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.

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.