THE PIRO PIPETTING ROBOT OPERATION MANUAL Version 1.5 · parts from DORNIER-LTF. If original parts are not used and inadequate spare parts are used for the replacement of faulty original

PIRO® Manual, Version 1.5.2

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THE PIRO®

PIPETTING ROBOT

OPERATION MANUAL

Version 1.5.2


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All rights reserved. No parts of this work may be reproduced in any form or by any means -

graphic, electronic, or mechanical, including photocopying, recording, taping, or information

storage and retrieval systems without the written permission of the authors.

Products that are referred to in this document may be either trademarks and/or registered

trademarks of the respective owners. The publisher and the author make no claim to these

trademarks.

While every precaution has been taken in the preparation of this document, the publisher

and the author assume no responsibility for errors or omissions, or for damages resulting

from the use of information contained in this document or from the use of programs and

source code that may accompany it. In no event shall the publisher and the author be liable

for any loss of profit or any other commercial damage caused or alleged to have been

caused directly or indirectly by this document.


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Intended Use The PIRO® Pipetting Robot is designed for the use of accurately transporting liquids within

the PIRO® system. With no exception, the instrument can only be used by trained personnel

and only with liquids and substances appropriate for the instrument. The standard deviations

for pipetting volumes determined by DORNIER-LTF can only be achieved by using the

pipetting tips recommended by DORNIER-LTF. Any use other than mentioned above does

not apply to the intended use of the system and DORNIER-LTF shall not be liable for any

damage resulting from such unintended use. An intended use also includes the compliance

with any safety and environmental laws and regulations as well as the instructions in the

manual to prevent any damage to the user and the environment. Any liability will also

extinguish when no or inadequate maintenance of the system has been performed. In doubt,

please contact DORNIER-LTF.

Intended Use of any Accessories on the PIRO® Pipetting Robot

Any accessories used on the PIRO® must have been recommended from DORNIER-LTF

and can only be used according to the description given by DORNIER-LTF. Any additional

equipment or accessory not recommended by DORNIER-LTF will not be regarded as an

intended use. For any damage resulting from such use, DORNIER-LTF will not take any

liability. The connection between the PIRO® and any accessory may only be done according

to the regulations outlined in the documents delivered or outlined in this manual. For any

damage resulting from inadequate connection of accessories DORNIER-LTF will not take

any liability. In doubt, please contact DORNIER-LTF GmbH.

Changes to the PIRO® Pipetting Robot

The PIRO® as a complete system is tested according to the safety regulations and can only

be used according to the intended use. Any changes to the PIRO® can only be performed

with the permission of DORNIER-LTF and only in the scale the permission has been granted

for. If the PIRO® will be changed without the permission of DORNIER-LTF, any liability and

warranty will be extinguished. For any damage resulting from changes to the PIRO®

DORNIER-LTF will not take any liability. In doubt, please contact DORNIER-LTF GmbH.

Original Parts from DORNIER-LTF

All parts used in the PIRO® are tested for safety and are optimised for the use of the PIRO®

in connection with all other parts used. Faulty parts can only be exchanged using original

parts from DORNIER-LTF. If original parts are not used and inadequate spare parts are

used for the replacement of faulty original parts, for example electrical parts, danger to staff

could arise from such a replacement. Non original parts could also influence the

performance of the PIRO® and its pipetting quality. For any damage to the user or the

instrument resulting from the use of non-original parts to the PIRO®, DORNIER-LTF will not

take any liability and the warranty will be extinguished. In doubt, please contact DORNIER-

LTF GmbH.


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Contents

1 The PIRO® Pipetting Robot ............................................................................................ 8

1.1 Unpacking the PIRO®.............................................................................................. 8

1.2 Box Contents ........................................................................................................ 10

1.3 The PIRO® Hardware ............................................................................................ 10

1.4 Pipetting Heads .................................................................................................... 11

1.4.1 Changing the Pipetting Head ......................................................................... 11

1.5 UV Radiation Procedure ....................................................................................... 11

1.6 HEPA-Filter ........................................................................................................... 11

1.7 Positioning the Robot ............................................................................................ 11

1.8 Lid ......................................................................................................................... 12

1.8.1 Functionality of the Lid ................................................................................... 12

1.8.2 Cleaning Acrylic Plexiglas .............................................................................. 12

1.9 Calibrations ........................................................................................................... 12

1.10 Safety ................................................................................................................... 12

1.11 Tips ....................................................................................................................... 13

1.12 Specifications ........................................................................................................ 13

1.13 Level Sensing ....................................................................................................... 14

1.14 The Computer ....................................................................................................... 14

1.15 Connecting the Robot ........................................................................................... 14

1.16 PIRO® Software Installation .................................................................................. 14

1.16.1 Updating the Software ................................................................................... 16

2 Introduction to the PIRO® Software .............................................................................. 17

2.1 Downloads ............................................................................................................ 17

2.1.1 PIRO® Software ............................................................................................. 17

2.1.2 PIRO® Manual ............................................................................................... 17

2.1.3 Other Documents ........................................................................................... 17

2.2 Starting the Software ............................................................................................ 17

2.3 Virtual Mode.......................................................................................................... 17

3 The PIRO® Workspace ................................................................................................ 18

3.1 Deck Layout .......................................................................................................... 18

3.2 Racks and Adaptors .............................................................................................. 18

3.2.1 Orientation of Rack and Plates ...................................................................... 19

3.2.2 Tip Racks ....................................................................................................... 19

3.2.3 Tips ................................................................................................................ 19

3.2.4 Tip Disposal ................................................................................................... 19


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3.2.5 Racks and Adaptors....................................................................................... 21

3.2.6 Plate Positions 4, 8, 12, 16 ............................................................................ 22

3.2.7 Tubes ............................................................................................................ 23

3.2.8 Setting up Plates in the Software ................................................................... 23

3.3 Position Offsets .................................................................................................. 24

3.4 Plate Functions ..................................................................................................... 25

3.5 Exchanging Existing Plates ................................................................................... 25

4 Main Menus ................................................................................................................. 26

4.1 File Menu .............................................................................................................. 26

4.2 Layout Menu ......................................................................................................... 27

4.2.1 Clipboard ....................................................................................................... 27

4.2.2 Tools .............................................................................................................. 27

4.2.3 Zoom ............................................................................................................. 28

4.2.4 Extras ............................................................................................................ 28

4.3 Options Menu ....................................................................................................... 29

4.3.1 Language Options ......................................................................................... 29

4.3.2 Robot Options ................................................................................................ 30

4.3.3 Graphics Options ........................................................................................... 31

4.4 Robot Menu .......................................................................................................... 31

4.4.1 Robot Controls ............................................................................................... 31

4.4.2 Report ............................................................................................................ 32

4.4.3 UV Program ................................................................................................... 33

4.5 Deck Window ........................................................................................................ 34

5 Feature Tree and Feature Table .................................................................................. 35

5.1 Feature Tree ......................................................................................................... 35

5.1.1 Triangles ........................................................................................................ 35

5.1.2 Info ................................................................................................................ 35

5.1.3 Warning ......................................................................................................... 36

5.1.4 Error .............................................................................................................. 36

5.1.5 Add ................................................................................................................ 36

5.1.6 Remove ......................................................................................................... 36

5.2 Feature Table ....................................................................................................... 36

5.2.1 Well Position Description ............................................................................... 36

5.2.2 Changing Names ........................................................................................... 37

5.2.3 Viscosity ........................................................................................................ 37

5.2.4 Mixing Options ............................................................................................... 37


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5.3 Plate Submenu ..................................................................................................... 38

5.3.1 Plates ............................................................................................................ 38

5.3.2 Tips ................................................................................................................ 38

5.3.3 Waste ............................................................................................................ 38

5.4 Transports ............................................................................................................ 38

5.4.1 Changing the Order of Transports .................................................................. 39

5.4.2 Setting up Transports ..................................................................................... 42

5.4.3 Auto Assignment of Transports in Empty Wells .............................................. 43

5.4.4 Function Colour Coding ................................................................................. 44

5.4.5 Transports and Selecting Wells ..................................................................... 44

6 Setting up a Run .......................................................................................................... 45

6.1 Diluent .................................................................................................................. 45

6.1.1 Setting up Diluents ......................................................................................... 45

6.2 Reagents .............................................................................................................. 45

6.2.1 Setting up Reagents ...................................................................................... 46

6.3 Mixes .................................................................................................................... 47

6.3.1 Setting up Mixes ............................................................................................ 48

6.3.2 Pipetting Mixes into Mixes.............................................................................. 49

6.3.3 The Pipetting Order........................................................................................ 49

6.4 General Orientation on Plates ............................................................................... 50

6.5 Samples ................................................................................................................ 51

6.5.1 Setting up Samples ........................................................................................ 51

6.5.2 Sample Options in the Deck Window ............................................................. 51

6.5.3 Skip Missing Samples .................................................................................... 52

6.5.4 Sample Groups .............................................................................................. 52

6.5.5 Sample Group Table ...................................................................................... 53

6.5.6 Creating Sample Groups from Samples located on different Plates ............... 54

6.5.7 Import ............................................................................................................ 55

6.5.8 Intelligent Pipetting ........................................................................................ 59

6.5.9 Barcode Reading ........................................................................................... 64

6.5.10 Normalisation of Sample Concentrations ....................................................... 65

6.5.11 Dilution Series of Samples ............................................................................. 67

6.6 Reactions and Fixed Reactions ............................................................................. 69

6.6.1 Setting up Reactions ...................................................................................... 69

6.6.2 Reaction Feature Table ................................................................................. 70

6.6.3 Setting up Fixed Reactions ............................................................................ 71


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6.6.4 Special Features of Fixed Reactions .............................................................. 72

6.6.5 Reaction Export ............................................................................................. 73

6.6.6 Sample-Multi-Dispensing ............................................................................... 74

6.7 Standards ............................................................................................................. 76

6.7.1 Setting up Standards ..................................................................................... 76

6.7.2 Ready to use Standard .................................................................................. 76

6.7.3 Creating a Standard ....................................................................................... 76

6.7.4 Standard Options in the Deck Window ........................................................... 77

6.7.5 Standard Group ............................................................................................. 78

6.8 Reserved .............................................................................................................. 79

6.8.1 Setting up Reserved Area .............................................................................. 79

6.9 Special Functions of the PIRO® Software .............................................................. 80

6.9.1 Automatic Tube Switching (ATS) ................................................................... 80

6.9.2 Automatic Fluid Transfer (AFT) ...................................................................... 81

7 Quick starting the PIRO® .............................................................................................. 82

8 Appendices .................................................................................................................. 83

8.1 PIRO® Alarm List .................................................................................................. 83


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1 The PIRO® Pipetting Robot The PIRO® PI-petting RO-bot is a precise pipetting system for performing a vast variety of pipetting operations. Due to its high flexibility, the PIRO® can be programmed to set up multiple mixes, standards, dilutions, and samples, using multiple groups of samples in many plate and tube formats. It automatically calculates the volume of mixes, reagents and other components required for each experiment. Import functions are implemented as well as export functions, which can be used for further processing. Reports are generated before and after each run and are automatically saved for future reference. For maximum precision the PIRO® uses high quality non-conductive tips with three tip volume sizes (50 µl, 200 µl, and 1000 µl) in order to achieve a perfect combination of volume capacity and pipetting speed. An innovative sensor technology provides a highly precise level sensing precision of very low volumes down to 5 µl. The tip moves to just below the surface of the liquid, rather than the bottom of the tube preventing carryover of material on the outside of the tip. Specially optimized pipetting performance steps increase accuracy and precision of all pipetting steps. The PIRO® software has been designed to be user friendly. The progress of any run can be followed in real time. On the screen, the software highlights the position of the pipetting head while moving. A progress bar and a continuous table update show the status of the current run. The screen layout corresponds to the plates set up on the robot. Any desired change can be performed by simply pointing to the corresponding image of the hardware on the screen. The PIRO® software can also be run in virtual mode which means runs can be set up, progress followed, and files stored, even if the robot is switched off or disconnected. The PIRO® software can be used on computers not directly attached to the PIRO® and due to the small size of PIRO® files they can easily be transferred to the computer running the PIRO® instrument. For further information or assistance, please do not hesitate to contact your distributor or the DORNIER-LTF team. DORNIER-LTF GmbH, Rickenbacherstraße 107, 88131 Lindau a.B., Germany.

Tel: +49 (0) 8382-273089-10, Fax: +49 (0) 8382-273089-29, www.dornier-ltf.com.

For general enquiries about DORNIER-LTF products and sales, contact: [email protected].

1.1 Unpacking the PIRO®

The packaging of the PIRO® has been carefully designed in order to ensure safe shipping

conditions. Please do not discard the packaging in case the PIRO® needs to be transported

again. The unpacking procedure is outlined below, to repack simply reverse the instructions.

http://www.dornier-ltf.com/

mailto:[email protected]

mailto:[email protected]


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Open the box and remove the foam protection from the top of the PIRO®.

Remove the sides of the cardboard box by lifting it up from the PIRO®. Lift the PIRO® on to a

table and carefully remove the plastic protection, whilst taking care not to scratch the lid of

the PIRO®. Remove the two small foam inserts located below the pipetting arm.

Remove the top two pieces of foam protection and cardboard box from the PIRO®. The

remaining piece of foam protection on the bottom of the PIRO® can now be removed.

Before connecting the PIRO® to the computer make sure, the deck is completely empty. The

PIRO® can now be connected and is ready for use.


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1.2 Box Contents

The PIRO® shipping box contains the following items listed below unless ordered differently:

1 x PIRO® (P100100, or /200, 300, 400)

1 x IEC Mains Cable (E100312)

1 x USB-cable (E100291)

1 x Software CD

1 x Manual (P100343)

Various racks consisting of:

1 x waste channel-1 (P100322)

1 x waste rack (P100321)

4 x tips racks (P100317)

2 x 96-well plate racks (P100318)

3 x standard racks (P100319)

3 x tube racks (P100323, P100487,

P100371)

5 x 96 tips (50 µl) (P100328)

5 x 96 tips (200 µl) (P100329)

For more details, please see delivery note

A computer is required to operate the PIRO® and can be ordered with the PIRO® if required.

The box content may vary, depending on the specifications of the order. Please check the

content while unpacking.

1.3 The PIRO® Hardware

An overview of the PIRO® hardware is shown below. The dimensions of the PIRO® are as

follows: 600 x 515 x 470 mm.


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1.4 Pipetting Heads

There are two choices of pipetting heads for the PIRO® available and each head has its own

electronics and calibration data:

1) Pipetting Head 200 2) Pipetting Head 1000 - in preparation

1.4.1 Changing the Pipetting Head

To change a pipetting head turn the

power off and move the y-arm

forward and the pipetting head

upwards to have an optimal

handling space. Hold the pipetting

head with your left hand and press

the unlock button on the bottom of

the head with your right hand index

finger and move the pipetting head

out of the lock. To install a new

pipetting head reverse the

procedure making sure both pins

are sitting in the appropriate holes

and fit properly to the connector.

The calibration data are stored in

the pipetting head and no new calibration has to be performed.

1.5 UV Radiation Procedure

The UV radiation procedure is implemented to allow the user to use UV in the PIRO® for

sterilization at the end of a run. The procedure can only be performed with a closed hood to

avoid any damage to the user. Though the hood is manufactured using UV-blocking acrylic

Plexiglas preventing transmission of UV light to the outside, for safety reasons do not look

into the UV light. Opening the hood will stop the UV radiation procedure immediately and the

procedure has to be restarted again.

In order to use the UV light it is best to remove all racks and disposables from the deck, so

the UV can access the complete deck area. To start the procedure, please see 4.3.2 and

4.4.3. The PIRO® arm will then move over the deck to avoid shadow effects.

1.6 HEPA-Filter

The PIRO® can be delivered with or without a HEPA filter. If a PIRO® has been installed

without a HEPA filter, it can also be upgraded by the distributor at a later stage. To active the

HEPA filter press the box ‘Activate HEPA filter’ located in the

‘Options’ menu. Once a PIRO® run is started the HEPA filter will then be activated.

1.7 Positioning the Robot

The PIRO® can be positioned on any bench, as long as the bench is reasonably stable and

not moving by the movement of the PIRO®. Make sure the lid can be opened completely as

otherwise scratches or damages of the lid might occur. The temperature and humidity should

be reasonably consistent and according to the specification (see 1.12). It is important to

make sure all four feet of the PIRO® evenly touching the surface. As outlined in the below


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chapter (see 3.2.4.1), if the waste channel-1 should be used but not the waste console (see

3.2.4.3), the front feet of the PIRO® have to be placed just before the edge of the table,

therefore allowing to place a waste bin below the waste channel-1.

1.8 Lid

The PIRO® is equipped with a lid to protect the reactions and reagents within the PIRO® but

also to protect the user. The lid is manufactured using UV-blocking acrylic Plexiglas.

1.8.1 Functionality of the Lid

The PIRO® will only work with a closed lid. Opening the lid during a run pauses the

instrument in order to protect the user. When the lid is closed, the PIRO® run has to be

actively resumed and the pause is recorded in the post-run report.

1.8.2 Cleaning Acrylic Plexiglas

Care should be taken when cleaning the lid. Use a soft cloth (e.g. cotton) never use paper

towels as they will leave streaks on the surface. Plexiglas has a very soft surface which can

easily be scratched. Dust can be blown off with a hairdryer or washed off using water and a

soft cloth. Then use a recommended cleaner to complete the job. Note: a mild solution of

dish detergent and warm water takes off stubborn dirt easily without harming the acrylic

Plexiglas. Please, never, use cleaners containing ammonia. Some recommended cleaning

methods are given below:

• Use only products specifically recommended for cleaning acrylic such as Novus

#1, Plexiclear or Brillianiz,

• Fill a spray bottle with an equal mixture of water and isopropyl alcohol. Use the

type of alcohol available in the first aid section of your grocery store,

• Mix 2/3 cup white distilled vinegar, 1 1/3 cups distilled water, and 5 drops

lavender essential oil, 5 drops rosemary essential oil in a spray bottle and clean

the Plexiglas surface.

1.9 Calibrations

The PIRO® does not need any calibration during the installation of the instrument as for all

plates and vessels in the plate database, the calibration has been performed in the factory

and the user can start working with the instrument straight after the installation.

.

All plates, racks, and positions implemented in the PIRO® software are pre-calibrated. If

alternative plate types with unusual formats need to be used, please contact the distributor,

as at this point of time, calibration of such unusual formats must be implemented by

DORNIER-LTF. As soon as new plate settings are available they will be implemented in a

software update for any user to be downloaded at following web page: www.dornier-ltf.com.

If the calibration of plates with formats available in the software needs to be adapted, please

see 3.3, which describe position offsets.

1.10 Safety

The PIRO® pipetting robot has moving parts, which the user needs to be aware of when

handling the PIRO®. For your own safety and to prevent damage of the instrument, please

take note of the following safety requirements:

Always use the packaging provided with the robot when shipping the instrument



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Do not interfere with the PIRO® while running (e.g. moving plates or tubes)

Do not lift the PIRO® by yourself to prevent damage to the user or the PIRO®

Keep the lid of the PIRO® closed whenever possible

NEVER apply any force (by hand or otherwise) in any direction to the robot arm

NEVER place any items on the y-arm of the instrument

NEVER put your hand under the pipetting head while it is descending

Only use recommended tips and tip racks

The PIRO® is for indoor use only

Use the Stop/Pause button located in the tool bar of the software to stop/pause the

robot before removing plates or tubes

1.11 Tips

The tips used on the PIRO® are clear, sterile, and filtered. Conductive tips are not needed to

perform level sensing (see 1.13). If 50 µl tips are incorrectly placed in a 200 µl tip position,

the pipetting head may become contaminated and will require a service.

Tips other than those recommended by DORNIER-LTF can be used on the PIRO® provided

that the tips have a compatible hub size and are of the same volume. However, DORNIER-

LTF does not guarantee the precision or accuracy of pipetting, if tips others than those

recommended are used. Performance may seriously be affected and any new tip must

undergo evaluation before using.

1.12 Specifications

Pipetting Tips

96 racked filtered tips, 50 μl, 200 μl, and

1000 μl (clear, non-conductive tips)

Pipetting Volume Range

0.5 μl - 180 μl using the 200 μl pipetting

head

X-, Y-, Z-axis Drive Resolution

Less than 0.0065 mm

Pipetting Speed

10 μl – 200 μl/sec, software adjustable

HEPA-Filter (optional)

High Efficiency Particulate Air filter system

provides positive pressure

UV Sterilization (optional)

UV light system for deck sterilization under

closed lid conditions

Precision, Accuracy using a 200µl head

CV < 1 %, 5 μl to 180 μl (dry-well transfer)

CV < 5 %, 2 μl to 5 μl (wet-well transfer)

CV < 10 %, 0.5 μl to 2 μl (wet-well transfer)

Temperature Range

4°C - 35°C

Electrical

100 - 250 VAC, 50 / 60 Hz, 60 Watts

Dimensions (W x D x H)

600 x 515 x 470mm

Weight

45 kg

Relative Humidity

40 - 70 %

Noise emission

42 dba


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1.13 Level Sensing

For maximum precision, the PIRO® is equipped with a liquid level sensor. The use of the

level sensor helps to prevent carryover of material on the outside of the tip. The liquid level

sensor is a very sensitive electronic device and is capable of detecting very small amounts

of liquid. The PIRO® level sensor relies on detection of pressure changes and allows the

robot to automatically detect when the pipetting tip is touching the surface of the liquid and

can therefore be used with non-conductive tips.

In a tip not touching fluid, no pressure can be measured. As soon as the tip reaches the

liquid level surface, pressure is built up. This build-up of pressure is measured by a pressure

sensor in the pipetting head, which then allows the pipetting head to lower the tip to a level

to aspirate the set volume. If a vessel is completely empty, a minimal z-axis value will be

recorded and an error message is displayed, allowing the user to add fluid to the tube.

In order for the above to work properly, there are two requirements for the tips: firstly, they

need to fit perfectly on the pipetting head and secondly, need to have a defined pore size of

the filter inside the tips.

1.14 The Computer

A personal computer running Microsoft Windows operating system is required to control the

PIRO® pipetting robot. The PC will be provided by DORNIER-LTF or the distributor. If a PC

is supplied by the user the following minimum specifications are required to run the PIRO®

software: Intel Core i3 2100, 2x 3.10 GHz or similar or more, 4 GB RAM, 250 GB Hard disc,

USB-drive, Graphic card: 1 GB memory, 1366 x 768 Pixel (widescreen), Monitor:

Widescreen. The software can be run with computers with lower specifications, but complex

calculations or processes might be slowed down, therefore influencing the graphical

presentation. In order to enhance the performance, the ‘use graphics card acceleration’ box

should ticked.

1.15 Connecting the Robot

To connect the PIRO® and the computer, please use the USB and the power cable supplied.

Connect the PIRO® to the power supply using the power cable socket at the rear of the

instrument. The USB connection on the PIRO® is found at the back of the instrument. Make

sure the PIRO® is switched off before connecting both the USB and power cables. The

PIRO® is now ready to be switched on.

1.16 PIRO® Software Installation


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For error free usage of the PIRO® software, make sure that all other software is shut down

and that the screen saver and power save mode are disabled. Insert the software CD or

USB stick and double click on the setup.exe icon to start the installation process. When

installing the PIRO® software for the first time, the setup will check if the required Microsoft

.NET framework is installed. If there is no Microsoft .NET framework or an older version

installed, the software will ask to download and install it before continuing with the PIRO®

software installation. Make sure the computer is connected to the Internet. Click ‘Install’ and

wait until it is finished. After installation of the Microsoft .NET framework, the setup of the

PIRO® software continues. Follow the on screen instructions.

Welcome to the DORNIER-LTF PIRO® Setup Wizard: This screen starts the installation

procedure. Click ‘Next’ to continue.

Select Installation Folder: This screen allows the user to select the installation folder. Click

‘Browse’ to alter the folder the software is installed in. ‘Disk Cost’ shows how much disk

space is needed and how much is available on existing hard drives. Choose between

‘Everyone’ and ‘Just me’ to select access rights for the software. Click ‘Next’ to continue with

the installation process.

Confirm Installation: Once the installation settings have

been selected, the software is installed by clicking ‘Next’.

Selecting ‘Back’ will take the setup back one step. To stop

the installation select ‘Cancel’.

Installation Complete:

Once the installation is

completed, click ‘Close’

and the software can

now be started. It is not

necessary to reboot the computer before starting the

PIRO® software for the first time. If the PIRO® is switched

on, a PIRO® run can be performed. If the PIRO® is

switched off the software will run in virtual mode (see 2.3).


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1.16.1 Updating the Software

Updating the software follows a similar procedure to the first installation. When the setup is

started, it will detect the previous version of the PIRO® software, which is automatically

removed before the new version is installed.

Select ‘Install’. A small window appears showing

that another version has been detected, click

‘Uninstall’ and wait until the process is done. Installation of the new software is performed as

described above.


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2 Introduction to the PIRO® Software The PIRO® software has been designed to be easy to use. For most applications there are

multiple ways of working to make the software as intuitive and easy to use as possible. The

following chapters will give a detailed overview of the software.

The PIRO® software has been designed for general pipetting operations, but its initial focus

is the setup of PCR and qPCR reactions. The software supports a variety of PCR and qPCR

plate formats and it is being continually updated with new plate types. If for any reason a

plate format cannot be supported, please contact your distributor.

2.1 Downloads

2.1.1 PIRO® Software

If the software has not been delivered with the PIRO® instrument, it can be downloaded from

the following web page: www.dornier-ltf.com. In order to do so, the user has to register and

once approved, will be able to download the software. We recommend registering for the

website, as DORNIER-LTF will provide regular updates (e.g. new features, new plate

libraries, fixing reported bugs). It is the users’ responsibility to keep the software updated to

ensure the best possible performance of the PIRO®.

2.1.2 PIRO® Manual

The most up-to-date version of the PIRO® manual can also be downloaded from the above

web page, once registered. It is provided as a pdf and free of charge.

2.1.3 Other Documents

In the future it is planned to add application notes and other useful material to the restricted

user area. It is therefore worthwhile regularly checking this area for updates.

2.2 Starting the Software

To start the software, double-click the PIRO® icon. The software will open the

default project (see 4.1). If the computer is connected to the PIRO® and the

PIRO® is switched on, the software will show it is connected on the bottom

right of the main screen. If the PIRO® is not connected, then ‘Not

connected’ is displayed and the PIRO® software will run in virtual

mode (see 2.3).

2.3 Virtual Mode

The virtual mode option allows the user to run the software without a connection to the

PIRO®. This is a useful option as it allows running files or templates to be set up, saved, or

analysed on a remote computer. Files can then be transferred to the computer the PIRO® is

attached to and run.



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3 The PIRO® Workspace

Shown above is the main area of the PIRO® workspace. This includes the feature tree (see

5.1), file menu (see 4.1), layout menu (see 4.2), options menu (see 4.3), and robot menu

(see 4.4). The feature tree is the top left window and contains detailed information about the

current run. When an item in the feature tree is selected, more details are shown in the

feature table which is located below the feature tree (see 5.2). The right side of the screen

shows the main workspace of the PIRO®.

3.1 Deck Layout

The figure below shows the empty deck layout of the PIRO®. The numbering starts in the top

left corner with number 1 and is followed through to number 16 on the bottom right corner of

the deck. Various racks and adaptors can be positioned on the PIRO® deck and these

options are discussed in more detail in the section below.

3.2 Racks and Adaptors

The PIRO® system is supplied with a standard selection of racks, but more racks and

adaptors are available. For further information, please check the customer area on our web

page or check the PIRO® software under ’Plates’ for an indication of available racks and


19

adaptors. All PIRO® loading racks are manufactured from stainless steel and/or high quality

aluminum, which is trowalised to give the rack a hard wearing finish. The racks are fully

autoclavable. To keep the racks cold, place the racks in a fridge or freezer before use.

DORNIER-LTF manufactured adaptors can be washed in bleach or soapy water. Please

note that some solvents or salts may stain the aluminum surface.

3.2.1 Orientation of Rack and Plates

PIRO® racks and plates have been designed so that they can only be placed on the deck in

the correct orientation using different sized holes on the bottom of each rack or plate.

3.2.2 Tip Racks

Tip racks can be placed in any suitable deck position. Right-clicking over the frame of any

plate will open a window allowing tips to be marked as available or unavailable, the tip type

to be changed, and removal of the tip rack.

Tip racks can be placed in the deck positions 1 to 12. However, in positions 4, 8, and 12,

only the first two rows of the tips can be reached by the pipetting head (see 3.2.6). Positions

13 to 16 are smaller and are not designed for use with tip racks.

3.2.3 Tips

The PIRO® has been calibrated for use with filtered tips from DORNIER-LTF and will

therefore guarantee the most accurate performance. For any tips, other than those available

in the PIRO® software, DORNIER-LTF will not take any responsibility with regards to the

performance. Tips not tested on the PIRO® might be too large, therefore allowing air to be

aspirated and inaccurate volumes pipetted or even fall off the pipetting head. It is strongly

recommended that only DORNIER-LTF tips are used with the PIRO®.

The software has options for 50 µl, 200 µl, and 1000 µl tips and all tips can be used with the

pipetting head 200. However, 1000 µl tips will only aspirate a maximum of 180 µl due to the

size of the pipetting head 200. If volumes up to 1000 µl need to be pipetted, then the

pipetting head 1000 needs to be used.

Before starting a run it is recommend indicating in the software the availability of the tips on

the deck. If the PIRO® however does not find any tip, it will move to the next tip position until

a tip has been found to continue the run.

3.2.4 Tip Disposal

The PIRO® has various options for the tip disposal: the external waste disposal through the

waste channel-1 or internal waste disposal into a waste rack.


20

3.2.4.1 The Waste Channel

The waste channel-1 is a metal tube

that leads to an opening located at

position 16 on the front right corner of

the PIRO®. Tips ejected into the waste

channel-1 will fall out of the bottom of

the PIRO® and can be collected externally either by placing a waste

container such as a bin or draw below the waste channel-1 or attaching a bag to the

underside of the waste channel-1 to catch the tips. DORNIER-LTF also provides a special

PIRO® table which has a built in section for collecting tips ejected through the waste

channel-1.

3.2.4.2 The Waste Racks

Tips may also be discarded into a waste rack positioned on the deck. The waste rack is equipped with a lid.

The metal waste rack is lined with a bag and can be discarded at the end of each run.

3.2.4.3 The Waste Console with Drawer

A further option to manage the tip waste is a

drawer positioned below the PIRO®. The console

is equipped with two drawers for the collection of

waste in the drawer at the right side and storing

accessories in the drawer on the left side. For

more details of the console please contact your

distributor.


21

3.2.5 Racks and Adaptors

Positions 1 to 12 are designed to hold SBS format racks. This include tip racks, 96- and 384-

well plates, adaptors, waste boxes, and many special plate formats (e.g. Eco-plates,

SmartCycler, Rotor-Gene plates).

3.2.5.1 Plate Racks

Three different SBS standard racks are shown above. From left to right one tip rack, Plate

Rack-1, SBS Grid 96 Wells, and Plate Rack-1, SBS Standard are shown.

Tip racks take all DORNIER-LTF tips. The standard and 96-well plate racks can

accommodate any SBS plate that fits within the footprint. The 96-well plate racks can also

take 96-well unskirted plates.

3.2.5.2 Adaptors

Adaptors are designed to fit on the 96-well and universal plate racks or other racks provided.

DORNIER-LTF provides a range of adaptors, such as the Adapter SBS 32 Well.

Adaptors are also available from alternative manufacturers such as Illumina (Eco-plate) or

Qiagen (33-well plate) which take 32 x 1.5 ml tubes. Additional plate formats are regularly

added to the PIRO® software. Once registered, software updates are available from

www.dornier-ltf.com.

3.2.5.3 Overview of Racks and Adapters

For an overview of on how racks, adapters, and plastic consumables fit together, please see

the document “Rack Adapter Compatibility List” located on the customer area of our web

page.



22

3.2.5.4 Tube racks (Positions 13 to 16)

Positions 13 to 16 at the bottom of the PIRO® deck are smaller than positions 1 to 12 and

have been designed to hold tube racks. Tube racks available take a variety of different

volumes like 0.2, 0.5, 1.5, 2, or 5 ml tubes. Other tubes and formats will be added to the

PIRO® software over time, as requested. For instructions on downloading PIRO® software

updates, please see 2.1. Some of the tube racks (TR) available from DORNIER-LTF are

shown below:

Tube racks are used to hold tubes containing diluents, reagents, mixes, standards, and

samples on the PIRO®’s work space.

If required, tube racks could also be set up on position 1 to 12 of the deck to allow for more

flexibility of using plates on different positions.

At position 16, there is a hole in the deck. Position 16 can be used in

conjunction with the waste channel-1 and allows used tips to be ejected

outside the PIRO® during a run. The PIRO® front feet have been

mounted about 15 cm inwards, allowing for the PIRO® to be placed

over the front of the table so a waste bin can be positioned below

position 16 of the deck. If waste tips are being collected in a waste rack

during a run, then position 16 can be used to hold a tube rack instead.

3.2.6 Plate Positions 4, 8, 12, 16

The PIRO® has 16 positions available on the deck. The availability of

positions 4, 8, 12, and 16 is limited due to the space needed for

positioning of the pipetting head. The light grey area shows the area

available for use, the dark grey area indicates where the pipetting head

is unable to reach. These positions can be used for the waste rack and

also for SBS racks where only a few positions are in use.

TR-28; 0.2 ml tapered

TR-5; 5 ml

TR-8; 2 ml V-bottom

TR-8; 2 ml free standing




23

3.2.7 Tubes

Many different types of tube can be used on the PIRO®. Some of the tubes that are

compatible with the PIRO® are shown below, along with the racks required to hold them:

3.2.8 Setting up Plates in the Software

Plates are added to the deck by right clicking on the arrester on the deck

position. A window will open containing multiple options. Hovering over ‘Add

plate’ opens a window allowing to select from ‘tip racks’, ‘plate racks’, or ‘tube

racks’. Hovering over each of these options brings up a different catalogue of plate and rack

types. Clicking on a plate or rack will add it to the deck.

TR-28; 0.2 ml tapered TR-8; 2 ml V-bottom

TR-8; 2 ml free standing

TR-10; 0.5 ml tapered TR-5; 5 ml tube



24

Details of the selected plate can be found by clicking on the plate (outside any position

indicating a well). The information appears in the feature table on the left side of the screen.

The ‘Properties’ tab contains details about the selected plate and the ‘Positions’ tab contains

information about the position, function, content, and other options available for each well on

the selected plate. Plates can also be added to the plate deck using the toolbar. Detailed

information on using the toolbar to add plates to the deck is given in the below chapter (see

4.2.2).

3.3 Position Offsets

The function ‘Position Offsets’ allows

the user to change the position of the

pipetting head relative to the plates or

tubes used. This is generally not

necessary if the exact plate described

in the software is used, but if a similar

plate is used, this option allows

adjusting the x, y, and z-values of the

pipetting head.

Having this option is useful for various

reasons:

Defining x- and y positions inside

the vessel for dispensing liquid,

for example closer to the wall

Defining z limitations for the tip if

a contact to a sediment should be

avoided

Defining z limitations to reduce

dead volume

Similar plates to what is in the plate library could be calibrated accordingly


25

In order to calibrate a plate, chose the plate where the adjustment should be done. Press the

‘real-time processing’ button and adjust positions by pressing the ‘up’ and ‘down’ arrows in

the x -, y-, or z offset tick boxes, depending on the direction it needs to be changed. Positive

and negative values could also be typed into the appropriate boxes. Repeat the processes

until the values are correct and the tip is above the desired position of the well. Press the

‘real-time processing’ button again to leave the ‘Position Offsets’ feature.

If plates are similar but not exactly the same, the depth of a well might be different compared

to the plate used. Similar to above, change the z values to move the head up or down. Make

sure the tip does not touch the bottom of the well, as otherwise the performance would be

influenced.

Following the adaptation of the x -, y-, or z values, the offset values are changed. To indicate

that a plate has adjusted x-, y-, or z-values, the plate now has a yellow rectangle in front of

the plate description in the feature tree (see red circle).

In order to use the plate with the adjusted values again, save the file as a template and use

the template. Be aware that the settings are only valid for the position of the plate where the

values have been adjusted for. If the same original plate is chosen from the plate library

again, the values need to be adjusted for this particular plate at its actual position again.

Moving a plate with altered x-, y-, or z values to another position will maintain the changes at

the new position. This means that position offset changes are attributes of the appropriate

plate.

It is also possible to perform these adjustments while the robot is running. The changes will

affect the adjustments immediately after pressing ‘Enter’, so any change can be observed

during the pipetting process.

WARNING: Please take caution not to introduce position alterations that might cause tip

crashes next to or inside the wells.

3.4 Plate Functions

Plate functions can be chosen independently of the plate and plate position. It is therefore

possible to have several different functions (e.g. reactions, samples, mixes) on the same

plate. This has various advantages; the speed of a run can be increased by choosing

positions close to each other, the transport ways can be optimised, and storage for future

runs can be facilitated.

3.5 Exchanging Existing Plates

The PIRO® software allows the plate type to

be changed even when the plates are in

use. This can be useful if the wrong plate

has been selected. This option can also be

used to change from 96- to 384-well plates

but not to plates where the number of wells

in the plate of origin is higher. In order to

change a plate, open the plate menu and choose a plate to replace the existing plate.


26

4 Main Menus

4.1 File Menu

The file menu tab is used to load and save projects as well as to import and export samples

and reactions.

New: Selecting ‘New’ enables opening a

new ‘Default project, ‘Empty project’, or

‘Template’. If no default project has been

saved then this option will be unavailable.

‘Default Project’ loads a project which has

been saved as a default project. Any

project may be saved as a default project.

Besides the deck layout and transports,

the default project will also save other settings like multi-

dispensing, level sensing etc. An ‘Empty project’ opens a file with

the deck completely empty and ‘From Template’ opens a

previously saved template.

Open Project: Selecting this function will open a previously saved

project.

Save Project: This function will save the current setup as a

project file. Choose a folder where to save the project and press

Save. Once saved, another click on ‘Save Project’ will overwrite

the previous project file.

Save As: A project can either be saved as a ‘Default project’ or a

‘Template’. Selecting ‘Save as’ opens a new window in which the

project file is to be saved in and can be selected or altered.

Template: Opening a template will open the previously saved template. Templates can only

be altered in a limited fashion, which ensures that runs can be repeated in controlled

conditions. There is no limit on the amount of project or template files that can be saved.

Project: A project contains all deck definitions (e.g. sample, mix, reaction setups) as well as

all defined options (e.g. multi-dispensing, level sensing) and all included settings. A project

name will be created automatically before executing a run. Once saved it can be easily

reopened and used for the same or similar runs.

For Import and Reaction Export please see 6.5.7 and 6.6.5, respectively.

Recent Files: This allows the user to quickly open

any of the last ten files that have been run on the

PIRO®. Files which have been saved, but not run do

not appear in the ‘Recent files’ list.


27

Exit: Selecting ‘exit’ closes the PIRO® software. If any changes have been made, the

software will ask to save them. Click ‘Yes’ to save, ‘No’ to exit or ‘Cancel’ to leave the

software open.

Right mouse clicking over any of the main

menu options will open the window shown on

the right.

Add to Quick Access Toolbar: Tabs will be added to the toolbar below the main tabs.

Show Quick Access Toolbar above/below the Ribbon: Will move the toolbar either above

or below the ribbon.

Minimize the Ribbon: Will minimize the ribbon; the ribbon can be restored by right clicking

in the menu ribbon.

4.2 Layout Menu

4.2.1 Clipboard

Paste, Cut, Copy: As the names say, the ‘Paste‘, ‘Cut‘, and ‘Copy‘ functions allow the user

to paste, cut, and copy. These functions can be used with the reagent

and mix function. This allows additional mixes to be created quickly by

copying and pasting mixes that have already been created and then

altering the content of the mix. This is particularly useful for users who create runs with many

mixes that contain only slight variation (e.g. magnesium chloride, primer, probe, or DNA

template titrations).

4.2.2 Tools

Pointer: The pointer is the default setting and allows free movement around the PIRO®

screen. The pointer function can be selected by clicking on ‘Pointer’ in the tools

menu or by double clicking in a grey section of the deck.

Delete: This option is used to delete plates from the deck to which no function has been

assigned. It is also used to delete the function of the selected area on a plate. If a

transport has been assigned to the function to be deleted, an error message will

appear indicating that the transport needs to be removed before the function can be deleted.

Move: This option allows plates to be moved from one position to another. Plates can be

moved even if various functions have been assigned to positions within the plate.

In addition, reactions, mixes, reagents, and diluents can also be moved, as well

as the whole deck for a better overview. Samples cannot be moved. To deselect the move

cursor function, press ‘Move’ again or click the ‘Pointer’ icon.


28

Undo, Redo: Those options allow the user to undo or redo any action taken during the run

setup. This is particularly useful if previous actions need to be checked or

corrected. This function can be used to go back to the very first change made

after opening the file. Even if the file has been saved in between the setup, the

undo button will still undo all the actions taken right back to when the file was initially

opened.

Plates: Selecting this function opens a catalogue of all the plates and racks available in the

PIRO® software. Plates will be continually added to the software and the latest

version of the PIRO® software, containing all the plates currently supported, can be

found in the restricted area of our web page: http://dornier-ltf.com/. The

plates menu can also be accessed by right clicking over the circle in the

main deck layout (for more information on selecting plates, please see 3.2).

Functions: In order to use the ‘Functions’ button, empty wells without

functions have to be selected. Once selected, the ‘Functions’

icon can be used to display a list of different functions that can

be assigned to the wells. The figure on the right side shows all

function options available.

4.2.3 Zoom

Zoom: The Zoom function allows the user to zoom into an area of interest and can be done

with the buttons shown or with the mouse wheel. This is particularly

useful when setting up 384-well plates or similar plates with small

distances. Pressing the ‘Fit to screen’ button restores the screen to

its original size.

4.2.4 Extras

Show Transports: Using ‘Show Transports’ shows the transports of the selected wells.

Different arrows are displayed, depending on the type of transport. A thin arrow

is shown, if the transport has been set up from one tube to another tube (one-

to-one). A thin arrow at the beginning with a thick ending indicates a transport

from one tube to many tubes (one-to-many). A thick arrow at the beginning and

the end will show a group transport. Many thin arrows ending up in one tube shows setup of

a mix or many diluents combined in one well. For more details on transport, please also see

5.4.

http://dornier-ltf.com/


29

Show Plate IDs

In order to give an ID to a plate choose ‘Properties’ as shown above and fill in the orange

field as shown below. This can be done manually as well as using a bar code reader (see

6.5.9), so each plate can have its own ID.

In order to see the ID on the screen, use the ‘Show plate ID’ from the menu.

Zip Log File and Project

Using the “Zip log file and project” function will automatically zip the project and log file which

can then be saved anywhere on the computer. This might be important for analyzing files.

4.3 Options Menu

4.3.1 Language Options

The software is designed to be used in many languages. Using the drop down menu shows

what languages are available and any user can choose the language suitable. At this point

of time, the English, German, and Russian languages are implemented, but this will increase

over time. If an alternative language is required, please contact your distributor to see for

options.


30

4.3.2 Robot Options

Use Multi-Dispensing

Turning on the multi-dispensing option enables multiple dispenses

to be performed from a single tip, saving both time and tips. If ‘Force

Tip Change’ is also selected, ‘Force Tip Change’ will take precedence over the multi-

dispensing option.

If the multi-dispensing function in the options menu is turned on, the PIRO® software also

has the option to turn off multi-dispensing function for selected plates. Select the plate where

the multi-dispensing options should be turned off, untick the box in the ‘Properties’ window

as shown below (orange circle) and no multi-dispensing will be done on this plate.

Enable Level Sensing

The PIRO® is equipped with a real-time liquid level sensor. When this

option is selected, the robot automatically locates the surface of the

liquid. We recommend that the level sensing option is enabled before starting a run. For

more details see 1.13. As shown above, level sensing for selected plates can also be turned

off if the level sensing has been enabled.

Level Sensing each (times)

This allows for choosing the amount of times the level

sensing can be performed when pipetting any new tube.

Typing in the number ‘1’ will perform level sensing each time, using e.g. ‘3’ will perform the

level sensing every third time.

Force Tip Change

With this option selected, tips will be changed after every

operation. When ‘Force tip change’ and ‘Use multi-dispensing’

are selected, ‘Force Tip Change’ has priority over multi-dispensing (see above). If mixes are

distributed to empty wells, which is by default done with the same tip and ‘Force tip change’

is enabled, the mix will be distributed with changing a tip every time. This could be of

advantage when wells already contain liquid and cross contamination should be avoided.


31

UV Program Duration

This option allows the user to choose the time in minutes

how long a UV program should be performed. The default

and recommended time is 15 minutes. For further details please see 1.5 and 4.4.3.

HEPA Filter

To active the HEPA filter press the box ‘Activate HEPA filter’. Once a

PIRO® run is started the HEPA filter will then be activated.

4.3.3 Graphics Options

Use Graphics Card Acceleration

This option allows for computers with lower performance to

be optimised. In order to do so, tick the box above. For

computers with specification mentioned above (see 1.14), this box should stay unticked,

otherwise an incorrect presentation might occur.

4.4 Robot Menu

4.4.1 Robot Controls

Stop

Will immediately stop and abort the run.

Pause

Pressing the pause button pauses the run. A window appears that informs the user how to

continue. This window also allows for the option to move the pipetting head to the home

position by ticking the box on the bottom left

corner of the window.

The run can also be paused by lifting the lid of the

PIRO®. When the lid is opened, the robot will

finish the operation it is currently performing

before pausing the run and moving to the home

position. Once paused, the run can be restarted

by pressing ‘Resume’.

The pause functions allows for various options, tips could be reloaded (see 3.2.2) and

changes would need to be marked in the software or plate position offsets could be adjusted

(see 3.3).


32

Start

Choosing ‘Start’ initiates the run to be started. Before a run is started, the following window

containing a checklist will appear:

Various colours are used to display potential issues arising from starting the run. Information

displayed in red indicates an error that needs to be corrected before the run can be started.

An error could be that no waste box or waste channel has been selected. The tick box

before the red information cannot be ticked. Orange warnings show potential errors, for

example if a reaction does not contain a mix. This tick box can still be ticked and the run

started. Black information show for example if a sample ID has not been given and if not

needed the boxes can be ticked and the run started. In the same window a Pre-Run Report

can be accessed showing all the operation to be done in the run set up. ‘Start Robot’ will

finally start the run. All boxes must

be ticked before the run can be

started.

4.4.2 Report

Show Post-Run Report

Using ‘Show Post-Run report’

creates a ‘Post-Run Report’ with all

details of the run. In order to save

the ‘Post-Run Report’ open the

report and select ‘Save file’, choose

a folder and click ‘Save’.

The user can define the file access via document security.

Document Security: Filling in a password will protect the document from any unauthorized

access. The created pdf file can only be opened with the defined password.


33

Document Info: Information about

the run can be entered here, if

needed.

Output Range: Defines the output

of the document.

Document Restriction: The author of the pdf

document can restrict further handling and

editing in a pdf viewer or editor (e.g. Adobe

Reader, Acrobat etc.) by entering a password in

the document restriction box.

The document restriction options that can be

selected are:

Allow printing

Allow content copying or extraction

Allow content editing

Allow annotations editing

If the document security and document restriction passwords are the same, then the

document restrictions will automatically be listed when the document is opened. Choosing

separate password for document security and restrictions will prevent this from happening.

4.4.3 UV Program

UV Program Start

To start the UV-radiation procedure, choose the time how long the procedure

should take (see 4.3.2). Press the icon shown on the left and start the UV

procedure. For further information, please also see 1.5.


34

4.5 Deck Window

Many of the PIRO® functions features can be accessed by right-clicking over the main deck

area. The deck window is shown on the right and cross references to other way to access

the feature are given.

Project: See 4.1. New

projects can be opened,

either empty, default, or

others.

Zoom: See 4.2.3.

Cut, Copy, Paste: See

4.2.1.

Show Transports: See

4.2.4.

Add Plate: See 4.2.2. This

function is similar to the

plates function in the

toolbar. However, only

plates that can be added to

the position selected are

shown.

Remove Plate: see 4.2.2.

When a plate is selected,

this option can be used to

remove the plate.

Add Function: See 4.2.2.

The additional greyed out

options shown in the deck

window are explained in the

appropriate chapters which

are the options about

samples, fixed reaction,

standards, and

normalisation.


35

5 Feature Tree and Feature Table

5.1 Feature Tree

The feature tree is the window located on

the top left corner of the main screen. It

features and summarizes the complete

setup of a PIRO® run. It also shows

symbols e.g. information, warnings, and

errors, as explained below.

A general function when highlighting any of

the main headings in the feature tree e.g.

samples, mixes, standards, is that at the

same time, the appropriate wells are

highlighted on the screen.

The ‘sample feature tree’ refers only to the

main heading and does not include the

subfolders, for example where the sample

groups are located. This is also the case for

mixes, reagents, standards, etc., see figure

on the right.

The explanation of symbols in the feature

tree is given below:

5.1.1 Triangles

Triangles appear in two colors: when white, more information can be found

under the triangle; when black, no extra information can be found by clicking on

the triangle. Clicking the black triangle next to the project heading will close the all folder

submenus including plate, tips, and waste folders. This allows for a better view of the actual

setup of the project.

5.1.2 Info

The info button appears in front of a task to indicate that there may be some information

missing from the task. If the user

decides the missing information is

not needed, the run can be started

regardless of the missing

information.


36

5.1.3 Warning

A warning sign is displayed

when tasks are missing

which are usually required to set

up a PCR run. In the displayed case, no mix has been added to the reaction. However, it

could be that a project has been designed not needing a mix, e.g. the run is simply

transferring liquids from one plate to another. In order to allow for such tasks, a warning

message is displayed for information only and the run may still be started.

5.1.4 Error

The error symbol indicates

that there is a problem with

the setup and prevents the run

from being started until it has

been fixed. Errors can include a

defined mix to which no volume has been added, a missing transport or any other serious

issue.

Hovering over the main mixes heading will only indicate that there is an error, the actual

error message will then be found in the mix subfolder with the error warning displayed in

front of the mix that has the error associated with it. In the above example it is mix 2. Once

the error has been fixed, the error message for this particular mix will disappear. If this is the

only mix error, then the error symbol in the main mix heading will also disappear, if there are

other mix errors then they must be fixed before the error message on the main mix heading

will disappear.

5.1.5 Add

The add symbol appears for example when sample groups should be added to

existing sample groups. This symbol is only used in the window when right-clicking

over a main feature tree item.

5.1.6 Remove

The remove symbol appears when right-clicking over a submenu of the main feature

tree and allows sample groups, mixes etc. to be removed from the feature tree.

5.2 Feature Table

The ‘feature table window’ is located below the feature tree and displays information

according to the task chosen in the feature tree. Described below are general features used

in various feature tables. Well position description, changing of names, and viscosity are

used in several feature tables, detailed descriptions of all other feature tables can be found

below.

5.2.1 Well Position Description

In any feature table a well position will be found but cannot changed. Well positions can only

be changed in the table layout. The first number of the well position description indicates the

plate position on the deck (see 3.1) and the second number gives the position of the well on

the particular plate. A position description 6.A5 therefore means plate 6 and position A5.


37

5.2.2 Changing Names

The name of any well with an assigned function can be altered in the feature tables. The

default names are the function followed by a number. To change a name, simply double-

click on the name and type in a different name. In some feature tables there is also an option

to choose names from a drop down menu.

In order to avoid confusion, the PIRO® software does not allow a mix and a reaction to have

the same name. A suggestion would be to add an ‘M’ (mix), ‘R’ (reaction) or a number

following the name, so the names could be clearly differentiated.

5.2.3 Viscosity

The PIRO® software allows

the robot pipetting speed to

be altered. Highly viscous

fluids containing glycerol or

other viscous substances

should be pipetted more slowly for optimal accuracy when pipetting.

The standard viscosity setting is ‘Normal’, which will pipette most liquids accurately. If a

liquid contains a high amount of alcohol (over 50 %) then the ‘Low viscous’ setting should be

used. Liquids containing glycerol as a

concentration of 20 % or greater should be

pipetted using the ‘High viscous’ setting.

5.2.4 Mixing Options

The PIRO® software allows the user to define

the mixing options. Various options in the

different menus are available either by

aspiration or dispensing. By typing a number

in the appropriate field tells the software how

often the fluid will be mixed. Shown in the

figure on the right is the mixing of reagent 4

and 3. Both are mixed two times on aspiration

and dispensing. In addition, the mix menu

also allows for a mixing option at the very

end, so the created mix is mixed properly

before dispensed in the reaction.

Mixing can also be performed

choosing the ‘Options’ after all

reagents have been added to the

mix.


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5.3 Plate Submenu

5.3.1 Plates

The plates feature tree gives information on all the

plates on the deck, including the tip racks. In the

example shown is a 50 µl tip rack named ‘96-tips Rack

50 µl’, the capacity of the rack is 96 and the availability

is 87 out of 96 tips. More details on the plate including

name, category, number of positions and more can be

found by clicking the plate of interest. The plate

feature table will open showing the plate details.

Once plates are set up and checked, it is

recommended to close that feature tree for a better

overview of the other processes.

5.3.2 Tips

The tips feature tree indicates the total

number of tips available in the current setup. The tips

feature table displays an overview of the tip type, total

capacity of tips, availability, usage, and the remaining

number of tips on each tip rack.

5.3.3 Waste

Clicking ‘Waste’ on the main screen will indicate where a waste channel-1 or a

waste box is positioned. There is no waste feature table available.

5.4 Transports

This option in the feature tree allows the user to check all transports set up in a particular

run.

The ‘Commands’ tab in the transport

feature table displays a summary of all the

actions set up in a particular run. The

number in the first column indicates the

order in which the action is performed the

second column gives the name, the third

column the ‘Type’ of action to be taken, and

the fourth column gives the details of the action. The fifth column gives the volume and the

sixth column how often this task I performed.


39

5.4.1 Changing the Order of Transports

While setting up a transport as well as after all transports have been setup, the order of the

transports can be changed.

As an example a run is setup with

two sample groups, two fixed

reactions, and two mixes. The

current order is Mix 2, Mix 1,

Sample group 1, and Sample

groups 2. The new order should be

as follows: Mix 1, Mix 2, Sample

group 2, and Sample groups 1 (see

the screenshots on the right).

In order to achieve this, choose ‘Transports’ in the feature tree, press the tick box ‘Enable

sorting the transport list manually’ and the box is highlighted in red.

To change the order of the

transports, select ‘Manually sorted’

and use the ‘Up’ and ‘Down’ buttons

to move the transports up or down.

Save the project or start the run and

the transports will be performed as

indicated in the ‘Manually sorted’

table. There will be no changes in

the ‘Transports’ table, as the

‘Manually sorted’ table has priority

over the ‘Transport’ table. The

‘Commands’ will list the transports

accordingly and cannot be changed.

Special Commands:

This option allows adding special

commands, which currently includes

a wait and a reset function. To

access this option press ‘Add’ and a

window appears to specify the parameters. Once done, the wait function has been included

in the list and can be edited or removed as the buttons are active.


40

A further special command is a function called ‘Reset reaction contents’. This function will

“forget” previously performed pipetting steps and assumes the reaction vessel is now empty.

This function makes sense to use when for example a first mix has been pipetted into the

reactions. The user now wants to add a second mix in the same reaction but wants to allow

for the multi-dispensing option to be performed saving tips. Normally the PIRO® software

would not allow this task, as there is already one mix in the reactions, therefore potentially

contaminating the reactions. Using the ‘Reset reaction contents’ function tells the PIRO® that

the reactions tubes are “empty” and will therefore allow for multi-dispensing. Using this

function will not reset sample pipetting operations.

Add new Transports: When adding new transports in the run, the details in the ‘Manually

sorted’ mode will be shown in orange and italic. Use the ’Update’ and ‘Confirm’ buttons to

finally include those reactions into the transport list.


41

The reactions can now be resorted again and the final reaction list looks like as shown

below:

When changing from ‘Transport’ to the ‘Manually sorted’ option all automated safety rules

build into the software will be overruled, as no feasibility checks are done. It is the users’

responsibility to ensure plausibility of the new transport order.


42

5.4.2 Setting up Transports

Brief overview: Setting up transports using the PIRO® software always follows the same

principle. Highlight the wells to be used, assign a function, and then drag the selected wells

into the destination required.

In detail: Select the wells, assign a function (see 4.2.1), and drag them to a new area. In the

case shown above, positions 9.1 to 9.6 have been assigned as samples and dragged to

plate 10. The software automatically assigns the corresponding number of wells in plate 10

to the reaction function.

In position 9.23 to 9.28, a reagent function has been assigned by the user and reagents

have been dragged into the mix position 13.3.

To set up a diluent, select the well, choose the dilution function, and drag the diluent into the

mix. The mix can now be dragged into the reaction. Once the mix has been dragged to the

reaction a one-to-many arrow will appear, showing the mix has been transferred to the

reaction area. The last figure on the bottom right shows two arrows.

The lower arrow shows the transfer of the mix to the reaction (one-to-many) and the upper

arrow shows the transport of the samples into the reactions. The upper arrow is a one-to-one

arrow, so each sample is transferred into a different well. For more details on transport

arrows, see 5.4.1.


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5.4.3 Auto Assignment of Transports in Empty Wells

The PIRO® software has several automated procedures which facilitate the setup of

transports. When a function has been chosen but no function of the target wells has been

defined, the PIRO® software will auto assign a function for the user, saving steps during the

setup of the transports.

Empty well: If an empty well or empty wells are chosen and moved, a sample function is

created in the well of origin and a fixed reaction created in the destination well.

Samples: If a sample or samples are dragged into an empty well, a fixed reaction will be

created according to the number of samples moved.

Reagents, Standards: If a reagents or standards are highlighted and dragged into an empty

well, a mix function is created and all reagents are transported into this mix.

Diluents: If a diluent or diluents are highlighted and dragged into an empty well, a mix

function is created and all diluents will be moved into this mix.

Mix: If a mix or various mixes are highlighted and dragged into an empty well, a fixed

reaction is created and the mix or mixes transported into this well.

Reaction or Reserved: If any of these three functions are highlighted and moved to an

empty well, no function in the target is created and a ‘forbidden’ sign will appear, indicating

that this is not a predefined setup option.


44

5.4.4 Function Colour Coding

The PIRO® software assigns a different colour to each function making it

easy to identify the function of individual wells.

Reagent PURPLE

Mix ORANGE

Diluent BLUE

Sample YELLOW

Reaction RED

Standard GREEN

Reserved GREY

No function WHITE

Examples of each function are shown below:

An empty circle: the well is empty.

An empty circle with colour on the outside:

a function has been assigned to the well

A grey filled circle with colour on the outside:

the well has been assigned both a function and a

transport.

A highlighted circle: the well has been selected.

A grey filled circle with colour on the outside

and a highlighted circle: a function as well as a

transport has been assigned and the reactions are highlighted.

An inner white circle: in addition to the functions

described above, the well has been selected in the

feature tree.

A line through the grey circle: Normalised sample

A line at the outside: Diluted sample, dilutions steps

are indicated by the amount of lines

Grids: Automatic Tube Switching

5.4.5 Transports and Selecting Wells

When wells should be selected for assigning a function,

position the cursor outside the first well to be used and

press the left mouse button. Move the cursor over the

wells to be used, release the left mouse button and the

wells are selected. If additional wells should be added

to the selection, use the ‘Ctrl’ button to select further wells.

In order to transport a selection, press inside the

selected well and move the selection to the position

required. The coloured outside of a well cannot be used

for the transport, as it is simply indicating what kind of

function is assigned to the particular well.


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6 Setting up a Run

This chapter gives details on how to set up a PIRO® run, using diluents, reagents, standards,

mixes, or samples. The feature tree and feature table are explained of each of the above

options. It also describes on how to set up templates for intelligent pipetting, perform cherry

picking of samples, or set the orientation of the plates. Serial dilutions and the normalisation

function are explained as well as the special functions like automatic tubes switching or

automatic fluid transfers.

6.1 Diluent

A diluent is usually used to dilute mixes in order to make them up to the final reaction

volume. Water is often used as the diluent, but the PIRO® software

allows other diluents to be used and multiple diluents can be assigned to

a run. This can be particularly useful when developing an assay, as it

allows multiple grades of water or different buffers to

be tested in one run.

The diluent feature table displays the well position, name, and volume of

each diluent.

6.1.1 Setting up Diluents

Select the wells to be used as diluents, and right-click over the selected

wells. Select ‘Add Function’ and choose ‘Diluents’. Diluents can also be

assigned using the function button in the layout menu. The diluent will

now be marked in blue.

6.2 Reagents

Selecting ‘Reagents’ in the feature tree highlights all the wells on

the deck, where reagents have been assigned. In this example,

ten reagents have been selected and three are shown in the

table. The number following ’Reagents’ indicates the number of

reagents set up in this run file (10). This does not necessarily

mean that all reagents are used in the run and no warning or

error message will be displayed if wells have been assigned as

reagents, but are unused.

As with any other function, reagents can be located in any position on the PIRO® deck. In

most cases, reagents are stored and used in 1.5 ml to 5 ml tubes and are therefore suitable

to be used in tube racks located from position 13 to 15. Reagents can also be assigned to a

32-well plate, which takes 1.5 ml tubes or any other suitable plate. The reagent table shows

the location (well), the name, and viscosity of each reagent (see 5.2.3).


46

6.2.1 Setting up Reagents

In order to set up reagents, select the wells to be used for reagents, then use the function

button in the layout menu (see 4.2) or right click over the selected wells and choose

‘Reagents’.

In this example, five

reagents on plate 9 have

been selected. To create a

mix using all five reagents,

simply select the reagents

and drag them into the

desired well. In this

example position 13.3 was

used and the PIRO®

software automatically

assigns this well as a mix.

The result will look like as

shown in the figure above

on the right.

Once the reagents have

been transferred to a mix,

the mix feature table will

open and the volume of

reagents required for each

reaction needs to be

specified. Once the

volumes have been

entered, the reagent setup

is completed.

Add/Remove: The ‘Add’

and ‘Remove’ functions

below the ‘Content of this

mix’ table can be used to

add or remove reagents

from the mix.


47

6.3 Mixes

The ‘mix

feature tree’

displays the

main heading of the mixes

as well as the submenus,

which are the various mixes

used in this project. The

number following ‘mixes’

indicates how many mixes

have been set up. Selecting

mixes in the feature free will

highlight all mixes set up on

the deck. Selecting one of

the mixes in the submenu

will highlight that particular

mix on the deck, as well as

all transports set up if the

transport button has been

activated. The number in the

submenu following each mix

will indicate how many

reagents have been used to

set up this mix.

Mix Submenu: This menu

allows for the name (see

5.2.2), ID, and viscosity (see

5.2.3) to be chosen. The

‘Volume added to the

reactions:’ means the volume

added to the reactions not

including the volume of the

sample. The sample volume

and the ‘Volume added to the

reactions:’ together is the

final reaction volume. ‘‘The

content of this mix’ shows the

reagents and diluents

included in this mix. Names

and volumes can be chosen

as well as the mixing option

for that particular mix (see 5.2.4).

Volume Calculation Submenu: This menu allows various volume calculations to be

selected. ‘Create Exact Volume’ allows the user to create a mix volume that is independent


48

of the volume calculated by the reaction set up. This option does not calculate if sufficient

volume to complete the run has been entered. ‘Calculate Volume from Reaction Count’

automatically calculates the volume of reagent required based on the reactions that will be

performed during the run. ‘Volume is Predefined / Premixed’, allows a mix to be used which

had previously been made either by the PIRO® run or manually.

Options Submenu: At this point of time the only option available under ‘Options’ is ‘Mixing

at end’. This allows the user mixing the mix created by the PIRO® after all reagents have

been added.

If ‘Volume is Predefined / Premixed’ is enabled, then in ‘Options’ the following window is

enabled, which allows ‘Mixing prior to usage’.

6.3.1 Setting up Mixes

Select a mix by assigning a mix function to a well, as for example shown below, see 6.6.1.

Drag the diluents and reagents into the well. Clicking Mix 1 in the feature tree displays the

transports in this project. To complete the setup, enter the ‘Volume added to Reaction’ and

the various diluent and reagent volumes to the feature table.

The mix is completed when no error message appears in front of the ‘Mix 1’ of the feature

tree, as shown above right.


49

6.3.2 Pipetting Mixes into Mixes

In most cases a mix is created and distributed in various reactions. But there are some

cases where the mix should be transferred in a further mix or mixes prior to the distribution

of the final mix into the reaction vessel. Shown below is an example and the way to set this

up in the PIRO® software.

On plate 9, 3 x 4 reagents have been set up. Reagents 1 to 4 have been transferred into mix

3 on plate 13, reagents 5 to 8 have been transferred into mix 2, and reagents 9 to 12 have

been transferred into mix 1. By simply dragging mix 3 into mix 2 and mix 2 into mix 1, the

final mix (mix 1) to be transferred into the reaction vessel has been created. Mixes do not

have to be prepared in a previous run in order to mix them with other mixes.

6.3.3 The Pipetting Order

The PIRO® has been assigned rules regarding the order of pipetting. When setting up a mix,

the largest volume will be pipetted first. If several reagents in a mix have the same volume,

the order will depend on the order in which the user entered them into the mix. For changing

the pipetting order, please see 5.4.1.


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6.4 General Orientation on Plates

If a specific orientation of e.g. various reactions is preferred it is necessary to choose the

orientation before setting up the run. Select the plate, in the case shown below, a 384-well

plate has been chosen. Open the drop down menu and choose ‘Horizontal’. The samples on

the plate will now be ordered horizontally.

This is independent of the orientation of the samples, they could be vertically as well as

horizontally orientated and the result on the 384-well target plate is horizontally orientated

reactions.

This is valid for ‘reactions’ (see 6.6.1), so the complete plate is orientated in a chosen

orientation. If ‘fixed reactions’ are used, than the orientation can be chosen plate

independently and several ‘fixed reactions’ could have different orientations on the same

plate (see 6.6.4.1).


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6.5 Samples

The sample feature tree displays the main heading of the

samples as well as the submenus, which are the sample groups.

The number following the ‘Samples’ indicates how many sample

groups have been set up by the user. Selecting

‘Samples’ highlights all samples set up on the deck.

Selecting a sample groups will highlight the

particular sample group on the deck, as well as

showing all transports assigned to that

particular sample. Right-clicking over ‘samples’ will open the option to add another sample

group. Right-clicking over a sample group will open the option to remove a sample group.

Sample groups can only be deleted, if the sample group has not been assigned to a

transport. If a transport has been assigned, the transport must be removed first by deleting it

in the feature tree. Once all transports have been removed, the sample group can be

deleted.

6.5.1 Setting up Samples

In order to set up samples for a run, select the wells by marking the area where the samples

should be located in (see 5.4.1). Having chosen ‘Samples’, the sample feature table is

showing the samples, inducing the position as described in the columns ‘Well’ and ‘Name’.

In this table, the position of the samples cannot be changed, but by clicking in the cells of

each of the other columns, the names can be altered as well as the ID and the concentration

filled in. If no ID has been assigned, the run will still start, but the software will display an info

sign (see 5.1.2), reminding the user to enter an ID, if required.

A special feature when setting up samples is that no target function has to be assigned if the

samples are to be transferred into a reaction. By dragging samples into an empty well, the

software automatically creates as many fixed reactions as samples have been transferred

(see 5.4.1).

6.5.2 Sample Options in the Deck Window

Right-clicking over

‘Samples’ opens the

following options in the

deck menu (see 4.5),

allowing for the below

actions to be taken:

Add samples to existing sample group: If several sample

groups have already been set up, choose the sample group, the

selected sample should be added to.

Remove samples from existing sample group: Individual

samples can be removed from a sample group.

Create sample group: A new sample group can be created.


52

Remove from sample group: The button below the sample table can be used to remove

samples from a sample group.

6.5.3 Skip Missing Samples

This function allows skipping samples even if the level sensing of the PIRO® does not detect

any liquid in the sample well. The run will continue with the next sample position without

asking for confirmation. If this function is ticked off, the robot will stop at the position where

no liquid has been detected and prompts the user to react, either ‘Ignore’ or ‘Continue’ the

run. This has been introduced as in some cases not all wells contain liquid but the PIRO®

should finish the run without intervention of the user.

Reasons for that could be that the extraction procedure was missing a sample which should

be ignored, or that in purpose a well has been left empty in order to keep the layout of the

samples, or for other user specific reasons. If this option was not turned on, then the PIRO®

would wait for a confirmation to either ignore or continue the run, the user would actually

need to be near the

PIRO® to confirm the

next action taken. With

the option turned on,

the PIRO® will finished

the run without asking

for confirmation

However, in the report

the PIRO® will record

the samples not

pipetted and the user

has a record of the

samples pipetted. This

function only makes

sense to use when level sensing is turned on.

In order to use this function, select ‘Project’ in the feature tree and tick the box ‘Skip missing

samples’. This function is related to the complete project. The PIRO® will now skip those

samples which do not have any liquid.

With this function turned on, the PIRO® only skips empty sample wells but does not skip

empty mix or reagent wells.

6.5.4 Sample Groups

A sample group is a group of samples. Multiple sample groups can be created in one run.

The same samples can be assigned to multiple sample groups, allowing different tasks to be

performed with the same sample. For example, seven samples can be set up; then five of

the seven samples are tested with mix 1 and four of the seven samples tested with mix 2.


53

Assigning samples to sample groups can be achieved in several different ways: mark empty

wells or select samples on the plate, right-click over the selected wells and choose ‘Create

Sample Group’ (see 6.5.2). The project table will then show a new sample group. A second

option is to right-click over ‘samples’ in the project table and choose ‘Add sample group’ (see

6.5). The desired samples then need to be added to the new sample group.

The figure above shows a table including

sample group 1 to sample group 8 and the

ticked boxes indicate the groups each

sample or various samples belongs to.

6.5.5 Sample Group Table

Various options are available in each

sample group table. The name of the group

and the samples can be changed in the

sample group table. The viscosity of the

sample can be addressed and the ID and

concentration can be entered. When a

sample is highlighted, the ‘Remove from

sample group’ button becomes active and a

sample can be removed from the sample

group.

Ticking the box ‘Return remaining quantity

for multi-dispensing’ means that the

remaining volume of the sample in the tip used is returned into the vessel where the sample

has been taken from. Depending on the height where the sample has been dispensed

during the ‘sample-multi-dispensing’ function, minimal remaining liquid on the outside of the

tip could be transferred back into the original sample vessel. The unticked box will discard

the remaining sample volume.


54

6.5.6 Creating Sample Groups from Samples located on different Plates

The PIRO® software is providing the option of creating sample groups from samples located

on different plates. This is of interest, when samples have been stored in different tube

formats or plates as shown below. 0.2 ml, 0.5 ml, 1.5 ml, 2 ml, and 5 ml tubes have been

used to create a sample group and have been transferred to a 96-well plate see below.

A sample group can also be created when the amount of samples is higher than the

available spaces on a given plate. Shown below are two 33-well plates. All 66 samples have

been allocated to one sample group and transferred into a 96-well plate. This also gives the

option of a sample group consisting of 4 x 96 samples to be transferred in one 384-well

plate.


55

6.5.7 Import

6.5.7.1 Import sample data

Existing sample data can easily be imported via the ‘Import’ in the file menu. Besides sample

names, ID, concentrations also indicators to determine further processing of the imported

samples can be imported (see 6.5.8). Select ‘Import sample data’/ ‘CSV Import’ and the

sample import window will open.

Prior to the sample import, a CSV file needs to be created and could contain the following

information:

Well position, name, ID, and concentration of

the sample. If the file is open in another

application during the import process, an error

message will appear saying ‘The entered file

cannot be opened’. Close the CSV file and

proceed.

Features of the file to be imported:

• The minimal file content consists of one column, e.g. the sample names or IDs. If no

column number for the ID is given, no import can take place but choosing the same

column number allows the import of the CSV file.

• Colum headers are not a necessity but allows for easier identification. There is no given

format for the headers of the columns to be used.

• Plate positions in the target plate are not mandatory, but allow the user to direct the

samples in certain positions. This could also be done by specifying the starting well

under ‘Start at position’ see below.

• In the CSV file there is no definite order of columns required this can be specified in the

boxes behind the sample import details.

• The CSV file can also contain columns not to be used for the import and would simply

not be specified in the boxes behind the sample import details.

• TXT files could also be imported but the file ending needs to be changed to .CSV

Prior to the sample import please make sure of the following points:


56

• The target plate provides enough space for the amount of samples to be imported

• The target positions are not in use by other samples or sample groups

• The names differ to the samples already present in the template

Open the file menu, select ‘Import’ / ‘Import sample data’ and click ‘CSV Import’. The sample

import filter window will open.

Select the browse button and open the folder containing the CSV file. Choose a

file by double clicking or select ‘Open’ to import the file.

Use the following encoding: Choose the geographic location.

Separator: Choose ‘Tabulator’, ‘Semicolon’, ‘Comma’ or ‘Other’. If for ‘Other’ is selected,

enter the separator used in the file to be imported. Tick the box ‘File has column headers’ if

the file has column headers.

The file contains column headers: This box should be selected if the columns have

column headers. If this box is ticked the first row of the CSV file will be ignored.


57

Import Range: Define the import range and type in the starting row. If a limit is to be set, tick

the ‘Limit to’ box and fill in the last row. Not ticking the box imports all rows defined in the

CSV file. Using the function ‘Rows’ allows defining specific rows to be imported, comma

separate the rows (e.g. 2, 4, 6) and press ‘Import’.

Sample Positions: Select how the

imported samples are to be

positioned in a defined plate setup.

Choose between:

‘Auto arrange within sample

function’, arranges all samples in one or more defined samples positions.

‘Read positions from file [column]’ imports all samples into predefined positions.

Choose between ‘Plate from Import File’ and other predefined plates.

‘Start at position [e.g: 5.a1]’ automatically arranges all samples from a defined

starting position.

Import Sample Name, ID, and Concentration need to be ticked and the column number of

the CSV file specified if any of those values are to be imported. If only one column should be

imported, then choose the same column number twice.

Sample Groups: Select ‘Sample Groups’ if imported samples are defining a new sample

group or if integration into existing sample groups should be performed:

‘Create new group’: Will add a new sample group to the setup

‘Add samples to

existing sample

group’: Select from

preexisting sample

groups

‘Read sample

groups from file’:

see 6.5.8.1

‘Insert samples

groups from file’:

see 6.5.8.1

If all settings are entered

correctly and no red cell or

text indicates an error, the

‘Import’ button becomes

active and sample data can

be imported. ‘Close’,

cancels the sample import

function.


58

6.5.7.2 Import volume data

This feature will allow in the future importing volume data and is currently under

development.


59

6.5.8 Intelligent Pipetting

The intelligent pipetting option allows a run to be set up directly from a database and once

set up, can be used repetitively for further runs simply by changing the import file. The three

steps required are explained below:

Set up a PIRO® template (see 6.5.8.1)

Setting up the CSV file for Import (see 6.5.8.2)

Importing the CSV file (see 6.5.8.3)

In the example below three parameters have been chosen to be tested (Virus-1, Virus-2, and

Virus-3) using three different mixes and sample groups.

6.5.8.1 Setting up a PIRO® Template for Intelligent Pipetting

a) Choose and select the plate layout to be used for the run. Mark an area and select

samples from the function menu (see 6.5.1). In this example the complete plate Nr. 9

has been selected. Unmark the samples. Right mouse click over samples in the

feature tree and select three different sample groups (see 6.5), which do not contain

any samples. Rename the sample groups accordingly; in this example the names

used are Virus-S1, Virus-S2, and Virus-S3. The sample groups need to have the

same name as the names in the database, see below.

b) Make sure that samples, mixes, and reactions do not have identical names, in the

example shown the samples are called Virus-S1, Virus-S2, and Virus-S3, the

reactions are called Virus-R1, Virus-R2, and Virus-R3 and the mixes are called Mix

Virus-1, Mix Virus-2, and Mix Virus-1.

c) Set up three fixed reactions areas (see 6.6.3), in the example shown below, plate 10

has been used. Name the fixed reactions accordingly; in this example the names

used are Virus-R1, Virus-R2, and Virus-R3.


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d) Set up three mixes (see 6.3.1) and name them accordingly, in our example the mixes

have been named Mix Virus-1, Mix Virus-2, and Mix Virus-1. In the reaction feature

table field called ‘Volume added to reaction’, add the volume to be used for each mix.

Continue until no error message is displayed in the mix feature tree anymore and

drag the mixes in the appropriate fixed reactions.

e) Select a fixed reaction. Press ‘Add’ to add a sample group to be used with this fixed

reaction. Select from the drop down menu and choose the appropriate sample group.

The file should look like as shown below:


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f) Save this file as a template.

6.5.8.2 Setting up the CSV-File for Import

To import the data create a file as shown in the example below:

The first column indicates the position where the sample should be imported to, the second

column indicates the names, and the third column indicates the ID of the sample. Columns

4, 5, and 6 show the tests which should be done for each of the samples. For sample ‘a’ all

three test should be done, for sample ‘b’, only Virus 2 should be tested and so on. As

mentioned above, it is important that the headings of columns 4, 5, and 6 are identical to the

sample group names in the PIRO® template. Once this Excel table is set up it is necessary

to save the file as a CSV file and it is not opened in any other program while importing (see

6.5.7).

On the bottom of the file above are also shown the total reactions of each test. This could be

helpful when setting up the amount of fixed reactions in the template.

Position Name ID Virus-S1 Virus-S2 Virus-S3

5.1 a 111 1 1 1

5.2 b 222 1

5.3 c 333 1 1

5.4 d 444 1 1

5.5 e 555 1

SUM 2 4 3


62

6.5.8.3 Importing a CSV-File

To import the CSV file open the above created template, select ‘Sample import’, ‘CSV

import’ and the sample import window will open (see 6.5.7). Select the file to be imported,

and select ‘Read sample groups from file’. Fill in the columns from the CSV file where the

description is given what test should be done with what sample. Press import and the PIRO®

run file is setup. Future run files can simply be set up by opening a template, importing the

samples from the CSV file and press start run.


63

6.5.8.4 Import Samples in Consecutive Order

In the above example, three different fixed reaction areas have been created in order to

separate the three different tests from each other (see 6.5.8.1c). Another option is to have

the reactions aligned in a consecutive order. For that to be achieved, proceed as outlined

above (see 6.5.8.1) but instead of setting up fixed reactions, simply set up one reaction area

(see 6.6.1) enough for all reactions to be done. In the

feature tree, right mouse click over ‘Reaction’ and

choose ‘Add reaction’. Importing the samples (see

6.5.8.3) will now order all the reactions in a consecutive order.


64

6.5.9 Barcode Reading

The PIRO® offers the option of

barcode reading the sample IDs. This

firstly facilitates the user with naming

the samples rather than typing in

every sample by hand. Secondly,

sample IDs are often needed for LIMS

integration. In order to do that, use a

manual hand scanner which can be

supplied by DORNIER-LTF and

connect it to the free USB port. Open the sample list of a project, click into the table of

sample name or sample ID and scan the barcode. No further software is required.

If the barcode reader has been supplied by DORNIER-LTF make sure the correct country

code setting are used. For more information the manual of the barcode reader has been

uploaded in the customer area of DORNIER-LTF.

The PIRO® also offers the option of barcode reading the Plate IDs, as shown in the figure

above. Select the orange field above and use the barcode reader to read the Plate ID.

In addition, ID for standards could also be given, for more details see 6.7.3.


65

Position ID conc

5.a1 a 1

5.a2 b 2

5.a3 c 3

5.a4 d 4

5.a5 e 5

5.a6 f 6

5.a7 g 7

5.a8 h 8

6.5.10 Normalisation of Sample Concentrations

The PIRO® software allows samples to be normalised. Samples with different concentrations

are diluted accordingly so all the samples will result with the same concentrations. The

samples are normalised within intermediate wells and can then be used for setting up

reactions or other procedures required in the same run. Normalized samples are shown with

a black line through a circle (see 5.4.4).

6.5.10.1 Import Sample Data

Sample concentrations can either be imported or directly typed into the software. Shown

below is an example of a CSV-file, where the position, ID, and concentration have been

given. By importing the CSV-file (see 6.5.7), the concentrations are imported and a new

sample group can be created.

6.5.10.2 Setting up a Normalisation

The next step is to create the normalisation procedure. Firstly, assign a diluent anywhere on

the deck, as without a diluent, no normalisation will take place. Next, select the samples to

be normalised, drag them over an empty well, right mouse click, and choose ‘Create

normalisation’ from the pop-up menu.

A new window appears showing the ‘Sample group 1 – Normalisation 1’ in the feature tree.

Fill in the requested information in the feature table, including a diluent as well as ‘Minimum

sample volume’ and ‘Minimum usable volume’. The ‘Minimum sample volume’ is the volume

of sample to be used from Sample Group 1 (undiluted samples) to do the normalisation with

and ‘Minimum usable volume’ is the volume required from the normalised samples for further

reactions.


66

Once the above information has been filled in, a reaction and mix can be created and the

normalised samples can be used to set up a run.

In the example above, the ‘Normalise to lowest concentration’ option has been used, which

means that the normalisation has been performed to the lowest sample concentration. The

other option is to ‘Normalise to target concentration’, where a different concentration can be

used to normalise the samples to. Samples with a lower concentration will then be ignored

but a warning message appears in the feature tree (see below on how to set that up).

The ‘Positions’ tab in the feature

table gives an overview of the

details on the dilutions to be

done. ‘Source conc.’,

‘Normalised conc.’, ‘Sample

volume’, ‘Diluent volume’, ‘Total

volume’ and ‘Diluent ratio’ are

indicated.

The ‘Options’ tab in the feature table allows for samples not to be aliquoted, as they have the

final concentration already. To allow for that, tick the box ‘No aliquot for undiluted samples’.

If the complete sample group will be used for further reactions, the undiluted sample will then

be used from the original sample well.


67

‘Mixing at the end’ allows

introducing a mixing step after

the normalisation by simply

indicating a number of times to

mix.

Samples could also be filtered

by a given concentration. To do

so, tick the box following ‘Filter

samples’ and set a range of

concentrations to be used. In the

example below, all samples with

concentrations above 7.00 and

below 2.00 have been excluded

from the normalisation step.

The orientation of the normalised samples can either be chosen automatically or set

manually.

Using the deck menu, two further options are available:

‘Add positions to

normalisation’ allows the

user to add more position

than currently available and

‘Remove position from normalisation’ allows the user to remove positions.

6.5.11 Dilution Series of Samples

With samples dilution series can be performed.

Before starting set up a diluent, as otherwise an

error message will appear. A dilution series cannot

be done without a diluent. Select the samples to

be diluted and drag them into an empty well. Right

mouse click over the new well and select ‘Create

dilution’.


68

If further dilutions should be created, repeat the above procedure, ending up in various

dilutions of the original sample, as shown above. With each dilution step an additional line is

added to the circle of the sample on the screen indicating the number of dilution steps done.

In the above example, five dilutions have been created. Names are also created

automatically as shown below, but can be changed if necessary.

When the dilution has been set up a window appears. Click on the ‘Dilution’ window to add

the information requested. The name can be changed, a concentration unit given and a

diluent chosen if several diluents have been created when setting up the run. In the lower

part of the window, the dilution ratio can be given as well as the final volume to be created.

The ‘Positions’ window shows the details of the dilutions to be done.

The ‘Options’ window

allows for specific

details to be changed

including filtering of

samples as well as

choosing the

orientation of the

samples. This

window is similar to

the window described

in normalisation (see

6.5.10.2).


69

6.6 Reactions and Fixed Reactions

Reaction wells are defined as carrying the final result of the pipetting steps in a certain

project. Reactions are the end of a pipetting project and are normally taken to be analyzed in

a further instrument.

Fixed Reactions: The PIRO® has an additional definition for reactions, called fixed

reactions. Fixed reactions help the user the setup of reactions,

and have their own special rules in a reaction area. If a

reaction has been set up as a reaction or fixed reaction can be

seen in the feature tree following the name of a particular

reaction; in the example shown above reaction 3 has been set up as a fixed reaction. There

are two numbers following a fixed reaction, the first number indicates how many fixed

reactions are in use and the second number indicates how many fixed reactions have been

set up. The orientation of fixed reactions on the plate can be changed, replicates can be

treated differently, and options to assign various mixes are realized. For more information on

fixed reactions, please see 6.6.2.

The reaction feature tree displays the main heading of the reactions and fixed reactions as

well as the submenus, which are the various reactions used in the project. The number

behind ‘Reactions’ indicates how many reactions have been set up. Clicking ‘Reactions’

highlights all reactions set up on the deck. Clicking on one of the reaction tabs in the

submenu only highlights those particular reactions on the deck, as well as all transports set

up for those reactions, if the transport button in the transport menu has been activated. The

number behind each reaction in the submenu indicates how many reactions have been used

in this run file.

6.6.1 Setting up Reactions

To set up a reaction area, mark the wells to be used

and either use the function button in the tools menu

or right-click over the chosen area and select ‘Add

Function’ then ‘Reactions’. A reaction area has now

been created.

In order to add samples to the reaction area, select

empty wells or define samples and drag them into the defined reaction area as shown

above. When using this function, be aware that the number of samples cannot exceed the

number of reactions. If ignored, samples will be missing in the reaction. The same procedure

can also be applied when cherry picking samples, meaning samples from various different

locations are chosen. Simply use the ‘Ctrl’ button on the keyboard, select the samples of

interest and drag them into the reaction area.


70

A sample volume now has to be entered into the feature table, as indicated by the red cell as

well as by the error message appearing in the feature tree. Once a sample volume has been

entered, the error message will disappear and in the case of the example shown below, a

warning, (see 5.1.3) message will be displayed, as no mix has been added to the reaction as

yet.

6.6.2 Reaction Feature Table

The reaction feature table has

three different submenus, reaction,

positions, and options.

Reaction: This window specifies

the ‘Name’ of the reaction, the

‘Reaction volume’, the ‘Content of

the reaction’, including the mixing

options available. All parameters

could be changed in this window,

except the ‘Reaction volume’,

which is the sum of the mix and the

sample per reaction.

When changing the volume of a mix, it also has to

be changed in the mix window. A warning sign will

appear if the two values do not match.

Position: This window gives details of the well

position, the ID, and the concentration of the

reaction, if required.


71

Options: This window gives several

options to the user. Ticking ‘User defined

orientation’ allows the user to automatically

let the software choose the default

orientation of the reactions. The orientation

options (see 6.6.4.1), are only available for

the ‘fixed reactions’. ‘Replicates’ can

simply be added by changing the number

in the replicate box.

‘Allow mix without samples’ is an option to

be used when no sample will be added to

the final reaction. As the software is mainly

set up for PCR users, a mix without a

sample is defined as an error, but this

option can be ticked to allow the reaction to be set up without the addition of a sample.

6.6.3 Setting up Fixed Reactions

The first option to set up fixed

reactions is by dragging samples or

selected empty wells into the desired

fixed reaction area (see 5.4.1). This

creates as many fixed reactions as samples have been dragged into the fixed reaction area.

The second option is to highlight an area where the fixed reaction is supposed to be then

right-click over the well and select ‘Create fixed reaction’. The same menu also allows

‘adding positions to an existing fixed reaction’. The third option to set up fixed reactions is to

choose ‘Options’ in the reaction table and tick the box called ‘Is fixed reaction’. This

transforms a reaction into a fixed reaction.


72

6.6.4 Special Features of Fixed Reactions

6.6.4.1 Orientation of Fixed Reaction

When using fixed reactions, the orientation of the samples in the reactions can be changed,

so for example horizontally orientated samples can be changed to vertically orientated

samples.

6.6.4.2 Replicates when using Fixed Reactions

If ‘fixed reactions’ are chosen the number of replicates to each ‘fixed reaction’ can differ. If

only ‘reactions’ have been used, all ‘reactions’ have to have the same number of replicates.

‘Fixed reactions’ therefore allow for more flexibility when setting up reactions in general.

6.6.4.3 Fixed Reactions and Intelligent Pipetting

Fixed reactions should also be used when a sample import or Intelligent Pipetting step is

performed. Due to the separation in fixed reactions each fixed reaction can have its own mix

or sample group assigned. If however, the reactions should be in a consecutive order, then

‘reactions’ instead of ‘fixed reactions’ should be used. For further details in regards to the

intelligent pipetting please see 6.5.8.4.


73

6.6.5 Reaction Export

Sample data and reaction results can be

exported as a CSV file. To export a

reaction, open the file menu, select

‘Reaction export’ and click on ‘CSV

Export’.

Two further options are available, which

is the direct export to samples files used

in the LightCycler and the Rotor-Gene.

In case multiple reaction plates have

been prepared by the PIRO®, different

export files for individual plates and

formats can also be selected.

This opens the ‘Reaction Export Filter’

Click the tab with the dots to

open the hard drive directory, name the

document, select where to save the file,

and select ‘Export’.

Use the following encoding: Choose

the preferred option:

Separator: Choose between ‘Tabulator’,

‘Semicolon’, ‘Comma’, or ‘Other’. For

‘Other’ type the separator to be used.

Tick the boxes if column headers and

plate info have to be exported as well.

Click on ‘Export’ to finish or ‘Close’ to

cancel.

An example of a reaction export is

shown below:


74

6.6.6 Sample-Multi-Dispensing

Warning: The ‘Sample-Multi-Dispensing’ feature could lead to cross-contamination of a

reaction. It is therefore important to verify prior to any run using the ‘Sample-Multi-

Dispensing’ feature if the outcome of the use of this feature is according to the aim of the

user.

The ‘Sample-Multi-Dispensing’ feature has been developed to multi-dispense samples into

reactions. According to the PIRO® rules, a reaction including various reagents is prepared

and the PIRO® will then use a new tip for every sample. This is the most secure way not to

allow for potential cross-contamination as the PIRO® will normally not multi-dispense

samples.

The advantage of using ‘Sample-Multi-Dispensing’ feature is that a large amount of tips

could be saved, so more cost effective runs could be done. Two options are available:

‘Enable Sample-Multi-Dispensing for replicates’: Using this option will multi-dispense

samples into reactions, where the reactions have the same mix, meaning that replicates are

prepared. If the tip containing the sample will touch the reaction, only a minimal amount of

mix on the outside of the tip could potentially be transferred into the next reaction. If this is

considered to be a contamination needs to be decided by the user.

‘Enable Sample-Multi-Dispensing for all reactions’: Using this option will multi-dispense

samples independent of the reaction content. This means that a sample will also be multi-

dispensed into reactions with different reaction content, therefore potentially contaminating

from the outside of the tip the next reaction with mix from the previous reaction. It therefore

needs to be taken great care if such a setup makes sense to apply for the user.


75

The advantage is that if the same sample should go into for example eight different reactions

that seven times less tips are used. This is a considerable amount of tips to be saved.

This function has been implemented as a PIRO® user has extensively tested this setup for

cross-contamination and no cross-contamination has been observed in the users’ setup.

DORNIER-LTF therefore decided to implement this option.

In addition and to allow for further flexibility, the PIRO® software has also the option to set

different x, y, and z-offsets when using the ‘Sample-Multi-Dispensing’ function. The general

offset values are normally pre-calibrated when the consumables have been set in the

software or if new consumables different to the software are used, the user has set such

values.

Using the ‘Sample-Multi-Dispensing’ function now also allows changing the x, y, and z-

offsets only for the ‘Sample-Multi-Dispensing’. This means that the mix will be pipetted

according to the initial settings, but the samples will be pipetted according to the new x, y,

and z-offsets chosen. As an example, the sample could therefore be dispensed above the

reaction or on the side of the wall of the reaction vessel. This will allow for contamination free

pipetting when using the ‘Sample-Multi-Dispensing’ function. However, the user has to take

great care that such settings are according to the expected results.


76

6.7 Standards

The standard feature tree displays the main heading of the standards

as well as the submenus, which are the various standards used in this

project. The number next to ‘Standards’ indicates how many

standards have been set up. Clicking ‘Standards’ highlights all the standards set up on the

deck. Using one of the standard tabs in the submenu highlights the standards in the

submenu on the deck, as well as all transports set up. The number next to each standard in

the submenu indicates how many standards have been used in the run file.

6.7.1 Setting up Standards

A diluent should be set up before setting

up any standards (see 6.1.1), as standards

cannot be set up without a diluent even in

virtual mode. If no diluent is set up, the

software only allows a ‘Ready to use

standard’ to be used.

To set up standards mark the wells to be

used, right click over the marked well and

choose ‘Create standard’ see below.

6.7.2 Ready to use Standard

A ‘Ready to use standard’ is one which

has been created prior to the run. The

‘Ready to use standard’ only allows the

concentration to be changed.

6.7.3 Creating a Standard

The PIRO® robot can be used to create

standards. Un-tick the box ‘Ready to use

standard’ and enter the required

information.

A master standard can either be placed on the same plate or rack, but can also be provided

on a different plate or a different sized tube. This means that the master sample does not

have to be aliquoted.

Minimum Volume to create [µl]: A volume of 50 µl is the default setting in the PIRO®

software, but the volume to be created can be adjusted. If the value is changed, the software

will automatically recalculate the volumes needed.

Diluent: If several diluents have been set up on the deck, the software allows the

appropriate diluent to be selected when setting up standards. This enables different diluents

to be evaluated in the same run.

Mixing: The ‘mixing’ option allows the number of times the mix should be mixed to be

defined by the user. This is particularly important for standards as the following standard

dilution has to be provided with a homogenous standard.


77

Change Tips: This option allows for tips to be changed when creating the standards.

Standard IDs: This field allows for IDs to be given, manually or with a barcode reader.

Standard Table: The standard table shows the details of the standards to be created.

Shown in the first column is the well position, the second column shows the dilution ratio to

be used. The original standard is called the ‘Master’ standard.

Concentration units: A concentration can be entered in the concentration column if

desired.

Diluent Added [µl], Sample Added [µl], Sample Taken [µl], and Created Vol [µl] indicate

the volumes the PIRO® will transfer during the run. The volumes can be altered by the user if

required.

6.7.4 Standard Options in the Deck Window

‘Create standard’ creates a

standard.

‘Add Positions to Existing

Standard’ adds a position to

an existing standard.

Create Standard Group: see 6.7.5


78

‘Add Standard Positions to Existing Standard Group’ adds standard positions to an

existing standard group.

‘Remove Standard Positions from Existing Standard Group’ removes standards from an

existing standard group.

6.7.5 Standard Group

The use of the standard group function has been developed to

provide an option to select only standards of interest as not all

standards are always used in a particular run, even though they have been created by the

PIRO®. The software therefore allows creating a group of standards derived from the

standards created originally. In many cases the user has a high concentrated master

standard but only wants to use the lower diluted standards. In other cases, the user would

like to use every second standard dilution, as the wide concentration range should be

covered. Performing a one-step 1:100 dilution is less accurate than doing a two-step 1:100

standard dilution.

To set up a standard group, create a standard as shown below. In our example, 12

standards have been created.


79

Now only choose the standards desired and drag them into a fixed reaction, in our example,

only every second standard should be used. A new standard group will automatically be

created. Any combination of standards can be chosen to create a new standard group.

6.8 Reserved

This option allows the user to reserve certain wells on the deck to prevent them from being

used. This function may be used if parts of a plate have been used during a previous run. In

some instruments, certain areas might not work

identically compared to others, which could be

excluded from usage as well. Certain output files

should look identically, so reserved areas could be

used without given up the option of an automatic

orientation provided by the software. In the

example shown on the right, positions C1, D1, and

E1 have been marked as reserved. Five standards

from plate 9 have been transferred to plate 10, starting at position A1. The

standards have automatically been added to positions A1, B1, F1, G1,

and H1. Positions C1, D1, and E1 are not used as they have been

marked as reserved wells.

6.8.1 Setting up Reserved Area

To set up a reserved area, select the wells to be used and use ‘Functions’

to create a reserved area. The reserved feature tree will then display the

number of reserved wells in the project as well as the position and names

of the reserved wells.


80

6.9 Special Functions of the PIRO® Software

6.9.1 Automatic Tube Switching (ATS)

The PIRO® has the option to automatically switch the tube when empty and use a second

alternative tube with the same content. This is possible for reagents, diluents, and mixes.

6.9.1.1 Automatic Tube Switching for Reagents and Diluents

Assign a position with the function ‘Reagent as described (see 6.2.1) and the feature window

opens. Two windows are shown titled ‘Assigned wells’ and ‘Unassigned wells’. Choose the

location where the alternative tube should be located by pressing the ‘+’ sign and

the available well positions will appear. In this case, position 13.2 has been

chosen. Press the arrows to transfer the well 13.2 to the window called ‘Assigned

wells’ and the position is defined. Alternative wells could be assigned as many as positions

are available. The alternative tube is displayed with a grid.

6.9.1.2 Automatic Tube Switching for Mixes

The same principal can be applied to mixes, however, before doing that choose ‘Volume is

predefined / premixed’. Open the ‘Options’ window and move the well to ‘Assigned wells’ as

described above and an alternative well containing the same mix is defined.

Switching level: The volume level when switching a tube can be defined by the user and is

measured in mm. Be aware that the level must be within the definitions of the tube size

which the software will recognize. If 1 mm is chosen, then the user needs to make sure the

calibration of the tubes is done correctly.


81

6.9.2 Automatic Fluid Transfer (AFT)

The “Automatic Fluid Transfer” option allows the user to carry over remaining fluid from a

reagent or mix into an alternative well with the same content. An ‘alternative well’ is

described above (see 6.9.1), meaning the same reagent or mix in a second or third tube. If

the ‘automatic tube switching’ is turned on, the remaining fluid from the first tube will be

aspirated and transferred into the second tube with the same content and so on, therefore

not wasting any fluid.

As with the ‘Automatic Tube Switching’ option, for mixes the option ‘Volume is predefined /

premixed needs to be chosen.


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7 Quick starting the PIRO®

Start the PIRO® software.

Choose the desired plates by using ‘Plates’ in the Layout Menu.

Highlight the reagents and use the ‘Function’ button in the Layout Menu to assign the

function. Drag the reagents into a well where the mix is supposed to be.

Assign a diluent function to a well and drag the diluent into the mix.

In the feature table, add the reagent volumes to the mix and specify the ‘Volume added to

reaction’.

Mark the wells containing the samples; assign a sample function and drag the samples into

the position where the reactions are supposed to be. The software will automatically assign

a reaction area. Specify the sample volume in the feature table.

Select the mix and drag into the reaction area.


83

8 Appendices

8.1 PIRO® Alarm List

Alarms Description Consequence

There are no tips with the needed volume left. User needs to

replace the empty tip rack.

Not enough tips for the project Change tips and push "resume"

There is not enough fluid left in the vessel to finish the

transport. Please make sure that there is enough fluid in each

vessel. You can continue or stop the run.

Not enough fluid for the project User decision, push "resume" or "stop"

The tip is lost during a job execution. Please check the project

for contamination. Robot run is stopped.

Tip lost You must check for contamination and

start the project from the beginning

The current job cannot be completed successfully because

there is no tip attached to the robot head. Maybe it has been

lost. Robot run is being stopped

Tip lost You must check for contamination and


An unexpected error happened during job execution. Unable

to continue the robot run. Please check the project for

contamination

unexpected error

You must check for contamination and


Disconnected while executing command Communication is interrupted Check USB cable. In addition you must

check for contamination and start the

project from the beginning

Communication lost Communication is interrupted. Check USB cable. In addition you must

check for contamination and start the

project from the beginning

Unable to reach x axis position Axis are blocked, light barriers are not

working or motor does not work

Call distributor

Unable to reach y axis position Axis are blocked, light barriers are not


Call distributor

Unable to reach z axis position Axis are blocked, light barriers are not


Call distributor

Tip switch is always active PIRO can not drop the tip or switch is

not working

Try to drop the tip manually and start

the project again. Change pipetting head

Pump limit switch is always active Limit switch is not working Change pipetting head

Pump switch not found Pump switch is not working Change pipetting head

Pump Capacity is too low Pipetting head has not enough volume

for the job

You must check for contamination and

start the project from the beginning. In

rare case change the pipetting head

The robot pump reports an invalid volume. Please check if the

pump is connected properly. If the pumpe is connected

properly, please contact the distributor. Pump serial number

________

Pipetting head is not working or lost its

memory

Change pipetting head


84

INDEX

A

Add ....................................................................... 36

Auto Assignment of Transports in Empty Wells ... 43

Automatic Tube Switching .................................... 80

C

Calibrations .......................................................... 12

Changing Names ................................................. 37

Changing the Order of Transports ........................ 39

Checklist .............................................................. 32

Cherry Picking ...................................................... 69

Cleaning Acrylic Plexiglas .................................... 12

Clipboard Menu .................................................... 27

Colour Coding ...................................................... 44

Computer ............................................................. 14

Copy ..................................................................... 27

Create Sample Group .......................................... 51

Cut ....................................................................... 27

D

Deck Layout ......................................................... 18

Deck Window ....................................................... 34

Default Project...................................................... 26

Delete ................................................................... 27

Diluent .................................................................. 45

Dilution Series ...................................................... 67

Document Info ...................................................... 33

Document Restriction ........................................... 33

Document Security ............................................... 32

Downloads ........................................................... 17

E

Enable sorting the transport list manually ............ 39

Error ..................................................................... 36

Exchanging Existing Plates .................................. 25

Exit ....................................................................... 27

F

Feature Table ....................................................... 36

Feature Tree ........................................................ 35

File Menu ............................................................. 26

Fixed Reactions ................................................... 69

Force Tip Change ................................................ 30

Functionality of the Lid ......................................... 12

Functions ............................................................. 28

H

HEPA-Filter .......................................................... 11

I

Import ................................................................... 55

Info ....................................................................... 35

L

Language ............................................................. 29

Layout Menu ......................................................... 27

Level Sensing ....................................................... 30

Level Sensing each (times) .................................. 30

Lid......................................................................... 12

M

Manual Orientation ............................................... 71

Mix Volume Calculation Submenu ........................ 47

Mixes .................................................................... 47

Mixing Options ...................................................... 37

Move ..................................................................... 27

Move to home position ......................................... 31

Multi-Dispensing ................................................... 30

N

New ...................................................................... 26

O

Open Project ........................................................ 26

Options Menu ....................................................... 29

Output Range ....................................................... 33

Overview of Racks and Adapters ......................... 21

P

Paste .................................................................... 27

Pause ................................................................... 31

Pipetting Heads .................................................... 11

PIRO® Manual ...................................................... 17

PIRO® Workspace ................................................ 18

Plate Functions ..................................................... 25

Plate Submenu ..................................................... 38

Plates ............................................................. 28, 38

Pointer .................................................................. 27

Position Offsets .................................................... 24

Positioning the Robot ........................................... 11

Post Run Report ................................................... 32


85

Project .................................................................. 26

Q

Quick starting the PIRO ....................................... 82

R

Racks ................................................................... 21

Racks and Adaptors ....................................... 18, 21

Reaction Export.................................................... 73

Reactions ............................................................. 69

Reagents .............................................................. 45

Recent Files ......................................................... 26

Remove ................................................................ 36

Remove Samples ................................................. 51

Reserved Area ..................................................... 79

Robot Menu ......................................................... 31

S

Safety ................................................................... 12

Sample Groups .................................................... 52

Sample-Multi-Dispensing ..................................... 74

Samples ............................................................... 51

Save As ................................................................ 26

Save Project ......................................................... 26

Separator ............................................................. 56

Setting up Transports ........................................... 42

Show Transports .................................................. 28

Software Installation ............................................. 14

Software Updates ................................................. 16

Special Racks ...................................................... 22

Standard Group.................................................... 78

Standards ............................................................. 76

Start ..................................................................... 32

Start Robot ........................................................... 32

Starting the Software ............................................ 17

Stop ...................................................................... 31

T

Template .............................................................. 26

The Waste Hole .................................................... 20

The Waste Rack ................................................... 20

Tip ........................................................................ 38

Tip Disposal .......................................................... 19

Tip Racks ............................................................. 19

Tips....................................................................... 19

Tools Menu ........................................................... 27

Transports ............................................................ 38

Triangles ............................................................... 35

Tubes ................................................................... 23

U

Undo, Redo .......................................................... 28

UV Program Duration ........................................... 31

UV Radiation Procedure ....................................... 11

V

Virtual Mode ......................................................... 17

Viscosity ............................................................... 37

W

Warning ................................................................ 36

Waste ................................................................... 38

Waste Console with Drawer ................................. 20

Well Position Description ...................................... 36

Z

Zoom .................................................................... 28