Rapid changes of miRNAs-20, -30, -410, -515, -134, and -183 and telomerase with psychological activity: A one year study on the relaxation response and epistemological considerations.

BACKGROUND AND AIM: Mental stress represents a pivotal factor in cardiovascular diseases. The mechanism by which stress produces its deleterious effects is still under study, but one of the most explored pathways is inflammation-aging and cell senescence. In this scenario, circulating microRNAs appear to be regulatory elements of the telomerase activity and alternative splicing within the nuclear factor kappa-light-chain-enhancer (NF-κB) network. Anti-stress techniques appeared to be able to slow down the inflammatory and aging processes. As we recently verified, the practice of the relaxation response (RR) counteracted psychological stress and determined favorable changes of the NF-κB, p53, and toll-like receptor-4 (TLR-4) gene expression and in neurotransmitters, hormones, cytokines, and inflammatory circulating microRNAs. We aimed to verify a possible change in the serum levels of six other micro-RNAs of cardiovascular interest, involved in cell senescence and in the NF-κB network (miRNAs -20, -30, -410, -515, -134, and -183), and tested the activity of telomerase in peripheral blood mononuclear cells (PBMCs). EXPERIMENTAL PROCEDURE: We measured the aforementioned molecules in the serum of patients with ischemic heart disease (and healthy controls) immediately before and after a relaxation response session, three times (after the baseline), in one year of follow-up. RESULTS: According to our data, the miRNA-20 and -30 levels and PBMCs-telomerase activity increased during the RR while the -410 and -515 levels decreased. During the RR sessions, both miRNA-134 and -183 decreased. CONCLUSION: The mediators considered in this exploratory work appeared to vary rapidly with the psychological activity (in particular when focused on relaxation techniques) showing that psychological activity should be part of the future research on epigenetics. Epistemological perspectives are also discussed.

Toward a Unified View of Cognitive and Biochemical Activity: Meditation and Linguistic Self-Reconstructing May Lead to Inflammation and Oxidative Stress Improvement.

Stress appears to be the basis of many diseases, especially myocardial infarction. Events are not objectively "stressful" but what is central is how the individual structures the experience he is facing: the thoughts he produces about an event put him under stress. This cognitive process could be revealed by language (words and structure). We followed 90 patients with ischemic heart disease and 30 healthy volunteers, after having taught them the Relaxation Response (RR) as part of a 4-day Rational-Emotional-Education intervention. We analyzed with the Linguistic Inquiry and Word Count software the words that the subjects used across the study following the progression of blood galectin-3 (inflammation marker) and malondialdehyde (oxidative stress marker). During the follow-up, we confirmed an acute and chronic decrease in the markers of inflammation and oxidative stress already highlighted in our previous studies together with a significant change in the use of language by the subjects of the RR groups. Our results and the precise design of our study would seem to suggest the existence of an intimate relationship and regulatory action by cognitive processes (recognizable by the type of language used) on some molecular processes in the human body.

Short-term biological variation and diurnal rhythm of cardiac troponin I (Access hs-TnI) in healthy subjects.

BACKGROUND: The availability of high-sensitivity cTnI (hs-cTnI) assays has improved the accuracy of cTn measurements at concentrations around and below the 99th percentile, allowing the evaluation of biological variation. METHODS: cTnI concentrations have been measured in blood samples of 35 reference subjects collected at time 0 (between 8 and 9 AM) and after 1,2,3 and 7 h using a high-sensitive assay (Access hs-TnI). Repeated measure ANOVA and lognormal transformation followed by Nested ANOVA were used to assess differences in cTnI concentration and to estimate biological variation components, respectively. Circadian variability was modelled by sine-wave functions fitting. RESULTS: At time 0, cTnI concentrations were significantly higher than those measured at other times in overall population, as well as in subjects subdivided by biological sex. The concentrations exhibit a strong circadian variability in males and females, with a predicted interval of around 5.4 h (R2 0.949 and 0.999 for males and females, respectively). CONCLUSIONS: Troponin I demonstrates a diurnal rhythm with decreasing values throughout daytime and the peak concentrations in the morning. The circadian variability is statistically significant, but not relevant from a clinical viewpoint. The intra-individual variation (CVI) is lower than that reported in the literature and the index of individuality lower than 0.6 suggests a scarce value of reference interval.

Clinical usefulness of Glycated Albumin in the diagnosis of diabetes: Results from an Italian study.

OBJECTIVES: Glycated Albumin (GA) has been proposed as a screening marker for diabetes in Asian countries in the last years. Nevertheless, few studies have been conducted in Caucasian population. The aim of this study is to evaluate the clinical usefulness of GA in diabetes diagnosis in Caucasian asymptomatic subjects considered at risk of diabetes based on medical history and Fasting Plasma Glucose (FPG). DESIGN AND METHODS: Three hundred and thirty-four Caucasian subjects having one or more risk factor for diabetes, and/or FPG ranging from 5.6 mmol/L to 6.9 mmol/L with no symptoms for diabetes were enrolled in this study. Plasma GA was measured by an enzymatic method (quantILab Glycated Albumin) on ILab Taurus instrument (Instrumentation Laboratory - A Werfen Company). RESULTS: GA median levels were 13.2% (IQR:12.2-14.4). Eighteen subjects (5.4%) were classified as diabetics based on their HbA1c. According to the ROC curve analysis, GA identified subjects with diabetes with a sensitivity of 72.2% (95% CI: 46.5-90.3) and a specificity of 71.8% (95% CI: 66.5-76.7) (AUC: 0.80; 95% CI: 0.75-0.84; P < 0.0001) at the cut-off of 14%. The cut-off of 13.5% was associated to a higher sensitivity 88.9% (95%CI: 65.3-98.6) and a specificity of 60.4% (95%CI, 54.8-65.9). CONCLUSIONS: This study confirms the clinical usefulness of GA for the diagnosis of diabetes in Caucasian subjects at risk for diabetes. More studies are required to clarify the role of GA in relation to the other diagnostic criteria for diabetes.

Thoughts modulate the expression of inflammatory genes and may improve the coronary blood flow in patients after a myocardial infarction.

BACKGROUND: Mental stress is one of the main risk factors for cardiovascular disease. Meditation and music listening are two techniques that are able to counteract it through the activation of specific brain areas, eliciting the so-called Relaxing Response (RR). Epidemiological evidence reveals that the RR practice has a beneficial prognostic impact on patients after myocardial infarction. We aimed to study the possible molecular mechanisms of RR underlying these findings. METHODS: We enrolled 30 consecutive patients after myocardial infarction and 10 healthy controls. 10 patients were taught to meditate, 10 to appreciate music and 10 did not carry out any intervention and served as controls. After training, and after 60 days of RR practice, we studied the individual variations, before and after the relaxation sessions, of the vital signs, the electrocardiographic and echocardiographic parameters along with coronary flow reserve (CFR) and the carotid's intima media thickness (IMT). Neuro-endocrine-immune (NEI) messengers and the expression of inflammatory genes (p53, Nuclear factor Kappa B (NfKB), and toll like receptor 4 (TLR4)) in circulating peripheral blood mononuclear cells were also all observed. RESULTS: The RR results in a reduction of NEI molecules (p < 0.05) and oxidative stress (p < 0.001). The expression of the genes p53, NFkB and TLR4 is reduced after the RR and also at 60 days (p < 0.001). The CFR increases with the relaxation (p < 0.001) and the IMT regressed significantly (p < 0.001) after 6 months of RR practice. CONCLUSIONS: The RR helps to advantageously modulate the expression of inflammatory genes through a cascade of NEI messengers improving, over time, microvascular function and the arteriosclerotic process.

Automated saliva processing for LC-MS/MS: Improving laboratory efficiency in cortisol and cortisone testing.

OBJECTIVES: The aim of this study was to implement in our routine practice an automated saliva preparation protocol for quantification of cortisol (F) and cortisone (E) by LC-MS/MS using a liquid handling platform, maintaining the previously defined reference intervals with the manual preparation. DESIGN AND METHODS: Addition of internal standard solution to saliva samples and calibrators and SPE on µ-elution 96-well plate were performed by liquid handling platform. After extraction, the eluates were submitted to LC-MS/MS analysis. The manual steps within the entire process were to transfer saliva samples in suitable tubes, to put the cap mat and transfer of the collection plate to the LC auto sampler. Transference of the reference intervals from the manual to the automated procedure was established by Passing Bablok regression on 120 saliva samples analyzed simultaneously with the two procedures. RESULTS: Calibration curves were linear throughout the selected ranges. The imprecision ranged from 2 to 10%, with recoveries from 95 to 116%. Passing Bablok regression demonstrated no significant bias. CONCLUSIONS: The liquid handling platform translates the manual steps into automated operations allowing for saving hands-on time, while maintaining assay reproducibility and ensuring reliability of results, making it implementable in our routine with the previous established reference intervals.

Salivary cortisol and cortisone by LC-MS/MS: validation, reference intervals and diagnostic accuracy in Cushing's syndrome.

BACKGROUND: The Endocrine Society recommends late-night salivary cortisol (LNS-F) as a first-line screening test for Cushing's syndrome (CS). In the parotid gland, 11ß-hydroxysteroid dehydrogenase type 2 inactivates cortisol (F) to cortisone (E), a known source of interference in the more frequently used immunoassays. A highly specific method is mandatory in determining salivary F and E: it is widely accepted that liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the best available technique for this purpose. METHODS: A LC-MS/MS method with SPE of saliva samples was developed and validated. Appropriate awakening and bedtime reference ranges were established. The diagnostic performance for F, E and the ratio at bedtime was evaluated in 25 cases of CS. RESULTS: The method was linear, with up to 55.4 nmol/L and 51.0 nmol/L, LLOQ of 0.51 nmol/L and 0.55 nmol/L, for F and E, respectively. Within-run and between-run imprecisions were <10% for both analytes. No ion suppression was observed. A cut-off of 2.4 nmol/L for LNS-F yielded a sensitivity of 100% and a specificity of 98% in the diagnosis of CS. CONCLUSIONS: The analytical performance of this method justifies its introduction into clinical practice, thus allowing clinicians the opportunity to further investigate CS and other endocrine diseases.

First evaluation of a multi-capillary electrophoresis CDT assay on Helena Biosciences' V8 analyser.

BACKGROUND: Carbohydrate-deficient transferrin measurement is currently used for the routine monitoring of excessive alcohol intake, thus playing a fundamental role in the management of alcohol consumption disorders as well as for medico-legal purposes. The aim of the present paper is to report the results obtained from the first performance evaluation of a new CDT quantification assay, and to assess its suitability in routine work. METHODS: We assessed the analytical performances of the multi-capillary electrophoresis analyser Helena Biosciences' V8 E-class and compared the results obtained with those from the HPLC BioRad Ready-Prep CDT assay. Furthermore, we evaluated the robustness of the system in a routine work conditions. RESULTS: Within laboratory imprecision CV% (n=40) using four commercially available quality control materials and two serum pool samples with different concentrations of CDT were <11.2%. The comparison made with the established method was CDT [V8capillary electrophoresis]=0.84 × CDT [HPLC]+0.03, with a Pearson coefficient of r=0.970, and with the Bland-Altman plot showing a significant bias -0.3 (-0.4 to -0.22, 95% CI). The obtained accuracy was highly satisfactory. CONCLUSIONS: The findings made in the present study indicated that the proposed analytical system is a valid alternative to other CDT screening assays currently proposed for routine use in clinical laboratories, since it is precise, accurate and robust, with a high throughput. Moreover, investment in the training of staff is of fundamental importance in ensuring correct interpretation of the electrophoretic pattern, thus providing reliable CDT results.

Cortisol and cortisone ratio in urine: LC-MS/MS method validation and preliminary clinical application.

BACKGROUND: The determination of urinary cortisol/cortisone ratio is of clinical utility in cases of Cushing's syndrome, apparent mineralocorticoid excess, and also provides information on 11ß-hydroxysteroid dehydrogenase (11ß-HSD) type 2 activity. It is therefore of utmost importance to ensure accurate cortisol and cortisone measurement and establish appropriate reference ranges. METHODS: After the isotopic dilution of urine, sample cleanups were obtained with on-line solid-phase extraction and cortisol and cortisone, separated using a Zorbax Eclipse XDB-C18 HPLC analytical column, were analyzed by tandem mass spectrometry with an electrospray ionization source in positive ion mode operation. RESULTS: The method was linear, with concentrations of up to 625 and 1125 nmol/L and lower limit of quantitation (LLOQ) of 5 and 6 nmol/L, for cortisol and cortisone, respectively. Within-run and between-run coefficients of variation were <5% and 6% for cortisol and 6% and 8% for cortisone, respectively. No ion suppression was observed. The non-parametric reference range for the cortisol/cortisone ratio was 0.14-1.09. CONCLUSIONS: A simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the measurement of cortisol and cortisone in urine. Our findings indicate that the proposed analytical method is suitable for routine purposes and useful in many pathological conditions.

Immunosuppressant therapeutic drug monitoring by LC-MS/MS: workflow optimization through automated processing of whole blood samples.

OBJECTIVES: Although, due to its high specificity and sensitivity, LC-MS/MS is an efficient technique for the routine determination of immunosuppressants in whole blood, it involves time-consuming manual sample preparation. The aim of the present study was therefore to develop an automated sample-preparation protocol for the quantification of sirolimus, everolimus and tacrolimus by LC-MS/MS using a liquid handling platform. METHODS: Six-level commercially available blood calibrators were used for assay development, while four quality control materials and three blood samples from patients under immunosuppressant treatment were employed for the evaluation of imprecision. Barcode reading, sample re-suspension, transfer of whole blood samples into 96-well plates, addition of internal standard solution, mixing, and protein precipitation were performed with a liquid handling platform. After plate filtration, the deproteinised supernatants were submitted for SPE on-line. The only manual steps in the entire process were de-capping of the tubes, and transfer of the well plates to the HPLC autosampler. RESULTS: Calibration curves were linear throughout the selected ranges. The imprecision and accuracy data for all analytes were highly satisfactory. The agreement between the results obtained with manual and those obtained with automated sample preparation was optimal (n=390, r=0.96). In daily routine (100 patient samples) the typical overall total turnaround time was less than 6h. CONCLUSIONS: Our findings indicate that the proposed analytical system is suitable for routine analysis, since it is straightforward and precise. Furthermore, it incurs less manual workload and less risk of error in the quantification of whole blood immunosuppressant concentrations than conventional methods.

Multicenter evaluation of hemoglobin A1c assay on capillary electrophoresis.

BACKGROUND: Hemoglobin A1c (HbA1c) measurement is currently used for the routine monitoring of long-term glycemic status, thus playing a fundamental role in the management of this disease. Since this marker has recently been recommended as an additional tool for diagnosing diabetes, it's of the utmost importance to ensure that the precision and accuracy of HbA1c methods are satisfactory. METHODS: We assessed the analytical performances of the Capillarys 2 Flex Piercing® analyzer and compared the results obtained with those from two other widely used HPLC instruments. Furthermore, we evaluated the convenience and ergonomics of the system in authentic routine work conditions in three centers. RESULTS: Within-laboratory (n=40) and between-laboratory (n=120) imprecision CV% using four blood samples with different concentrations of HbA1c were <3.4% and <3.1% using IFCC units and <2.1% and <2.0% using NGSP units, respectively. The obtained trueness (<3 mmol/mol, <0.3%) was highly satisfactory, nor was HbA1c measurement compromised by the presence of the commonly present hemoglobinopathies. The comparison made with established methods revealed excellent agreement (r>0.985). CONCLUSIONS: The evaluated method is precise, accurate and robust, with a high throughput. It also allows the identification of the most frequent Hb variants and therefore may be a valid alternative to other methods currently proposed for routine use in clinical laboratories.

Quality indicators in laboratory medicine: a fundamental tool for quality and patient safety.

OBJECTIVES: The identification of reliable quality indicators (QIs) is a crucial step in enabling users to quantify the quality of laboratory services. The current lack of attention to extra-laboratory factors is in stark contrast with the body of evidence pointing to the multitude of errors that continue to occur in the pre- and post-analytical phases. DESIGN AND METHODS: Different QIs and terminologies are currently used and, therefore, there is the need to harmonize proposed QIs. RESULTS: A model of quality indicators (MQI) has been consensually developed by a group of clinical laboratories according to a project launched by a working group of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). The model includes 57 QIs related to key processes (35 pre-, 7 intra- and 15 post-analytical phases) and 3 to support processes. CONCLUSIONS: The developed MQI and the data collected provide evidence of the feasibility of the project to harmonize currently available QIs, but further efforts should be done to involve more clinical laboratories and to collect a more consistent amount of data.