Laura Macias-Muñoz

Introduction This study assesses the accuracy of neutrophil activation markers, including neutrophil extracellular traps (NETs) and calprotectin, as biomarkers of disease activity in patients with established rheumatoid arthritis (RA). We also analyse the relationship between NETs and various types of therapies as well as their association with autoimmunity. Methods Observational cross-sectional study of patients with RA receiving treatment with biological disease-modifying antirheumatic drugs or Janus kinase inhibitors (JAK-inhibitors) for at least 3 months. Plasma calprotectin levels were measured using an enzyme-linked immunosorbent assay test kit and NETs by measuring their remnants in plasma (neutrophil elastase-DNA and histone-DNA complexes). We also assessed clinical disease activity, joint ultrasound findings and autoantibody status [reumatoid factor (RF), anti-citrullinated peptide/protein antibodies (ACPAs) and anti-carbamylated protein (anti-CarP)]. Associations between neutrophilic biomarkers and clinical or ultrasound scores were sought using correlation analysis. The discriminatory capacity of both neutrophilic biomarkers to detect ultrasound synovitis was analysed through receiver-operating characteristic (ROC) curves. Results One hundred fourteen patients were included. Two control groups were included to compare NET levels. The active control group consisted of 15 patients. The second control group consisted of 30 healthy subjects. Plasma NET levels did not correlate with clinical disease status, regardless of the clinic index analysed or the biological therapy administered. No significant correlation was observed between NET remnants and ultrasound synovitis. There was no correlation between plasma NET and autoantibodies. In contrast, plasma calprotectin positively correlated with clinical parameters (swollen joint count [SJC] rho = 0.49; P < 0.001, Clinical Disease Activity Index [CDAI] rho = 0.30; P < 0.001) and ultrasound parameters (rho > 0.50; P < 0.001). Notably, this correlation was stronger than that observed with acute phase reactants. Conclusion While NET formation induced by neutrophils may play a role in RA pathogenesis, our study raises questions about the utility of NET remnants in peripheral circulation as a biomarker for inflammatory activity. In contrast, this study strongly supports the usefulness of calprotectin as a biomarker of inflammatory activity in patients with RA.

Objectives: Chronic liver disease and related complications, cirrhosis and hepatocellular carcinoma, are associated with high mortality. Curative treatments, partial hepatectomy or liver transplantation, have limited applicability in patients with cirrhosis due to the poor liver regenerative capacity. Thus, we need to find new diagnostic and therapeutic alternatives, to block the disease progression and to improve the survival of patients. In this context, preclinical studies have demonstrated the key role of the protein kinase B (Akt) in liver dysfunction, but the status of Akt and its targets in patients with chronic hepatopathy remains unknown. Aims: To determine the activation status of the Akt pathway and their association with liver functionality in cirrhotic patients. Methods: This retrospective study includes liver tissue samples from 36 hepatectomized patients with (n=27) and without (n=9) cirrhosis. Multiplex analysis of proteins involved in the Akt/mTOR pathway was performed using a Luminex panel and Western blot. Conventional liver function tests were determined in serum before resection surgery. Results: Akt and forkhead box protein O1 (FoxO1) are overexpressed in the liver of cirrhotic patients: (2.1 vs. 1.0 densitometric relative units (DRU); p. 4.4 DRU; p<0.01, respectively). FoxO1 showed the best correlation with markers of liver injury (aspartate aminotransferase (ASAT): r=0.51, p<0.05; alanine aminotransferase (ALAT): r=0.49, p<0.05), and was the only enzyme in the Akt pathway identified as an independent predictor of ASAT and ALAT levels. Conclusions: The intrahepatic expression of FoxO1 could have clinical utility as a potential prognostic marker for patients with advanced liver disease.

Article Reporting magnesium critical results: clinical impact on pregnant women and neonates was published on August 29, 2022 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 0, issue 0).

Background: Calprotectin is a calcium‐binding protein that can be measured in serum, plasma, and feces. Increased serum and plasma calprotectin concentrations have been found in chronic inflammatory rheumatic disorders. An analytical and clinical evaluation of the DiaSorin Liaison® fecal Calprotectin assay using LIAISON® XL was performed.Methods: The protocol included an analytical and clinical evaluation in which imprecision, the linearity of dilution, differences between serum and plasma samples and method comparison with CalproLab™ ELISA kit were assessed. Serum calprotectin concentrations in active (n = 26) and remission (n = 23) rheumatoid arthritis (RA) patients were compared.Results: The intra‐day and inter‐day analytical imprecision CVs ranged from 2.9% to 4.0% and 2.7% to 10.4%, respectively. Correlation between measured and expected values was high (R > 0.99), indicating good linearity. The Wilcoxon signed‐rank test showed that serum and plasma matched samples presented statistically significant differences (p < 0.001) being the highest concentrations of calprotectin observed in serum samples. Deming regression equation was as follows: Diasorin calprotectin (μg/ml) = −0.32 (95% CI: −0.65 ‐ −0.05) +1.58 (95% CI: 1.42–1.79).* Calprolab calprotectin (μg/ml). Significantly higher serum calprotectin levels were found in RA patients with active disease when compared to patients with low disease activity or in clinical remission (mean ± SD) [(3.35 μg/ml ± 1.55) vs. (1.63 μg/ml ± 0.52), p < 0.001] and these levels correlated well with all disease activity indices.Conclusions: The DiaSorin Liaison® fecal Calprotectin assay adapted for serum samples showed adequate technical performances and the clinical performances were similar to other assays.

Objectives: The communication of critical results (CR) is considered an essential role in clinical laboratories to ensure patient safety. This is especially relevant to outpatients, who are non-hospitalized and more difficult to locate. In our laboratory, there is a specific protocol for CR management that sets up the communication pathway to adequately provide these results to clinicians. The aim of this study is to evaluate the impact of CR reporting on outpatient care. Methods: This is a retrospective study focused on CR for biochemistry parameters in a clinical laboratory of a Spanish tertiary hospital during 2019. A total of 156 CR were determined and properly provided to clinicians. We collected CR, age, gender, and the requesting department. We also collected the medical action data resulting from the communication of the CR. Results: Seventy-six outpatients (49%) were properly treated because of effective CR communication. Hypoglycemia was the most frequent event (33%), however, the greatest clinical impact was observed for patients with hyponatremia (100%), hyperkalemia (62%), hypokalemia (60%), and hypercalcemia (57%). Based on these findings, we evaluated new glucose alert thresholds depending on whether or not the outpatient was diabetic (1.7 and 2.2 mmol/L, respectively). Based on these new thresholds, we established a CR reporting protocol with 69% effectiveness in outpatients. Conclusions: We demonstrate that CR communication in outpatients has a significant clinical impact. To increase the effectiveness of the CR reporting protocol, we propose to adjust alert thresholds according to pathology, department, and patient population.

Background: Mother’s own milk does not provide enough nutrients to feed a preterm baby born before 32 weeks’ gestation; therefore, human milk fortifiers are needed. However, human milk fortifiers increase the osmolality, and enteral administration of high osmolality fluids has been associated with gastrointestinal symptoms. For this reason, it is necessary for laboratories to have a validated system in order to measure human milk osmolality. Research aim: The aim of this study was to validate the OM-6050 Station System for measuring the osmolality of fortified mother’s milk samples. Methods: Osmolality was measured using the osmometer OM-6050 Station System. Milk samples from healthy mothers ( N = 3) unfortified and with two fortifiers (Almirón Fortifier ^® or NAN FM85 ^® ), as well as a nutritional supplement (Duocal MCT ^® ) were used in the validation study through precision and linearity analysis. Results: In the precision study the mean intra-assay coefficient of variation was 1.2% and 1.7% for mother’s milk and fortified mother’s milk, respectively. The mean inter-assay coefficient of variation was ≤ 1% in both cases. In the linearity study the regression analysis had a linear response to fortified mother’s milk osmolality between 294 mOsm/kg and 539 mOsm/kg. Conclusion: The osmometer OM-6050 Station was reliable for determining the osmolality of fortified and unfortified mother’s milk. It may be useful in the clinical practices within Neonatal Intensive Care Units.

Introduction: Chronic liver disease is associated with high mortality. Liver transplantation is the definitive treatment for patients with end-stage liver disease, improving their survival and quality of life. However, chronic rejection of the graft and the imbalance between the demand and the availability of organs limit its applicability. Therefore, finding therapeutic and/or diagnostic alternatives for these patients is a priority. In this context, preclinical studies in rodents have demonstrated that Akt plays a key role in liver dysfunction. Even with all this evidence, the activation status of Akt and its downstream targets in the liver of patients with chronic hepatopathy is still unknown. Hence, the present study aims to determine the activation status of the molecules involved in the Akt signaling pathway in livers of cirrhotic patients. Materials and Methods: In this study, 36 liver tissue samples from a cohort of 27 cirrhotic patients and 9 patients without cirrhosis were included. A total of 10 proteins involved in Akt/mTOR pathway (GSK3β, IGF1R, IRS1, mTOR, p70S6K, IR, PTEN, GSK3α, TSC2, and RPS6) were analyzed using a multiplex immunoassay based on Luminex® technology. Results: Significant differences were found in several Akt/mTOR target proteins between the groups of cirrhotic patients vs. non-cirrhotic: FoxO1 (9.5 vs. 4.4; p p p p p p <0.05). Moreover, the individual influence of FoxO1 on these parameters was confirmed by multiple regression analysis. It was the only enzyme in the Akt signaling pathway identified as a positive independent predictor of increased ASAT and ALAT levels. Conclusion: FoxO1 is overexpressed in the liver of cirrhotic patients after partial hepatectomy. FoxO1 levels are also associated with the degree of liver injury, showing a positive correlation with current biomarkers used in clinical practice to detect liver injury.

Resumen: Introducción: El estradiol en suero (E2) se emplea para el diagnóstico y/o seguimiento de una amplia variedad de patologías. El objeto del presente estudio es evaluar el ensayo enhanced estradiol (eE2) de Siemens en el sistema Atellica IM 1600 (Siemens Healthineers) y llevar a cabo una comparación de muestras con el analizador Advia Centaur XP (Siemens Healthineers). Métodos: Los coeficientes de variación (CV) intradía e interdía se evaluaron utilizando pools de muestras séricas y materiales de control de calidad. El límite de cuantificación se determinó mediante el empleo de seis muestras de suero con concentraciones decrecientes de E2. La linealidad fue evaluada a través del análisis de dos muestras diferentes de suero. La exactitud se determinó midiendo tres materiales de referencia certificados. Comparamos los sistemas Atellica y Centaur XP con el método de regresión de Passing–Bablok y el gráfico de Bland–Altman utilizando 119 muestras de suero con concentraciones de entre 45 y 10.059 pmol/L. Resultados: La imprecisión intradía e interdía fue <6,6%. La exactitud fue <6,0% en todos los valores de E2 evaluados excepto para la concentración de 114 pmol/L (22%). El análisis del límite de cuantificación resultó en una imprecisión interdía <20%. Se observó una alta correlación entre los valores de dilución de E2 observados y esperados (r=0,99), con un porcentaje de recuperación que oscila entre el 77 y el 93%. El estudio comparativo mostró un buen nivel de concordancia, sin hallarse un sesgo significativo. Conclusiones: Este estudio indica que el ensayo eE2 tiene una imprecisión y una exactitud aceptables, mostrando una buena correlación con el sistema Centaur XP.

Background and Aims: Despite the availability of new‐generation drugs, hepatocellular carcinoma (HCC) is still the third most frequent cause of cancer‐related deaths worldwide. Cerium oxide nanoparticles (CeO₂NPs) have emerged as an antioxidant agent in experimental liver disease because of their antioxidant, anti‐inflammatory, and antisteatotic properties. In the present study, we aimed to elucidate the potential of CeO₂NPs as therapeutic agents in HCC. Approach and Results: HCC was induced in 110 Wistar rats by intraperitoneal administration of diethylnitrosamine for 16 weeks. Animals were treated with vehicle or CeO₂NPs at weeks 16 and 17. At the eighteenth week, nanoceria biodistribution was assessed by mass spectrometry (MS). The effect of CeO₂NPs on tumor progression and animal survival was investigated. Hepatic tissue MS‐based phosphoproteomics as well as analysis of principal lipid components were performed. The intracellular uptake of CeO₂NPs by human ex vivo perfused livers and human hepatocytes was analyzed. Nanoceria was mainly accumulated in the liver, where it reduced macrophage infiltration and inflammatory gene expression. Nanoceria treatment increased liver apoptotic activity, while proliferation was attenuated. Phosphoproteomic analysis revealed that CeO₂NPs affected the phosphorylation of proteins mainly related to cell adhesion and RNA splicing. CeO₂NPs decreased phosphatidylcholine‐derived arachidonic acid and reverted the HCC‐induced increase of linoleic acid in several lipid components. Furthermore, CeO₂NPs reduced serum alpha‐protein levels and improved the survival of HCC rats. Nanoceria uptake by ex vivo perfused human livers and in vitro human hepatocytes was also demonstrated. Conclusions: These data indicate that CeO₂NPs partially revert the cellular mechanisms involved in tumor progression and significantly increase survival in HCC rats, suggesting that they could be effective in patients with HCC.

Background: Serum estradiol (E2) levels may be used in the diagnostic and/or monitoring of a broad variety of clinical conditions. The aims of this study were to evaluate the Siemens enhanced estradiol assay (eE2) on Atellica IM 1600 (Siemens Healthineers) and to perform a sample comparison with the Siemens ADVIA Centaur XP (Siemens Healthineers). Methods: Within-day and between-day coefficient of variation (CV) were determined using serum sample pools and quality control materials. Six serum samples with decreasing concentrations of E2 were used to assess the limit of quantification. Linearity was evaluated using two different serum samples. Accuracy was evaluated by measuring three certified reference materials. Passing–Bablok regression and Bland–Altman plot were used for comparing Atellica and Centaur XP in 119 serum samples ranging from 45 to 10,059 pmol/L. Results: Within-day and between-day imprecision was R = 0.99), with recoveries ranging from 77 to 93%. Comparison study showed good agreement and no significant bias. Conclusions: The study shows that Atellica eE2 assay presents acceptable imprecision and accuracy and correlates well with Centaur XP.