Von Willebrand factor is an independent predictor of short-term mortality in acutely ill patients with cirrhosis.

BACKGROUND AND AIMS: Levels of von Willebrand factor (VWF) are elevated in patients with cirrhosis, and correlate well with disease severity. In patients with decompensated cirrhosis (DC), plasma VWF is associated with mortality. The value of VWF in predicting short-term mortality risk in patients with acute-on-chronic liver failure (ACLF) is, however, unclear. METHODS: We included patients with DC (n = 111) and ACLF (n = 105). We measured VWF levels and correlated these with other laboratory parameters and prediction models for mortality. Also, we assessed the predictive value of VWF in the prediction of 90- and 30-day mortality in patients with DC and ACLF, respectively, and compared this to the predictive value of clinically used prediction models. Finally, we determined the optimal cut-off value for VWF in patients with ACLF. RESULTS: Sixteen of 111 (14%) patients with DC and 35 of 105 (33%) with ACLF died within 90 and 30 days, respectively. VWF was associated with mortality and correlated closely with other prediction models. In patients with ACLF, VWF levels had a discrimination for 30-day mortality comparable with these models and accurately identified ACLF patients with high 30-day mortality risk. CONCLUSIONS: Levels of VWF associate closely with risk of mortality in patients with DC and ACLF, and may have predictive utility as a laboratory marker of prognosis. Further research is warranted to assess the additional value of VWF in the prediction of mortality and associated complications in chronic liver failure syndromes.

In vivo generation of thrombin in patients with liver disease without apparent evidence of activation of the intrinsic or extrinsic pathway of coagulation.

BACKGROUND: Patients with liver diseases are in a hypercoagulable state, as evidenced by enhanced in vitro thrombin generating capacity and elevated plasma levels of markers of in vivo thrombin generation. However, it is unknown by which mechanism in vivo activation of coagulation occurs. OBJECTIVES: We aimed to clarify the mechanisms underlying enhanced in vivo thrombin generation to provide a rationale for targeted anticoagulant therapy. PATIENTS/METHODS: Overall, 191 patients diagnosed with stable or acutely decompensated cirrhosis, acute liver failure or injury, acute-on-chronic liver failure, or sepsis without underlying chronic liver disease were recruited from King's College Hospital, London, from 2017 to 2021 and compared with reference values of 41 healthy controls. We measured levels of markers of in vivo activation of coagulation and activation of the intrinsic and extrinsic pathways, their respective zymogens, and natural anticoagulants. RESULTS: Thrombin-antithrombin complexes, prothrombin fragment 1+2 (F1+2), and D-dimer levels were increased in acute and chronic liver disease, proportional to disease severity. Plasma levels of free activated factor XII (FXIIa), C1-esterase-inhibitor (C1inh)-FXIIa, C1inh-factor XI, C1inh-plasma kallikrein, factor-VIIa-antithrombin-complexes, and activated FVII were reduced in acute and chronic liver disease, even after adjusting for zymogen levels, which were also substantially reduced. Natural anticoagulants antithrombin and protein C were profoundly reduced in liver patients. CONCLUSIONS: This study provides evidence of enhanced thrombin generation in liver disease without detectable activation of the intrinsic or extrinsic pathway. We propose that defective anticoagulant mechanisms highly amplify the low-grade activation of coagulation by either pathway.

Microfluidic system for near-patient extraction and detection of miR-122 microRNA biomarker for drug-induced liver injury diagnostics.

Drug-induced liver injury (DILI) results in over 100 000 hospital attendances per year in the UK alone and is a leading cause for the post-marketing withdrawal of new drugs, leading to significant financial losses. MicroRNA-122 (miR-122) has been proposed as a sensitive DILI marker although no commercial applications are available yet. Extracellular blood microRNAs (miRNAs) are promising clinical biomarkers but their measurement at point of care remains time-consuming, technically challenging, and expensive. For circulating miRNA to have an impact on healthcare, a key challenge to overcome is the development of rapid and reliable low-cost sample preparation. There is an acknowledged issue with miRNA stability in the presence of hemolysis and platelet activation, and no solution has been demonstrated for fast and robust extraction at the site of blood draw. Here, we report a novel microfluidic platform for the extraction of circulating miR-122 from blood enabled by a vertical approach and gravity-based bubble mixing. The performance of this disposable cartridge was verified by standard quantitative polymerase chain reaction analysis on extracted miR-122. The cartridge performed equivalently or better than standard bench extraction kits. The extraction cartridge was combined with electrochemical impedance spectroscopy to detect miR-122 as an initial proof-of-concept toward an application in point-of-care detection. This platform enables the standardization of sample preparation and the detection of miRNAs at the point of blood draw and in resource limited settings and could aid the introduction of miRNA-based assays into routine clinical practice.

Fibrin clot quality in acutely ill cirrhosis patients: Relation with outcome and improvement with coagulation factor concentrates.

BACKGROUND & AIMS: Patients with liver disease may acquire substantial changes in their hemostatic system, which are most pronounced in patients who are critically ill. Changes in the quality of the fibrin clot in critically ill patients have not been studied in detail. Here we assessed markers of fibrin clot quality and effects of coagulation factor concentrates in patients with acutely decompensated (AD) cirrhosis and acute on chronic liver failure (ACLF). METHODS: We measured plasma levels of fibrinogen, factor XIII, prothrombin and performed thrombin generation assays in 52 AD patients, 58 ACLF patients and 40 controls. In addition, we examined the effects of coagulation factor concentrates on functional assays of fibrin quality. RESULTS: We found increased thrombin generating capacity in both AD and ACLF in comparison with healthy controls. Plasma levels of prothrombin, fibrinogen, and factor XIII were lower in patients compared to controls, appeared lower in ACLF compared to AD patients, and were related to clinical outcomes. Fibrinogen concentrate, but not factor XIII or prothrombin complex concentrate, improved clot quality in vitro. Prothrombin complex concentrate increased the resistance of the clot to break down. CONCLUSIONS: We have demonstrated elevated thrombin generation but decreased plasma levels of prothrombin, fibrinogen and FXIII in acutely ill patients with cirrhosis. In addition, we showed that fibrinogen concentrate and PCCs, but not factor XIII concentrate, improve clot properties in patient plasma. Whether there is true clinical benefit from coagulation factor concentrates in prevention or treatment of bleeding requires further study. LAY SUMMARY: Patients with liver diseases are at risk of bleeding, but mechanisms involved in this bleeding risk are incompletely understood. We studied components that determine the stability of the blood clot and found that concentrations of certain proteins involved in clot stability are present in low levels in acutely ill patients with liver disease. We furthermore demonstrated that some clinically available drugs improve the stability of blood clots from these patients in a test tube.

Colorectal Cancer CTCs Detection Using Two FCM Protocol Approaches.

BACKGROUND: The presence and quantification of circulating tumor cells (CTCs) could minimize the mortality among cancer patients by tailored, personalized, and targeted therapy and could also help in the field of investigation about new therapeutic targets. Identification of CTCs has been performed using molecular techniques and more advanced automated techniques such as CellSearch® and Amnis®. Our aim was to test the possibility of identifying CTCs in colorectal cancer patients using a lower cost and less complex flow cytometry-based method. METHODS: Besides the CellSearch® system for CTCs enumeration and Amnis® Imaging Flow Cytometers, which are both commercially available, we tested and developed two FCM protocol approaches after immunomagnetic enrichment for CTCs enumeration in the blood of colorectal cancer patients. The CTCs numbers were assessed at baseline before anesthesia and the curative surgery and day one after the curative surgery. Blood from healthy donors was used as negative control. The research was performed by the Cyflow® Space cytometer (Partec, Münster, Germany). RESULTS: In the patient group, the enumeration using the direct protocol with a threshold 3 cells/mL, showed a mean of CTCs before surgery of 32, while after surgery it was 20, with sensitivity (SN) of 49.2% and specificity (SP) of 58.3%. On the other hand, using the intracellular protocol with a threshold of 1 cell/mL in patients' group, the mean of CTCs before surgery was 65 and after surgery it was 60, with a sensitivity (SN) of 62.7% and specificity (SP) of 70%. In the intracellular protocol the most significant correlation identified was for the expression of CKs with adenocarcinomas (r = 0.256, p = 0.044), with the existence of lymph node infiltration (r = 0.380, p = 0.008), and with the stage of the disease (r = 0.391, p = 0.003). For the direct protocol, considerable correlation was found with the samples of the right colon (r = 0.369, p = 0.002). CONCLUSIONS: Our findings demonstrate that we established two FCM low cost protocol approaches to detect and enumerate CTCs in colorectal cancer patients. In both FCM protocols (direct and intracellular) we observed a statistically significant increase (p ˂ 0.05) of CTCs number in the patient group. No statistical significance (p ˂ 0.05) of CTCs before and after surgery in patient group in both protocols was observed.

Evaluation of serum levels of IL-6, TNF-alpha, IL-10, IL-2 and IL-4 in patients with chronic hepatitis

BACKGROUND: Changes in various cytokine activities have been reported during both HBV and HCV infections, while an imbalance of pro-inflammatory and anti-inflammatory cytokine production influences their immunopathogenesis. The aims of the present study are (a) to measure serum levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), interleukin-2 (IL-2) and interleukin-4 (IL-4) in a sample of patients affected either by chronic HBV infection or by chronic HCV infection and in healthy controls (b) to correlate serum levels of IL-6, TNF-α, IL-10, IL-2 and IL-4 with biochemical markers of liver disease and (c) to evaluate differences of the aforementioned cytokines between HBV and HCV patients, as well as between patients and healthy controls. METHODS: The study population consisted of 50 patients with chronic hepatitis B, 40 patients with chronic hepatitis C and 30 healthy controls aged between 28 and 75 years. Biochemical markers of liver disease were evaluated by routine methods approved by IFCC. Serum concentrations of IL-6, TNF-α, IL-10, IL-2 and IL-4 were determined with the Human Cytokine/Chemokine Panel I Merck Millipore. RESULTS: HBV patients showed statistically significant difference in TNF-α and IL-2 levels, versus healthy controls. HCV patients showed statistically significant difference in TNF-α, IL-10 and IL-2 levels versus healthy controls. IL10 and IL-2 levels were significantly different between HBV and HCV patients. CONCLUSIONS: This study evaluated the serum cytokine levels (IL-6, TNF-α, IL-10, IL-2 and IL-4) of chronic hepatitis B or C patients, as well as the differences in such levels between patients and healthy controls. Correlations of cytokine levels with biochemical markers of liver disease were also observed, reflecting the degree of activity of the inflammatory process in the liver

ANTECEDENTES: Tanto en las infecciones por VHB como por VHC se han notificado cambios en las diversas actividades de las citocinas, y es conocido que su inmunopatogénesis está influida por un desequilibrio en la producción de citocinas pro y antiinflamatorias. Los objetivos del presente estudio son: a) medir las concentraciones séricas de interleucina 6 (IL-6), factor de necrosis tumoral α (TNF-α), interleucina 10 (IL-10), interleucina 2 (IL-2) e interleucina 4 (IL-4) en una muestra de pacientes afectados, ya sea por una infección crónica por VHB o por VHC y en controles sanos; b) correlacionar las concentraciones séricas de IL-6, TNF-α, IL-10, IL-2 e IL-4 con los marcadores bioquímicos de enfermedad hepática, y c) evaluar las diferencias de las citocinas antes mencionadas entre los pacientes con VHB y con HCV, así como entre los pacientes y los controles sanos. MÉTODOS: La población del estudio consistió en 50 pacientes con hepatitis B crónica, 40 pacientes con hepatitis C crónica y 30 controles sanos de edades comprendidas entre 28 y 75 años. Se evaluaron los marcadores bioquímicos de la enfermedad hepática mediante métodos rutinarios aprobados por la IFCC. Las concentraciones séricas de IL-6, TNF-α, IL-10, IL-2 e IL-4 se determinaron mediante el Panel I de citocinas/quimiocinas humanas de Merck Millipore. RESULTADOS: Los pacientes con VHB mostraron diferencias estadísticamente significativas en las concentraciones de TNF-α e IL-2, comparadas con las de controles sanos. Los pacientes con HCV mostraron diferencias estadísticamente significativas en TNF-α, IL-10 e IL-2 respecto a las concentraciones de los controles sanos. Las concentraciones de IL-10 e IL-2 fueron significativamente diferentes entre los pacientes con VHB y con HCV. CONCLUSIONES: Este estudio evaluó las concentraciones séricas de citocinas (IL-6, TNF-α, IL-10, IL-2 e IL-4) de pacientes con hepatitis crónica B o C, así como las diferencias en dichas concentraciones entre pacientes y controles sanos. También se observaron correlaciones de las concentraciones de citocinas con marcadores bioquímicos de la enfermedad hepática, lo que refleja el grado de actividad del proceso inflamatorio en el hígado

Evaluation of the lipid profile in type 2 diabetes mellitus patients in Greece.

BACKGROUND: Diabetes mellitus (DM) is one of the most serious global health problems. In Greece, DM constitutes a public health problem and is highly associated with decreasing levels of physical activity, increasing obesity rates, population ageing, and unhealthy lifestyle and dietary behaviors. MATERIALS: In this study we evaluated the sera from 800 type 2 diabetic patients recruited during a three year period of time and 200 age matched controls without any clinical history of diabetes. For each subject we measured levels of fasting glucose (GLU), total cholesterol (TCHOL), triglycerides (TRG), high density lipoproteins (HDL-C), and glycosylated hemoglobin (GHbA1c) and calculated levels of low density lipoproteins (LDL-C). The aims of our study were to find characteristics of lipid parameters in the population under study, to find gender differences in the parameters, to evaluate correlations between pairs of lipid parameters, and to compare the lipid parameters between patients and healthy controls focusing on patient gender. For this purpose we analyzed the data using descriptive statistics, x-square test, logistic regression and ROC curve analysis. RESULTS: According to our results, 70.0% of diabetic patients presented at least one lipid abnormality. Elevated LDL-C, elevated TCHOL, elevated TRG, and reduced HDL-C levels were noted in 28.37%, 36.37%, 39.01%, and 30.12% of the patients, respectively. The combination of elevated TRG and reduced HDL-C was the most preva- lent of the combined lipid abnormalities. Moreover, there are statistically significant differences in the levels of HDL-C, TCHOL, TRG, and GLU between men and women. In contrast, no differences were observed in levels of GHbA1c. CONCLUSIONS: We identified an important linear relationship between LDL-C and TCHOL (LDL-C = -28.69 + TCHOL * 0.75, adjusted R2 = 76.96%. Finally, we calculated optimal thresholds for GLU and GHb1Ac levels using two methodologies: overall accuracy maximization or sensitivity-specificity minimization for the identification of patient from healthy controls. Differences in the optimal thresholds between men and women were not observed.

Detection of antinuclear antibodies (ANA), antibodies to double stranded DNA (anti-dsDNA) and antibodies to extractable nuclear antigens (anti-ENA) in Greek patients.

BACKGROUND: The diagnosis of autoimmune diseases depends on clinical history, physical examination, and laboratory detection of specific autoantibodies directed against nuclear or cytoplasmic antigens. The aim of this study was to investigate the types and prevalence of serum ANA, anti-dsDNA, and anti-ENA antibodies in a population examined at the Immunology Laboratory of the Naval Hospital of Athens and their correlation with patient age and gender. METHODS: We evaluated the sera of 3000 patients, both male and female, aged between 18 and 75 years old, born in different parts of Greece. All requests for ANA detection were performed by enzyme linked immunoassay (ELI SA). All ELISA borderline, weak positive and pbsitive results were run on the indirect immunofluorescence assay (IFA) on Hep-2 cells. Anti-dsDNA antibodies were detected by IFA on Crithidia luciliae substrate slides. Antibodies to Sm, RNP, SS-A(Ro), SS-B(La), Jo-1, and Scl-70 were determined by an immunodot qualitative test. RESULTS: 206 patients were positive for ANA, representing a prevalence of 6.87%. The positive samples demonstrated the expected variety of titers and reactivity patterns. 9 samples (4.37%) presented anti-dsDNA positive result and 44 (21.36%) presented reactivity to various ENA autoantibodies. All the examined autoantibodies presented a higher prevalence among women. CONCLUSIONS: In this study we estimated the prevalence of ANA, anti-dsDNA, and anti-ENA antibodies in samples of 3000 sera. We followed the strategy of performing immunofluorescence testing in order to confirm positive ELISA results, proposed by many scientists. We also evaluated autoantibody titers and fluorescence patterns, and we examined the correlation of autoantibody presence with patient age and gender.

Evaluation of carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule EpCAM (GA733-2), and carbohydrate antigen 19-9 (CA 19-9) levels in colorectal cancer patients and correlation with clinicopathological characteristics.

BACKGROUND: Colorectal cancer (CRC) is a major public health problem and one of the leading causes of death worldwide. The aim of our study was: a) to determine the CEA, CA 19-9, EGFR, and EpCAM (GA733-2) levels both in healthy volunteers and in colorectal cancer patients, b) to evaluate the ELISA method for EGFR and EpCAM (GA733-2) measurement, and c) to correlate the tumor marker levels with clinicopathological findings in the CRC patients group. METHODS: Our study was conducted on 50 blood samples obtained from CRC patients and 40 blood samples from healthy individuals. CEA and CA 19-9 measurements were performed using electrochemiluminescence immune-assay technology, while EGFR and EpCAM (GA733-2) measurements were performed by an in-house enzyme immunoassay. RESULTS: CEA, CA 19-9, and EpCAM (GA733-2) levels were higher in the CRC patients group than in the control group. EGFR levels were lower in the patients group than in the control group. The mean levels of CA 19-9 and EpCAM (GA733-2) vary at different colon cancer stages. CEA, CA19-9, and EpCAM (GA733-2) vary according to performance status. CONCLUSIONS: CEA, CA 19-9, and EpCAM (GA733-2) showed similar specificity (80%, 80% and 84%, respectively). EGFR showed the lowest sensitivity and specificity. CA 19-9 was the marker with the highest sensitivity. The need for convenient tumour marker tests with high sensitivity is of great importance for early diagnosis and monitoring of CRC.

Genotype distribution in chronic hepatitis C patients in Greece.

BACKGROUND: Hepatitis C is a major public health problem. HCV infection contributes to progressive liver disease, cirrhosis, and hepatocellular carcinoma. HCV has high genetic heterogeneity and is classified into various genotypes and subtypes. Regional differences exist in their distribution. The aim of this study was to investigate the relative frequency of HCV genotypes in Greek patients with chronic infection. METHODS: We evaluated 82 patients with chronic HCV infection, both males and females, belonging to different risk groups. We performed viral load measurement and HCV genotyping in all specimens. RESULTS: HCV genotype 3 was the most prevalent (41.5%) followed by genotype 1 (34.1%), 2 (12.2%), 4 (10.9%), and 5 (1.2%). Genotype 6 was not detected in any patient. Most prevalent subtypes were 3a (32.9%), 1b (26.8%), and 2a (6.1%). Fourteen subjects revealed mixed infections within types. There were no cases with mixed infections across types. CONCLUSIONS: Our data indicate that genotypes 3a and 1b are the most prevalent in Greek patients. Genotype 3a is predominant in younger patients and also in male patients. Moreover, HCV genotype distribution is in continuous temporal change in Greece.