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CHIP-BASED DIGITAL PCR: AN ACCURATE AND SENSITIVE METHOD FOR ROUTINE
CHIMERISM MONITORING AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION
Elise Gourri1, Urs Schanz2, Beat M. Frey1, Christoph Gassner1
1 Blood
Donor
1500 –
850 –
400 –
FYB
FYA
DOB
DOA
JKB
JKA
107T
107G
After hematopoietic stem cell transplantation
(HSCT) recipient and donor populations of
leukocytes cohabit in the peripheral blood of the
patient, resulting in “chimerism”. Monitoring
chimerism is crucial to follow the outcome of the
disease and to make informed clinical decision
concerning further therapeutic interventions.1
Current chimerism monitoring is performed
using Short-Tandem-Repeats analysis (STR)
and quantitative real-time PCR (RT-PCR).
However, both methods suffer from limited
sensitivity and reproducibility. Chip-based digital
PCR (dPCR) represents a new alternative for
accurate
and
reproducible
chimerism
monitoring.
Recipient
Background
1490A
1490G
Transfusion Service Zurich, SRC, Department of Molecular Diagnostics and Research & Development (MOC), ZurichSchlieren, Switzerland, 2 University Hospital Zurich, Department of Hematology, Zurich, Switzerland.
 Control
200 –
100 –
50 –
1500 –
850 –
400 –
 Control
200 –
100 –
50 –
Figure 2: PCR-SSP Instand external proficiency testing. Pre-HSCT recipient and donor
DNAs genotyping using PCR-SSP. Adequate SNPs for chimerism assessment for which
donor and recipient are oppositely homozygous were chosen (SNPs shown: 1490G/A =
rs1490413, 107G/T = rs10776839, JK*A/B, DO*A/B, FY*A/B; selected SNPs circled in blue).
70%
60%
After DNA extraction from peripheral blood,
original samples were analyzed for specific SNP
genotypes by PCR using Sequence Specific
Priming (PCR-SSP).
Chip-based Quantstudio 3D chip-based dPCR
(Applied Biosystems, Thermo Fisher Scientific,
Life Science Group, Zug, Switzerland) allows for
SNP detection in approximately 20’000 separate
RT-PCRs per sample, with statistical presence
of only 0, 1, or 2 DNA molecules per each 755
pL reaction well (Figure 1). Fluorescence of
each well is separately measured and delivers
absolute counts of the two different alleles.
% of recipient DNA
Methods
50%
40%
Expected Value
dPCR Value
30%
20%
10%
0%
Post 1
Post 2
Post 3
Post 4
Post 5
Figure 3: Instand external proficiency testing. Chimerism assessment of 5 post-HSCT
probes. The dPCR results strongly match with the expected values. Error bars: 95 %
confidence intervall.
Figure 1: Chip for digital PCR. Each chip contains
20’000 reactions wells. After thermal cycling, every
single reaction tube of the chip is read with a
fluorescence reader. © Applied Biosystems
% of recipient DNA
7%
6%
5%
4%
STR analysis
dPCR value
3%
2%
1%
0%
F34
Results
Using artificial DNA mixes, we estimated our
limit of detection to range at approximately 0.5%
for the minor DNA. Typing samples of Instand’s
external proficiency testing (EPT) for post-HSCT
chimerism, was performed on 1 pre-HSCT
patient and donor sample each and on 5 postHSCT samples, using only 2 diffe-rent SNP
assays. Instand recognized the quality of our
result with 20/20 points (Figure 2).
F49
F40
F52
F55
F58
F22
Figure 4: Post-HSCT samples chimerism status according to STR analysis and dPCR.
Seven out of a total of 15 investigated peripheral blood derived DNA samples presented a
chimerism status below 5% DNA of the recipient. In 4 cases (circled in blue), results of STR
analysis and dPCR differed by more than the 95% confidence intervalls obtained by dPCR
(error bars in red) .
Using 15 DNA sample triplets, each one consisting of one pre-HSCT, one donor
and one post-HSCT specimen each, dPCR results were consistent with previous
STR results in 11 out of 15 triplets. Discrepancies were only observed for 4 triplets,
where chimerism was estimated to range below 5% by STR analysis (Figure 4), a
method with known limited sensitivity2.
Conclusion
According to the high performance of our method in the Instand EPT and based on the results of 15 patient/donor/post-HSCT sample
triplets, chip-based dPCR appears as an accurate and routinely applicable technique for exact chimerism determination with excellent
sensitivity.
Acknowledgments We thank Sophie Leroy and Dominique Dewolf (Applied Biosystems, Thermo Fisher Scientific, Life Science Group, Zug, Switzerland) for their support and sponsoring. References 1 Khan et al., Bone Marrow Transpl 2004;34:1-12. 2 Stahl et al., Exp Hematol 2015;43:462-468.
49. Jahrestagung der Deutschen Gesellschaft für Transfusionmedizin und Immunhämatologie, 7. – 10. September 2016, Nürnberg, Deutschland