Original Paper

Blood Purif 2016;41:34–40 Received: July 10, 2015

Accepted: September 8, 2015
DOI: 10.1159/000440975
Published online: October 20, 2015

The Role of Cell-Free Plasma DNA in

Critically Ill Patients with Sepsis
Anna Clementi a, e Grazia Maria Virzì a, c Alessandra Brocca a, c, d Silvia Pastori a, c
       

Massimo de Cal a, c Stefano Marcante b Antonio Granata e Claudio Ronco a, e

       

a
  IRRIV-International Renal Research Institute, b Intensive Care Unit, San Bortolo Hospital, and c Department of
   

Nephrology, Dialysis and Transplant, San Bortolo Hospital, Vicenza, d Department of Medicine DIMED, University
 

of Padova Medical School, Padova, and e Department of Nephrology and Dialysis, San Giovanni Di Dio Hospital,
 

Agrigento, Italy

Key Words stay. The cfDNA concentrations at admission were higher in

Cell-free DNA · Sepsis · Apoptosis · Outcome
Abstract
Background: The identification of highly reliable outcome
predictors in severe sepsis is important to define disease se-
verity, predict bedside prognosis and monitor response to
treatment. Cell-free plasma DNA (cfDNA) has been recently
proposed as a possible prognostic marker of clinical out-
come in septic patients. In this study, we investigated the
prognostic value of cfDNA in patients with sepsis and its pos-
sible correlation with caspase-3, IL-6 and IL-18 levels. Meth-
ods: We enrolled 34 patients admitted to the intensive care
unit (ICU). Out of these 34, 27 patients were septic and 7 were
non-septic. cfDNA was extracted from plasma and quanti-
fied by real time PCR. Plasma levels of caspase-3, IL-6 and IL-
18 were measured by ELISA. Results: We observed signifi-
cantly higher levels of cfDNA in septic patients. No signifi-
cant differences were found between cfDNA levels in
patients with Gram+, Gram– and fungal infections. Out of
the 27 septic patients, 12 developed acute kidney injury (AKI)
requiring continuous renal replacement therapy (CRRT), and
cfDNA levels resulted to be higher in this group. Out of the
27 septic patients, 11 had a negative outcome during the ICU
non-survivors than in survivors. Caspase-3, IL-6 and IL-18 lev-
els were significantly higher in septic patients when com-
pared to these levels in non-septic patients and correlated
with cfDNA levels. Conclusion: cfDNA can be considered a
good prognostic marker of clinical outcome in septic pa-
tients. Its levels increase in case of AKI complicating sepsis,
in particular if CRRT is needed, and are associated with poor
outcome. Caspase-3, IL-6 and IL-18 levels are higher in septic
patients and correlate to cfDNA concentrations.
© 2015 S. Karger AG, Basel
Introduction
Sepsis refers to severe systemic inflammation in re-
sponse to invading pathogens [1]. Based on the latest ep-
idemiological survey, about 750,000 cases of severe sepsis
per year occur in the United States alone [2], with a mor-
tality rate varying between 30 and 60% as shown by dif-
ferent studies [2, 3].
The identification of highly reliable outcome predic-
tors in severe sepsis is important to define disease sever-
ity, predict bedside prognosis and monitor response to
treatment. Several biomarkers have been proposed to be
144.82.108.120 - 8/12/2016 4:17:56 AM

© 2015 S. Karger AG, Basel Dr. Grazia Maria Virzì

0253–5068/15/0413–0034$39.50/0 Department of Nephrology, Dialysis and Transplantation
International Renal Research Institute Vicenza (IRRIV), San Bortolo Hospital
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E-Mail karger@karger.com
Via Rodolfi 37, IT–36100 Vicenza (Italy)
www.karger.com/bpu
E-Mail graziamaria.virzi @ gmail.com
UCL
of potential use for sepsis, including inflammatory cyto-
kines, cell-surface markers, acute-phase proteins, coagu-
lation factors, and apoptosis mediators [4, 5].
Recent research suggested that cell-free plasma DNA
(cfDNA), composed of circulating extracellular DNA
fragment originating from necrotic and apoptotic cells [6,
7], may have prognostic utility in several clinical condi-
tions, such as trauma, cancer, stroke, myocardial infarc-
tion, and sepsis [8–12].
cfDNA is released from a number of cells, including
neutrophils, eosinophils, and macrophages, as a result of
either apoptosis or other forms of cellular damage. Apop-
tosis plays a pivotal role in the pathogenesis of sepsis, and
increased plasma levels of nucleosomes, in which frag-
mented DNA is packed during apoptosis, have been
found in patients with severe sepsis and septic shock [13].
Compelling evidence has demonstrated that in higher
organisms, apoptosis is executed by a family of cysteine
proteases, known as caspases that cleave after an aspartate
residue in their substrates [14]. Caspases are essential for
apoptosis, and several studies indicate that both the ex-
trinsic and intrinsic pathways are likely to be involved in
sepsis-induced lymphocyte apoptosis [15]. Importantly,
caspases have been recognized as important factors in hu-
man diseases where excessive apoptosis and uncontrolled
inflammation are hallmarks of pathology [16].
In this study, we investigated the prognostic value of
cfDNA in critically ill patients with sepsis and its possible
correlation with caspase-3, IL-6 and IL-18 levels.
Materials and Methods
Patients
We enrolled 34 patients admitted to the Intensive Care Unit
(ICU) of San Bortolo Hospital, Vicenza. Out of the 34, 27 patients
were septic and 7 were non-septic (patients with trauma). Sepsis
was defined based on the Surviving Sepsis Campaign guidelines
[17]. Acute kidney injury (AKI) was defined based on AKI Net-
work criteria [18]. Estimated glomerular filtration rate (eGFR) was
calculated with the 4-variable standardized MDRD formula [19].
A negative outcome was defined as death during the ICU stay. The
SOFA at the time of admission was estimated and recorded for
each patient. Clinical characteristics, laboratory data and dialysis-
related parameters were recorded for all patients.
Sample Collection
Blood samples were collected from all 34 patients into EDTA-
containing tubes at ICU admission and processed within 30 min
after venipuncture. Samples were subsequently centrifuged for
10 min at 3,500 rpm. Plasma was carefully removed without touch-
ing the cell pellet and re-centrifuged at 13,000 rpm for 10 min. Af-
ter centrifugation, the supernatant was placed into a clean poly-
propylene tube and stored at –80 ° C until use.
   
cfDNA in Septic Patients
Isolation and cfDNA Extraction
Within the first 30 min after drawing fresh plasma samples
from all patients, DNA was isolated from 250 μl of plasma using a
DNA isolation kit (ArrowDNA Kit; NorDiag, Holliston, Mass.,
USA) by automatic extractor NorDiag Arrow (NorDiag, Holliston,
Mass., USA), according to the manufacturer’s protocol. The DNA
was eluated in 50 μl of elution buffer and cfDNA concentration was
measured using a Picodrop Spectrophotometer (Bulldog Bio,
Portsmouth, N.H., USA).
Real-Time Quantitative PCR
Plasma cfDNA was measured using real-time quantitative assay
in RotorGene 6000 (Qiagen, Milan, Italy) for the β-actin gene pres-
ent in all nucleated cells. The amplification and product-reporting
system is based on the increase in fluorescence of Eva Green
(Biotium, Hayward, Calif., USA) coupled to an amplification reac-
tion. The primers sequences were as follows: forward (5′–3′) GCGC
CGTTCCGAAAGTT; reverse (5′–3′): CGGCGGATCGGCAAA.
A volume of 10 μl of cfDNA (template) was added to each reac-
tion mixture (total of 15 μl), which consisted of 4.95 μl of deionized
water, 1.25 μl of Eva Green (Biotium, Hayward, CA), 5 μl of Real
Time PCR Buffer, magnesium free (TakaRa, Shiga, Japan); 1.5 μl
of magnesium (TakaRa, Shiga, Japan); 0.5 μl of dNTP mixture
(TakaRa, Shiga, Japan); 5 μM of each primer and 0.3 μl of Takara
Ex Taq R-PCR (TakaRa, Shiga, Japan).
Each run included an activation step at 95 ° C for 3 min followed
by 50 amplification cycles of 30 s denaturation, 30 s annealing at
65 ° C (touchdown 1 ° C for 10 cycles) and 30 s elongation, and a final
elongation step at 72 ° C for 5 min, with fluorescence acquired on the
green channel. The PCR products were heated to 99 ° C and cooled
for heteroduplex formation, and melt was monitored by fluores-
cence emission using the appropriate denaturation range (50–99 ° C).
For each run, one aliquot of stock DNA was serially diluted with
deionized water to generate a 6-point standard calibration curve
(200,000–20,000–2,000–200–20–2 pg/ml). A conversion factor of
6.6 pg of DNA per diploid cell was used. Results are expressed as
genome equivalents (GE)/ml; 1 GE/ml equals 6.6 pg DNA.
We performed PCR runs two times in triplicate with samples
and standard curves. A blank reaction and several negative con-
trols were included in every run. The melt-curve analysis showed
a single product-specific melting temperature with a mean of
93.5 ° C for the β-actin gene.
Determination of Caspase-3 Activity
Plasma samples were assayed for cell free-caspase-3. Caspase-3
concentration was measured by human caspase-3 instant enzyme-
linked immuno-sorbent assay (ELISA) kit (eBioscience, San Diego,
Calif., USA) with a fluorometric assay at 450 nm by VICTORX4
Multilabel Plate Reader (PerkinElmer Life Sciences, Waltham
Mass., USA). Each experiment was performed in triplicate. Cas-
pase-3 concentrations (ng/ml) were calculated from the standard
curve according to the manufacturer’s protocol. Standard samples
ranged from 0.16 to 10.0 ng/ml. Human caspase-3 instant ELISA
kit sensitivity is 0.12 ng/ml.
Cytokines ELISA
Quantitative determination of IL-18 and IL-6 in plasma was per-
formed using the human instant ELISA kit (eBioscience, San Diego,
Calif., USA) according to the manufacturer’s instructions. Optical
density was read by using a VICTORX4 Multilabel Plate Reader
144.82.108.120 - 8/12/2016 4:17:56 AM
Blood Purif 2016;41:34–40 35
DOI: 10.1159/000440975
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 Table 1. Demographic and clinical data of septic and non-septic patients at ICU admission

Septic patients Non-septic patients p value

Age, years 62±18 64±21 NS

Male, % 70 71 NS
Creatinine, mg/dl, IQR 1.9 (0.9–4.0) 0.7 (0.7–0.9) <0.01
Urine output, ml/24 h, IQR 1,870 (462–3,090) 2,250 (1,603–2,750) 0.04
Urea, mg/dl, IQR 91 (58–178) 36 (30–53) <0.01
Na, mmol/l, IQR 139 (136–146) 142 (138–142) NS
K, mmol/l, IQR 3.9 (3.6–4.3) 3.6 (3.4–4.0) NS
White blood cells, ×103 cell per μl, IQR 10.6 (6.2–15.2) 11.6 (8.3–13.6) NS
Platelets, ×103 cell per μl, IQR 94 (57–139) 157 (135–192) NS
Procalcitonin, ng/ml, IQR 10.9 (1.8–25.0) 0.33 (0.1–0.5) 0.05

NS = Not significant.

(PerkinElmer Life Sciences, Waltham, Mass., USA) at 450 nm. The
concentration values for these molecules were calculated from stan-
dard curves. Standard samples ranged from 78 to 5,000 pg/ml for
IL-18 and from 3.1 to 200 pg/ml for IL-6. Human IL-18 instant
ELISA kit sensitivity is 9.2 pg/ml. Human IL-6 instant ELISA kit
sensitivity is 0.92 pg/ml. All tests were performed in triplicate.
Statistical Analysis
Statistical analysis was performed using the STATA software
package. A p value of <0.01 was considered statistically significant.
Categorical variables were expressed as percentages; continuous
variables were expressed as mean ± SD (normally distributed vari-
ables) or median and interquartile range (IQR; non-normally dis-
tributed variables). The Mann–Whitney U test or t test was used
for making comparisons between the two groups when appropri-
ate. Spearman’s rho correlations were calculated to verify the cor-
relation between variables. We applied a linear transformation of
cfDNA to manage the data in a better way.
Results
was caused by Gram+ infections in 26% of these patients,
Gram– infection in 33% and fungal infection in 22%. In
19% patients, the cause of sepsis was unknown.
The mean age of 7 non-septic patients was 64 ± 21
years and 71% of these patients were male. Forty three
percent of subjects belonging to the non-septic group had
diabetes and 29% had hypertension. The median admis-
sion serum creatinine of non-septic subjects was 0.74 mg/
dl 0.7 (IQR 0.7–0.9), the median eGFR was 103 ml/
min/1.73 m2 (IQR 86–118). No non-septic patients re-
quired CRRT. The median length of ICU stay was 8 days
(IQR 2–25). No patients had a negative outcome during
the ICU stay. Four out of the 7 non-septic patients re-
quired a surgical intervention: 2 neurosurgery, 1 vascular
surgery and 1 chest surgery.
Demographic and clinical data of these patients were
recorded and blood and urine biochemical parameters
were analyzed (table 1).

Subjects Baseline Characteristics cfDNA Levels in Septic Patients

We enrolled 34 patients admitted to the ICU: 27 pa-
tients were septic and 7 were non-septic (patients with
trauma). The median SOFA score was 7.5 (IQR 4.5–10.5)
in these patients at the time of admission.
The mean age of 27 patients with sepsis was 62 ± 18
years and 70% of these patients were male. Fifty two per-
cent septic subjects had diabetes mellitus and 44% had
hypertension. The median serum creatinine at ICU ad-
mission was 1.9 mg/dl (IQR 0.9–4.0) with a median eGFR
of 36 ml/min/1.73 m2 (IQR 17.8–86). Out of the 27 septic
patients, 12 developed AKI requiring continuous renal
replacement therapy (CRRT). The median number of
ICU stay was 8 days (IQR 4–18). Furthermore, 11 out of
the 27 had a negative outcome during the ICU stay. Sepsis
Plasma cfDNA was extracted and quantified in all
samples.
In these 34 ICU patients, no statistically significant cor-
relation between SOFA scores at admission and cfDNA
levels was observed (Spearman’s rho = 0.47, p = 0.07), but
a positive trend was evident.
Furthermore, we observed significantly higher levels
of cfDNA in patients with sepsis (5,331 GE/ml; IQR
1,751–16,113 vs. 238 GE/ml; IQR 208–1,001 in non-sep-
tic patients; p < 0.01; fig.  1). No significant differences
were found between the levels of cfDNA in patients with
Gram+, Gram– and fungal infections.
Moreover, cfDNA levels resulted to be higher in the 12
septic patients who developed AKI requiring CRRT
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36 Blood Purif 2016;41:34–40 Clementi/Virzì/Brocca/Pastori/de Cal/

DOI: 10.1159/000440975 Marcante/Granata/Ronco
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 10
10
9
9
8 8

log (cfDNA) GE/ml

log (cfDNA) GE/ml

7 7
6 6
5 5
4 4
3 3
2 2
1
1
0
0
Septic Non-septic
AKI requiring CRRT AKI no CRRT

Fig. 1. Plasma cfDNA in patients with and without sepsis. The lev- Fig. 2. Plasma cfDNA in septic patients who developed AKI requir-
els of cfDNA were significantly higher in patients with sepsis than ing CRRT and not. cfDNA levels resulted to be higher in septic
in patients without sepsis (p < 0.01). patients who developed AKI requiring CRRT compared with sep-
tic patients who did not require CRRT (p < 0.01).

(CRRT patients: 13,060; IQR 8,692–79,367,316 vs. non-

CRRT: 1,891; IQR 1,203–4,324; p < 0.01; fig. 2).
Of the 27 septic patients, 11 had a negative outcome 10
during ICU stay. The cfDNA concentrations at admission 9
were higher in ICU non-survivors than in survivors (neg- 8
log (cfDNA) GE/ml

ative outcome: 18,678; IQR 6,175–157,747 vs. positive 7

outcome: 1,891; IQR 1,497–6,319; p < 0.01; fig. 3). 6
5
4
Caspase-3 Levels in Septic and Non-Septic Patients
3
The median value of plasma caspase-3 was 3.8 ng/ml
2
(IQR 3.5–5.1) in septic patients. The median level of plas-
1
ma caspase-3 was 0.4 ng/ml (IQR 0.2–1.5) in non-septic 0
patients. The level of caspase-3 in the septic group was Survivors Non-survivors
significantly increased when compared to non-septic
subjects (p < 0.001; table 2). A significant positive correla-
tion was observed between caspase-3 and cfDNA levels
(Spearman’s rho = 0.75, p < 0.01) in our study population. Fig. 3. Plasma cfDNA in survivors and non-survivors. The cfDNA
concentrations at admission were higher in ICU non-survivors
Cytokines Concentration than in survivors (p < 0.01).
We examined the inflammatory state of all the en-
rolled patients. Plasma pro-inflammatory cytokines lev-
els (IL-18 and IL-6) were measured by ELISA in all 34 Table 2. Cytokine levels in plasma in septic and non-septic patients
ICU patients at admission time. Cytokines levels are re-
Septic group Non-septic p
ported in table 2. group value
IL-18 and IL-6 levels in plasma were significantly ele-
vated in the septic group compared to the non-septic pa- Caspase-3,
tients (p < 0.001). A significant positive correlation was ng/ml 3.8 (3.5–5.1) 0.4 (0.2–1.5) 0.098
observed between both pro-inflammatory cytokines and IL-18, pg/ml 239.8 (133.9–598.3) 54.4 (48.2–69.6) 0.002
IL-6, pg/ml 87.1 (63.6–170.5) 13.1 (12.3–60.4) 0.005
cfDNA levels (IL-18 Spearman’s rho = 0.89, IL-6 Spear-
man’s rho = 0.80; both, p < 0.05) in our study population.
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cfDNA in Septic Patients Blood Purif 2016;41:34–40 37

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 Discussion
To our knowledge, this is the first study to evaluate
cfDNA levels in critically ill patients with sepsis, correlat-
ing its concentrations to caspase 3, IL-6 and IL-18 levels.
cfDNA is comprised of acellular DNA fragments circulat-
ing in peripheral blood, with cell apoptosis being pro-
posed as a potential source of this type of DNA [6, 7].
Recently, high concentrations of cfDNA have been re-
ported in several clinical conditions, such as trauma, can-
cer, stroke, myocardial infarction, and sepsis [8–12]. El-
evated levels of nucleosomes [13] and cfDNA have also
been detected in the blood of septic [12, 20] and critically
ill patients [21, 22].
Initial observations indicated that cfDNA consisted of
DNA fragments ranging from <0.5 to 21 kb [23]. Subse-
quently, it was demonstrated that most of DNA circulates
as mononucleosomes, protecting DNA from degrada-
tion; hence, internucleosomic degradation of chromatin,
characteristic of apoptosis, was suggested as a potential
source of cfDNA [7]. A correlation between cfDNA levels
and plasma levels of typical cellular apoptosis markers in
patients with lung cancer has been demonstrated [24].
Therefore, cfDNA can be considered a direct marker of
cell apoptosis, which plays a pivotal role in the patho-
physiology of sepsis [25].
In our study, cfDNA levels were significantly higher
in critically ill patients with sepsis compared to the levels
in non-septic patients and in patients who developed
AKI requiring CRRT. There were no significant differ-
ences in terms of cfDNA levels among patients with
Gram+, Gram– and fungal infections. The mechanisms
underlying apoptotic cell death in sepsis remain elusive.
Imbalance between oxygen delivery and consumption
resulting in anaerobic glycolysis and lactate production
are central features in severe septic infection. This may
lead to oxygen deprivation, endothelial cell damage and
subsequent apoptotic cell death, which are involved in
the development of AKI [26–28]. For this reason, pa-
tients with sepsis and/or AKI requiring CRRT may pres-
ent increased levels of cfDNA, which is considered a
marker of apoptosis.
Moreover, our data suggest that non-survivors criti-
cally ill patients have higher admission levels of cfDNA
compared to those of the survivors. Our findings are sup-
ported by previous studies where preliminary data from
ICU patients suggested that admission plasma DNA con-
centrations may be higher in non-survivors than in sur-
vivors [20, 22]. Saukkonen et al. [21] demonstrated that
the maximum cfDNA concentrations in the first days of
38 Blood Purif 2016;41:34–40
DOI: 10.1159/000440975
intensive care were independently associated with hospi-
tal mortality in critically ill patients. More recently, the
same authors suggested that plasma cfDNA levels may be
considered an independent predictor for ICU mortality
in patients with severe sepsis and septic shock [12].
Unfortunately, in our study population, no significant
correlation between SOFA score at admission and cfDNA
level was observed, but a positive trend was evident. We
hypothesized that this might be due to the small sample
size of study population. Larger studies may demonstrate
the utility of cfDNA in ICU patients and its possible rela-
tionship with prognostic ICU scores, such as SOFA, SAPS
II and APACHE.
In our study, plasma caspase-3 levels were significant-
ly higher in septic patients compared to the levels in non-
septic patients and correlated with cfDNA. Caspases are
essential for apoptosis and drive two distinct pathways. In
the extrinsic one, apoptosis is activated by the binding of
a ligand to a death receptor (e.g. FAS), which in turn ac-
tivates caspase-8 [16]. Active caspase-8 then initiates
apoptosis directly by cleaving executioner caspases, such
as caspase-3, -6 and -7 or indirectly by triggering the in-
trinsic pathway, where apoptosis is induced by caspase-9
able to cleave and activate executioner caspases [16].
Several studies indicate that both the extrinsic and in-
trinsic pathways are likely to be involved in sepsis-in-
duced lymphocyte apoptosis [15]. Caspase-3 is involved
in the apoptotic cascade in patients with sepsis, and this
may explain the higher levels of this protein in septic pa-
tients and its correlation with cfDNA levels.
Moreover, our data demonstrated significantly higher
levels of IL-6 and IL-18 in patients with sepsis and a pos-
itive correlation between them and cfDNA concentra-
tion. Elevated IL-6 concentrations have been found in
many acute conditions, such as burns, major surgery and
sepsis [29]. Plasma levels of IL-6 are stably elevated in
these conditions and correlate with many indicators of
disease severity, such as clinical scores, the occurrence of
multiple organ failure and septic shock [30]. IL-6 has a
variety of biological effects, including the activation of B
and T lymphocytes and the coagulation system, the in-
duction of fever, and the mediation of the acute phase
response. Moreover, the inflammatory process during in-
fection is enhanced by the activation of molecular plat-
forms called inflammasomes, which are protein complex-
es suspended in the cytoplasm. Inflammasomes are in-
volved in the development of inflammation by the
activation of caspase-1, which transforms inactive forms
of IL-1β, IL-18 and IL-33 into their active forms [31].
With active participation of inflammasomes, these cyto-
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kines are released from the cell to the extracellular space
and regulate the immune response.
Our data confirmed the higher levels of IL-6 and IL-18
levels in septic patients compared to non-septic patients
and their correlation to cfDNA concentrations.
Nevertheless, we acknowledge the limitations of the
small sample size of our study population and these pre-
liminary results can be considered hypothesis-generating.
Moreover, this prospective study is limited by the single
sample collection. In particular, we measured cfDNA only
before CRRT session: the exact kinetics and clearance of
cfDNA during CRRT treatment remain unknown. Be-
cause of these limitations, we cannot establish a causal re-
lationship between cfDNA levels and inflammation,
apoptosis and oxidative stress, but we can hypothesize a
possible association. These findings are provocative and
the design of the study does not allow us to make conclu-
sions about causality.
Conclusion
cfDNA can be considered a direct marker of cell apop-
tosis, which plays a pivotal role in the pathophysiology of
sepsis. Its levels increase in case of AKI complicating sep-
sis and are associated with poor outcomes. Caspase-3,
IL-6 and IL-18 are involved in the apoptotic cascade in
septic patients and their levels are correlated to cfDNA
concentrations. These data obtained from this small study
provide the basis for larger studies that may clarify the ef-
ficacy and the utility of cfDNA in ICU and septic patients
and the contribution of inflammation and apoptosis in
these populations.
Disclosure Statement
This statement is to certify that all authors have seen and ap-
proved the manuscript being submitted. We warrant that the ar-
ticle is the authors’ original work. We warrant that the article has
not received prior publication and is not under consideration for
publication elsewhere. On behalf of all co-authors, the correspond-
ing author shall bear full responsibility for the submission. This
research has not been submitted for publication nor has it been
published in whole or in part elsewhere. We attest to the fact that
all authors listed on the title page have contributed significantly to
the work, have read the manuscript, attest to the validity and le-
gitimacy of the data and its interpretation, and agree to its submis-
sion to blood purification. All authors agree that author list is cor-
rect in its content and order and that no modification to the author
list can be made without the formal approval of the Editor-in-
Chief, and all authors accept that the Editor-in-Chief’s decisions
over acceptance or rejection or in the event of any breach of the
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