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Quantification of Post-transplantation Chimerism: Algorithm for Correcting Systematic Errors

Quantification of Post-transplantation Chimerism: Algorithm for Correcting Systematic Errors. Moshe Israeli Computational Biology Undergraduate Program, Department of Life Science, Bar-Ilan University Project Supervisor: Prof. D. Kristt, M.D.

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Quantification of Post-transplantation Chimerism: Algorithm for Correcting Systematic Errors

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  1. Quantification of Post-transplantation Chimerism: Algorithm for Correcting Systematic Errors Moshe Israeli Computational Biology Undergraduate Program, Department of Life Science, Bar-Ilan University Project Supervisor: Prof. D. Kristt, M.D. Laboratory of Immunogenetics and Histocompatibility/ Tissue Typing - Rabin Medical Center, Petach-Tikva.

  2. Bone Marrow Transplantation • Cancer of blood cells • Genetic metabolic diseases • Anemias • Immune deficiency Bone Marrow

  3. Chimerism Cells from two different individuals in the same body

  4. chromosome cell nucleus Double stranded DNA molecule Individual nucleotides STR Markers STR

  5. AATG Short Tandem Repeats(STRs) REPEAT 7 repeats Locus Polymorphic DNA loci that contain a shortrepeated nucleotide sequence probability of a random match: ~1 in 3 trillion

  6. Donor pre-Tx Recipient post-Tx Recipient pre-Tx % CHIMERISM = D:R

  7. Rationale for the Research • Tools ‘imported’ from another field – forensic tests. • New Assay, introducing noveltools and methods of analysis. • No standardization between laboratories across the world.

  8. Chimerism Research around the world

  9. Marker performance Equipment sensitivity Calculation formula DNA concentration Percent Chimerism Size difference between donor and recipient’s alleles Unbalanced PCR amplification Donor and recipient’s allele constellation Anecdotal Sources of Variability in Chimerism Testing

  10. Project Goals • Quantification of sources of variability for % Chimerism • Devising corrective algorithm

  11. The Formula What is the correct mathematical formula for Chimerism calculation?

  12. Formula for D:R • The traditional formula:.

  13. Insensitive Inaccurate The Traditional Formula % Donor Chimerism

  14. Constant The New Formula • Chimerism ratio varies among samples, but the constant Total of DNA enables reliable comparison between them. • The new formula is:

  15. Total Cell count in sample: 1*104 The New Formula

  16. Are all markers equal in terms of accuracy and dependability?

  17. Approaches • Chimerism Simulation – treating heterozygosity as a 50% Chimerism • 5 yr Longitudinal Followup

  18. Chimerism Simulation – treating heterozygosity as a 50% Chimerism Homozygous Locus Heterozygous Locus

  19. Marker Performance Simulated Chimerism

  20. Approach 2 – Longitudinal Marker Performance

  21. Allelic Size Differences

  22. Marker performance Equipment sensitivity Calculation formula DNA concentration Percent Chimerism Size difference between donor and recipient’s alleles Unbalanced PCR amplification Donor and recipient’s allele constellation Anecdotal Sources of Variability in Chimerism Testing

  23. Conclusions and Recommendations • Constant Total amount of DNA. • Marker quality - reliability constant, α. • Size Difference – • f(AiB) Identify informative loci

  24. Improve post-tx patient monitoring • Increase laboratory reliability. • Provide clinicians with accurate data. • EFI, Israeli Transplant Society, ISHI. • Future research: • Blood. • ADMO, ASHI. Formula for Quantitative Mixed Chimerism Calculation:

  25. Acknowledgments • Project Supervisor: Prof. D. Kristt, M.D. Department of Interdisciplinary Studies, Bar-Ilan University. And. Laboratory of Immunogenetics and Histocompatibility/ Tissue Typing - Rabin Medical Center, Petach-Tikva. • Dr. Tirza Klein – Director of The Laboratory of Immunogenetics and Histocompatibility, RMC. • Dr. Jerry Stein and Dr. Isaac Yaniv of the BMT Unit, Department of Pediatric Hemato-oncology, Schneider Childrens’ Medical Center.

  26. TheEnd

  27. Chimerism Monitoring • Function of graft • Prediction of negative events . • Disease relapse • Graft rejection. • Graft-versus-host disease. The quantitative estimation of the proportion of donor versus recipient cells in the patient is related to as thepercent of mixed chimerism.

  28. Materials & Methods • DNA Extraction from patient's blood cells. • PCR amplification of STR markers. • Sequencing of the amplified products. • Sequencer output analysis using the ABI Genescan program.

  29. Recipient Before Transplant Donor Day one One month Three months

  30. ChimerTrack

  31. (D1+D2)/(D1+D2)+(R1+R2)

  32. ChimerTrack REPORT PAGE

  33. Four Methods of Analysis • One person’s DNA. • One patient’s Chimeric DNA. • Population of patients. • Artificial Chimeras.

  34. Informative Loci Which STR locus can be used?

  35. DONOR POST PRE-TX Recipient DONOR POST PRE Informative Loci

  36. Allele D1 Allele R1 Allele D2 Allele R2 USE 1 Separate Separate Separate Separate Yes 2 Separate Separate Shared Shared Yes 3 Separate Shared Shared Shared No 4 Shared Separate Shared Shared No 5 Homozygous- separate Separate 0 Separate Yes 6 Separate Homozygous- Separate Separate 0 Yes 7 Homozygous- Separate Homozygous- Separate 0 0 Yes Informative Loci

  37. Allelic Constellation Who’s peak is first – donor or recipient?

  38. Allelic Constellation

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