Quantification of Posttransplantation 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
Bone Marrow
• Cancer of blood cells
• Genetic metabolic diseases
– Anemias
– Immune deficiency
Chimerism
Cells from two different
individuals in the same body
STR Markers
chromosome
cell nucleus
Double stranded
DNA molecule
STR
Individual
nucleotides
Short Tandem Repeats
(STRs)
REPEAT
AATG
7
repeats
Locus
Polymorphic DNA loci that contain a short
repeated nucleotide sequence
probability of a random match: ~1 in 3 trillion
% CHIMERISM = D:R
Donor pre-Tx
Recipient postTx
Recipient preTx
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
Anecdotal Sources of Variability in
Chimerism Testing
Marker
performance
DNA
concentration
Unbalanced
PCR
amplification
Equipment
sensitivity
Calculation
formula
Percent
Chimerism
Donor and
recipient’s
allele
constellation
Size
difference
between
donor and
recipient’s
alleles
Project Goals
•Quantification of sources of
variability for % Chimerism
•Devising corrective algorithm
The Formula
What is the correct mathematical formula
for Chimerism calculation?
Formula for D:R
• The traditional formula:.
D1  D2
1 ( D  D  R  R )
1
2
1
2
%Chimerism 
n
n
D1  D2
11
1

  50%
D1  D2  R1  R2 (1  1)  (1  1) 2
The Traditional Formula
% Chimerism as a function of Donor
Cells Count
100
90
80
70
60
50
40
30
20
10
0
Insensitive
Inaccurate
0
2000
4000
6000
8000
Amount of Donor Cells
10000
12000
The New Formula
• Chimerism ratio varies among samples, but the
constant Total of DNA enables reliable
comparison between them.
• The new formula is:
%Chimerism 
D1  D2
D1  D2

( D1  D2 )  [Total  ( D1  D2 )] Total
Constant
The New Formula
D1  D2
1 ( Total )
%Chimerism 
n
% chimerism as a function of Donor cell count
n
100
90
80
70
60
% chimerism 50
40
30
20
10
0
0
1000
2000
3000
4000
5000
6000
Amount of donor cells
Total Cell count in sample: 1*104
7000
8000
9000
10000
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
re
en
26
G
4
re
en - 34
5
G
D
re 187
en
- 2 18 S
12 43
51
D
8Bl
17 21
ue
28 2 D S11
9
Bl
8
u e - 34 S1
23 1 D 17
42S 9
2
13
74
Bl
u
38
D
Bl
e
1
ue
15 6 S
11 7-2 539
409
1
Ye
V
4
l lo 2 D WA
3
w
Ye
21 S1
Ye l low 5- 3 358
5
l lo
w 165 4 F
G
10
6- 205 A
14
TH
0
0
D
19 2
S4
33
G
Marker Performance
Simulated Chimerism
Marker Result in One Person's Sample
100
90
80
70
% Chimerism 60
50
40
30
20
51.2
50.7
51.5 49.8 52.5
50.9
50.0
50.1
50.1 52.3
STR Marker
Approach 2 – Longitudinal Marker
Performance
Standard Deviation of Marker Outcome
10
9
8
7
6
4
3
2
1
0
99
19
8/
/0
00
01
20
1/
/0
02
11
20
5/
/0
02
30
20
7/
/0
02
11
20
8/
/0
02
21
20
0/
/1
02
13
20
0/
/1
03
31
20
4/
/0
03
06
20
7/
/0
03
21
20
9/
/0
03
12
20
1/
/1
03
24
20
2/
/1
03
02
20
2/
/1
03
09
20
2/
/1
04
29
20
1/
/0
04
11
20
1/
/0
04
25
20
2/
/0
04
15
20
3/
/0
04
08
20
4/
/0
15
Sample Date
STDEV
5
Allelic Size Differences
Percent
Donor Chimerism
Effect of Allelic Size Differences on
% Chimerism
50%
40%
30%
20%
10%
0%
4
5
7
Size Difference
26
Anecdotal Sources of Variability in
Chimerism Testing
Marker
performance
DNA
concentration
Unbalanced
PCR
amplification
Equipment
sensitivity
Calculation
formula
Percent
Chimerism
Donor and
recipient’s
allele
constellation
Size
difference
between
donor and
recipient’s
alleles
Conclusions and
Recommendations
Identify informative loci
•
•
•
•
Constant Total amount of DNA.
Marker quality - reliability constant, α.
gC
Size Difference –
abs ( D  R )
f(AiB)
Size
Size
Formula for Quantitative
Mixed Chimerism Calculation:
gC
D1  D2
[ i  f ( AiB )i  (
)(
)]

abs ( DSize  RSize )
Total
i 1
%Chimerism 
n
n
•Improve post-tx patient
monitoring
•EFI, Israeli Transplant
Society, ISHI.
•Increase laboratory reliability.
•Future research:
•Provide clinicians with
accurate data.
•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
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
(D1+D2)/(D1+D2)+(R1+R2)
ChimerTrack REPORT PAGE
Four Methods of Analysis
•
•
•
•
One person’s DNA.
One patient’s Chimeric DNA.
Population of patients.
Artificial Chimeras.
Informative Loci
Which STR locus can be used?
Informative Loci
DONOR
POST
DONOR
POST
PRE
PRE-TX Recipient
Informative Loci
Allele D1
Allele R1
Allele
D2
Allele
R2
US
E
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
HomozygousSeparate
Separate
0
Yes
7
Homozygous- Separate
HomozygousSeparate
0
0
Yes
Allelic Constellation
Who’s peak is first – donor or recipient?
Allelic Constellation
One Person "Chimerism simulation"
100
90
80
70
60 % Chimerism 50
"Two
40 Components"
30
20
10
0
20
15
10
Sample Number
5
0