Digital PCR (dPCR) and quantitative PCR (qPCR) methods were compared for their ability to detect mixed chimerism and predict relapse in leukemia patients after stem cell transplantation. Research results showed that dPCR using the QuantStudioTM 3D Digital PCR System produced similar results to qPCR when recipient DNA levels were above 1%, but was more sensitive for detecting lower levels of 0.2% or less. dPCR was able to detect increasing recipient chimerism earlier than qPCR in some samples, potentially allowing for earlier prediction of relapse. The false positive rate for dPCR was close to the 0.01% level considered full donor chimerism.

1. Figure 1. QuantStudio™ Digital PCR System
A) B)
C)
During leukemia treatment mixed chimerism occurs in
which both recipient and donor cells are present in the
bone marrow or peripheral blood after transplantation.
Chimerism analysis is performed to monitor peripheral
blood or bone marrow in the recipient after allogenic
stem cell transplantation to monitor for leukemic relapse.
Observation of increasing mixed chimerism after
transplantation is associated with a higher risk of relapse
in acute leukemia. Previously, a quantitative PCR (qPCR)
technique, using INDEL polymorphisms, was found to
predict relapse in 88.2% vs. 44.4% of individuals analyzed
by VNTR markers with a median anticipation period of 58
days and a sensitivity of 0.01% vs. 3%. Here we present
results from research experiments performed to
determine if a digital PCR (dPCR) method is able to
predict relapse earlier and with greater accuracy than the
qPCR method using retrospective leukemia samples.
Research results showed that dPCR using data generated
by the QuantStudio™ 3D Digital PCR System and the qPCR
method yielded similar percent recipient chimerism
values when recipient DNA was present above the 1%
level. Furthermore, dPCR using the system was found to
be more sensitive than the qPCR method based on the
ability to detect the recipient DNA in a relapsed individual
about 2 months earlier where the percent recipient
chimerism was 0.2% or less. The false positive rate was
close to the complete chimerism value of 0.01% for
peripheral blood samples.
Quantification of donor/recipient chimerism in
leukemia samples by digital PCR
Kathleen C. Hayashibara1, Antonio Jiménez-Velasco2
1Life Technologies, 180 Oyster Point Boulevard, South San Francisco, CA, 94080, USA. 2Hematology Department, Carlos Haya Hospital, Málaga, SPAIN.
ABSTRACT
INTRODUCTION
RESULTS
CONCLUSIONS
Digital PCR is an analytical technique for quantification of
nucleic acid samples based on PCR amplification of single
template molecules, without a standard curve.
In these experiments the number of negative PCR
reactions is used to estimate the number of target
molecules based on Poisson distribution statistics.
Mixed chimerism is a state in which both recipient and
donor cells are present in bone marrow or peripheral
blood after transplantation.
Chimerism analysis is performed to monitor peripheral
blood or bone marrow in the recipient after allogenic
stem cell transplantation to avoid leukemic relapse.
Increasing mixed chimerism after transplantation is
associated with a high risk of relapse in acute leukemia.
A qPCR technique was found to predict relapse in 88.2%
vs. 44.4% of individuals analyzed by VNTR markers with a
median anticipation period of 58 days and a sensitivity of
0.01% vs. 3%.
The goal of this project is to compare results from the
QuantStudio™ 3D Digital PCR System (Figure 1) to results
from qPCR to determine if relapse can be predicted
earlier and with greater accuracy by digital PCR.
Pre-
Transplantation
Chemotherapy or
Radiation Therapy
Stem Cells
Post-
Transplantation
Mixed Chimera
No Relapse
Potential
Relapse
Example
1
Example
2
Complete Chimera
Sealed System
AmplifyLoad ReadMix
We thank Patricia Hegerich, Paco Cifuentes, Maria Jesus Garcia-Ortiz and
Juan-Antonio Barba for project coordination and helpful discussions.
REFERENCES
ACKNOWLEDGEMENTS
TRADEMARKS/LICENSING
The QuantStudio™3D Digital PCR Systemis For Research Use Only. Not for
use in diagnostic procedures.
1 mm
DNA Samples
Genomic DNA was purified from fresh whole peripheral
blood (PB), bone marrow (BM), or umbilical cord blood using
standard procedures from 48 DNA samples isolated from 7
donor/recipient pairs. Recipient DNA was analyzed before
and after stem cell transplantaion (SCT) at various intervals.
Some were knownto have relapsed while others had not.
Assays
Identical nucleotide sequences for were used for both qPCR
(5′ Exonuclease-Based Real-Time PCR Assay) and dPCR
experiments using Custom TaqMan®Assays. Polymorphisms
were selected where donors possessed the deletion (null
allele) and recipients possessed the insertion.
qPCR
For all runs 100 ng of gDNA was used in 20 ml reactions using
standard procedures. The b-Globin reference was analyzed
in the same run in a separate reaction. The normalized ratio
of target vs. reference was used to calculate the fraction of
DNA from the recipient(1).
dPCR
The concentrationof purified DNA was sufficiently low
(<20ng/ml) so that dilution was not necessary. Each chip was
loaded and run on a GeneAmp® 9700 PCR System for 40
cycles using standard procedures. Target assays were labeled
with FAM as indicatedin blue (above) and the b-Globin
reference assay was labeled with VIC and the target and
reference assays were run in duplex on each chip. The b-
Globin gene is not located on the same chromosome as the
target loci. Some reactions therefore contained target only
(detected by FAM), some contained the b-Globin gene only
(detected by VIC), and some contained both (FAM + VIC)
producing 3 clusters of data points.
The number of reactions containing each target was
calculated in external software using Poisson distribution
estimates using the number of non-target containing
reactions relative to the total number of reactions. The
recipient chimerism was calculatedas shown below using the
copies/ml calculated by the QuantStudio™ 3D Digital PCR
System software for the target and reference.
Recipient Chimerism = (Target/Reference)Sample /(Target/Reference)Pre-SCT
(If the recipient was heterozygous for the insertion then the
target/reference ratio was normalized by multiplying by 2.)
The recipient chimerism values were compared with those
determined by qPCR. The software was also used to
calculate 95% confidence intervals of the copies/ml of target
and reference measurements on each chip.
1. Jiménez-Velasco et al., (Leukemia (2005) 19, 336–343).
• The QuantStudio™ 3D Digital PCR System produced
similar percent recipient chimerism values to qPCR
when recipient DNA was present above the 1% level.
• The QuantStudio™ 3D Digital PCR System is more
sensitive than qPCR because it is able to detect the
presence of recipient DNA earlier than qPCR in samples
where percent recipient chimerism is 0.2% or less.
• The false positive rate based on analysis of donor
samples was close to the complete chimerism value of
0.01% for peripheral blood samples.
• Increasing mixed chimerism was detected in recipients
1-3 and 5-7 and not in recipient 4 consistent with
known outcomes.
A) The QuantStudio™ 3D dPCR Chip. B) Magnified chip surface. C) Workflow.
MATERIALS AND METHODS
Target Chromosome
MID-1039 5
MID-2113 10
MID-R271 22
GSTT1 22
MID-2113 10
SRY Y
GSTM1 1
MID-1732 10
b-Globin Reference 11
Insertion target only
(from recipient)
Both insertion (from recipient)
and b-Globin(from donor or recipient)
in one reaction
Neither target present b-Globintarget only
(from donor or recipient)
Digital PCR was able to detect increasing amounts of
recipient chimerism in all cases where increasing amounts
were also detected by qPCR. However, dPCR was also able to
detect increasing recipient chimerism in samples that failed
to yield signal in qPCR experiments.
Recipient Pre-SCT Donor +62 Days
+147 Days +174 Days +192 Days
Reactions containing recipient DNA appear in blue (reactions with target
alone) or green (reactions with both target and reference DNA molecules).
At day 192 the presence of recipient DNA (blue and green cluster) is readily
apparent. Recipient DNA was detected as early as day 62 post-SCT.
dPCR%RecipientChimerism
0.0%
0.1%
1.0%
10.0%
100.0%
Pre-SCT Donor 62 147 174 192
Samples are from a recipient known to have undergone relapse. Digital PCR
detected recipient DNA in all post-SCT samples consistentwith relapse.
Relapse was also confirmed by qPCR. However, no recipient DNA was
detected until day 192 and an additional sample beyond 192 days was
required for confirmation by qPCR.
Recipient/
Donor Pair
(Target) Description
% Target/
Reference+
dPCR
% Recipient
Chimerism+
dPCR
95% Confidence
Interval
Upper
dPCR
95%
Confidence
Interval
Lower
qPCR
% Recipient
Chimerism
1*
(MID-1039)
Pre-SCT 55.34 100.00 100.07 99.87
Donor 0.01 0.01 0.07 0.00
60 0.48 0.87 1.06 0.72 0.59
83 2.97 5.37 5.71 5.05 4.51
94 5.19 9.38 9.83 8.95 8.21
125 5.64 10.19 11.07 9.39 9.28
151 0.07 0.13 0.49 0.03 0.07
2
(MID-2113)
Pre-SCT 48.57 100.00 101.98 98.07
Donor 0.00 0.00 0.00 0.00
55 0.02 0.05 0.10 0.02 0.10
61 0.05 0.10 0.16 0.06 0.08
91 1.98 4.08 8.66 1.92 0.76
127 43.71 90.00 107.50 75.41 72.94
3 **
(R271)
Pre-SCT 55.33 100.00 100.00 100.00
Donor 0.02 0.03 0.06 0.01
62 0.11 0.20 0.37 0.10 0.00
147 0.04 0.08 0.17 0.04 0.00
174 0.09 0.16 0.21 0.12 0.00
192 2.80 5.06 5.32 4.82 11.45
4 ***
(GSTT1)
Pre-SCT 53.73 100.00 100.54 99.51
Donor 0.01 0.01 0.03 0.00
14 0.10 0.18 0.28 0.12 0.10
21 0.03 0.05 0.09 0.03 0.02
34 0.04 0.07 0.12 0.05 0.04
69 0.01 0.02 0.06 0.01 0.02
5
(SRY)
Pre-SCT 51.46 100.00 100.24 99.77
Donor 0.00 0.00 0.00 0.00
317 0.04 0.07 0.10 0.05 0.05
331 0.01 0.03 0.05 0.02 0.03
376 0.02 0.04 0.07 0.03 0.02
417 0.06 0.11 0.15 0.09 0.06
477 2.14 4.17 4.31 4.02 4.09
6
(GSTM1)
Pre-SCT 51.65 100.00 100.38 99.54
Donor 0.01 0.03 0.18 0.00
26 0.01 0.02 0.04 0.01 0.01
40 0.01 0.02 0.05 0.01 0.04
59 0.14 0.27 0.43 0.17 0.13
61 0.12 0.23 0.37 0.14 0.29
73 1.85 3.58 3.78 3.38
7
(MID-1732)
Pre-SCT 97.79 100.00 100.01 99.99
Donor 0.00 0.00 0.01 0.00
23 6.72 6.87 7.27 6.50 6.79
48 1.40 1.43 1.49 1.38 1.88
108 0.83 0.85 0.90 0.79 0.95
140 1.29 1.32 1.39 1.25 1.14
154 1.74 1.78 1.90 1.67 1.12
184 1.22 1.25 1.44 1.08 1.81
+ These percentages were calculated in external software.
*This recipient was treated with inmunosupprants to avoid Graft-Versus-
Host-Disease (GVHD) complications. This therapy decreases the Graft-
Versus-Leukemia (GVL) effect. The immunosupression therapy continued
through day 125.
**dPCR was able to detect recipient DNA earlier than qPCR.
***This receipient did not relapse. Both dPCR and qPCR show lack of
significant increase in recipient chimerism.
Comparisonof % Recipient Chimerism from dPCR and qPCR
Leukemia cells in red, non-leukemia in blue