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

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

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

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

8. Chimerism Research around the world

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

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

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

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

13. The Traditional Formula % Donor Chimerism Inaccurate Insensitive

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

15. Total Cell count in sample: 1*10 4

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. Allelic Imbalance - AIB Unbalanced PCR amplification of participant’s alleles

23. Allelic Imbalance - AIB

24. Percent of accuracy as a function of AIB

25. DNA Concentration

26. Sequencer Sensitivity

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

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

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

30. 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.

31. The End

32. Lecture Outline Background. Rationale for the Research. Project Description. Conclusions and Recommendations.

33. CLASSIC CHIMERAS MINOTAURSPHINX Composite of one or more animals

34. 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 the percent of mixed chimerism.

35. 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.

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

37. ChimerTrack

38. (D 1 +D 2 )/(D 1 +D 2 )+(R 1 +R 2 )

39. ChimerTrack REPORT PAGE

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

41. Informative Loci Which STR locus can be used?

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

43. Informative Loci Allele D1Allele R1Allele D2 Allele R2 US E 1 Separate Yes 2 Separate Shared Yes 3 SeparateShared No 4 SharedSeparateShared No 5 Homozygous- separateSeparate0 Yes 6 SeparateHomozygous- Separate Separate0Yes 7 Homozygous- Separate 00Yes

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

45. Allelic Constellation