EDTA-MEDIATED INHIBITION OF NUCLEASE PROTECTS CELL-FREE DNA FROM EX-VIVO
DEGRADATION IN BLOOD SAMPLES
J. A. Vasques1, T. H. Santa Rita2, C. F. Chianca2, L.F. R. Velasco1, L. F. Abdlla1, S. S. S. Costa1, G. B. Barra1.
1- Sabin Laboratory, Brasília, Brazil, 2- University of Brasília, Brasília, Brazil.
INTRODUCTION
Abstract:
Background:
The cell-free DNA (cfDNA) in bloodstream is becoming an important analyte. Several studies have shown that its
measurement is useful for monitoring patients with cancer and other health conditions. Moreover, the fetal cfDNA in
the maternal circulation is currently the method of choice for non-invasive determination of fetal genetic traits.
However, cfDNA is unprotected and it may be susceptible to known blood high nuclease activity. In this study, we use
Y chromosome-specific sequence (DYS-14) levels in maternal serum and EDTA-plasma to investigate the ex-vivo
impact of this nuclease activity on cfDNA quantity.
The analysis of cell-free DNA in blood circulation has become an important analyte, for example, this year's AACC selected three studies that approach this subject. Several studies connected the
alteration in its quantity to diseases like cancer and other health conditions. Besides that, nowadays, the analysis of fetal cell-free DNA in the mother's blood circulation is the method of choice for a noninvasive investigation of the fetus genetic traits. Some examples of non-invasive analysis of the fetus are: sex-determination, RH fator status, aneuploidy detection, twin zygosity, and also the fetus
genome sequencing. However, because it doesn't have any cell protection, the fetal cell-free DNA is unprotected and it can be susceptible to the well known DNAse activity present in the blood. This way,
Methods:
This study used banked (-20°C) EDTA-plasma and serum paired samples (n=34) from women with a male fetus
(gestational age, 12 ± 4 weeks). Informed consent was obtained from all participants and institutional review board
approved the study. Experiments were done with sets of 9-10 paired samples, except the -20°C/4°C/24°C assay (EDTA
plasma and serum sample were not paired). Nucleic acid extraction was performed by using Nuclisens easyMAG
(bioMerieux). DYS-14 assays were performed on a StepOne Real-time PCR Systems (Applied Biosystems) by using of
hydrolysis probe chemistry and absolute quantification. The results were shown as median (Genomic
Equivalents/mL). Statistical analyses were Wilcoxon's test or Friedman's test. For exogenous nuclease assay samples
were treated or not with 25U of DNase I (Fermentas) for 1 hour at 37°C before DYS-14 assay. For endogenous
nuclease assay a hydrolysis probe and a passive reference (ROX) were added to the crude serum or EDTA-plasma,
fluorescence increase were measured for 10 hours at 37°C in the Real-time PCR System. For serum nuclease
inhibition assay a serial dilution of EDTA from 0.000005 to 50 mM was used.
it's important to guarantee stability to the sample after being taken, considering the means of transportation and storage, so that there's no pre-analytic effects over the analysis. These considerations
motivated us to compare the fetal cell-free DNA stability in serum and EDTA-plasma. So, the main purpose of this study is to assess the impact ex-vivo in the nucleases activities in the blood over fetal
cell-free DNA. Since the fetus is the source of fetal cell-free DNA, specifically the placenta, it's expected that the quantity of this material, collected through venipuncture from the mother's arm, is
constant, despite of the type of tube or anticoagulant used to collect the sample.
METHODS
In order to make these tests, we used 34 samples of EDTA serum and plasma of pregnant women with male fetuses, with gestational age between 8 to 16 weeks. All participants signed a consent form and
Results
DYS-14 quantity was higher in EDTA-plasma than in serum (24.77 versus 18.13 GE/ml, P=0.0137). This difference
increased after specimen exposure to 37°C for 24 hours, 22.22 GE/ml for EDTA-plasma versus 5.18 GE/ml for serum,
P=0.002. Next, the samples were subject to -20°C, 4°C or 24°C for 24 hours and no difference was observed on DYS14 concentration in EDTA-plasma, 36.24, 38.02 and 37.31 GE/ml (p=0.328), respectively. In serum, DYS-14
concentration was reduced at 24°C (11.60 GE/ml) compared to -20°C (18.65 GE/ml) and 4°C (17.90 GE/ml), p =
0.0002. Furthermore, DNAse-I treatment did not alter the DYS-14 amount in EDTA-plasma (untreated 16.07 versus
treated 17.57 GE/ml, p=0.42) but completely eliminates it in serum, (untreated 9.21 versus treated 0.0003 GE/ml,
p=0.0039). Additionally, hydrolysis probe degradation could be detected in serum (12.7-fold) but not in EDTA-plasma
(0.28-fold). Finally, hydrolysis probe and DYS-14 degradation in serum was inhibited from 0.5 mM of EDTA.
the study was approved by the institutional review board. 4 tests were made: In the first test, serum and EDTA-plasma samples were submitted to different temperatures. We proceeded with DNA
extraction and then with absolute quantification of DYS-14 sequences (standard curve parameters – dilution 1:5; slope -3,575; Y-inter 22,273; R2 0,998; efficiency 90%). The results were presented in a
Genomic Equivalents per milliliter box plot graphic. This graphic is an example of the standard curve we used and it shows the parameters related to the efficiency of the quantification reaction by PCR in
real time. In the second test, In order to assess the action of the exogenous nucleases and to compare the degradation of cfDNA in the two types of tubes used, serum and EDTA-plasma samples were
treated with DNAse I before the DNA extraction and DSY-14 quantifications. In the third test, the endogenous DNAse activity was assessed in both materials. For that, a Taqman hidrolysis probe for qPCR
was added in the serum or plasma samples. This probe is a 20pb single-strand DNA sequence that contains one fluorescent molecule (FAM) in one end and one mitigating on the other end (DABCYL).
And when it degrades by the nucleases, it becomes fluorescent which is detected by the qPCR equipment. A passive reference dye (ROX) was also added to the serum and EDTA-plasma samples. Then,
Conclusions
We found that cfDNA is subject to a temperature-triggered degradation in serum but not in EDTA-plasma. Moreover,
exogenous and endogenous nucleases were active only in serum. EDTA probably by chelation of divalent ions inhibits
blood nucleases conferring an ex-vivo protection to cfDNA. Ultimately, we developed a real-time fluorescence
method to detect nuclease activity in clinical samples.
the variation of fluorescence, expressed as delta Rn (as it’s used by the software in the qPCR equipment), was measured for 8 hours at 37°C. Fourth and last test, we assess the dose-response effect of
EDTA over the serum’s endogenous nuclease activity. For that, increasing doses of EDTA were added to the serum and the DNAse activity was measured the same way as the previous experiment. The
statiscal methods used were Wilcoxon’s test and Friedman’s test.
RESULTS
EDTA-Plasma
or serum
qPCR DYS-14
(absolute
quantification)
Results shown
as median
(GE/mL)
EDTA-Plasma
or serum
Treated or
not treated
with DNAse I
(1hr)
Extraction
and DYS-14
quantificatio
n
Results shown
as median
(GE/mL)
60
40
P=0,002
40
30
20
10
20
Plasm
50
B)
50
Serum
A)
40
40
30
20
10
B)
0.3
20
10
0.1
2
4
6
8
0.0
2
I
D
N
A
se
N
A
se
-10
-0.1
-0.1
Serum 1
Time (hr)
Plasma 2
60
40
80
60
P=0,0002
The next graphic is the effect of adding DNAse I in the samples.
Ten paired samples of serum and EDTA-plasma were treated with
exogenous DNAse I and it was observed nothing happened with
EDTA-plasma (A), but complete degradation of cell-free DNA in the
serum (B)
C)
factors
0.4
0.2
We wondered what would be the reason for the tube with EDTA
anticoagulant to be protecting the cell-free DNA. It’s known that
EDTA chelates the divalent ions of Ca+ and takes them out of the
environment, making the coagulation factors to be inactive, and that
nucleases depend on Ca+ to work, and for that reason, they don’t
work in this material too (A). The opposite happens in the serum
tube, where the Ca+ is free so that the coagulation factors can be
active, and for that matter, so does the nucleases (B).
0.2
Serum
C)
0.1
0.8
EC50 = 1x10-4 M
0.0
2
20
4
6
0.0
8
2
Time (hr)
22
°C
4°
C
Plasma 3
24 hours
In the first graphic (A), we used 10 paired samples of serum and
EDTA-plasma. They were not submitted to any temperature variation.
We found a larger basal quantity of cell-free DNA in the EDTA-plasma
than in the serum. Next (B), we exposed the same 10 samples to
temperature of 37°C for 24 hours and, as seen in figure B, the quantity
of cell-free DNA decreased even more in the serum, compared to the
EDTA-plasma. To investigate if the temperature can influence the
quantity of cell-free DNA, we submitted 9 EDTA-plasma samples to 20°C, to 4°C and to 22°C (C). The same was done to the serum (D). It
was observed that in the serum, the quantity of cell-free DNA was
influenced by the increase of temperature and that the same didn’t
happen with plasma.
Serum 3
Probe degradation
FAM
FAM
Nuclease activity
(Serum/Plasma)
Q
E)
6
8
-0.1
Fluorescense
Hidrolyse probe
4
Time (hr)
Plasma 4
Serum 4
Probe degradation
F)
0.4
0.4
0.3
0.3
0.2
0.2
0.6
0.4
0.2
0.0
-10
-8
-6
-4
-2
0
Log[EDTA], M
Delta Rn
-2
0°
C
22
°C
4°
C
Serum 2
0.3
40
0
-2
0°
C
D)
0.6
-0.2
24 hours
8
0.4
20
0
6
Probe degradation
Delta Rn
80
D)
4
+
Plasm 1
Delta Rn
P=0,3285
Coagula on
factors
Nucleases
0.1
Delta Rn
100
Fetal DNA (GE/ml)
Fetal DNA (GE/ml)
C)
No Addi ve
Ca+2
X Coagula on
Nucleases X
0.2
0.0
+
Serum
Serum
EDTA
0.3
Probe degradation
Plasm
Plasm
Serum
B)
Ca+2
0.2
30
-D
I
D
N
A
se
A
N
-D
Se
ru
m
Pl
as
m
Se
ru
m
Pl
as
m
se
-10
A)
0.5
Plasma
0.4
0
0
0
Endogenous Dnase
Assay
EDTA from 0.000005
to 50 mM
Serum
Probe degradation
Time (hr)
0
Fluorescence
(DeltaRn)
EDTA-Plasma and
serum
Probe degradation
Delta Rn
P=0,013
80
A)
Fetal DNA (GE/ml)
B)
Fetal DNA (GE/ml)
100
Fetal DNA (GE/ml)
Fetal DNA (GE/ml)
A)
24 hours at 37°C
50
Addition of
hidrolyse probe
and a reference
dye (ROX)
EDTA dose-response assay
Serum
Plasm
Basal
Endogenous DNAse assay
Delta Rn (Final measure)
Extraction
Nuclisens
easyMAG
(bioMerieux)
Exogenous DNAse I assay
Delta Rn
DYS-14 quantification assay
0.1
0.0
0.0
2
Q
From this result, we develop a test to assess if there was any
difference in the activities of exogenous DNAses between both
materials.
0.1
4
6
8
2
Time (hr)
6
8
Time (hr)
-0.1
-0.1
Plasma 5
4
Serum 5
Blank
It was observed that the nucleases were active in the serum but
not in the EDTA-plasma samples (A-F).
REFERENCES
1. Crowley E, Di Nicolantonio F, Loupakis F, Bardelli A (2013) Liquid biopsy: monitoring cancer-genetics in the blood. Nature reviews Clinical oncology. doi:10.1038/nrclinonc.2013.110
2. Chiu RW, Lo YM (2013) Clinical applications of maternal plasma fetal DNA analysis: translating the fruits of 15 years of research. Clinical chemistry and laboratory medicine : CCLM / FESCC 51 (1):197-204. doi:10.1515/cclm-2012-0601
3. Lo YM, Hjelm NM, Fidler C, Sargent IL, Murphy MF, Chamberlain PF, Poon PM, Redman CW, Wainscoat JS (1998) Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. The New England journal of medicine 339 (24):1734-1738. doi:10.1056/NEJM199812103392402
4. Yu SC, Jiang P, Choy KW, Chan KC, Won HS, Leung WC, Lau ET, Tang MH, Leung TY, Lo YM, Chiu RW (2013) Noninvasive prenatal molecular karyotyping from maternal plasma. PloS one 8 (4):e60968. doi:10.1371/journal.pone.0060968
5. Qu JZ, Leung TY, Jiang P, Liao GJ, Cheng YK, Sun H, Chiu RW, Chan KC, Lo YM (2013) Noninvasive prenatal determination of twin zygosity by maternal plasma DNA analysis. Clinical chemistry 59 (2):427-435. doi:10.1373/clinchem.2012.194068
6. Lo YM (2013) Noninvasive fetal whole-genome sequencing from maternal plasma: feasibility studies and future directions. Clinical chemistry 59 (4):601-603. doi:10.1373/clinchem.2012.191098
To prove this EDTA effect, in the last test we added increasing
doses of this anticoagulant and we got an inhibition of DNAse in the
serum, EDTA dependent, showing that the EC50 of this inhibition is
10-4M order, meaning that the degradation of the hidrolysis probe
was inhibited (C).
CONCLUSION
1. Cell-free DNA is subject to degradation by the variation of
temperature in the serum but not in the EDTA-plasma.
2. Nucleases are active only in the serum, very likely because of the
EDTA effect of chelate divalent ions of calcium, giving na ex-vivo
protection to cell-free DNA.
3. We develop a fluorescence method in real-time to detect the
nucleases activities in clinical samples.