Influence of pre-analytical and analytical factors on osteoprotegerin measurements

IntroductionOsteoprotegerin (OPG), an osteoclastogenesis inhibitor implicated in bone remodelling, has emerged as a potential biomarker for cardiovascular disease. In order to implement OPG determination in the clinical laboratory, it is crucial to identify the most appropriate specimen type, preparation and measurement conditions. The present study focuses on identifying the pre-analytical variables that may influence OPG measurements.MethodsSerum and plasma (in EDTA, heparin and citrate) were collected from 45 healthy volunteers (men (n = 21, 46.7%), women (n = 24, 53.3%)). OPG was analysed by ELISA. The influence of the centrifugation speed, the number of freeze–thaw cycles, delay in sample processing, thermo-stability and endogenous interfering agents (haemolysis, triglycerides, bilirubin, cholesterol and RANKL) were studied.ResultsOPG concentrations were significantly lower (p < 0.0001) in serum (1015 ± 357 pg/mL) than in all plasma samples (1314 ± 448 pg/mL in EDTA, 1209 ± 417 pg/mL in heparin and 1260 ± 498 pg/mL in citrate).Increasing centrifugation speed (200 g to 3000 g) did not change serum OPG concentration (p = 0.88). However, OPG concentration significantly increased when centrifuged serum samples were stored at 48 h at room temperature (p < 0.0001). Repeated freeze–thaw cycles did not modify OPG levels until 4 cycles (p < 0.0001). Increasing time before processing the samples (2 h and 6 h) raised OPG concentrations both at room temperature (p < 0.0001) or 4 °C (p < 0.001).Positive concentration-dependent interference of triglycerides was found in the analysed pooled samples; however, OPG concentrations were falsely diminished with haemoglobin interference. Bilirubin, cholesterol and RANKL did not interfere with OPG measurements.     

Correlation between concentrations of 8-oxo-7,8-dihydro-2′-deoxyguanosine in urine,plasma and saliva measured by on-line solid-phase extraction LC-MS/MS

Background8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is the most frequently measured biomarker of oxidative stress. Chromatographic-based methods for 8-oxodGuo in urine are well established; however, the 8-oxodGuo measurement in plasma and saliva has been problematic.MethodsWe firstly and successfully applied an on-line solid-phase extraction (SPE) LC-MS/MS following manual SPE pretreatment to quantify the 8-oxodGuo both in plasma and saliva. Urine, plasma and saliva specimens were simultaneously collected from 50 healthy adults and measured for 8-oxodGuo.ResultsMean baseline levels of 8-oxodGuo in plasma and saliva were 21.7 ± 9.2 and 5.1 ± 2.6 pg/ml, respectively, being far lower than that in urine (6.2 ± 4.8 ng/ml). The 8-oxodGuo levels obtained in this study for plasma and saliva were, however, up to several hundred times lower than those reported by commercial ELISA kit in the literature. Furthermore, the 8-oxodGuo levels in plasma and saliva were significantly correlated with the 8-oxodGuo levels in urine (Spearman correlation coefficients, r = 0.33, P = 0.02 for plasma and r = 0.56, P = 0.0015 for saliva). 8-OxodGuo in plasma was also correlated with the 8-oxodGuo in saliva (r = 0.52, P = 0.0041).ConclusionsSignificantly correlations were observed between plasma, saliva and urine, giving the possibility of using other body fluids in addition to urine for assessing whole body oxidative stress.     

Plasma osteopontin concentration correlates with the severity of hepatic fibrosis and inflammation in HCV-infected subjects

BackgroundThe association between OPN level and the histological severity of hepatic fibrosis and inflammation in hepatitis C virus (HCV) induced liver fibrosis remains unknown.Methods120 chronic HCV-infected subjects and 75 controls were enrolled in this study. Assessment of liver histology was performed based on liver biopsy. Plasma OPN levels were determined.ResultsSignificant differences were noted in the mean plasma OPN levels between subjects with extensive fibrosis and those with mild fibrosis (4.29 ± 1.01 ng/ml vs. 2.15 ± 0.63 ng/ml, respectively; p < 0.001). Similarly, the subjects with higher histological activity index (HAI) score had elevated OPN levels than those with mild HAI score (4.41 ± 1.11 ng/ml vs. 2.25 ± 0.94 ng/ml, respectively; p < 0.001). The correlation between the plasma OPN levels and the severity of liver fibrosis degree and HAI score were noted (r = 0.945, and r = 0.788, respectively both p < 0.001). Logistic regression analysis showed that serum OPN was an independent risk factor contributing to extensive liver fibrosis and inflammation (p = 0.0018 and p < 0.001, respectively) in patients with HCV subjects.ConclusionThe plasma OPN level is correlated with the severity of liver fibrosis and inflammation, suggesting OPN could be used as a biomarker to evaluate the severity of liver damages in HCV subjects.     

Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection

ObjectivesTo assess the effect of different anticoagulants (EDTA, citrate and heparin) on the isolation procedure of human neutrophils and in the subsequent alterations of calcium levels and respiratory burst induced by phorbol myristate acetate (PMA).Design and methodsIsolation of human neutrophils from whole blood was performed by the gradient density centrifugation method. PMA-induced neutrophil burst was measured by chemiluminescence. Intracellular calcium ([Ca²⁺]_i) was measured using Fluo-3 AM, a calcium-sensitive dye.ResultsEDTA provided the highest number of isolated neutrophils/mL of blood (1.7 × 10⁶ ± 1.5 × 10⁵) when compared with citrate (0.46 × 10⁶ ± 0.95 × 10⁵) and heparin (0.66 × 10⁶ ± 0.15 × 10⁵). EDTA originated less degree of PMA-induced activation (370 ± 30%) relatively to citrate (830 ± 98%) and heparin (827 ± 77%). [Ca²⁺]_i was lower with EDTA (122 ± 11 nM) when compared with citrate and heparin (150 ± 13 and 230 ± 30 nM).ConclusionThe anticoagulant used during blood collection interfered differently with the yield of isolated neutrophils as well as on their calcium levels and reactivity to PMA.     

The role of hypercytokinemia in the pathophysiology of tumor lysis syndrome (TLS) and the treatment with continuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter (PMMA-CHDF)

ObjectiveTo examine the role of hypercytokinemia in the pathophysiology of tumor lysis syndrome (TLS) and the efficacy of continuous hemodiafiltration in the treatment of TLS.Design and settingRetrospective observational study in a general intensive care unit of a university hospital.PatientsFour patients with hematological disorder developing TLS after the treatment of anti-tumor chemotherapy.InterventionsContinuous hemodiafiltration using a polymethylmethacrylate membrane hemofilter (PMMA-CHDF) was performed at the onset of TLS. Blood samples were collected daily after ICU admission, and clinical parameters and blood levels of cytokines were evaluated.Measurements and resultsAll four patients underwent induction anti-tumor chemotherapy, during which they developed hyperuricemia, hyperkalemia, and acute renal failure. Two of them also developed multiple organ failure. Serum levels of tumor necrosis factor (TNF) -alpha, interleukin-6 (IL-6), and IL-10 prior to the initiation of PMMA-CHDF were 102 ± 85 pg/mL, 1097 ± 546 pg/mL, and 98 ± 83 pg/mL, respectively (mean ± SD). After three days of PMMA-CHDF treatment, corresponding blood levels were 37 ± 55 pg/mL, 326 ± 511 pg/mL, and 9 ± 8 pg/mL, respectively. Thus, all cytokine levels were significantly decreased by three days of PMMA-CHDF treatment (p < 0.05, paired t-test). Following three days of PMMA-CHDF treatment, blood urea nitrogen (BUN) and serum creatinine (Cre.) were significantly decreased (pre/post BUN 42.3 ± 15.4/16.5 ± 8.4 mg/dL, p < 0.05, pre/post Cre. 2.7 ± 1.2/1.2 ± 0.6 mg/dL, mean ± SD, p < 0.05). Furthermore, the clinical condition of each patient was improved after the treatment of PMMA-CHDF, and all of four patients were survived.ConclusionHypercytokinemia plays a pivotal role in the pathophysiology of TLS and PMMA-CHDF may be an effective therapeutic modality for TLS patients not only as renal replacement therapy but also as a cytokine modulator.     

Association between myeloperoxidase polymorphisms and its plasma levels with severity of coronary artery disease

ObjectivesMyeloperoxidase (MPO) polymorphism ?463 has been related to higher cardiovascular risk. This study was conducted to test whether the MPO promoter polymorphism ?463A/G and MPO plasma levels are associated with coronary artery disease (CAD) severity.Design and methodsPatients submitted to elective coronariography were enrolled, CAD severity was assessed and blood samples collected to identify the MPO polymorphism and its plasma levels.ResultsGenotypes were determined in 118 patients. Among these patients, 12 (10%) were homozygous for AA, 69 (58%) for GG and 37 (32%) were heterozygous. Mean MPO plasma levels were 8.6 ± 4.7 ng/mL for AA, 8.6 ± 7.0 ng/mL for AG and 9.4 ± 5.6 ng/mL for GG genotypes. The CAD severity was not associated with MPO genotypes (p = 0.43), however, patients with higher CAD score presented higher MPO levels (p = 0.02).ConclusionWe found no association between MPO polymorphism and CAD severity, although a relation was observed for MPO plasma levels and extension of CAD.     

Stability of plasma adrenocorticotrophic hormone (ACTH): Influence of hemolysis,rapid chilling,time, and the addition of a maleimide

ObjectivesThe aim of this study was to examine the effects of hemolysis, rapid chilling, time, and the addition of a maleimide on the stability of human plasma ACTH measurements.Design and methodsPartially hemolyzed EDTA blood (n = 10), initially at 37 °C, was centrifuged at 4 °C either immediately or after rapid chilling in ice/water. Plasma ACTH was then measured either immediately, or after 1 h at 22 °C with or without the addition of 2 mM N-phenyl maleimide (NPM).ResultsFor 0.2% hemolysis compared to no hemolysis, the mean (±SEM) loss with immediate centrifugation and immediate ACTH measurement was 11 ± 1%. This loss was significantly (p < 0.002) reduced to 6 ± 1% by an initial rapid chilling of the samples. For analysis after 1 h at 22 °C, the addition of NPM decreased the loss of ACTH from 15 ± 2% to 2 ± 2% (p < 0.002).ConclusionRapid chilling, prompt analysis, and addition of NPM can each reduce the interference of hemolysis in the measurement of plasma ACTH concentrations.     

Reduced serum vaspin concentrations in obese children following short-term intensive lifestyle modification

BackgroundRecently, visceral adipose tissue-derived serpin (vaspin) was identified as a potential insulin sensitizing adipokine, however, the factors determining the levels of circulating vaspin levels have not been fully understood. We investigated the association between adiposity, insulin resistance, lipid profiles and inflammatory markers including vaspin levels, and the effects of short-term intensive lifestyle modification on circulating vaspin levels in overweight or obese children.MethodsA total of 50 (25 boys, 25 girls) overweight or obese children aged 11 to 13 years (average age: 12.0 ± 0.9 y, BMI: 25.35 ± 86 kg/m²) who complied with inclusion criteria participated in our study. To determine the association between adiposity, insulin resistance, lipid profiles and inflammatory markers including vaspin levels, cross-sectional analyses were performed. Thereafter, subjects underwent a tightly controlled seven-day intensive lifestyle modification including physical activity, dietary modification, and behavioral modification education in residence of a local university dormitory.ResultsThere was a negative correlation between vaspin concentration and fasting insulin (r = ?.325, p < 0.05) and homeostasis model assessment of insulin resistance (HOMA-IR) (r = ?.331, p < 0.05) when percent body fat was controlled. Multivariate linear regression analysis found serum vaspin level to be an independent predictor of insulin and HOMA-IR. Short-term intensive lifestyle modification significantly decreased vaspin levels by 39.28% (pre: .84 ± 1.0, post: .51 ± 1.0 ng/ml, p < 0.001) while adiponectin levels increased by 11.2% (pre: 6.50 ± 2.89, post: 7.28 ± 2.98 ng/ml, p < 0.01). In addition, short-term lifestyle modification significantly improved HOMA-IR (pre: 3.58 ± 1.93, post 1.30 ± 1.9, p < 0.001) and lipid profiles.ConclusionsSerum vaspin level is one of the predictors for insulin resistance and was significantly reduced following short-term lifestyle modification.     

The plasma levels of relaxin-2 and relaxin-3 in patients with diabetes

ObjectivesRelaxin-2 has been found to alleviate fibrosis in experimental diabetic cardiomyopathy. In addition, the levels of serum relaxin-3 were increased and correlated with all the component traits of metabolic syndrome. We investigated the levels of plasma relaxin-2 or relaxin-3 and their relationship to component traits in patients with diabetes.Design and methodsWe studied 33 newly diagnosed type 2 diabetes patients and 38 age-matched healthy subjects. Blood samples were taken at study entry, and relaxin-3, relaxin-2, fasting blood glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides, serum insulin and hemoglobin A_1c (HbA_1c) levels were measured.ResultsRelaxin-2 levels were significantly lower in patients with diabetes than in controls: the median plasma relaxin-2 concentration was 34.68 pg/mL (range, < 29.00–50.81 pg/mL) in patients with diabetes and 45.80 pg/mL (range, < 37.42–54.46 pg/mL) in controls (p = 0.0150). However, no differences in relaxin-3 levels were observed between the diabetes group and controls (p = 0.6550). The plasma levels of relaxin-2 or relaxin-3 were not correlated with systolic blood pressure (BP), diastolic BP, total cholesterol, LDL-C, HDL-C, triglyceride, fasting blood glucose, fasting insulin and HbA_1c in patients with diabetes. Additionally, there was no correlation between the plasma concentrations of relaxin-2 and relaxin-3 in patients with diabetes (r_s = 0.225; p = 0.208).ConclusionsWe conclude that the plasma levels of relaxin-2 in diabetes patients were lower than in controls, however, there are no difference in plasma relaxin-3 concentrations between controls and patients with diabetes. Relaxin-2 or relaxin-3 levels are not related to component traits in patients with diabetes.     

Enhanced levels of soluble and membrane-bound CD137 levels in patients with acute coronary syndromes

BackgroundIncreasing evidence shows that costimulatory molecules of the tumor necrosis factor superfamily such as CD40/CD40 ligand and OX40/OX40 ligand have been implicated in atherosclerosis. We investigated whether the expression levels of the tumor necrosis factor superfamily members CD137 in serum and membrane-bound were related to acute coronary syndromes (ACS).MethodsThirty normal controls and 210 patients, including 70 with stable angina (SA), 80 with unstable angina (UA), and 60 with acute myocardial infarction (AMI), were enrolled in our study. The expression of CD137 in peripheral monocytes was analyzed by flow cytometry. Serum soluble CD137 (sCD137) and C-reactive protein levels were measured by commercially available ELISA.ResultsThe expression of CD137 in peripheral monocytes in patients with UA [14.2 ± 3.5 mean fluorescence intensity (MFI)] and AMI (15.1 ± 4.4 MFI) was significantly higher than those in patients with SA (6.5±2.4 MFI) and controls (7.1 ± 3.5 MFI). sCD137 in patients with UA (16.7 ± 4.9 ng/ml) and AMI (19.1 ± 4.3 ng/ml) were significantly higher than those in patients with SA (3.4 ± 1.4 ng/ml) and controls (3.9 ± 1.3 ng/ml) (p < 0.001). C-reactive protein level in serum in patients with UA (13.8 ± 3.3 ng/ml) and AMI (15.5 ± 4.7 ng/ml) were also higher than those in patients with SA (1.4 ± 0.4 ng/ml) and controls (1.3 ± 0.3 ng/ml). It was interesting that percutaneous transluminal coronary angioplasty (PTCA) induced a marked rise in sCD137 levels in SA patients, while CD137 expression in peripheral monocytes showed no difference between SA patients with PTCA before and after. A positive correlation was found between sCD137 and serum C-reactive protein levels (r = 0.681; p < 0.0001).ConclusionPatients with ACS showed increased soluble and membrane-bound CD137 expression. sCD137 level showed a significantly positive correlation with CRP level in patients with ACS. The relation between sCD137 and ACS needs further researches.     

Correlation of endothelin 1 plasma levels with plasma antioxidant capacity in elderly patients treated for hypertension

Experimental studies confirmed that reactive oxygen species increase endothelin-1 (ET-1) synthesis, and modulate ET-1 signaling pathway resulting in vasoconstriction and vascular remodeling.The aim of this study was to evaluate the relationship between plasma ET-1 concentration and antioxidant status in patients with essential hypertension and type 2 diabetes mellitus.Methods78 hypertensive patients, 53.8% diabetic, mean age 72.1 ± 7.07 were examined. The plasma concentration of glucose, creatinine, uric acid, bilirubin, cholesterol, insulin, HbA1c and ET-1 were measured. Antioxidant status was assessed by Ferric Reducing Ability of Plasma (FRAP), vitamin C concentration and erythrocyte superoxide dismutase (SOD) activity.ResultsWith diabetes ET-1 concentration was higher (1.35 ± 0.51 vs 1.12 ± 0.46 pg/mL, p = 0.04). The negative correlations between ET-1 concentration and FRAP (r = ? 0.50, p < 0.0001), vitamin C (r = ? 0.296, p = 0.01) and SOD (r = ? 0.44, p = 0.001) were found. Concentration of ET-1 correlated positively with SBP (r = 0.33, p = 0.005) but not with DBP. The relationship between DBP and ET-1 only in subjects with DBP > 110 mm Hg and FRAP < 0.40 mmol/L was found. In multiple regression analysis plasma ET-1 levels were associated independently with FRAP (beta = ? 0.583, p = 0.003) and plasma vitamin C (beta = ? 0.407, p = 0.04).ConclusionsIn hypertensive and diabetic patients higher plasma endothelin-1 level was independently associated with lower plasma antioxidant status measured by FRAP and decreased vitamin C concentration, which may be a result of increased oxidative stress in these diseases.     

Association between irisin and homocysteine in euglycemic and diabetic subjects

ObjectivesThe aim of study was to explore whether a relationship exists between homocysteine and irisin in type 2 diabetes (T2D) patients—a population with a high risk of developing cardiovascular disease—and euglycemic controls.Design and methods69 T2D patients and 75 control subjects (adjusted by body mass index (BMI)) were included in the study. Irisin and homocysteine concentrations and anthropometric and biochemical variables were determined.ResultsLevels of homocysteine were significantly higher (11.0 ± 3.0 vs 12.4 ± 4.2 μmol/l) and levels of irisin were lower (279 ± 58 vs 263 ± 38 ng/ml) in T2D patients. When both T2D and controls were considered, irisin was found to correlate only with homocysteine (r = − 0.215; p = 0.011). Moreover, a decreasing trend in irisin levels was observed according to homocysteine tertile (p = 0.034).ConclusionsOur results provide evidence of an association between irisin and homocysteine, which may be due to nicotinamide metabolism. The clinical significance of this relationship is unclear, but our findings may prompt further mechanistic research to investigate the role played by irisin in vascular disorders.     

Biological variability of blood omega-3 biomarkers

ObjectivesWe conducted a pilot study to estimate the biological variability and effects of a prior meal on the omega-3 fatty acid (FA) content of 3 blood FA pools.Design and methodsWe measured FA levels in red blood cells (RBCs), plasma and plasma phospholipids (PL) obtained from 20 healthy volunteers tested weekly over 6 weeks.ResultsThe within-subject coefficients of variation were 4.1% ± 1.9%, 15.9% ± 6.4%, and 14.5% ± 8.4%, respectively (RBC vs. others, p < 0.001). RBC omega-3 FA content had the lowest biological variability and was not altered in the fed state.ConclusionsFrom the perspective of variability and of the sample types tested, RBCs may be the preferred sample type for assessing omega-3 FA status.     

Anticoagulants affect matrix metalloproteinase 9 levels in blood samples of stroke patients and healthy controls

ObjectivesMatrix metalloproteinase-9 (MMP-9) represents a promising marker for acute stroke management. In clinical studies MMP-9 has been quantified by ELISA using differing protocols. We aimed to establish a valid protocol by evaluation of preanalytics.Design and methodsBlood from stroke patients (n = 28) and healthy controls (n = 28) was drawn into tubes containing different anticoagulants (EDTA, citrate, lithium-heparin (heparin) and heparin with proteinase inhibitors) and processed after 0, 60 and 240 min. MMP-9 plasma protein and mRNA from mononuclear leukocytes were determined.ResultsIn regard to anticoagulants used, samples showed different MMP-9 protein baseline values and kinetics. Stable MMP-9 protein concentrations were only measured from EDTA samples. Particularly in samples with proteinase inhibitors protein and mRNA concentrations increased over time. Kinetics did not differ between patients and controls.ConclusionsPreanalytics plays a key role for determination of MMP-9. EDTA seems to be a valid anticoagulant for MMP-9 protein measurement.     

Plasma ascorbic acid preparation and storage for epidemiological studies using TCA precipitation

Objectives:To introduce a procedure to validate an ascorbic acid method using trichloroacetic acid (TCA) for plasma stabilization at different storage temperatures.Methods:EDTA and heparin plasma were precipitated with TCA (1:5) containing 0.54 mol/L EDTA, or without. Samples were stored at ? 20 °C and ? 70 °C and their stability was tested at room temperature for 24 h.Results:A significant 40% loss (p < 0.001) of plasma ascorbic acid was found when EDTA samples with added EDTA were stored at ? 20 °C for 2–4 weeks compared with storage at ? 70 °C. Ascorbic acid in heparin plasma without added EDTA was most unstable and samples left at room temperature for 24 h lead to almost a total loss of ascorbic acid. Addition of EDTA to the TCA solution improved stability of samples of both plasma types at room temperature.Conclusion:The recommended procedure for ascorbic acid determination in plasma stabilized with TCA is immediate storage at ? 70 °C and inclusion of EDTA into the TCA solution.     

Receptor for advanced glycation end products (RAGE) and glyoxalase I gene polymorphisms in pathological pregnancy

ObjectivesThe aim of the study was to analyze polymorphisms of receptor for advanced glycation end products (RAGE) gene, and glyoxalase I gene and soluble RAGE, sRAGE, in physiological and pathological pregnancy.Design and methodsPolymorphisms of RAGE gene (? 429 T/C, ? 374 T/A, 557 G/A, 2184 A/G) and glyoxalase I gene (A419C) and sRAGE serum levels were determined in 284 women with pathological and physiological pregnancy.ResultsNo differences in distribution of genotype and allelic frequencies of studied polymorphisms were found. GA genotype of RAGE 557 G/A polymorphism (known as Gly82Ser) is associated with lower sRAGE serum levels in healthy pregnant women compared to GG genotype (483 ± 104 vs. 692 ± 262 pg/mL, p = 0.008). sRAGE correlates negatively with ALT in patients with pregnancy intrahepatic cholestasis (r = ? 0.536, p = 0.05).ConclusionsWe did not show any association of RAGE and glyoxalase I gene polymorphisms with pathological pregnancy, however further studies are needed to confirm the results.     

Soluble receptor for advanced glycation end-products (sRAGE) and polymorphisms of RAGE and glyoxalase I genes in patients with pancreas cancer

ObjectivesThe receptor for advanced glycation end-products (RAGE) takes part in the pathogenesis of many diseases, including diabetes mellitus and cancer. AGE-precursors are detoxified by glyoxalase (GLO). sRAGE, soluble RAGE, is an inhibitor of pathological effects mediated via RAGE. The aim was to study sRAGE and polymorphisms of RAGE (AGER) and GLO genes in patients with pancreas cancer (PC).Design and MethodsThe studied group consisted of 51 patients with PC (34 with impaired glucose tolerance—IGT, 17 without IGT), 34 type 2 DM and 154 controls. For genetic analysis, the number of patients was increased to 170. Serum sRAGE was measured by ELISA and all polymorphisms (RAGE ?429T/C, ?374T/A, 2184A/G, Gly82Ser and GLO A419C) were determined by PCR-RFLP and confirmed by sequencing.ResultsSoluble RAGE is decreased in patients with PC compared to patients with DM and controls (975 ± 532 vs. 1416 ± 868 vs. 1723 ± 643 pg/mL, p < 0.001). Patients with PC and IGT have lower sRAGE levels compared to patients with PC without IGT (886 ± 470 vs. 1153 ± 616 pg/mL, p < 0.05). No relationship of sRAGE to the stage was found. We did not show any difference in allelic and genotype frequencies in all RAGE and GLO polymorphisms among the studied groups.ConclusionThis is the first study demonstrating decreased sRAGE in patients with pancreas cancer. Its levels are even lower than in diabetics and are lowest in patients with PC and IGT. Our study supports the role of glucose metabolism disorder in cancerogenesis. Further studies are clearly warranted, especially with respect to potential preventive and therapeutic implications.     

Increased plasma levels of soluble CD40 ligand correlate with platelet activation markers and underline the need for standardized pre-analytical conditions

ObjectivesTo investigate whether sCD40L dosage might represent a useful tool to explore in vivo platelet function.Design and methodssCD40L and sP-selectin levels and light transmission aggregometry (LTA) were analyzed in 69 healthy donors. Immunoassays were performed on platelet-depleted citrate plasma samples. The effects of in vitro aspirin treatment on the release of sCD40L were investigated in 15 subjects following platelet stimulation. The effects of a 1-month therapeutic course of low-dose aspirin on sP-selectin and sCD40L levels were also investigated.ResultsA significant correlation was observed between sCD40L and sP-selectin (p < 0.01). In vitro aspirin treatment remarkably decreased sCD40L levels following platelet activation by exogenous agonists. sCD40L directly correlated with LTA (Rho = 0.62, p < 0.0001). In vivo aspirin treatment significantly reduced both sP-selectin and sCD40L levels (both p < 0.01) in a direct correlation (Rho = 0.66, p < 0.05).ConclusionsCitrated plasma samples reflect sCD40L released from platelets, thus yielding the most valid estimates of in vivo circulating levels of this platelet activation markers.     

Measurement of Type B natriuretic peptide in heparin and K2 EDTA plasma

BackgroundEthylene diamine tetraacetic acid (EDTA) plasma is the only suitable specimen recommended by the manufacturers to be used in the determination of BNP. It appears crucial to evaluate if more conventional heparin plasma samples could be reliably used for BNP determination. Aim of this study was to evaluate the use of heparin plasma sample for BNP determination.MethodsVenous blood from 42 consecutive patients admitted at the division of cardiology was collected in two test tubes, with K₂-EDTA (Group 1) and lithium heparin with gel separator (Group 2) and analysed within 20 min of blood collection.ResultsStatistical analysis showed a significant difference between Group 1 and Group 2 (p < 0.0001). Sample collected in K₂-EDTA showed a significant underestimation when compared to lithium–heparin.ConclusionsOur data showed that BNP could not be dosed on different collection tubes without altering the results. In our experimental conditions, interestingly we found that BNP levels are significantly lower if measured in EDTA plasma.