Association between lower plasma adiponectin levels and higher plasma thrombin generation parameters in men with type 2 diabetes: role of plasma triglycerides.

OBJECTIVE: Previous studies showed a significant association between lower plasma adiponectin levels and higher risk of adverse cardiovascular outcomes in patients with and without type 2 diabetes mellitus (T2DM). Presently, it is uncertain whether lower plasma adiponectin levels are associated with greater plasma thrombin generation in patients with T2DM. PATIENTS AND METHODS: We studied 82 middle-aged men with non-insulin-treated T2DM [mean age ± SD: 64.1 ± 8 years; median duration of diabetes: 12.5 (inter-quartile range 6-19) years; mean hemoglobin A1c 7.0 ± 0.7%], consecutively attending our diabetes outpatient service over a 6-month period. Using the newly developed fully automated thrombin generation analyzer ST Genesia®, we measured the plasma parameters lag time (LT), time to peak (TP), peak height (PH) and endogenous thrombin potential (ETP) in all participants. RESULTS: In univariable linear regression analyses, lower plasma adiponectin levels were significantly associated with higher plasma thrombin generation parameters, as reflected by higher values of PH (Pearson's r coefficient = - 0.228, p = 0.039) and EPT (r = - 0.293, p = 0.007). Plasma adiponectin levels were not significantly associated with other thrombin generation parameters (LT and TP). Notably, the significant associations of plasma adiponectin levels with thrombin PH and EPT values persisted after adjustment for age and adiposity measures, but they were lost after additional adjustment for plasma triglycerides. CONCLUSION: Our findings show for the first time the existence of a significant association between lower levels of plasma adiponectin and greater plasma thrombin generation (as assessed by the ST Genesia® analyzer) in men with non-insulin-treated T2DM, which appears to be largely mediated by plasma triglycerides.

Screening for non-alcoholic fatty liver disease using liver stiffness measurement and its association with chronic kidney disease and cardiovascular complications in patients with type 2 diabetes.

AIM: Despite the high prevalence and serious clinical implications of non-alcoholic fatty liver disease (NAFLD) in patients with type 2 diabetes mellitus (T2DM), NAFLD is usually overlooked during routine diabetes care. This study explored the proportion of NAFLD cases and increased liver fibrosis (LF), and the association between LF and either chronic kidney disease (CKD) or cardiovascular complications in T2DM patients. METHODS: The study included 137 patients with non-insulin-treated T2DM and no known liver disease consecutively attending our diabetes outpatients' service who underwent liver ultrasonography and liver stiffness measurement (LSM) using vibration-controlled transient elastography (FibroScan®). RESULTS: The proportion of patients with hepatic steatosis on ultrasonography was 73.7%, and the proportion with significant LF was 17.5% with an LSM cut-off ≥7kPa or 10.2% with an LSM cut-off ≥8.7kPa. The presence of CKD (estimated GFR <60mL/min/1.73m2 and/or abnormal albuminuria) increased significantly across LSM tertiles (from around 15% in tertile 1 to 45% in tertile 3). Cardiovascular complications (previous ischaemic heart disease, ischaemic stroke, permanent atrial fibrillation) also tended to increase across LSM tertiles (from around 15% to 30%). After adjusting for established risk factors and potential confounders, LSM tertile 3 remained significantly associated with an approximately threefold higher risk of prevalent CKD (adjusted OR: 3.28, 95% CI: 1.22-8.90; P=0.019), but not for cardiovascular complications. CONCLUSION: These results suggest that NAFLD and significant LF (as assessed by FibroScan®) are very commonly seen in T2DM outpatients with no known liver disease attending a secondary-care diabetes service, and that increased LF is associated with a greater proportion of chronic vascular complications, especially CKD.

Increased Gene Expression of RUNX2 and SOX9 in Mesenchymal Circulating Progenitors Is Associated with Autophagy during Physical Activity.

Lack of physical exercise is considered an important risk factor for chronic diseases. On the contrary, physical exercise reduces the morbidity rates of obesity, diabetes, bone disease, and hypertension. In order to gain novel molecular and cellular clues, we analyzed the effects of physical exercise on differentiation of mesenchymal circulating progenitor cells (M-CPCs) obtained from runners. We also investigated autophagy and telomerase-related gene expression to evaluate the involvement of specific cellular functions in the differentiation process. We performed cellular and molecular analyses in M-CPCs, obtained by a depletion method, of 22 subjects before (PRE RUN) and after (POST RUN) a half marathon performance. In order to prove our findings, we performed also in vitro analyses by testing the effects of runners' sera on a human bone marrow-derived mesenchymal stem (hBM-MSC) cell line. PCR array analyses of PRE RUN versus POST RUN M-CPC total RNAs put in evidence several genes which appeared to be modulated by physical activity. Our results showed that physical exercise promotes differentiation. Osteogenesis-related genes as RUNX2, MSX1, and SPP1 appeared to be upregulated after the run; data showed also increased levels of BMP2 and BMP6 expressions. SOX9, COL2A1, and COMP gene enhanced expression suggested the induction of chondrocytic differentiation as well. The expression of telomerase-associated genes and of two autophagy-related genes, ATG3 and ULK1, was also affected and correlated positively with MSC differentiation. These data highlight an attractive cellular scenario, outlining the role of autophagic response to physical exercise and suggesting new insights into the benefits of physical exercise in counteracting chronic degenerative conditions.

Association between non-alcoholic fatty liver disease and bone turnover biomarkers in post-menopausal women with type 2 diabetes.

AIM: Information is lacking on the association between non-alcoholic fatty liver disease (NAFLD) and bone mineral density (BMD) or circulating bone turnover biomarkers in post-menopausal women with type 2 diabetes (T2DM). METHODS: We recruited 77 white post-menopausal women with T2DM, who consecutively attended our diabetes outpatient service during a 3-month period. Liver ultrasonography and transient elastography (Fibroscan®) were used for diagnosing and staging NAFLD. A dual energy X-ray absorptiometry, and serum levels of 25-hydroxyvitamin D3 [25(OH)D], parathyroid hormone and multiple bone turnover biomarkers (periostin, sclerostin, dickkopf-related protein-1 [DKK-1], C-terminal telopeptide of type 1 collagen [sCTX], procollagen type 1 N-terminal propeptide [P1NP], receptor activator of nuclear factor-kB ligand [RANKL]) were also measured. RESULTS: Overall, 10 patients had NAFLD with clinically significant fibrosis (i.e., liver stiffness measurement > 7 kPa), 52 had NAFLD without fibrosis and 15 patients were free from steatosis. Although the three patient groups had comparable values of BMD, after adjustment for age, waist circumference, HOMA-insulin resistance and serum 25(OH)D levels, patients with NAFLD and significant fibrosis had significantly higher sclerostin levels (54.1 ± 16.4 vs. 36.1 ± 11.9 vs. 42.3 ± 14.7 pmol/L) and lower levels of serum DKK-1 (26.6 ± 17.8 vs. 49.0 ± 22.4 vs. 42.9 ± 19.4 pmol/L), RANKL (0.04 ± 0.03 vs. 0.08 ± 0.06 vs. 0.11 ± 0.06 pmol/L) and sCTX (0.16 ± 0.09 vs. 0.29 ± 0.17 vs. 0.40 ± 0.28 ng/mL) compared to other groups. Serum periostin and P1NP levels did not significantly differ between the groups. CONCLUSION: In post-menopausal women with T2DM, the presence of NAFLD and clinically significant fibrosis was strongly associated with a low bone turnover, which may reflect the presence of qualitative bone abnormalities.

Fully automated chemiluminescence vs RIA aldosterone assay in primary aldosteronism work-up.

Aldosterone and renin measurement is a cornerstone for primary aldosteronism (PA) diagnosis, but different thresholds are used according to different assays. A fully automated chemiluminescence (CL) immunoassay for renin and aldosterone was recently proposed, showing good performance for PA screening by aldosterone to renin ratio (ARR). This study aimed to define the accuracy of this assay in the screening and in the most popular confirmatory test of autonomous aldosterone production, the intravenous saline loading test (ivSLT). We compared aldosterone results obtained by CL vs radioimmunoassay (RIA) in hypertensive patients investigated for PA (102 baseline and 85 after ivSLT). An excellent correlation was observed between RIA and CL in the entire population for aldosterone (r=0.922) and ARR (r=0.977). For ARR, Deming regression proved a good accordance between methods and, consistent with the fit model, our previous institutional ARR cut-off of 32 (pg ml-1)/(pg ml-1) corresponded to 20 pg ml-1 mU-1 l-1 in CL assay. However, the correlation was weaker in the low end of aldosterone concentrations (r=0.676 for aldosterone <100 pg ml-1), with a concordance of ivSLT results in only 68% of patients. CL assay displays a diagnostic performance very similar to RIA for ARR screening, but it is substantially inferior in the setting of confirmatory tests of autonomous aldosterone secretion, that is, ivSLT.

Reference miRNAs for colorectal cancer: analysis and verification of current data.

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

Sodium citrate blood contamination by K2 -ethylenediaminetetraacetic acid (EDTA): impact on routine coagulation testing.

INTRODUCTION: The potential cross-contamination of additives between primary blood tubes is a well-known problem during sample collection. The aim of this study was to assess the impact of citrated blood contamination with different amounts of dipotassium ethylenediaminetetraacetic (K2 EDTA blood) on activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen. METHODS: Blood was collected from 15 ostensibly healthy volunteers into four 0.109 m citrate blood tubes followed by one K2 EDTA blood tube. The citrate tubes of each subject were pooled and divided in five aliquots. The whole blood of the K2 EDTA tube was then added in scalar amounts to autologous citrated blood aliquots, to obtain K2 EDTA contamination ranging from 0% to 43%, and thus mimic potential pre-analytical contamination. RESULTS: A statistically and clinically significant prolongation was observed for both APTT and PT between 29% and 43% K2 EDTA contamination, whereas the decrease of fibrinogen values became statistically and clinically significant at 43% K2 EDTA contamination. CONCLUSION: The results of this investigation show that contamination of citrated blood with as much as 29% of K2 EDTA blood generates a significant bias in results of routine clotting assays. This has serious implications for patient safety and management.

Allopurinol prevents cardiac and skeletal muscle damage in professional soccer players.

Xanthine oxidase (XO), a free radical-generating enzyme, is involved in tissue damage produced during exhaustive exercise. Our aim was to test whether allopurinol, a powerful inhibitor of XO, may be effective in preventing exercise-induced tissue damage in soccer players. Twelve soccer players were randomized into two experimental groups. One received allopurinol, before a match of the premier Spanish Football League, and the other placebo. Allopurinol prevented the exercise-induced increase in all the markers of skeletal muscle damage analyzed: creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and myoglobin. Creatine kinase-MB isoenzyme and highly sensitive troponin T, specific biomarkers of myocardial injury, increased significantly in the placebo but not in the allopurinol-treated group after the football match. We also found that the exercise-induced lipid peroxidation, as reflected by malondialdehyde measurements, was prevented after allopurinol administration. However, inhibition of XO did not prevent the increment in the activity of alanine aminotransferase found after the match. No changes in the serum gamma glutamyltransferase activity was found after the match on either the placebo and the allopurinol groups. These two enzymes were determined as biomarkers of liver injury. Allopurinol represents an effective and inexpensive pharmacological agent to prevent tissue damage in soccer players.

Epigenetic alteration: new insights moving from tissue to plasma - the example of PCDH10 promoter methylation in colorectal cancer.

BACKGROUND: Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical-pathological features. METHODS: A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay. RESULTS: The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3-97.8%) and 5.9% (0-80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234). CONCLUSION: Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

Brand of dipotassium EDTA vacuum tube as a new source of pre-analytical variability in routine haematology testing.

This study assesses the use of different dry K2 (dipotassium) EDTA vacuum tubes and whether or not they might represent a bias in haematological testing. Blood was collected in three dipotassium EDTA vacuum tubes from different manufacturers: Venosafe, Vacuette and Vacutainer. Samples were analysed on an Advia 2120i analyser. Significant differences among results and biases were compared with current quality specifications. Significant differences were found for haematocrit (HCT), mean corpuscular volume (MCV), white blood cell count (WBC) and platelet distribution width (PDW) when comparing Venosafe vs. Vacuette; for MCV, WBC and PDW when comparing Venosafe vs. Vacutainer; and for HCT and MCV when comparing Vacuette vs. Vacutainer. Clinically significant variations were observed for HCT and PDW in Venosafe vs. Vacuette; PDW in Venosafe vs. Vacutainer; and HCT and MCV in Vacuette vs. Vacutainer. The use of dipotassium EDTA vacuum tubes from different manufacturers represent a clinically relevant source of variation for HCT, MCV and PDW.

Transillumination: a new tool to eliminate the impact of venous stasis during the procedure for the collection of diagnostic blood specimens for routine haematological testing.

INTRODUCTION: The collection of diagnostic blood specimens for routine haematological testing (RHT) is traditionally performed with tourniquet. However, the transillumination devices based on cold near-infrared LEDs have been formerly proposed as a valuable tool for identifying reliable venous accesses, especially in patients with difficult or small veins, such as children. This study was aimed to evaluate whether a transillumination device can advantageously replace the use of the tourniquet during the procedure for collection of blood specimens for RHT and thereby eliminating the discomfort and risk of spurious results caused by excessive or prolonged venous stasis. METHODS: Two hundred and fifty volunteers were divided into five groups (G1, G2, G3, G4 and G5) to compare the results of RHT between blood sample collected with transilluminator device (left arm) and with tourniquet application (right arm) for 30 s(G1), 60 s(G2), 90 s(G3), 120 s(G4) and 180 s(G5). RESULTS: No significant increases were observed in any of the haematological parameters tested in G1 when compared with blood collected by the transilluminator device. From G2 to G5, significant increases were observed for the platelet count, red blood cell count, haemoglobin, haematocrit, white blood cell count, neutrophils, monocytes and eosinophils. From G3-G5, further increases were observed for lymphocytes. Clinically significant variations were, however, observed for basophils in G2; red blood cell count, haemoglobin, haematocrit and basophils in G3 and eosinophils in G3 only. CONCLUSION: As such, considering that inappropriate use of the tourniquet is commonplace, we conclude that transillumination devices can represent a suitable tool to eliminate the venous stasis and to improve the quality of phlebotomy procedures.

Acute variation of leucocytes counts following a half-marathon run.

There is growing interest on haematological changes following elite and recreation sports. However, no information is available on leucocyte variations after an intermediate-distance run. Complete blood cell count was assessed on 17 trained, middle-aged males before a 21-km half-marathon, at the end and 3, 6, 24 h thereafter. Statistically significant variations by one-way analysis of variance were observed for all the parameters tested. Haematocrit, haemoglobin and red blood cells and platelet count increased significantly by the end of the run and returned to pre-run values 3 h thereafter. White blood cells, neutrophils, monocytes and basophils counts increased after the run, reached the peak at 3 h and returned to the baseline after 24 h. Conversely, the eosinophils count significantly decreased after the run, reached a nadir at 3 h and slowly returned to pre-run values at 24 h. The lymphocytes count significantly increased after the run, decreased 3 h thereafter and returned to pre-run values after 6 h. These results show that an intermediate-distance run, which is a popular form of recreational sports worldwide, is associated with transitory leucocytosis, neutrophilia, monocytosis and basophilia, but it also induces acute eosinopenia, all changes being completely reversed 24 h thereafter.

Right or wrong sample received for coagulation testing? Tentative algorithms for detection of an incorrect type of sample.

Inappropriate blood collection potentially comprises the major pre-analytical problem for coagulation testing. Inappropriate samples are most difficult to detect when received as secondary aliquots, common for referred tests. This study aimed to identify a simple, quick and inexpensive process to help laboratories distinguish the type of sample, should there be suspicion of inappropriate collection. Samples from 15 patients [selected on the basis that four different primary tubes were available: serum, citrated plasma, ethylene diamine tetraacetic acid (EDTA) plasma, lithium-heparin plasma], were tested for common electrolytes that might substantially differ according to the type of sample. In citrated plasma, potassium, chloride, calcium and magnesium were significantly decreased compared with serum and lithium-heparin plasma, while sodium was markedly increased. In EDTA plasma, sodium and chloride were significantly decreased compared with both serum and lithium-heparin plasma, potassium was always >14 mmol/l, whereas magnesium and calcium were virtually undetectable. These data allowed development of two algorithms for differential identification of citrated plasma vs. other samples with 100% sensitivity and specificity, the former based on the sequential measurement of potassium, calcium and sodium, the latter on potassium and sodium. These simple assays can supplement classical coagulation test methods to identify most inappropriate blood collections and validate sample rejection.

Thrombin generation assay: a useful routine check-up tool in the management of patients with haemophilia?

Severity assessment of patients with haemophilia A (HA) is traditionally based on FVIII activity (FVIII:C). Clinical phenotype of HA patients often differs between individuals with the same FVIII:C determined with clotting and chromogenic assays. The aim of this study was to assess the influence of the FVIII:C on thrombin generation (TG) assay parameters both in vitro and ex-vivo postinfusion plasma. For in-vitro approach, influence of FVIII:C was evaluated on TG parameters in several dilutions of a normal plasma pool with commercial FVIII-depleted-plasma (FVIIIDP) and in others experiments, adding increasing amounts of different commercial FVIII concentrates (Fanhdi, Haemate-P, Hemofil-M and Kogenate Bayer) to FVIIIDP. In a series of 50 postinfusion samples, from HA patients of different severity, we assayed TG and FVIII:C (chromogenic and clotting). In vitro experiments, the 50% of maximum TG peak (TGMP) was achieved using only 5% FVIII:C and the TGMP was obtained with 40% of normal VIII:C. Impaired response compared with normal plasma was found in FVIIIDP using addition of increasing amounts of different commercial FVIII concentrates. An overall good correlation between the two FVIII assays was observed (y = 0.9115x - 0.273, r = 0.975, P < 0.001); TGMP and the Lag-Phase-Time (LPT) provided some discrepant results when compared with the total range of FVIII:C measurements. In contrast, correlations for TGMP, LPT and endogenous thrombin potential were improved in samples restricted to FVIII:C <5%. We conclude that TG parameters tentatively could be a tool to tailor the global haemostatic capacity in haemophilic patients.

Influence of temperature and time before centrifugation of specimens for routine coagulation testing.

The accurate standardization of the preanalytical phase is of pivotal importance for achieving reliable results of coagulation tests. Because information on the suitable storage conditions for coagulation testing is controversial, we aimed at investigating the sample stability with regard to the temperature and time before centrifugation. The activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen and D-dimer were assayed in specimens collected from 26 consecutive patients on antivitamin K therapy on the ACL TOP analyzer. Three primary 3.6-ml siliconized evacuated tubes containing 0.109 mol/l buffered trisodium citrate were sequentially collected from each patient. These three tubes were mixed, pooled and divided into seven identical aliquots. The first aliquot was immediately centrifuged according to the standard protocol [1500 g for 15 min at room temperature (RT)] and analyzed. The other aliquots were left for 3, 6 and 24 h, respectively, at RT or 4 degrees C, and then centrifuged and analyzed. Test results were compared with those obtained on the reference specimen. Statistically significant prolongations were observed for aPTT in all the samples. Such differences exceeded the analytical quality specifications for desirable bias in the samples stored for 24 h. A significant reduction, yet comprised within the desirable bias, was observed for PT and fibrinogen in uncentrifuged specimens stored at RT for 3 and 6 h. No significant biases could be recorded in D-dimer. In conclusion, a 6-h storage of uncentrifuged specimens at either RT or 4 degrees C may still be suitable to achieve results of routine coagulation testing comprised within the analytical quality specifications for desirable bias.