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.

Bone Marrow Transplantation Cancer of blood cells Genetic metabolic diseases –Anemias –Immune deficiency Bone Marrow

Chimerism Cells from two different individuals in the same body

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

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

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

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.

Chimerism Research around the world

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

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

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

Formula for D:R The traditional formula:.

The Traditional Formula % Donor Chimerism Inaccurate Insensitive

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

Total Cell count in sample: 1*10 4

Are all markers equal in terms of accuracy and dependability?

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

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

Marker Performance Simulated Chimerism

Approach 2 – Longitudinal Marker Performance

Allelic Size Differences

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

Allelic Imbalance - AIB

Percent of accuracy as a function of AIB

DNA Concentration

Sequencer Sensitivity

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

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

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.

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.

The End

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

CLASSIC CHIMERAS MINOTAURSPHINX Composite of one or more animals

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.

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.

Recipient Before Transplant Donor Day one One month Three months

ChimerTrack

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

ChimerTrack REPORT PAGE

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.

Informative Loci Which STR locus can be used?

Informative Loci DONOR POST PRE DONOR POST PRE-TX Recipient

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

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

Allelic Constellation