quantification of post- transplantation chimerism: algorithm for correcting systematic errors moshe...
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Quantification of Post-Quantification of Post-transplantation Chimerism: transplantation Chimerism:
Algorithm for Correcting Algorithm for Correcting Systematic ErrorsSystematic Errors
Moshe IsraeliComputational 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 TransplantationBone Marrow Transplantation
• Cancer of blood cells
• Genetic metabolic diseases– Anemias– Immune deficiency
Bone MarrowBone Marrow
STR MarkersSTR Markers
STRSTR
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
Short Tandem RepeatsShort Tandem Repeats ((STRsSTRs))
AATGREPEAT
7 repeats
Polymorphic DNA loci that contain a shortshort repeated nucleotide sequence
Locus
probability of a random match: ~1 in 3 trillion
Rationale for the ResearchRationale 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.
Percent Percent ChimerismChimerism
Marker performance
DNA concentration
Equipment sensitivity
Size difference between
donor and recipient’s
allelesDonor 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 GoalsProject Goals
Formula for D:RFormula for D:R
• The traditional formula:.
n
RRDDDD
Chimerism
n
1 2121
21 )(
%
%502
1
)11()11(
11
2121
21
RRDD
DD
The Traditional FormulaThe Traditional Formula
% Chimerism as a function of Donor Cells Count
0102030405060708090
100
0 2000 4000 6000 8000 10000 12000Amount of Donor Cells
% D
onor
Chi
mer
ism
Inaccurate
Insensitive
• Chimerism ratio varies among samples, but the constant Total of DNA enables reliable comparison between them.
• The new formula is:
Total
DD
DDTotalDD
DD
Chimerism
21
2121
21
)]([)(
%
ConstantConstant
The New FormulaThe New Formula
The New FormulaThe New Formula
Total Cell count in sample: 1*104
% chimerism as a function of Donor cell count
0
10
20
30
40
50
60
70
80
90
100
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
Amount of donor cells
% chimerism
nTotalDD
Chimerism
n
1
21 )(%
Are all markers equal in terms Are all markers equal in terms of accuracy and dependability?of accuracy and dependability?
ApproachesApproaches
• Chimerism Simulation – treating heterozygosity as a 50% Chimerism
• 5 yr Longitudinal Followup
Chimerism Simulation – treating Chimerism Simulation – treating heterozygosity as a 50% Chimerismheterozygosity as a 50% Chimerism
Homozygous Locus
Heterozygous Locus
Marker Performance Marker Performance Simulated ChimerismSimulated Chimerism
51.5 49.8 52.550.0
50.750.9
51.250.1
50.1 52.3
2030405060708090
100
% Chimerism
STR Marker
Marker Result in One Person's Sample
Approach 2Approach 2 – Longitudinal Marker – Longitudinal Marker PerformancePerformance
Standard Deviation of Marker Outcome
0
1
2
3
4
5
6
7
8
9
10
Sample Date
STDEV
Allelic Size DifferencesAllelic Size Differences
Effect of Allelic Size Differences on % Chimerism
0%
10%
20%
30%
40%
50%
26754Size Difference
Pe
rce
nt
Do
no
r C
him
eri
sm
Percent Percent ChimerismChimerism
Marker performance
DNA concentration
Equipment sensitivity
Size difference between
donor and recipient’s
allelesDonor and recipient’s
allele constellation
Unbalanced PCR
amplification
Calculation formula
Anecdotal Sources of Variability in Chimerism Testing
Conclusions and Conclusions and RecommendationsRecommendations
• Constant Total amount of DNA.• Marker quality - reliability constant, α.• Size Difference – • f(AiB)
)( SizeSize RDabs
gC
Identify informative loci
Formula for Quantitative Formula for Quantitative Mixed Chimerism Calculation:Mixed Chimerism Calculation:
n
TotalDD
RDabsgC
AiBf
Chimerism
n
i SizeSizeii
1
21 )]())(
()([
%
•Improve post-tx patient monitoring
•Increase laboratory reliability.
•Provide clinicians with accurate data.
•EFI, Israeli Transplant Society, ISHI.
•Future research:
•Blood.
•ADMO, ASHI.
AcknowledgmentsAcknowledgments• 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.
Chimerism MonitoringChimerism 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 & MethodsMaterials & 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.
Four Methods of AnalysisFour 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 LociInformative Loci Allele D1 Allele R1 Allele
D2Allele 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
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