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

ChimerismChimerism

Cells from two different

individuals in the same body

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

% %CHIMERISM = D:RCHIMERISM = D:R

Recipient pre-Tx

Recipient post-Tx

Donor pre-Tx

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.

Chimerism Research around the worldChimerism Research around 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

What is the correct mathematical formula for Chimerism calculation?

The FormulaThe Formula

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.

The End

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.

Recipient Before Transplant

Donor

Day one

One month

Three months

ChimerTrackChimerTrack

(D(D11+D+D22)/(D)/(D11+D+D22)+(R)+(R11+R+R22))

ChimerTrackChimerTrack REPORT PAGE REPORT PAGE

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 LociWhich STR locus can be used?

Informative LociInformative Loci

DONOR

POST

PRE

DONOR

POST

PRE-TX Recipient

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

Allelic ConstellationAllelic Constellation

Who’s peak is first – donor or recipient?

Allelic ConstellationAllelic Constellation

One Person "Chimerism simulation"

0102030405060708090100

05101520

Sample Number

% Chimerism - "Two

Components"