GENDX Azure Cielo 6 Real Time PCR System User Manual

Azure Cielo 6 Real-Time PCR System compatibility with the GenDx KMRtype ® and KMRtrack ® Workflows

Azure Cielo 6 Real Time PCR System

Kai Leng Tan1 , Bram Luiken 2 , Eleni Draina 2 , Vanna Sombatsaphay 1 1 Azure Biosystems, 6747 Sierra Ct, Suite AB, Dublin, CA 94568, USA and 2 GenDx, Yalelaan 48, 3584 CM Utrecht, The Netherlands

Introduction

A bone marrow or hematopoietic stem cell transplantation is a treatment for leukemia that is intended to replace the patient’s diseased bone marrow cells with healthy cells from a donor. After the transplantation, it is important to monitor the patient’s chimeric state (now often a mixture of the donor and recipient genotypes) to ensure that the donor cells survive and stably outnumber the recipient’s cells. An increase of the recipient genotype relative to the donor type indicates the patient’s cells are multiplying faster than the transplanted healthy cells, crowding out the healthy blood cells and signaling a relapse of leukemia. Early detection of increasing mixed chimerism allows for earlier intervention and earlier intervention is associated with improved outcomes.

The KMRtype ® and KMRtrack ® kits from GenDx are designed for high-sensitivity monitoring of donorrecipient chimerism. The KMRtype kit is used to identify markers, called informative markers, that differentiate the donor cell genotype from the recipient cell genotype. KMRtype is a multiplex assay that requires a real-time PCR system such as the Cielo 6 that offers multiple fluorescent channels. KMR products are only IVD registered in EEA, Ukraine and Jordan.
Once informative markers are identified for a donorrecipient pair using KMRtype, two of those markers are used with the KMRtrack assay to monitor post-transplant samples for chimerism. KMRtrack is a singleplex assay that assesses the genotype of the patient’s cells at the two informative markers, thereby determining whether and at what level recipient chimerism is present. KMRtrack can also reveal whether chimerism is increasing when used

Figure 1. Overview of KMRtype workflow. The steps in blue are described in the application note. Steps in white are described in the KMRengine Instructions For Use  (IFU) available from GenDx.to assess the chimeric state of patient samples collected at successive time points after the transplantation. The GenDx KMRtype and KMRtrack  kits contain all assay components and reagents required to set up multi-well plate assays that are carried out using a realtime quantitative PCR instrument. A software package from GenDx, KMRengine, is used both to set up the plate layouts for the KMRtype and KMRtrack real-time PCR assays and to analyze the output of the quantitative real-time PCR assays. KMRengine identifies informative markers based on KMRtype results and quantifies the chimerism present based on KMRtrack results. In this application note, we describe how to integrate the Cielo 6 Real-Time PCR system into the KMRtype and KMRtrack workflows (Figures 1 and 2) based on testing using a  simulated chimerism case employing cell- line gDNA.

Figure 2. Overview of the KMRtrack workflow. The steps in blue are described in the application note. Steps in white are described in the KMRengine IFU available from GenDx.

Materials and methods

KMRtype Genotyping
Genomic cell-line DNA samples, representing a donor and recipient pair, were obtained from GenDx. Each sample was genotyped for 30 targets/markers using the first 10  mixes of the KMRtype kit. The plate layout and laboratory protocol were generated by KMRengine and the run was set up in a 96-well plate accordingly. Reactions were  carried out in low-profile, opaque, white, 0.1 ml 96-well plates (Azure Biosystems AIQ322), sealed with Ultra Clear Optical Sealing Sheets (Azure Biosystems AIQ325). The  plate layout file was exported toward the Azure Cielo Manager as a .TXT file. Duplicate runs were performed to ensure consistency of results.

1. Run plate on Cielo 6 system
   Cielo Manager software was connected to Cielo 6 system via WI-FI, ethernet or USB. A new experiment was created on the Cielo Manager software. To create a new  protocol, Thermal Profile was selected under Experiment Setup from the EXPERIMENT AREA menu to the left of the screen and the Protocol Manager tab was  selected in the menu bar at the top of the screen.
   New Protocol was created and named. Dye tab was selected and dyes were assigned to each channel as recommended in the KMRengine IFU (Figure 3):
   Channel 1 FAM
   Channel 2 HEX
   Channel 4 ROX Figure 3. Dye setup window. Note some channels are “on” but unused.
   Next, the Well Selection tab was opened and it was confirmed that all channels were set to ON for all wells (Figure 4).
   Then, the Cycling Settings tab was opened and the cycling parameters were set according to the instructions in the KMRengine IFU (Table 1, Figure 5).
   The Auto Save function was turned on by toggling the circle to the bottom right of the PROTOCOL window. The file save location was selected by clicking “Browse”  and the data file automatically saved as an .AZE file when the run was completed.
   The assay was run by clicking the green Run button to the right of the Cycling Settings window. Figure 4. Well selection window Step| | Temp| Time
   ---|---|---|---
   2-step cycling
   (40 cycles)| | 95 °C| 5 min
   Denaturation| 95 °C| 15 sec
   | Annealing/Elongation| 62 °C| 1 min

Table 1. Cycling Settings

2. Import plate set up from KMRengine
   The data file (.AZE) was opened in Cielo Manager version 1.0.1.62 for data analysis. The Experiment Details screen was opened by selecting Experiment Details under  Experiment Setup from the EXPERIMENT AREA menu on the left side of the screen. On the Experiment Details screen, Import Plate Layout (.TXT) was selected  from the dropdown menu that appears when OPEN is selected in the menu bar at the top of the screen (see Figure 6). The file that had been exported from KMRengine  was then located in the navigation window and selected for import.
   The imported plate layout can be viewed by selecting Plate Setup from the menu on the left side of the screen (Figure 7). Figure 6. Importing a plate layout file from KMRengine to Cielo Manager

3. Set analysis settings
   Analysis settings were set by selecting Graphical Displays under Analysis in the EXPERIMENT AREA menu to the left of the screen and opening the Amplification  Curve tab in the menu bar at the top of the screen. Clicking the SETTINGS icon at the top right of the screen opened a popup window to access analysis setting.  Clicking the General tab of the popup window menu bar opened a screen in which the Threshold Value for each channel was set to 50000 and saved by clicking OK   (Figure 8).
   Next, on the Baseline Settings tab from the popup window menu, all channels were set to Manual baseline mode with a start value = 3 and an end value = 15 and saved by clicking OK (Figure 9).

4. Export results from the Cielo 6 to KMRengine
   Data were then exported by selecting Export under Results in the EXPERIMENT AREA menu to the left of the screen. On the Export screen, the TXT tab was  selected and the button Export txt to GenDx KMRengine was clicked (Figure 10).
   The results were imported into KMRengine following the software instructions and analyzed. The informative markers found can be used to set up monitoring experiments for the genotyped recipientdonor pair.

KMRtrack Chimerism Analysis

The KMRtrack kit was used with samples provided by GenDx. One time point (emulated post-transplant recipient sample) was analyzed for two different artificial chimerism  samples with two markers each. Selected markers were previously found to differentiate the “recipient” and “donor” genotypes. The samples and
markers used are shown in Table 2.
The experimental design and plate layout were created in KMRengine and the plate layout exported as a .TXT file. Reactions were carried out in low- profile, opaque, white,  0.1 ml 96-well plates (Azure Biosystems AIQ322), sealed with Ultra Clear Optical Sealing Sheets (Azure Biosystems AIQ325).

Table 2. Samples and markers used in KMRtrack chimerism analysis

1. Run plate on Cielo 6 system
   Cielo Manager software was connected to Cielo 6 system via WI-FI, ethernet or USB. A new experiment was created on the Cielo Manager software. To create a new  protocol, Thermal Profile was selected under Experiment Setup from the EXPERIMENT AREA menu to the left of the screen and the Protocol Manager tab was  selected in the menu bar at the top of the screen.
   New Protocol was created and named. Dye tab was selected and dyes were assigned to each channel as recommended in the KMRengine IFU (Figure 11): Channel 1 FAM
   Next, the Well Selection tab was opened and it was confirmed that all channels were set to ON for all wells (Figure 12).
   Subsequently, the Cycling Settings tab was opened and the cycling parameters were set according to the KMRengine IFU (Table 3, Figure 13).
   The Auto Save function was turned on by toggling the circle to the bottom right of the PROTOCOL window.
   The file save location was selected by clicking “Browse” and the data file automatically saved as an .AZE file when the run was completed. Figure 11. Dye setup window. Note some channels are “on” but unused. Figure 12. Well selection window. Note some channels are “on” but unused.
   The assay was run by clicking the green Run button to the right of the Cycling Settings window.

2. Import plate set up from KMRengine
   The data file (.AZE) was opened in Cielo Manager version 1.0.1.62 for data analysis. The Experiment Details screen was opened by selecting Experiment Details under  Experiment Setup from the EXPERIMENT AREA menu on the left side of the screen. On the Experiment Details screen, Import Plate Layout (.TXT) was selected  from the dropdown menu that appears when OPEN is selected in the menu bar at the top of the screen (see Figure 14). The file that had been exported from  KMRengine was then located in the computer’s file system and selected for import.Step| | Temp| Time
   ---|---|---|---
   Initial denaturation| 95 °C| 5 min
   2-step cycling(40 cycles)| Denaturation| 95 °C| 15 sec
   Annealing/Elongation| 62 °C| 1 min

Table 3. Cycling Settings Figure 13. Cycling settings window Figure 14. Importing a plate layout file from KMRengine to Cielo Manager
The imported plate layout was viewed by selecting Plate Setup from the menu on the left side of the screen (see Figure 15).

3. Set analysis settings
   Analysis settings were set by selecting Graphical Displays under Analysis in the EXPERIMENT AREA menu to the left of the screen and opening the Amplification  Curve tab in the menu bar at the top of the screen. Clicking the SETTINGS icon at the top right of the screen opened a popup window to access analysis setting.  Clicking the General tab of the popup window menu bar opened a screen in which the Threshold Value for each channel was set to 50000 and saved by clicking OK  (Figure 16). Next, on the Baseline Settings tab from the top menu of the popup window, all channels were set to Manual baseline mode with a start value = 3 and an end value = 15 and saved by clicking OK (Figure 17).

4. Export results from the Cielo 6 to KMRengine
   Data were then exported by selecting Export under Results in the EXPERIMENT AREA menu to the left of the screen. On the Export screen, the TXT tab was elected and the button Export txt to GenDx KMRengine was clicked (Figure 18).
   The results were imported into KMRengine following the software instructions. KMRengine software used the ΔΔCq method with the experimental Cq values to calculate the percentage of recipient chimerism.

Results

KMRtype
Genomic cell-line DNA samples, representing a donor and recipient pair, were genotyped for 30 markers each on the Cielo 6 using the KMRtype kit (Figure 19). Two runs  using the same samples were performed, by different operators on different days. The resulting data were successfully transferred to KMRengine for analysis (Figure 20). The  genotyping results found in both runs were 100% concordant with each other as well as with the expected results for these known samples (Figure 21).

KMRtrack
One time point (emulating a post-transplant recipient sample) was analyzed for two different artificial chimerism samples. Each “patient” sample was analyzed using two  informative markers demonstrated by the KMRtype kit to differentiate the DNA of “donor” from the “recipient”. Experiments for both “patients” were combined on one  plate.

Figure 19. KMRtype output Figure 20. KMRengine import and analysis of the 2nd run of the typing plates

Upon importing the data into KMRengine, all negative controls passed and the variation between triplicate reactions was below 2% C.V., yielding a valid result for all wells included (Figure 22).
The ΔΔCq method was then used to assess chimerism in each sample, using the generated Cqs for each marker normalized to the reference of its sample and then to a 100%  recipient reference sample. If chimerism would increase over time, this would indicate an increase in the relative amount of recipient cells and a high potential for relapse. The  current two experiments show only a starting point of such graphs. An example of the output of the monitoring assay is shown in Figure 23.

Figure 21. Resulting Cq values compared between runs and to pre-type generated by GenDx Figure 22: KMRengine import and analysis of the monitoring plate.

The results found for each marker using the Cielo 6 as well as the previous reference from GenDx are shown in Table 4.   Figure 23. Monitoring output from the analysis of sample Rec14S at one time point

Marker| ΔΔCq| % Recipient Chimerism| % Recipient Chimerism
(reference GenDx)
---|---|---|---
(Emulated) recipient 13
KMR016| 3.57| 8.39| 7.6
KMR035| 4.73| 3.77| 3.12
Mean % reported| —| 6.08| 5.36
( Emulated) recipient 14
KMR038| 9.66| 0.12| 0.08
KMR057| 10.16| 0.09| 0.09
Mean % reported| —| 0.11| 0.08

Table 4. Chimerism analysis results

Conclusion

The Cielo 6 Real-Time PCR system is easily integrated into the KMRtype and KMRtrack workflows and delivers reliable results in both genotyping and monitoring. It is  straightforward to import plate layout files from KMRengine to the Cielo 6, and to export qPCR data from the Cielo 6 to KMRengine for analysis. In the experiments carried  out in this note using known test samples provided by GenDx, all qPCR results obtained using the Cielo 6 agreed with the expected results, confirming the Azure Cielo 6 is  compatible with both the KMRtype genotyping and the KMRtrack monitoring kits.

USA : For in vitro diagnostic (IVD) use. The Azure Cielo 3 Dx and Azure Cielo 6 Dx Real-time PCR systems are registered with the U.S. FDA as Class II 510(k) exempt devices.
Europe: For IVD use. The Azure Cielo 3 Dx and Azure Cielo 6 Dx systems are CE marked under the In Vitro Diagnostic Medical Devices Directive (98/79/EC). The CE  IVD-authorized Azure Cielo Dx Systems are for distribution and use in the following countries: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czechia, Denmark, Estonia,  Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Liechtenstein, Lithuania, Luxembourg, Malta, the Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, and the United Kingdom.

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References

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