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Technote #
Title
1 How can I alter the reporter Channel (RP1) Voltage/Sensitivity
Date Updated Category
17-08-07 Calibration / Control Verification
Products
[STarStation 2.0] [STarStation 2.3]

The Luminex is a dynamic fluorescent detection system in which the signal output must be standardized through a process known as calibration. Calibration is essential for both optimal performance and day-to-day reproducibility of results. The STarStation calibration routine consists of an algorithm that automatically sets the peak of a sample of standardized beads to a specific channel or "target" value. This algorithm is used for calibrating all four channels on the system (DD, CL1, CL2 and RP1). The calibration process uses calibration microspheres to adjust voltage settings for optimal and consistent microsphere classification and reporter readings over time and across different instruments.

Users that wish to modify the "normal" reporter detector sensitivity of their Luminex system established during Reporter Microsphere calibration ( e.g. when using an Alexa 532 Reporter fluorochrome, or specific assays which recommend using the HIGH RP1 Target calibration setting) should refer to the instructions outlined in the following options:

Instructions for Modifying the Reporter Detector Sensitivity - STarStation Version 2.3 (Luminex 100 IS 2.3 and LX200 Systems)

Instructions for Modifying the Reporter Detector Sensitivity - STarStation Version 2.0 (Luminex 1.7 and IS version 2.2 Systems)

STarStation 2.3 Users: How to Modify the Reporter Detector Sensitivity of your Luminex System

1) STarStation version 2.3 users can modify the sensitivity of their system via altering the target value for the RP1 parameter prior to performing a CAL2 calibration.This process is outlined below:

a) Launch STarStation 2.3 and display the Reagent Manager (hold the CTRL key and Press R). When the Reagent Manager is displayed activate the CAL2 tab.

b) Press the New button and create a new entry for a CAL2 microsphere lot. Multiply the RP1 target value for your current active CAL2 reagent by the conversion factor 4.55.

So for example, if the lot of Reporter Calibration Microspheres you currently use is A5192 (which has an RP1 target value of 3515) the HIGH RP1 target value you need to enter into the Reagent Manager will be 3515 x 4.55 = 15993.

We recommend that you name the lot something like "A5192HIGH" to differentiate this from lot A5192 which is used with the standard RP1 target Value.

Note: When entering a lot number/name into the STarSTation 2.3 Reagent Manager it is important that there is no space in the lot number/name, e.g. to signify a high rp1 for lot A5192 enter the name "A5192HIGH" and NOT "A5192 HIGH".

c) Click the Save button.

d) When users require to increase the reporter detection sensitivity of their system they should simply perform a calibration using the correct lot of microspheres using the HIGH RP1 target value.

To perform a CAL2 calibration use the Calibrate Reporters button on the Calibrate tab of the STarStation Instrument Controls. Remember to use the correct lot of microspheres toghether with the HIGH RP1 target value (see image below).

Note: Any attempt to perform reporter control (CON2) operations when the system has high RP1 target settings will result in a failure of the CON2 operation. The system will maintain the higher sensitivity settings until calibrated using CAL2 with the standard RP1 target value.

e) The CAL2 Calibration operation will proceed and continue for up to 180 seconds, during this time the system alters the voltage on the RP1 detector in an attempt to place the mean detected fluorescence generated by the calibration microspheres into the channel on the detector which corresponds with the target value you specified for the calibration. If the mean RP1 fluorescence is not placed in or near the target channel in this time the calibration will fail. If calibration fails you should immediately attempt calibration again (you can use the same well as you used for the first calibration operation and you should not need to use additional CAL2 reagent). The second time the calibration procedure starts it will begin at the RP1 voltage it reached at the end of the previous calibration attempt.

f) Following Calibration the current RP1 voltage setting for your instrument can be viewed via the Status tab of the STarStation Instrument Controls (see image below).

STarStation 2.0 Users: How to Modify the Reporter Detector Sensitivity of your Luminex System

STarStation 2.0 users have two options available to them for modifying the Reporter detector sensitivity of their system.

Option 1: change the RP1 target value prior to performing CAL2 (Reporter Microsphere) Calibration (this indirectly modifies the voltage on the reporter fluorescence detector since the voltage is only modified when the modified RP1 target value is used to perform a Reporter (CAL2) Calibration).

Option 2: change the RP1 detector voltage directly by specifying the voltage setting required for the Reporter fluorescence (RP1) detector.

Modifying the Reporter Fluorescence Detector Sensitivity Indirectly (STarStation version 2.0)

ACS recommends that users first attempt to modify the reporter detector sensitivity using the indirect method (entering elevated RP1 target values into the STarStation 2.0 Reagent Manager and calibrating with these HIGH RP1 target values) before using the direct method provided by the STarStation User Specified Reporter Voltage functionality outlined below. Using the indirect high-RP1 target method of increasing reporter fluorescence sensitivity is recommended since this method is specific to the users system.

1) Launch STarStation 2.0 and display the Reagent Manager (use the shortcut sequence CTRL + R)

2) When the Reagent Manager is displayed activate the Calibration tab.

3) Click the New CAL2 buttonand create a new entry for a CAL2 microsphere lot. Multiply the RP1 target value for your current live CAL2 reagent by the conversion factor 4.55.

So for example, if the Reporter Calibration Microsphere lot you currently use is A5192 (which has an RP1 target value of 3515) the HIGH RP1 target value you need to enter into the Reagent Manager will be 3515 x 4.55 = 15993.

ACS recommend that you name the lot something like "A5192HIGH" to differentiate this from lot A5192 which you use to perform a "standard" CAL2 calibration.

When the details have been entered for the new "HIGH RP1" lot click the OK button.

4) When users require to increase the reporter detection sensitivity of their system they should simply perform a CAL2 calibration using the Calibrate Reporters Button on the Calibrate tab of the STarStation Instrument Controls. Remember to use the correct lot of microspheres together with the HIGH RP1 target value (see image below).

5) Note:It is may not always be possible to successfully calibrate using the HIGH RP1 target values on earlier Luminex systems using STarstation 2.0.

Users might encounter difficulty in changing from a low to a high RP1 target value during CAL2 calibration. As a result, the CAL2 calibration may fail a large percentage of time when switching from a low to a high RP1 target value.

Outlined below is a procedure which allows the calibration to start at a specific PMT voltage close to the final result, thereby facilitating the calibration process. If a successful HIGH RP1 calibration has been performed skip to step 6).

Procedure to Follow If HIgh RP1 Calibration Fails using STarSTation 2.0

i) Open the Luminex Library Calibration history which is stored in a file called CalHistory.csv located in theC:/Program Files/Luminex/LXRLibrary/Log folder.

ii) Locate the RP1 Detector voltage column in the log and review the previous entries. Locate and record the most recent successful "high" CAL2 Calibration RP1 voltage entry (i.e. high calibration refers to the RP1 target value of approximately 16894, which should result in a corresponding PMT voltage of 600-800 depending on the instrument, in the example illustrated in the figure below the value you would use is shown in bold red outline).

iii) Open the LxrLibrary Test Application by double clicking the Test Program option via the Windows Start button->Programs->Luminex->LxrLibrary->Test Program.

iv) When the LxrLibrary Test Application launches, enter the value 1015 into the Property ID field. Press the Get button, the current instrument RP1 Gain voltage setting should be displayed in the Property Value field.

v) Without closing the LxrLibrary Test Application , proceed with CAL2 calibration in STarStation using the "high" RP1 target value. As soon as calibration starts switch back to the LxrLibrary Test Application.

vi) Enter the most recent successful "high" calibration RP1 detector voltage or the RP1 gain value recorded when the most recent calibration attempt failed in in the Property Value field and press Set.

If you have not performed a HIGH RP1 calibration previously or do not know what the most recent successful "high" calibration RP1 detector voltage is proceed to step vi) otherwise skip to step vii).

vi) As calibration starts in STarStation the RP1 Gain or voltage will jump from the value established during the last successful calibration to something in excess of 800V. You can monitor this by pressing the Get button repeatedly to display the current RP1 gain value in the Property Value field. Continue to monitor the RP1 gain you will find that the voltage setting now starts to decrease. Contiue to monitor this decrease (by pressing the Get button) until the point at which calibration will pass or fail (you will need to refer to the Calibration dialog in STarStation to ascertain this).

If Calibration fails, record what the RP1 voltage setting was at the time the calibration process failed (following calibration failure press the Get Button and the RP1 gain value will be displayed in the Property Value field ).

Now repeat the calibration in STarStation and use the value voltage recorded above to set the instrument in step vi)

vii) Once you have successfully completed CAL2 calibration, return to the Luminex Test Application and press the Close button to exit the program.

If STarStation 2.0 users have difficulty in successfully calibrating their system using the HIGH RP1 target value they can follow the instructions for directly modifying the RP1 detector voltage.

Note: Any attempt to perform reporter control (CON2) operations when the system has high RP1 target settings will result in a failure of the CON2 operation. The system will maintain the higher sensitivity settings until calibrated using CAL2 with the standard RP1 target value.

6) The current RP1 voltage setting for your instrument can be viewed via the Status tab of the STarStation Instrument Controls (see image below).

Modifying the Reporter Fluorescence Detector Sensitivity Directly (Available in STarStation version 2.0 only)

Note that the Reporter PMT detector voltage is normally set during CAL2 calibration, and should only be adjusted by users who know the precise voltage setting that they want to specify. If you change the RP1 detector voltage, you will no longer have day-to-day and unit-to-unit reproducibility of results (additionally all Reporter Control (CON2) operations performed when the system has HIGH RP1 calibration settings will fail).

Users can directly set the Reporter Detector Voltage (sensitivity) setting of their Luminex system prior to running an assay. A field called "User Specified Voltage) is available in the STarStation acquisition wizard (see figure 1) that allows the user to set the RP1 detector voltage value (up to a maximum of 1000V) before an assay is started.

Note: The STarStation User Specified Voltage function only sets the RP1 detector voltage on a per session basis, the RP1 detector voltage will be reset to the calibrated value after the microplate is read and the results are generated.


Figure 1

By default the acquisition wizard in STarStation 2.0 does not display the User Specified Voltage Control.

A software utility (figure 2) is required to be able to set the RP1 voltage manually.


Figure 2

To display the User Specified Voltage Control on the Acquisition wizard

1) Download the toggle voltage utility here and copy to your PC.

2) Ensure that the STarStation 2.0 Acquisition wizard is not running and launch the toggle voltage utility.

3) Select the "Allow User to Set RP1 Voltage" option and click the OK button.

 

Note that altering the RP1 voltage will influence all assays in a multiplex and will have the affect of increasing bakground MFI readings for all assays.

Please note that the "normal" RP1 detector voltage will vary from calibration to calibration on individual systems, this variation in RP1 detector voltage is related to the variation in reporter laser output power. RP1 detector voltage increases as the reporter laser ages. The RP1 detector voltage can be located in STarStation via the status tab of the instrument controls. (Note: If you have set the RP1 voltage directly the current RP1 voltage will differ from the RP1 voltage determined at the time of the Last Reporter Calibration).

Figures 3 and 4 below illustrate how modifying the RP1 voltage influences the detected RP1 MFI. Reference to figure 3 indicates that increasing the RP1 detector voltage increases the detected median fluorescent intensity (MFI) of the system.

STarQuality target and background beads were used as samples. Readings of each microsphere type were performed using a range of RP1 detector voltages.
On the instrument used to perform the tests the calibration set voltage was 627 V.

This information is provided for illustration purposes only. Users should determine the required RP1 detector voltage setting themselves by running samples (e.g. CON2 microspheres or ACS StarQuality Target beads) over a range RP1 voltages to determine the voltage setting which is most applicable.


The target beads had an MFI value of 8900 (at the "normal" RP1 voltage) and this could be influenced significantly by altering the RP1 voltage without increasing the background readings too much. At the maximum voltage of the RP1 detector (1000 V) the MFI of the target beads is more or less at the maximum (32767 is the highest MFI , since this is the highest channel number on the RP1 detector).

Voltage Background Bead MFI (Events Counted) Target Bead MFI (Events Counted)
500
0 (90)
1801 (101)
627 (calibration set voltage)
5 (59)
8913 (96)
700
11 (55)
19323 (99)
800
51 (12)
28683.00(49)
1000
155 (48)
32429 (80)

Figure 3


Figure 4


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