Module n° : 608
Title : Chimerism and DNA fingerprinting
Lecturer : Friedel Nollet
Molecular Biology and Cytometry Course, May 5-6 2011, Mol
Hematopoïetische stamceltransplantatie
myeloablative SCT
graft falen/rejectie
conditionering
& stamcel
engraftment
herval
donor
stamcellen
(graft)
patiënt
graft versus stroma
rejectie
patiënt na SCT
prognostic significance of chimerism testing
Koldehoff et al., Am J. Hemato (2006) 81:735-746
Microsatelliet
Types of STR Repeat Units
• Dinucleotide
(CA)(CA)(CA)(CA)
• Trinucleotide
(GCC)(GCC)(GCC)
• Tetranucleotide
(AATG)(AATG)(AATG)
• Pentanucleotide
(AGAAA)(AGAAA)
• Hexanucleotide
(AGTACA)(AGTACA)
DNA fingerprinting
persoon X
persoon Y
Chimerisme test
patient
donor
sample (29.1% donor)
why chimerisme testing?
•  to follow engraftment : aplasia after transplantation
can be caused by
•  graft failure (<5% donor)
•  versus graft-versus-stroma effect (~100% donor chimerism)
•  conditioning for second transplant
•  to monitor immunotherapy (donor lymphocyte
infusions) for the treatment of relapse
•  detection of relapse (!not a sensitive technique)
•  confirm monozygosity of twins (syngeneic transplant)
Non-myeloablative SCT
Non-myeloablative transplant protocol
Childs et al. 1999, Blood 94 (9), 3234-3241
Methods of chimerism testing
•  FISH for sex-mismatched transplantations
•  RBC antigens
•  Enzyme polymorphisms
•  DNA polymorphic markers
-  HLA antigens
-  RFLP (Restriction Fragment Length Polymorphism)
-  VNTR (Variable Number of Tandem Repeats)
-  Minisatellites (> 6 nt)
-  Microsatellites (STR, short tandem repeats, 2-5 nt)
nucleus
aatttttgta
tcaccatgtt
tattttaagg
gatagaacac
ctaacgATAG
ATAGATAGAT
TGATAGtttt
ctatagtaaa
gtgcaattct
aatcgttata
ttttttttag
ggtcaggctg
ttaatatata
ttgtcatagt
ATAGATAGAT
AGATAGATAG
tttttatctc
catttaatta
gtcaatgagg
attcttaaga
agacggggtt
actatggagt
taaagggtat
ttagaacgaa
AGATAGATAG
ATAGACAGAT
actaaatagt
ccaatatttg
ataaatgtgg
atatatattc
CEL
NNNNNNN-(ATAG)n-NNNNNN
HISTONEN
DNA
chromosoom
donor
patiënt
donor
patiënt
Procedure
PB or BM
(1) if necessary, T-cell isolation
(2) DNA-extraction from PB / BM (T-cells)
(3) PCR-amplification of markers (AmpFlSTR or Powerplex16)
(4) Electrophoresis and detection (ABI PRISM 3130)
(5) Interpretation
Changes made to the protocol
1. Amount of DNA template was increased :
from 1-2.5 ng DNA to
25 ng DNA (corresponds to ~3000 cells)
2. Number of PCR cycles was decreased :
from 10+22 to 10+18
10+22 cycles
10+20 cycles
10+18 cycles
10+16 cycles
Interpretation of results
•  analysis of donor and recipient before
transplantation to determine donor and recipient
alleles
• 
donor
donor
patiënt patiënt
A1 A2
% donor =
A3
A4 (oppervlaktes onder piek)
A2+A4
A1+A2+A3+A4
From : Nollet F, Billiet J, Selleslag D, Criel A.
Bone Marrow Transplant. 2001 Sep;28(5):511-8.
Standardisation of multiplex fluorescent short tandem
repeat analysis for chimerism testing.
Stutter peaks
D
slippage of TAQ
polymerase
=> minor product peak at n-4
R
(for tetranucleotide repeats)
SAMPLE
exclusion for stutter interference
D
Peak Area
R < D : exclude
R > D : include
R
stutter is always < 13%
mean ~7%
SAMPLE
RD
name patient
precision and accuracy
intralaboratory
TYPE-I
TYPE-II
precision and accuracy
intralaboratory (2)
Accuracy
observed percent chimerism for each mock sample differs
< 1,7% (TYPE-I)
< 2,7% (TYPE-II)
from the prepared mixing ratio
Precision
standard deviation for marker used to determine percent
chimerism
< 2,1% (TYPE-I)
< 4,2% (TYPE-II)
precision and accuracy
interlaboratory (2)
Average
absolute deviation:
Range:
0.60%
0.86%
49.1-50.2% 38.8-40.2%
0.72%
8.6-11.4%
Other Human Identification systems
TYPE-I markers
EQC chimerism testing
Centers for Molecular Diagnostics, Belgium
Detection limit of chimerism analysis
Detection limit
Imprecision increases at low/high chimerism values.
5% < TYPE-I markers < 95%
Definitions:
Full Chimerism (FC) : >95% donor cells
Mixed Chimerism (MC) : 5% <-> 95% donor cells
We were able to detect even <1% populations
(dependent upon marker).
However, there is a persistent recipient T-cell compartment after
transplantation (especially of graft was T-cell depleted).
Advantages for STR Markers
•  Small product sizes are generally compatible with
degraded DNA and PCR enables recovery of
information from small amounts of material
•  Multiplex amplification with fluorescence detection
enables high power of discrimination in a single test
•  Commercially available in an easy to use kit format
Real-Time PCR Chimerism
Performance Targets
•  Sensitivity
–  Detection of 0.05% of minor component in a
mixed DNA sample when starting with 250 ng of
DNA
–  Sensitivity is limited only by the amount of input
DNA in the reaction
•  Need enough copies of the minor component to avoid
stochastic sampling error
•  Ability to detect a 2-fold change
real time PCR primers and probes to detect indels
Koldehoff et al., Am J. Hemato (2006) 81:735-746
Real-time PCR vs STR PCR
Alizadeh Blood Blood. 2002 Jun 15;99(12):4618-25.
Article 33bis
EQC UKNEQAS
•  nov 2011 :
68 participants
number of markers used for calculation