Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.

Aims: Characterization of PSA in extracellular microvesicles (EVs) and its reactivity to commercial methods. Materials and methods: EVs derived from serum of 47 prostate cancer (PCa) patients, 27 benign prostatic hyperplasia (BPH) patients and 42 healthy controls were analyzed. EVs isolation and quantification of PSA immunoreactive to total (ev-T-PSA) or free (ev-F-PSA) PSA immunoassays, were performed using commercial assays. PSA in CD81+ or CD63+ EVs was determined directly in serum by an immunocapture-ELISA (IC-ELISA). Results: Ev-T-PSA immunoreactive to Elecsys assay was detected in all samples. Median T-PSA ev/srm ratio was 2.20 % (Q1-Q3: 0.80-4.00 %), although in some samples this ratio reached 59 %. T-PSA ev/srm ratio was higher in those samples with serum T-PSA below 4 µg/L than in those exceeding that cut-off (p < 0.001). T-PSA ev/srm ratio was lower in PCa patients compared to healthy controls and BPH patients (p < 0.001). Elecsys immunoassays detected higher concentrations of ev-T-PSA and ev-F-PSA than Immulite (p < 0.001). PSA was detected by IC-ELISA more intensely in CD81+ EVs than in CD63+ EVs, and ev-T-PSA correlated with PSA+ CD63+ (p < 0.001) but not with PSA+ CD81+. Conclusion: EVs-bound PSA is another form of circulating PSA whose measurement could be easily performed in clinical laboratories by automated immunoassays.

Objectives Laboratory Medicine is a crucial discipline that contributes to the diagnosis, management and monitoring of patients. This branch of medicine faces two major challenges: New technologies and increased demand. There is limited information available of the state of affairs in Laboratory Medicine in Spain. This study provides a picture of clinical laboratories and clinical laboratory professionals. Methods The Spanish Society of Laboratory Medicine distributed a questionnaire among the 250 most representative centers (the ones with the largest volume of determinations and training programs), of which 174 (69.6%) returned the questionnaire providing data for 2019. Results Laboratories were classified according to the number of determinations. In total, 37% identified themselves as small (<1 million determinations per year); 40% considered themselves medium-sized (1¿5 million determinations per year) and 23% considered they were large laboratories (>5 million determinations). The level of specialization of laboratory physicians and laboratory performance were higher in large laboratories. Most requests (87%) and determinations (93%) corresponded to biochemistry and hematology. As many as 63% of physicians had an indefinite contract, and 23% were older than 60 years. Conclusions Laboratory medicine is a consolidated discipline that is gaining relevance in Spain. It adds value to the diagnosis, prognosis and follow-up of diseases, and to treatment response monitoring. The results of this study will help us address challenges such as the need for specialized training for laboratory professionals; the emergence of technological innovations; exploitation of Big Data; optimization of quality management systems and patient safety.

Background: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. Results: Here we describe a method that, using just a few microliters of patient's plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV zeta-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. Conclusions: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.

Objectives: To assess the impact of the COVID-19 pandemic on the activity of clinical laboratories in Spain.Methods: A descriptive, observational, retrospective, multicenter study.Results: Between March and December 2020, there was a statistically significant decrease in the number of test requests (-17.7%, p=< 0.001) and total tests performed (-18.3%, p < 0.001) with respect to the same period in 2019. A decrease was observed in the number of requests from primary care (-37.4%) (p < 0.001) and in the number of foecal occult blood (-45.8%); qualitative urine (-30.1%); PSA (-28.5%); TSH (-27.8%); total cholesterol (-27.2%) and HbA(1c) (-24.7%) tests performed, p < 0.001. A significant increase was found in the number of requests from ICUs (76.6%, p < 0.001) and number of IL-6 (+22,350.9), D-dimer (+617.2%), troponin (+46.8%) and arterial blood gas (+3.9%) tests carried out, p < 0.001. During the first months of 2021, there were significant changes in the number of requests for qualitative urine (-8.7%, p < 0.001), PSA (-6.3%, p=0.009), IL-6 (+66,269.2, p < 0.001), D-dimer (+603.6%, p < 0.001), troponin (+28.7%, p < 0.001), arterial blood gas (+26,2%, p=0.014) and ferritin (+16.0%, p=0.002) tests performed.Conclusions: There were changes in the origin and number of test requested to clinical laboratories in Spain. The number of requests for the evaluation and monitoring of COVID-19 patients increased, whereas requests for the control of non-COVID patients and for population screening decreased. Long-term analysis reveals that the volume of tests performed for the control of chronic diseases returned to normal over time, whereas the increase observed in the volume of tests performed for the management of COVID-19 patients is maintained.

Scarce data have been reported about cellular immunity and longevity for different COVID-19 vaccination schedules. We carried out a prospective study enrolling 709 healthcare workers receiving two doses of mRNA-1273, BNT162b2, ChAdOx1, ChAdOx1/BNT162b2 or ChAdOx1 single dose to compare humoral and cellular immunogenicity across 9 months. Higher SARS-CoV-2 spike antibody levels were observed among individuals with hybrid immunity with one dose of any vaccine in comparison to uninfected individuals receiving two doses (mRNA-1273: 20,145 vs 4295 U/mL; BNT162b2: 15,659 vs 1959 U/mL; ChAdOx1: 5344 vs 2230 U/mL), except for ChAdOx1/BNT162b2 heterologous schedule (12,380 U/mL). Naturally infected individuals did not increase substantially the titers after the second dose and showed higher levels throughout the 9 months follow-up. The mean elimination half-life of antibodies among COVID-19 naive participants was 98, 111, 60 and 36 days, for mRNA-1273, BNT162b2, ChAdOx1/ChAdOx1 and ChAdOx1/BNT162b2, respectively. Cellular immunity was preserved in 96%, 98%, 88% and 92% of uninfected individuals who received mRNA-1273, BNT162b2, ChAdOx1/ChAdOx1 and ChAdOx1/BNT162b2 after 6/9 months. Individuals with specific T cells showed robust long lasting protection, especially when m-RNA based vaccines are inoculated. These data may influence the validity of the vaccination passport and the need for booster vaccinations.

Objectives Retrospective studies frequently assume analytes long-term stability at ultra-low temperatures. However, these storage conditions, common among biobanks and research, may increase the preanalytical variability, adding a potential uncertainty to the measurements. This study is aimed to evaluate long-term storage stability of different analytes at Methods Twenty-one analytes commonly measured in clinical laboratories were quantified in 60 serum samples. Samples were immediately aliquoted and frozen at <-70 degrees C, and reanalyzed after 11 +/- 3.9 years of storage. A change in concentration after storage was considered relevant if the percent deviation from the baseline measurement was significant and higher than the analytical performance specifications. Results Preanalytical variability (CVP) due to storage, determined by the percentage deviation, showed a noticeable dispersion. Changes were relevant for alanine aminotransferase, creatinine, glucose, magnesium, potassium, sodium, total bilirubin and urate. No significant differences were found in aspartate aminotransferase, calcium, carcinoembryonic antigen, cholesterol, C-reactive protein, direct bilirubin, free thryroxine, gamma-glutamyltransferase, lactate dehydrogenase, prostate-specific antigen, triglycerides, thyrotropin, and urea. As nonnegligible, CVP must remain included in reference change value formula, which was modified to consider whether one or two samples were frozen. Conclusions After long-term storage at ultra-low temperatures, there was a significant variation in some analytes that should be considered. We propose that reference change value formula should include the CVP when analyzing samples stored in these conditions.

Breath tests used to evaluate carbohydrates malabsorption require baseline H2 and CH4 levels as low as possible. Test cancellation is recommended when exceeding certain cut-offs (H2 ¿ 20 ppm and CH4 ¿ 10 ppm). Although following preparation protocols, many patients have baseline levels above those cut-offs. We investigated if light walking can reduce baseline H2 and CH4 levels. We retrospectively analyzed baseline H2 and CH4 levels from 1552 breath tests. Baseline levels (B1), especially in H2, were lower when obtained at later hours of the day. In those with baseline levels above cut-off, re-sampling (B2) after light walking for one hour, decreased H2 levels 8 ppm (Q1-Q3: 1-18 ppm), and 2 ppm (Q1-Q3: 0-3 ppm) for CH4. Consequently, 40% of tests with elevated B1 levels, presented B2 levels below mentioned cut-offs. Ten percent of tests considered negative when using B1 for calculations, turned positive when using B2 instead. All positive tests when using B1 values, remained elevated when using B2. Re-sampling after light walking for one hour could allow test performance in those with previous elevated baseline levels, avoiding diagnosis delays. Using the second sample for delta calculations identifies positive patients for malabsorption that would have been considered negative.

Phase I/II translational trial evaluating the molecular determinants of pazopanib plus interferon alpha efficacy in advanced renal cell carcinoma. The expression levels of TNF-alpha, endoglin and PD-L1 correlated with response at eight weeks after treatment initiation. This suggests a crucial role of vascular remodelling and inflammatory-mediated immune cell infiltration for an optimal response to pazopanib plus interferon alpha combination. Introduction: The therapeutic repertoire available for advanced renal cell carcinoma (RCC), including tyrosine kinase inhibitors (TKIs) and immunotherapy, required for molecular biomarkers for response. Patients and Methods: This was a phase I to II trial on the combination of pazopanib with interferon-alpha (INF-2A) as first-line treatment for advanced RCC. The primary endpoint was recommended phase II dose (RP2D) and efficacy in terms of objective response rate (ORR, RECIST 1.1 criteria). Secondary endpoints included safety and a translational study of molecular biomarkers in serum and exosomes from peripheral blood samples at three-time points: baseline, 8 weeks of treatment, and progression of the disease Results: Between July 2011 and July 2017, 53 eligible patients were treated and followed up (I, n = 20; II, n = 33). Pazopanib 800 mg + INF-2A 3 MIUs showed a manageable safety profile; therefore, it was selected for dose expansion. Overall, grade 3/4 toxicities were reported in 24 (72.7%) patients. The ORR was 27.2%. The 12-month OS rate was 83.6% (median not reached), and after 30.9 months of follow-up, 24 (72.7%) patients were still alive, CCL2, IL8, TNF-alpha, and PD-L1 were significantly overexpressed after treatment initiation, while TGF-beta 1 and CCL5 were significantly decreased. TNF-alpha, endoglin, and PD-L1 expression are correlated with the response after treatment initiation. Conclusion: The trial did not reach its pre-specified target ORR. However, OS was longer than expected with pazopanib monotherapy. Changes in the molecular profile suggest a crucial role of vascular remodeling and infIammatory-mediated immune cell infiltration in optimal response to pazopanib plus INF-2A. (C) 2022 Elsevier Inc. All rights reserved.

Nueva edición de la obra en el campo de la bioquímica clínica que año tras año se está convirtiendo en una herramienta fundamental para la toma de decisiones del clínico, ya que gran parte de ellas se basan en los datos proporcionados por el laboratorio que necesitan por una interpretación adecuada teniendo en cuenta los diferentes factores preanalíticos y analíticos que puedan influir en ellos. Uno de las novedades más importantes de la bioquímica clínica es que ha ido incorporando progresivamente técnicas propias de la biología molecular y la proteómica.

2ª edición 2014

Liquid biopsy refers to the molecular analysis in biological fluids of nucleic acids, subcellular structures, especially exosomes, and, in the context of cancer, circulating tumor cells. In the last 10 years, there has been an intensive research in liquid biopsy to achieve a less invasive and more precise personalized medicine. Molecular assessment of these circulating biomarkers can complement or even surrogate tissue biopsy. Because of this research, liquid biopsy has been introduced in clinical practice, especially in oncology, prenatal screening, and transplantation. Here we review the biology, methodological approaches, and clinical applications of the main biomarkers involved in liquid biopsy.

Complejo de glucoproteínas asociadas con la distrofina, distrofias musculares de Duchenne y Becker, técnicas bioquímicas diagnósticas de las distrofias musculares de Duchenne y Becker, otras distrofias.