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KimerDx overview Chimerism Monitoring qPCR KimerDx solution Chimerism EFI 2010 workshop KMRtype Genotyping Monitoring Chimerism KMRtrack Monitoring Current Method KMRengine workflow Earlier detection Workflow Advantages Available products For Research Use only 1 M15-030 Chimerism Monitoring Chimerism Recipient Complete Donor Chimerism Mixed chimerism Healthy cell Diseased cell Donor cell Relapse Donor For Research Use only 3 Relapse probability Monitoring Chimerism Increasing mixed host chimerism ►Leads to lower probability of survival ►Higher probability of relapse Koldehoff, et al, A J Hematology 2006 For Research Use only 4 STR for chimerism analysis • Short Tandem Repeats – – – – – Limited sensitivity Tedious analysis Multiple pieces of equipment Peak imbalances Not intended for BMT monitoring For Research Use only 5 Earlier detection with qPCR STR LOD qPCR LOD Jimenez et al, Leukemia, 2005 For Research Use only 6 qPCR vs STR • STR = Poor sensitivity (1-5%) – Confirms relapse, no anticipation • qPCR = high sensitivity (0.05%) STR qPCR n=8 n=27 – Detects recipient cells months before relapse Koldehoff, et al, A J Hematology 2006 For Research Use only 7 qPCR for chimerism monitoring • Early detection through high sensitivity STR LOD qPCR LOD • Automatable • Rapid, objective analysis - workflow • Platform independent For Research Use only 8 qPCR For Research Use only qPCR • Quantitative PCR – DNA amplification – Monitored in real-time – Using fluorescence DNA Detection Fluorescence For Research Use only 10 qPCR DNA Probe Primer Polymerase dNTPs 11 qPCR signal detection Light source Detector Emission filter Excitation filter Signal For Research Use only 12 qPCR signal detection Plateau phase Lineair phase Exponential phase Cq cycle For Research Use only 13 qPCR 1 Cq cycle = 2 fold [DNA] For Research Use only 14 qPCR quantification • Absolute Sample – Standard curve Standard • Relative – Ratios of Cqs – Internal control (reference gene) – Uniform Efficiency Sample Reference gene For Research Use only 15 Sensitivity • Minimal detectable chimerism level • Dependent on amount of input DNA • Detection limit = 10 copies Sensitivity* Cells DNA Heterozygous: 10Input cells % # ng 0.05 20 0005 cells 132 Homozygous: 0.10 10 000 66 1 000 6.61.00 pg per cell 2.00 500 5.00 200 7 3 1 Example: 132 ng of DNA 132 / 0,0066 10 / 20 000 0.0005 * 100% = 20 000 cells = 0,0005 = 0.05% chimerism of 0.05% will be detectable For Research Use only 16 EFI 2010 Workshop For Research Use only EFI 2010 Workshop • Clinical relevance • EFS proposed study – Selected patients: • significant increase in mixed chimerism (STR) • followed by relapse • Samples available: • 6 patients, more than one sample before relapse were available (21 total) For Research Use only Participants in EFI Workshop Country Turkey Croatia Italy United Kingdom Poland Tunisie Poland Spain Italy Italy Poland Netherlands France France City Ankara Zagreb Turin Manchester Wroclaw Tunis Warsaw Salamanca Milano Florence Warsaw Maastricht Grenoble Rennes Group Hematology Laboratory Medicine University Tissue Typing Center, University Hospital Center Immunologia dei Trapianti, Universitaria S.Giovannia Battista Transplantation Laboratory, Manchester Royal Infirmary Department of Clinical Immunology, L. Hirszfeld Institute of Immunology and Experimental Therapy HLA Laboratory, Centre de transfusion Sanguine, Tunis Department of Immunogenetics, Institute of Haematology and Transfusion Medicine Hospital Universitario de Salamaca Tissue Typing Lab Department of Clinical Physiopathology, Medical Genetics Unit Medigen molecular diagnostics Transplantation Immunology, Tissue Typing Laboratory, University Hospital Maastricht HLA Laboratory, EFS Rhon Alpes HLA Laboratory, EFS Bretagne For Research Use only Confidential Information For Research Use only Workshop Results • For 6 patients more than one sample before relapse were available • Increasing mixed chimerism could be measured by qPCR assay before the STR related diagnosis (from day -140 to -60) For Research Use only Our Solution For Research Use only Chimerism Post-transplant Pre-transplant Recipient Mixed chimerism 2 weeks 4 weeks 2 months 6 months 0.1% 0.5% 2% Relapse 6 Marker 1 Healthy cell Diseased cells Donor cells Donor Genotyping KMRtype Marker 2 4 2 0 2 4 8 24 Monitoring: KMRtrack For Research Use only 23 KimerDx Quantitative Chimerism Monitoring • KMRtype® • KMRtrack® • KMRengine® Genotyping Monitoring Analysis Software For Research Use only 24 Genotyping workflow Recipient PreTransplant Donor Genotyping KMRtype Informative For Research Use only 25 Informative marker selection Patient specific markers Donor specific markers For Research Use only 26 KMRtype Assay set up 30 markers tri-plexed Reference gene No template control Sample Reference gene No template control For Research Use only 27 KMRtype Genotyping Recipient PreTransplant Donor Genotyping KMRtype For Research Use only 28 Monitoring workflow Recipient Recipient PreTransplant PreTransplant Recipient PostTransplant Monitoring KMRtrack Recipient PostTransplant For Research Use only 29 KMRtrack Assay set up KMRtype sample Reference gene No template control For Research Use only 30 Experimental files Export file Import file KMRengine qPCR instrument Template file [email protected] For Research Use only 31 KMRengine For Research Use only 32 KimerDX • Provides Earliest Detection of Adverse Events • Workflow is simple and can be automated • Saves valuable hands on time in the lab For Research Use only 33 Workflow advantages For Research Use only Workflow advantages KimerDx Other KMRengine analyses data (seconds) Manual data analysis (30 min per sample) Developed for BMT monitoring Not intended for BMT monitoring Sensitivity 0.05% Poor sensitivity (1-5%) Predicts relapse 3-4 months earlier Confirms relapse, no anticipation Multiple donor Very difficult to do multiple donor analysis Platform independent Restricted to platforms or Multiple instruments needed Software supports experimental set up Lot of manual calculations and experimental set up Software stores and analyses data Minimal data analysis Automatability (Qiagility robot & Rotor-Gene qPCR system) Not automatable For Research Use only 35 Workflow advantages Time to 5 Timepoints for 10 Samples 2500 2050 Time (Minutes) 2000 1500 1000 850 500 250 90 0 Total Time STR Total Time qPCR Setup PCR CE setup Hands On STR CE run Hands On qPCR Analysis For Research Use only 36 Workflow advantages For Research Use only 37 Workflow advantages STR LOD qPCR LOD STR qPCR n=8 n=27 For Research Use only 38 Workflow advantages For Research Use only 39 Available products For Research Use only KMRtype • 30 marker mixes in 1 box • Reference Gene Assay • qPCR Buffer & Enzyme (24 typings) (288 rxn) (288 rxn) For Research Use only 41 KMRtrack • 30 single markers, full set • Reference Gene Assay • qPCR Buffer & Enzyme (48 rxn) (288 rxn) (288 rxn) For Research Use only 42 KimerDx KMRengine • • • • Compatible with all major qPCR machines Supports experimental set up Analyse multiple donors Stores data For Research Use only 43 KimerDx Developments • • • • Continuous quality improvement Moving towards CE mark Domain expertise In house R&D and software development team For Research Use only 44