Metabolic characterization of the natural progression of chronic hepatitis B
- Johannes C. Schoeman1, 2,
- Jun Hou3,
- Amy C. Harms1, 2,
- Rob J. Vreeken1, 2, 4,
- Ruud Berger1, 2,
- Thomas Hankemeier†1, 2 and
- Andre Boonstra†3Email author
© The Author(s). 2016
Received: 17 February 2016
Accepted: 17 May 2016
Published: 10 June 2016
Worldwide, over 350 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing progressive liver diseases. The confinement of HBV replication to the liver, which also acts as the central hub for metabolic and nutritional regulation, emphasizes the interlinked nature of host metabolism and the disease. Still, the metabolic processes operational during the distinct clinical phases of a chronic HBV infection—immune tolerant, immune active, inactive carrier, and HBeAg-negative hepatitis phases—remains unexplored.
To investigate this, we conducted a targeted metabolomics approach on serum to determine the metabolic progression over the clinical phases of chronic HBV infection, using patient samples grouped based on their HBV DNA, alanine aminotransferase, and HBeAg serum levels.
Our data illustrate the strength of metabolomics to provide insight into the metabolic dysregulation experienced during chronic HBV. The immune tolerant phase is characterized by the speculated viral hijacking of the glycerol-3-phosphate–NADH shuttle, explaining the reduced glycerophospholipid and increased plasmalogen species, indicating a strong link to HBV replication. The persisting impairment of the choline glycerophospholipids, even during the inactive carrier phase with minimal HBV activity, alludes to possible metabolic imprinting effects. The progression of chronic HBV is associated with increased concentrations of very long chain triglycerides together with citrulline and ornithine, reflective of a dysregulated urea cycle peaking in the HBV envelope antigen-negative phase.
The work presented here will aid in future studies to (i) validate and understand the implication of these metabolic changes using a thorough systems biology approach, (ii) monitor and predict disease severity, as well as (iii) determine the therapeutic value of the glycerol-3-phosphate–NADH shuttle.
Worldwide, over 350 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing progressive liver diseases, including fibrosis, liver failure, or hepatocellular carcinoma (HCC), over the course of several decades [1, 2]. Chronic HBV infection can be divided into four progressively distinct clinical phases based upon serum levels of HBV DNA, alanine aminotransferase (ALT), and HBV envelope antigen (HBeAg). These four phases are the immune tolerant (IT), immune active (IA), inactive carrier (IC), and HBeAg-negative (ENEG) phases. The increased ALT levels in the IA and ENEG phases reflect hepatic injury due to viral activity and immune activity. Even though these phases are used in clinical practice for deciding on therapeutic interventions , not much is known about the underlying metabolic mechanisms associated with each and/or the progressive nature of the disease. HBV replicates in the liver, which also acts as the central hub for metabolic and nutritional regulation, emphasizing the interlinked nature of the metabolism and the disease. Hence, a clear metabolic understanding of the progressive driving forces and diverse clinical outcomes is important in the management of chronic hepatitis B—for instance, to provide prediction markers for disease progression and candidate targets for therapeutic intervention. In vitro systems are not reliable models for chronic HBV infection since they do not represent the persistence, complexity, and progressive nature of the disease. Animal models do not appropriately mimic the pathogenic and immunological responses experienced during human HBV infection, hindering effective translational studies [4, 5]. Thus, the use of material obtained from patients is crucial to study and understand chronic HBV infection.
Metabolomics is an established, continuously improving tool used to study the metabolome through targeted or untargeted approaches, providing static observational and/or longitudinal readouts from a dynamic system [6–9]. Targeted metabolomics, which focuses on a predefined biology-driven subset of metabolite classes, is ideal for low concentration metabolites, whereas untargeted metabolomics is more suitable for measuring high concentration metabolites . Metabolomics data aid in identifying and understanding the metabolic perturbations leading to dysregulated homeostasis and stress, e.g., during viral infection [11–14]. Subsequently, the contribution of these metabolic perturbations to the pathogenicity of the virus can be determined. Several metabolomics studies have been done to assess the metabolic host–pathogen interactions during chronic HBV infection. Targeted metabolomics approaches have identified the phospholipid [15, 16], triglyceride [15, 17, 18], sphingomyelin [15, 19], and free fatty acid [16, 20] metabolic pathways as affected during chronic HBV infection. Untargeted approaches have also been used to investigate metabolic changes experienced during chronic HBV infection [21–23] and have primarily focused on HBV-related cirrhosis and HCC [15, 24–26].
Here we present the first discovery metabolomics study of chronic HBV infection, shedding light on the progressive metabolic alterations over the different clinical phases. We used targeted metabolomics platforms to illuminate the phospholipid, triglyceride, sphingomyelin, amino acid, acyl-carnitine, and signaling lipid profiles to characterize the progressive nature of chronic HBV infection. We identified the most profound changes to occur in choline glycerophospholipids, plasmalogens, very long chain triglyceride species, and urea cycle intermediates across the progression of the four clinical phases of chronic HBV infection. Our data support the viral hijacking and persistent impairment of the glycerol-3-phosphate–NADH shuttle, modulating the host lipid profile. These events are central to understanding the metabolic perturbations reflective of the natural progression of chronic HBV infection.
Sixty-nine chronic HBV patients and 19 healthy controls (HCs) were included in this discovery study. Blood was prospectively collected in SST tubes at the Erasmus MC and centrifuged to separate the serum and stored at −80 °C until analysis. Patients were treatment naïve and excluded if they had other chronic infections (HIV, hepatitis C virus) or a body mass index of 31 or more. This study was conducted in accordance with the guidelines of the Declaration of Helsinki and the principles of Good Clinical Practice. The ethical review board of the Erasmus MC approved the study and informed consent was obtained from all patients who were asked to donate blood.
Definition of chronic HBV clinical phases
Serum ALT was measured on an automated analyzer, qualitative serum HBV surface antigen (HBsAg), HBeAg, and anti-HBeAg antibodies were measured on an Architect Abbott analyzer, and serum HBV DNA levels were measured using the COBAS AmpliPrep-COBAS Taq-Man HBVv2test (CAP-CTM; Roche Molecular Systems, Indianapolis, IN, USA). Based on serum HBV DNA, ALT levels, and HBeAg presence at the time of sampling, patients were categorized into four clinical HBV phases according to international guidelines . IT patients (n = 18) had detectable serum HBeAg and repetitive normal ALT values (<40 U/L). IA patients (n = 12) and ENEG patients (n = 19) had repetitive or intermittent abnormal serum ALT (>40 U/L) values and HBV DNA levels >2000 IU/mL. IC patients (n = 20) were HBeAg-negative and had both repetitive normal ALT values (<40 IU/L) and HBV DNA levels below 20,000 IU/mL.
Targeted liquid chromatography–mass spectrometry metabolomics
Metabolomics platforms: volumes, sample preparation, and analytical instruments
Targeted metabolomics platform
Volume of serum used (μL)
Sample prep method
Metabolite class coverage
Platform targets (n)
Quality control passed
Percentage of missing data
Biogenic amine 
Protein precipitation and AccQTag derivatization
Amino acids, catecholamines and polyamines
Positive lipid 
Isopropyl alcohol extraction
Phospholipids, cholesterol esters, di/triglycerides and sphingomyelins
Negative lipid 
Free fatty acids and phospholipids
Oasis HLB solid-phase extraction
Hydroxylated fatty acids, prostaglandins, and thromboxanes
Acylcarnitines, TMAO, choline, betaine
Quality control (QC) samples consisted of equally pooled volumes of all study samples. A set of QC samples was then included during the analyses of the experimental groups on the individual metabolomic platforms and evenly distributed through the randomized samples prior to liquid chromatography–mass spectrometry (LC–MS) analyses. In addition, independent duplicate samples (10–15 %) were randomly selected where sample volume allowed.
After LC–MS analyses, peak integration was done using the instrument’s software and the relative ratios between metabolites and their corresponding internal standards were determined. Using the QC samples and duplicate samples, a double QC approach was applied to include metabolites that were reliably measured by the individual metabolomics platforms by reporting and using only those metabolites for which both duplicate samples and QC samples showed a relative standard deviation <30 %. After QC, a data set comprising 88 cases and 314 metabolites was constructed, with all missing data being replaced by half the minimal intensity value of the corresponding metabolite.
Statistical data analyses
SPSS 21.0 (SPSS Inc., Chicago, IL, USA) was used for Fisher’s exact tests on the patient cohort characteristics presented as frequencies and ANOVA on the continuous values. A combination of univariate and multivariate bioinformatics approaches was performed using the R script-based online tool Metaboanalyst 3.0, a comprehensive tool suitable for analyzing metabolomics data [28, 29]. The metabolomics data sets were log transformed and auto-scaled prior to bioinformatics analyses. For the analyses between HCs and IT patients, significant metabolites were identified per metabolomics platform based on the following criteria: (i) a p value <0.05 using the independent student t-test; and (ii) a fold change ≥1.20 or ≤0.80, indicating a 20 % increase or decrease. The false discovery rate’s q values are reported for every reported p value. Multivariate principal component analysis was also done to visualize the natural distribution of the data.
The multivariate ANOVA test was used to identify the significant changes (p < 0.05) across the four clinical phases of chronic HBV infection in conjunction with K-means clustering. The K-means clustering was performed in MATLAB using kmeans and “correlation” as the distance measure on the metabolites in order to examine changes in their levels during the course of chronic HBV infection, using all 314 metabolites and 88 patients. Its algorithm partitions the metabolites into K mutually exclusive clusters (k is the number of desired clusters), taking into account the measurement of each metabolite under multiple conditions, in this case HCs plus all four clinical phases. Within each cluster, metabolite measurement patterns are as close to each other as possible, while as far away from those in other clusters as possible. The partition was repeated with metabolites reassigned among clusters at each iteration until the sum of point-to-centroid distance reached a minimum . The pattern analysis was performed sequentially with k assigned with 12, 16, 20, and 24 each time.
Baseline characteristics of the study population
Baseline characteristics of the patient cohort
Healthy control (HC)
Immune tolerant (IT)
HBeAg-positive active hepatitis (IA)
Inactive carrier (IC)
HBeAg-negative active hepatitis (ENEG)
Number of patients
Age, years (SE)
7 (17.1 %)
13 (31.7 %)
5 (12.2 %)
14 (34.2 %)
2 (4.9 %)
6 (14.6 %)
5 (12.2 %)
7 (17.1 %)
6 (14.6 %)
17 (41.5 %)
BMI, kg/m2 (SE)
44 (63.77 %)
16 (36.4 %)
9 (22.7 %)
8 (18.21 %)
10 (22.7 %)
9 (13.04 %)
0 (0.0 %)
3 (33.3 %)
2 (22.2 %)
4 (44.4 %)
8 (11.59 %)
0 (0.0 %)
0 (0.0 %)
5 (62.5 %)
3 (37.5 %)
8 (11.59 %)
2 (25.0 %)
0 (0.0 %)
4 (50 %)
2 (25.0 %)
Log HBV DNA, IU/ml (SE)***
HBV genotype A/B/C/D**
ALT, IU/l (SE)***
Metabolic characterization of the IT phase of chronic HBV infection versus the HCs
Significant serum metabolites identified between HCs and the IT phase
Targeted metabolomics platform
Metabolite species identified as significant between the HCs and IT phase
Trend in the IT phase
C32:1, C32:2, C34:1, C34:3, C34:4, C36:3, C36:4, C36:6, C38:3
Plasmalogen phosphatidylcholines (pPC)
C34:2, C36:2, C36:3, C38:6
Plasmalogen phosphatidylethanolamines (PE)
sn1: C14:0, C15:0, C16:1, C18:1, C18:2, C18:3 (ω3ω6), C20:3 (ω3ω6), C20:3 (ω9), C20:4, C22:4, C22:5 (ω6)
sn2: C14:0, C16:1, C18:1, C18:2, C18:3 (ω3ω6), C20:3 (ω3ω6), C20:4
C18:0, C18:1, C18:2#, C20:3 (ω3ω6), C20:4, C22:5# (ω3)
Plasmalogen lysophosphatidylcholines (pLPC)
C16:0, C16:1, C18:1, C18:2
C42:1, C44:2, C46:1, C46:2, C46:3, C48:1, C48:2, C48:3, C48:4#, C50:0, C50:1, C50:2, C50:3, C50:4, C50:5, C51:1, C51:2, C51:3, C52:1, C52:2, C54:1, C54:2, C55:2, C56:5, C58:5
Amino acids metabolites
4-Hydroxyproline, pipecolic acid
C18:1/14:0, C18:1/21:0, C18:1/23:0, C18:1/25:0, C18:1/25:1
Free fatty acids and Acyl-carnitines
Free fatty acids (FFA)
Oleylcarnitine (C18:1)#, Linoleylcarnitine (C18:2)
Cholestrol esters (CE)
11-HDoHE#, 14-HDoHE#, 10-HDoHE#
5-HETrE, 8,9-DiHETre,#, 12,13-DiHOME,#
Metabolomics characterization of the natural course of a chronic HBV infection
Serum metabolites identified as changing significantly over the progression of chronic HBV infection
Targeted metabolomics platform
Metabolite species identified as changing significantly during the progression of chronic HBV
Cluster 6: stable increased
Cluster 8: stable decreased
sn1, C20:3 (ω3ω6), sn2, C20:3 (ω3ω6)
Cluster 10: increasing over the four phases
Isovalerylcarnitine, 2-methylbutyroylcarnitine, stearoylcarnitine
Phenylalanine, glutamic acid, methionine
C54:2, C54:3, C54:4, C55:3, C56:3, C56:4, C56:5, C56:6, C58:5
Free fatty acids
Cluster 13: reflective of ALT levels
Amino acid metabolite
Cluster 15: increasing over the four phases
C42:1, C52:2, C55:2
C18:1, C20:3 (ω3ω6), C20:4
Cluster 23: reflective of HBV DNA levels
Free fatty acids
C20:3 (ω3ω6), C22:4
Metabolite clusters showing a “stable” increased or decreased trend over the four clinical phases of chronic HBV
Clusters 6 (Fig. 3a) and 8 (Fig. 3b) represent metabolites significantly altered in the IT phase compared with HCs and whose alteration persisted throughout the progression of chronic HBV infection with stable increased or decreased trends. Cluster 6 contained seven plasmalogen phospholipid and lysophospholipid species, of which three were significantly up-regulated in the IT phase compared with the HCs, having sustained increased levels across the four clinical phases. We named this cluster the “ROS-plasmalogen cluster”. Four non-enzymatic route oxylipins (10-, 11-, 13-, and 14-HDoHEs) grouped within cluster 6 indicate maintained oxidative stress experienced during the progression of chronic HBV infection. Interestingly, the choline catabolic metabolites betaine and sarcosine were also in the ROS-plasmalogen cluster, which may suggest some degree of choline metabolism perturbation. The amine metabolites carnitine and ornithine were identified as significantly changing over the clinical phases (Table 4). Carnitine is the free fatty acid transporter metabolite and indicates significant dysregulation during chronic HBV infection (p < 0.001). The IT and IC phases had lower carnitine levels compared with the IA and ENEG phases, reflecting increased energy requirements during these phases. Ornithine is a urea cycle intermediate together with citrulline (cluster 9, p = 0.006), indicating a dysregulated urea cycle, worsening with the progression of chronic HBV infection as both had their highest levels in the ENEG phase.
Cluster 8 represents the opposite trend, showing a stable decline of metabolite levels across the four clinical phases compared with HCs. Cluster 8 was labeled the “choline phospholipid cluster” as it comprises 14 phosphatidylcholines and 21 lysophosphatidylcholines. The strong initial reduction of choline phospholipids, together with the subsequent maintenance of the reduced levels over the four clinical phases, hints towards an imprinted metabolic alteration. Only the sn1 and sn2 lysophosphatidylcholine C20:3 species showed significant fluctuations during the progression of the clinical phases (Table 4). Three amines, including saccharopine, glutathione and γ-glutamyl glutamine, had stable decreased levels compared with HCs. The glutathione levels reflect diminished antioxidant capacity during chronic HBV, supporting the increased levels of lipid peroxidation products observed within cluster 6.
Metabolite clusters showing a trend for increasing over the clinical phases of chronic HBV
From the 24 clusters, clusters 10 (Fig. 3c) and 15 (Fig. 3d) were the most dense, containing 47 and 42 metabolites, respectively. Both clusters showed an increasing trend across the four clinical phases of chronic HBV infection, with their defining difference observed when taking the HCs into account.
Cluster 10 shows subtle changes between the HCs and the IT phase but significantly increased during the progression of chronic HBV infection to levels well above HC levels in the ENEG phase. Cluster 10 is dominated by 13 amino acids and 17 triglyceride species, composed of long and very long acyl species with a combined acyl chain length of between C54 and C60. Thus, this cluster is called the “very long chain triglyceride cluster”. During the progression over the four clinical phases, 9 of the 17 triglycerides were identified as significantly increasing, revealing a perturbed triglyceride metabolism (Table 4). The essential amino acids methionine and phenylalanine were identified as significantly changing over the clinical phases and have both been identified as markers of hepatic injury and had their highest levels in the ENEG phase [31–33]. Choline, identified as significant and in cluster 10, is an integral part of glycerophospholipid and sphingolipid metabolism, acting as a functional lipid moiety. The increasing choline levels oppose that of its elevated catabolic metabolites betaine and sarcosine (cluster 6).
Cluster 15 includes metabolites that were significantly reduced in the IT phase compared with the HCs, after which metabolite levels started to increase back to HC levels. More than half of the 42 metabolites in cluster 15 are triglycerides, with a combined acyl chain length ranging from C42 to C55, effectively labeling this the “long chain triglyceride cluster”. These results show a biphasic triglyceride response during chronic HBV infection. Three of the five clustered lysophosphatidylethanolamines species, including C18:1, C20:3, and C20:4, were subsequently identified as significantly increasing over the four clinical phases, with levels almost resembling the HCs in the ENEG phase. These results also show that lysophosphatidylethanolamines (cluster 15) respond differently during chronic HBV infection to lysophosphatidylcholines (cluster 8).
Metabolite clusters correlating to ALT and HBV DNA levels
During chronic HBV infection, ALT levels (Fig. 1a) were highest during the IA and ENEG phases, whereas HBV DNA levels (Fig. 1b) were high in the IT, IA, and ENEG phases and lowest in the IC phase. Clusters 13 (Fig. 3e) and 23 (Fig. 3f) represented metabolites correlating to ALT and HBV DNA levels, respectively.
Kynurenine identified in cluster 13 correlated to ALT levels and had significantly higher levels in the IA and ENEG phases compared with the IT and IC phases (Table 4). Kynurenine and its precursor amino acid tryptophan (cluster 15) were both also identified as significantly down-regulated during the IT phase compared with the HCs. Due to these fluctuating levels during the progression of chronic HBV, we inspected the tryptophan/kynurenine ratio relating to indoleamine 2,3-dioxygenase (IDO) activity. The tryptophan/kynurenine ratio had a p value of 0.038 across the five groups with a reduced IDO ratio in the IT, IA, and IC phases of chronic HBV infection compared with an ENEG phase ratio comparable to that of the HCs, implying altered IDO regulation during the first three phases of chronic HBV infection.
Cluster 23, reflecting HBV DNA levels during chronic HBV infection, consisted solely of five acyl-carnitines and four free fatty acids. The long chain free fatty acids C20:3 (ω3/ω6) and C22:4, together with decanoylcarnitine, had significantly lower levels in the IC phase compared with the other clinical phases, possibly correlating to HBV-induced secreted phospholipases [34, 35].
The present study is the first targeted and biology driven metabolomics profiling of chronic HBV infection, characterizing the natural progression through its distinct clinical phases. With the liver being the central organ in nutritional regulation and metabolism, it is not surprising that chronic HBV infections have been shown to induce multiple metabolic alterations in lipid metabolism of the host [15–21]. However, these studies have not addressed and positioned metabolic changes in relation to the progression of chronic HBV disease.
During the natural progression of chronic HBV, we measured increased choline, methionine, and very long acyl chain triglyceride levels together with reduced phosphatidylcholine and lysophosphatidylcholine levels, indicating a perturbed choline metabolism. Dietary choline and methionine depravation is strongly linked to the development of steatosis, non-alcoholic fatty liver disease, cirrhosis, and HCC [38–41]. During conditions of choline restriction, the reduced levels of phosphatidylcholine, a critical component of the very low density lipoprotein particle, impair hepatic lipoprotein synthesis and result in the accumulation of free triglycerides within hepatocytes [42, 43]. Decreased phosphatidylcholine species in the presence of high choline levels during the IA, IC, and ENEG phases support the permanent G3P–NADH shuttle hijacking hypothesis, impairing lipoprotein synthesis during chronic HBV, while also explaining the accumulation of triglycerides. Even with decreased levels of long chain free fatty acids in the IC phase, no attenuation of long chain triglyceride levels was observed in this phase. The stable elevated levels of betaine, sarcosine, and methionine indicate enhanced choline catabolism, while increased levels of methionine are also reflective of hepatic injury [32, 33]. Previous studies demonstrated that host factors were responsible for the development of steatosis rather than viral factors [44, 45]. Our metabolomics data suggest that initiation of steatosis may be a consequence of HBV hijacking of the host’s glycerophopholipid metabolism, as liver fat content closely correlates with serum triglyceride levels [46, 47].
Collectively, the data presented here comprise the first metabolic study on the natural progression of chronic HBV infection using patient samples. We found impaired choline glycerophospholipid metabolism across the four chronic HBV clinical phases, together with increasing triglyceride and urea cycle intermediate levels as a liver metabolic fingerprint of the progression of chronic HBV infection. This metabolic fingerprint relates to HBV’s hijacking of the G3P–NADH shuttle, a key player in the plasmalogen, choline, and glycerophospholipid metabolic pathways, and its potential as a therapeutic target deserves further investigation. Elegant work done by Zeissig et al.  demonstrated the role of lysophospholipids as endogenous antigenic lipid species able to illicit protective immunological responses, which enhanced HBV clearance during acute infection. This may imply that, in addition to redirecting host lipid metabolism to produce the plasmalogens, hijacking of glycerophospholipid metabolism during acute HBV infection could act as a switch to determine HBV clearance or persistence. Furthermore, the diabetes drug metformin, which inhibits mitochondrial GPDH , part of the G3P–NADH shuttle, was found to inhibit HBV protein production and replication . These findings substantiate the therapeutic value of the G3P–NADH shuttle in chronic HBV infection.
The present study provides many insights and leads to design follow-up studies and, at the same time, highlights the need for a systems biology approach to better understand chronic HBV infection. We identified liver-related metabolic and injury perturbations, which reflect the natural progression of the disease. The altered glycerophospholipid metabolism in the IT phase attributed to the HBV hijacking of the G3P–NADH shuttle has an intimate relationship with the persistent lipid dysregulation observed in the IA, IC, and ENEG clinical phases. Increased levels of the very long chain triglycerides in the IA phase and urea cycle intermediates in the IC phase highlight the risk for developing secondary liver complications during chronic HBV infection. These metabolites might prove useful as markers of disease progression and severity.
ALT, alanine aminotransferase; DHAP, dihydroxyacetone phosphate; ENEG, HBeAg-negative; G3P, glycerol-3-phosphate; GPDH, glycerol-3-phosphate dehydrogenase; HBeAg, HBV envelope antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HC, healthy control; HCC, hepatocellular carcinoma; IA, immune active; IC, inactive carrier; IDO, indoleamine 2,3-dioxygenase; IT, immune tolerant; LC–MS, liquid chromatography–mass spectrometry; QC, quality control.
This study was supported by the NWO-ZonMW (grant number 435002027) and the Virgo consortium, funded by the Dutch government project number FES0908. We would also like to thank the technical staff, including L. Lamont, B. Gonzalez Amoros, and H. Elfrink at the Netherlands Metabolomics Centre, Leiden University for performing the metabolomics sample analyses.
Availability of data and materials
The metabolomics datasets supporting the conclusions of this article are available in the online MetaboLights data repository, under accession numbers MTBLS253, MTBLS279, and MTBLS280.
AB and TH served as principal investigators and conceived and designed the study; JCS, JH, and ACH performed the experiments; JH and AB assisted in acquisition of the samples and clinical data; JCS, JH, RB, RJV, TH, and AB contributed to the interpretation of the data; JCS wrote the first draft of the manuscript with input from all authors; and all authors approved the final manuscript revisions.
The authors declare that they have no competing interests.
Consent for publication
All authors give consent for the publication of the manuscript in Genome Medicine.
Ethics approval and consent to participate
This study was conducted in accordance with the guidelines of the Declaration of Helsinki and the principles of Good Clinical Practice. The ethical review board of the Erasmus MC approved the study and informed consent was obtained from all patients who were asked to donate blood.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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