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OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2â¯% for androstenedione, 1.6-10.8â¯% for DHEAS, and 4.3-8.7â¯% and 2.6-7.1â¯% for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20â¯%. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6â¯% for androstenedione (p<0.001), 7.2 vs. 4.9â¯% for DHEAS (p<0.001), 6.4 vs. 7.6â¯% for female testosterone (p<0.001) and 6.8 and 7.4â¯% for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5â¯% and -4.9 to 18.7â¯% for androstenedione, -10.9 to 4.8â¯% and -3.4 to 3.5â¯% for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6â¯% for testosterone in females, and -7.0 to 8.5â¯% and -7.5 to 11.8â¯% for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.
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Androstenodiona , Sulfato de Deshidroepiandrosterona , Testosterona , Femenino , Humanos , Masculino , Persona de Mediana Edad , Androstenodiona/sangre , Androstenodiona/análisis , Calibración , Sulfato de Deshidroepiandrosterona/sangre , Sulfato de Deshidroepiandrosterona/análisis , Sulfato de Deshidroepiandrosterona/normas , Cromatografía Líquida con Espectrometría de Masas/métodos , Cromatografía Líquida con Espectrometría de Masas/normas , Espectrometría de Masas en Tándem/normas , Espectrometría de Masas en Tándem/métodos , Testosterona/sangre , Testosterona/análisis , Testosterona/normasRESUMEN
OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.
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Cortisona , Humanos , Cromatografía Liquida/métodos , Cortodoxona , Corticosterona , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.
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Hidrocortisona , Progesterona , Aldosterona , Calibración , Cromatografía Liquida/métodos , Humanos , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodosAsunto(s)
Antibacterianos , Lipoglucopéptidos , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Lipoglucopéptidos/uso terapéutico , Antibacterianos/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Masculino , Femenino , Persona de Mediana Edad , Recurrencia , Anciano , Monitoreo de Drogas , Pruebas de Sensibilidad Microbiana , Adulto , Osteomielitis/tratamiento farmacológico , Osteomielitis/microbiología , Vancomicina/análogos & derivadosRESUMEN
A macro-thyroid-stimulating hormone (macro-TSH) is an infrequent yet noteworthy phenomenon in the thyroid field. A 69-year-old patient presented with persistently elevated thyroid-stimulating hormone (TSH) levels ranging from 30 to 50 mIU/L, paradoxically accompanied by normal thyroid hormone levels and normal thyroid ultrasound, with no findings on pituitary magnetic resonance. Laboratory studies were conducted to investigate potential interferences impacting the accuracy of TSH measurements. After excluding other potential causes, polyethylene glycol (PEG) precipitation technique was used, which led us to the diagnosis of macro-TSH. This result was confirmed through chromatography. Macro-TSH, although rare, emerged as the key contributor to the patient's unexplained increase in TSH levels. This case highlights the importance of considering macro-TSH as a potential etiology in cases characterized by unexplained TSH elevation, offering insights into diagnostic protocols and expanding our understanding of thyroid function anomalies.
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BACKGROUND AND OBJECTIVES: Some disease-modifying treatments impair response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in multiple sclerosis (MS), potentially increasing the risk of breakthrough infections. We aimed to investigate longitudinal SARS-CoV-2 antibody dynamics and memory B cells after 2 and 3 messenger RNA (mRNA) vaccine doses and their association with the risk of COVID-19 in patients with MS on different treatments over 1 year. METHODS: Prospective observational cohort study in patients with MS undergoing SARS-CoV-2 mRNA vaccinations. Antispike (anti-S) immunoglobulin G (IgG) titers were measured by chemiluminescence microparticle immunoassay. Frequencies of spike-specific memory B cells were measured on polyclonal stimulation of peripheral blood mononuclear cells and screening of secreted antibodies by ELISA. RESULTS: We recruited 120 patients with MS (58 on anti-CD20 antibodies, 9 on sphingosine 1-phosphate (S1P) receptor modulators, 15 on cladribine, 24 on teriflunomide (TFL), and 14 untreated) and collected 392 samples up to 10.8 months after 2 vaccine doses. When compared with untreated patients, anti-CD20 antibodies (ß = -2.07, p < 0.001) and S1P modulators (ß = -2.02, p < 0.001) were associated with lower anti-S IgG, while TFL and cladribine were not. Anti-S IgG decreased with months since vaccine (ß = -0.14, p < 0.001), independently of treatments. Within anti-CD20 patients, anti-S IgG remained higher in those with greater baseline B-cell counts and were not influenced by postvaccine anti-CD20 infusions. Anti-S IgG increase after a 3rd vaccine was mild on anti-CD20 and S1P modulators. Spike-specific memory B-cell responses were weaker on S1P modulators and anti-CD20 than on TFL and influenced by postvaccine anti-CD20 infusions. The frequency of breakthrough infections was comparable between DMTs, but the risk of COVID-19 was predicted by the last measured anti-S IgG titer before infection (OR = 0.56, 95% CI = 0.37-0.86, p = 0.008). DISCUSSION: Postvaccine anti-S IgG titers decrease over time regardless of MS treatment and are associated with breakthrough COVID-19. Both humoral and specific memory B-cell responses are diminished on S1P modulators. Within anti-CD20-treated patients, B-cell count at first vaccine determines anti-S IgG production, whereas postvaccine anti-CD20 infusions negatively affect spike-specific memory B cells.
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COVID-19 , Esclerosis Múltiple , Humanos , SARS-CoV-2 , COVID-19/prevención & control , Inmunoglobulina G , Cladribina , Leucocitos Mononucleares , Estudios Prospectivos , Antígenos CD20 , ARN MensajeroRESUMEN
Background: Doll therapy (DT) is a non-pharmacological intervention for the treatment of the behavioural and psychological symptoms of dementia (BPSD). We designed a single-blind randomized controlled trial of the 30-day efficacy of DT in reducing the BPSD, professional caregivers' distress and patients' biomarkers of stress, and in improving the exploration and caregiving behaviours. Methods: We randomly assigned 134 women with moderate-to-severe dementia living in nursing homes (NHs) to a DT intervention (DTI, 67) or a sham intervention with a cube (SI, 67). Results: From the first to the 30th session, the DTI group showed a significant decrease in the Neuropsychiatric Inventory-NH (NPI-NH) total score and in the NPI-NH-Distress score compared to the SI group (both p < 0.001). We observed a greater interest in the doll than in the cube, a greater acceptance of a separation from the nurse among DTI participants, and caregiving and exploratory behaviours towards the doll. There were no differences between the groups in the stress biomarkers. Conclusions: Consistent with attachment theory, our findings support the 30-day efficacy of DT, as this non-pharmacological intervention promotes perceptions of security by creating a situation in which patients feel confident and engaged in a caregiving relationship with the doll and reduces the challenging behaviours that are stressful for professional caregivers.
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BACKGROUND: Cannabis oils from FM2®, Bedica®, Bediol®, Bedrocan®, Bedrolite® and Pedanios 22/1® are largely used for medical purposes such as spasticity, chronic pain and appetite stimulating. Several studies showed cannabinoids action on CB1 and CB2 receptors reduces the hyperalgesic phase in inflammatory pain, leading to an improvement of conditions. The active compounds of these galenic preparations show a high variability making titration mandatory. For this reason, the exact oil composition knowledge is fundamental for personalizing therapy. This amis at adapting the correct dose to the patient, improving safety and efficacy of the galenic formulation, choosing the best preparation for each patient. PURPOSE: The aim of this study was to investigate oil preparations variability among different galenic laboratories in order to highlight the importance of titration activity. METHODS: Cannabis pharmacological active compounds titration has been performed in a large cohort of galenic laboratories in Italy. CBD, CBN, THC, THCA and CBDA quantification was carried out by a previous validated method in UHPLC-MS/MS. RESULTS: A number of 4318 samples of Cannabis oil from 83 pharmacies between January 2021 and February 2022 were evaluated. All galenic preparation specialities showed statistically significant differences among galenic laboratories (p-value < 0.001). THCA and CBDA concentrations were investigated as percentage of the extration yelds for total THC and CBD: these compounds had different values in the same specialities among distinct galenic laboratories. Moreover, seasonal variability in analytes concentrations was observed. CONCLUSION: This study described a wide range of oily samples from a large number of galenic laboratories, compared to published papers. In conclusion, knowledge of the exact oil composition is fundamental in the perspective of personalized therapy. Further studies aiming at the correlation between galenic composition and cannabinoids pharmacokinetics, clinical outcomes and toxic effects could be useful to improve our knowledge.
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Cannabinoides , Cannabis , Analgésicos , Cannabis/química , Cromatografía Líquida de Alta Presión/métodos , Dronabinol , Humanos , Espectrometría de Masas en Tándem/métodosRESUMEN
Sofosbuvir (SOF) is an HCV NS5B polymerase inhibitor, and GS-331007 is its major metabolite. The aim of this study was to investigate whether clinical and pharmacological factors could influence GS-331007 intracellular (IC) concentrations in peripheral blood mononuclear cells (PBMCs) associated with a sustained virological response in patients treated with SOF and ribavirin (RBV). Drug levels were analyzed using liquid chromatography at different days of therapy, whereas variants in genes encoding transporters and nuclear factors were investigated using real-time PCR. This study enrolled 245 patients treated with SOF; 245 samples were analyzed for pharmacogenetics and 50 were analyzed for IC pharmacokinetics. The GS-331007 IC concentration at 30 days was associated with its plasma concentration determinate at 30, 60 and 90 days of SOF-therapy and with daclatasvir concentrations at 7 days of therapy. No genetic polymorphism affected IC exposure. In linear multivariate analysis, ledipasvir treatment, baseline albumin and estimated glomerular filtration rate were significant predictors of IC exposure. This study presents data on an IC evaluation in a cohort of patients treated with SOF, also considering pharmacogenetics. These results could be useful for regions where SOF-RBV treatment is considered the standard of care; moreover, they could further deepen the knowledge of IC exposure for similar drugs in the future.
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Introduction: Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the androgen receptor (AR), through ACE2 receptor and TMPRSS2, to enter nasal and upper airways epithelial cells. Genetic analyses revealed that HSD3B1 P1245C polymorphic variant increases dihydrotestosterone production and upregulation of TMPRSS2 with respect to P1245A variant, thus possibly influencing SARS-CoV-2 infection. Our aim was to characterize the HSD3B1 polymorphism status and its potential association with clinical outcomes in hospitalized patients with COVID-19 in Southern Switzerland. Materials and Methods: The cohort included 400 patients hospitalized for COVID-19 during the first wave between February and May 2020 in two different hospitals of Canton Ticino. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue blocks, and HSD3B1 gene polymorphism was evaluated by Sanger sequencing. Statistical associations were verified using different test. Results: HSD3B1 polymorphic variants were not associated with a single classical factor related to worse clinical prognosis in hospitalized patients with SARS-CoV-2. However, in specific subgroups, HSD3B1 variants played a clinical role: intensive care unit admission was more probable in patients with P1245C diabetes compared with P1245A individuals without this comorbidity and death was more associated with hypertensive P1245A>C cases than patients with P1245A diabetes without hypertension. Discussion: This is the first study showing that HSD3B1 gene status may influence the severity of SARS-CoV-2 infection. If confirmed, our results could lead to the introduction of HSD3B1 gene status analysis in patients infected with SARS-CoV-2 to predict clinical outcome.
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BACKGROUND: There are data on the safety of cancer surgery and the efficacy of preventive strategies on the prevention of postoperative symptomatic COVID-19 in these patients. But there is little such data for any elective surgery. The main objectives of this study were to examine the safety of bariatric surgery (BS) during the coronavirus disease 2019 (COVID-19) pandemic and to determine the efficacy of perioperative COVID-19 protective strategies on postoperative symptomatic COVID-19 rates. METHODS: We conducted an international cohort study to determine all-cause and COVID-19-specific 30-day morbidity and mortality of BS performed between 01/05/2020 and 31/10/2020. RESULTS: Four hundred ninety-nine surgeons from 185 centres in 42 countries provided data on 7704 patients. Elective primary BS (n = 7084) was associated with a 30-day morbidity of 6.76% (n = 479) and a 30-day mortality of 0.14% (n = 10). Emergency BS, revisional BS, insulin-treated type 2 diabetes, and untreated obstructive sleep apnoea were associated with increased complications on multivariable analysis. Forty-three patients developed symptomatic COVID-19 postoperatively, with a higher risk in non-whites. Preoperative self-isolation, preoperative testing for SARS-CoV-2, and surgery in institutions not concurrently treating COVID-19 patients did not reduce the incidence of postoperative COVID-19. Postoperative symptomatic COVID-19 was more likely if the surgery was performed during a COVID-19 peak in that country. CONCLUSIONS: BS can be performed safely during the COVID-19 pandemic with appropriate perioperative protocols. There was no relationship between preoperative testing for COVID-19 and self-isolation with symptomatic postoperative COVID-19. The risk of postoperative COVID-19 risk was greater in non-whites or if BS was performed during a local peak.
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Cirugía Bariátrica , COVID-19 , Diabetes Mellitus Tipo 2 , Obesidad Mórbida , Prueba de COVID-19 , Estudios de Cohortes , Humanos , Incidencia , Obesidad Mórbida/cirugía , Pandemias , Complicaciones Posoperatorias/epidemiología , SARS-CoV-2RESUMEN
Therapeutic drug monitoring (TDM) of immunosuppressive drugs is crucial in organ-transplanted patients to prevent rejection or toxic effects due to inadequate dosage. Mycophenolic acid (MPA) is a commonly used immunosuppressant in this setting. Nowadays, MPA concentrations are monitored by Enzyme Multiplied Immunoassay Technology (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Tandem Mass Spectrometry (LC-MS/MS). This study evaluates the concordance between TDM results for MPA obtained through CE-IVD EMIT and LC-MS/MS assays in plasma samples. LC-MS/MS quantification was based on a commercial kit and the analytical performance in terms of accuracy was tested through external proficiency tests and inter-laboratory comparison with a home-made HPLC-UV method. Both these evaluations confirmed the reliability of the LC-MS/MS method (1.6 % and 9.0 % of bias, respectively). Conversely, the comparison between EMIT and LC-MS/MS showed overestimation by EMIT of 33.5 %. This bias resulted concentration-dependent, ranging from 46.4 % in the concentration range of 1-2 mg/L, to 21.4 % over 4 mg/L. Considering the theoretical clinical impact of this overestimation, a fraction comprised between 12.4 % and 31.4 % of samples which resulted over three different minimum effective concentration values by EMIT (no indication for dose adjustment) had discordant indications by LC-MS/MS (dose adjustment needed). Concluding, this study highlights a clinically relevant systematic overestimation of MPA concentration by EMIT, supporting the switch to LC-MS/MS techniques for TDM purpose. However, further prospective studies are needed in order to evaluate the clinical impact of switching the TDM activity from EMIT to LC-MS/MS in a larger cohort in a long period.
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Monitoreo de Drogas/métodos , Inmunosupresores/farmacocinética , Ácido Micofenólico/farmacocinética , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Técnica de Inmunoensayo de Enzimas Multiplicadas , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/análisis , Ácido Micofenólico/administración & dosificación , Ácido Micofenólico/análisis , Trasplante de Órganos/métodos , Reproducibilidad de los Resultados , Espectrometría de Masas en TándemRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), with a clinical outcome ranging from mild to severe, including death. To date, it is unclear why some patients develop severe symptoms. Many authors have suggested the involvement of vitamin D in reducing the risk of infections; thus, we retrospectively investigated the 25-hydroxyvitamin D (25(OH)D) concentrations in plasma obtained from a cohort of patients from Switzerland. In this cohort, significantly lower 25(OH)D levels (p = 0.004) were found in PCR-positive for SARS-CoV-2 (median value 11.1 ng/mL) patients compared with negative patients (24.6 ng/mL); this was also confirmed by stratifying patients according to age >70 years. On the basis of this preliminary observation, vitamin D supplementation might be a useful measure to reduce the risk of infection. Randomized controlled trials and large population studies should be conducted to evaluate these recommendations and to confirm our preliminary observation.
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Infecciones por Coronavirus/sangre , Neumonía Viral/sangre , Vitamina D/análogos & derivados , Factores de Edad , Anciano , Anciano de 80 o más Años , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/diagnóstico , Suplementos Dietéticos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/diagnóstico , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , SARS-CoV-2 , Suiza , Vitamina D/administración & dosificación , Vitamina D/sangreRESUMEN
BACKGROUND: Doll therapy is a non-pharmacological intervention for people with dementia aimed to reduce distressing behaviours. Reliable results on the efficacy of Doll therapy for people with dementia are needed. The concept of attachment theorised by Bowlby has been proposed to explain the Doll therapy process, but it has not been proven to influence the response to doll presentation. METHODS/DESIGN: This single-blind, randomised controlled trial will involve people with dementia living in nursing homes of the Canton Ticino (Switzerland). Participants will be randomised to one of two interventions: Doll Therapy Intervention or Sham Intervention with a non-anthropomorphic object, using a 1:1 allocation ratio. The two interventions will consist of 30 daily sessions lasting an hour at most, led by a trained nurse for an hour at most. We will enrol 64 participants per group, according to power analysis using an estimated medium effect size (f = 0.25), an alpha level of 0.05, and a power of 0.8. The primary goal is to test the efficacy of the Doll Therapy Intervention versus the Sham Intervention as the net change in the following measures from baseline to 30 days (blinded outcomes): the Neuropsychiatric Inventory-Nursing Home administered by a trained psychologist blinded to group assignment, the professional caregivers' perceived stress scale of the Neuropsychiatric Inventory-Nursing Home, patients' physiological indices of stress (salivary cortisol, blood pressure and heart rate) and interactive behaviours. The secondary goal is to assess the relationship between attachment styles of people with dementia (detected by means of the Adult Attachment Interview to the patients' offspring) and their caregiving behaviours shown during the Doll Therapy Intervention. DISCUSSION: This is the first single-blind, randomised controlled trial on the efficacy of Doll therapy for dementia and an explanatory model of the response of people with dementia to doll presentation. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03224143. Retrospectively registered on 21 July 2017.
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Demencia/terapia , Casas de Salud , Ludoterapia/métodos , Cuidadores/psicología , Femenino , Humanos , Enfermeras y Enfermeros/psicología , Psicología , Método Simple Ciego , Suiza , Resultado del TratamientoRESUMEN
Doxorubicin treatment was found to augment the expression of the extracellular matrix (ECM) protein fibulin-1 in cultured human breast cancer cell lines and in MDA-MB-361 tumors grown in athymic mice. Doxorubicin was also found to augment tumor expression of the fibulin-1-binding proteins fibronectin and laminin-1. Growth of breast cancer cell lines on Matrigel, an ECM extract containing fibulin-1 and laminin-1, resulted in lower levels of doxorubicin-induced apoptosis as compared with controls. Moreover, tumors formed by injection of athymic mice with MDA-MB-361 cells mixed with Matrigel were significantly more doxorubicin resistant and displayed lower levels of apoptosis compared with those that formed in the absence of Matrigel. Monoclonal antibodies against fibulin-1 reversed Matrigel-dependent doxorubicin resistance. Furthermore, small interfering RNA-mediated suppression of fibulin-1 expression in breast cancer cells resulted in a 10-fold increase in doxorubicin sensitivity as compared with control cells. Together, these findings point to a role for fibulin-1 in breast cancer chemoresistance.
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Antibióticos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Unión al Calcio/fisiología , Doxorrubicina/farmacología , Animales , Neoplasias de la Mama/patología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
Nowadays the practice of therapeutic drug monitoring, consisting in the measurement of drugs concentrations in biological matrices in order to guide possible posological adjustments, is becoming more and more important for the management and optimization of several treatments, especially when drugs with narrow therapeutic indexes are administered. Although TDM on plasma samples is currently considered the gold standard, this practice shows some limitations: it requires venous blood sampling, centrifugation and, if necessary, shipment with refrigeration; moreover, drug concentrations in plasma or blood do not necessarily reflect the ones in the target tissues or cells. Therefore, in the recent years great attention has been given to alternative matrices for TDM purpose, in order to reduce invasiveness, costs or to obtain better information about drug concentrations at the active sites. This evolution is strongly sustained by the spreading use of liquid chromatography coupled with mass spectrometry (LC-MS) techniques for the analysis of small molecules, which is constantly increasing sensitivity and specificity of TDM assays. In this review, we present and summarize recently published LC-MS applications providing alternatives to plasma testing, in order to avoid blood withdrawal, plasma separation, refrigerated shipment or, if possible, to obtain better information about drug exposure in target cells. By analyzing the last 5 years of literature, reported in PubMed website, LC-MS/MS applications have been reported, with particular focus on the ones with higher probability to enter in the near future in clinical practice. Microsampling strategies and alternative biological matrices, from urine to tissue samples, have been included.
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Cromatografía Liquida/métodos , Monitoreo de Drogas/métodos , Espectrometría de Masas en Tándem/métodos , HumanosRESUMEN
Clinical endocrinology has always had a close relationship with laboratory medicine. In fact, the quantification of hormones is of great importance for diagnosis, treatment, recurrence and patient's prognosis of endocrine disorders. This review dealt with the role of the laboratory in diagnosing endocrine pathologies related to adrenal gland, pituitary, gonads and thyroid. The measurements of the main hormones (17-hydroxyprogesterone, androstenedione, dehydroepiandrosterone sulphate, testosterone, estradiol, cortisol, aldosterone, metanephrines, thyroglobulin and insulin-like-growth factor-1) were described considering analytical characteristics but also some aspects of preanalytical and postanalytical steps. Traditionally, hormonal quantification is performed with immunoassays (IMAs), which have several advantages (i.e. limited training of technicians, high throughput, widely spread worldwide), but also some limitations on the accuracy of the results due mainly to cross-reactivity of IMA antibodies (i.e. steroid measurements) and protein interferences (i.e. heterophilic antibodies, antithyroglobulin antibodies in Tg measurements, etc.). In order to meet the need for clinicians to obtain ever more accurate results from laboratories, in recent decades, mass spectrometry (MS) has been developed. MS, in particular Liquid Chromatography-Tandem MS (LC-MS/MS), is a more complex but also more flexible technique able to guarantee greater specificity. It also provides multi-parameter quantification in the same analytical session delineating steroid profiles and, in some cases, defining the appropriate reference range for each hormone. Similar to IMAs, LC-MS/MS shows some inter-method variability, limiting the goal of standardization. However, several studies have recently demonstrated the possibility of reaching harmonization of this technology with good expectations for the future.
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Enfermedades del Sistema Endocrino/sangre , Endocrinología/métodos , Hormonas/análisis , Espectrometría de Masas/métodos , Biomarcadores/análisis , Biomarcadores/sangre , Endocrinología/instrumentación , Hormonas/sangre , Humanos , Espectrometría de Masas/estadística & datos numéricosRESUMEN
PURPOSE: Since HCV infection may lead to hepatocellular carcinoma (HCC) and vitamin D (deficiency) is related to cancer, we investigated if SNPs in genes involved in vitamin D pathway could predict HCV-related HCC presence in patients treated with new anti-HCV drugs. METHODS: Patients with chronic hepatitis C and treated with direct-acting antivirals were enrolled. SNPs in VDR, CYP27B1, CYP24A1 and GC genes were assessed through real-time PCR. 258 patients were analyzed. RESULTS: HCC was present in six patients, all taking sofosbuvir, all males and five/six had cirrhosis. HCV-RNA log levels at baseline were statistically different between patients with and without HCC. VDR FokI T > C SNP resulted associated with HCC: all the CC patients were free from HCC. An association between HCC presence and undetectable HCV-RNA at 1 month of therapy was suggested; cirrhosis was related to HCC. HCC risk factors were age, ribavirin administration, IL28Brs12979860CC and previous treatments; VDR FokICC, sex and insulin resistance were protective factors. CONCLUSIONS: These data highlighted vitamin D pathway gene SNPs and HCC relationship in the Italian population; further studies are required.
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Carcinoma Hepatocelular/genética , Hepatitis C Crónica/complicaciones , Neoplasias Hepáticas/genética , Polimorfismo de Nucleótido Simple , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Antivirales/uso terapéutico , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/metabolismo , Femenino , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/virología , Humanos , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/metabolismo , Modelos Logísticos , Masculino , Persona de Mediana Edad , Receptores de Calcitriol/genética , Vitamina D3 24-Hidroxilasa/genéticaRESUMEN
LC-MS is becoming a standard for many applications, thanks to high sensitivity and selectivity; nevertheless, some issues are still present, particularly due to matrix effect (ME). Considering this, the use of optimal internal standards (ISs, usually stable-isotope labeled) is important, but not always possible because of cost or availability. Therefore, a deep investigation of the inter-lot variability of the ME and of the correcting power of the chosen IS (isotope-labeled or not) is mandatory. While the adoption of isotopically labeled ISs considered as a 'gold standard' to mitigate ME impact on analytical results, there is not consensus about the standard technique to evaluate it during method validation. In this paper, currently available techniques to evaluate, reduce or counterbalance ME are presented and discussed. Finally, these techniques were summarized in a flowchart for a robust management of ME, particularly considering the concept of 'internal standard normalized ME'.
Asunto(s)
Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Métodos Analíticos de la Preparación de la Muestra , Marcaje Isotópico , Estándares de ReferenciaRESUMEN
Electrospray Ionization and collision induced dissociation tandem mass spectrometry are usually employed to obtain compound identification through a mass spectra match. Different algorithms have been developed for this purpose (for example the nist match algorithm). These approaches compare the tandem mass spectra of the unknown analyte with the tandem mass spectra spectra of known compounds inserted in a database. The compounds are usually identified on the basis of spectral match value associated with a probability of recognition. However, this approach is not usually applied to multiple reaction monitoring transition spectra achieved by means of triple quadrupole apparatus, mainly due to the lack of a transition spectra database. The Surface Activated Chemical Ionization-Electrospray-NIST Bayesian model database search (SANIST) platform has been recently developed for new potential metabolite biomarker discovery, to confirm their identity and to use them for clinical and diagnostic applications. Here, we present an improved version of the SANIST platform that extends its application to forensic, pharmaceutical, and food analysis studies, where the compound identification rules are strict. The European Union (EU) has set directives for compound identification (EU directive 2002/657/EC). We have applied the SANIST method to identification of 11-nor-9-carboxytetrahydro-cannabinol in urine samples (an example of a forensic application), circulating levels of the immunosuppressive drug tacrolimus in blood (an example of a pharmaceutical application) and glyphosate in fruit juice (an example of a food analysis application) that meet the EU directive requirements. Copyright © 2016 John Wiley & Sons, Ltd.