Qiagen PyroMark Q24 MDx User manual

Version 1 January 2016
Sample & Assay Technologies
PyroMark®Q24 MDx User Manual
IVD
MAT
1063078EN
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, GERMANY
R4

Trademarks: QIAGEN®, DNeasy®, EpiTect®, PyroMark®, Pyrosequencing®, Pyrogram®, QIAamp®, QIAprep®, Q-Solution®, RNeasy®(QIAGEN
Group); Adobe®, Reader®(Adobe Systems Incorporated); Intel®, Pentium®(Intel Corporation); Microsoft®, Windows®(Microsoft Corporation); Millex®,
Millipore®, Milli-Q®(Merck KGaA); Sepharose®(GE Healthcare). Registered names, trademarks, etc. used in this document, even when not
specifically marked as such, are not to be considered unprotected by law.
HB-0332-004 © 2011–2016 QIAGEN, all rights reserved.

Contents
PyroMark Q24 MDx User Manual 01/2016 Contents-1
Contents
1Safety Information 1-1
1.1 Proper use 1-1
1.2 Electrical safety 1-2
1.3 Biological safety 1-3
1.4 Chemicals 1-4
1.5 Mechanical hazards 1-4
1.6 Heat hazard 1-5
1.7 Consumables 1-5
1.8 Symbols on the PyroMark Q24 MDx and
PyroMark Q24 products 1-6
2Introduction 2-1
2.1 About this user manual 2-1
2.2 General information 2-2
2.2.1 Technical assistance 2-2
2.2.2 Policy statement 2-2
2.2.3 Version management 2-2
2.3 Intended use of PyroMark Q24 MDx 2-3
2.3.1 Requirements for PyroMark Q24 MDx users 2-3
3General Desc ription 3-1
3.1 PyroMark Q24 MDx definitions 3-1
3.2 Pyrosequencing principle 3-1
3.3 PyroMark Q24 MDx principle 3-3
3.4 PyroMark Q24 MDx Instrument 3-4
3.4.1 Process chamber 3-5
3.4.2 Dispensing unit 3-5

Contents
Contents-2 PyroMark Q24 MDx User Manual 01/2016
3.5 Sample preparation with the PyroMark Q24 MDx
Vacuum Workstation 3-6
3.6 Analysi s sof tware 3-6
4Installation Procedures 4-1
4.1 System delivery and installation 4-1
4.2 Requirements 4-1
4.3 Installation of the analysis software 4-3
4.3.1 Installing or upgrading PyroMark Q24 MDx
Software 4-3
4.3.2 Uninstalling PyroMark Q24 MDx Software 4-4
5Operating Procedures 5-1
5.1 Instrument administration 5-1
5.1.1 Setting date and time 5-1
5.1.2 Copying unsaved runs 5-1
5.1.3 Copying recently saved runs 5-2
5.1.4 Copying log files 5-2
5.1.5 Extracting damaged runs 5-3
5.1.6 Viewing acknowledgements, version, and contact
information 5-3
5.1.7 Upgrading the instrument software 5-3
5.1.8 Run an external application 5-4
5.2 Setting up a run 5-4
5.2.1 Starting PyroMark Q24 MDx Software 5-4
5.2.2 Setting up an assay 5-4
5.2.3 Setting up a run 5-5
5.2.4 Managing instrument methods 5-7
5.3 Sample prepar ation 5-8
5.3.1 PyroMark Q24 MDx Vacuum Workstation function
test 5-9
5.3.2 DNA amplification 5-10
5.3.3 Immobilizing the PCR product to beads 5-10

Contents
PyroMark Q24 MDx User Manual 01/2016 Contents-3
5.3.4 Separation of DNA strands and release of
samples into the PyroMark Q24 Plate 5-11
5.3.5 Annealing of sequencing primer to samples 5-16
5.4 Preparation of PyroMark Gold Q24 Reagents 5-16
5.5 Processing a run on the PyroMark Q24 MDx
Instrument 5-17
5.5.1 Instrument software 5-18
5.5.2 Starting the instrument 5-18
5.5.3 Starting the run 5-19
5.5.4 Monitoring the run 5-20
5.5.5 After the run 5-21
5.5.6 Analyzing the run 5-24
5.5.7 Viewing the analysis results 5-25
5.5.8 Analysis reports 5-27
5.6 Finishing work and shutting down 5-28
5.6.1 Shutting down the instrument 5-28
5.6.2 Emptying the waste container and troughs 5-29
5.6.3 Checking the instrument 5-30
5.7 Backup of PyroMark Q24 files 5-30
6Maintenance 6-1
6.1 Checking the performance of the PyroMark Q24 MDx 6-1
6.2 Maintenance of the PyroMark Q24 MDx Instrument 6-2
6.2.1 Cleaning the instrument 6-2
6.2.2 Cleaning the heating block and light guides 6-3
6.3 Maintenance of the PyroMark Q24 MDx Vacuum
Workstation 6-5
6.3.1 Cleaning the PyroMark Q24 MDx Vacuum
Workstation 6-5
6.3.2 Testing and replacing the filter probes 6-6
6.3.3 Replacing the rubber seal 6-7
6.3.4 Replacing the tubing 6-8
6.3.5 Replacing the waste filter 6-9

Contents
Contents-4 PyroMark Q24 MDx User Manual 01/2016
7Troubleshooting 7-1
7.1 Analysis-related errors 7-2
7.2 Analysi s sof tware-related errors 7-4
7.3 Instrument-related errors 7-5
7.4 Vacuum workstation-related errors 7-6
7.5 Verification of correct installation and operation 7-7
8Glossary 8-1
Appendix A A-1
Technical data A-1
Environmental conditions A-1
Waste Electrical and Electronic Equipment (WEEE) A-5
FCC declaration A-6
EC declaration of conformity A-7
Appendix B B-8
Assay design and validation B-8
Appendix C C-1
Liability clause C-1
Index Index-1

Safety Information
PyroMark Q24 MDx User Manual 01/2016 1-1
1Safety Information
Before using the PyroMark Q24 MDx, it is essential that you
read this user manual carefully and pay particular attention
to the safety information. The instructions and safety
information in the user manual must be followed to ensure
safe operation of the system and to maintain the system in a
safe condition.
The following types of safety information appear throughout
this manual.
WARNING
The term WARNING is used to inform you about situations
that could result in personal injury to you or other
persons.
Details about these circumstances are given in a box like
this one.
CAUTION
The term CAUTION is used to inform you about situations
that could result in damage to the system or other
equipment.
Details about these circumstances are given in a box like
this one.
The advice given in this manual is intended to supplement,
not supersede, the normal safety requirements prevailing in
the user’s country.
1.1 Proper use
WARNING
Risk of personal injury and material damage
Improper use of the PyroMark Q24 MDx may cause
personal injuries or damage to the system.
PyroMark Q24 MDx must only be operated by qualified
personnel who have been appropriately trained.
Servicing of the PyroMark Q24 MDx must only be
performed by QIAGEN Field Service Specialists.
Perform the maintenance as described in Section 6.
QIAGEN®charges for repairs that are required due to
incorrect maintenance.

Safety Information
1-2 PyroMark Q24 MDx User Manual 01/2016
WARNING
Risk of personal injury and material damage
The PyroMark Q24 MDx Instrument is too heavy to be
lifted by one person. To avoid personal injury or damage
to the instrument, do not lift the instrument alone.
WARNING
Risk of personal injury and material damage
Do not attempt to move the PyroMark Q24 MDx during
operation.
1.2 Electrical safety
Disconnect the line power cords from the power outlets
before servicing.
WARNING
Electrical hazard
Any interruption of the protective conductor (earth/ground
lead) inside or outside the instrument or disconnection of
the protective conductor terminal is likely to make the
instrument dangerous.
Intentional interruption is prohibited.
Lethal voltages inside the equipment
When the equipment is connected to line power, terminals
may be live, and opening covers or removing parts is likely
to expose live parts.
To ensure satisfactory and safe operation of the PyroMark
Q24 MDx, follow the advice below:
The instrument’s line power cords must be connected to
line power outlets that have protective conductors
(earth/ground).
Keep mains plugs easily accessible in case the
equipment needs to be disconnected quickly from mains
power.
Use only power supplies and cords supplied with the
system.

Safety Information
PyroMark Q24 MDx User Manual 01/2016 1-3
1.3 Biological safety
When handling biological material, use safe laboratory
procedures as outlined in publications such as Biosafety in
Microbiological and Biomedical Laboratories, HHS
(www.cdc.gov/od/ohs/biosfty/biosfty.htm).
WARNING
Biological materials
Handle biological material with the greatest of care and in
accordance with the required safety regulations. Always
wear safety glasses, 2 pairs of gloves, and a lab coat.
The responsible body (e.g., laboratory manager) must take
the necessary precautions to ensure that the surrounding
workplace is safe, and that the operators are suitably
trained and not exposed to hazardous levels of infectious
agents as defined in the applicable Material Safety Data
Sheets (MSDSs) or OSHA,* ACGIH,†or COSHH‡
documents.
For more information, visit
www.qiagen.com/support/msds. aspx.
Venting for fumes and disposal of wastes must be in
accordance with all national, state, and local health and
safety regulations and laws.
* OSHA: Occupational Safety and Health Administration (United States of America).
† ACGIH: American Conference of Government Industrial Hygienists (United States of America).
‡ COSHH: Control of Substances Hazardous to Health (United Kingdom).

Safety Information
1-4 PyroMark Q24 MDx User Manual 01/2016
1.4 Chemicals
WARNING
Hazardous chemicals
The Denaturation Solution used with the vacuum
workstation contains sodium hydroxide, which is irritating
to eyes and skin.
Always wear safety glasses, gloves, and a lab coat.
The responsible body (e.g., laboratory manager) must take
the necessary precautions to ensure that the surrounding
workplace is safe and that the operators are not exposed
to hazardous levels of toxic substances (chemical or
biological) as defined in the applicable Material Safety
Data Sheets (MSDSs) or OSHA,* ACGIH,†or COSHH‡
documents.
For more information, visit
www.qiagen.com/support/msds. aspx.
Venting for fumes and disposal of wastes must be in
accordance with all national, state, and local health and
safety regulations and laws.
* OSHA: Occupational Safety and Health Administration (United States of America).
†ACGIH: American Conference of Government Industrial Hygienists (United States of America).
‡COSHH: Control of Substances Hazardous to Health (United Kingdom).
1.5 Mechanical hazards
The lid of the PyroMark Q24 MDx Instrument must remain
closed during operation of the instrument. An audible
warning signal will alert you if the lid is opened when it is not
safe.
WARNING
Moving parts
To avoid contact with moving parts during operation of the
PyroMark Q24 MDx Instrument, the instrument must be
operated with the lid closed.
Do not remove the cover panels since there are no user-
serviceable parts inside. If there is a problem with the
PyroMark Q24 MDx Instrument, contact QIAGEN
Technical Services immediately.

Safety Information
PyroMark Q24 MDx User Manual 01/2016 1-5
WARNING
Sharp needles
Do not touch the sharp needles at the bottom of the
reagent cartridge. Handle the needles with care. Small
particles and fibers may obstruct the needles.
CAUTION
Light guide maintenance
Use lint free tissues to clean the space between the heating
block and the light guide block inside the instrument. Do
not use paper tissues.
1.6 Heat hazard
WARNING
Hot surface
The plate holder and the heating block (for annealing) can
reach temperatures of up to 80°C (176°F). Avoid touching
them when they are hot.
CAUTION
Risk of overheating
To ensure proper ventilation, maintain a minimum
clearance of 10 cm (3.94 in.) at the sides and rear of the
PyroMark Q24 MDx Instrument.
Slits and openings that ensure the ventilation of the
PyroMark Q24 MDx Instrument must not be covered.
1.7 Consumables
CAUTION
Unsupported consumables
Do not connect or use any consumables, accessories, or
external equipment other than that specified.

Safety Information
1-6 PyroMark Q24 MDx User Manual 01/2016
1.8 Symbols on the PyroMark Q24 MDx and
PyroMark Q24 products
Symbol
Location
Language
Description
Type plate
on the back
of the
instrument
EN
CE mark
Type plate
on the back
of the
instrument
EN
CSA listing mark for
Canada and the USA
Type plate
on the back
of the
instrument
EN
FCC mark of the
United States Federal
Communications
Commission
Type plate
on the back
of the
instrument
EN
RCM mark for Australia
(supplier identification
N17965)
Type plate
on the back
of the
instrument
EN
RoHS mark for China
(the restriction of the
use of certain
hazardous substances
in electrical and
electronic equipment)
Type plate
on the back
of the
instrument
EN
Waste Electrical and
Electronic Equipment
(WEEE)
Type plate
on the back
of the
instrument
EN
In vitro diagnostic
medical device

Safety Information
PyroMark Q24 MDx User Manual 01/2016 1-7
Symbol
Location
Language
Description
Reagents,
solutions,
cartridge,
plate
EN
Identification of
production batch/lot
<N>
Reagents,
solutions
EN
Contains reagents for
<N> tests
Reagents,
solutions,
cartridge,
plate
EN
Catalog number
Reagents,
solutions,
cartridge,
plate
EN
Material number
Reagents,
solutions,
cartridge,
plate
EN
Component
Reagents,
solutions,
cartridge,
plate
EN
Contains
Reagents,
solutions,
cartridge,
plate
EN
Number
Reagents,
solutions,
cartridge,
plate
EN
Use-by date
Reagents,
solutions,
cartridge,
plate
EN
Temperature limitation

Safety Information
1-8 PyroMark Q24 MDx User Manual 01/2016
Symbol
Location
Language
Description
Instrument,
VPW,
reagents,
cartridge
EN
Read the manual
Type plate
on the back
of the
instrument
and all other
products
EN
Legal manufacturer
Cartridge
EN
Keep away from
(sun)light
Cartridge
EN
Method number
Cartridge
EN
Instrument parameters
Inside the
instrument
EN
Warning, consult user
manual

Introduction
PyroMark Q24 MDx User Manual 01/2016 2-1
2Introduction
Thank you for choosing the PyroMark Q24 MDx. We are
confident it will become an integral part of your laboratory.
Before using the PyroMark Q24 MDx, it is essential that you
read this user manual carefully and pay particular attention
to the safety information (see Section 1). The instructions and
safety information in the user manual must be followed to
ensure safe operation of the system and to maintain the
system in a safe condition.
2.1 About this user manual
This user manual provides information about the PyroMark
Q24 MDx in the following sections:
1. Safety Information
2. Introduction
3. General Description
4. Installation Procedures
5. Operating Procedures
6. Maintenance Procedures
7. Troubleshooting
8. Glossary
Appendices
The appendices include the following:
Technical data
Environmental conditions
WEEE recycling requirements
Assay design and validation
Warranty terms

Introduction
2-2 PyroMark Q24 MDx User Manual 01/2016
2.2 General information
2.2.1 Technical assistance
At QIAGEN, we pride ourselves on the quality and
availability of our technical support. Our Technical Service
Departments are staffed by experienced scientists with
extensive practical and theoretical expertise in sample and
assay technologies and the use of QIAGEN products. If you
have any questions or experience any difficulties regarding
the PyroMark Q24 MDx or QIAGEN products in general,
please do not hesitate to contact us.
QIAGEN customers are a major source of information
regarding advanced or specialized uses of our products. This
information is helpful to other scientists as well as to the
researchers at QIAGEN. We therefore encourage you to
contact us if you have any suggestions about product
performance or new applications and techniques.
For technical assistance and more information, please see
our Technical Support Center at www.qiagen.com/Support or
call one of the QIAGEN Technical Service Departments or
local distributors (see back cover or visit www.qiagen.com).
2.2.2 Policy statement
It is the policy of QIAGEN to improve products as new
techniques and components become available. QIAGEN
reserves the right to change specifications at any time.
In an effort to produce useful and appropriate
documentation, we appreciate your comments on this user
manual. Please contact QIAGEN Technical Services.
2.2.3 Version management
This document is the PyroMark Q24 MDx User Manual,
version 1.0, revision R4.

Introduction
PyroMark Q24 MDx User Manual 01/2016 2-3
2.3 Intended use of PyroMark Q24 MDx
PyroMark Q24 MDx is a system for detecting changes in
specified variable positions in DNA that may have clinical
relevance.
The PyroMark Q24 MDx is intended for in vitro diagnostic
use in Europe.
The PyroMark Q24 MDx Instrument is intended to be used
only in combination with QIAGEN kits indicated for use with
the PyroMark Q24 MDx Instrument for the applications
described in the kit handbooks.
The PyroMark Q24 MDx System is intended for use by
professional users, such as technicians and physicians
trained in molecular biological techniques and the operation
of the PyroMark Q24 MDx System.
2.3.1 Requirements for PyroMark Q24 MDx users
The table below covers the general level of competence and
training necessary for transportation, installation, use,
maintenance, and servicing of the PyroMark Q24 MDx.

Introduction
2-4 PyroMark Q24 MDx User Manual 01/2016
Task
Personnel
Training and
experience
Delivery No special requirements No special requirements
Installation
QIAGEN Field Service
Specialists only
Routine use
(running
protocols)
Laboratory technicians
or equivalent
Appropriately trained
and experienced
personnel familiar with
use of computers and
automation in general
Assay design
and validation
Scientist or equivalent
Appropriately trained
and experienced
personnel familiar with
molecular biological
techniques
Preventive
maintenance
Laboratory technicians
or equivalent
Appropriately trained
and experienced
personnel familiar with
use of computers and
automation in general
Servicing and
annual
preventive
maintenance
QIAGEN Field Service
Specialists only

General Description
PyroMark Q24 MDx User Manual 01/2016 3-1
3General Description
The PyroMark Q24 MDx uses proven real-time sequence-
based Pyrosequencing®technology for sequence-based
detection and quantification in genetic analysis and
epigenetic methylation studies. The system can analyze up to
24 samples simultaneously. An easy-to-use protocol is used
to prepare samples.
3.1 PyroMark Q24 MDx definitions
PyroMark Q24 MDx: Instrument, software, and
installation
PyroMark Q24 MDx Instrument: Instrument only
PyroMark Q24 MDx Software: Software only
PyroMark Q24 MDx Vacuum Workstation: Vacuum
Workstation only
PyroMark Q24 MDx System: All of the above, plus any
PyroMark Kits
3.2 Pyrosequencing principle
Pyrosequencing uses sequencing by synthesis for accurate
and quantitative analysis of DNA sequences.
1. A sequencing primer is hybridized to a single-stranded,
PCR-amplified DNA template.
2. The template is incubated with enzymes and substrates.
3. The first of four nucleotides is added to the reaction. If
the nucleotide is complementary to the base in the
template strand it will be incorporated into the DNA
strand by the DNA polymerase.
4. Each incorporation event is accompanied by release of
pyrophosphate (PPi) in an equimolar quantity to the
amount of nucleotide incorporated.

General Description
3-2 PyroMark Q24 MDx User Manual 01/2016
5. ATP sulfurylase quantitatively converts PPi to ATP in the
presence of adenosine 5' phosphosulfate.
6. This drives the conversion of luciferin to oxyluciferin by
luciferase, generating visible light in amounts
proportional to the amount of ATP. Light is detected
using charged coupled devices (CCDs) and seen as a
peak in the Pyrogram®. Each light signal is proportional
to the number of nucleotides incorporated.
7. Apyrase, a nucleotide-degrading enzyme, continuously
degrades unincorporated nucleotides and ATP. When
degradation is complete, another nucleotide is added.
8. Nucleotides are added one at a time.
Note: Deoxyadenosine alfa-thio triphosphate (dATPαS)
is used instead of natural deoxyadenosine triphosphate
(dATP) since it is used efficiently by the DNA polymerase,
but not recognized by the luciferase.
9. As the process continues, the complementary sequence is
built up and the nucleotide sequence is determined from
the peak in the Pyrogram.