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This Owner’s Handbook provides a guide to working with the Hidex 300 SLTM liquid scintillation counter. It is valid for software versions as follows:

Hidex 300 SL embedded software, v. 1.44, 1.77 and 1.82 Hidex 300 SL MikroWin driver, v. 5.04 MikroWin 300 SL, v. 5.53

A number of optional features and modes of operation described in the handbook may not be available on your instrument.

Hidex Oy operates a policy of continuous development; therefore we reserve the right to make alternations and improvements to the Hidex 300 SL liquid scintillation counter without prior notice.

The Hidex 300 SL liquid scintillation counter is designed for use in research work only. It is not intended for clinical diagnostic use.

Hidex and Hidex 300 SL are trademarks of Hidex Oy.

MikroWin 300 SL is trademark of Mikrotek Laborsysteme GmbH.

Microsoft, Windows and Excel are registered trademarks of Microsoft Corporation.

Document Number

24th October 2014

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1 INTRODUCTION 9 1.1 Hidex 300 SL in brief 9 1.2 Purpose of this handbook 10 1.3 Important general warnings 10

2 UNPACKING AND CONNECTING HIDEX 300 SL 11 2.1 Equipment supplied (425-201) 11 2.2 Connecting the power and computer 11 2.3 Installing MikroWin 300 SL software 12

2.3.1 System Requirements 12 2.3.2 Installation of software 12 2.3.3 Installation of driver 12 2.3.4 Setting the COM Port 13 2.3.5 Instrument Configuration 14 2.3.6 Updating the Driver 14 2.3.7 Installation settings 14 2.3.8 Default folders and files 15

3 GETTING STARTED WITH HIDEX 300 SL 17 3.1 Introduction 17 3.2 Switching on 17 3.3 Setting assay parameters 18

3.3.1 Setting counting windows or ROI’s 19 3.3.2 Tray sizes and types 19 3.3.3 Vials to be measured 20

3.4 Saving the parameters 20 3.5 Loading/unloading the vial tray 20 3.6 Measurement 21

3.6.1 Start 22 3.6.2 Stop 24 3.6.3 Adding more vials and continuing with counting 24

3.7 Handling the data 24 3.8 Exporting results 25

3.8.1 Export driver settings 25 3.8.2 Data export 27 3.8.3 Template specific export driver setup 28

4 DETAILS OF ASSAY PARAMETERS 30 4.1 Counting 30

4.1.1 Counting time 30 4.1.2 Max counts 30 4.1.3 Activity Type 31 4.1.4 Coincidence time 31 4.1.5 Ionized delay (seconds) 31

4.2 ROI 31 4.2.1 ROI number 31 4.2.2 ROI type 31

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4.3 Data 32 4.3.1 CPM 32 4.3.2 DPM 32 4.3.3 TDCR 32 4.3.4 Chemi 32 4.3.5 Counts 32 4.3.6 Dead Time 32 4.3.7 Counting Time 32 4.3.8 End Time 32 4.3.9 QPE 33 4.3.10 QPI 33 4.3.11 Lumi Counts 33 4.3.12 Temperature 33

4.4 Repeats 33 4.4.1 Vial repeat 33 4.4.2 Range repeat 33

4.5 Spectra 33 4.5.1 Beta 33 4.5.2 Beta Triple 34 4.5.3 Alpha 34 4.5.4 Alpha Triple 34 4.5.5 2D Plot 34

4.6 Advanced Options 34 4.7 External Standard 34

4.7.1 Use External Standard 34 4.7.2 Counting time (seconds) 34

4.8 Cooling 35 4.8.1 Cool during Measurement 35 4.8.2 Switch Cooling off after Measurement 35

4.9 Guard 35 4.9.1 Use guard 35

4.10 Alpha Specific Parameters 35 4.10.1 Alpha Mode 35 4.10.2 Alpha delay time 35 4.10.3 Alpha y offset 35 4.10.4 PLI limit 35 4.10.5 Y gain 35 4.10.6 Gate Time 36 4.10.7 Spill to Beta 36

4.11 Screen CPM 36 4.11.1 Screen Type 36 4.11.2 Target CPM Low 36 4.11.3 Target CPM High 36 4.11.4 Target Sigma 37

4.12 Delay before measurement 37 4.12.1 Use delay 37 4.12.2 Delay (hh:mm:ss) 37

5 INTRODUCTION TO DATA REDUCTION FEATURES OF MIKROWIN 300 SL 38 5.1 Measurement data window 38 5.2 Calculation Matrices 39

5.2.1 Organization 39

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5.2.1 Presentation of the Results 40 5.2.2 Calculation formula for new matrices 41 5.2.3 Calculations based on raw data 43 5.2.4 Associating data with matrices in repeat counting 44

5.3 Setting the tray layout 46 5.3.1 Work with replicates 46 5.3.2 Define multiple groups 46 5.3.3 Overlay of tray 47

5.4 Controls 47 5.4.1 Definition of Controls 47 5.4.2 Control calculation 47

5.5 Quench curve 48 5.5.1 Standard concentrations 48 5.5.2 Curve fitting algorithm 48

5.6 Classifications 49 5.6.1 Threshold calculation 49

5.7 Printing 51 5.7.1 Setting up the Report 52

5.8 Saving data and templates for further use 53 5.8.1 Saving data 53 5.8.2 Saving templates 54

5.9 Other 54 5.9.1 Viewing the data 54 5.9.2 Exporting the data 55 5.9.3 Printing out the data 55

6 DOUBLE-LABEL COUNTING 56 6.1 Constant quench samples 56

6.1.1 Define counting efficiencies 56 6.1.2 Future assays 57 6.1.3 Calculation formulae used in the counting 58 6.1.4 Principle of ROI settings in double label counting 58

6.2 Double label counting with the external standard 59 6.2.1 Creation of double label quench curves with the external standard. 59 6.2.2 Counting unknown double labelled samples 63

7 SUPER LOW LEVEL OPTION 65 7.1 Operation 65

8 ALPHA/BETA SEPARATION COUNTING 66 8.1 Alpha/beta separation calibration with MikroWin 68 8.1.1 Alpha Specific Parameters 69

Alpha delay time 69 Alpha y offset 69 PLI limit 69 Y gain 69 Gate Time 69 Spill to Beta 69

9 USING THE REPORT RENDER MACRO 70 9.1 Importing spectra and measurement data 70

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9.2 How to optimize ROIs 72 9.3 Exporting spectra and measurement data, spectrum subtraction 72 9.4 Problems with using Report Render Export selected data button 74 9.5 Programmatic access to Visual Basic Project is not trusted 74 9.6 Unable to set the XValues property of Series class 75

10 COOLING MODULE OPTION 76 10.1 Nature and purpose of the module 76 10.2 Cooling settings 76

11 ORGANIZER 77 11.1 Using Organizer to enter sample information 77 11.2 Creating batches 78 11.3 Adding to batches and prioritizing measurement 79 11.4 Using Toggle Mode 80

REFERENCE SECTION 81 12.1 Principle of liquid scintillation counting 81 12.2 Čerenkov counting 81 12.3 Phosphorescence 81 12.4 Quench correction 81

12.4.1 Quench correction with TDCR method 81 12.4.2 Quench correction with standard quench set 82 12.4.3 Storing a quench curve of a standard quench set 84 12.4.4 Correction of quench in Hidex 300 SL with internal standardization 86

12.5 End CPM criteria 86

13 TROUBLESHOOTING 87 13.1 Connection Troubleshooting 87 13.2 Recovering if a vial remains inside the detector chamber 88

14 ROUTINE MAINTENANCE 89 14.1 Cleaning and maintaining the instrument 89

14.1.1 Outer case 89 14.1.2 Sample vial compartment 89

14.2 Other periodic maintenance tasks 89

GLOSSARY 91

INDEX 94

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1 Introduction

1.1 Hidex 300 SL in brief The Hidex 300 SL is a liquid scintillation analyzer with an automatic sample changer and a triple photomultiplier tube detection assembly. The Hidex 300 SL is used to measure liquid scintillation samples in all typical scintillation vials, from 5 to 20 mL, loaded in sample racks.

Temperature stabilization, available using the instrument’s cooling module option (see page 74), supports measurement at a controlled temperature optimal for the scintillation cocktails used. The instrument allows automatic calibration of sample activities using the TDCR absolute counting method or, as a further option, via an external standard method. The Hidex 300 SL runs with embedded software controlled by MikroWin 300 SL user interface software. This software controls all the instrument functions as well as providing data processing and analysis, and reporting functions.

Note! The Hidex 300 SL liquid scintillation analyzer is intended for professional laboratory research use by trained personnel, who understand the nature of liquid scintillation analysis.

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1.2 Purpose of this handbook This handbook has been designed to give new users the knowledge they need to benefit from all features of the Hidex 300 SL. It provides a systematic explanation starting with unpacking and connecting the instrument, through routine operation to the advanced features of the instrument.

If your instrument is not yet connected, please start by reading section 2, Unpacking and connecting the instrument.

To learn to measure your samples quickly and efficiently, please proceed to section 3, Getting started with Hidex 300 SL, on section

se 17, which provides the minimum information you need. It is recommended that all users read this section of the manual.

More comprehensive general information is to be found in the next two sections, 4, Details of Assay parameters on page 30, and 5, Introduction to data reduction features of MikroWin 300 SL on page 38.

Sections 6-10 provide more specialist information: Double label counting on page 56, Alpha/beta separation counting on page 66, Cooling module option on page 76, and Organizer on page 77.

The Reference section (section 12) on page 81 and Troubleshooting (section 13) on page 87 can be referred to as needed.

A brief Routine maintenance section (section 14) on page 89 helps you to keep your Hidex 300 SL clean and in good working order.

1.3 Important general warnings Caution! Do not remove the instrument cover. Only Hidex-trained service personnel are authorized to do this.

Caution! Use proper laboratory practices when handling any hazardous materials. Samples can be potentially infectious. Dispose of all used vials, disposable gloves, syringes, disposable tips, etc. as biohazardous waste.

Caution! Always wear gloves when performing any kind of maintenance or service, especially if it involves potential contact with spilled fluids or fluid residues of any kind.

Note! The Hidex 300 SL instrument weighs about 135 kg (298 lbs). Four persons are required to lift the instrument, one gripping each lift bar.

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2 Unpacking and connecting Hidex 300 SL

2.1 Equipment supplied (425-201) For model version 425-201 the pack contains the following items:

Hidex 300 SL counter Owner’s Handbook on CD Hidex CD with QA report, parameter template library, newest MikroWin driver, Report Render Spectrum Analysis software and Service software. Power supply 100-240V/24V RS-232 cable MikroWin 300 SL Instrument control/data acquisition software Sample Rack (x 2)

2.2 Connecting the power and computer Caution! Before connecting the power, ensure that the power switch on the left hand side of the back panel of the instrument is in the Off position.

Caution! Never operate the instrument from a power outlet without an earth connection.

Remove all packaging materials from the items listed above. Place the instrument near your computer, on a firm surface at table height. The instrument weighs about 135 kg (298 lbs).

Assuming your computer has a serial port, connect the serial cable from the instrument to this.

If your computer does not have a direct serial port you may need to install a USB serial port adapter.

Connect the adapter to a free USB port in the computer. Connect the serial cable from the instrument to the adapter. If you experience difficulties with connection, please refer to Connection Troubleshooting on page 87.

Connect the power box (transformer unit, supplied with the instrument) to the Hidex 300 SL instrument, and connect the power box to the mains using an earthed connection.

Power switch

Power connection

Computer connection socket

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2.3 Installing MikroWin 300 SL software

2.3.1 System Requirements MikroWin 300 SL requires Microsoft Windows 7 or a more recent Windows operating system. To make good use of MikroWin 300SL, the personal computer requires a minimum of 2 GB (better 4 GB or more) available memory. Also a Monitor resolution of 1280 x 1024 points or higher is required. Depending on installation options, a MikroWin 300SL installation requires 20…40 MB of available disk space.

2.3.2 Installation of software With the computer already switched on and running, insert the MikroWin 300 SL CD (PC interface software for Hidex 300 SL software) into the drive. If the AutoPlay mode of the drive is active, the MikroWin 300 SL installation program will start automatically.

As the program installs follow the instructions on the screen.

MikroWin 300 SL software is delivered with a hardlock protection system. After completing the installation you need to attach the hardlock (dongle) to a USB port on your computer.

Note: Do not attach the hardlock before completing the installation of the software.

2.3.3 Installation of driver Start the MikroWin 300 SL software, for example, by clicking the desktop icon.

Then switch on the Hidex 300 SL instrument (power switch at the back of the instrument).

Select Driver from the Installation menu of MikroWin.

Although you will see a default driver for Hidex 300 SL listed under the Reader tab, you should update this to the latest driver version, provided on the Hidex CD and MikroWin Driver. For instructions on this, see Updating the driver (page 14).

When you have installed the latest driver version, select it from the listing under the Reader tab.

Click Driver Setup and supply the required settings.

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2.3.4 Setting the COM Port Choose the appropriate COM Port. If you do not know which one it is, you can find out from the System properties box under your computer’s Windows Control Panel. For information, refer to Connection Troubleshooting on page 87.

Click Get Instrument Configuration to obtain these settings

Use Windows Control Panel to establish which COM port is in use

Use Windows Control Panel to establish which COM port is in use

Click Get Instrument Configuration to obtain these settings

Use Windows Control Panel to establish which COM port is in use

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2.3.5 Instrument Configuration Click Get Instrument Configuration to transfer the appropriate information on the configuration of the instrument to the computer.

2.3.6 Updating the Driver The procedure described below only needs to be performed if you already have a Hidex 300 SL driver installed and you need to replace it with a newer version. As mentioned above, you should anyway replace the default driver supplied with a new instrument, with the driver that is supplied on the Hidex CD and MikroWin Driver. Hidex and MikroTek are constantly reviewing the drivers for MikroWin 300 SL software and, as a result, further new driver versions may become available from time to time.

First remove the existing driver as follows.

Select Drive from the Installation menu as described above.

Select the Hidex 300 SL driver and select Delete Driver.

Use Windows Explorer to go to ProgramData\MikroWin 300 SL\ and delete the file hidex2.xdl.

Save the new driver file in a temporary folder.

Note that if the file is saved directly in the Program Files\MikroWin300 SL\ folder the installation will not work.

Again select Driver from the Installation menu.

This time, choose Add Driver.

Browse to the folder you have saved the new driver in, select the driver and click OK.

The Hidex 300 SL driver should appear as one of the installed reader drivers listed in the Installation Driver dialog box.

With the Hidex 300 SL driver selected, click Driver Setup and then proceed with identifying the COM port, getting the instrument configuration, and selecting the needed advanced parameters, as described in sections 2.3.4 and 2.3.5 above.

2.3.7 Installation settings The Installation Settings dialog allows you to personalize your counting system and to specify various default settings.

To open the Installation Settings dialog, choose Settings from the Installation menu.

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Under the General tab you can specify measurement units and plate size, and also check various options.

Under other tabs you can set default folders for your files (see following section) manage user access, specify font types and manage how system use is logged.

2.3.8 Default folders and files Choosing the Directories tab allows you to define default folders for both measurement data files and template files that contain counting parameters. These folders are used for file operations (load, save) whenever a path has not been selected. For both data and template files, an individual folder and its path are specified. In addition, a further path for transfer operations (e.g. export files) can be specified.

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Measurement Directory allows you to specify a folder (and its path) for data file (*.dat) operations. This folder serves as the default for opening and saving data files.

Template Directory allows you to specify a folder (and its path) for template file (*.par) operations. This folder serves as the default for opening and saving template files. If no special folder is chosen, all defined and saved templates will be stored in this folder.

Transfer Directory allows you to specify a folder (and its path) for file exports (e.g. export of results, storage of external curve fit data).

Instead of using the Installation Settings dialog as described above, you can click the Export toolbar button to open the File Export dialog directly.

Click the Browse button to open the Browse for Folder dialog.

Parameter template files delivered along with the Hidex CD should be copied under the Para-folder. The quench curve .MW5fit-files delivered along with the Hidex CD should be copied under the Transfer-folder

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3 Getting started with Hidex 300 SL

3.1 Introduction Before starting, you need to know what nuclide you wish to measure, and the parameters you want to use to measure it.

Your Hidex 300 SL instrument will support:

Liquid scintillation counting for detection of beta or alpha emitting nuclides, including all commonly used beta labels. Among these are labels such as 125I and 51Cr that also emit gamma radiation and are thus often regarded as being only gamma labels

Alpha/beta separation as an optional feature

Super low level counting as an optional feature

3.2 Switching on Assuming your Hidex 300 SL instrument is already set up with PC or laptop connected, you may power up the computer and the instrument in either order.

After you have switched your computer on you should start the MikroWin 300 SL application, for example using the desktop icon. Ensure that the dongle is plugged into one of the USB ports (see Installation of software on page 12).

When you switch the instrument on, the green LED in the front panel of the instrument is illuminated and the instrument is ready for use.

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3.3 Setting assay parameters Hidex provides a selection of MikroWin template files that you can use for different types of measurements and calculations. When you start the program the default template file is loaded. If you wish, you can change this by selecting another one from those available. Click the Open Template button. You will see a list of the available templates in the Para directory (under Type, the files will be marked as PAR). Choose one of them and click Open. Although the template you have chosen will include all of the measurement parameters, you may modify these, and save the new settings as a new template file. How to do this is briefly explained below. If you wish to specify different results calculations in your template, then please see Calculation matrices on page 39.

When you have MikroWin 300 SL running, with the default template or the new one you have chosen, you will see a view as above.

Open the Hidex 300 SL Options box by clicking the Settings button or choosing Settings from the side menu (under Template and Reader).

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The Hidex 300 SL Options box shows the settings associated with the template file that you have chosen. The settings cover measurement technology criteria, tray type and vials to be measured, as well as other parameters. You may accept these settings or specify new values. More detailed information on template files and settings is given under Details of assay parameters on page 30.

3.3.1 Setting counting windows or ROI’s The term, region of interest (ROI) means a counting window.

Choose the number of ROI’s. There can be from 1 to 4 of them and they can be either preset nuclide windows or “Free” windows.

The ROI is selected from the drop-down menu. For each of the listed nuclides, there are default values for the low and high limits of the counting window.

Alternatively you can select Free from the list. Free allows any window to be set.

3.3.2 Tray sizes and types Two types of vial tray or rack may be used with Hidex 300 SL. The tray with smaller vial capacity takes up to 40 vials in 5*8 format. The tray with larger capacity follows standard microplate 8*12 format, and takes up to 96 vials. In practice the two tray types are themselves similar in size.

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3.3.3 Vials to be measured The vials to be measured are selected by “painting” or dragging with the mouse over the tray area required. While you are dragging them, the selected vials will appear shaded in blue. After the selection is complete they will be filled with black circles.

All vials can be selected by clicking the # symbol in the upper left corner of the tray layout.

You can clear the tray layout with Clear All.

3.4 Saving the parameters In order to save all settings made in a new assay, parameters should be saved with a new name.

Choose Save As from the File and Template menus.

In the Save As dialog, select the desired file type in the Save as type field. Choose MikroWin Para File to activate the template file format (*.par).

Now enter the name of the new assay in the File name field. Do not enter any file name extension because the application adds the necessary extension by itself.

Click the Save button. All the assay information, including the settings you make for printing and possible export of the data (see Exporting results on page 25) will be saved to disk, and can be reloaded every time you need it.

3.5 Loading/unloading the vial tray

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Before unloading the tray, make sure that the instrument is in the ready state (green LED on). Pull the tray holder out and remove the tray.

3.6 Measurement Prepare your samples in an appropriate way, by mixing them with scintillation cocktail. Information on the liquid scintillation technology is to be found in the Reference section on page 81.

Before starting measurement make sure that all vials have been properly capped. It is important that the caps are tightly closed, because the vials are going to be lifted with a pneumatic grip by their caps.

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3.6.1 Start

Under Tray ID you need to supply an identifier that will serve as the name of a file for storage of the measurement data after a run.

If you wish you can specify preset characters that will appear automatically. To preset characters choose Automation from the Installation menu and make your settings in the Installation Automation box that appears.

You can choose from various menu items or make your own free-text setting. Having made your choice, exit the dialog by clicking OK.

Click the Start button to start measurement. During the reading the measurement status is shown in the Status Information field. Also, while the reading is going on, the Start button is replaced with a Cancel button. Click this if you need to terminate the reading prematurely.

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The data is stored immediately using the given file name and the measurement values can be viewed as counting progresses in the Measurement Values window. In the software Live spectrum is available from Results window Graphics Spectra. The spectrum will be automatically updated in the vial position.

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3.6.2 Stop Measurement will automatically terminate when all the specified measurement repeats have been completed. If an empty vial position is detected you will be invited to confirm that you have finished counting and can do this by clicking Yes. If you have used the Organizer feature (see page 77) to run a number of different parameters, and an empty vial is detected, the current parameter will be stopped and the next parameter in line will be automatically started. If there have not been any errors during the run, the current measurement will be seen in the measurement bar. In case of errors at a certain position, that cell in the tray representation of the measurement data is marked with a red triangle. This marker is also shown in all results matrices at the same position. Errors will also be marked with suspended measurement, e.g. after clicking the red Cancel button.

3.6.3 Adding more vials and continuing with counting Since counting is temporarily suspended, the inclusion of an empty vial position can provide a useful opportunity to load additional vials. The vial positions for the new vials must be selected. When the End Counting confirmation box (see above) is being displayed, you can pull the tray holder out even though the red LED is on. Add additional vials to the next measuring positions in the tray and close the door. Click Cancel to count the newly added vials. Additional vials may be added even while measurement is in progress, provided the instrument robotics is not moving.

3.7 Handling the data After completing the measurement the instrument returns to ready mode. You can choose to view the measurement data in MikroWin 300 SL, print it out, or export it to other programs such as MS Excel. Further evaluation of the data can also be done using the MikroWin program itself. The MikroWin Help Card assists you in the efficient use of the program’s features as you perform the data reduction steps.

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Hidex provides a selection of MikroWin template files that you can use as templates for different types of calculations or you can create a new template with your own calculations specified as explained under Calculation matrices on page 39.

3.8 Exporting results In order to be able to transfer your results to other programs or host systems, you need to set the export driver. This is done in the same way as setting the reader driver, described earlier.

3.8.1 Export driver settings To access export settings choose Driver from the Installation menu. In the Installation Driver dialog select the Export tab. You will see a list of drivers. The three most common export drivers used are line export, matrix export and raw data export drivers. The biggest differences between these drivers is that line and matrix export drivers export what is defined in the calculation matrices while the raw data export driver exports all data points chosen by well or by list in the export driver dialog (see below).

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In this example, let’s select Line Export driver. Then click Driver Setup to access the settings.

In the Line Export Driver dialog, choose a header or footer for the exported data. This can include, for example, date, time operator information etc. You can see the options available by clicking the Add button.

Matrices show the calculations and result data. Calculation matrices hold formulae such as sum, average, CV% etc. Result matrices show the corresponding results for the same matrix. How to specify and edit matrices is explained later, under Calculation matrices on page 39. Here, select which matrices are to be exported. Specify the operation mode and export target as indicated above.

Having made your settings, click OK in the Line Export Driver dialog.

Export file format

Target folder

Choose to export general statistics (instrument setup can be viewed with every set of data)

Select the matrix for export, and click Add to bring new matrices to list

Header/footer can be used to enter text in export file

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The corresponding driver dialogs for Matrix Export and RawData Export are:

3.8.2 Data export

Measured data can be exported to other software applications either manually or automatically.

If you wish to export the data manually, click the Export button or choose Export… from the File menu.

Select the driver from the drop-down list of drivers.

Click OK.

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If you wish to have automatic export of data after a measurement, the required export driver needs to be configured for automatic export before the run.

From the View menu chose Export and Configuration.

In the Configuration dialog you can toggle the manual or automatic export option for the selected driver.

Select the required Export Driver.

Click Enable / Disable Automatic to change the usage to automatic and click OK.

3.8.3 Template specific export driver setup The Configuration dialog also provides the possibility to set template specific driver configurations for use instead of the default configuration. Under Config you can see whether the driver is using its default configuration (default) or a template dependent configuration (user).

To change from default to user, click the Export Driver Configuration button.

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The driver configuration dialog is then displayed so that you can make the needed modifications. All settings made in this dialog will be part of, and stored into the template currently loaded when the template is saved.

To restore the default configuration, use the button, Reset to Default Configuration; the configuration of the selected driver will be reset to the default driver configuration, and data export will be performed independently of the template currently loaded.

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4 Details of assay parameters

The Options box, as already explained (see page 18), is obtained by clicking the Settings button or choosing Settings from the side menu (under Template and Reader).

4.1 Counting

4.1.1 Counting time Counting time is given in seconds. Counting the sample is stopped, at the latest, when this time is reached. However counting may stop earlier if the Screen CPM condition is met or the count value set under Max counts is exceeded.

4.1.2 Max counts This is a second count terminator, in counts. Note that actual end count value may be greater. A time check is done only once a second and the actual count number is used. The counts are then measured according the settings for ROI #1 .

Data that is collected from the counter to form measurement data matrices is listed here.

Counting time is in seconds from 1 to 9999.

A value other than 0 sets a condition that stops counting of a vial even though counting time is not complete.

From 1 to 4 ROI’s (counting windows) can be set. They can be ”Free” or preset nuclide windows.

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4.1.3 Activity Type Three settings are possible for Activity Type: Low, Standard and Alpha. Both Low and Standard are for beta activity measurements.

Low, which is always required when Guard is used, is optimized for the highest sensitivity and has a count rate from 1 to 999,999 CPM. In MikroWin 300 SL driver software version 1.07 and subsequent versions the CPM and DPM values produced are to one decimal place.

Standard is optimized for broader dynamic range and has a count rate of 1 to 3,999,999. CPM and DPM values are rounded to integer values.

Alpha is selected when alpha separation is enabled. When alpha/beta separation is being performed it is necessary to select at least one ROI for alpha counts.

4.1.4 Coincidence time This determines the period of time when two or three PMTs must detect photon(s) in order for them to be registered as a coincidence pulse. The time starts when the first pulse arrives; a pulse from the other detector(s) must arrive before the coincidence time has elapsed. Triple coincidence requires that all three detectors detect a pulse within the time window. The default time is 35 ns.

4.1.5 Ionized delay (seconds) The period of time over which the sample is kept in the ionized air flow can be set here.

4.2 ROI

4.2.1 ROI number ROI (the region of interest) is a section of the MCA from which counts are collected. Up to four ROIs can be determined. Each ROI has a number of selections.

4.2.2 ROI type The field to the right determines from which MCA the counts are collected.

The ROI type choices are:

Beta BTriple Alpha ATriple

Selection of Beta will mean that counts are calculated from the beta spectrum. If alpha/beta counting is available, Beta or Alpha selection determines from which spectrum (Alpha or Beta) counts are calculated. By selecting BTriple and/or ATriple you can also collect just the triple coincidence counts, either from beta, or alpha, or both spectra.

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As the triple counts determine the statistical precision of the TDCR method this (triple counts) ROI can be used as a CPM source in the End CPM criteria (see page 36). The requirement is that this ROI must be ROI_1, therefore results have to be collected from the other ROI.

4.3 Data Here you can see the results that are sent from the counter to the PC, and the calculations that you can see in the measurement data window. Each set of data represents a matrix.

Using the raw data export driver all data can be exported to another program.

4.3.1 CPM This is the figure for accumulated counts multiplied by the dead time.

4.3.2 DPM This is the CPM figure divided by TDCR. It is assumed that the ROI used in obtaining TDCR covers the whole active area of the nuclide.

4.3.3 TDCR TDCR stands for triple to double coincidence ratio and its role as an index of quench is explained under Quench correction with TDCR method on page 81.

4.3.4 Chemi This is the estimated chemiluminescence. Chemiluminescence is liable to appear in the region of channels 5 to 150 of the MCA, so if the ROI is above this, chemiluminescence would not be expected to represent a problem.

4.3.5 Counts The total accumulated counts within the set ROI and within the counting time. These are raw counts, not corrected for dead time.

4.3.6 Dead Time Dead time, a correction factor applied to the raw count figure to compensate for missed counts.

4.3.7 Counting Time The time actually used for counting. Counting time is given in seconds. It can be less than the set Counting time if counting terminates on the basis of Max counts or a set Screen CPM value.

4.3.8 End Time The time at which counting stopped, in seconds, counted from previous midnight.

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4.3.9 QPE QPE is the approximate border line in the spectrum where the left window consists of 99 % of the total counts of the whole spectrum. The value is typically fractional, i.e. it is between two channels.QPE is used with External Standard when creating Quench curve (page 56). It is also used with Unquench Standard Set when couting efficiency of Hidex 300 SL.

4.3.10 QPI Internal Quench parameter. Technically this is the centre of gravity of the coincidence counts spectrum of the sample.

4.3.11 Lumi Counts Luminecence counts per second when the instrument is used as luminometer.

4.3.12 Temperature Temperature is specified in °C. If you have optional cooling module in the Hidex 300 SL instrument see page 74.

4.4 Repeats

4.4.1 Vial repeat Defines how many times the vial is measured, the default is 1 meaning that the sample is measured only once. The vial is not moved from the detector during measurements therefore the differences in results of the same sample should reflect only the statistical uncertainty of radioactive decay and the instrument stability. In the current software version spectra are not available with Vial repeat.

4.4.2 Range repeat This is another form of repeat counting where the same tray can be counted a number of times. Range repeat and repeat selections can be combined. In the current software version spectra are not available with Range repeat.

4.5 Spectra Selects which spectra are sent to MikroWin 300 SL. The spectra are then visible under Results and Spectra. The spectra can also be exported as a .csv file. The exported spectral files are stored in the MikroWin300 SL\transfer folder with the .csv extension. The name of a spectrum is constructed from the assay name that has been given and the vial position.

In addition to allowing viewing of traditional spectra on two axes, MikroWin 300 SL can also show the 2-dimensional alpha/beta separation plot.

In the current software version importing spectra to MikroWin 300 SL prevents the use of repeats.

The different spectra available are:

4.5.1 Beta Prints the spectrum of which triple and double counts are collected.

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4.5.2 Beta Triple Prints the spectrum of which triple coincidence counts are collected.

4.5.3 Alpha Prints the spectrum of which the counts categorized as alpha are collected.

4.5.4 Alpha Triple Prints the spectrum of counts that have caused triple coincidence, and that are categorized as alpha pulses.

4.5.5 2D Plot Prints a 2-dimensional alpha/beta separation plot.

4.6 Advanced Options Clicking Advanced Options opens a separate dialog allowing you to adjust the settings for the various options and special features covered by the driver setup and listed in the driver setup dialog (see the section on installing the driver page 12, and the illustration there). In the image below, External Standard, Cooling, Guard and Alpha beta, are optional modules supplied for the Hidex 300 SL. Selection for these are not normally shown if these options are not installed.

4.7 External Standard This selection is available if your instrument has the optional external standard feature. Please refer to the page 59 for more information on External Standard.

4.7.1 Use External Standard If this box is ticked external standard counting time selection is enabled. All samples are counted twice, first with the external standard automatically positioned under the detector chamber for the selected time. QPE (Quench Parameter External) can be plotted against efficiency of standards with the curve fit function of MikroWin 300 SL. Please refer to the Quench correction section in this manual (page 81) for more information on quench correction methods.

4.7.2 Counting time (seconds) Counting time for external standard is given in seconds.

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4.8 Cooling This selection is available if your instrument has the optional cooling feature. Please refer to page 74 for more information on cooling.

4.8.1 Cool during Measurement Switch the cooling on when you start measurement.

4.8.2 Switch Cooling off after Measurement Switch the cooling off after measurement to save on power consumption.

4.9 Guard This selection is available if your instrument has the optional guard feature. When using Guard, the activity type must be set to Low. Please refer to page 35.

4.9.1 Use guard For the use of super low level option please activate the guard by selecting this.

4.10 Alpha Specific Parameters This selection is available if your instrument has the optional Alpha Beta measuring feature. Please refer to the page 65 for more information on Alpha Beta separation counting.

4.10.1 Alpha Mode The available options are Counting and Raw_Data. Counting is used for all measurement runs, while Raw_Data is intended for service use only. For the recommended Hidex cocktail, setup has already been performed at the factory.

4.10.2 Alpha delay time A period of time in which both, the beta and alpha contribute the pulse (measured from the pulse start). Alpha and beta pulses are separated based on their length. The first part (up to the value used) is saved as beta and period of time after this as alpha.

4.10.3 Alpha y offset This adjustment moves the 2D plot (without PLI limit) in vertical dimension. Alpha y offset was called alpha tail offset with earlier MikroWin 300 SL drivers than 1.08.

4.10.4 PLI limit PLI stands for pulse length index. It moves the PLI limit in vertical dimension and therefore changes alpha/beta classification. The higher it is the less counts are collected to the alpha spectrum. If the Show PLI box is checked then the PLI limit is represented on 2D plots by a horizontal line, usually between the alpha and beta spectra. The selection does not affect alpha/beta classification.

4.10.5 Y gain This adjustment stretches the 2D plot (without PLI limit) in vertical dimension.

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4.10.6 Gate Time A period of time in which only alpha contribute the pulse (measured after alpha delay time). Gate Time is only shown with MikroWin 300 SL driver 1.08 and Hidex 300 SL embedded software, v. 1.56 or later.

4.10.7 Spill to Beta If this option is selected alpha pulses that are over 31 in the Y axis of the 2D plot are not included as alpha spectrum but forced to Y pos 1 and contribute beta spectrum and results. This feature could be used in cases when the alpha activity is very small compared to the beta activity and beta spills to alpha side.

4.11 Screen CPM

4.11.1 Screen Type The Screen CPM selection allows additional conditions to stop counting of the sample, before the set counting time is reached. The End CPM choices are:

None

Upper

Lower

Middle

Lower or upper

4.11.2 Target CPM Low Lower border limit.

4.11.3 Target CPM High Higher border limit.

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4.11.4 Target Sigma Confidence interval. 1 sigma means app. 1-1/es confidence level.

See page 86 for actual count end criteria.

Counts are taken from a full ROI of 0 to 1023 channels.

aaaaaaaaxxxxxxbbbbbbbbxxxxxxcccccccc

----------------low------------------high------------

End CMP regions: a = if lower b = if middle c = if upper a or c = lower or upper x = uncertain region, this is a function of accumulated counts.

4.12 Delay before measurement

4.12.1 Use delay Determines the period of time the instrument waits before actually starting to measure. The principal use of this is to make sure that chemi luminescence has disappeared before counting.

Note! When delay is in progress the instrument LED is green. When the actual counting starts the LED will turn to ORANGE.

4.12.2 Delay (hh:mm:ss) Delay time before measurement is set in form of hours, minutes and seconds.

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5 Introduction to data reduction features of MikroWin 300 SL After performing your assay, you can view the data in the measurement data window or graphical windows and print it out, export the data to other programs, such as MS Excel for evaluation, or you can use MikroWin 300 SL software itself for data reduction. A number of MikroWin 300 SL protocols are supplied for this or you can create your own protocol with your required data evaluation (see page 39).

5.1 Measurement data window When counting proceeds in MikroWin, data is collected under the measurement data window. It shows the result for each vial after counting of that particular vial has finished.

The measurement data window shows the raw data values collected from the counter. Each of the data options listed on the Options menu (see page 30) is represented here as an individual plate. Since you would normally be interested in viewing a number of different plates - as an example, you might choose CPM, DPM and TDCR - you can switch from one to the next by selecting from the list in the lower part of the display.

In order to see the above-mentioned list of matrices that interest you, you need to choose the matrices, as will be explained below under Calculation Matrices on page 39.

There are two drop down menus, Active Tray and Active Label.

Active Tray is relevant when repeat measurements are selected. If 10 repeats are selected then each label has 10 plates of data. In our example all the data selections become individual labels. CPM is referred to as Label 1, DPM is now Label 2 and TDCR is Label 3. Each label has several plates of data. Each repeat is one plate.

Active Label is relevant when spectral import is selected. In this case one label is the spectra. Spectrum has 1024 channels and each channel is stored as a plate under the label, therefore there are 1024 plates of data. Any other data selected e.g. CPM and TDCR also have their own label and 1024 plates of data. This means that Spectra import and repeat cannot be selected simultaneously. If repeat is selected only spectra export is possible.

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Over the next pages you will learn about the main data reduction features available for users of MikroWin 300 SL Full version.

5.2 Calculation Matrices Matrices are used to calculate and show the result data. Information on them is found under the Matrices section of the Template side bar menu.

5.2.1 Organization Click Organization to open the dialog box, which has four tabs.

Under the Organization tab you can see the matrices already defined. They are listed by name and number.

If you would like to define a new matrix, click Add Matrix.

Click Edit Matrix and supply a name for the new matrix.

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The new matrix name will also appear in the bar at the bottom of the display.

As a rule each matrix performs a particular calculation relevant to the name you have assigned. Please consider carefully which results should be shown in each matrix.

To use the new matrix you have created, see the section, Calculation formula for new matrices on page 41.

To delete an existing matrix, select it and click on Delete Matrix.

5.2.1 Presentation of the Results Under the View tab, you can review and adjust the settings associated with the results, which appear in the Results window or printout report.

You can select the font used for each matrix and define the amount of characters used for the result display.

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The Combination tab allows combination of matrices so that you see the values for each vial together.

Choose, for example, CPM as the main matrix in the Matrix field on the left (lower left picture). You might then wish to view DPM, TDCR and Chemi values too. Click Add Matrix to place these additional matrices in the Combination field.

The Sequence tab (lower right picture) allows you to choose the operating sequence.

5.2.2 Calculation formula for new matrices The values that are available for use as raw data matrices (see page 32) are as follows.

CPM

DPM

TDCR

Chemi

Counts

Dead Time

Counting Time

End Time

QPE

QPI

Lumi Counts

Temperature

These names are recognized by the software as formulae, so to apply them, you simply associate the value name with the vial.

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Let’s say that for a particular template we have set a new calculation matrix for QPI (see above, page 39).

Type QPI into the Calculation Formula of Position A01 field and press Enter on your computer keyboard, so that the value appears for position A01.

Drag that square so that all of the required positions are filled with the same value, or simply click the # mark in the corner so that all positions are filled.

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5.2.3 Calculations based on raw data You can also add formulae or other values for the calculation matrices. To the right of the Calculation Formula field is an Add Formula button (see, for example, the previous image). Click on this button to open a dialog used to choose a formula like SID for Sample ID (found under the group, Tray-Based Variables).

To perform calculations you must refer to the raw data, and you must refer to it by the matrix number.

For example, let’s say the raw data settings in your template are as follows, and you wish to calculate the activity in Bq.

Data item Matrix number Counts MA1 CPM MA2 TDCR MA3 DPM MA4 Chemi MA5

The calculation involves subtracting the background from the CPM value and dividing by the TDCR value.

The background is a numerical value (in CPM) for the appropriate label, so you simply create a matrix for it and specify the known value as the formula.

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Then create another new matrix and specify the detailed calculation formula using matrix numbers. These numbers are formed from the abbreviation “MA” and the order number of the items listed under Calculation Formula of Position xxx on the left of the display.

Data item Formula Matrix number Counts Counts MA1 CPM CPM MA2 DPM TDCR MA3 TDCR DPM MA4 Chemi Chemi MA5 Background 6 MA6 Activity Bq (MA2-MA6)/MA3/60 MA7

5.2.4 Associating data with matrices in repeat counting You may often wish to count the same sample several times and perform statistics on the values obtained.

Let’s say, for example, that you want to count your vials twice, obtaining CPM and TDCR as raw data values each time.

Make the appropriate settings under Repeats in the Options dialog.

Then under the Organization tab create 4 matrices for statistics. For both CPM and TDCR we are going to calculate the mean and standard deviation, so the four statistics matrices can be named, Mean CPM, St Dev CPM, Mean TDCR, and St Dev TDCR.

In the picture on the right this operation is almost complete: just select “Untitled”, click Edit Matrix, and type the name, St Dev TDCR.

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To obtain the formula for the statistical calculations, click the Add Formula button and choose the items needed from under Multitray functions.

First we choose the mean or MAVE(MEA) and overwrite MEA as CPM. Then we click Add Formula again and choose standard deviation or MSD(MEA). In this way we set the calculation matrices as MAVE(CPM), MSD(CPM), MAVE(TDCR) and MSD(TDCR).

Associate the calculation matrices with the relevant vial positions as explained in section, Calculation formula for new matrices, on page 41.

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5.3 Setting the tray layout The Tray Layout section of the Template side menu allows you to access the views, Partition, Grouping and Overlay.

5.3.1 Work with replicates The Partition view allows you to combine vials, so that the individual values are added together to produce one value in either Mean or Median form. Different control positions should not be combined. The Tray Partition dropdown list is used to select the type of partition.

Select the particular partitioning type that corresponds to your needs. Just click the position where you require the new partition to occur. You will see that the graphical representation changes each time a position is clicked.

5.3.2 Define multiple groups The Grouping view allows you to define a multiple number of tests on one sample tray. All calculations are performed within each group.

With the aid of the Tray Grouping dropdown list you may select the group of your choice. If a group has been selected it may be assigned to the part of the sample rack simply by clicking the vial required. Directly afterwards it will be visualized in the vial of your choice.

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5.3.3 Overlay of tray In the view you will see the Tray Overlay dropdown list. First choose the overlay type you require like PosCon for a positive control, or Sample for an unknown sample. Click the vial or group of vials using the mouse. You will now see that the desired vials have been assigned with the specified control. In a similar fashion other controls may be assigned to the sample rack.

Note: If a Blank sample is used and determined, the measurement value of this sample will automatically be subtracted from the results of the other samples.

5.4 Controls Go now to the Controls section of the Template side menu and choose Control Positions.

5.4.1 Definition of Controls On the upper part of the sample rack you can find the Control Type options box. If you have already chosen predefined sample types you will notice that the defining (marking) of controls has automatically been performed for you.

If you have entered your own non-standard control or you have other special requirements then select in the options box on the upper side of the sample rack a control type and mark the relevant vials with the mouse.

5.4.2 Control calculation Now select the Calculation item from the side menu to obtain the Template Controls dialog.

This option enables you to define a source matrix for each individual control. Each control can be calculated on the basis of a particular matrix.

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If, for example, the matrix ReaderValues has been defined as source matrix of the positive controls, the positive controls defined in position A1 to D1 will be calculated as the mean value of the four readings obtained in matrix ReaderValues.

This mean value is then accessible as program variable PC. For each control used it is necessary to define a source matrix.

5.5 Quench curve Also on the Template side menu you will see a section marked QuenchCurve.

Choose Standard Positions.

5.5.1 Standard concentrations On the tray click a vial that is defined as a standard. Choose the concentration from the Standard Concentration dropdown menu. You will see that the concentration value is assigned to the selected vial.

Continue in the same way with the other standards. Vials that are not used as standards are classified as Unused.

5.5.2 Curve fitting algorithm

Select the Algorithm item from the side menu to open the Algorithm tab of the Template QuenchCurve dialog.

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First you need to select the Source Matrix to be used for the curve fitting. After this select the algorithm of your choice. The entry boxes XY-Scale and XY-Units will modify the visual appearance of the curve obtained.

Under the Intercept, View and Additions tabs, you can add intercepts, specify the appearance of the curve, and choose various additional options.

5.6 Classifications

5.6.1 Threshold calculation Threshold limits are needed in order to provide a quantitative basis for any flag used (e.g. POS, neg). Three matrices may have limits set, and these limits are grouped as Threshold Set 1, Threshold Set 2 and Threshold Set 3.

Select Threshold Calculation from under Classifications in the side menu.

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For your chosen matrix, choose the type of flag and then enter the threshold limits into the entry boxes on the right hand side. Start with the highest limit first and then work downwards for each limit. For n limits you have n+1 qualitative flags. A maximum of 10 flags may be calculated.

For example if threshold limits for TDCR needs to be defined first create a matrix for it, for example, “TDCR check”. Then select the TDCR check and click the Add Formula button (found in the upper part of the display when Template is the selected left menu view).

Make your selection from the available Threshold Functions formula. TRH(Max) is used to identify the matrix for Threshold Set 1, TRH2(Max) is used for Threshold Set 2 and TRH3(Max) for Threshold Set 3.

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Define that Threshold Set 1 refers to TDCR. Change the TRH(MAx) to TRH(MA4) as matrix 4 is TDCR. Now threshold limits for each flag used can be specified in the Threshold Set 1 as shown above.

5.7 Printing Under the View menu you will find the Printout submenu, which allows access to the functions needed in reporting results.

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5.7.1 Setting up the Report

Select the Compilation item from View and Printout to obtain the Template Printout dialog.

This option allows you to structure the printout in a form appropriate to the test being used. The report is split into single components (e.g. Header, Filenames, Measurement Data, etc.). Under the Compilation tab, the Selected Items list box shows the actual single components to be printed out in the report.

Select an entry in the Available Items list box and then click the Add button to transfer it to Selected Items. Other details of the report are set using the other three tabs.

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The sample report in the illustration below shows the sample type, CPM, TDCR and QPE values for the three samples measured.

5.8 Saving data and templates for further use

5.8.1 Saving data In order to save all entries made to the test, Select the Save As ... item from the Data item under the File menu.

In the Save As dialog enter a name without an extension in the Filename field. The program will automatically assign an extension for you. Click the OK button.

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5.8.2 Saving templates Templates can also be saved.

Select Save As item from the Template item of the File menu.

Templates are automatically saved as *.par template files.

5.9 Other

5.9.1 Viewing the data You can view the raw data in the measurement data window.

This window allows you to view the raw data values of each individual kinetic time point or technology (if multiple technologies were used for the measurement). You can also view the data in graphical windows .

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Depending on your assay, the graphical views available are bar graphics, colour graphics, quench curve, spectra and control history.

Curve spectra can be examined individually or superimposed on the same axes. Simply click the icon in the left corner above the spectra to toggle between these two views.

5.9.2 Exporting the data Data export can be done manually by clicking the Export button or automatically by selecting Export Setup from the File menu. See Exporting results on page 25.

5.9.3 Printing out the data See Setting up the Report on page 52.

Click to toggle between individual and super-imposed spectra views

Click to toggle between individual and super-imposed spectra views

Click to toggle between individual and super-imposed spectra views

Click to toggle between individual and super-imposed spectra views

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6 Double-label counting Double label counting is a common application in liquid scintillation. It is used in situations where samples contain two or more different nuclides. A typical case is a sample with H-3 and C-14.

Double label counting has a critical limitation, which must be carefully taken in account. If samples have constant quench, i.e. counting efficiency stays the same for standards and unknowns, then a simple calculation can be employed. If samples have variable quench levels, then quench sets and an external standard are required. For an explanation of quench and related terms, please see section 8.4 on page 81.

6.1 Constant quench samples A template file with ready-made calculations is provided in the template database. The file for samples labelled with C14 and H3 has the name Double label H-3 C-14.par.

Note: The template file can be applied with other isotope pairs by changing the ROI settings accordingly (See xxx)

6.1.1 Define counting efficiencies You need to prepare two samples with known amount of activity, one with just H-3 and one with just C-14 in it. You should use samples within the range 50000 to 500000 DPMs of activity to achieve good statistics. We recommend the use of the NIST traceable Hidex internal standard capsules, which are available for aqueous and non-aqueous samples. Both are available for H-3 and C-14 under the following product codes:

462-011 Internal standard capsules, 40 pcs, H-3 water soluble 462-012 Internal standard capsules, 40 pcs, H-3 organic soluble 462-013 Internal standard capsules, 40 pcs, C-14 water soluble 462-014 Internal standard capsules, 40 pcs, C-14 organic soluble

The samples should be prepared in exactly the same way as the unknown samples and be of the same quench level. These samples are placed in the sample rack in positions A1 and A2.

Place H-3 in A1 and C-14 in A2.

You need to enter the known activities of the standards (in DPM) into the template file calculation matrix under DPM of Standards.

Enter the H-3 DPM value into A1 and the C-14 DPM value into A2.

Now you may begin counting. Enter your unknown samples starting at position A3 for the first of them. Give your assay a name and start counting by pressing Start.

Results are displayed as DPM in the Result Matrices. Counting efficiencies for the

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different windows are displayed under User Statistics.

6.1.2 Future assays If the quench level changes from one sample batch to another, then you need to perform this standardization for all runs. If the quench level stays the same you may now enter the known counting efficiencies to the template file and you do not need to prepare standards again. Open the Options Definitions box by choosing the Definitions item from the Options menu. Enter the counting efficiencies for the definitions.

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Now store the template file with a new name, double label H-3 C-14 with predefined efficiencies.par.

Next time you count a batch of unknown samples you can open the template-file and start counting right away.

6.1.3 Calculation formulae used in the counting The calculations used in the approximation of double label counting are as defined.

Components are abbreviated with H-3 = A and C-14 = B. Divide the CPM’s in the two windows by their known DPM’s, to get EA1, EA2, EB1 and EB2 (=efficiency of A in window 1, efficiency of A in window 2, efficiency of B in window 1 and efficiency of B in window 2, respectively).

The unknowns are counted and CPMs registered in the two windows. The following pair of equations is true.

CPMw1 = EA1*DPMA + EB1*DPMB

CPMw2 = EA2*DPMA + EB2*DPMB

Where CPMw1 and CPMw2 are the CPMs for windows 1 and 2, respectively.

From this pair, DPMA and DPMB (= DPM of 3-H and DPM of C-14) can be solved.

DPMA = (EB2*CPMw1 - EB1*CPMw2) / (EA1*EB2 - EA2*EB1)

DPMB = (EA1*CPMw2 - EA2*CPMw1) / (EA1*EB2 - EA2*EB1).

6.1.4 Principle of ROI settings in double label counting

With double label counting it important to use correct window settings for optimized measurement. The principle on selecting the counting windows (ROI is the following):

As an example window settings for H-3 and C-14 double label counting are presented below for a parameter template with four (4) ROI windows.

ROI 1. from 5 to peak position of the lower energy isotope (e.g. 5 – 130) ROI 2. from end channel of ROI 1 +1 to QPE of the lower energy isotope (e.g. 131 - 300) ROI 3. from end channel of ROI 2 +1 to QPE of higher energy isotope(e.g. 301 - 600) ROI 4. from 5 to QPE of higher energy isotope (e.g. 5 - 600) The same principle can be adjusted for other isotope as needed.

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6.2 Double label counting with the external standard An external standard source provides a useful method for counting double labelled samples such as those containing both H3 and C14. If the samples have constant quench e.g. HPLC eluate it is easier to prepare one pure H3 sample and one pure C14 and count them with the TDCR method. If samples with these two labels have variable quench the external standard is needed. The Hidex 300 SL has an optional external standard, which uses a europium-152 source with 74kBq. The external standard is moved automatically to a position just under the sample to obtain the quench parameter QPE. After this the samples are measured a second time to obtain the actual sample results.

Note: The quench parameter QPE is used with an external standard. Technically it is the upper channel of the window, starting from channel 0 that consists of 99 % of total counts caused by the radioactive external standard.

6.2.1 Creation of double label quench curves with the external standard. To perform double label counting you will need double label quench curves for the nuclide pair you are using. The most common pair is H3 and C14; if your instrument came with a pre-installed external standard please contact Hidex for the double label quench curves.

To create the double label quench curves you will need a quench set. This you can prepare yourself or you can contact your local Hidex distributor if you would rather purchase one.

The procedure for creating double label curves is described as follows. Two template files are used:

The first of the files listed in the image above is designed for creating a quench file (as an MW5FIT file in the transfer folder); the second one is for using this quench curve.

Open the template file, 300SL Save new double label quench curve.par

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Position the vials as shown in the picture, with the H-3 quench set from position A1 to B2, and the C-14 set from position C1 to D2

Click Advanced Options and make sure that External Standard is selected.

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Choose Settings from the Installation menu and press ALT+S to access Special Settings.

Change the value of ShowAllWells to Yes.

According to the settings we have now made, the first window/ROI is selected between channels 5 and 200, the second window/ROI is selected between channels 201 and 650.

The idea of the method is to detect with 2 Quench sets (here: H-3 and C-14) the individual quench levels in the 2 windows:

the H-3 efficiency in the H-3 window (5…200)

the H-3 efficiency in the C-14 window (201 – 650)

the C-14 efficiency in the H-3 window (5…200)

the C-14 efficiency in the C-14 window (201 – 650)

The external standard is used to detect the QPE – level of each sample, which is the information on the quench level.

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Open the Calculation tab of the Template Definitions dialog by clicking Calculation from the Template side menu.

Enter the half-life corrected DPM values of the quench standards (H3 STD and C14 STD)

Start counting. The data file name should reflect the quench curves as this will be the name of the quench curve when it is later used.

After the counting is finished the curve fit view should look like this:

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The quench curve file, obtained by choosing the Curve Fit tab is stored as an MW5FITfile in the transfer folder. In the diagram the QPE value is plotted against efficiency; 4 curves are visible: Gr1 (blue), Gr2 (red), Gr3 (green) and Gr4 (brown).

For Gr1 the following Efficiency calculation applies:

)650...5(3

)200...5(331

cpmtotalH

cpmHEff H

Gr

for Gr2 the following Efficiency calculation applies:

STDDPMH

cpmtotalHEff H

Gr 3

)650...5(332

for Gr3 the following Efficiency calculation applies:

)650...5(14

)650...201(14143

cpmtotalC

cpmCEff C

Gr

for Gr4 the following Efficiency calculation applies:

STDDPMC

cpmtotalCEff C

Gr 14

)650...5(14144

with:

cpmH-3(5…200): count rate of H-3 in H-3 window 5…200

cpmtotalH-3(5…650): count rate of H-3 in full window 5…650

DPMH-3STD: activity of H-3 standard

cpmC-14(201…650): count rate of C-14 in C-14 window 201…650

cpmtotalC-14(5…650): count rate of C-14 in full window 5…650

DPMC-14STD: activity of C-14 standard

6.2.2 Counting unknown double labelled samples

After you have received your 300SL New double label quench curve.mw5fit file you can start counting the samples.

Each measurement is done (automatically) in 2 steps. In the first step the measurement takes place with the external standard (typically the time is 30 seconds) for detection of the QPE value (quench level); in the second step the measurement is performed without external standard. It is assumed that the count rate, caused by the external standard is high compared with the count rate of the sample. If this is not the case, the measurement time for the external standard must be increased.

Open 300SL Use saved double label curves.par

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Open the Additions tab of the Template QuenchCurve dialog by clicking the appropriate item on the Template side menu.

Choose Browse and go to your Transfer folder or other folder where the 300SL New double label quench curve.mw5fit file is located and open that file. (If this has been done earlier and saved with the template file it does not need to be done again.)

Note: You might wish to change either the MW5FIT file or the curve fitting method. You should note that quench curves are not visible until measurement has been started. You can make alterations to the curve fitting algorithm only by accessing the original data

Enter your data file name.

Under the result section the results will appear. You will see the CPM in the two selected ROIs. Calculated total CPM caused by H3 and C14 and DPM of individual nuclide. Running pure sample H3 and C14 quench series will give you information on how much spillover you will see at different quench ranges.

If you have done your own double label curves you must use the appropriate ROIs suitable for those nuclides.

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7 Super low level option The Super Low Level version of the Hidex 300 SL is equipped with a plastic scintillator detector placed under the instrument. When a cosmic ray passes through the sample it excites the cocktail and emits false pulses that cannot be separated from the normal pulses. When the guard is activated the cosmic rays also cause a pulse in the separate guard detector and these pulses are neglected. Cosmic rays are mostly observed in the higher energy range of the MCA. Therefore the guard has highest efficiency in background reduction on the higher channels. For extra reduction in background at the lower energies the Super Low Level model is equipped with extra lead shielding compared with the standard model.

7.1 Operation For the use of super low level option please activate the guard in the MikroWin Advanced parameter options. The Guard can be operated with counting mode, low and alpha beta separation as well as Triple mode ROI.

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8 Alpha/beta separation counting If your counter includes the alpha/beta separation option, you can obtain excellent results for a number of key radionuclides with decay chains involving both alpha and beta emission. Alpha/beta separation is performed on the basis of the pulse length index (PLI); long alpha pulses are measured in the alpha channel, the shorter beta – pulses in the beta channel. The successful use of the alpha/beta separation feature depends on careful optimization of settings for the nuclide being measured, the cocktail and the sample-to-water ratio. Alpha/beta measurement has been set in the factory. When you are counting alpha/beta you need to set separate ROIs for the alpha and beta emissions, and then set the alpha specific parameters that have been delivered with the Hidex 300 SL. It is important to change the default values to factory values to get the best measurement results. Depending of sample it is possible to change also these values if needed. (For example, PLI limit i.e. counts above the limit are to be classified as alpha, counts below the limit as beta). After counting has been performed using these settings, the spectra can be viewed in Mikrowin Spectra graphics or be exported to the Report Render macro (see page 70), which allows you to optimize the settings.

Start by making the appropriate counting settings in the Options dialog (see below).You need to set ROI’s for both beta and alpha. Click Advanced Options and make the additional settings shown below.

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The factory-set alpha/beta parameters can be found in the QA report sent with the instrument.

Start measurement by giving a name to your data file and clicking Start. After the measurement has ended a file with YOURDATANAME.csv is created to your default transfer folder.

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8.1 Alpha/beta separation calibration with MikroWin AB calibration is done with a alfa active sample. The factory set alpha/beta parameters can be found in the QA report sent with the instrument which can be used as the starting point for optimized calibration.

PLI optimization may be performed using the 2D plot generated by MikroWin or also with Report Render.

With Mikrowin the 2D plot is obtained by specifying the desired spectra data then selecting 2D _Plot_<AS> from the Spectra Selection drop down menu.

Clicking the icon in the top left corner of the image on the right will then open a full screen view of the 2D spectrum.

The calibration is done when the PLI line is correct place between the beta belt and alpha islets

It is important also to remember that the PLI-line is represented as a line 20 counts wide. These artificial counts might interfere when optimizing PLI, so it is mandatory to exclude the PLI-line. Uncheck the PLI checkbox in the Advanced Options box.

For the best settings this optimization procedure also needs to be done with the background sample; the optimum setting is then achieved, if the FOM (Figure of Merit) is maximum.

These settings can now be used for measurements; do not forget to activate the PLI window using the checkbox in the Advanced Options box.

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8.1.1 Alpha Specific Parameters This selection is available if your instrument has the optional Alpha Beta measuring feature.

Alpha delay time A period of time in which both, the beta and alpha contribute the pulse (measured from the pulse start). Alpha and beta pulses are separated based on their length. The first part (up to the value used) is saved as beta and period of time after this as alpha.

Alpha y offset This adjustment moves the 2D plot (without PLI limit) in vertical dimension. Alpha y offset was called alpha tail offset with earlier MikroWin 300 SL drivers than 1.08.

PLI limit PLI stands for pulse length index. It moves the PLI limit in vertical dimension and therefore changes alpha/beta classification. The higher it is the less counts are collected to the alpha spectrum. If the Show PLI box is checked then the PLI limit is represented on 2D plots by a horizontal line, usually between the alpha and beta spectra. The selection does not affect alpha/beta classification.

Y gain This adjustment stretches the 2D plot (without PLI limit) in vertical dimension.

Gate Time A period of time in which only alpha contribute the pulse (measured after alpha delay time). Gate Time is only shown with MikroWin 300 SL driver 1.08 and Hidex 300 SL embedded software, v. 1.56 or later.

Spill to Beta If this option is selected alpha pulses that are over 31 in the Y axis of the 2D plot are not included as alpha spectrum but forced to Y pos 1 and contribute beta spectrum and results. This feature could be used in cases when the alpha activity is very small compared to the beta activity and beta spills to alpha side.

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9 Using the Report Render macro

The Report Render Excel macro accepts your spectra and measurement data and provides flexible facilities to work with spectra.

With Office 2007 and more recent Office versions, Report Render version number 100413 should be used.

With the older Office versions, version 091104 should be used.

9.1 Importing spectra and measurement data

Open Report Render in Excel and enable the use of macros.

Press the Import Data button and browse to the transfer folder.

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The importing of data may take some time. The measurement data imported will be in the form that you specified in the MikroWin Options Read data settings, and the spectra will be displayed, one 2D graph being shown at a time (see following picture).

The figure shows a typical Rn-222 spectrum, obtained using the Hidex 300 SL. The trace on the upper left is beta emission, and that on the upper right is alpha emission. The lower plot is the 2D plot from which the upper graphs are derived. In the 2D plot the x-axis is energy, the y-axis is the pulse length (PLI) and the z-axis (represented by the different colours) is intensity.

The PLI limit value that has been set determines the exact criterion for whether a pulse is classified as alpha or beta emission. Pulses with longer PLI values are categorized as alpha pulses, while those with shorter PLI values are categorized as beta pulses. In the picture here, the PLI limit is represented by the black horizontal line.

Alpha channel

Beta channel

PLI limi

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In both the 1D and 2D plots shown above, the counts have been allocated to the spectra solely on the basis of the ROI settings and the PLI limit that you set in the Options and Advanced Options dialogs. On the basis of what you see in the plots you can now move on to optimize both ROI and PLI settings.

9.2 How to optimize ROIs

ROI selection can be used to optimize the ROI windows for the given PLI. The optimum ROI should be as narrow as possible, in order to reduce background in combination with the highest efficiency. To collect just the counts for the Rn-222 peak in the alpha channel, we might change the alpha settings from 750 - 900 to 760 - 830.

9.3 Exporting spectra and measurement data, spectrum subtraction Select samples for export and then click Export selected data (green button).

Now select reference spectrum for subtraction. Select sample so that it is active and then click the Reference Spectrum Subtraction button. The reference sample is now marked with purple colour. In this example the reference sample is the red tritium spectrum.

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In the picture below the lower spectrums can be seen after subtraction of the selected tritium reference spectrum.

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9.4 Problems with using Report Render Export selected data button Report Render version 100413 is designed to work with Excel 2007 and later versions. If you have an older Excel version you should be using Report Render version 091104. Report Render version can be seen under Hidex logo in Report Render.

9.5 Programmatic access to Visual Basic Project is not trusted If this Microsoft Visual Basic Run-time error ‘1004’ occurs with using Excel 2007 you need to allow ‘Trust access to Visual Basic Project’. To do this click the Microsoft Office button in the upper left corner of Excel. Select Excel options Select Trust Center and then Trust Center Settings…

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Select Macro Settings and then click selection to Trust access to the VBA project object model. Now click OK to finish selections. Export selected data option in Hidex Report Render should now work.

If this error occurs with using Excel 2002/2003 you need to allow ‘Trust access to Visual Basic Project’ Go to Tools -> Macro -> Security and Trusted publishers tab. Select Trust access to Visual Basic Project.

9.6 Unable to set the XValues property of Series class This Microsoft Visual Basic Run-time error ‘1004’ reveals a capability problem. Please use another version of Report Render.

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10 Cooling module option

10.1 Nature and purpose of the module The Hidex 300 SL instrument can be equipped with an optional cooling module. This consists of a cooling unit with a fan. The inside of the counter is cooled by means of cool air directed from the back of the cooling module. The air inflow to, and hot air outflow from the module must not be blocked.

The cooling unit and the blue cooling power adjustment are shown (left).

The main purpose of the module is to stabilize the temperature inside the instrument and thus reduce variation in cocktail performance. Hidex cocktails are optimized for use at roughly 20 degrees centigrade. The cooling power can be increased or decreased with the blue adjustment lever if the cooling is too weak or too strong.

10.2 Cooling settings Cooling settings - Cooling On and Cooling Off - may be made from the instrument menu.

To add cooling to a template file first open the template Options (see page 18), then go to the Advanced Options dialog (see page 34) and, in the Cooling pane, check the Cool during Measurement option.

To save on power consumption you can also choose to turn cooling off automatically after counting has ended.

The cooling of the unit needs to be set to the requested degree by increasing or decreasing the cooling power using the blue lever on the right hand side of the unit. It may take some hours to balance the temperature. Please note that the cooling capacity is related to the ambient temperature and access of cooled air. Under normal conditions the cooling unit can cool the internal temperature of the instrument by about 5 degrees.

Hot air from the cooler is blown out from the left side of the instrument. Please do not block this fan.

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11 Organizer Organizer is a new feature that provides the flexibility to handle complex tasks and to save you time. To open, click the Organizer button at the top left of the display.

11.1 Using Organizer to enter sample information Organizer allows you to enter a great deal of sample information in such a way that you do not need to make a large number of matrices.

If you want to add sample info to a tray on which just one kind of measurement will be done (for example, all of the vials measured as 3H), first enter the Tray ID and press Enter. Choose the desired template to be measured. Input all the sample info that you want.

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Save the tray and exit Organizer. You can then enter the same Tray ID as used in Organizer. In the main view and start the measurement in the usual way.

11.2 Creating batches

Hidex 300 SL allows you to make different measurements from the same list at the same time. You do this by batching your samples using the Organizer feature. After supplying the Tray ID you need to choose one of the default templates in order to specify whether the tray is for 40 or 96 vials. Choose either default_tray40 or default_tray96 from the drop-down menu.

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After entering the IDs for your samples, you need to specify a template file for each one. You can choose from the available template files by right-clicking in the respective cell and then dragging the mouse over the Lookup item.

Having made your selections click Save Tray. Run the tray by clicking Run Tray.

The Batch Processor dialog will open. You can start the measurement from this by clicking Run Batch.

11.3 Adding to batches and prioritizing measurement Sometimes, you may wish to add additional samples and give them priority over those already loaded. If your job is already running when another job with higher priority comes along, press Pause Batch.

Click the Organizer button and enter the tray ID to find the correct tray.

Add the new parameters to the specific vial position. Remember to save and then click Run Tray. Choose the new vial positions to be measured first. The list will then continue in the normal manner.

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11.4 Using Toggle Mode Clicking the Toggle Mode button in the Batch processor dialog allows you to switch between two alternative views or modes. In the previous picture the mode shows all samples listed on separate lines. In the alternative mode, below the sample positions are grouped so that all samples of each particular assay are grouped together. The results will then be saved according these groups.

Note that Toggle Mode cannot be changed during the batch run.

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Reference section

12.1 Principle of liquid scintillation counting Hidex 300 SL measures low intensity light. Radionuclides do not produce light directly. For those that emit beta radiation, the light is produced when the sample (with the radionuclide) is mixed with a scintillator – normally a liquid, and consisting of a solvent and at least one fluor. The light comes in bursts (from a few to several hundred photons) and is collected on the photocathodes of photomultiplier tubes (PMT).

12.2 Čerenkov counting LSC should be used to measure beta labelled samples with emissions having energy less than 2000 keV. In practice most common beta labels belong to this category. High-energy nuclides, e.g. P-32, Cl-36 Sr/Y-90 etc. with beta energies exceeding about 265 keV, can also be counted directly in aqueous solutions by detecting Čerenkov light pulses without liquid scintillator.

12.3 Phosphorescence Many substances are naturally phosphorescent and may emit light, which interferes with that from the sample and scintillator. To avoid problems with phosphorescence do not expose the instrument, vials or samples to direct sunlight or strong laboratory illumination. The triple coincidence counting mode is highly resistant to phosphorescence and can be used to directly count sample with high phosphorescence.

12.4 Quench correction In liquid scintillation counting, because the sample is mixed with a scintillator, the sample’s chemical and physical properties may affect the scintillation process, resulting in variable light production. This phenomenon is called quenching and its main forms are chemical quench, in which light production is affected by the sample’s chemical composition, and colour quench, in which light transmission from scintillating atoms to the PMT varies due the absorption in the scintillation cocktail. The latter varies also according to the sample volume while the chemical quench does not. In addition to these, optical quenching occurs if dirty scintillation vials are used.

Quench correction should be applied if the quench varies from sample to sample. If it is constant the correction is not needed and it suffices that the result value is multiplied by a factor.

After correction of variable or constant quench, results are expressed as DPM rather than CPM.

12.4.1 Quench correction with TDCR method The Hidex 300 SL is the first commercial liquid scintillation analyser to facilitate the triple to double coincidence ratio counting.

In TDCR triple and double coincidences are measured and the ratio of these coincidences is calculated. Triple coincidences are more affected by quench (chemical and color) than double coincidences (or the total of all coincidences).

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For pure beta-emitting radionuclides the ratio of triple to double (all) coincidences is directly proportional to the overall efficiency. The proportional factor is 1 (+/- 15 %). This can be proved by experimental results and mathematics.

Hence, in TDCR the quench level is (as different from systems with 2 PM`s at 180° facing the sample) automatically included in the ratio of the triple to double coincidences.

Measuring TDCR enables the determination of activities for pure beta-emitting radionuclides with high accuracy. The level of quench is directly taken care of by TDCR.

TDCR does not require an external source and does not require a vial of known activity – therefore it is an “absolute” measurement.

TDCR is automatically deployed in the Hidex 300 SL. DPM is available as part of the data selection. In this situation it is CPM divided by the TDCR factor.

The TDCR value is also available from the data selection and can be used as an indicator of quench in standards. A standard curve of quench set can be stored with the curve fit option and this can be employed to gain a more accurate DPM value.

12.4.2 Quench correction with standard quench set

Standard quench sets can be purchased from your local Hidex representative. As an example we can consider the H-3 quench set. Open the Hidex 300 SL H-3 template file using the stored quench curve.par. This template file has stored information on the quench curve of the standards and automatically fits the TDCR value to the standard curve to determine the real efficiency of the counter.

The quench curve needs to be imported to the template.

Under QuenchCurve in the Template side menu click on Additions.

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Click the Browse button. Go to your MikroWin Transfer folder to open the appropriate MW5FIT file, here it is TDCR quench correction curve.mw5fit.

You might wish to change either the MW5FIT file or the curve fitting method. You should note that quench curves are not visible until measurement has been started. You can make alterations to the curve fitting algorithm only by accessing the original data.

With original data file you can check the curve fitting method from the Algorithm tab of the Template QuenchCurve dialog.

In general the Cubic Spline method is found to be the best fitting method for quench curves.

Select Smoothed Cubic Spline and Click OK, and your curve fit view should be of the TDCR quench curve.

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Enter a data file name and start counting. The quench curve will then become visible in Results window and Quench curve Graphics. The DPM matrix will then show the quench curve corrected activities of your unknown samples.

12.4.3 Storing a quench curve of a standard quench set To create your own standard curve you may either obtain a ready made quench set from Hidex or prepare one yourself. As an example we can consider the creation of a standard curve using a ready made H-3 quenched standards set. Open the template file, Hidex 300 SL H-3 20mL save TDCR quench curve.par. Make sure your sample trays include the correct standard set. Place the standards in the positions from A1 to B2. Make sure your reader settings are set as shown below.

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You can increase counting time to increase the precision of the results.

Choose the Calculation Matrix with DPM of Standards and enter the half-life corrected activity as DPM into positions A1 to B2.

Enter a name for your data, for example, H3 TDCR quench curve; it is advisable to choose a name clearly indicating that the data represents a TDCR quench curve.

Start counting.

When all the positions in the rack are counted a quench curve is generated. You can view the curve under Quench Curve.

The curve plots TDCR number as a function of efficiency for the known standards. Two artificial points TDCR 0 and Efficiency 0 and TDCR 1 and Efficiency 1 are added, because triple to double coincidence theory states that they must be included.

In your MikroWin Transfer folder (if you followed the example above) you now have a file called H3 TDCR quench curve.MW5fit. This file holds the information of the particular quench set you have used in the specific instrument that you have been working with. In practice TDCR quench curves are quite generic and do not need to be redone several times. All Hidex instruments are calibrated in the factory for H3 and C14 TDCR quench curves. Please contact your local distributor to obtain the curves retained by the manufacturer for your instrument.

Store the data file name with an appropriate name as that will be the name of the quench curve.MW5fit file in your preset transfer folder. The fit file can now be used by selecting it as your curve fit master plate in your template files.

You can create a quench curve for any isotope. It is important to note that the MCA ROI window must be set to accommodate the energy of the isotope in question.

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Also either the QPE (if your instrument has an optional external standard) or QPI can be used as quench indicator in all the above cases. TDCR is found to be the most reliable quench correction method as it is not dependant on the spectral position.

12.4.4 Correction of quench in Hidex 300 SL with internal standardization An unknown sample is first measured and its CPMx value is recorded. A known amount of the nuclide (internal standard) is then added to the cocktail and the sample is recounted. The efficiency value is:

E = (CPMsd+x - CPMx) /DPMsd

The DPM value of the unknown sample can now be calculated:

DPMx = CPMx /E where:

E = counting efficiency

CPMx = the recorded CPM value of the sample

CPMsd+x = the recorded CPM value of the sample mixed with the internal standard

DPMsd = the activity of the internal standard

NIST traceable internal standards for H-3 and C-14 are available from your local Hidex representative.

This is the most reliable method available to determine the sample’s true activity.

12.5 End CPM criteria

NONE

UPPER if((MeasuredCounts - UncertainCounts) > TargetCounts_High)

LOWER if((MeasuredCounts + UncertainCounts) < TargetCounts _Low) "MIDDLE if (((MeasuredCounts + UncertainCounts) < TargetCounts _High) AND ((MeasuredCounts - UncertainCounts) > TargetCounts _Low))

!OR! if(((dMeasuredCounts - dUncertainCounts) > TargetCounts _High) OR ((MeasuredCounts + UncertainCounts) < TargetCounts _Low))

Where:

dTargetCounts_Low = Protocol[TargetCPM_Low] * counted time, in minutes dTargetCounts_High= Protocol[TargetCPM_High] * counted time, in minutes UncertainCounts = TargetSigma * sqrt(dMeasuredCounts)

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13 Troubleshooting

13.1 Connection Troubleshooting You can use the Device Manager obtained via the Control Panel to check whether devices are working correctly, and find out which port on your computer a particular device is connected to. If you are using a USB serial port adapter and have problems with the driver, you can use the Device Manager to update the driver.

From the Control Panel choose Device Manager.

If the device is not working you will not be able to see it as listed below under Ports.

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Under Universal Serial Bus controllers, select CBUSB Ver 2.0, and double click to open the Properties box.

Click Update Driver and follow the instructions in the wizard.

Following successful installation, the COM port to which the device is connected will be shown under Ports (COM & LPT) in Device Manager.

13.2 Recovering if a vial remains inside the detector chamber

Due to power failure during counting a vial may be left inside the counter detector chamber. There is a simple procedure to remove the vial.

Choose Recover from the Instrument menu. As part of the recovery process the vial will be expelled and you will be invited to remove the tray.

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14 Routine maintenance Caution! While the instrument is powered up, the cover must not be removed.

Caution! Do not touch or loosen any screws or parts other than those specifically designated in the instructions. Doing so may cause misalignment and will invalidate the service warranty.

14.1 Cleaning and maintaining the instrument

14.1.1 Outer case For reliable daily operation, ensure that the instrument remains free of dust and liquid spills. Clean the outer case of the instrument from time to time using a soft cloth dampened in a warm, mild detergent solution. Abrasive cleaning agents should not be used.

Any saline solutions, solvents, acids or alkaline solutions spilt on the instrument’s outer surface should be immediately wiped away. In the event of spillage of biohazardous material, a mild sterilizing solution should be used.

14.1.2 Sample vial compartment The vial compartment should be kept clean to avoid dust and dirt from entering the measurement chamber. Clean the compartment surface at least once a week using a soft cloth or tissue paper soaked in a mild detergent solution or 70% ethanol.

Wipe up spills immediately. Do not use formaldehyde. If you have spilt biohazardous materials in the sample vial compartment, decontaminate according to the procedure described below.

Caution! Do not autoclave any part of this instrument.

Caution! Do not use alkaline or chlorite solutions for cleaning any parts of the measurement chamber, as this may damage the instrument.

14.2 Other periodic maintenance tasks Decontamination should be performed in accordance with normal laboratory procedures. It must be performed when infectious substances have been in direct contact with any part of the instrument, or, for example before shipping an instrument for service. Performing the decontamination procedure is also strongly recommended before moving the instrument from one laboratory to another. Decontamination is not required for the proper functioning of the instrument.

The decontamination procedure should be performed in a vial-ventilated room by authorized trained personnel wearing disposable gloves, protective glasses and clothing.

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Suitable substances that may be used as decontaminants are:

Ethanol 70%

Virkon solution 1 - 3%

Glutaraldehyde solution 4%

Chloramine T

Microcide SQ 1:64

Prepare the decontaminant: for example, 200 mL 4% glutaraldehyde solution.

Empty the sample vial compartment. Leave it open.

Set the power switch on the instrument to OFF and then switch off the mains power supply box and disconnect the mains power supply cable.

Disinfect the outside of the instrument using a cloth dampened with 70% ethanol.

Place the instrument in a large plastic bag. Place a cloth soaked in the prepared solution into the bag. Ensure that the cloth does not come into contact with the instrument.

Close the bag firmly and leave the instrument in the bag for at least 24 hours.

Remove the instrument from the bag.

Clean the outside of the instrument using a mild detergent.

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Glossary

Beta label Radionuclide that emits beta particles, and that may be used as a label or “tag” on a substance being measured.

Bq A Becquerel - the activity of a quantity of radioactive material in which one nucleus decays per second.

Čerenkov pulses Light emitted by a charged particle when the particle passes through a transparent, nonconducting, material at a speed greater than the speed of light in the material. High-energy nuclides, e.g. P-32, Cl-36 Sr/Y-90 etc. with beta energies exceeding about 265 keV, can be counted directly in aqueous solutions without liquid scintillator by detecting Čerenkov light pulses.

CPM Counts per minute, or the number of atoms that have been measured as decayed. Were counting 100% efficient, CPM would be identical to DPM (disintegrations per minute). Since it is not, quench correction is applied to convert CPM to DPM.

Decontamination Removal or neutralization of radiological, bacteriological, chemical or other contamination.

Device Configuration Information on the configuration of the device, in this case the Hidex 300 SL instrument required by the driver program.

Disinfection The destruction of pathogenic bacteria, usually with an antiseptic chemical or disinfectant.

DPM Disintegrations per minute, or the number of atoms in a given quantity of radioactive material that decay in one minute. One Bq is equal to 60 DPM.

Driver (Device driver) A program that controls a particular type of device, in this case the Hidex 300 SL instrument, serving as a link between the instrument and the MikroWin 300 SL software running on the computer.

Efficiency (In this handbook meaning counting efficiency) CPM expressed as a percentage of DPM. To determine the efficiency, the amount of quench has to be known.

Fluor Fluorescent molecule that forms the scintillating component of a liquid or solid scintillator.

Initialization Initialization tests are so-called self- tests, which are carried out before operation to ascertain that the necessary instrument adjustments have been carried out.

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Isotope See nuclide.

Label Usually within this handbook a label can be taken to mean a radionuclide with which the substance being measured is marked. An alternative use is as one of two identifiers to allow the association of data with matrices. In this sense, label is abbreviated to “lb” and the other identifier, plate is abbreviated to “p”. LED Light-emitting diode.

LSC (Liquid scintillation counting) The detection of light (scintillation) that is produced by the action of beta particles from the sample on a liquid scintillator (called a scintillation cocktail) mixed with the sample.

Matrix (In this handbook) a data grid corresponding to a set of vials for presentation of calculation formulae or results.

MCA (Multi channel analyzer) An electronic circuit to sort the incoming pulses to a number of “bins”, or channels, according to one or more criteria. The Hidex 300 SL has a number of MCAs which are fully programmable, they are used either as a single dimensional MCA (the criterion is pulse height) or a two dimensional one (the criteria are pulse height and pulse length). The latter is used only in an alpha/beta separation circuitry where the ratio of these two is the final criterion whether the pulse is saved to the (single dimensional) beta MCA or alpha MCA. MCAs are also used to separate decays that are detected in one, two or three detectors.

NIST National Institute of Standards and Technology – a US federal technology agency that develops and promotes measurement, standards, and technology.

Nuclide Any of two or more types of atom possessing the same chemical properties but different atomic weights due to the different number of neutrons in their nuclei. While the term, isotope is commonly used when wishing to emphasize or distinguish between forms of the same element, nuclide is more suitable when referencing only one nucleus or several nuclei of different elements.

Phosphorescence Fluorescence emission from the sample, vial or cocktail that can interfere with measurement. The problem may be solved by avoiding working in direct sunlight or under strong laboratory illumination.

Photomultiplier tube (PMT) A photoelectric cell that converts light into electric current and amplifies the current.

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Plate (In this handbook) one of two identifiers to allow the association of data with matrices. Plate is abbreviated to “p”, and the other identifier, label is abbreviated to “lb”. Positron A particle having the same mass and magnitude of charge as an electron but exhibiting a positive charge; a positive electron.

QPE External standard quench parameter.

QPI Internal standard quench parameter

Quantum yield (Q) The ratio of the number of emitted photons to the number of excited molecules. Fluorophores differ in quantum yield, the higher the Q value, the more fluorescent the compound is. The theoretical maximum of 0=1 is for a highly fluorescent compound, and 0=0 corresponds to a non- fluorescent compound. RFU or rfu Relative Fluorescence/Fluorometric Units. The arbitrary units in which fluorescence intensity is reported. RH Relative humidity.

Quench Phenomenon in liquid scintillation counting, in which the sample’s chemical and physical properties affects the scintillation process, resulting in variable light production.

ROI Region of interest – a counting window.

TDCR Triple to double coincidence ratio – triple and double coincidences are measured and the ratio of these coincidences is calculated. Triple coincidences are more affected by quench (chemical and color) than double coincidences.

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Index

A

Active Plate/Active Label menus · 38

Additions Quench Curve tab · 49, 82

Advanced Options dialog · 34, 60, 66, 68, 72, 76 Alpha/beta separation · 17, 66

optimizing ROIs · 66 Automation

in file naming · 22

C

Calculation formulae in double label counting · 58

Caution · 10, 11, 89 Cerenkov counting · 81 Colour quench · 81 Comm Port

in MW driver installation · 13 Computer · 11, 14, 91 Connections · 11 Control calculation · 47 Control positions · 47 Cooling option · 76 Counting time · 30, 35 Curve fitting · 48

D

Default folders and files · 15 Device Configuration

in MW driver installation · 14 Dongle · 12, 17 Double label counting · 56

with external standard · 59

E

Export button · 27 Export driver types · 25 Exporting results · 25 External standard

Option · 34

F

Free counting · 19

G

Graphical windows · 54 Green LED · 17, 21 Grouping

in Tray layout · 46

H

Hardlock protection MikroWin 300 SL · 12, 17

Hidex 300 SL back of instrument · 11 cleaning and maintenance · 89 instrument weight · 10 introduction · 9

I

Installation Settings · 14, 16 Intercept

Quench Curve tab · 49 Isotope · See Nuclide

L

Label · 19, 92 Loading vials · 21 LSC (liquid scintillation counting) · 81, 93

M

Matrix associating data in repeat counting · 44 creating · 41 deleting · 40

MCA · 92 Measurement directory · 16 MikroWin 300 SL · 9, 12, 18

driver installation · 12

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driver updating · 14 hardlock protection · 12, 17

N

Nuclide · 17, 19, 66, 86

O

Organizer · 10, 24, 77, 78 Overlay

in Tray Layout · 46, 47

P

PAR suffix · 20 Parameter files · See Template files Partition

in Tray Layout · 46 Phosphorescence · 81 Photomultiplier tube · 81 Photon · 81 PLI

pulse length index · 35, 66, 68, 69, 72 Power box

(Transformer unit) · 11 Power failure

recovery after · 88 Powering up · 11, 17 Printing report · 52

Q

QPE · 33 QPI · 33 Quench correction · 81, 93 Quench curve · 33, 48

R

Radioactivity detection · 17, 21, 81 Red LED · 24 Repeat counting · 33, 44 Report Render macro · 66, 70, 72, 74

Rn-222 spectrum · 71 ROI · 19, 30, 31, 32, 37, 72, 93

S

Saving data and templates · 53 Scintillator · 81, 91 Screen CPM · 30, 32, 36 Serial port · 11, 87 Settings dialog · 18, 19, 30 Super low level counting · 17, 65

T

Temperature control · 9, 76 Template directory · 16 Template files · 19, 20, 25, 53, 54, 58, 59, 64 Threshold Calculation · 49 Transfer directory · 16 Tray type · 19, 35 Troubleshooting

connection · 87

U

Unpacking the instrument · 11 USB · 11, 12, 87, 88

V

Vial tray loading into instrument · 21

Vials loading additional · 24 removing from detector chamber · 88 selecting for measurement · 20

View Quench Curve tab · 49

Viewing data · 54

W

Warnings · 10, 89