Liquid biopsies: DNA methylation analyses in circulating cell-free DNA

Analysis of patient's materials like cells or nucleic acids obtained in a minimally invasive or noninvasive manner through the sampling of blood or other body fluids serves as liquid biopsies, which has huge potential for numerous diagnostic applications. Circulating cell-free DNA (cfDNA) is explored as a prognostic or predictive marker of liquid biopsies with the improvements in genomic and molecular methods. DNA methylation is an important epigenetic marker known to affect gene expression. cfDNA methylation detection is a very promising approach as abnormal distribution of DNA methylation is one of the hallmarks of many cancers and methylation changes occur early during carcinogenesis. This review summarizes the various investigational applications of cfDNA methylation and its oxidized derivatives as biomarkers for cancer diagnosis, prenatal diagnosis and organ transplantation monitoring. The review also provides a brief overview of the technologies for cfDNA methylation analysis based on next generation sequencing.     

Effects of capture on biological parameters in free-ranging bighorn sheep (Ovis canadensis): evaluation of drop-net, drive-net, chemical immobilization and the net-gun

Blood samples and physiological data were collected from 634 bighorn sheep captured between 1980 and 1986 in the western United States. Bighorn sheep were evaluated for physiological parameters (temperature, pulse and respiration), selected biochemical parameters (cortisol, creatine phosphokinase (CPK), serum glutamic oxaloacetic transaminase (SGOT), lactic dehydrogenase (LDH), alkaline phosphotase (AP), potassium, sodium, chloride, creatinine, blood urea nitrogen (BUN), selenium, glucose, total protein, plasma pH and plasma PCO2), and selected hematological parameters (packed cell volume (PCV), hemoglobin (HB), red blood cell count (RBC), and white blood cell count (WBC]. These parameters were compared among bighorn sheep captured by four different methods: drop-net (n = 158), drive-net (n = 249), chemical immobilization (n = 90) and the net-gun (n = 137). Biological parameters affected by stress, including temperature, respiration, cortisol, CPK, SGOT, potassium, glucose and WBC revealed significant differences among capture methods (P less than 0.05). Some blood parameter differences, including temperature, respiration, cortisol, glucose and WBC could be explained partially by the distribution of age and sex within capture method groups. Drop-net and net-gun methods of capture appeared to produce the least amount of alteration to biological parameters related to capture stress or compromise and capture mortality. Drive-net was similar to the former methods while chemical immobilization caused the greatest changes in the above physiological, biochemical and hematological parameters.     

宁乡花猪原代和第一代血液生理生化指标比较

目的比较原代宁乡花猪与其第一代仔猪血液生理生化指标的变化。方法常规方法测定宁乡花猪F0代16头,F1代16头血液18项生理指标和13项生化指标。结果F0代与F1代部分血液生理指标如白细胞（WBC）、红细胞（RBC）、血红蛋白（HGB）、红细胞积压（HCT）等13项有显著性差异（P〈0．05）;部分血液生化指标如总胆红素（TBIL）,尿素氮（BUN）,总蛋白（TP）,白／球蛋白（ALB／GLB）,葡萄糖（GLU）,有显著性差异（P〈0．05）;其他指标均无统计学差异。结论2代宁乡花猪之间部分血液生理生化指标有显著性差异。得到的数据可以为实验动物化研究提供相应的基础生理参考数据。     

The Role of Reticulocyte Hemoglobin Content for the Diagnosis of Functional Iron Deficiency in Hemodialyzed patients

The effectiveness of reticulocyte hemoglobin content (CHr) had been reported to detect early functional iron deficiency especially among Chronic kidney disease (CKD) patients. CHr is more superior to classic biochemical indices in reflecting transient iron-deficiency status, therefore improving diagnosis and treatment. This study was conducted to determine the sensitivity of CHr in the diagnosis of functional iron deficiency (FID) in hemodialyzed patients. One hundred hemodialyzed patients along with 60 healthy controls were recruited and blood specimens were collected. Venous blood was used for hematological and biochemical investigations collected via 3 ml lavender-top tubes for hematological tests including CBC, blood film, ESR and CHr, and red-top tube for biochemical tests including TIBC, SF and CRP. A statistically significant decrease was noted in CHr values between hemodialysis patients and the control group (24.8 ± 2.0 pg vs. 30.9 ± 1.3 pg, p<0.001). CHr values showed a significant correlations with RBCs, Hb- hemoglobin, Hct- hematocrit level, MCV- mean corpuscular volume, MCH- mean corpuscular hemoglobin, MCHC, RDW- red cell distribution width , SI-Serum Iron, TIBC- Total iron binding capacity and TSAT- Transferrin saturation. The present study showed that CHr in comparison to the conventional hematological and biochemical markers commonly used to diagnose iron deficiency.     

Diurnal variations and effects of fasting on blood constituents in minipigs.

We examined the diurnal variations and the effects of 48-hour fasting on hematological and serum biochemical values to obtain basic physiological data on ten male and seven female G?ttingen minipigs 6 to 16 months of age. For all hematological parameters examined (RBC, HCT, HGB, WBC and PLT), there was no diurnal variation in either sex, but red blood cell parameters were affected by fasting, with an increase in males and a decrease in females. Three serum biochemical parameters (GOT, UN and i.p.) for males and four (GOT, UN, Ca and i.p.) for females exhibited diurnal variation. These variations were eliminated by fasting. The BIL level in males was increased by fasting, and the color of serum was yellowish. Fe concentration in both sexes and CRE and Mg levels in males were decreased by fasting. These findings are basic data for various experiments on minipigs, and indicate that great care is required in establishing feeding times and fasting intervals and in the analysis of results of experiments on minipigs.     

Genome-wide detection of additional fetal chromosomal abnormalities by cell-free DNA testing of 15,626 consecutive pregnant women

Cell-free DNA(cfDNA) testing for common fetal trisomies(T21, T18, T13) is highly effective. However, the usefulness of cfDNA testing in detecting other chromosomal abnormalities is unclear. We evaluated the performance of cfDNA testing for genome-wide abnormalities, and analyzed the incremental yield by reporting extra abnormalities. We performed genome-wide cfDNA testing in 15,626 consecutive pregnancies prospectively enrolled in this study. cfDNA testing results were reported and counseling was given depending on the presence of extra chromosomal abnormalities. cfDNA testing identified 190 cases(1.2%)of chromosomal abnormalities including 100 common trisomies and 90 additional abnormalities. By expanding the cfDNA reporting range to genome-wide abnormalities, the false positive rate increased to 0.39%(P0.001) and positive predictive value(PPV) was reduced to 65.58%(P=0.42). However, the detection yield increased from 0.44% to 0.65%(P=0.014), and cfDNA testing detected 38.61%(39/101) additional abnormalities with no ultrasound and biochemical screening findings. cfDNA testing outperformed biochemical screening by showing 60 times higher true positive rate and fewer false negative results.Genome-wide cfDNA testing significantly increased the diagnostic yield by detecting extra abnormalities, especially those without diagnostic indications. Genome-wide cfDNA testing has fewer false positive and false negative results compared with biochemical screening.     

Age and gender related changes in hematological parameters of thoroughbred foals

Abstract

Hematological and biochemical profiles commonly are required in equine medicine. We studied hematological parameters including red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (PCV), differential leukocyte counts, mean cell volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in thoroughbred foals at different ages and for both sexes. Sixty healthy thoroughbred foals, 1 day, 3 days and 1 year old were used. Each age group consisted of 10 male and 10 female animals. We found significant differences related to age in RBC values of females, PCV, MCV values of males, WBC, neutrophil percentages, lymphocyte percentages, monocyte percentages of females, and eosinophil percentages and basophil percentages. Significant differences related to gender were found only with regard to PCV at 1 year and WBC at 1 day. The hematological parameters of thoroughbred foals up to one year old may be useful for evaluating and monitoring the health of these animals.     

Hematological,biochemical, respiratory,cardiovascular and electroneurophysiological parameters in African green monkeys (Cercopithecus aethiops sabaeus). Its use in non‐clinical toxicological studies

Background The purpose of this study is to better characterize the hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in young adult Cercopithecus aethiops sabaeus of both sexes. The rhesus and cynomolgus monkeys are widely used as experimental primate models. However, only few articles have been published testing toxicological effects of pharmaceuticals on African green monkey. Methods The present study was carried out with the recompilation of all parameters recorded before the first drug administration in five sub‐chronic or chronic toxicological studies performed on 66 Cercopithecus aethiops sabaeus, born in Cuba. Results This study provides hematological, biochemical, respiratory, cardiovascular and electroneurophysiological data for both choosing animals to be included into experiments and monitoring these parameters during the study. Conclusions We conclude that this study provides valuable integrated data for determining the health status, including electroneurophysiological parameters, data not previously reported for this species, of the African green monkey.     

Effects of Hydration Level and Heat Stress on Thermoregulatory Responses,Hematological and Blood Rheological Properties in Growing Pigs

Heat stress is one of the major limiting factors of production efficiency in the swine industry. The aims of the present study were 1) to observe if hemorheological and hematological parameters could be associated to physiological acclimation during the first days of heat stress exposure and 2) to determine if water restriction could modulate the effect of thermal heat stress on physiological, hematological and hemorheological parameters. Twelve Large White male pigs were divided into an ad libitum and a water restricted group. All pigs were submitted to one week at 24°C (D-7 to D-1). Then, at D0, temperature was progressively increased until 32°C and maintained during one week (D1 to D7). We performed daily measurements of water and feed intake. Physiological (i.e., skin temperature, rectal temperature, respiratory rate), hematological and hemorheological parameters were measured on D-6, D-5, D0, D1, D2 and D7. Water restriction had no effect on physiological, hematological and hemorheological parameters. The first days of heat stress caused an increase in the three physiological parameters followed by a reduction of these parameters suggesting a successful acclimation of pigs to heat stress. We showed an increase in hematocrit, red blood cell aggregation and red blood cell aggregation strength during heat stress. Further, we observed an important release of reticulocytes, an increase of red blood cell deformability and a reduction of feed intake and blood viscosity under heat stress. This study suggests that physiological acute adaptation to heat stress is accompanied by large hematological and hemorheological changes.     

Changes in hematological and serum biochemical parameters in common marmosets (Callithrix jacchus) after inoculation with dengue virus

Background Marmosets are susceptible to dengue virus (DENV) infection. However, blood parameter data and clinical signs of DENV-infected marmosets are limited. Methods Blood hematological and serum biochemical values were obtained from twelve DENV-inoculated and four mock-infected marmosets. Additionally, body temperature and activity level were determined. Results Five DENV-inoculated marmosets demonstrated thrombocytopenia, nine demonstrated leucopenia, and five demonstrated an increase in the levels of AST, ALT, LDH, and BUN. Additionally, seven DENV-inoculated marmosets demonstrated clinical signs including fever and decreases in activity. None of the four mock-inoculated marmosets demonstrated changes in either hematological or biochemical parameters. Conclusions Marmosets inoculated with DENV exhibited clinical signs and changes in hematological and biochemical parameters. The results suggest that blood parameter data and clinical signs could potentially be useful markers for understanding the progress of DENV infection in studies using marmosets.     

Correlative clinical biochemistry and hematological profiles of laboratory-bred Bolivian squirrel monkeys (Saimiri sciureus)

Twenty-five clinical and biochemical parameters were determined on 17 normal, male, laboratory-bred Bolivian squirrel monkeys (Saimiri sciureus). Parallel hematologic parameters were conducted. The correlation of biochemical and hematological observations and the distribution characteristics, range, and standard error of the mean were determined. The purpose of the present communication is to establish baseline biochemical values for laboratory-bred squirrel monkeys and to present a correlative comparison between selected cellular elements and major blood electrolytes.     

血浆循环DNA水平与血常规指标的关联分析

目的:通过观察血浆循环DNA(cfDNA)浓度与血常规指标间的关系,探索cfDNA的来源。方法:用实时荧光定量PCR测定选定人群的血浆cfDNA浓度,用ADVIA120血细胞分析仪测定血常规指标,用双变量关联分析和逻辑回归分析血浆cfDNA浓度与性别、年龄及血常规指标间的相关性。结果:男性的血浆cfDNA浓度(23.23±3.186)略高于女性(18.55±2.037),但差异无显著性(P=0.2183);在各年龄段中,61~70岁年龄段的cfDNA浓度(23.20±4.274)最高,其次为70岁以上年龄段(20.92±2.089),51~60岁年龄段最低(15.81±1.747),但三者之间的差异没有显著性(P=0.3276);双变量关联分析表明血浆cfDNA浓度与过氧化物酶活性指数、血红蛋白及红细胞比积呈正相关(P<0.05),与性别、年龄及其他血常规指标无关;逻辑回归分析提示血浆cfDNA浓度升高只与平均血小板浓度和过氧化物酶活性指数呈正相关(P<0.05)。结论:血浆cfDNA增加可能与平均血小板浓度升高及过氧化物酶含量或活性有关,血小板前体细胞(即巨核细胞)可能是cfDNA的一个重要来源。     

Cholinesterase inhibition and its association with hematological,biochemical and oxidative stress markers in chronic pesticide exposed agriculture workers

The present study investigated the pesticide induced adverse health effects, hematological and biochemical alterations among agriculture workers. A cross sectional study of 51 agriculture workers and 54 unexposed subjects was carried out to evaluate hematological and biochemical alterations in blood. Pesticide exposed individuals were reported adverse clinical outcomes, including tingling, muscle pain, headache, skin disease, etc. A significant alterations in the level of hematological parameters, liver and renal dysfunctions markers and lipid profile suggested hematological, hepatic and renal dysfunctions. A significant decrease in the activity of acetylcholinesterase, reduced glutathione, superoxide dismutase, catalase and increased level of lipid peroxidation was also observed in these agriculture workers. Correlation coefficient analysis showed a positive correlation of chronic exposure with most of the hematological and biochemical parameters. The results demonstrate that the chronic exposure of pesticides cause reduction in the acetylcholinesterase activity and enhanced the risk of adverse clinical outcomes in agriculture workers.     

Hemato-biochemical responses of Van fish (Alburnus tarichi Guldenstadt, 1814) during sublethal exposure to cypermethrin

Cypermethrin is a highly active type II pyrethroid that is widely used to control harmful insects. The present study was conducted to investigate the possible effects of the sublethal concentration of cypermethrin (0.05 μL/L) on the biometric, hematological, and biochemical parameters of Van fish (Alburnus tarichi Guldenstadt 1814) for 24, 48, 72, and 96 h. Behavioral and physiological changes were observed in the fish treated with cypermethrin. Hematological parameters revealed a significant decrease in the erythrocyte (RBC), hemoglobin (Hb), and hematocrit (Hct) values (p ≤ .05). Serum enzymes, aspartate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), and lactate dehydrogenase (LDH, EC 1.1.1.27) activities and serum cortisol level were significantly increased (p ≤ .05). The results showed that changes in the biometric, biochemical, and hematological values of the Van fish exposed to cypermethrin can be used as biomarkers for monitoring toxicity.     

Shorea roxburghii Leaf Extract Ameliorates Hyperglycemia Induced Abnormalities in High Fat/Fructose and Streptozotocin Induced Diabetic Rats

This study investigated the hypoglycemic effect of the methanol extract of Shorea roxburghii leaves (SRL) in high fat diet/high fructose solution (HFDHF) and streptozotocin (STZ) induced type 2 diabetes mellitus (T2DM) in rats as well as evaluating its ameliorative potentials in altered biochemical and hematological parameters in the treated rats. T2DM was induced in Sprague Dawley (SD) rats by feeding with HFDHF for 4 weeks and administering STZ (35 mg/kg, i. p.). Diabetic rats were given SRL extract at doses of 100 and 400 mg/kg for 30 days. The food and water intake were monitored on a daily basis, while the fasting blood glucose (FBG) levels and body weight were measured weekly. Biochemical and hematological parameters as well as histopathological studies of the pancreas were also evaluated. SRL significantly decreased FBG and improved the body weight, food and water intake of treated diabetic rats. Furthermore, biochemical and hematological parameters including liver and kidney function enzymes, lipid profiles, white blood and red blood cells parameters were markedly ameliorated by SRL. Histopathological analyses of the pancreas indicated reconstitution of β‐cells architecture in SRL treated rats. The results of this study suggest that SRL has antidiabetic potential and can be considered for the treatment of T2DM.     

Biochemical and hematological parameters and histological alterations in fish Cyprinus carpio L. as biomarkers for water pollution with chlorpyrifos

The possible effects of the chlorpyrifos (CPF) on the fish Cyprinus carpio L. exposed to sub-lethal concentrations (52, 79, and 158 µg/L) of CPF for 6 weeks were evaluated, and an analysis was made of their hematological parameters, biochemical parameters, and histology of various organs such as liver, gill, and muscle. At three concentrations, packed cell volume (PCV), mean corpuscular hemoglobin, Red blood cells (RBC) Hb, T3, T4, and total protein were significantly decreased in fish treated with CPF, whereas the parameters White blood cells (WBC), creatinine, Glutamic Oxaloacetic Transaminase (GOT) and Glutamic Pyruvic Transaminase (GPT) presented a significant increase at the two higher concentrations. However, the sub-chronic exposure to CPF resulted in histological lesions and caused clear damage to liver, muscles, and gill tissues of the fish Cyprinus carpio. Thus, we may conclude that the altered biochemical and hematological parameters can be used as efficient biomarkers in monitoring the toxicity of CPF in aquatic organisms. At the same time, histopathology proves as a reliable and easy tool for toxicological studies.     

Multiparametric Analysis of Cell-Free DNA in Melanoma Patients

Cell-free DNA in blood (cfDNA) represents a promising biomarker for cancer diagnosis. Total cfDNA concentration showed a scarce discriminatory power between patients and controls. A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications.The aim of the present study was to identify a sequential multi-marker panel in cfDNA able to increase the predictive capability in the diagnosis of cutaneous melanoma in comparison with each single marker alone. To this purpose, we tested total cfDNA concentration, cfDNA integrity, BRAF^V600E mutation and RASSF1A promoter methylation associated to cfDNA in a series of 76 melanoma patients and 63 healthy controls. The chosen biomarkers were assayed in cfDNA samples by qPCR. Comparison of biomarkers distribution in cases and controls was performed by a logistic regression model in both univariate and multivariate analysis. The predictive capability of each logistic model was investigated by means of the area under the ROC curve (AUC). To aid the reader to interpret the value of the AUC, values between 0.6 and 0.7, between 0.71 and 0.8 and greater than 0.8 were considered as indicating a weak predictive, satisfactory and good predictive capacity, respectively. The AUC value for each biomarker (univariate logistic model) was weak/satisfactory ranging between 0.64 (BRAF^V600E) to 0.85 (total cfDNA). A good overall predictive capability for the final logistic model was found with an AUC of 0.95. The highest predictive capability was given by total cfDNA (AUC:0.86) followed by integrity index 180/67 (AUC:0.90) and methylated RASSF1A (AUC:0.89).An approach based on the simultaneous determination of three biomarkers (total cfDNA, integrity index 180/67 and methylated RASSF1A) could improve the diagnostic performance in melanoma.     

封闭群五指山小型猪血液生理生化指标的测定

目的对2-12月龄封闭群五指山小型猪的部分血液生理和生化指标进行测定和分析。方法常规方法测定五指山小型猪血液的19项生理和12项生化指标,统计分析各指标间的性别差异,并与近交系五指山小型猪以及其他小型猪的同类指标进行对比分析。结果封闭群五指山小型猪绝大多数血液生理、生化指标性别间差异不显著,仅生理指标中的粒细胞分类计数和生化指标中的总胆固醇差异显著。同近交系五指山小型猪同类指标比较,生理指标中血红蛋白和血小板相关指标存在差异,生化指标中有6项差异显著。同其他种类小型猪同类指标差异较与近交系五指山小型猪间差异明显。结论封闭群五指山小型猪血液生理、生化指标稳定,许多生理生化指标接近人类,可能逐步替代犬、猴用于药物非临床安全评价和生物医学研究。     

Protective efficacy of ellagic acid on glycoproteins, hematological parameters, biochemical changes, and electrolytes in myocardial infarcted rats

The cardioprotective property of ellagic acid in rats has been reported previously. The present study reveals the protective role of ellagic acid in biochemical parameters including serum iron, plasma iron binding capacity, uric acid, glycoprotein, and electrolytes along with hematological parameters. Rats were subcutaneously injected with isoproterenol (ISO) (100 mg/kg) for 2 days to induce myocardial infarction. ISO-induced rats showed a significant increase in their levels of serum iron, serum uric acid, and blood glucose, and a significant decrease in their levels of plasma iron binding capacity, serum total protein, albumin/globulin ratio, and heart glycogen, when compared with normal control rats. The altered hematological parameters were also observed in ISO-induced rats when compared with normal control rats. Pretreatment with ellagic acid at doses of 7.5 and 15 mg/kg produced significant beneficial effect by returning all the above-mentioned biochemical and hematological parameters to near normal levels.     

Blood-based DNA methylation signatures in cancer: A systematic review

DNA methylation profiles are in dynamic equilibrium via the initiation of methylation, maintenance of methylation and demethylation, which control gene expression and chromosome stability. Changes in DNA methylation patterns play important roles in carcinogenesis and primarily manifests as hypomethylation of the entire genome and the hypermethylation of individual loci. These changes may be reflected in blood-based DNA, which provides a non-invasive means for cancer monitoring. Previous blood-based DNA detection objects primarily included circulating tumor DNA/cell-free DNA (ctDNA/cfDNA), circulating tumor cells (CTCs) and exosomes. Researchers gradually found that methylation changes in peripheral blood mononuclear cells (PBMCs) also reflected the presence of tumors. Blood-based DNA methylation is widely used in early diagnosis, prognosis prediction, dynamic monitoring after treatment and other fields of clinical research on cancer. The reversible methylation of genes also makes them important therapeutic targets. The present paper summarizes the changes in DNA methylation in cancer based on existing research and focuses on the characteristics of the detection objects of blood-based DNA, including ctDNA/cfDNA, CTCs, exosomes and PBMCs, and their application in clinical research.