Chapter 2: Kit content o Table 1 adjusted, information on KMRtrack Starter kit corrected

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2 Updates of the Instructions for Use, RUO Edition 3.1 Chapter 2: Kit content o Table 1 adjusted, information on KMRtrack Starter kit corrected IFU RUO Edition 3.1, 2017/02 2 OF 18

3 CONTENT 1 Key to symbols 4 2 Kit content 5 3 Shipping and storage 7 4 Technical assistance 8 5 Warning and precautions 8 6 Principle 9 7 Procedure 9 8 Equipment and reagents 10 9 Protocols 11 Protocol 1. Genotyping procedure 11 Protocol 2. Chimerism monitoring Appendix a: contamination control Troubleshooting guide Limited license agreement 17 Ordering information 18 DISCLAIMER GenDx has made every effort to ensure that this IFU is accurate. Information in this IFU may be subject to change without notice. GenDx reserves the right to make improvements to this IFU and/or to the products described in this IFU, at any time without notice. If you find information in this manual that is incorrect, misleading, or incomplete, we would appreciate your comments and suggestions. Please send them to info@gendx.com. COPYRIGHT This publication, including all photographs, illustrations, is protected under international copyright laws, with all rights reserved. Neither this manual, nor any of the material contained herein, may be reproduced without written consent of the author. Copyright 2017 IFU RUO Edition 3.1, 2017/02 3 OF 18

4 1 KEY TO SYMBOLS Material number Components Batch code / Lot number Catalogue number Consult Instructions For Use Contains reagents for N tests Legal manufacturer Store at -20 C VOL Store at -20 C Contains reagents in a volume of N µl IFU RUO Edition 3.1, 2017/02 4 OF 18

5 2 KIT CONTENT KMRtype Core and Extended Genotyping kits contain KMRtype Mixes only (Table 2). The KMRtype Full Genotyping kit contains all reagents necessary to perform genotyping experiments (Table 1). The KMRtrack Core and Extended Monitoring kit contain KMRtrack assays only. Each KMRtrack assay can be ordered individually (Table 3) and chromosomal locations are indicated in Figure 1. The qpcr reagents composed of KMRassay Reference Assay (REF901) and KMRassay qpcr Buffer and Enzyme can be purchased separately. Table 1. List of available products, for Research Use Only Product Description Size Catalog # Core Mix 1 10 (30 assays) 24 samples Extended Mix (9 assays) 24 samples KMRtype Mix 1 10 (30 assays) with Reference Full 24 samples Assay and qpcr Buffer & Enzyme Core Primairy set of 30 assays 48 rxn/marker Extended Secondary set of 9 assays 48 rxn/marker KMRtrack Core set of 30 assays with Reference Starter kit 48 rxn/marker Assay and qpcr Buffer & Enzyme Reference Assay rxn KMRassay qpcr Buffer & Enzyme 288 rxn The Core and Extended kits for KMRtype and KMRtrack differ in number of assays that they contain. With the Core kit you have the availability of 30 assays, multiplexed in 10 mixes. These 10 KMRtype mixes allow the reactions for one sample to fit on one row of a 96 well plate, making genotyping more efficient. The additional 9 assays of the Extended kit, multiplexed in 3 mixes, can be applied in cases where no informative marker was obtained with the Core kit. There is also the option to apply both the Core and Extended kit at the same time, genotyping your samples for 39 assays in one go. IFU RUO Edition 3.1, 2017/02 5 OF 18

6 Table 2. List of available KMRtype mixes KMRtype Kit KMRtype Mix Fluorophore KMRtrack name KMR KMR035 KMR036 KMR KMR038 KMR039 KMR KMR041 KMR042 KMR KMR051 KMR011 KMR KMR052 Core KMR053 KMR KMR013 KMR054 KMR KMR016 KMR056 KMR KMR028 KMR046 KMR KMR048 KMR030 KMR KMR019 KMR049 KMR KMR034 KMR029 KMR017 Extended 12 KMR004 KMR031 KMR KMR010 KMR020 IFU RUO Edition 3.1, 2017/02 6 OF 18

7 Table 3. List of available KMRtrack assays, including chromosomal information KMRtrack name Chromosome Chromosome Catalogue number KMRtrack name location location Catalogue number KMR004 18q KMR039 17p KMR009 17p KMR040 7q KMR010 5q KMR041 5q KMR011 Xp KMR042 2q KMR013 6q KMR043 1p KMR014 12q KMR044 Xq KMR016 17q KMR045 10q KMR017 Yp KMR046 22q KMR019 20q KMR047 18q KMR020 1p KMR048 14q KMR028 20q KMR049 18q KMR029 2q KMR050 1p KMR030 9q KMR051 4q KMR031 11p KMR052 10q KMR033 12q KMR053 7p KMR034 1q KMR054 Yp KMR035 2q KMR055 11q KMR036 5q KMR056 1p KMR037 22q KMR057 8q KMR038 5q Figure 1. Location KMR markers on human chromosome IFU RUO Edition 3.1, 2017/02 7 OF 18

8 3 SHIPPING AND STORAGE KMRtype and KMRtrack products are: Shipped with cool packs or on dry ice and should be stored at -20 C upon arrival. Stable until the kit expiration date (kit label) when stored at -20 C. 4 TECHNICAL ASSISTANCE For technical assistance and more information: support@gendx.com Website: Phone: +31 (0) or contact your local GenDx distributor ( 5 WARNING AND PRECAUTIONS Product Use Limitations The KMRtype and KMRtrack products are for Research Use Only and not to be used in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. Product Application The KMRtype and KMRtrack products are designed for chimerism analysis and monitoring by means of qpcr. To ensure the best performance, please use the KMRtype and KMRtrack products with the materials, reagents, and equipment recommended in section 8 Equipment and Reagents. Use of materials other than specified, must be validated by user! The qpcr multiplexes used for genotyping that are contained in the KMRtype products are not designed, optimized or otherwise validated for use in quantitative analysis of chimeric mixtures. GenDx does not recommend the use of these multiplexes for quantifying mixtures and does not support the use of these reagents for that purpose. The KMRtrack products are specifically designed, optimized and validated for use in chimeric mixture analysis. These reagents have been specifically formulated for optimal performance. Altering the recommended concentrations of reaction components could lead to inaccurate results. Use of alternative reactions conditions is not recommended or supported by GenDx. Please take special note of Appendix A: Contamination control. Safety Information When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. IFU RUO Edition 3.1, 2017/02 8 OF 18

9 6 PRINCIPLE KMRtype and KMRtrack products are primer probe systems dedicated for genotyping samples and quantifying chimeric DNA mixtures by means of qpcr. First, pre-transplant DNA samples of the recipient and donor(s) are genotyped to enable the selection of genetic markers which uniquely differ between the samples, also called informative markers. This process is performed using the KMRtype reagents where 3 markers are multiplexed together in each mix. Second, post-transplantation DNA samples can be monitored for the presence of a reference DNA, which could either be recipient or donor DNA. The reference DNA can be quantified in a chimeric mixture using the informative markers obtained during genotyping. Multiple post-transplantation DNA samples can be measured in order to monitor the chimeric status of the transplant over time. This process is performed using the KMRtrack reagents where 1 marker is applied per reaction (singleplex). The whole process from start to finish can be registered using KMRengine. This chimerism-dedicated software package allows users to: - Register details such as name and date of birth of recipient and donor(s) - Design experiments with KMRtype and KMRtrack for which KMRengine will generate a detailed protocol and sample setup - Analyse the obtained data and calculate the percentage of reference DNA - Record results in reports and overviews - Follow the chimeric state of a transplant over time with a detailed data overview and graph 7 PROCEDURE 1. Experimental design genotyping. Information on recipient and donor(s) is registered and the protocol and sample setup is designed using KMRengine. 2. Genotyping. This is performed by qpcr using: - KMRtype mixes - KMRassay Reference Assay (REF901) - KMRassay qpcr Buffer & Enzyme - Pre-transplant DNA sample of recipient and donor(s) 3. Genotyping data analysis. Informative markers are identified for each sample using KMRengine. 4. Experimental design monitoring. Information on post-transplant samples is registered and the protocol and sample setup is designed using KMRengine. 5. Monitoring. This is performed by qpcr using: - KMRtrack assays that are informative for the reference DNA - KMRassay Reference Assay (REF901) - KMRassay qpcr Buffer & Enzyme - Post-transplant chimeric DNA sample 6. Monitoring data analysis. The proportion of the reference DNA in the chimeric mixture is calculated using KMRengine. IFU RUO Edition 3.1, 2017/02 9 OF 18

10 8 EQUIPMENT AND REAGENTS KMRtype and KMRtrack products have been validated on Life Technologies Viia7 qpcr platform. Other qpcr machines may require different profiles and may require user validation. Please go to our website for the up to date list of compatible qpcr machines. Table 4. Equipment and reagents Equipment and reagents Catalogue number Supplier General equipment and reagents Pipettes and tips (hydrophobic filters) N.A. Multiple Ice or cooling block N.A. Multiple Optical 96-wells reactions plates N.A. Multiple Eppendorf tubes N.A. Multiple PCR tubes, strips or plates N.A. Multiple Optical adhesive seals N.A. Multiple Micro centrifuge N.A. Multiple Microtiter Plate Centrifuge N.A. Multiple Vortex N.A. Multiple MilliQ water N.A. Multiple Compatible qpcr machine N.A. Multiple Sample Genotyping KMRtype Core Genotyping kit (24 samples, RUO) GenDx KMRtype Extended Genotyping kit (24 samples, RUO) GenDx KMRtype Full Genotyping kit (24 samples, RUO) GenDx KMRassay Reference Assay 901 (288 reactions, RUO) GenDx KMRassay qpcr Buffer & Enzyme (288 reactions, RUO) GenDx Chimeric Monitoring KMRtrack Core Monitoring kit (48 reactions, RUO) GenDx KMRtrack Extended Monitoring kit (48 reactions, RUO) GenDx KMRtrack Starter Monitoring kit (48 reactions, RUO) GenDx KMRtrack Assays (48 reactions, RUO) GenDx.com GenDx KMRassay Reference Assay 901 (288 reactions, RUO) GenDx KMRassay qpcr Buffer & Enzyme (288 reactions, RUO) GenDx Plate setup and analysis KMRengine GenDx.com GenDx IFU RUO Edition 3.1, 2017/02 10 OF 18

11 9 PROTOCOLS PROTOCOL 1. GENOTYPING PROCEDURE Important notes before starting: Hematologic malignancies are often aneuploidy. In case of isochromosomal defects, selection of informative recipient markers that target the deleted chromosomal region should be avoided. It is therefore also highly recommend to select several markers for monitoring instead of depending on one marker. Blood samples should be collected in tubes with ACD or EDTA as an anticoagulant. Do NOT use heparinized samples, as this has an inhibitory effect on a PCR. Purified DNA should have an A260/A280 ratio between 1.7 and 1.9 as determined with a Nanodrop. If necessary, DNA should be diluted in 10mM Tris, ph 8.0 containing 0.1mM EDTA (TE buffer-low EDTA) or nuclease-free H2O before use. Use optical qpcr plates for your reactions. Protocol: 1. Design your experiment in KMRengine. 2. Set up all reactions in a pre-pcr lab. 3. Reactions can be set up without ice under ambient conditions. 4. Briefly vortex and then quickly centrifuge all tubes before opening to ensure that the solution is well mixed and at the bottom of the tube. 5. Prepare a qpcr Master Mix as indicated in Table 5. Table 5. Composition of the qpcr Master Mix for genotyping Component 1x KMRassay qpcr Buffer, 4x 5 μl KMRassay Enzyme, 50x 0.4 μl Nuclease free H 2O 0.6 μl Total Volume 6 μl 6. Mix the qpcr Master Mix thoroughly, centrifuge briefly, and add 6 µl of qpcr Master Mix into each well. 7. Dispense 5 µl of the 4x KMRtype mixes to the respective wells as indicated in the KMRengine Assay Layout view. 8. Add 5 µl of 4x KMRassay Reference Assay to the wells designated for positive control and no-template control reactions. 9. Add 9 µl of 1.1 ng/µl sample DNA to all wells as indicated in the KMRengine Assay Layout view. Except the no-template control reactions, add 9 µl of H 2O to these wells. 10. The total volume per well amounts to 20 µl. IFU RUO Edition 3.1, 2017/02 11 OF 18

12 11. Cover and seal the plate with Optical Adhesive film, making certain that all wells are completely sealed. 12. Vortex the plate to mix the contents of each well. 13. Centrifuge the plate briefly to collect the contents at the bottom of the wells. 14. Load the plate into your qpcr machine. 15. Open a pre-configured template which contains the necessary experimental properties and run conditions as described in Table 6. Table 6. Cycling protocol for amplification Step Temp Time Initial denaturation 95 C 5 min 2-step cycling Denaturation 95 C 15 sec Annealing/Elongation 62 C 1 min 40 cycles 16. Import the Sample Setup sheet generated by KMRengine. 17. Make sure the appropriate channels are calibrated and selected for each sample. Names may differ between qpcr systems. With KMRtype you need to measure 4 fluorophores: o o Orange560 (also corresponding with JOE, VIC and HEX) o Red610 (also corresponding with ROX) o Quasar670 (also corresponding with Cy5) The KMRengine Sample Setup sheet will specify which channels KMRengine recommends for your run and qpcr system. 18. Save the file with a unique name and start the qpcr run. 19. After the qpcr run is finished, normalize the data using the qpcr software and export your data to a.txt or.csv file (file format is qpcr system specific). Make sure that the channel names are kept the same as in the Sample Setup sheet from KMRengine. Note: Each qpcr system will have its own analysis settings for normalization and its own way of exporting data to specified file formats. If you are unsure on how to proceed, please support@gendx.com with your qpcr system specifics. 20. Import the qpcr export file into KMRengine for analysis. Analysis of genotyping data is recommended to be performed by KMRengine, for more details about how to run results through KMRengine please read the KMRengine Instructions for Use or manual. IFU RUO Edition 3.1, 2017/02 12 OF 18

13 PROTOCOL 2. CHIMERISM MONITORING Important notes before starting: It is highly recommend to apply two or three markers (if possible) for monitoring. Blood samples should be collected in tubes with ACD or EDTA as an anticoagulant. Do NOT use heparinized samples, as this has an inhibitory effect on a PCR. Purified DNA should have an A260/A280 ratio between 1.7 and 1.9 as determined with a Nanodrop. If necessary, DNA should be diluted in 10mM Tris, ph 8.0 containing 0.1mM EDTA (TE buffer) or nuclease-free H2O before use. Use optical qpcr plates for your reactions. Protocol: 17. Design your experiment in KMRengine. 18. Set up all reactions in a pre-pcr lab. 19. Reactions can be set up without ice under ambient conditions. 20. Briefly vortex and then quickly centrifuge all tubes before opening to ensure that the solution is well mixed and is at the bottom of the tube. 21. Prepare a qpcr Master Mix for triplicate reactions as indicated in Table 7. Table 7. Composition of the qpcr Master Mix for chimeric mixture analysis Component 1x KMRassay qpcr Buffer, 4x 5 μl KMRassay Enzyme, 50x 0.4 μl Nuclease free H 2O 0.6 μl Total Volume 6 μl 21. Mix the qpcr Master Mix thoroughly, centrifuge briefly, and add 6 µl of qpcr Master Mix into each well. 22. Dispense 5 µl of the 4x KMRtrack assays to the respective wells as indicated in the KMRengine Assay Layout view. 23. Add 5 µl of 4x KMRassay Reference Assay to the wells designated for positive control and no-template control reactions. 22. To the wells indicated for pre-transplant DNA, add no less than 10 ng pure DNA. To the wells indicated for post-transplant DNA, add the amount of DNA sufficient to achieve your desired sensitivity limit (See Table 8 for the relationship between input DNA/cells and sensitivity). 24. Add 9 µl of DNA to all wells, as indicated in the KMRengine Assay Layout view, with the amounts of DNA as determined in step 21. Except the no-template control reactions, add 9 µl of H 2O to these wells. 23. The total volume per well amounts to 20 µl. IFU RUO Edition 3.1, 2017/02 13 OF 18

14 Table 8. Relationship between minimal detectable chimerism level through DNA input, number of needed cells and sensitivity. Input DNA (ng) # Cells Sensitivity % # Cells Input DNA (ng) Sensitivity % Sensitivity % # Cells Input DNA (ng) , ,05 0, , ,07 0, , ,13 1, , ,29 2, , ,67 5, , , Cover and seal the plate with Optical Adhesive film, making certain that all wells are completely sealed. 25. Vortex the plate to mix the contents of each well. 26. Centrifuge the plate briefly to collect the contents at the bottom of the wells. 27. Load the plate into your qpcr machine. 28. Open a pre-configured template which contains the necessary experimental properties and run conditions as described in Table 9. Table 9. Cycling protocol for amplification Step Temp Time Initial denaturation 95 C 5 min 2-step cycling Denaturation 95 C 15 sec Annealing/Elongation 62 C 1 min 40 cycles 29. Import the Sample Setup sheet generated by KMRengine. 25. Make sure the appropriate channels are calibrated and selected for each sample. Names may differ between qpcr systems. With KMRtrack you need to measure 2 fluorophores: o o Quasar670 (also corresponding with Cy5) The KMRengine Sample Setup sheet will specify which channels KMRengine recommends for your run and qpcr system. 30. Save the file under a unique name and start the qpcr run. 31. After the qpcr run is finished, normalize the data using the qpcr software and export your data to a.txt or.csv file (file format is qpcr system specific). Make sure that the channel names are kept the same as in the Sample Setup sheet from KMRengine. Note: Each qpcr system will have its own analysis settings for normalization and its own way of exporting data to specified file formats. If you are unsure on how to proceed, please support@gendx.com with your qpcr system specifics. 32. Import your qpcr export file into KMRengine for analysis. Analysis of monitoring data is recommended to be performed by KMRengine, for more details about how to run results through KMRengine, please read KMRengine Instructions for Use or manual. IFU RUO Edition 3.1, 2017/02 14 OF 18

15 10 APPENDIX A: CONTAMINATION CONTROL IMPORTANT: It is extremely important to include at least one negative control in every qpcr setup that lacks template nucleic acid to detect possible contamination. General physical precautions Separate the working areas for setting up the qpcr master mix and DNA handling, including the addition of starting template, PCR product analysis, or plasmid preparation. Ideally, use separate rooms. Use a separate set of pipettes for DNA. Use of pipette tips with hydrophobic filters is strongly recommended. In case of contamination, laboratory benches, apparatus, and pipettes can be decontaminated by cleaning them with a 1% Trigene disinfectant. Afterwards, the benches and pipettes must be rinsed thoroughly with 70% ethanol. General chemical precautions Another approach to prevent amplification of contaminating DNA is to treat individual reaction mixtures with DNAseI or restriction enzymes that cut between the binding sites of the amplification primers used, before adding the template DNA sample. IFU RUO Edition 3.1, 2017/02 15 OF 18

16 11 TROUBLESHOOTING GUIDE Sensitivity limit of monitoring reactions To demonstrate that the desired sensitivity was possible with the reagents and cycling protocol used, we recommend to use a control sample of known concentration, which is at the desired sensitivity limit, to be executed in parallel with the unknown samples. Use of multiple markers in monitoring We suggest using multiple markers, since the use of a single marker may yield erroneous results. Using two or more markers for monitoring will provide more confidence in the levels of chimerism measured. If two or more markers yield significantly different results, the user may want to consider repeating the experiment. Negative control is positive In case the Cq of the negative control well is < 38.0, the negative control failed due to positive amplification in that well. To view the Cq data for each well refer to the Data View tab within the Results window of KMRengine. Check all negative controls on plate, there are two possible scenario s: 1. If all are positive, there might be DNA contamination in the qpcr Master Mix or the solution used to dilute the DNA. Start with fresh reagents and repeat. 2. If it is the only positive negative control it could be that accidently DNA was pipetted into the well designated as negative control. Set up plate again being careful not to repeat the same issue. Positive control is negative Cq of Reference Assay is > To view the Cq data for each well refer to the Data View tab within the Results window of KMRengine. Check all wells on plate that contain the Reference Assay, there are two possible scenario s: 1. If all are negative, there might be an issue with the Reference Assay. Check that the reagent was properly stored and that it is not out of date. 2. If it is the only negative positive control it would be that accidently the Reference Assay was not pipetted into the well designated as positive control. Set up plate again being careful not to repear the same issue. Atypical well An atypical result observed for an individual assay such as the Cq of assay wells that are outside of the specified range when compared to the Cq of the controls. This could have two reasons: 1. Poor DNA quality. Verify that it is not degraded or set up a new reactions with freshly extracted DNA. 2. Reaction plate not sealed properly prior to amplification. Ensure that the plate is sealed with Optical Adhesive Film. To ensure reaction wells have not had evaporation during PCR, check plate wells for consistency of volume after amplification. If wells have inconsistent volumes run a new plate. For all other questions and remarks regarding this IFU we refer you to our technical support, support@gendx.com. Some additional information like frequently asked questions, product information and updates can be found on our website, IFU RUO Edition 3.1, 2017/02 16 OF 18

17 12 LIMITED LICENSE AGREEMENT Use of this product signifies the agreement of any purchaser or user of the GenDx KMRtype and KMRtrack products with the following terms: The KMRtype and KMRtrack products may be used solely in accordance with the IFU KMRtype genotyping kit and KMRtrack monitoring assays, and only with components described in the IFU. GenDx grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the GenDx KMRtype and KMRtrack IFU and additional protocols available at Other than expressly stated licenses, GenDx makes no warranty that KMRtype/KMRtrack products and/or its use(s) do not infringe the rights of third-parties. This kit and its components are licensed for one-time use and may not be re-used, re-furbished, or re-sold. GenDx specifically disclaims any other licenses, expressed or implied other than those expressly stated. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. GenDx may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components. For updated license terms, see Trademarks: KMRtype, KMRtrack and KMRengine are registered trademarks of KimerDx B.V. GenDx is a registered trademark of Genome Diagnostics B.V. All other trademarks are the property of their respective owners.( IFU RUO Edition 3.1, 2017/02 17 OF 18

18 ORDERING INFORMATION GenDx products are supported either directly or by your local GenDx distributor or reseller. Please contact your local GenDx distributor or GenDx Customer Support team at or for US customers) for any product information or quote request. GenDx Alexander Numan Building Yalelaan CM Utrecht, the Netherlands Phone: +31 (0) Fax: +31 (0) www: GenDx, all rights reserved. IFU RUO Edition 3.1, 2017/02 18 OF 18

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