Section16:Instrumentation

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Instrumentation

There are two microplate scintillation counters available for detection of photons from radioactive assays - Microbeta (Trilux) and TopCount, both available from Perkin Elmer Life and Analytical Sciences (PE LAS). Each instrument is listed below with basic set up information for counting protocols. Other imaging instruments are capable of reading radioactive assays in microplates, but are not discussed in this document.

Microbeta Trilux

General Concepts

A Microbeta Trilux comes with either 6 or 12 detectors. Each detector is comprised of two photomultiplier tubes (PMT’s), one on top of the sample and one on the bottom. The PMT’s operate using conventional coincidence circuitry as shown in the diagram below.

Each detector counts only a portion of a 96-well microplate (16 wells per detector on the 6-detector Microbeta model, ~9 wells on the 12-detector model). The area of the plate counted by each detector of a 6- or 12-detector model is shown in the diagram below.

Although the use of multiple detectors can increase throughput, since the performance of PMT’s are not identical, a calibration procedure (Normalization) is required. An identical sample is counted by all the detectors and a relative efficiency (fractional value) is determined. If an activity (DPM) for the sample is known, this can be inputted into the software and the detectors are normalized to this activity. This will result in the efficiency factors being lower than if the detectors are normalized against each other. As an example, the typical efficiency relative to activity for [3H] with SPA beads is 0.20 – 0.30. When the detectors are normalized against each other, the relative efficiencies should be 0.9 – 1.0

Modes of Normalization

There are two ways to normalize the Trilux with a single sample in well G11:

1. Relative to the detector with the highest reading (CCPM = CPM)
2. Relative to the activity inputted in well G11 (CCPM = DPM)

The basic principle for each of these modes is shown at the end of this section. The sample to be used for normalization must be in well G11. Both normalization protocols are set up the same way, with one additional step for mode 2, when results in DPM are desired. There are other features for Standardization (e.g. using quench curves) or Easy DPM, Paralux, etc. that are not discussed in this document.

Setup of a Normalization Protocol

These instructions are meant to serve as a guide only. Consult other sources of information (engineers, previous users, or manufacturer) for detailed assistance. All screenshots are from Microbeta Workstation Software (version 3.00; Perkin Elmer Life and Analytical Sciences).

1. Click on the Protocols button at the top of the Microbeta software toolbar.
2. Select Normalizatons followed by the Open button.

   
   

3. Click on the New button to create a new normalization protocol.
4. Select the appropriate label from the pull-down menu in the pop-up dialog box and click OK. Do not check SPA unless you want to use Paralux counting mode (consult instrument manual).

   
   

   In many cases, particularly with YSi SPA beads (as recommended by GE Healthcare), you should select Other and use the manual energy spectrum window settings shown in the table below in Step 6. The default settings were designed for PVT SPA beads.
5. Under the General tab, type in a name for the protocol and select a number for the protocol from the pull-down list (only unused, available protocol numbers are listed).
   
   If it is desired to express results in DPM: Check the Isotope activity box and input a number for the activity (in DPM) that is in well G11. This activity should be determined by counting an identical aliquot in a liquid scintillation counter (for 3H) or a gamma counter (for 125I) that has a known efficiency (DPM = CPM/Efficiency). In this example, replicate aliquots of YSi SPA beads were counted in a liquid scintillation counter with an average of 140,782 DPM. An identical aliquot was placed in well G11 of a microplate for normalization. The value 140,782 is entered into the area on the General tab as shown below.

   
   

6. If Other was selected as the label, the energy spectrum window settings may need to be manually defined. By default it will appear under the Other tab as a window from 5 to 1024, an open energy spectrum window. GE Healthcare has performed studies to maximize window settings for SPA applications.
   
   Uncheck the box next to Use defaults. The window settings for Low and High can now be changed.
   
   The table shown below indicates the suggested settings for several isotopes and types of SPA beads and Cytostar-T plates. The figure following the table shows the Other tab, after new window settings have been inputted for tritium YSi SPA beads and the Microbeta (Table from GE Healthcare).

   
   

   Under the Other tab:

   
   

   

   
   

7. Click OK to save the normalization protocol.

Setup of a General Counting Protocol

A Normalization protocol is linked to a General Counting protocol, to define the counting parameters (i.e. isotope, window settings, etc.) and the detector efficiencies (relative to the highest detector reading or relative to DPM activity) needed to correct raw counting data.

1. Click on the Protocols button at the top of the Microbeta software toolbar.
2. Select General followed by the Open button.

   
   

3. Click on the New button to create a new General counting protocol.
4. In the Edit Counting Protocol window, type in a name for the protocol in the Identification space. Select a protocol number from the pull-down list to the right of the Identification name. Only unused protocol numbers will appear in this pull-down list.

   
   

5. Select the isotope from the pull-down list. Once the isotope is selected, Normalization protocols that have been created using that isotope will appear in the Normalization pull-down area. Select the appropriate Normalization protocol to link to the General counting protocol. Note that an underscore (_) before the name of a Normalization protocol indicates that the Normalization plate has not been counted yet. Once the Normalization data has been stored, an (n) will appear before the name of the Normalization protocol.

   
   

6. Change the Counting time if desired (default is 1 min). The other tabs in the Edit Counting Protocol window (Corrections, Counting Control, Other) usually do not need to be modified unless special counting circumstances are being used.
7. Click OK to save the General counting protocol. Click Yes on the dialog box that pops up.
8. From the Protocol group General window, the General counting protocols can be edited.

   
   

9. The Protocol button allows editing of the protocol parameters (i.e. counting time)
10. The Plate map button allows selection of microplate wells to count (default set to entire plate)
11. The Output button allows selection of file and printing options. There are a couple of changes that should be made in the output as outlined below:

    If the instrument is connected to a network and does not have a dedicated printer attached to the PC controller, it may be desirable to deselect the printing option. Quality printouts of the data directly from the instrument to a network laser printer are difficult. Deselect Generate print output in the Print tab.
    
    Under the File 1 tab, it is advisable to change the path where data files are electronically stored. By default, they are stored in the Results subdirectory where the Microbeta software is stored. This can be dangerous, as the Normalization parameters are also stored in that subdirectory. Accidental deletion or moving of Normalization protocol results files will render the Normalization protocols useless. To prevent this, direct General counting output to a different subdirectory.
    
    Under the File 1 Items tab, if you do not want the electronic data file to have the data expressed as 96 numbers in a column (for a 96-well plate), deselect the Column section box. The data file will have results in plate format only (8 x 12 array for 96-well microplates).

The suggested outline shown above is for general counting conditions. Consult instrument owners or the manufacturer for advanced counting options such as cross-talk correction, background correction or manual setting of count windows.

The following two pages demonstrate the linking of a Normalization protocol to a General counting protocol for a 12-detector Trilux using either a relative detector efficiency set up or an efficiency relative to a known activity. Similar linking occurs for a 6 detector instrument.

TopCount

General Concepts

The TopCount is different than the Microbeta because it uses a single photomultiplier tube (PMT) counting from the top of the microplate instead of one PMT on top and one on bottom. Consequently, the TopCount determines background from true photon events using a time-resolved discrimination method of scintillation counting. This means that appropriate scintillators (known as slow scintillators) must be used for proper signal detection. The TopCount is available in 6- and 12-detector models.

Diagram of TopCount Pulse Discrimination (from PE LAS). Appropriate slow scintillators must be used to allow the photon energy to dissipate in a time resolved manner (multiple pulses detected during resolving time). Single pulses detected by the PMT during the resolving time would be eliminated as background noise. The TopCount uses a single PMT positioned on top as shown below. Diagrams from Perkin Elmer Life and Analytical Sciences (Document TCA-003).

Normalization of the TopCount is similar to the Microbeta, except that Well A10 is used by the detectors as the common read well. In addition, the TopCount NXT software does not have a provision to enter in an activity (in DPM) for the amount being normalized to on the plate. Therefore, results are always reported in corrected CPM, with the detectors normalized relative to each other. Efficiency of the TopCount must be determined manually and the correction factor applied to determine DPM activity. Further information about normalization procedures and applications for the TopCount can be obtained from the manufacturer.

A stepwise procedure for setting up a counting protocol on a TopCount NXT is shown on the following page.

Setup of Counting Assay

1. Click on the Assay Wizard icon located in the tool bar at the top of the software window (hold the mouse over a button to obtain a description of each icon).
2. Select Create a New Assay.
3. Define the assay name and number; select CPM as the Assay Type; select the desired plate type if requested.
4. Accept the default selection of Unknowns, unless you need to add Totals and Blanks for additional calculations.
5. Select Counting Options including delays and repeats, and select the Radionuclide from the drop-down list. The following table lists preset window settings on the TopCount NXT:

   Name	Scintillator	Energy Range	Efficiency Mode	Channels
   				Region A	Region B
   Polyvinyltoluene (PVT) SPA
   3H-PVT-SPA	Liq/Plastic	Low	Normal	1.5-35.0	1.5-256.0
   125I-PVT-SPA	Liq/Plastic	Low	Normal	1.5-100.0	1.5-256.0
   33P-PVT-SPA	Liq/Plastic	Low	Normal	2.9-256.0	2.9-256.0
   35S-PVT-SPA	Liq/Plastic	Low	Normal	2.9-256.0	2.9-256.0
   Yttrium Silicate (YS) SPA
   3H-YS-SPA	Glass	Low	High Sens.	0.0-50.0	0.0-256.0
   125I-YS-SPA	Glass	Low	High Sens.	0.0-100.0	0.0-256.0

6. Define printed and ASCII file outputs, as well as post-processing user application programs.
7. Select Instrument Correction Factors.
8. Establish Instrument Correction Factors.
9. Define Sample Map and finish Setup.
10. The first time the Assay Protocol is selected, it will be expecting a normalization plate with a sample of activity in well A10. Future runs will count plates using the stored normalization parameters.