – Test ID Selected

On the Create Data File from Test dialog box, select the Test ID from the drop-down list
of Test IDs available. Once the user selects the Test ID, the software automatically
generates a Data File name for the output data file. The Data File, by default, is placed
in the folder where the Pyros EQS application executable resides. The default name
assigned to the Data File by the software is the Test ID suffixed by the date of data
collection for that test.
Click “OK” to create the Data File. Click “Cancel” to cancel the operation.
Note: To successfully create a data file from a pre-existing test, the user must have local
Administrator rights on the computer with the EQS application installed. Otherwise, the
generated file will not be generated in the EQS application folder, and an error message
will occur.

5.2.3 Logout
File  Logout will log the user out of the Pyros EQS software without exiting the
application completely.

Logout Confirmation Screen

The dialog box above will be displayed when the user selects File  Logout. Click “Yes”
to log out of the software. Click “No” to return to the “Main Application” screen.
Once a user logs out, the software will display the Login dialog box (See Section 4.1).
Any registered Pyros EQS user can then enter valid login credentials to gain access to
the software.

5.2.4 Exit
File  Exit will log the user out of the application and close the Pyros EQS software.

5.3 Options Menu

Main Application Screen – Options Menu

5.3.1 Select Default Settings

Default Settings are resident within Data Groups. In each of the Data Groups for which
they have “Read + Write” privileges, a user can create multiple Default Settings sets. It
is strongly recommended that, upon first use of the software, a user create at least one
default setting set, with information specific to the computer, test method, and
instrument in use. For additional information on creating Default Settings, please refer
to Section 6.2.
If more than one default dettings set has been created on the system, other than the
factory supplied Default Settings, the software will display a selection dialog box for
Default Settings.

Select Default Settings

Select the Default Setting from the drop-down list and click “OK” to begin using this
default setting.

Note: It is strongly recommended that the user create default settings for each workstation.
Additionally, if the system includes a Pyros Kinetix Flex and the user anticipates running
both chromogenic and turbidimetric assays, a default settings group should be created
for each assay type. Specific assay parameters are recommended in the LAL Reagent
Product Insert.

5.3.2 Store Curve for Analysis

The Store Curve for Analysis function allows the user create a Keyboard standard curve
and store it for use in future test analysis.

Standard Curve for Analysis Screen

For additional information on the various parameters for this screen, which is similar to
the “Standard Curve for Analysis” screen in Data Analysis, please refer to Section 7.2. A
notable difference is that the “Use This Curve” button remains disabled. Specify the
information pertaining to the desired line equation, endotoxin concentration range,
potency and related curve parameters on the Standard Curve Info and Lots tabs, and
click “Save” after specifying a Stored Curve ID and Data Group. The curve parameters
will be stored for future use. The user can now apply this curve to any test that they
want to analyze by selecting the “Stored Curve ID” on the “Test Information” screen
(during Data Collection and Data Analysis) or on the” Standard Curve for Analysis”
screen (during Data Analysis).

5.3.3 Well Uniformity Test (Pyros Kinetix Only)

The Well Uniformity Test option is only available for use with the Pyros Kinetix tube
reader. The checkbox is a quick shortcut to a partially configured “Test Information”
screen, used when performing a Well Uniformity Test for a Pyros Kinetix instrument.
Selecting this menu item takes the user directly into the “Test Information” screen for

Data Collection, with the Well Uniformity Format applied by default. The Well
Uniformity Test and format-related buttons are automatically configured and disabled.

Test Information Screen – Well Uniformity Test

To run the Well Uniformity Test, check and confirm the parameters on the Hardware
tab. The user will also have to click the “Show More Info” button, click the “Apply
Curve” checkbox under Stored Curve, and select the “Well Uniformity Stored Curve”, so
that the results of the well uniformity assay may be calculated. Once the instrument is
setup and the tubes have been prepared for the test, click “OK”.
Once the Well Uniformity Test has been performed, a special Well Uniformity Report
can be generated during Data Analysis. This report is explained in Section 7.5.8.

5.4 Admin Menu

Main Application Screen - Admin Menu

The Admin menu option allows the user to perform Administrative functions for their Pyros
EQS installation. The menu items and their functions are explained in detail below.
Note: Admin system settings (5.4.1, 5.4.3) can only be modified by an Administrator.
Individual system Default Settings (5.4.4) may only be modified by an Administrator or
Supervisor.

5.4.1 Change Admin Settings

At the “Main Application” screen select Admin  Change Admin Settings. This opens
the “Administrator Settings” screen. Two tabs are available: Additional Field
Designation and Tube Reader Parameters.

5.4.1.1 Additional Field Designation

The Additional Field Designation function can be used by an Administrator to create
additional information fields. Once new fields have been created for a particular set of
Default Settings, they will be available under the Custom Fields tab in the specified
default settings or the “Test Information” screen, once a user has selected the Default
Settings (Sections 5.4.4.5 and 6.2.4).

Administrator Settings – Additional Field Designation Tab

5.4.1.2 Tube Reader Parameters

Tube Reader Parameters define the physical manner in which data is collected and the
controls placed on data collection. These fields may be altered, but will significantly
impact the quality of the assay results. Please consult with an ACC representative
before altering any of these fields.
Cycle Range for A/D Zeroing Begin – End: (6) – (12) The values entered in these fields
define the range of data points used in the averaging of A/D values, which are in turn,
used to calculate the "Time 0" A/D value. This is the "Before reaction" A/D value, from
which all OD calculations are made. Default values of 6 and 12 cycles equate to a range
from 60 to 120 seconds.
Valid A/D Count Range Min – Max: Default range is 800 - 2520 for Pyros Kinetix [Non-
Flex] and 800 - 3500 for Pyros Kinetix Flex [Flex] instruments. These recorded values
define the Min and Max acceptable A/D values for each respective tube reader. Pyros
EQS will only perform this check during the zeroing period, the range for which is set in
the above field range. If the A/D for a given well falls outside of the acceptable range
during the zeroing period, the A/D error is recorded, and will be displayed in Data
Analysis, under View  Test Errors. See Section 7.4.2 for additional information.
Minimum OD Change for Data Point Storage (1.5) the value displayed describes the
smallest increment of change, in mAbs units, that will be stored. The value that Pyros
EQS compares to the minimum change setting is the absolute value of the current A/D

value, minus the A/D value from the previous read. If the value calculated is greater
than or equal to the minimum OD change value, the data point is stored. If the number
of consecutive readings that have not met or exceeded the described amount of
change equals the value entered in the Maximum number of cycles between data
readings field, the data point is stored. This insures that the maximum number of cycles
between data readings is not exceeded. The default value is 1.5 mAbs units.
Number of Points Above/Below Threshold for Onset Time Regression (3) In this field,
the user may edit the total number of points used to perform the linear regression to
calculate the adjusted onset time. Note that the "adjusted onset time" for a given well
should be calculated after the "baseline adjustment" has been applied for that well.
Default is 3 points.
Maximum Number of Cycles between Temperature Readings (6) Allows the user to
edit the frequency of temperature reads. Default is 6 cycles, which equates to 60
seconds.
Maximum Number of Cycles between Data Readings (6) Allows the user to edit the
maximum number of cycles allowed between stored data points. Default is 6 cycles,
which equates to 60 seconds.

Administrator Settings Screen - Tube Reader Parameters Tab

Sudden OD Change Detection

% Change to Suspect Sudden OD (5.0%) When Pyros EQS is performing the test for
minimum change for data point storage, it also applies a check for a maximum change
in A/D value. Instead of being described in mAbs units, it is described as a maximum
percent change between stored A/D data points. A percent A/D change of greater than

the user defined value indicates an abnormally rapid change in OD. Typical causes of
such rapid changes are non-LAL related, usually accidental movement of the tube or
instrument. If the percent change between stored A/D data points is greater than the
user-defined value, the tube will be "monitored" to see if the rapid change is a single
event (moved tube or bumped instrument) or a continuing event (LAL-endotoxin
reaction). The default is 5.0%.
No. of Data Points to Monitor after Suspect Status (3) When a tube reflects an
abnormally fast change in OD, it will be monitored for the number of data points
specified in this field. The default is 3 readings.
% Change in A/D Value to Remove Suspect Status (0.5%) This field is used to check
whether the A/D value of a sample continues to change after a sudden change. If the
A/D value continues to change, it is assumed to be the result of a reaction; if it does
not, it is likely that the sudden change in OD was not due to a reaction in the tube but
to some other event. Such events include a bubble bursting or bumping a tube or the
instrument.
The value in the field is the percent change in the average of the A/D values (number
selected in the previous field - see above) that are required to remove the suspect
status. If the change is less than the value, a sudden OD change is recorded and
reported. The default is 0.5%.
Logo for Reports This feature allows an Administrator to specify a .JPG image file to be
used by Pyros EQS for display in the top right corner of all reports.

5.4.2 Change Password

From the “Main Application” screen, choose Admin  Change Password. This will open
the “Change Password” screen. This screen allows only the default (factory created and
shipped) Admin user to change their password. Pyros EQS takes a significant step
forward in simplifying security and application access by using Windows authentication
for all users wanting to use the application. Only those users with valid Windows
operating system accounts can use Pyros EQS, once these users have been authorized
within Pyros EQS.

Change Password Screen

5.4.3 Change Security Settings
From the “Main Application” screen, choose Admin  Change Security Settings. This
will bring up the “Security Settings and Audit Trail” screen. The options on this screen
allow an Administrator to enable/disable Audit Trail and Secondary Authentication
features of the Pyros EQS system. Note that these features are enabled/disabled at the
database level and hence, applicable across all Client Applications of Pyros EQS
installed in a Network environment. Audit Trail features essentially enable the system
to track changes made throughout the course of setting up a test, running it, analyzing
it and reporting it. Sections 2.3.1 and 2.3.2 detail the functionality of each option.

Audit Trail Activation is a system-wide feature that is enabled or disabled at the

database level. In a Network deployment, enabling or disabling any of the features on
the “Security Settings and Audit Trail” screen will impact all the Pyros EQS Client
Application installations. It is recommended that all users log-off and restart their
systems to have Audit Trail settings appropriately reflected. In a Standalone
environment, the scope of these features is now restricted to the single installation of
Pyros EQS only. See Section 2.3.1, Security Settings for more detailed explanations.
Audit Trail functionality can be enabled by clicking on and checking the “Enable Audit
Trail” button.

Security Settings and Audit Trail Screen

The Limit User Access to Audit Trail button, if checked and enabled, will prohibit
Technician level users from generating Audit Trail Reports. These reports will now be
available only to Administrator and Supervisor level users. This functionality cannot be
enabled if the Enable Audit Trail checkbox is not checked.
This window also contains the Security Settings that govern the login functionality of
the software and govern the length of time permitted before the user must re-
authenticate. This helps to ensure no other user can make changes to the test prior to,
during or after data collection and analysis. Individual departments or corporations
must make their own determinations of appropriate level of control. See Section 2.3.1,
Security Settings for more detailed explanations.

5.4.4 Edit Default Settings

Note: Pyros Kinetix Flex users are strongly encouraged to create default settings sets for
turbidimetric and chromogenic assays, as several important parameters may change.
For additional information, please refer to the product insert accompanying your LAL
reagent, consult your local sales representative or contact ACC Technical Services at
(800) 848-3284 / [email protected].

Default Settings – Test Parameters – General Tab

Default settings contain all the necessary fields for test preparation. Default settings do
not have to be used, but they make test preparation easier by filling in all of the test
parameters automatically. From the “Main Application” screen, choose Admin  Edit
Default Settings. If Custom Fields will be used to define other items used in an assay
(e.g. pipette serial no., pipette tip lot #s, etc.), an Administrator must first create these
fields (see Section 5.4.1.1) before they will be displayed for data entry (last tab in
screen above).
Note for Network Version Users:
It is strongly recommended that the Administrator rename the Settings Set ID field
from “Default Settings” to the System ID of the workstation being used. This will help
the user create their own set of Default Settings that can be applied to tests that they
perform using Pyros EQS. To do this, go to the Hardware tab of the “Default Settings
Test Parameters” screen and note, or enter, the System ID. Click back on the General
tab of the same screen and edit the Settings Set ID field to the System ID name.
Users without Administrative privileges can create Default Settings from the “Test
Information” screen. For more information about creating new Default Settings, please
see Section 6.2.

5.4.4.1 General Tab

The General tab allows the user to specify parameters governing Data Collection.
Settings Set ID designates a name for the edited default settings.
Data Group designates the area of the database in which the test will be saved. Choose
a group from the pull down menu. New Data Groups can only be created by an
Administrator by using the Admin  Setup User functionality from the “Main
Application” screen. See Section 5.4.5 for more information on creating new Data
Groups and controlling “Read + Write” access to Data Groups.
Valid Temperature Range (Min: 36.5, Max: 37.5) defines the acceptable lower and
upper range of temperature that the test can run at. The Pyros Kinetix and Pyros
Kinetix Flex machines are calibrated to 37°C ± 0.5°C, so the tightest valid range is
36.5°C to 37.5°C.

Range for Spike Recovery (Min: 50, Max: 200) defines the acceptable limits for spike
recovery in samples. Please refer to the documents listed below for more information
on setting appropriate limits for a particular application.
Note: In the USP Bacterial Endotoxin Test <85>, the positive product control spike
recovery limits are 50 - 200% of the added endotoxin. Associates of Cape Cod,
Inc. product inserts list these limits and are FDA-approved documents. These
more recently approved documents thus supersede the stated positive product
control spike recovery limits published in the 1987 FDA Guideline (± 25%) and
the 1991 FDA Guidance (± 50%).
Recommended references
- Limulus Amebocyte Lysate, Pyrotell-T Product Insert
- Limulus Amebocyte Lysate, Pyrochrome Product Insert
- Current version of the US Pharmacopeia, Bacterial Endotoxin Test <85>
- U. S. Department of Health & Human Services, FDA, Interim Guidance for
Human and Veterinary Drug Products and Biologicals: Kinetic LAL Techniques,
July 15, 1991
- U. S. Department of Health & Human Services, FDA, Guideline on Validation of
the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for
Human and Animal Parenteral Drugs, Biological Products, and Medical Devices,
December, 1987
The following parameters relate to Onset Time, where Onset Time is defined as the
amount of time, in seconds, required for each reaction to pass the predetermined
Threshold OD.
Threshold OD (mAbs) (Default 20) defines the OD limit, from which the onset time of
the reaction is determined. Once a well's OD reaches this predetermined mark, an
onset time is generated and then used for endotoxin calculation at the conclusion of an
assay.
Max. OD Stored (mAbs) (Default 70) defines the OD limit at which the software stops
collecting data for a particular well. Example: If the Threshold OD is set to 20 or 30,
then a Max OD stored value of 70 is appropriate. Note that the more data points
stored, the larger the database becomes, potentially affecting the speed of data
retrieval.
Maximum Test Time (mins) (Default 100) defines the maximum length (in minutes)
that an individual test will run (and this function is specific to the last tube inserted).
Depending on the range of the standard curve being run, this time may be increased
or decreased. Other options available to the user may end the test sooner, but the
test will never go longer than this set time. The time in minutes is editable while a test
is running, so additional time can be added to the run time of the assay if needed.
Auto-end at 1/X  up to maximum test time (unselected, 4) is an auto-end option,
based upon the lowest concentration of the standard series. The lowest standard
concentration, also referred to as  (lambda), is considered to be the sensitivity of the
assay. Activation of this feature causes the software to apply a maximum run time to
the assay, which is equivalent to the theoretical onset time for the fraction of , as
specified by the user, unless the maximum run time is reached first.
Baseline Adjustment (Default period Min: 300 seconds, Max: 500 seconds) when
checked (by default) activates the baseline adjustment feature. If unchecked, the
baseline adjustment feature is not active. The default time period of 300-500 seconds

is suitable for most applications (see baseline adjustment in Section 6.2.1). The option
to accept the defaults or to enter alternate adjustment parameters is provided under
the “Show more info” icon in the “Test Information” screen, which can be accessed
during either Data Collection (see Section 6.2) or Data Analysis (see Section 7.4.1).
See Appendix A.2 for additional information.

5.4.4.2 Options Tab

The Options tab contains several fields that help define the way the data will be
collected and reported.

Default Settings – Test Parameters – Options Tab

Flag Correlation Coefficient if less than ___ (absolute value) is an option that will flag
and report a pass/fail condition on the Pass/Fail Report (Section 7.5.7) if this check
box is checked and the calculation of the standard curve yields a correlation
coefficient less than the user-defined value.
Significant Digits (3) will determine the number of significant digits (non-zero digits)
that the user deems appropriate for display of Endotoxin Concentration and Spike
Concentration values. Note that this parameter will have no impact on the values used
internally by Pyros EQS for calculations. Pyros EQS will always use the maximum
permissible number of decimal places as permitted by the development tools and/or
operating system.
Display Coefficient of Variation Based on: will result in the ability to choose a
Coefficient of Variation (also known as CV) type based on either calculated Endotoxin
Concentration values or Onset Times and have them reported in the Data Summary
Report (Section 7.5.2), Pass/Fail Report (Section 7.5.7) and the Data Trending Report
(Section 8.1). CV values are an indication of how well replicates correspond to each
other.
Flag Coefficient of Variation if greater than ___ % is an option that will flag and report
a pass/fail condition on the Pass/Fail Report (Section 7.5.7) if this check box is
checked. If this feature is enabled, it will flag the CV value if the CV value is greater
that the specified percentage limit on the Data Summary Report (Section 7.5.2).

Extrapolate beyond Range of Standard Curve is an option that uses the calculated
linear regression of the standard curve to extrapolate endotoxin values beyond the
highest and lowest endotoxin standards. These extrapolated values are not valid test
results but may be useful for determining future dilutions or other analysis by the
user. This feature, if enabled, simply reports the actual value of endotoxin measured
on the Individual Well Results Report (Section 7.5.4), even if the value is outside the
range of the standard curve.
Auto Test ID is a feature that, if enabled, will have Pyros EQS auto-generate the Test
ID for each test that the user prepares in the software. A user will have the option to
either retain the Test ID automatically generated by the application, or, enter their
own Test ID when setting up tests.
Print Sign-off footer on reports allows the user to print a standard footer on all
reports:
“Data Collected By __________________ Data Reviewed By __________________”
Override PPC requirement if enabled by a Supervisor, negates the requirement for
calculation of spike recovery in the Data Summary and Individual Sample Reports.
When enabled, this feature overrides the requirement for a Product Positive Control
(PPC) to be present for a sample with respect to reporting endotoxin concentrations.

5.4.4.3 Hardware Tab

The Hardware tab defines a user’s physical equipment setup.

Default Settings – Test Parameters – Hardware Tab

Computer ID identifies the name of the computer being used to collect data. The
contents of this field are automatically filled in by Pyros EQS by reading the computer
name from the Windows Operating System. The contents of this field are non-editable.
Instrument Type identifies the type of kinetic assay to be performed, turbidimetric or
chromogenic.
Instrument defines the type of machine being employed. A Pyros Kinetix instrument is
used for turbidimetric testing only, while Pyros Kinetix Flex instruments allow either
turbidimetric testing at a wavelength of 660 nm or chromogenic testing at a

wavelength of 405 nm. This information is included in the heading of all reports, as well
as the wavelength the assay was run at for Pyros Kinetix Flex users.
Serial Number is the serial number appearing on the instrument. This information is
included in the heading of all reports.
System ID is any name chosen that generally includes a particular computer and any
other equipment dedicated to a system. This information is included in the heading of
all reports.
Serial Port defines the port on the back of the computer used to connect and
communicate with the Pyros Kinetix or Pyros Kinetix Flex instrument in use. If this port
is not defined correctly, the computer will not be able to establish communication with
the intended instrument.
Note: Selecting “N/A” as the Serial Port allows tests to be run in Simulation Mode.
This mode permits assays to be run without an instrument physically connected
to the computer. In this mode, Pyros EQS uses an input data file. This file has to
be created using the Create Data File from Test functionality (described in
Section 5.2.2). Test files created using other applications cannot be used with
Pyros EQS. To run a test in Simulation Mode, a user needs to rename the Data
File created by Pyros EQS to PYROSOFT.tst (or PYROSOFT if the computer has
auto recognition mode enabled) and place it in the Pyros EQS \Input folder on
the C drive of the host computer. The Pyros EQS folder could be a different
path depending on where the Pyros EQS executable is installed on the
computer.

5.4.4.4 Lots Tab

The Lots tab allows the user to specify LAL and Endotoxin Lots, their associated Units
and potency information, a LAL Reconstitution Buffer Lot and Water Lot. The lots
entered in this tab will be available in editable dropdown menus of screens associated
with well configuration in Data Collection.

Default Settings – Test Parameters – Lots Tab

LAL Reconst. Buffer Lot designates the buffer lot that will be used for LAL
reconstitution. If none is used, the user can leave this field blank and/or enter “N/A.”
Water Lot designates the water lot that will be used for LAL reconstitution and/or
dilutions.
LAL Lot is the lysate lot to be used.
Endotoxin Lot is the lot of endotoxin to be used.
Units define the units of endotoxin to be used. Units may be defined as EU/mL, IU/mL,
ng/mL or pg/mL.
Potency relates mass units of endotoxin to actual endotoxin units (EU) for a given lot
combination of LAL and CSE. If EU/mL (or IU/mL) were chosen in the Units field, then
the application will not use the potency information provided. The user can still enter
all the potency information, which is Potency, Potency Units, Valid Concentration
Range (Min and Max), Valid Concentration Range Units, but this information will be
used by Pyros EQS as reference only. If ng/mL or pg/mL were chosen then enter the
appropriate numerical potency value. The units of potency will automatically be
displayed. Potencies are available on Certificates of Analysis from Associates of Cape
Cod, Inc. Certificates of Analysis can be obtained online at http://www.acciusa.com,
under Certificates of Analysis or contact an ACC Customer Service Representative to
receive the certificate by email or FAX. See The 1987 FDA Guideline for more
information about potency.

5.4.4.5 Custom Fields Tab

The Custom Fields tab will not be available unless an Administrator has defined one or
more fields in Section 5.4.1.1. Only those fields defined by the Administrator will be
visible on this tab. In the example displayed below, the Administrator has only setup
three fields: Pipette 10uL, Pipette 5uL, and Customer ID. These fields are available for
any other specific information the user may wish to include with their test such as
sample lots, chemical lots, etc.

Default Settings – Test Parameters – Custom Fields Tab

5.4.5 Setup User

This section describes the User and Data Group Management functionality of Pyros
EQS. This functionality is accessible through the Admin  Setup User option on the
“Main Application” screen. There are three levels of access rights that can be given to a
new user: Administrator, Supervisor or Technician.

Setup Users – User and Data Group Management – User Management Tab

Pyros EQS takes a significant step forward in simplifying security and application access
by using Windows authentication for all users utilizing the application. Only those users
with valid Windows operating system accounts can use Pyros EQS, once these users
have been authorized by an Administrator within Pyros EQS.
Pyros EQS is shipped with Administrator rights already set under the user name
“admin” and password “password”. This password should be changed as soon as the
Administrator has logged in. Each individual user should be given a unique user name
and password. This allows the audit trail to be generated for individual users and limits
the use of certain aspects of the software based on the access level of the user
account. Only an Administrator can setup additional Administrator, Supervisor and
Technician accounts.
The Setup User menu option under the Admin menu on the “Main Application” screen
is automatically disabled if the logged-in user does not have Administrator level
privileges.
To setup users and/or data groups, choose Admin  Setup User from the “Main
Application” screen. A screen similar to the one shown below will be displayed. Upon
fresh install of the Pyros EQS Network or Standalone versions, no users will be listed
under the User List and only the Default Data Group will be listed.

Setup Users – User and Data Group Management – User Management Tab

The screen has two tabs – User Management and Data Group Management. The User
Management tab allows an Administrator level user to setup additional users, activate
or deactivate users on the Pyros EQS system and assign “Read” or “Read + Write”
privileges for specific data groups to particular users. The Data Group Management tab
allows an Administrator level user to setup Data Groups and assign “Read” or “Read +
Write” privileges to these data groups for existing users. Note that Pyros EQS uses
Windows Operating System authentication. Therefore, it is critical to ensure that users
added to the Pyros EQS system have legitimate accounts on the Windows Operating
System and that a user’s credentials be properly supplied when creating the user
accounts in Pyros EQS.
To setup a new user, click on the User Management tab and then select “Add New
User”. A blank row will be added under User List. Click on each field starting with First
Name and enter the user’s credentials. Note that Login ID should be entered prefixed
by the domain name or computer name, as appropriate for the network environment.
For example, if an account on the Windows Operating System was johndoe and the
Windows domain was MWWS002, the user would enter MWWS002\JOHNDOE in the
Login ID field. The Access Level field is a drop-down list that allows a user to choose
between Technician, Supervisor and Admin. Admin is the highest level of access to the
system whereas Technician is the lowest level of access to the system. Supervisor is an
intermediate level of access. A Supervisor cannot create or modify users nor change or
delete system settings. Check the “Active” box to enable a user account.
Note: The first time an Administrator attempts to add a new user, the application
may display the message “ERROR: User FirstName is Mandatory and cannot be
empty.” This is a known behavior of the application. Simply click “OK” and click
in the First Name field to activate it for entry.
Privileges to existing Data Groups can be given to the user created by selecting either
“Read” or “Write” check boxes, as appropriate, for each data group that an
Administrator wants to give full or restricted access to the user. Note that clicking on
the “Write” check box will display a message indicating that the “Read” privilege will be

automatically assigned. Pyros EQS does not allow an Administrator to give a user the
“Write” privilege without giving that user the “Read” privilege as well.
Continue adding as many users as required and click on “Save Changes” to save all
changes to the database. At any given point in time, the Administrator may revert to
the previously saved status by clicking “Refresh”. To avoid losing valuable work, it is
recommended that the Administrator select “Save Changes” after adding each user.
Clicking “Close” will close the “User and Data Group Management” screen.
The Administrator may modify created users by clicking on any of the fields, making
changes, and then selecting “Save Changes” to save the edits.
To create data groups, or modify privileges to several users to a single data group more
easily, select the Data Group Management tab of the “User and Data Group
Management” screen.

Setup Users – User and Data Group Management – Data Group Management Tab

To create a new data group, click “Add New Group”. A blank row will be added under
the Data Group List. Click on the row under Data Group Name and enter a name for the
data group. All existing users will be listed on the right hand pane under User Privileges.
Assign desired privileges to users by clicking on “Read”, or “Write”, or neither of the
check boxes. Selecting “Read” will give read-only privileges to that particular user to
the created data group. Selecting “Write” will give “Read + Write” privileges to that
particular user to the created data group. Selecting neither will ensure that the user has
no privileges at all to the created data group. Click “Save Changes” to commit all
changes and additions to the database. To revert back to the previous state and discard
any changes made in the current session, click “Refresh”.
Note that any additions/changes to users and data groups will only take effect at the
next login session.

5.4.6 Administrator Override

The Administrator Override menu item in turn has three distinct override functions.
These are explained in detail below.

5.4.6.1 Sign-off Override

Sign-off Override unlocks Supervisor Signed-off tests. Once a test has undergone
Supervisor Sign-off, that test is available to users in read-only mode. No edits to any
signed-off test parameters can be made. This override function provides an opportunity
to unlock such tests by performing a Sign-off Override. The test then becomes available
for editing and test parameters may be modified as required. Any subsequent edits will
be captured in the Audit Trail, if this functionality has been activated.
On selecting the “Sign-off Override” menu item, Pyros EQS will search the database for
all tests that have undergone Supervisor Sign-off, and the following dialog box will be
displayed:

Sign-off Override

Select the test that should be overridden for Supervisor Sign-off from the drop-down
list of Test IDs and click “OK”.

5.4.6.2 Test Complete Override

In the rare event that data collection for a test has begun and the assay has gone into
an unknown state (where the user is unable to manually terminate the test through use
of the Close  Terminate Test options), Pyros EQS will allow an Administrator level
user to manually terminate the test via an override.
Selecting this menu item brings up a dialog box with a list of Test IDs whose status in
the database indicates that they are presently in Data Collection mode. Select the test
that the user would like to manually override and mark as having completed and click
“OK”.

Test Complete Override

Note: Before attempting a forceful Test Complete Override operation, it is

recommended that a user attempt to terminate the test in progress through
the Close  Terminate Test option available on the “Collect Data” main screen.
If an attempt to terminate the test using this option has already been made,
and the “Collect Data” main screen for the test continues to be displayed, the
Test Complete Override function may be used by the Administrator of the
system to mark the test as having completed in the Pyros EQS database.

5.4.6.3 Unlock Assay

Pyros EQS may be installed in a Network configuration, in which multiple Client
computers can connect to a central Database repository. The software is designed such
that once a test is opened on one client computer system, no other user will be able to
open the same test thereby preventing scenarios where multiple users are attempting
to setup, edit, or analyze the same test. A test, once opened by a user for setup, edit or
analysis, is “locked” by the software until it is closed by the user. In such a
circumstance, if the computer fails for any reason, the test will continue to remain in a
locked state and no other user will be able to open the test for Data Collection or Data
Analysis. The Unlock Assay function is specifically built to address this scenario.
This function, when accessed, will display a dialog box listing all the Test IDs that are
presently in locked state. Select the Test ID that needs to be unlocked and click “OK”.

Note: Only Administrator level users can use this function. It is strongly recommended
that the Administrator verify that a particular Test ID, when listed in a locked
state, is indeed not in use by any user across the system, and only then proceed
to unlock the test and make it available to users for data collection or analysis.

5.5 Help Menu

Main Application Screen – Help Menu

5.5.1 Pyros EQS Help
The Pyros EQS Help menu option brings up an Adobe® PDF version of this User Manual.
A user can browse through the entire file, search for specific text, and print relevant
sections/pages, as required.

5.5.2 About
The Help  About menu option brings up the following screen. It primarily displays the
application name, version number and contact information for Associates of Cape Cod,
Inc. offices.

Help  About Screen

5.6 System Tray Icon

After Pyros EQS has been installed, a small system tray icon will appear in the lower righthand
corner of the desktop. This has been designed to provide the user with additional information
regarding the system environment and Pyros EQS activity.
The system tray icon polls the service at an interval of one minute. The service pings the
database at 1 minute intervals if the system is idle or at 30 second intervals if there is assay
data being collected.

5.6.1 Blue System Tray Icon

A blue system tray icon indicates that Pyros EQS is idle; no assay data is being collected.

5.6.2 Green System Tray Icon

A green system tray icon indicates that Pyros EQS is collecting assay data. This icon will
appear green regardless of whether there is an active Pyros EQS window open on the
desktop. A green icon also indicates that the system is able to communicate and get
data from the instrument.

5.6.3 Red System Tray Icon

A red system icon tray indicates that instrument, database or service connectivity has
been lost. See Section 9.2.11 for additional information.

5.6.4 System Tray Icon Displays ‘X’

In the event that the Pyros EQS database is down, an ‘X’ will appear superimposed over
the system tray icon, regardless of the color. This icon will be displayed until
connectivity to the database has been restored.

Section Six: Data Collection

6.1 Data Collection
This section discusses each step involved in setting up a test. It is important to note that the
Pyros EQS software is Oracle database-driven. In a database environment, raw data is collected
and stored. There are Test IDs, but individual files are not created for each test. All test
information is stored in the database. Each time a user selects a report or plot, it is generated
anew from the data stored in the database. This database environment is necessary to comply
with Part 11 requirements and allows trending of data across multiple tests, users, etc.
From the desktop, double-click on the “Pyros EQS” icon or click on the “Start” menu and go to
All Programs  Associates of Cape Cod  Pyros EQS.

Pyros EQS Desktop Icon

Note: During installation, a user is given the option to change the folder where Pyros EQS is
installed. If a user has changed the install directory, they will find the Pyros EQS icon in
their chosen folder.
When Pyros EQS is launched for the first time the software will display a Login dialog box:

Pyros EQS Login Screen

At this login screen, enter "admin" in the User Name field, "password" in the Password field,
and click “OK”. Pyros EQS will now display the “Main Application” screen.
Note: As the above password is a "published" password, the Administrator must change it to a
new password to prevent unauthorized access to the system. Passwords are case-
sensitive, therefore upper and lower case letters must be used as required.
Although the Admin user has the access rights and ability to collect data, it is not appropriate
for the Admin user name to be employed for routine data collection. Customers should see
Section 5.4.5 for instructions to setup users for routine data collection.

Pyros EQS Main Application Screen

6.2 Test Information Screen

Click on the “Collect Data” icon to enter the Data Collection part of the application.
Note: The first time a user clicks the Collect Data icon, a registration screen will
appear. Follow the directions to register a system. For more information refer to
Section 3.2.2.5.

Test Information Screen – General Tab

The Test Information screen will appear as displayed above. Note that there are three
tabs: General, Options, and Hardware.
Note: A fourth tab, Custom Fields, appears only if Additional Fields have been defined
by the Administrator. For additional information on the Additional Fields, please
refer to Section 5.4.1.1.

If a set of Default Settings have been entered in the Admin menu, many of the fields in
these tabs will be pre-filled from the default settings. (Section 5.4.4)
In addition to the OK and Cancel options at the bottom of the “Test Information”
screen, the user is also given the option to save any changed settings as default for
future assays. This is discussed in greater detail below.
The Save as Default button allows a user to create multiple Default Setting sets which
contain information specific to their testing needs. Many of the parameters that will be
specified on the “Test Information” screen (all four tabs – General, Options, Hardware
and Custom Fields, if applicable), can be stored for repeat use as Default Settings. Once
all of the relevant information has been entered on these tabs, click the “Save as
Default” button at the bottom of the “Test Information” screen.
The Save as Default function will allow the user to:
 Create a new Default Setting
 Update the currently selected Default Setting, except the factory supplied Default
Settings
Note: The Save as Default button will not allow a user to update Default Settings other
than the Default Settings currently in use. To select a different set of Default
Settings for use, choose the Options  Select Default Settings menu option on
the “Main Application” screen.
Below the entry field for the Test ID, the user can select the Data Group in which to
store the assay. Clicking this field will list all the Data Groups to which the current user
has “Read + Write” privileges. Choose the desired Data Group into which to create
Default Setting. The Default Setting ID will default to the name of the Default Setting
presently in use. If a user has not created any Default Settings yet, the factory default
setting Default Settings will be displayed.
Note: The factory supplied Default Settings set with the ID Default Settings is non-
editable. A user can use it as a starting point to create their Default Settings and
name them as convenient.
To create a new Default Setting, enter a name for the Default Setting ID and click “OK”.
If another Default Setting with the same name exists, the application will display an
error message stating “Default Setting <default setting ID> already exists. Please type a
different Default Setting ID.” If the save operation is successful, a message stating
“Default Settings saved successfully” will be displayed.
Note: If the user enters a name of a pre-existing Default Setting ID and selects “OK” to
save the changes, the application will overwrite the prior values/settings.

Save as Default – Update In-Use Default Setting

To update the in-use Default Setting, simply click “OK”. The operation, once successful,
will display a message stating “Default Settings updated successfully.”

6.2.1 General Tab

Test ID designates the desired name for the assay being performed.
Technician is the user currently logged in. This information is automatically captured by
the application from the login particulars and displayed on this screen. It cannot be
edited.
Data Group designates the area of the database the test will be saved to. Choose a
group from the pull down menu. Data Groups can be set up by an Administrator. Refer
to Section 5.4.5 for additional information regarding setting up Users and Data Groups.
Data Collection Date is a fixed field displaying the actual time and date when Data
Collection commenced. This field is automatically populated by the application and
cannot be edited.
Apply Format, when checked (enabled), allows a user to apply a pre-saved format to
the new test being setup.
Note: When applying a Stored Curve, ensure that the lots used in the Stored Curve
appropriately match those intended to be used in the test. Mismatch in any of
the lots specified – Water Lot, LAL Reconst. Buffer Lot, LAL Lot, or Endotoxin Lot
– can adversely impact test analysis and result in both the analysis screen and
reports indicating “Diff. Lot”.

The user can select the Type from one of two types of pre-saved formats. These are:
 Existing Test ID a user can apply the test configuration data from a previous assay
 Stored Format a user can use an existing Stored Format

Test Information Screen – Format Applied

Depending on a user’s selection in the Type drop-down list, the appropriate entries will
be listed in the Name drop-down. They can then select one of the entries listed under
Name and select to apply it to the test being setup. The above screen capture image
depicts an application of a Stored Format named “test”.

Test Termination Parameters contains two criteria, one of which can be used to
determine completion of the test:
Max Test Time should be specified in minutes and defines the maximum length of a
test (based on when the last tube was inserted). This field is editable during data
collection, and can be increased if a test requires more time for all replicates of the
standard curve to onset.
Auto-end, when checked (enabled), allows the user to specify a fraction of  (Lambda)
that will be used by the application to determine maximum run-time for the test. When
all of the Endotoxin standards have reached Onset OD, Pyros EQS will apply this value
to the lowest Endotoxin standard concentration () and use the standard curve
regression line to calculate the amount of time the test should continue before ending.
The last inserted tube will run for the duration computed by the software. For example,
if an auto-end time was computed as 5,200 seconds, and the last tube had an insertion
delay of 500 seconds, the total duration of the assay would be 5,700 seconds.
Note: In LAL testing,  (lambda) is the detection limit or sensitivity of the test. For a
given test, the detection limit () is the lowest standard concentration used to
construct the standard curve.
Test Comments is an open field in which a user may enter any other information
pertinent to the test.
Well Uniformity Test, if checked (enabled), allows the user to run a special Well
Uniformity assay. Note that this test can only be performed on a Pyros Kinetix
instrument and does not apply to Pyros Kinetix Flex instruments. See Section 5.3.3 for
additional information.
Show More Info The bottom portion of the General tab has options that typically
remain unchanged and are infrequently modified. The screen may be expanded to
display these options by clicking on the “Show More Info” button and hide these
options by clicking on the “Hide More Info” button. These options are explained below.

Test Information Screen – General Tab – Show More Info (Expanded)

Note: The red exclamation mark is an indicator that the field contents did not pass the
field validation criteria. Move the mouse over the exclamation mark for an
explanation on what the software is expecting in the associated field. This
indicator appears throughout all the dialog boxes of the software.
Apply Curve allows the user to apply a stored Standard Curve to analyze the current
test.
LAL Reconst. Buffer Lot allows a user to specify the lot information for the buffer (if
any) that is being used to reconstitute the LAL in this test.
Threshold OD is the Optical Density value that the well must cross for the application
to determine that the well has reacted.
Max OD Stored is the Optical Density limit in mAbs, which signifies the end of data
collection for that particular well. Example: If the Threshold OD is set to 20, then a Max
OD stored value of 70 is generally appropriate. Note that as the database becomes
larger the rate of data retrieval may be affected.
Baseline Adjustment (checked and enabled by default), allows a user to specify the Min
and Max range in seconds to be considered for Baseline Adjustment. This feature
corrects for any drift in the baseline (background) OD reading prior to a reaction
occurring. The average stored OD between the user-specified duration, (default of 300-
500 seconds), is calculated for each well and subtracted from every reading post
completion of Zeroing Period (60 seconds to 120 seconds) to arrive at the Adjusted OD
value. See Appendix A.2 for more information.
Note: Baseline adjustment is considered invalid for a given well if the onset time for
that well is less than or equal to the Max time value specified in the baseline
adjustment range. If this state exists for a well, Pyros EQS will indicate
“Baseline!” in the Onset Time column and Endotoxin concentrations will not be
displayed. If the well(s) in such invalid state are Standards, Pyros EQS will not
perform regression, and therefore not calculate Endotoxin concentrations. If the
standard concentrations are such that this occurs, the baseline adjustment
period can either be turned off by unclicking the checkbox, or moved forward
from the default time of 300 – 500 seconds. The adjustment range must be a
minimum of 200 seconds, and the earliest baseline adjustment period that can
be used is the 130 - 330 second data period, as the 130 second block is the first
recorded point after the zeroing period for each well.
Valid Temperature Range defines the acceptable lower and upper range of
temperature that the test can run at. Pyros Kinetix and Pyros Kinetix Flex instruments
are calibrated to 37°C ± 0.5°C, thus the tightest valid range is 36.5°C to 37.5°C.
Range for Spike Recovery defines the acceptable limits for the spike recovery. Please
refer to the documents listed below for more information on setting appropriate limits.
Note that measured endotoxin values from samples with onset times greater than
those for the lowest standard concentration are below the valid range of the standard
curve. These are reported as less than the lowest standard endotoxin concentration
and a value of zero is assigned to these replicates when subtracted from the endotoxin
concentration of the PPC when calculating endotoxin spike recovery. If one replicate
has an onset time greater than the lowest standard concentration but the other does
not, the software returns a “Check wells” message and the user must determine how to
use the data or whether a retest is required.

Note: In the USP Bacterial Endotoxin Test (USP 24, <85>), the positive product control
spike recovery limits are 50 - 200% of the added endotoxin. The Associates of
Cape Cod, Inc. product inserts list these limits and are FDA-approved
documents. The positive product control spike recovery limits published in the
1987 FDA Guideline (± 25%) and the 1991 FDA Guidance (± 50%) are thus
superseded.
- Limulus Amebocyte Lysate, Pyrotell-T Product Insert
- Limulus Amebocyte Lysate, Pyrochrome Product Insert
- Current version of the US Pharmacopeia, <85>
- U. S. Department of Health & Human Services, FDA, Interim Guidance for
Human and Veterinary Drug Products and Biologicals: Kinetic LAL Techniques.
July 15, 1991
- U. S. Department of Health & Human Services, FDA, Guideline on Validation of
the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for
Human and Animal Parenteral Drugs, Biological Products, and Medical Devices.
December 1987

6.2.2 Options Tab

The Options tab contains several fields that help a user define the way their data will be
collected and reported.

Test Information Screen – Options Tab

Check Negative Control Validity if onset time > theoretical onset time for 1/__  if
enabled, will include a check for the Negative Control in the Pass/Fail Report. In order
for the software to report a “Pass” a) at least 1 Negative Control must be configured in
the test, b) the run time for the Negative Control must be greater than the theoretical
onset time of lambda, and c) if it crosses the threshold OD, the onset time must be
greater than the theoretical onset time for the fraction of lambda, as indicated in this
field. If activated, and any of the above criteria are not met, the Negative Control
validity will indicate “Fail” on the Pass/Fail Report.
Other fields present on this screen are discussed in Section 5.4.4.2.

6.2.3 Hardware Tab

The Hardware tab allows computer- and instrument-specific information to be
specified. This tab is discussed at length in Section 5.4.4.3.

Test Information Screen – Hardware Tab

6.2.4 Custom Fields Tab

The Custom Fields tab will not be available unless an Administrator has defined one or
more fields for the selected Default Settings in Section 5.4.1.1. Only those fields
defined by the Administrator will be visible on this tab.

Test Information Screen – Custom Fields Tab

6.3 Instrument Communication

Once all the test information has been entered and the “OK” button selected, the
software will attempt communication with the instrument via the serial port specified
on the Hardware tab of the “Test Information” screen.

For Pyros Kinetix Flex instruments, a wavelength calibration service must occur before
communication with the instrument is established (instrument responds to commands
sent by the software). A “Setting instrument wavelength” screen will appear and cycle
through four messages: “Setting calibration mode”, “Sending wavelength select
command”, “Sending wavelength set value command”, and “Exiting calibration mode”.
At any time, if desired, the user can click “Abort” to return to the “Test Information”
screen. If the contents of this window are not updated and if the status continues to
say “Awaiting response on port” followed by the port name (COM1, COM2, etc.)
specified on the Hardware tab, it means that the software is unable to get a response
from the instrument. Please check that the instrument is connected properly to the
computer, the instrument is turned on, and the serial port specified on the Hardware
tab is indeed the port on which the instrument is physically connected. Once the
“Exiting calibration mode” is complete, the software will continue to the “Device
Status” screen.

Pyros Kinetix Flex – Setting Instrument Wavelength

If a Pyros Kinetix tube reader was selected during assay setup, the Pyros EQS
application will display the “Device Status” screen immediately after selecting “OK” on
the “Test Information” screen. This window will update itself once communication with
the instrument is established (instrument responds to commands sent by the
software). The screen messages reflect the status of communication with the
instrument, the instrument type in use, the temperature of the instrument, and
whether any tubes are present in the instrument. At any time, if desired, the user can
click “Abort” to return to the “Test Information” screen. If the contents of this window
are not updated and if the Status continues to say “Awaiting response on port”
followed by the port name (COM1, COM2, etc.) specified on the Hardware tab, it
means that the software is unable to get a response from the instrument. Please check
that the instrument is connected properly to the computer, the instrument is turned
on, and the serial port specified on the Hardware tab is indeed the port on which the
instrument is physically connected. These dialog boxes will look similar to the ones
shown below.

Device Status Screen – Awaiting Instrument Response

Device Status Screen – Instrument Communication

Device Status Screen – Instrument with Tubes Detected

If the target temperature has not been reached and/or the presence of tubes is
detected in the instrument, the software will continue to display this screen until the
target temperature is reached and/or all tubes have been removed. At that point, this
display will be replaced by the one shown below, indicating that the instrument is
ready for use.

Instrument Communication Confirmation Screen

Clicking the “OK” button will bring up the “Collect Data” main screen.

6.4 Collect Data Main Screen

The “Collect Data” main screen is depicted below. It is essentially a grid displaying key
well-specific information such as well run-times, onset values and endotoxin
concentrations. Tubes may now be added to the Pyros Kinetix or Pyros Kinetix Flex
instrument. Insertion of the first tube will trigger the start of data collection; the assay
run-time will be displayed and automatically incremented on the top left of the window
title bar. The instrument temperature will also be automatically displayed and updated
in the top right of the window title bar. See the LAL Reagent Product Insert for more
information on reaction tube preparation.

Collect Data Main Screen

The image above indicates that the first 6 tubes were simultaneously inserted to begin
the test and that 13 seconds have elapsed so far in the test.
Each well is individually timed, so tubes may be added minutes after the start of the
test. Well descriptors may be entered at any time. Once a test has ended, well

descriptors for wells that had tubes detected during Data Collection may still be added
or modified. Wells which have no data associated with them cannot be edited once
Data Collection has completed and the test is in Data Analysis mode.
The following buttons appear at the top of this screen:
Neg. Ctl. allows the user to setup Negative Controls.
Std. Curve allows a user to setup Standard Curves and Positive Controls.
Sample allows unknown Samples to be defined.
View menu allows Graphic Circle, Numerical Data, Reagent Lots Report, Standard Curve
Regression Plot and individual (selected) Well Info to be reviewed.
Options menu allows Assay Termination, Printer Configuration and Formats to be
saved.
Edit menu, if a configured well is selected, allows editing of the Selected Well, All
Replicates, or, Entire Series. If a non-configured well is selected, the user will be
prompted to Define Selected Well (as Negative Control, Standard Curve, or Sample).
Test Info displays and allows editing of selected parameters of the Test Information
screen.
Print Test Setup generates and allows printing of the Reagent Lots Report.
Close allows a user to close the Collect Data main screen and either allows the test to
continue to run in the background or to completely terminate the test.
Note: It is important to remember to ensure that the proper lots are specified when
setting up the controls – Negative Control, Standard, Positive Control (if
applicable) and Samples. Mismatch in any of the lots specified (Water Lot, LAL
Lot, or Endotoxin Lot) can affect test analysis and result in both the analysis
screen and reports indicating “Diff. Lot.”

6.5 Negative Control Setup

The - Neg Ctl button opens up the Negative Control setup dialog box. These fields allow
wells to be setup as Negative Controls and the associated information pertinent to
those Negative Controls entered.

Negative Control Setup

Starting Well specifies the starting well for this series of Negative Controls.
No. of Controls specifies the number of consecutive wells that will be used by this
series of Negative Controls.
LAL Lot is the lot of lysate being used with these Negative Controls. This is an editable
dropdown menu.
Water Lot is the lot of LAL Reagent Water (LRW) employed to generate these Negative
Controls. This is an editable dropdown menu.
Control Type defines the type of Negative Control being run. A user can choose
between Water (default) and Product. Product Negative Controls should be included,
along with water negative controls, if a product standard curve is employed. More
information regarding Product Standard Curves can be found in the 1987 FDA
Guideline. Refer to the product insert for information on appropriate Negative
Controls.
Product Information fields are enabled only if the Control Type is chosen as “Product”:
Type refers to either (a) an endotoxin free example of the product being tested, or, (b)
a buffer or some other solution the technician may be using to dilute their standards
and samples (i.e. anything other than LRW).
Lot refers to the lot number (if there is one) of the product being used to prepare the
Negative Controls.
Concentration refers to the concentration of the product being used to prepare the
Negative Controls.
Click “OK” to save the Negative Control assignments or “Cancel” to discard all
information specified on this dialog box.

6.6 Standard Curve Setup

The Std. Curve button opens the “Standard Curve Setup” screen which allows the user
to specify standards and positive controls. This screen defines the wells and associated
information for the endotoxin standards. The maximum sensitivity of a kinetic LAL
assay will depend upon the LAL reagent used. See product package insert for
specifications. For more information refer to the Certificate of Analysis for the
particular combination of LAL/CSE. Certificates of Analysis can be found on our website
at http://www.acciusa.com under Certificates of Analysis or can be obtained via email
or fax by contacting a Customer Service Representative at (800) LAL-TEST (525-8378) or
(508) 540-3444.
Curve Information is where key Standard Curve parameters are specified.
Curve No. is automatically filled by the software to the lowest value available for
identification of the curve. If no curves have been specified, this number starts at 1.
This number may be changed to suit a user’s needs.
Starting Well specifies the first well to be occupied by tubes belonging to this curve.
Concentrations specify the number of standard concentrations that will be included in
the curve. A minimum of three standards is required for the software to generate a
regression line.

Standard Curve Setup Screen

Replicates specify the number of tubes that will be used for each standard
concentration.
Highest Concentration defines the highest concentration of the standards being used
and defines the starting point of the automatic generation of the standard curve series.
Dilution Factor is the dilution employed to create the standard curve. The Highest
Concentration will be used in conjunction with this number and the previously
designated Concentrations and Replicates to generate the standard curve series
automatically.
Group By option has two choices for grouping by Concentration or Replicates. Grouping
by concentration yields a dilution scheme where replicate concentrations remain
together (0.001, 0.001; 0.01, 0.01; 0.1, 0.1) whereas grouping by replicates allows the
series to appear twice (0.001, 0.01, 0.1; 0.001, 0.01, 0.1). Concentration is the default
and most commonly used grouping.
Start With allows a user to choose whether the Lowest or Highest standards start the
standard series on the Collect Data screen.
Positive Control has a separate functionality that is discussed in Section 6.7 Positive
Controls on Standard Curves. Do not check this box while setting up a standard curve.
Spike Recovery Range applies only if a Positive Control is being setup and does not
apply to a standard curve. Until Positive Control is enabled, this field will remain
grayed-out (disabled). This is discussed in Section 6.7 Positive Controls.
Lot Information is where the user specifies various lot-related details.
Water Lot is the lot number of the water used to reconstitute the LAL and/or perform
endotoxin dilutions for this standard curve.
LAL Lot is the lot number of the LAL being used with this standard curve.

Endotoxin Lot is the lot number of the endotoxin used to generate the standard curve
plus the appropriate units associated with the lot.
Potency relates mass units of endotoxin with endotoxin units (EU) that describe
standard endotoxin activity. For additional information about potency, please refer to
the 1987 FDA Guideline. Certificates of Analysis containing potency information can be
obtained from our website at http://www.acciusa.com under Certificates of Analysis or
via email or Fax by contacting a Customer Service Representative at (800) LAL-TEST,
(888) 395-2221 or (508) 540-3444.
Potency Units describe the relationship of endotoxin mass units with EUs. For example,
Potency = 10, Units = EU/ng would describe a relationship where 1ng of endotoxin has
10 units of activity.
Standard Curve Type allows additional curve-specific information to be specified.
Curve Type permits a user to select either Water or Product. If Product is chosen as
Curve Type, the following fields become enabled. They remain inaccessible if Curve
Type is Water. Water indicates LRW was used for endotoxin dilution whereas Product
indicates some other solution, typically a buffer or an endotoxin-free example of the
sample being tested.
Product Type allows the type of liquid used to dilute the endotoxin to generate the
standard curve to be specified.
Product Lot is the lot number of the liquid used to dilute the endotoxin standards.
Product Concentration is the concentration of the liquid used to dilute the endotoxin
standards.
Final Results In if checked (enabled), will enable reporting of endotoxin concentrations
for this standard in the units displayed. The units displayed are derived based on the
units specified for the Endotoxin Lot and the units specified for Potency.
Valid Concentration Range allows a user to specify the Min and Max concentration
range in the units specified, in which the standard curve concentrations must lie. At
least 3 standards must lie within the Valid Concentration Range specified. Otherwise,
the software will prompt the user and indicate that the standard cannot be used for
analyzing the test results.
Notes field is for any other information the user wishes to enter relating specifically to
the standard curve. This information will appear on the Report Cover Sheet.
Once all fields have been specified, click “OK” to accept parameters specified, “Cancel”
to discard changes.
6.7 Positive Controls
The Positive Control setup dialog box can be accessed by clicking on the “Std Curve”
button on the “Collect Data” main screen. The dialog box is the same that is used to
setup standards. To define a Positive Control, simply check (enable) the “Positive
Control” check box. See the 1987 FDA Guideline and 1991 Interim Guidance for Human
and Veterinary Drug Products and Biologicals for more information about positive
controls when reading a test from an archived curve.
All fields except the following automatically get disabled and are grayed out. The Curve
No. automatically defaults to Positive Control and is non-editable.
Starting Well specifies the first well to be occupied by tubes belonging to this Positive
Control.
Replicates specify the number of tubes that will be used by this Positive Control.

Highest Concentration designates the concentration of the Positive Control being set
up.

Standard Curve Setup Screen – Positive Control Setup

Positive Control is the flag indicating that the user is setting up a Positive Control and
not a Standard Curve. Un-checking (disabling) this flag will allow the user to setup a
Standard Curve.
Spike Recovery Range is the range of recovery in percentage applicable for the Positive
Control being setup, to be considered valid. If the Spike Recovery for the Positive
Control lies outside this specified range, the Positive Control will not be reported as
“Pass” on the Pass/Fail Report.
Lot Information, Standard Curve Type, and Notes fields have the same meaning as
mentioned above in Section 6.6 – Standard Curve Setup.
6.8 Sample Setup
The Sample button brings up the “Sample Setup” screen. This is used to setup unknown
samples in a user’s test.
Sample Information is a set of fields that help the user define a sample uniquely and
specify its concentration. The Type, Lot and Description fields together constitute a
unique identifier for each sample.
Type is a field used to enter the type of sample the user is entering. It is used for their
organizational purposes and is also important for data trending purposes.
Lot is the lot number of the sample the user is running.
Description is the actual name of the sample and the way it will appear on the “Data
Collection” and “Data Analysis” screens, and on all reports.

Starting Well is the first well number where the first replicate of the sample series
begins.
Replicates Unspiked is the number of replicates of the sample.
Replicates Spiked is the number of replicates of the spiked sample, otherwise known as
the positive product control (PPC).
Spike Conc is the known concentration of endotoxin added to the sample to spike the
sample. This is used to ensure that a sample does not have inhibitory or enhancing
effects on the lysate that could create a false result. See the LAL Reagent Product Insert
for additional information.

Sample Setup Screen

Dilution Information is where the user specifies pertinent information related to the
sample’s dilution.
No. of Dilutions determines the number of different sample dilutions using the Serial
Dilution Factor.
Initial test dilution is used to define the dilution of the highest concentration of a
sample in a series. For example, if a sample requires a 1:100 dilution to be tested with
LAL, then the initial dilution is 1:100. If value entered in the No. of Dilutions field is
greater than 1, the additional dilutions will be calculated by applying the Serial Dilution
Factor to the initial test dilution.
Start With gives the user the option of setting up the sample dilution series from
highest to lowest or vice-versa.

Endotoxin Limit is a pass/fail test that compares the final (dilution-adjusted) mean
concentration of endotoxin found in the sample with this defined value. It is reported
on the Pass/Fail Report. The endotoxin limit value and units are both specified here.
Product Conc. before Dilution specifies the concentration and units of the product,
prior to any adjustment for dilution. This is a numeric field defaulting to blank with
units being (mg/ml, Units/ml, %, or ml/device). When the user specifies the product
concentration before dilution and its associated units, and if the Give results per Unit of
Product checkbox is checked (enabled), the software will report results based upon
units of product.
No. of Vials Pooled allows the user to specify the number of vials (or other units)
pooled for testing. This is an optional field. If this is specified and the Correct Endotoxin
Limit for Pooled Vials checkbox is checked (enabled), the software will reduce the
Endotoxin Limit by this factor in its calculations.
Lot Information contains editable dropdown menus for entry of LAL Lot, Water Lot,
Endotoxin Lot, and Potency. These should match those entered for the Standard Curve
and assay controls.
Notes field is for any other information the user wishes to enter relating specifically to
the sample.
Once all fields have been specified, click “OK” to accept parameters specified, “Cancel”
to discard changes.

6.9 View Menu

Clicking the “View” button brings down a pull-down menu with the following options –
Graphic Circle, Numerical Data, Standard Curve Regression Plot, Reagent Lots Report,
and Well Info. Each of these is explained below.

Data Collection – View Menu

6.9.1 Graphic Circle

Clicking the “Graphic Circle” option brings up the following screen for a test run on a
Pyros Kinetix instrument. Users of a Pyros Kinetix Flex will see this screen in the same
configuration as the Flex instrument in use.

Graphic Circle Screen

This screen allows the user to see which wells have tubes inserted in them (wells
without tubes appear as empty white circles, such as the ones from well number 45 to
47 and 57 through 96 in the image above), and for each well with a tube, see mini-
plots of the reaction on this screen itself.
White wells indicate that no tube has been inserted in the well.
Yellow wells indicate that a tube is inserted in the well and recognized by the software.
Green wells indicate that the tube has completed the LAL reaction and has crossed the
Threshold OD.
Orange wells indicate that a Sudden OD change has been detected in the tube.
Red wells indicate that a fatal error (A/D low/high or Tube Removed) has been
detected.
Blue wells indicate that the well is currently selected by the user.
To view the actual raw data points as they are collected the user may either select all
wells by using the “Select All” button at the upper right hand portion of the screen, or
the user may select an individual well by clicking on it. The user may select multiple
wells by keeping the CTRL key pressed and then individually selecting the wells that
they want to view. Selected well(s) will appear blue, once wells have been selected,
click the “Plot” button at the upper right hand portion of the screen to bring up the raw
data plots.

Graphic Circle – Well Charts

To return to the Graphic Circle screen click the “Close” button in the toolbar. To return
to the “Collect Data” main screen from the Graphic Circle screen, click the “Data”
button at the top right of the screen or the red “X” button at the top right corner.

6.9.2 Numerical Data

The Numerical Data option displays the Data Block for the last reading recorded by the
software. This is used primarily for advanced troubleshooting and is not generally
important to the Data Collection process. The values displayed on-screen are the raw
Analog to Digital (A/D) values received from the instrument and not the OD values.

Numerical Data Screen

The user can click anywhere in the highlighted area which displays the Data Block, and
use the Page Up/Page Down keys to scroll through the data blocks collected from the
instrument. This screen automatically updates itself as a new data block is received
from the instrument, typically every 10 seconds. The user can freeze the display at the
current block if necessary by clicking “Freeze”. Once frozen, the display can be returned
to auto-update mode by clicking the same button which is now labeled “Thaw”. A
single data block may be printed by clicking “Print” or continuously print the data
blocks being received to the default printer setup by clicking “Continuous Print”.
Clicking “Close” will close the Numerical Data screen. Additional information on data
blocks is included in Section 9: Troubleshooting.

6.9.3 Standard Curve Regression Plot

This menu option is used to bring up the Standard Curve Regression Plot. When clicked,
it brings up the following dialog box.

Standard Curve Regression Plot Selection

This window, under Standard Curve, will list all the standard curves specified in the test.
In the above example depicted, only a single curve has been specified in the test. The
curve number is suffixed by the Endotoxin Lot used as the standard.
The user can click on the curve listed to select the curve and then click “OK” to bring up
the plot.
Note: The standard curve regression plot for the selected curve cannot be plotted until
all replicates of the curve have reached onset and the curve has completed. An
error message will be displayed if the selected curve has not completed and
regression cannot be computed. During Data Collection, only one curve can be
selected at any given point in time to display its regression plot. Selection of
multiple standards will cause the message “Please select only one standard for
regression plot in data collection mode” to be displayed.

Standard Curve Regression Plot with Onset Times

When the selected curve is complete and “OK” is clicked, the plot will be displayed as
depicted above. The image above also displays a smaller window overlaid on the plot
listing the concentrations that are part of the curve and their theoretical onset times.
This window can be displayed by clicking on the “Onset Times” button at the top of the
plot screen. Clicking “OK” on the window will close the Onset Times table. Clicking
“Close” at the top of the screen will close the Standard Curve Regression Plot itself and
return the user to the “Collect Data” main screen.
The Print button can be used to print the plot whereas Rescale can be used to rescale
the plot.
Note: When “Print” is selected for the Standard Curve Regression Plot, Individual Well
Plot, or Multiple Plot windows, the plot is printed as it is seen on the screen. In
order to provide additional flexibility for users printing from a variety of
different screens, the user will be prompted with a dialog box for changes in X
and Y scaling. By default, the value for both scales is 0.78 (for a screen resolution
of 800 x 600 pixels).

6.9.4 Reagent Lots Report

The Reagent Lots menu item in the View menu brings up the Reagent Lots Report
listing the defined test well numbers with their respective Sample Lot, Sample
Description, Standard/Spike Concentration, Units, Sample Dilution, LAL Lot and
Endotoxin Lot information.

Reagent Lots Report

6.9.5 Well Info

View Well Info is used to display the individual well information screen for the
selected/highlighted well from the “Collect Data” main screen. Depending on the type
of the well – Negative Control, Standard Curve (or Positive Control), or Sample, the
appropriate screen will be displayed.
In the example screen capture image depicted below, the highlighted well, number 12,
belongs to a standard curve series, and as such, clicking View Well Info brings up the
Standard Curve well information screen for well number 12.
Note that this is a display-only screen and does not permit edits to any of the well
parameters. To edit well parameters, use the Edit button at the top of the “Collect
Data” main screen.

View Well Info – Standard Curve Information

6.10 Options Menu

The Options menu presents the user with the following sub-menu items: Terminate
Test, Printer Setup, and Save Format. Each of these is explained below.

6.10.1 Terminate Test

The Terminate Test option works similarly to the Close button on the menu bar at the
top of the “Collect Data” main screen. It presents the user with two primary choices –
Continue Test or Terminate Test (see also Section 6.14).

Terminate Test Screen

Clicking “Continue Test” will close the “Collect Data” main screen but allow Data
Collection to continue in the background. This option is useful if the user wants to
edit/save default settings or perform other Administrative functions without disrupting
the test. To return to the test, click “Collect Data” on the main screen.
Clicking “Terminate Test” will forcefully terminate the test and make it available for
analysis.
Closing Comments is where the user can enter specific notes, especially if the test is
being terminated early and it is necessary to document the reason.
Clicking “Cancel” returns the user to the Collect Data main screen.
Note: When the “Close” option is selected, clicking on the “Terminate Test” button
should close the test within 60 seconds. In some instances, the test may not
terminate. This may occur in the case of sample replicates that have not reached
onset, but are at a time period beyond the onset times of all replicates of the
lowest standard in the standard curve. In this circumstance, the Test Complete
Override option must be used to close the test. At the Pyros EQS - Close Data
Collection icon, the user should select the “Continue Test” button. This will
return the user to the “Main Application” screen. If the user has Administrator
privileges, click on “Admin”, scroll down to Administrator Override, and highlight
“Test Complete Override.” When clicked, this will bring up a dialog box with a
list of test IDs that are still in data collection mode. Clicking on the test will close
it. If the user does not have Administrator privileges, log out of the Pyros EQS
application, have a user with Administrator privileges log in, and proceed as
above.

6.10.2 Printer Setup

This menu option brings up the standard Windows Printer Setup dialog box and allows
the user to select a default printer for the software and configure printer specific

parameters. The printer selected in the drop-down box for Name will be recognized as
the default printer for the software once the user clicks “OK” on this screen.

Printer Setup

6.10.3 Save Format

This Options menu item allows a user to save the existing test parameters as a new
Stored Format in a specific Data Group.
Enter New Format Name is where a user specifies the name to associate with the
format being stored.
Data Group is a drop-down box listing the data groups to which an Administrator has
configured the user to have “Read + Write” privileges. The user will select a Data Group
in which they will create the new Stored Format.

Save Format Screen

Note: Stored Format names need to be unique across Data Groups. If a user enters a
format name that is already in use, the software will indicate this by way of a
red exclamation mark next to the field.

6.11 Edit
The Edit button selects the highlighted well from the “Collect Data” main screen and
presents a dialog box that allows a user to choose between editing the single well, all
replicates of that well, or, the entire series. Entire series refers to a Standard Curve,
Positive Control or Sample series.

Edit Well Selection Screen

The window displayed by clicking one of the “Selected Well”, “All Replicates”, or “Entire
Series” buttons, depends on the type of the well selected, whether Standard or Sample.
The relevant screen is then displayed. The “Edit Well Selection” screen does not appear
if the selected well is a Negative Control.
Note: If the user chooses to edit a specific well, or a specific set of replicates, within a
sample series (e.g. only the spiked wells of a specific sample dilution), the
application will determine that the set of wells associated with that particular
sample is a broken series. Further attempts to edit the entire series will be
prevented. If such a scenario occurs, please go to Section 9.2.21.

6.12 Test Info

The Test Info button brings up the “Test Information” screen with certain fields
disabled. The user can alter most of the information related to the test in progress,
except information that uniquely identifies the test such as the Test ID and Data Group
combination, and information specific to the hardware on which the test is being run
such as Instrument Type, Instrument, Serial Number and Serial Port. Parameters such
as Max Test Time, Threshold OD can easily be modified and a Stored Format can be
applied.

Test Information Edit Screen

The behavior of this screen and additional information on specific parameters can be
found in Section 6.2.

6.13 Print Test Setup

The Print Test Setup button allows the user to generate and print the Reagent Lots
Report. For additional information, refer to Section 6.9.4 above.

6.14 Close
The Close button allows the user to close the “Collect Data” main screen either with the
test continuing to run in the background, or, by terminating the test manually. For
additional information, please refer to Section 6.10.1 above.

6.15 Right-Click Functionality

On the “Collect Data” main screen, the user can click the right mouse button to bring
up a shortcut menu that functions largely the same as the right mouse click
functionality in a standard Microsoft Office application such as Microsoft Excel®.
The following menu items are present on the right mouse click menu and behave as
described:
 Cut
Removes well descriptors of row(s). This action does not shift rows up.
 Copy
Creates a copy of the selected wells in the clipboard. The selected wells do not
change or move.
 Insert
Inserts the contents of the clipboard. Pre-existing wells below the point of insertion
are shifted down. If there is nothing in the clipboard, a blank well is inserted.
 Paste
Inserts contents of clipboard, and writes over existing wells.
 Clear
Functions like Delete, but no well shift takes place.
 Delete
Removes contents of selected wells. Contents not retained in clipboard. Existing
wells shifted up.
 Undo
Undoes previous action
Note: Certain operations, such as Delete, are 2-step operations. The first step clears
the contents of the wells (well descriptors) and the second steps deletes and
shifts wells. As such, when such an operation is performed, attempting to
recover to original state requires a 2-step Undo process.

Right-Click Menu

Section Seven: Data Analysis

7.1 Data Analysis
This section describes all of the available options for data interpretation and reporting. There
are several levels of analysis available for tests including Endotoxin Concentration
determination, Pass/Fail Reports, and Audit Trail Reports.

Data Collection Completed Dialog Box

After the user completes Data Collection for a test, Pyros EQS will display a message indicating
that Data Collection has completed and will ask the user if they would like to switch to the Data
Analysis mode. If the user clicks “OK” on the dialog box, the “Analyze Data” main screen will be
displayed. Clicking “Cancel” will cause the application to return the user to the “Main
Application” screen from which a user can Collect Data, Analyze Data, run Audit Trail (if
previously activated), Trend Data, or perform Administrative tasks.

Main Application Screen – Cursor over Analyze Data

If the user selects “Analyze Data” from the “Main Application” screen, a dialog box will prompt
the user to select a Test ID. The user can select a Test ID from any of the Data Groups to which
he/she has “Read” and/or “Write” access.

Analyze Data – Select Test ID Screen

Once the user selects an assay and clicks “OK,” the “Analyze Data” main screen is displayed:

Analyze Data Main Screen

The layout of the “Analyze Data” main screen is similar to the “Collect Data” main screen, but
the menu buttons at the top of the screen differ. Functionality for each of the menu items is
described in greater detail throughout the rest of this section.
Computed individual well-level test data have been computed in the Onset Time and Endotoxin
columns. The Onset Time column reports the onset time for the wells. If a well has not reacted
or the software is unable to calculate the onset time, no onset time will be reported. In cases
where a well has not reacted for the duration of the test, the Endotoxin column will report the
run-time for which a tube was present in the well and indicate that Endotoxin was “Not
detected by N secs”, where N is the run-time for that particular well.
Note: If one or more wells of the standard curve has not reached onset time, no standard
curve has been configured in the test, and/or no Stored Curve has been applied to
analyze the test, an error message of either “Unable to create resident standard curve
for analysis” or “Error in calculating regression parameters for standard 1” will appear.
Upon clicking “OK” a message of “Assay calculations could not be performed as the
standard curve parameters could not be determined” will appear. Upon clicking “OK” no
values or messages will appear in the Endotoxin column. Using the Exclude/Include
feature under options to exclude the non-onsetting standard wells will allow a standard
curve to be configured provided there are at least 3 standard concentrations which have
attained onset.

7.2 Standard Curve for Analysis

The Std Curve button brings up the “Standard Curve for Analysis” screen. This screen
has three tabs – Standard Curve Info, Lots and Analysis Tools. The functionality of each
of these tabs is described below.

7.2.1 Standard Curve Info

The Standard Curve Info tab displays information regarding the Standard Curve(s). The
Standard Curve No field lists all the curves that have been defined in the test using the
curve numbers. If only one curve had been defined, only a single curve number will be
observed in the drop-down box. In addition to resident standard curves that are part of
the assay, there is always the option to manually enter standard curve parameters
using the Keyboard option. Selecting this entry opens up most fields on this screen for
editing purposes and allows a user to specify parameters to create a Keyboard curve for
temporary analysis of the test. Note that a Keyboard curve, unless explicitly saved as a
Stored Curve by specifying a Stored Curve ID, is only stored by the software in memory
for the duration of the analysis session. Once the user logs out or exits the software,
the Keyboard curve parameters will be deleted.
Note: If a standard is improperly configured, and/or each standard has not reached
onset, the software will not be able to calculate a regression line. If a user
attempts to bring up this screen, the software will display an error message to
inform the user that it was unable to compute regression.

Standard Curve for Analysis

Stored Curve ID allows the technician to use a stored standard curve for analyzing a
test. These stored curves are also referred to as Archived Curves in the 1987 FDA
Guideline and 1991 FDA Guidance. All stored curves belonging to the selected data
group, are listed here. The technician may also use this field to specify a stored curve
name. After choosing the appropriate data group, click “Save” to save the parameters
on this screen under the assigned Stored Curve ID.
Note: When applying a Stored Curve, note that the lots used in the Stored Curve must
match those used in the test. Mismatch in either LAL Lot, or Endotoxin Lot – can
adversely impact test analysis and result in both the analysis screen and reports
indicating “Diff. Lot”.
Data Group lists all the data group names to which the user has access. Data Group
setup and user access configuration are functions performed by the Administrator.
Line Equation box displays the Slope, Y-Intercept, and Correlation Coefficient for the
standard curve regression analysis. For a resident standard curve in use, these
parameters are non-editable, as illustrated in the image above. These parameters
become editable only if the user selects the Keyboard option under Standard Curve No.
All standard curve information for resident standard curves is carried over from the
original configuration of the standard curve and is displayed on this screen. The
following are the parameters that reflect how the curve was setup.
Endotoxin Concentration Range with its Min and Max values and Units.
Final Results In check box and the associated units, which are automatically computed
based on the information provided about endotoxin limit(s) and endotoxin standards.
Potency value and Units.
Valid Concentration Range for the potency from Min to Max values and Units.

Threshold OD reflects the threshold OD specified for the test on the “Test Information”
screen. The user can change the threshold OD value on the “Standard Curve for
Analysis” screen to suit their needs. The modified value will be reflected on the “Test
Information” screen.
Baseline Adjustment checkbox and its accompanying Min and Max range (in seconds)
reflect the status of this flag and the range specified on the “Test Information” screen.
The user may choose to enable/disable Baseline Adjustment or alter its range to suit
their analysis purposes. Note that the adjustment range must be a minimum of 200
seconds, and the earliest baseline adjustment period that can be used is the 130-330
second data period, as the 130 second block is the first recorded point after the zeroing
period for each well. See Section 6.2.1, Baseline Adjustment for more information.
Standard Curve Type will display whether the standard curve selected was a “water”
standard curve or a “product” standard curve. See Section 6.6 for more information.
Description will display any notes that may have been entered by the user while setting
up the standard curve. See Section 6.6 for more information.
Wells Excluded lists specific wells which belong to the resident standard curve being
used for analysis and have been explicitly excluded on the “Analyze Data” main screen
by using the Options  Exclude/Include menu item. For additional details on
excluding/including wells during analysis, please refer to Section 7.6.2.
Use this Curve when clicked will use the selected resident/stored/keyboard curve for
analyzing the current test.
Cancel simply closes the “Standard Curve for Analysis” screen.
Save allows the user to save the parameters displayed on-screen to the Stored Curve ID
specified. An existing stored curve cannot be overwritten so it is important that the
user specifies a unique stored curve ID to save the curve parameters.
Clear, when clicked, clears all the fields on this screen and defaults curve selection to
Keyboard. The technician may now specify all the relevant curve parameters and apply
the curve for analysis, or simply specify a stored curve ID and save the curve for use in
the future.

7.2.2 Lots
The Lots tab on the “Standard Curve for Analysis” screen carries over information from
the resident standard curve selected for analysis on the Standard Curve Info tab or
from the Stored Curve selected for analyzing the current test. In both these cases, the
information on this tab is purely for display purposes and cannot be edited. If, however,
the user selects the Keyboard option on the Standard Curve Info tab, these fields will
become editable and will allow a user to specify lot information. Note that the lot
information plays an important role in test analysis and incorrect lots can lead to
erroneous results. A user should therefore ensure that the information is entered
correctly.

Standard Curve for Analysis – Lots Tab

Once entered, a user may choose the “Save” option, to save this information as a new
Stored Curve.

7.2.3 Analysis Tools

The Analysis Tools tab of the “Standard Curve for Analysis” screen has two key
components to it. The right half of the screen has the Theoretical Onset Time table
which lists the Concentrations from the standard series (Resident, Stored, or Keyboard)
and the corresponding Theoretical Onset Times for each of the concentrations. The
number of rows displayed will correspond to the number of concentrations in the
curve. For Stored and Keyboard curves, the interim concentration values are not
available to the software and only endpoint concentrations (Minimum and Maximum)
will be listed.

Standard Curve for Analysis – Analysis Tools Tab

Note: Selecting a Keyboard Curve and navigating to the Analysis Tools tab may result
in an error message “Error in parameters for curve chosen”. This error occurs if a
Keyboard Curve has been defined, but the Use This Curve button has not been
clicked to indicate to the software that this curve should be used for analysis.
After selecting a curve, or defining a Keyboard Curve, click on the “Use This
Curve” button to avoid errors.
Clicking on “Report” will generate a Theoretical Onset Time Report as displayed below.

Theoretical Onset Time Report

The left half of this tab gives the user a calculator to perform interpolation between
Endotoxin Concentration and Theoretical Onset Time values. Entering a value in one of
the fields and clicking “Calculate” will generate the other value based on the standard
curve regression being used for analysis.

Standard Curve for Analysis – Interpolation Feature

Clicking “Reset” will clear both the Endotoxin Concentration and Onset Time fields.

Clicking “Report” will generate an Interpolation Report as displayed below.

Interpolation Report – Onset Time Calculated

7.3 Graphics
The Graphics button on the “Analyze Data” main screen allows the user to look at Data
Plots and Standard Curve Regression Plots. These are explained in detail below.

Graphics Menu

7.3.1 Data Plots

The first option when the user clicks on the “Graphics” button is Data Plots. The Data
Plots option allows the user to generate different types of well plots.
Clicking on the “Data Plots” menu item brings up the following dialog box.

Graphics – Data Plots Well Selection

This dialog box defaults to selecting All Wells to be plotted in a Single Plot. The Plot
Type can be either Single Plot or Multiple Plots. Single Plot will display all selected wells
in a single X-Y plot as shown below, whereas Multiple Plot will display each selected
well in a small XY plot of its own. 12 wells are automatically grouped together on each
page, when the Multiple Plot option is chosen.
If the All Wells option is unselected, the user may choose specific wells to plot by
entering well numbers separated by commas in the Select Wells field. For example, if
only well numbers 1, 2, 3 and 8 are to be plotted, enter 1,2,3,8 in this field. All of the
following methods to specify individual wells are acceptable:
1,2,3,8
1, 2, 3, 8
1-3,8
1-3, 8
1-3, 8, 10, 11-13
Presence or absence of spaces is immaterial. The commas and dashes are the
separators recognized by the software.
Wells Excluded lists well numbers which have been excluded from analysis on the
“Analyze Data” main screen using the Options  Exclude/Include functionality.

Graphics – Data Plots – Single Plot

Graphics – Data Plots – Multiple Plots

The user can Print and Rescale either of the Single or Multiple Well Plots by clicking on
the respective buttons of the same labels. Clicking the red “X” in the top right corner

will close the plot window. On the Multiple Plot window, depending on the number of
wells selected for plotting, the user will be able to navigate from one screen to another
using the Previous and Next icons.
Note: When “Print” is selected for the Standard Curve Regression Plot, Individual Well
Plot, or Multiple Plot windows, the plot is printed as it is seen on the screen. In
order to provide additional flexibility for users printing from a variety of
different screens, the user will be prompted with a dialog box for changes in X
and Y scaling. By default, the value for both scales is 0.78 (for a screen resolution
of 800 x 600 pixels).

7.3.2 Standard Curve Regression Plot

The second option when the user clicks on the “Graphics” button, is Standard Curve
Regression Plot. This option allows the user to choose one or more of the resident
standard curves and plot their regression lines.
Clicking on this option brings up a curve selection dialog box as shown below.

Graphics – Standard Curve Selection Dialog Box

All resident standard curves will be listed in this dialog box. The curve number is listed,
one per row, along with the Endotoxin Lot number, separated by a hyphen. To plot a
curve regression line, select one or more curves. To select just one curve, click on the
curve number and then click “OK”. To select multiple curves, CTRL-click on each curve
to be plotted. If multiple curves are selected, they must be of the same endotoxin
units; otherwise the software will display an error message indicating that the units do
not match. Clicking “OK” brings up the plot as shown below. Clicking “Cancel” exits the
dialog box.
Note: If users are running curves with concentrations below three decimal places (i.e.
<0.001), the x-axis of the graph may display “0.000.” This is a known behavior of
the graphing module of the software, and does not affect calculations.

Graphics – Standard Curve Regression Plot – Single Curve Plotted

The line equation parameters for each curve are displayed above the regression lines.
The Close button at the top will close the plot window.
Print and Rescale buttons allow the user to print the plots and rescale them,
respectively.

7.4 View
The View menu lets the user look at some of the reports and test parameters. The
contents of the menu can be seen highlighted in the picture below. Each menu item is
explained in detail in this section.

View Menu

7.4.1 Test Info

Clicking View  Test Info brings up the following window. This window allows the user
to review the parameters used to setup the test. However, since the test has already

been performed and Data Collection has completed, the technician will not be allowed
to edit most of the test information parameters on this screen.

View – Test Information

Cancel closes the screen.

Refer to Section 6.2 for more information on the “Test Information” screen.

7.4.2 Test Errors

This option generates the Test Error Report. Any errors that were noted during the test
will be displayed on this report. The following types of errors are noted by recording
the run-time at which they were observed by the software:
 Missing Data Block
 Bad Data Block
 A/D Value Out of Range
 Sudden OD Change
 Temperature Out of Range
 Tube Removal

Test Errors Report

Of the list of errors reported on the Test Errors Report, A/D Value out of Range and
Tube Removal are fatal to the well in question. Once a fatal error is detected, no
further data collection and calculations are performed on the well(s) in question.
Additional details on error conditions can be found below.

Missing Data Block Time (sec) lists the time that the system computer did not receive
the block of data that was expected. A missing data block occurs when the computer
does not receive a data block at the time it is expected. Data blocks should be
transmitted every 10 seconds. The software allows for a tolerance of +2 seconds for
each subsequent data block to be received, from the time it receives a data block. A
lost data block may occur if the tube reader becomes disconnected from the computer.
Another cause is a discrepancy between the clock in the instrument and the clock in the
computer. 30 consecutive missing data blocks will terminate the test. See Section
9.1.13 for additional information on missing data blocks.
Note: Occasional bad or missing data blocks do not necessarily invalidate the test. The
decision to regard the test as invalid must be made by the individuals
responsible for test results in accordance with the user’s company policy.

Bad Data Block Time displays the time (seconds) that a data block arrives with an
incorrect format. Bad data blocks are not used in calculations. This type of error is
usually caused by a communication problem. Check the cable between the tube reader
and computer. 30 consecutive bad data blocks will terminate the test. Bad data blocks
may be caused by one of the following:
1. Faulty or poorly connected serial cable between the tube reader and the computer
– check connections and replace the cable if necessary.
2. Faulty serial connection board in the system computer – try an alternate computer.

3. Failing serial board inside the tube reader – make arrangements to return the unit
to Associates of Cape Cod, Inc. for repair.
See Section 9.1.12 for additional information on bad data blocks and Section 9.1.14 for
information on data block format.

A/D Out of Range displays the time block (seconds) at which the A/D value is either too
low or too high. For a value to be flagged as out of range A/D, the count must be either
below 800 or above 2520 for a Pyros Kinetix, or below 800 or above 3500 for Pyros
Kinetix Flex instruments. The Administrator can access this range on the “Tube Reader
Parameters” screen, but it is not recommended that the range be adjusted. When a
problem occurs with the A/D values, it is normally specific to one or several wells. This
is not fatal to the run but is fatal to data collection for the offending well(s). Possible
causes include:
1. The sample is too turbid.
2. The meniscus or other foreign object is blocking the light path.
3. The reaction tube is scratched or dirty.
4. The amplifier gain is in need of adjustment.
If the sample is too turbid, repeat the test (not to exceed MVD) at a higher dilution.
Causes 2 and 3 can be corrected by using the remaining wells of the instrument to
retest the sample in question again. In the case of cause 4, the tube reader must be re-
calibrated. See Section 9.1.10 for additional information on A/D out of range errors.

Sudden OD Change displays the time (seconds) at which an acute change in OD

occurred. This is a response not characteristic of an LAL reaction. When a Sudden OD
change occurs, the time and well number are recorded. A user may view this change by
looking at an Individual Well Plot. Sudden OD changes can occur when a tube is moved,
the instrument is bumped, or a bubble bursts inside the tube. This might occur if:
1. The product is coming out of solution – check the pH of the product. Adjust the pH
if necessary of dilute the sample further (not to exceed the product MVD).
2. There is vibration of the bench top under the tube reader.
3. Fluctuations in lamp intensity – the lamp may be beginning to fail. Contact
Associates of Cape Cod, Inc. to arrange for repair and re-calibration.
4. A power surge – Pyros Kinetix and Pyros Kinetix Flex instruments should be
attached to a surge protector.
5. Poor connection from the tube reader to the computer – check the serial cable
connection between the instrument and the computer.

Temp Out of Range lists the time at which the temperature of the Pyros Kinetix or
Pyros Kinetix Flex failed to achieve or exceeded the specified temperature limits. H
indicates a High and L indicates a Low value for the temperature recorded. Note for
Pyros Kinetix Flex instruments with more than one heating unit, each heating unit will
be listed on the report; those in specification will exhibit an N for normal temperature.

Tube Removed is the time at which the tube in the specified well was removed from
the well. Once a tube removal is detected, re-insertion of a tube in the well is not
recognized by the software.

7.4.3 Well Info

The View  Well Info menu option allows a user to take a look at all parameters for
the selected/highlighted well. To view well information for a particular well, click on
that well (row) in the “Analyze Data” main screen and then click on View  Well Info.
Depending on the type of well selected, the appropriate window and well information
will be displayed.
Different windows are displayed for Negative Controls, the Standard Curve, and
Samples. Each of these windows is shown below.

View – Negative Control

View – Standard Curve

View – Sample

7.4.4 Reagent Lots

The View  Reagent Lots option brings up the Reagent Lots Report. During Data
Collection, this report is also available through the Print Test Setup button.

Reagent Lots Report

7.4.5 Temperatures
The View  Temperatures option brings up the Temperature Report. This report
displays the minimum, maximum and mean temperatures recorded for the duration of
the test, and also lists each of the temperature values of each heating unit in the
instrument recorded during the test. The temperature values are listed based on the
Maximum Number of Cycles between Temperature Readings parameter configured by
the Administrator in the Tube Reader Parameters dialog box. If the temperature falls
outside the specified range for the test, those values will also be listed on this report,
though not flagged in any manner.

Temperature Report – Pyros Kinetix

7.4.6 Tube Insertion Delay

This option brings up the Tube Insertion Delay Report, shown below. This report lists
each of the active wells and the corresponding insertion delay (in seconds) from the
time the first tube was inserted.

Tube Insertion Delay Report

In the example shown above, the tubes in wells number 1 to 6 were inserted to start
the test. The tubes in wells 7 -10 were inserted with a delay of 10 seconds after tube 1.

7.5 Reports
Clicking the “Reports” button displays the menu items as shown in the highlighted
section of the picture below. Each of these items is explained in detail in this section of
the manual.

Report Menu Items (Highlighted)

Note that the reports generated in Pyros EQS can easily be exported to an alternative
file format. To export a particular report, go to the View or Reports menu and select
the desired report. In the toolbar at the top of the window, click on the first icon
(placing the cursor over the icon will reveal a screen tip “Export Report”).

Export Report – Screen Tip

A Save As dialog box will appear. The user can select the desired drive, file name and
type of file to be saved from the various menus. Pyros EQS allows a user to export the
report in various convenient formats:

Note: Once information collected in Pyros EQS has been exported outside the
application, the 21 CFR Part 11 compliance features associated with Pyros EQS
no longer apply.

7.5.1 Report Cover Sheet

The Report Cover Sheet contains summary information about the test being analyzed.
Data presented on this report is grouped by category. The following information can be
found on this report:
 Test ID
 Report Date
 Data Group in which test resides in the database

 Computer ID on which test was conducted

 Technician (the user who initiated the test)
 Instrument type and serial numbers on which test was performed. For Pyros Kinetix
Flex instruments, the wavelength the test was read at will also be displayed.
 Minimum, Mean and Maximum temperatures during the course of the test
 Data Collection Date and Time
 Lot Information (Water Lot, LAL Reconstitution Buffer Lot, LAL Lot, Endotoxin Lot)
 Sign-off status indicating whether test has been signed-off by Supervisor
 Key test parameters [Threshold OD, Baseline Adjustment Range (if active), Spike
Recovery Range]
 Standard Curve Information
 Test Comments
 Report GUID (Global Use Identifier: see text box below for additional information)
Much of the key test information has been grouped together on this Report Cover
Sheet so as to not have to repeat it on each individual report. The recommended
approach is to first analyze the test, then print out key reports and group them with the
Report Cover Sheet in order to have all the relevant test analysis information available
together.

Report Cover Sheet

If Additional Fields have been defined on the Custom Fields tab of the “Test
Information” screen (see Section 5.4.1.1), these would be listed on the Report Cover
Sheet in a separate box with the heading Additional Test Information below the
Standard Curve information box.

If Test Comments had been separately entered for the test, they would appear below
the Additional Test Information box (if Additional Fields were defined), or, below the
Standard Curve box in the absence of additional fields.

Report GUID
Pyros EQS has a feature which will prove to be very useful in collating reports belonging
to the same analysis of a test. This is the Report GUID which appears on all reports. The
Report GUID is a 32-character sequence generated by Pyros EQS based on changes to
test information, including information specified using the “Test Information” screen
(Refer to Section 6.2) and well descriptor information. In essence, the Report GUID
reflects the state of a test at any given point in time.
This identifier looks like this: d4870fc6-59ac-461a-920a-4af86c9eb9bb.
Should any test meta-data be modified, Pyros EQS will automatically generate a new
Report GUID, which will be visibly different from the previously generated GUID. In
other words, if the user input data for the test in question does not change, all reports
generated by Pyros EQS for the same test will bear the exact same Report GUID – thus
helping collate reports belonging to a single analysis set.

Note: The application does not perform field comparison changes on some screens. A
new GUID may be generated when a user clicks “OK” or “Save” even if no visible values
were changed. To avoid such scenarios, users should select “Cancel” to exit a dialog box
where no changes have been made.

7.5.2 Data Summary

The Data Summary Report lists the general test information which includes the Test ID,
the name of the Technician who performed the test, the Data Group in which the test
resides in the database, the data collection date and time, report date and time, as well
as instrument type, and wavelength for Pyros Kinetix Flex instruments, and serial
number. In addition, information specific to this report such as the calculated values of
the standards used to construct the standard curve applied to sample analysis, well
descriptors, mean calculated concentrations, spike recovery analysis, and the CV% for
replicate correlation is listed in the report. The report also lists the endotoxin limit
values and units as specified for the samples.

Data Summary Report

Additional Information on Data Summary Report contents:

Negative Controls will appear on the Data Summary Report if:
 Check Negative Control Validity feature is enabled, and,
 Negative Controls are found to reach onset.
Water Negative Controls will appear as WaterNegCtl
Product Negative Controls will appear based on Product Type and Product Lot.
Negative Control wells with the same Product Type but different Product Lot will be
treated as separate Negative Controls.
Results are not necessarily reported by well order.

Lot Mismatch
In the case of an LAL lot mismatch, the Mean Concentration column on the report
would indicate ‘Diff. Lot’.
In case of Endotoxin Lot/Unit mismatch, the Mean Spike Concentration column on the
report would indicate ‘Diff. Lot’.
Effect of “PPC Override”
If PPC is not configured in the test, or, if all PPC wells are excluded from analysis, and:
 If PPC Override check box is not checked, the Mean Spike Concentration column
would report ‘Invalid’ whereas the Mean Spike – Un-spiked, Spike Recovery and
Mean Final Concentration columns would be blank.
 IF PPC Override check box is checked, the Mean Spike Concentration, Measured
Spike – Un-spiked and Spike Recovery columns would be left blank whereas Mean
Final Concentration would get displayed.
Effect of “Flag CV if greater than” flag

 If the flag is checked, and if the CV% value is greater than the limit specified on the
Test Information screen, the CV value will be flagged on this report with a # mark.
 If the flag is un-checked, CV value will be displayed.
Effect of “Display Coefficient of Variation” flag
If this flag is not checked, CV values for Standard and Samples will not be reported on
the Data Summary Report.
Give Results per Unit of Product
If this check box is enabled, Mean Final Concentration column will display values in
respective units of the product. If disabled, the Standard Curve unit will be used.
Footnotes on Report
<!>  indicates replicates have not incubated long enough for valid results
#  indicates CV value is greater than specified CV limit

7.5.3 Individual Sample

Individual Sample Reports may be generated for each sample included in the assay.
Each sample report lists general information about the test – Test ID, user name, Data
Group in which the test resides in the database, data collection date and time, report
date and time, as well as instrument type, and wavelength for Pyros Kinetix Flex
instruments, and serial number. In addition, information specific to this report –
endotoxin recoveries, spike recovery analysis and Pass/Fail results – is also listed.

Individual Sample Report – Sample Selection Screen

Choosing the Individual Sample Report menu option from the Reports menu brings up
the above sample selection screen. The column on the left contains an arrow pointer
but does not indicate row selection. A user must explicitly select a row (sample) by
clicking in the pointer column and then click “OK” to generate the report for the chosen
sample, as shown below.

Individual Sample Report

Note: Multiple samples cannot be selected together. Sample reports must be

generated separately, for each sample selected.
Additional Information on Individual Sample Report contents
Endotoxin Limit  User-specified limit value displayed here
Maximum Valid Dilution
The Maximum Valid Dilution is the maximum dilution of a sample at which the
endotoxin limit can be determined. Conseqently, it is the maximum allowable dilution.
It is a unitless dilution factor. If the units in the equation do not cancel out, “Invalid”
will be displayed on the report.

When the Correct Endotoxin Limit for Pooled Vials checkbox is selected in the Sample
Setup window the MVD is calculated using the Adjusted Endotoxin Limit instead of the
Endotoxin Limit (Refer to Section 6.8)
The Endotoxin Limit is multiplied by Product Concentration when the limit is specified
in a Unit of Product. When the limit is specifed in the Unit of the Standard Curve this is
not applicable.
Initial Concentration
The Initial Concentration is the Product Conc. before Dilution (user entered value).
Vials Pooled  User input value
Adjusted Endotoxin Limit
The Adjusted Endotoxin Limit is the user specified Endotoxin Limit divided by the
number of Pooled Vials (if specified). The Adjusted Endotoxin Limt will be converted to
the reported endotoxin unit as specifed by “Give Results per Unit of Product”. In the

event that the Endotoxin Limit can not be converted to the reported endotoxin unit the
Adjust Endotoxin Limit will display “Unit Error”.
Pass / Fail Value
In order for a sample in a test to “pass”, the test must have all valid controls and must
meet the user specified criteria for a valid standard curve, such as Correlation
Coefficient and Coefficient of Variation. Once this is confirmed, the results for
individual samples are evaluated against criteria specified for the samples themselves,
such as Coefficient of Variation, Spike Recovery. If all criteria for a valid test have been
satisfied and the measured endotoxin concentrations of all of the replicates of a sample
are less than the Endotoxin Limit the sample is marked as having passed.
Lot Mismatch
If the LAL lot of the sample does not match that of the standard, the Mean Measured
Endotoxin Concentration column would display ‘Diff. Lot’ and the Pass/Fail column will
appear blank.
Errors
If any un-spiked replicates encounter the following errors, the Mean Measured
Endotoxin Concentration column will display Check Wells and the Pass/Fail column
would be left blank.
 Tube removal
 A/D out of range (during zeroing period)
 Invalid Baseline Adjustment
Effect of “PPC Override”
If a PPC is not configured for the selected sample, or, if all of the PPC wells are excluded
from analysis, and:
 If PPC Override check box is not checked, the Spike Recovery and Mean Final
Endotoxin Concentration columns would be left blank.
 If PPC Override check box is checked, the Spike Recovery column would be left
blank and the Mean Final Endotoxin Concentration would be displayed.

7.5.4 Individual Well Results

The Individual Well Results Report includes general test information – Test ID, user
name, Data Group in which the test resides in the database, data collection date and
time, report date and time, as well as instrument type, and wavelength for Pyros
Kinetix Flex instruments, and serial number. In addition, the Individual Well Results
Report also lists each well with its descriptor, standard/spike concentration value and
units, adjusted onset time, dilution (for samples), and endotoxin measured.

Individual Well Results Report

Note: If the Pyros EQS software is not able to generate or print an Individual Well
Results Report, see Section 9.2.22.

7.5.5 Assay Audit Trail

The Assay Audit Trail Report lists the report data time interval, report print date and
time, date and time of audit event occurrence, user name, Test ID, pre- and post-
change values, and comments. Edits to test parameters are captured in the Assay Audit
Trail and reported here.

Assay Audit Trail Report

7.5.6 Raw Data/OD

The Raw Data/OD Report is generated for all wells that contain tubes during the assay.
The report includes general test information at the top – Test ID, user name, Data
Group in which the test resides in the database, data collection date and time, report
date and time, as well as instrument type, and wavelength for Pyros Kinetix Flex
instruments, and serial number. As with all other data reports, it also contains the
Report GUID (See Section 7.5.1).
This report is organized by well number. For each active well, this report displays the
Analog to Digital value reported by the instrument, the corresponding Optical Density
(OD) value, and the time at which these values were reported. The frequency at which
the values are listed on this report is governed by the following parameters setup by
the Administrator on the Tube Reader Parameters dialog box.
 Cycle Range for A/D Zeroing
 Maximum number of cycles between Data Readings

Until the Zeroing period has been completed, every single raw data value will be listed
in this report. Post completion of the zeroing period, the number of data points that
will be skipped before a point is listed on this report will be governed by both the
Maximum number of cycles between Data Readings parameter, and the Minimum O.D
change for data point storage parameter. If there is not enough O.D. change (default of
1.5 mABs) for the data point to be stored, by default every 7th value will be listed on
this report after the zeroing period, due to the number (default 6) of the Maximum
number of cycles between Data Readings parameter.

Raw Data/OD Report

Note: Selecting one or more wells on the Analyze Data main screen and then
generating this report by clicking Reports  Raw Data/OD will not generate the
report for just the selected wells. This report, as mentioned above, is generated
for all wells containing tubes during the test. To print out pages specific to wells
of interest, please note down the page numbers and specify just those page
numbers in the dialog box that will pop-up after the printer icon is selected.

7.5.7 Pass/Fail
The Pass/Fail Report indicates the status of a variety of conditional tests available
during test setup. Some of these tests are:
 Standard Curve Correlation Coefficient
 Positive Control Spike Recovery
 Flag Coefficient of Variation if greater than
 Range for Spike Recovery
 Endotoxin Limit

Individual standard and sample well descriptors will be listed on this report, along with
the conditional test being performed; the specifications for the test; the actual value as
noted by the software, and the status Pass/ Fail. Results are not necessarily reported by
well order.

Pass/Fail Report

Additional Information on Pass/Fail Report contents

Conditional Test Listing

Conditional tests are listed on the reports if checkbox is activated under options in the
collect data box:
 Correlation Coefficient
 Negative Control Validity
 CV Limit (Standards)
 CV Limit (Positive Control)
 CV Limit (Sample)
 Positive Control Spike Recovery Validity
 Range of Spike Recovery
 Endotoxin Limit
CV is reported based on Endotoxin Concentration or Onset Time, as specified in Default
Settings.

Correlation Coefficient Validity

If more than one standard curve is defined in the test, the Correlation Coefficient
Validity will be reported for only the default standard curve. The default standard
curve, unless explicitly selected using the “Standard Curve for Analysis” screen is the
standard with the lowest curve number. In such situations, the additional standard(s)
would be treated as unknown samples.

Negative Control Validity

This test will appear on the Pass/Fail Report only if the Check Negative Control Validity
check box has been checked. Pass Criteria: Negative Control has no detectable
endotoxin or the detected endotoxin is less than the user defined fraction of lambda.

Positive Control Spike Recovery

This test appears only when a Positive Control has been configured in the assay. Pass
Criteria: Positive Control Spike Recovery value is within the user defined Positive
Control Spike Recovery Range.

Endotoxin Lot Mismatch

This results in the Actual Value column displaying “Diff. Lot” and Pass/Fail column
displaying blank for Spike Recovery Test.

LAL Lot Mismatch

This results in the Actual Value column displaying “Diff. Lot” and Pass/Fail column
displaying blank for the CV Test.

CV Limit (Standard/Sample/Positive Control)

This test appears only if the check box for Flag Coefficient of Variation if Greater Than is
checked and an applicable multi-replicate Standard, Sample and/or Positive Control is
defined. Pass Criteria: The CV % is less than or equal to the user defined limit.

Range of Spike Recovery (Sample)

This test appears when Sample Spikes are described. Pass Criteria: Sample Spike
Recovery value is within the user defined Spike Recovery Range and the Sample Spike
Found Endotoxin Concentration value is within the range of the standard curve.

Note: If Sample Spikes are not defined the Sample Actual Value and Endotoxin Limit
Pass / Fail result will not be reported. Check the Override PPC requirement to
get a reported Sample Actual Value and Endotoxin Limit for samples without a
spike.

Endotoxin Limit (Sample)

The test will appear on the Pass/Fail Report only if an Endotoxin Limit has been
entered. The specification for this test is entered by the user for a specific sample
during assay setup. Pass Criteria (Endotoxin Limit): Found Endotoxin Value is less than
the user defined Endotoxin Limit and all of the following are valid (if applicable)
 Standard Curve Correlation Coefficient
 Standard Curve CV%
 Negative Control
 Positive Control
 Sample Spike Recovery
 Sample CV%

7.5.8 Well Uniformity (Pyros Kinetix Only)

The Well Uniformity feature available in Pyros EQS generates its own special report.
This report can be accessed under Reports. The Well Uniformity feature is specific to
the Pyros Kinetix instrument and does not apply to Pyros Kinetix Flex instruments. The
Well Uniformity Report stands by itself and as such, unlike other reports, has an
extensive report header summarizing well uniformity test information. This header lists
the computer name on which the test was performed, user performing the test, test ID,
test date and time, report date and time, and goes on to list key test parameters such
as Baseline Adjustment Range, Spike Recovery Limits, Water, LAL and Endotoxin Lots
used, instrument Minimum, Average and Maximum temperatures recorded during the
test, whether there were test errors, and whether the test has undergone Supervisor
Sign-off.
In addition, analysis information pertaining to the standard curve used for analyzing the
Well Uniformity Test is also listed. This information contains the line equation
parameters for the curve and the LAL and Endotoxin lots used by the curve.
Given that the Well Uniformity Test is a system test which uses a stored format for the
test and a stored Well Uniformity curve for analysis (default curve used for analysis),
these parameters will remain constant unless specifically modified by the user.
The report is shown below.

Well Uniformity Report – Page 1

Well Uniformity Report – Page 2

The second page of this report summarizes the data and lists the average endotoxin for
all 96 wells, the calculated minimum and maximum limit values (75% and 125% of the

average), Minimum endotoxin recorded, Maximum endotoxin recorded, Results:

Pass/Fail (Pass is reported if all endotoxin concentrations fall within the +/- 25%
average), CV% and Standard Deviation.

7.5.9 Potency
The Potency Report can only be generated if multiple standard curves are present in
the test. This report is relevant only when calculating the potency for control standard
endotoxin, using a reference standard endotoxin curve. Pyros EQS allows a user to
specify a single Reference Standard Curve and up to 4 Control Standard Curves. The
report is as shown below.

Potency Report

The Potency Report lists the user name, Test ID, Data Group in which test resides in the
database, data collection date and time, and instrument type, and wavelength for
Pyros Kinetix Flex instruments, and serial number as the header for the report. The
report has two halves. The upper half contains information pertaining to the Reference
Standard and the bottom half contains information about the Control Standard. The
LAL and Endotoxin lots used for Reference and Control standards are also listed. Note
that if the Control Standards have different Endotoxin lots, this report can only be
generated if wells using different lots are excluded. That is, at any given point in time,
only two lots (one Reference Standard lot and one Control Standard lot) are being
analyzed.
For each concentration of the Reference standard, the software lists the mean Onset
Time and the Mean Measured Concentration. The Mean Measured Concentration is
calculated using the Mean Onset Time. For each concentration of the Control
standards, the software lists the mean onset times for each replicate set, the mean
onset time and log of mean onset time, for all the replicates for the same

concentration, and the calculated potency. The average calculated potency and
correlation coefficient are also listed on the report.
7.6 Options
The Options button on the “Analyze Data” main screen allows a user to perform
multiple functions. Each of these is described in detail below.

7.6.1 Test Comments

Clicking “Test Comments” opens the following screen which allows a user to specify
general comments for the test being analyzed. Test Comments are cumulative in that
once a user enters a comment in the bottom half of the screen and clicks “OK”; the
comments will be stored along with the test in the database and cannot be edited or
deleted. Stored comments will appear in the upper half of the screen as shown in the
screen capture below.

Options – Test Comments

7.6.2 Exclude/Include
This option allows a user to exclude specific wells from analysis. To exclude/include
wells, select one or more wells on the “Analyze Data” main screen and then click on
Options  Exclude/Include. The selected wells, if included, will now be excluded from
analysis and a Yes will appear in the Exclude? column on the “Analyze Data” main
screen. Likewise, if selected wells had already been excluded from analysis, they will
now be included in the analysis and the Yes removed from the Exclude? column.

Options – Exclude/Include Wells

Note: To select multiple non-contiguous wells, hold the CTRL key on the keyboard and
click on each desired well on the Analyze Data main screen.
Note: There are serious regulatory implications that must be considered prior to
exclusion of Out of Specification (OOS) wells. Users are cautioned to perform
investigations for any observed OOS results, and only exclude wells after
documenting the justification for doing so.

7.6.3 Save Format

This menu item under the Options menu allows a user to save the existing test
parameters under a new Stored Format under a specific Data Group.
Enter New Format Name is where a user specifies the name to associate with the
format being stored.
Data Group is a drop-down box which will list only those data groups to which the
Administrator has configured a user to have read and write privilege. The user will
select a Data Group under which they want to create the new Stored Format.

Options – Save Format

Note: Stored Format names need to be unique across Data Groups. If the user enters a
format name which is already in use, the software will convey this by displaying
a red exclamation mark next to the field.

7.6.4 Supervisor Sign-off

The Supervisor Sign-off option allows a user with the appropriate access rights to sign-
off and lock test analyses. This menu option is enabled only if the logged-on user is a
Supervisor or Administrator.
To sign-off an assay and lock its state, login with Supervisor privilege level, and open
the assay to be signed-off by clicking on “Analyze Data” from the “Main Application”
screen and selecting the Test ID. Clicking Options  Supervisor Sign-off generates the
following dialog box.

Options – Supervisor Sign-off Dialog Box

When the user clicks “Yes”, Pyros EQS will lock all test parameters and display the
following confirmation message.

Supervisor Sign-off Success

Clicking “OK” closes the test and re-opens it immediately in Read-only mode. In this
mode, several buttons will be disabled. No changes to test parameters will be possible.
The user can however, view plots and print reports.

Supervisor Signed-off Test – Opens in “Read-only” Mode

7.6.5 Printer Setup

This menu option brings up the standard Windows Printer Setup dialog box and allows
the user to select the default printer for the software and configure printer specific
parameters. The printer selected in the drop-down box for Name will be recognized as
the default printer for the software once the user clicks “OK” on this screen.

Note: In some instances, selection of a non-default printer to print a specific report

will not carry over to additional print jobs in the same session. This is a known
behavior of the software; if the user wishes to continue using a specific printer
other than the default, they must select it for each print job.

Options – Printer Setup

7.7 Edit
The Edit button uses the highlighted well from the “Analyze Data” main screen as the
selected well and presents a dialog box that allows the user to pick between editing
just the single well, all replicates of that well, or the entire series. Entire series refers to
a Negative Control, Standard Curve, Positive Control or Sample series.

Options – Edit

The window displayed upon clicking the Selected Well, All Replicates, or Entire Series
button depends on the type of well selected, whether Standard, Positive Control or
Sample. The relevant screen is then displayed.

7.8 Test Info

The Test Info button gives the user access to the “Test Information” screen that was
used to setup the test for Data Collection. Parameters (fields) that would otherwise
alter the duration of the test will remain disabled. See Section 6.2 for more
information.

7.9 Print
The Print button allows the user to quickly select multiple reports for printing. A user
can check the boxes corresponding to the desired reports and click “OK”, which will
print the selected reports. If All Single Sample Reports is unchecked, and multiple
samples are included in the test, these samples will be listed under the Select Single
Sample Reports window. They can be manually selected and Individual Sample Reports
generated for them.

Print – Document Selection Dialog Box

7.10 Close
Clicking the Close button causes the analysis of the current Test ID to be terminated,
and returns the user to the “Main Application” screen.

Section Eight: Data Trending

8.1 Data Trending
The Trending Report header includes all of the fields used to select the range of data compiled
in the report. The report covers pertinent test information including test IDs, test dates, serial
numbers, technicians, lot numbers, sample descriptors, calculated data, whether there were
test errors and/or test comments.
Trending analysis allows the user to compare sample types, LAL lots, instruments and
technicians over multiple tests stored in the database. In this manner, an individual may look
for reproducibility, growing errors or reduction of errors over time, equipment and technician
performance over time, etc.
To use the Trending functionality of Pyros EQS, click the “Trend Data” icon from the “Main
Application” screen to open the “Data Trend Report Parameters” screen. Each field that the
user selects further restricts the amount of information that will be reported. The user does not
need to select an item for every field, just the item(s) to be trended.

Note: The application can only perform data trending on assays that have been
electronically signed off by a supervisor or Admin level user. Please see Section
7.6.4 for more information. Certain invalid assays are available for data trending
once a test has been signed off and locked. Invalid assays include the following:
negative controls with onset times less than the onset time of the lowest
concentration on the standard curve, and standard curves with correlation
coefficients below 0.980. To prevent invalid assays from possible inclusion in
data trending, the standard curve wells should be excluded in data analysis
using the Exclude/Include option, rendering the assay unable to be locked.

Sample dilutions with invalid spike recoveries in an otherwise valid assay are
also available to be trended in a locked assay. For these sample dilutions with
invalid spike recoveries, both the unspiked and spiked replicate wells should be
excluded from analysis using the Exclude/Include option. This operation should
be performed before the test is signed off into a locked state. This renders the
invalid sample dilutions unavailable for data trending.

Data Trend Report Parameters Screen

Start Date and End Date identify the desired date range during which the data to be trended,
was collected.
Data Type Plot allows the user to select between Evenly Spaced and Date Proportional.
Depending on the selection, either the data points will be plotted evenly spaced, or
proportional based on the data collection dates for the items being trended.
Data Group defines the data group or area of the database where the tests to be compared are
stored. Data Groups may be chosen from the pull down menu.
Sample Type defines the sample type to be trended. Sample types are created and associated
with samples when defining sample wells. See Section 6.8 for more information on creating
sample types.
Note: For maximum data trending value, we advise the user(s) to take special care when
defining specific sample types for testing.
Technician identifies the Technician performing tests for which the user wishes to compare
data.
LAL Lot defines the LAL lot the user wishes to view.
Instrument defines the instrument used to collect data that the user wishes to trend.
Once a date range, plot type and one or more of the qualifying parameters have been selected,
click “Report” to generate the Trending Report. Note that attempting to trend over a large
period of time, or over an extensive data set, can result in a time delay while generating the
Trending Report.
Examples of the Trending Report and the two types of plots – Evenly Spaced and Date
Proportional – are shown below.

Trending Report – Page 1 of 2

Trending Report – Page 2 of 2

Trending Plot – Data Points Evenly Spaced

Trending Plot – Data Points Date Proportional

Section Nine: Troubleshooting

Note: For technical assistance, please contact the Technical Services department of Associates of
Cape Cod, Inc. via phone at (800) 848-3248 / (508) 540-3444 or email at
[email protected].

9.1 Tube Reader

Problems (in bold print) are followed with possible solutions and references to other sections of
the manual where appropriate.
Note: Canned air should NEVER be used to remove debris from a tube well in either a Pyros
Kinetix or Pyros Kinetix Flex tube reader, as it will likely cause debris to become lodged
within the light path. The well(s) may be vacuumed out using a micro-vacuum, which is
available at computer or electronics stores.

9.1.1 No Power
Pyros Kinetix
No power to the LEDs on the front panel of the instrument. Front panel LEDs are not
illuminated.
 Check that the power cord is plugged into power supply and into the power outlet.
 Confirm that the power supply is connected to the tube reader. DO NOT plug the
power supply into the Pyros Kinetix instrument when the power switch is in the on
position.
 Confirm that the power supply is switched on.
Pyros Kinetix Flex
No power to the LEDs and LCD display on the top of the instrument.
 Check that the power cord is plugged into the power outlet and the power switch is
in the on position.
 DO NOT plug the power cord into the instrument when the power switch is in the
on position.

9.1.2 Front Panel LED Not Red on Instrument Power-up (Pyros Kinetix Only)
 Turn off the instrument, wait 15 seconds, then power up again.
Note: The Pyros Kinetix has a single LED indicator on the front which is temperature
sensitive, even when it is not in communication mode. The front LED should
start Red, unless the unit has been turned on previously and is up to
temperature.

9.1.3 Tube Reader Not Detected

Pyros EQS software consistently fails to detect the tube reader
 Confirm that the tube reader power switch is turned on, and LED(s) are lit.

 Confirm that both ends of the RS 232C cable have been inserted into the tube
reader and the computer correctly.
 Check that the serial cable is attached to the correct serial or USB port of the
computer.
 Verify that the correct default port is set. From the “Main Application” screen,
under Admin  Edit Default Settings, choose the Hardware tab and update the
field labeled Serial Port.
 Verify the correct COM port is set (in Device Manager, under System Properties).
 For a Pyros Kinetix, it is possible the tube reader has not been properly reset. Turn
off the power to the instrument's power supply for 1 minute. Then, power the unit
back up and retry initialization.
 If a USB  COM converter or USB cable is employed:
 Ensure that the device drivers have been installed.
 Ensure that the correct COM port is set (in Device Manager, under System
Properties).

9.1.4 Tubes Not Detected in the Software During Data Collection

Sample well LEDs turn green when reaction tubes are inserted into the sample wells
but the computer monitor does not show that tubes have been inserted.
 Check on the computer monitor that the incubator is at operating temperature.
 Wait for up to 10 seconds (one data cycle) to let software refresh the data on the
screen.
 Check that the cable connections between the instrument and the computer are
secure.
 For a Pyros Kinetix it is possible the tube reader has not been properly reset. Turn
off the power to the reader's power supply for 1 minute. Then, power the unit back
up and retry initialization.
 In a Pyros Kinetix, the light source may have burnt out (tube detect light source).
Contact Associates of Cape Cod, Inc. to arrange to have the unit serviced and
recalibrated.

9.1.5 Tube-Detect LEDs On but No Tubes Inserted

Pyros Kinetix
 In a Pyros Kinetix If no tubes inserted, yet all well LEDs are turned on, the tube
detect light source has burnt out. Contact Associates of Cape Cod, Inc. to arrange to
have the unit serviced and recalibrated.
 In a Pyros Kinetix if some (but not all) of the well LEDs are on when no tubes have
been inserted, verify that no dust or debris has fallen into the well(s) in question.
 If cleaning the light path(s) does not solve the problem, the tube-detect reference
voltage needs adjustment.
 If problem cannot be solved, contact Associates of Cape Cod, Inc. to arrange for
repair of the instrument.

Pyros Kinetix Flex

 A well switch may not be operating correctly. Contact Associates of Cape Cod, Inc.
to arrange for repair of the instrument.

9.1.6 Tubes Inserted; LEDs Not On

LEDs on top of the tube reader do not turn on when tubes are inserted.

Pyros Kinetix
 If the tube reader does not detect one or more tubes as they are inserted, try
rotating the problem tube while it is still inserted. An improperly formed tube,
which has a slight dimple at its base, may cause the tube to go undetected.
 If the problem is not caused by malformed tubes, and is position specific, the
reference voltage may need adjustment.
 If problem cannot be solved, contact Associates of Cape Cod, Inc. to arrange for
repair of the instrument.
Pyros Kinetix Flex
 A well switch may not be operating correctly. Contact Associates of Cape Cod, Inc.
to arrange for repair of the instrument.

9.1.7 Tubes Loose/Tight

Tubes are too loose, too tight or cannot be inserted in wells.
 Inspect the well in question for debris.
 If the tube fits part way into the well, but will not go all the way down, the tube
may be deformed or incorrectly sized. The wells of Pyros Kinetix and Pyros Kinetix
Flex instruments are designed for 8x75 mm round-bottomed Borosilicate tubes
(TK100 in the ACC catalog).

9.1.8 Number Ring Lifted (Pyros Kinetix Only)

The Number Ring of the Pyros Kinetix has lifted from the Delrin block.
As part of CE compliance on the Pyros Kinetix, four springs were inserted below the
retention pins for the aluminum well number ring. The springs were inserted as a
grounding bridge between the number ring and the lower half of the instrument
chassis. Occasionally, lifting of the number ring may be experienced on instruments
that are not kept powered up at all times. This is due to the contraction of the Delrin as
the powered off instrument cools to room temperature. It is recommended that the
instruments be left powered up at all times.

9.1.9 Inconsistent Results or Poor Replication

 Check that the outside surfaces of the reaction tubes and the vortex mixer used to
mix the samples are clean and dry.
 Check that tubes sit at the bottom of the well after insertion. Good technique is
important for good replication.
 Avoid excessive vortexing of the reaction tube prior to insert, as foaming may cause
inconsistent results.

 Avoid touching or bumping a previously inserted tube, as tube movement

subsequent to insertion may contribute to poor replication.

9.1.10 A/D values out of range

A/D Values are out of range and either ‘A/D Hi’ or ‘A/D Lo’ scenarios are observed.
If an A/D out of range error occurs, the time, in seconds, and whether the value is too
low (L) or high (H) is recorded. In order to be flagged as an out of range A/D value, the
count must be either below 800 or above 2520 in a Pyros Kinetix instrument, or below
800 or above 3500 in Pyros Kinetix Flex instruments. When a problem occurs with the
A/D values, it is normally specific to one or several wells. This is not fatal to the run,
and may not be fatal to data collection for the offending well(s). During the zeroing
period (70 to 120 seconds for each well), if the mean A/D value is outside the
acceptable range, a Low or High A/D message will be received. Possible causes
include:
1. Sample too turbid.
2. Meniscus or other foreign object blocking light path (user may have forgotten to
add sample or LAL reagent).
3. Reaction tube is scratched or dirty.
4. Amplifier gain in need of adjustment.
If the sample is too turbid, repeat the test (not to exceed MVD) at a higher dilution.
Causes 2 and 3 are corrected by using remaining wells for the sample at that dilution,
or repeating the testing of the sample in question at the problematic dilution. For
cause 4 the tube reader must be calibrated.

9.1.11 Temperature Out of Range

The Temperature Out of Range error is displayed in the Test Errors Report (Section
7.4.2) if the unit temperature falls outside of the acceptable range that was specified in
Default Settings. The time, in seconds and the nature of the error (e.g. the temperature
is too low (L) or too high (H)) are recorded. For Pyros Kinetix Flex instruments with
more than one heating unit, each heating unit will be listed on the report. Those in
specification will exhibit an N for normal temperature.
Use View  Temperatures (Section 7.4.5) to see the temperature values that were
recorded. If a temperature out of range error is noted in the Test Errors Report, a
complete temperature calibration should be performed. See the calibration
documentation provided with the instrument for more details.

9.1.12 Bad Data Blocks

A Bad Data Block error message is recorded (time in seconds) if a data block arrives
with an incorrect format. If the data block format does not follow the requirements of
the software, this message will appear. The requirements are as follows:
1. A $ must be one of the first 3 characters received to denote the start of a data
block.
2. No more than 4 characters between the : delimiters.
3. 0-9 are the only acceptable characters.
4. Number of data points per block verified by counting number of colons (:).

If one or more of these conditions are not met, the data block is considered to be
corrupt, and is not used in calculations. This is usually caused by a communication
problem of some type. Bad data blocks may be caused by one of the following:
1. Faulty or poorly connected serial cable between the tube reader and the computer
- check connections, replace cable if necessary.
2. Faulty serial communications board in system computer - try alternate computer.
3. Failing serial board inside the tube reader - make arrangements to return
instrument to Associates of Cape Cod, Inc. for repair.
Note: Occasional Bad or Missing data blocks do not necessarily invalidate the test. The
decision to regard the test as invalid must be made by the individuals
responsible for test results in accordance with the customer’s company policy.

9.1.13 Missing Data Blocks

When the Missing Data Block message occurs, the message field indicates the time (in
seconds) that the system computer did not receive the block of data that was
expected. A missing data block occurs when the computer does not receive a data
block at the time it is expected. Data blocks should be transmitted every 10 seconds,
and the computer checks that a data block is received every ten seconds. A lost data
block may occur if the tube reader becomes disconnected from the computer.
Another cause is a discrepancy between the clock in the instrument and the clock in
the computer. This type of synchronization issue will likely become apparent on assays
left open for long periods of time (4 hours or more). The clocks will be resynchronized
when the next data block is received.
Note: Occasional Bad or Missing data blocks do not necessarily invalidate the test. The
decision to regard the test as invalid must be made by the individuals
responsible for test results in accordance with the customer's company policy.

9.1.14 Interpretation of Data Blocks

Pyros Kinetix and Pyros Kinetix Flex instruments communicate with the computer by
sending packets of information that are referred to as “data blocks”. Each data block
consists of twelve rows and a status header line. In a properly formatted data block
each row will begin with a $, end with a # and be colon delimited. Each of the
characters between the colons should be a numerical value between 0 and 9.
Header Information:
The header line contains sixteen values, but the Pyros EQS software only interprets
the first 9. A sample status line might look like this:
$1:1:1:1:370:1:370:1:370:0:0:0:0:0:0:0:#
The line can be interpreted as follows:
1. The first three values indicate the number of units present. 1:1:1 indicates the
presence of a Pyros Kinetix.
2. The next number indicates temperature status for the first heating unit. A 0
indicates that the unit is below temperature, a 1 indicates that the unit is within the
acceptable range of temperature, and a 2 indicates that the unit is above the
acceptable range of temperature.

3. The fifth value in the header is the temperature (°C) for the block but without the
decimal place. Thus, 370 indicates 37.0°C.
4. In the case of the Pyros Kinetix, the temperature status and reading are repeated a
second and third time. The remaining seven values in the header line are ignored
by the software.
Data Block Body:
The body of the data block contains twelve rows, each containing eight values. Each of
the eight values is the Analog to Digital (A/D) value for a well within one of the
instrument(s) present. A/D value will not exceed 4 characters in length. The first
position in the first row equates to the first well in the Pyros Kinetix. The rows
correspond to wells in the Pyros Kinetix instrument as follows:
Row 1 Wells 1 – 8
Row 2 Wells 9 – 16
Row 3 Wells 17 – 24
Row 4 Wells 25 – 32
Row 5 Wells 33 – 40
Row 6 Wells 41 – 48
Row 7 Wells 49 – 56
Row 8 Wells 57 – 64
Row 9 Wells 65 – 72
Row 10 Wells 73 – 80
Row 11 Wells 81 – 88
Row 12 Wells 89 – 96
Non-zero A/D values indicate that tube detection has been triggered for the well, and
that light is reaching the photo-detector. At the start of the assay, it is expected that
the A/D value for a well containing a tube will be between 800 and 2520 counts if
using a Pyros Kinetix instrument and 800-3500 for Pyros Kinetix Flex instruments. For
positions where no tube is inserted, the value should be 0. The letters EOB for "end of
block" signifies the end of a data block.

9.2 Pyros EQS

9.2.1 Signed-off Assay Requires Editing

Administrator Override: Sign-off Override. Users with Supervisor access rights have the
ability to sign-off on a test. This results in their electronic signature being applied to
reports along with the date and time. It also renders the test ID unchangeable. In the
event that the sign-off status needs to be removed, an Administrator level user may
override the sign-off. To do this, select Admin (from menu)  Administrative Override
 Sign-Off Override and then select appropriate test ID.
See Section 5.4.6.1 for more information.

9.2.2 Assay Will Not Close Using the Terminate Test Icon
Administrator Override: Test Complete Override. If a test will not close by clicking on
the “Terminate Test” icon within 60 seconds, the test can be closed by using the Test

Complete Override function, to close the test, an Administrator must force the Test to
be marked as complete. To do this, click on the “Continue Test” icon, the user will be
returned to the “Main Application” screen. If the user has Administrator privileges,
select Admin (from menu)  Administrative Override  Test Complete Override
select the appropriate Test ID, and click “OK”. If the user does not have Administrator
privileges, the user must log out of Pyros EQS, a user with Administrator privileges
must log in, and proceed as above. Note that the test will be closed in a signed off
state, if additional editing is required, the signoff override function must be used to
allow the test to be edited.
See Section 5.4.6.2 for more information.

9.2.3 Assay Closed Abnormally and Not Visible in Data Analysis

Administrator Override: Assay Unlock. Pyros EQS is a multi-user system. To avoid
conflicts, the software prevents simultaneous access to a test by multiple users. The
software locks a test once it is opened by a user either for Data Collection or Analysis.
The test is unlocked once the user closes the test. In the adverse event of the client
computer failing (power failure, operating system failure, etc.), or an abnormal test
closure, the test may remain in the locked state. An Administrator would then need to
unlock the test before it can be accessed by any other user. To do this, choose Admin
 Administrative Override  Unlock Assay from the “Main Application” screen. From
the drop-down list, select the appropriate Test ID that needs to be unlocked and click
“OK”.
See Section 5.4.6.3 for more information.

9.2.4 Service Packs

Pyros EQS uses the Oracle Relational Database for data storage. To ensure proper
functioning of Pyros EQS, it is important that the computer have the appropriate
Operating System, Service Packs and Oracle Client software. This information is
provided in Section 3 of this User Manual. Prior to installing any new Service Packs or
updates from Microsoft, please verify with Associates of Cape Cod, Inc. that it is
appropriate to do so. Also review the Pre-Install Checklist in Appendix B for additional
information.

9.2.5 Login Failure

Pyros EQS relies on Windows to authenticate user names and passwords. If a user
experiences the “Invalid User Name/Password” message multiple times, when trying
to login to the software, the User ID may become locked out of the Windows account,
depending upon the End User Corporate Policy on Passwords and Network Access
rights. The End User’s Systems Administrator may have a security policy in place to
lock accounts after a certain number of unsuccessful attempts. In such case, please
contact the IT department to get the Windows account unlocked.
Note: This does not apply to user ID ‘admin’ which is a special Pyros EQS user and
not a Windows user.

9.2.6 Enabling/Disabling Audit Trail

Pyros EQS has multiple background processes running on the computer system.
Enabling/Disabling the Audit trail is an important event that affects the components on
the workstation (if a Standalone Version has been installed) or the components on all
the client workstations (if a Network Version has been installed). All workstations
should be restarted after either enabling or disabling the Audit Trail functionality.

9.2.7 Keyboard Curve

Pyros EQS allows the user to define a Keyboard curve on the “Standard Curve for
Analysis” screen. Users can perform analysis based on the selected curve by navigating
to the Analysis Tools tab. Navigating to this tab may produce an error message “Error in
parameters for curve chosen”. This error occurs when a user has defined a Keyboard
curve but not clicked on the Use this Curve button to indicate to the software that this
curve should be used for analysis. Click on the “Use this Curve” button to avoid this
error.
See Section 5.3.2 and Section 7.2 for more information.

9.2.8 Report ID Generation

Pyros EQS allows users to track changes made to a test through the Report ID. The
Report ID is printed on each report with the label Report ID. A new Report ID is
generated every time the user opens a screen that affects test parameters and selects
“OK”. For example, opening the “Test Information” screen and clicking “OK” tells the
software that changes have been made on the screen and the software accordingly
generates a new Report ID. If no changes have been made on any screen, it is highly
recommended that the user press the “Cancel” button to close the screen and not the
“OK” button. Clicking the “OK” button saves changes to the database and generates a
new Report ID.
See Section 7.5.1 for more information.

9.2.9 Multiple Plot Printing

The “Multiple Plot” screen allows the user to print plots. The Print button only
generates a Print Preview. Since Pyros EQS only generates the preview and allows
printing of a single screen (page) of multiple plots, clicking the buttons at the top to
display more than one page on a single screen have no effect. To print all pages,
navigate to each page and click “Print” on each page.
See Section 7.3.1 for more information.

9.2.10 Creating First User

Pyros EQS allows the Administrator to create users through the “User and Data Group
Management” screen. On clicking the “Add New User” button, the user may encounter
a warning message “User First Name is Mandatory and it cannot be empty.” This is a
benign message and appears only when the first user is created. The message can be
ignored by clicking “OK” and continuing to create the new user.

9.2.11 System Tray Icon is Red

The System Tray icon is red in the following situations:
 ACCI Pyros EQS service is not running. Please start the service by clicking Start 
Control Panel  Administrative Tools  Services. If a user does not have the
privileges to access Administrative Tools, they may have to request the IT
Administrator do this for them.
 Data collection is in progress and missing or bad raw data blocks are received by
the software.
For additional information about the behavior of the Pyros EQS System Tray Icon,
please see Section 5.6.

9.2.12 Oracle Errors

Pyros EQS reports all database/network error(s) with any of the following titles:
 Database connectivity error
 Database error
Please make sure that the database server is running and that the workstation is
connected to the network and has access to network resources, including being able
to connect to the database server.
A few common Oracle error messages that might be encountered are:
 ORA-03114: Not connected to Oracle
 ORA-03113: End-of-file on communication channel
 ORA-12545: Connect failed because target host or object does not exist
 ORA-12150: TNS: unable to send data
 ORA-12152: TNS: unable to send break message
 ORA-12153: TNS: not connected
 ORA-12154: TNS: could not resolve the connect identifier specified
 ORA-12157: TNS: internal network communication error
 ORA-12514: TNS: listener does not currently know of service requested in connect
descriptor
All the above errors are primarily a result of connection with the database server
having been lost. Please contact the End User’s Systems Administrator to have the
connection restored.
It is important to note that if database connectivity is lost during Data Collection, the
raw data will not be lost. The raw data will be stored locally, until the database
connection is restored.

9.2.13 Test Started but Collect Data Screen Remains Blank

The software may not have refreshed the on-screen display of data. Click Close 
Continue to close the “Collect Data” main screen. From the “Main Application” screen,
click File  Exit to exit the Pyros EQS software. Launch the software again and login.
The software should indicate that Data Collection for the test is in progress. Click “OK”
on the dialog box to go to the “Collect Data” main screen. Wait for up to 10 seconds to

see if the screen is refreshed and displays data for the ongoing test. If not, this may be
an indication of no tubes in the instrument. This can be verified by:
 Making sure that there are raw data file(s) in the \Pyros EQS \Data\Capture folder.
 Open any raw data file (in Notepad or Wordpad) and verify the A/D values. The
null blocks demonstrate that no inserted tube has been detected by the software.

9.2.14 Test ID Does Not Appear in the Selection List

The Test ID does not appear in the selection list in following situations:
 Data collection for the Test is running in background.
 Data collection for the Test is in progress on some other Pyros EQS client.
 Test is open on some other Pyros EQS workstation for analysis.
 Test is locked in the database – Please refer to Sections 9.2.2 and 9.2.3.

9.2.15 Axes Origin for Plots

Plots in Pyros EQS can be re-scaled by clicking the “Re-scale” button. However, if new
origin (from) value is not in the multiples of the current plot series, this origin is
omitted from the plot. The plot re-scales correctly but the origin point label is not
visible. To make the origins visible, please specify the origin values in multiples of the
axes intervals. For example, if the time displayed on the X-axis has intervals of 1,000
seconds (0, 1000, 2000, 3000 and so on), specify a re-scale value in multiples of 1,000
and the origin will be properly displayed. An interim value specified as origin (say, 500,
or 678) will not be displayed on the plot itself though the plot will take that point as the
origin for the purposes of plotting.

9.2.16 Field Validation Errors – Red Exclamation

Pyros EQS indicates a field input error with a red exclamation (!) mark next to it. The
error message can be displayed by moving the cursor over the exclamation mark. Once
the user clicks on the exclamation mark, the software interprets the action as the error
message having been read and the error is no longer displayed. Correct the error by
changing the field value, or, move the cursor to another field and back again, in order
to re-read the error message.

9.2.17 Spaces in Field Values

Pyros EQS does not trim spaces (between or at the end) before it accepts a user-
entered value. For example, if the user enters “Domain1 \user1” as the user id when
creating a new user, instead of entering it as “Domain1\user1”, this could cause the
Windows authentication to fail. Administrators are requested to take note and enter
exact field values without extra spaces between or at the ends.

9.2.18 License Registration – Serial Number Entry

The “License Registration” screen allows the customer to register the license. The
Instrument Serial Number is required to complete the registration process. The Pyros
Kinetix serial number is of the format NA-NNNN where N represents a digit from 0 to 9
and A represents a letter from A to Z. In order to register Pyros Kinetix Flex

instruments, provide the last six digits of the serial number. Please note that in position
A, the software only accepts capital letters.

9.2.19 Data Group Creation

Pyros EQS allows new Data Group creation on the “User and Data Group
Management” screen. If no users exist (other than the default Admin user), new Data
Group creation fails with the message “There are no changes to save.” This is a known
limitation of this screen. It is recommended that at least one user is created. Save
changes by pressing “Save Changes” and then proceed to creating Data Groups.

9.2.20 Series is “Broken” After Editing

When wells are not edited as a complete series, a dialog box containing a “Series is
broken. Series level parameters cannot be modified” message may appear. In order to
proceed to make well edits, the wells must be re-edited in a manner that restores the
integrity of the series. The following procedure will accomplish this function.

1. Highlight the series, well, or wells to be edited.

2. Right click on the mouse and select “cut”.
3. Cut the selected wells.
4. Click on the well or wells to be edited and select “edit” from the toolbar.
5. Define the selected wells as negatives, standards, or samples.
6. Enter the descriptor information. Upon getting the following message: “Duplicate
well descriptor found. Do you want to load values from original descriptor,” click
“Yes”.
7. Enter descriptor information such as description, replicates unspiked, replicates
spiked, spike concentration, number of dilutions, serial dilution factor, and initial
dilution.
8. Click “OK”. If “Duplicate well descriptor exists. This will merge current series with
original. Do you want to proceed with merging?” message appears click “Yes”.

9.2.21 Pyros EQS Unable to Generate an Individual Wells Results Report

Occasionally a report will be unavailable for viewing or printing. If the software is
unable to generate or printout an Individual Well Results Report, usually accompanied
by the error message “Error in File C;\DOCUME 1\lab\LOCALS 1\Temp\{GUID #}.rpt:
The request could not be submitted for background processing.” The report must be
deleted from the computers temporary file storage folder, and generated anew to be
viewed and/or printed.

1. On the computer toolbar select “Start”.

2. Select “Run”.
3. Type in “%temp%” this will open the temp files folder.
4. Select the file using the GUID # from the error message.
5. Right click and delete the file.
6. Close the temp file folder.
7. Log into Pyros EQS, select “Analyze Data”, select the assay file, select “Individual
Wells Results”.

9.2.22 Backup and Restore

Please see Oracle’s website for Backup and Restore information and to determine
which method is appropriate for your needs.

Appendix A: Program Calculations

A.1 Calculation of Optical Density from Numerical Data
The digital (A/D) values representing light received by the photodetector for each tube location
are recorded and averaged during an initial zeroing period (default is 60-120 seconds). These
values are the reference values for all subsequent calculations and should be in the range 800-
2520 for a Pyros Kinetix instrument and 800-3500 for Pyros Kinetix Flex instruments. They can
be viewed by selecting View  Raw Data in Data Analysis. The mean of the 7 digital values is
taken to represent 100% transmission (transmittance = 1). All subsequent readings are convert-
ed to transmittance and then to optical density before storage in the data file. The relationship
between transmittance (T) and optical density (OD; in absorbance units) is:
OD = -log10T
Example: If the mean digital value, determined from the zeroing period, is 1937, this is taken to
be a transmittance of 1. Thus, the initial OD = -log10T= 0. A subsequent digital value of 1850
represents a transmittance of 1850/1937 = 0.9550 and OD = -log10 0.9550 = 0.02 absorbance
units (or 20 milliabsorbance units).
A.2 Baseline Adjustment
Baseline adjustment is performed to ensure that the average OD for each sample is zero, prior
to any increase in optical density resulting from the LAL-endotoxin reaction. The adjustment is
calculated independently for the data from each tube location. Baseline adjustment is
performed in Data Analysis and does not change the raw OD values that are stored in the data
file. By contrast, the initial zeroing (see A.1 above) is performed in Data Collection before any
OD values are stored in the file.
Raw or baseline adjusted OD values can be plotted (see Section 7.3.1, Data Plots).
To perform baseline adjustment on the data for a given tube location, the program calculates
the mean of the stored OD values collected during the specified time period (default is 300-500
seconds). This value is then subtracted from every OD value collected during the test. Baseline
adjustment ensures that the mean stored OD is zeroed during the adjustment period and that
the OD plots of all tubes start from the same point (0).
A.3 Linear Regression
Linear regression analysis is used at two points in the Data Analysis program for the Pyros
Kinetix tube reader: (1) calculation of the adjusted onset time, and (2) calculation of line
equations for standard curves.
a. Onset Time
The onset time is the time at which the OD value reaches the preset OD threshold. It is
displayed on the “Data Collection” screen and can be viewed and printed in Data Analysis.
The onset time is estimated using the three OD readings taken before and the three
readings taken after the threshold is reached. The slope and y intercept for the six points
are calculated by regression analysis. The adjusted onset time (estimate of X) is then
determined by substituting the threshold OD for Y in the equation for a straight line. The
regression interpolates between data points as shown below. This calculation is performed
for each sample, after baseline adjustment.

Determination of Onset Time

b. Standard Curve
The standard curve for a kinetic assay is calculated by performing a regression of the log10 of
the corrected onset time against the log10 of the endotoxin concentration. The slope, the Y
intercept and the range of the standard endotoxin concentrations define the standard
curve. The endotoxin concentrations of unknowns (antilogs of estimates of X) are
calculated by substituting the log10 of the onset time for Y in the standard line equation. The
antilog of the resulting value gives the endotoxin concentration of the unknown in the
same units used for the standard curve (see equations below).
Note: Standard curve parameters are only valid over the range of standard endotoxin
concentrations used to construct the curve. It is strongly recommended that
extrapolated values not be used in the release of product.
The standard curve for an endpoint assay is a simple linear regression and does not require
a log-log calculation adjustment.

c. Equations and their Application

1. Equation for a Straight Line

Y = a + bX
Where:
Y= the value of the dependent variable for a given value of the independent variable
(X).
a= the y intercept.
b= the slope of the line (this value will always be negative in kinetic LAL tests).
X= the value of the independent variable (X).

2. Linear Regression Equation

Where X and Y are the means of X and Y values respectively

∑ ̅∑
( )
∑ ̅∑

( ) ̅ ̅

3. Correlation Analysis Calculation of Endotoxin Concentrations - Kinetic Reactions

∑ ̅∑
√(∑ ̅ ∑ ) (∑ ̅∑ )

To calculate the endotoxin concentration of unknown samples from the adjusted time,
the equation for a straight line is used in the following form:
Where:

X= log10 endotoxin concentration. It is expressed in the same units as the standards

used to generate the standard curve described by a and b (the y intercept and
slope respectively).
Y= log10 of the adjusted onset time in seconds.
a= y intercept of the standard curve calculated by linear regression analysis. b= slope
of the standard curve calculated by linear regression analysis.

Thus: Endotoxin concentration = antilog10X

Appendix B: Pre-Install Checklist

Database Configuration
Operating System:
Supported operating system 
Power-saving (Hibernate, Standby, Disk spin-down, Screensaver) modes disabled 
Hardware:
Supported processor architecture 
Meets or exceeds memory requirements 
Meets or exceeds disk space requirements 
For Remote Databases:
Network connected with a static IP address 
Firewall / Antivirus appropriately configured 
Other:
CPU, Memory, Disk I/O intensive applications evaluated, if present 
Installation to be performed using an account with admistrative privileges 

Application Configuration
Operating System:
Supported operating system 
Power-saving (Hibernate, Standby, Disk spin-down, Screensaver) modes disabled 
Hardware:
Supported processor architecture 
Meets or exceeds memory requirements 
Meets or exceeds disk space requirements 
For Remote Databases:
Connected to network 
Other:
Previous Pyros EQS Application versions removed 
Pyros EQS Database pre-installed 
Firewall / Antivirus appropriately configured 
1 or more Serial or USB ports available 
CPU, Memory, Disk I/O intensive applications evaluated, if present 
Installation to be performed using an account with Admistrative privileges 
Notes:

B. 3 Pyros EQS Registration Checklist

End-user Information
Company Name: ____________________________ 
Address: ____________________________ 
City: ____________________________ 
State/Province: ____________________________ 
Zip/Postal Code: ____________________________ 
Country: ____________________________ 
Contact First Name: ____________________________ 
Contact Last Name: ____________________________ 
Contact Email: ________________________________________________ 
Contact Phone/Fax: _________________________________________________ 

Purchase Information
Pyros EQS Version: ____________________________ 
Order Date: ____________________________ 
PO Number*: ____________________________ 
Catalog Number: ____________________________ 

Equipment Information
Instrument Type: ____________________________ 
Serial Number: ____________________________ 
If the non-compliant application was purchased, a single registration code will be visible in the License
Registration dialog box (see Section 3.2.2.5). If the 21 CFR Part 11 compliant version was purchased,
make sure that the Compliance License Purchased checkbox is checked, and two registration numbers
should appear:

Registration Code: _

Once this form is completed, please send the information to the ACC Customer Service department:
Email: [email protected] / Fax: (508) 540-8680/Tel: (888) LAL-TEST (525-8378) or (508) 540-3444
Note: Do NOT close the License Registration dialog box and/or exit the application until the
appropriate authorization codes have been received and entered. The registration codes are
randomly generated and will change if the dialog box is closed and reopened, thus rendering
the authorization codes provided by ACC for the prior registration code(s) nonfunctional.
*If this application was purchased through an ACC licensed distributor, please include the distributor
name.

Thank you for choosing Associates of Cape Cod, Inc. for your LAL testing needs. We pride
ourselves on providing the best quality reagents and the highest level of support for all of our
products. If you have any questions, comments, or would like to participate in a customer
survey, please contact our Customer Service department at (800) LAL-TEST (525-8378) or (508)
540-3444.

We regularly publish LAL Updates, newsletters designed to address current questions and
concerns confronting members of the LAL community. You can subscribe, free of charge, to
future editions of the newsletter online at http://www.acciusa.com/lal/newsletter.html, where
an archive of past articles is also available.

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