Placental cartography of NADPH oxidase (NOX) family proteins: Involvement in the pathophysiology of preeclampsia.

The placenta is an essential organ for fetal development. During the first trimester, it undergoes dramatic changes as it develops in an environment poor in oxygen (around 2-3%). From about 10 gestational weeks, oxygen levels increase to 8% in the intervillous chamber. These changes are accompanied by modulation of the activity of NADPH oxidase, a major source of production of reactive oxygen species in the first trimester of pregnancy. The NOX complex is composed of seven different proteins (NOX1-5 and DUOX1-2) whose placental involvements during physiological and pathological pregnancies are largely unknown. The aim of the study was to produce a cartography of NOX family proteins, in terms of RNA, protein expression, and localization during physiological pregnancy and in the case of preeclampsia (PE), in a cohort of early-onset PE (n = 11) and late-onset PE (n = 7) cases. NOX family proteins were mainly expressed in trophoblastic cells (NOX4-5, DUOX1) and modulated during physiological pregnancy. NOX4 underwent an unexpected and hitherto unreported nuclear translocation at term. In the case of PE, two groups stood out: NOX1-3, superoxide producers, were down-regulated (p < 0.05) while NOX4-DUOX1, hydrogen peroxide producers, were up-regulated (p < 0.05), compared to the control group. Mapping of placental NOX will constitute a reference and guide for future investigations concerning its involvement in the pathophysiology of PE.

Analytical performance of Abbott C-16000 analyser haemolysis index and its potential use in measuring plasma cell-free haemoglobin.

Purpose: Measurement of the haemolysis index (HI) is usually performed in clinical chemistry laboratories in order to inform about whether biological analyses are influenced by in vivo or in vitro haemolysis of the specimen. Our aim was to evaluate the analytical performance of Abbott C-16000 analyser HI measurement in order to determine whether this could be used to reliably measure cell-free haemoglobin (fHB) in plasma samples. Methods: The repeatability, reproducibility, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) of C-16000 HI measurement were determined as well as the potential interference of bilirubin, triglycerides and myoglobin. C-16000 HI values of biological samples with various ranges of fHB were compared to those measured using the established reference method, second-derivate spectroscopy. Results: Results: C-16000 HI determination showed excellent linear correlation with the reference method (y = 1.0043x ­ 1.248, R² = 0.998), a broad analytical measurement range (400-20,000 mg/L; y = 0.9904x + 72.972, R² = 0.999), clinically relevant LLOD (56 mg/L) and LLOQ (84 mg/L), good repeatability (coefficient of variation (CV) = 1-15%) and good reproducibility (CV = 5-7%). No interference was observed with myoglobin at concentrations as high as 35,447 mg/L, unconjugated and conjugated bilirubin (at concentrations up to 500 mg/L and 375 mg/L, respectively) or triglycerides up to 6.8 mmol/L. However, a significant underestimation of fHB concentrations was observed at higher triglyceride levels. Conclusion: This study demonstrates that Abbott C-16000 analyser HI is reliable and accurately measures plasma fHB concentrations under pathophysiological conditions except when there are high blood concentrations of triglycerides.

[Detection and identification of urinary crystals using an automated urine particle analyzer coupled with a digital particle imaging device]. / Détection et identification de cristaux dans les urines à l'aide d'un analyseur de cytologie urinaire couplé à un dispositif d'imagerie numérique.

Polarized light microscopy (POM) remains the gold standard for crystalluria analysis. However, such method is time consuming and requires well-trained staff. Here, to address this issue, we tested the Sysmex UF-4000 analyzer coupled to a UD10 module as an automated flow cytometry-digital particle imaging workflow to assess (i) the ability of the system to detect and identify the crystals species and (ii) the quality of the images provided by the UD-10 module (n = 40) for each urine sample analyzed. First, systematic analysis of 76 samples by POM and the UF-4000/UD-10 analyzer showed that only attentive examination of the 40 photos was able to confidently detect crystalluria-positive samples with no misses and thus serve to discriminate positive-test crystalluria from negative-test crystalluria. These first results were confirmed by sensitivity analysis and the negative predictive value calculated on 200 samples for the results provided by the UF-4000 (39% and 46%) and after examination of the 40 UD-10 photos (100% for the both values). Digital images can therefore serve to screen crystalluria without missing crystals. A part of samples were treated by POM whereas it was not necessary (positive predictive value: 78%). Finally, we compared the crystal identification performances of the Sysmex UF4000/UD10 workflow and the 'gold standard' POM method on 131 urine samples containing crystals. Only calcium oxalate dihydrate crystals were identified by the Sysmex UF-4000. A close examination of the digital photographs enabled exact identification of crystals in 84.7% of the samples, suggesting however that POM is still require as soon as crystals are observed on the photographs. We conclude that a SYSMEX UF-4000 coupled with a UD-10 module can be used in practice with close examination of the photographs to discriminate positive crystalluria from negative crystalluria.

Clinico-biological markers for the prognosis of status epilepticus in adults.

Prediction of mortality, functional outcome and recovery after status epilepticus (SE) is a challenge. Biological and clinical markers have been proposed to reflect the brain injury or to monitor critical ill patients' severity. The aim of this study was to characterize short-term and long-term prognostic factors for SE patients hospitalized in intensive care unit. Patient's outcome was assessed using the modified Rankin Scale at discharge and after 6-12 months. We first assessed the univariate prognosis significance of 51 clinical, demographic or biochemical markers. Next, we built multivariate clinico-biological models by combining most important factors. Statistical models' performances were compared to those of two previous published scales STESS and mSTESS. Eighty-one patients were enrolled. Thirty-five patients showed a steady state while 46 patients clinically worsened at discharge: 14 died, 14 had persistent disability at 6-12 months and 18 recovered. Logistic regression analysis revealed that clinical markers (SE refractoriness, SE duration, de novo SE) were significant independent predictors of worsening while lipids markers and progranulin better predicted mortality. The association of clinico-biological variables allowed to accurately predict worsening at discharge (AUC > 0.72), mortality at discharge (AUC 0.83) and recovery at long-term (AUC 0.89). Previous scales provided lower prediction for worsening (AUC 0.63, STESS; 0.53, mSTESS) and mortality (AUC 0.56, STESS; 0.62, mSTESS) (p < 0.001). We proposed new clinico-biological models with a strong discrimination power for prediction of short- and long-term outcome of hospitalized status epilepticus patients. Their implementation in electronic devices may enhance their clinical liability.

Comparison of blood gas results obtained on Abbott i-Stat® and on Radiometer ABL 800 Flex® analyzers Impact for the clinical decision

Background: Point of care testing (POCT) tests are needed to assess severity and to help for triage in hospital and in prehospital settings. Before their use, the analytical performances of POCTs have to be compared with central laboratory reference methods. In this study, we describe the comparability of results obtained by either the Abbott i-STAT® System POCT handheld device or the blood gases analyzer of the central laboratory of our hospital. Methods: Sample blood from 37 septic patients admitted to the intensive care unit (ICU) were assayed by Abbott i-STAT® System POCT and Radiometer ABL800 Flex® lab analyzer. Studied parameters were as follows: pH, pO2, pCO2, base excess (BE), HCO3- and lactate. Comparability was evaluated using Bland-Altman method. The clinical value for possible mismatch issued of values differences was also assessed. Results: Quite acceptable correlations in results of POCT and laboratory analyzer were observed with R² most of time above 0.85. Bland-Altman analysis showed a bias of 1.26% for Abbott i-STAT® System POCT vs laboratory. Conclusion: Abbott i-STAT® System POCT handheld device is comparable to Radiometer ABL800 Flex® lab analyzer and concordant with laboratory analysis. Abbott i-STAT® System POCT handled device could be used in the prehospital settings in order to evaluate the severity of sepsis.

Contexte: Les dispositifs médicaux de biologie délocalisée peuvent être utiles pour évaluer la gravité et aider au triage des patients à la fois en milieu hospitalier mais aussi en milieu préhospitalier. Avant leur utilisation, les performances analytiques de ces dispositifs médicaux doivent être comparées aux méthodes de référence des laboratoires hospitaliers. Dans cette étude, nous décrivons la comparabilité des résultats obtenus soit par le dispositif portable Abbott i-STAT® avec ceux fournis par l'analyseur de gaz du sang du laboratoire central de notre hôpital. Méthodes: Des échantillons de sang provenant de 37 patients septiques admis en réanimation ont été analysés par le système Abbott i-STAT® et l'analyseur de laboratoire Radiometer ABL800 Flex®. Les paramètres étudiés étaient les suivants : pH, pO2, pCO2, excès de base (BE), HCO3- et lactate. La comparabilité a été évaluée par la méthode de Bland-Altman. La valeur clinique de l'éventuelle inadéquation issue des différences de valeurs a également été évaluée. Résultats: Des corrélations tout à fait acceptables entre les résultats du système Abbott i-STAT® et de l'analyseur de laboratoire ont été observées, avec un R² le plus souvent supérieur à 0,85. L'analyse Bland-Altman a montré un biais de 1,26 % pour le système Abbott i-STAT® par rapport aux résultats fournis par l'analyseur du laboratoire. Conclusion: Le dispositif de biologie délocalisée Abbott i-STAT® est comparable et concordant avec l'analyseur de laboratoire Radiometer ABL800 Flex®. Le dispositif Abbott i-STAT® System POCT pourrait être utilisé en milieu préhospitalier afin d'évaluer la gravité de patients atteints de sepsis.

[Plasmatic and urinary pyroglutamic acid measurement by capillary zone electrophoresis in chronic poisoning with acetaminophen in children]. / Dosage plasmatique et urinaire de l'acide pyroglutamique par électrophorèse capillaire de zone dans le cadre d'intoxication chronique par le paracétamol chez l'enfant.

An increase of pyroglutamic acid, or 5-oxoproline plasmatic concentration was reported in metabolic acidosis observed after chronic intake of some drugs, as acetaminophen. We developed a simple, fast and reproducible method by capillary zone electrophoresis using a commercial Anion Analysis Kit® to quantify pyroglutamic acid, in plasma after acetonitrile precipitation, and after simple dilution in urines. Fumaric acid was used as internal standard in both. In less than 7 min, the method separates pyroglutamic acid from other organic and inorganic anions. The method is linear between 0.25 and 10 mmol/L in plasma, and 0.15 and 10 mmol/L in urines. The quantification limits are 0.25 mmol/L and 0.15 mmol/L for plasma and urines, respectively. For repeatability and intermediate precision, the variation coefficients are less than 15% and the bias values are between ± 10%. For the 2 matrices, the recoveries are between 88% and 101%. The method does not interfere with physiological organic and inorganic anions. Pyroglutamic acid concentrations measured in 9 children were between 0.45 and 3.96 mmol/L in the plasma and between 0.15 and 3.2 mmol/L in the urine. No correlation between pyroglutamic acid and acetaminophen concentrations were found, regardless of the biological media. In conclusion, our method measures pathophysiological concentrations of pyroglutamic acid and highlights the increase in other organic acids that may explain metabolic acidosis due to chronic acetaminophen intake.

Human Placental NADPH Oxidase Mediates sFlt-1 and PlGF Secretion in Early Pregnancy: Exploration of the TGF-ß1/p38 MAPK Pathways.

Preeclampsia, a hypertensive disorder occurring during pregnancy, is characterized by excessive oxidative stress and trophoblast dysfunction with dysregulation of soluble Fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) production. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (Nox) is the major source of placental superoxide in early pregnancy and its activation with the subsequent formation of superoxide has been demonstrated for various agents including Transforming Growth Factor beta-1 (TGF-ß1), a well-known p38 MAPK pathway activator. However, the bridge between Nox and sFlt-1 remains unknown. The purpose of this study was to explore the possible signaling pathway of TGF-ß1/Nox/p38 induced sFlt-1 production in human chorionic villi (CV). METHODS: Human chorionic villi from first trimester placenta (7-9 Gestational Weeks (GW)) were treated with TGF-ß1 or preincubated with p38 inhibitor, SB203580. For NADPH oxidase inhibition, CV were treated with diphenyleneiodonium (DPI). The protein levels of phospho-p38, p38, phospho-Mothers Against Decapentaplegic homolog 2 (SMAD2), and SMAD2 were detected by Western blot. The secretion of sFlt-1 and PlGF by chorionic villi were measured with Electrochemiluminescence Immunologic Assays, and NADPH oxidase activity was monitored by lucigenin method. RESULTS: We demonstrate for the first time that NADPH oxidase is involved in sFlt-1 and PlGF secretion in first trimester chorionic villi. Indeed, the inhibition of Nox by DPI decreases sFlt-1, and increases PlGF secretions. We also demonstrate the involvement of p38 MAPK in sFlt-1 secretion and Nox activation as blocking the p38 MAPK phosphorylation decreases both sFlt-1 secretion and superoxide production. Nevertheless, TGF-ß1-mediated p38 activation do not seem to be involved in regulation of the first trimester placental angiogenic balance and no crosstalk was found between SMAD2 and p38 MAPK pathways. CONCLUSIONS: Thus, the placental NADPH oxidase play a major role in mediating the signal transduction cascade of sFlt-1 production. Furthermore, we highlight for the first time the involvement of p38 activation in first trimester placental Nox activity.

[SARS-CoV-2 and neurological disorders: the revelance of biomarkers?] / SARS-CoV-2 et atteintes neurologiques : quelle est la place des biomarqueurs ?

Soon after the pandemic, numerous publications described cases of neurological disorders associated with the SARS-CoV-2 infection. The range of neurological symptoms is becoming increasingly more extensive as the pandemic progresses. However, it is not yet well established whether the manifestations are due to direct viral damage to the nervous system or indirect consequences of the infection. This review presents an inventory of the biochemical markers studied in the context of neurological disorders related to SARS-CoV-2. By reflecting various physiopathological mechanisms, these biomarkers allow both a better understanding of the pathophysiology of Covid-19 and a contribution to the diagnosis of neurologic troubles; they could participate in the prognostic evaluation of patients.

Prehospital lactate clearance is associated with reduced mortality in patients with septic shock.

BACKGROUND: Assessment of disease severity in patients with septic shock (SS) is crucial in determining optimal level of care. In both pre- and in-hospital settings, blood lactate measurement is broadly used in combination with the clinical evaluation of patients as the clinical picture alone is not sufficient for assessing disease severity and outcomes. METHODS: From 15th April 2017 to 15th April 2019, patients with SS requiring prehospital mobile Intensive Care Unit intervention (mICU) were prospectively included in this observational study. Prehospital blood lactate clearance was estimated by the difference between prehospital (time of first contact between the patients and the mICU prior to any treatment) and in-hospital (at hospital admission) blood lactate levels divided by prehospital blood lactate. RESULTS: Among the 185 patients included in this study, lactate measurement was missing for six (3%) in the prehospital setting and for four (2%) at hospital admission, thus 175 (95%) were analysed for prehospital blood lactate clearance (mean age 70 ± 14 years). Pulmonary, digestive and urinary infections were probably the cause of the SS in respectively 56%, 22% and 10% of the cases. The 30-day overall mortality was 32%. Mean prehospital blood lactate clearance was significantly different between patients who died and those who survived (respectively 0.41 ± 2.50 mmol.l-1 vs 1.65 ± 2.88 mmol.l-1, p = 0.007). Cox regression analysis showed that 30-day mortality was associated with prehospital blood lactate clearance > 10% (HRa [CI95] = 0.49 [0.26-0.92], p = 0.028) and prehospital blood lactate clearance < 10% (HRa [CI95] = 2.04 [1.08-3.84], p = 0.028). CONCLUSION: A prehospital blood lactate clearance < 10% is associated with 30-day mortality increase in patients with SS handled by the prehospital mICU. Further studies will be needed to evaluate if prehospital blood lactate clearance alone or combined with clinical scores could affected the triage decision-making process for those patients.

Pre-Hospital Lactatemia Predicts 30-Day Mortality in Patients with Septic Shock-Preliminary Results from the LAPHSUS Study.

BACKGROUND: Assessment of disease severity in patients with septic shock (SS) is crucial in determining optimal level of care. In both pre- and in-hospital settings, the clinical picture alone is not sufficient for assessing disease severity and outcomes. Because blood lactate level is included in the clinical criteria of SS it should be considered to improve the assessment of its severity. This study aims to investigate the relationship between pre-hospital blood lactate level and 30-day mortality in patients with SS. METHODS: From 15 April 2017 to 15 April 2019, patients with SS requiring pre-hospital Mobile Intensive Care Unit intervention (MICU) were prospectively included in the LAPHSUS study, an observational, non-randomized controlled study. Pre-hospital blood lactate levels were measured at the time of first contact between the patients and the MICU. RESULTS: Among the 183 patients with septic shock requiring action by the MICU drawn at random from LAPHSUS study patients, six (3%) were lost to follow-up on the 30th day and thus 177 (97%) were analyzed for blood lactate levels (mean age 70 ± 14 years). Pulmonary, urinary and digestive infections were probably the cause of the SS in respectively 58%, 21% and 11% of the cases. The 30-day overall mortality was 32%. Mean pre-hospital lactatemia was significantly different between patients who died and those who survived (respectively 7.1 ± 4.0 mmol/L vs. 5.9 ± 3.5 mmol/L, p < 10-3). Using Cox regression analysis adjusted for potential confounders we showed that a pre-hospital blood lactate level ≥ 4 mmol/L significantly predicted 30-day mortality in patients with SS (adjusted hazard ratio = 2.37, 95%CI (1.01-5.57), p = 0.04). CONCLUSION: In this study, we showed that pre-hospital lactatemia predicts 30-day mortality in patients with septic shock handled by the MICU. Further studies will be needed to evaluate if pre-hospital lactatemia alone or combined with clinical scores could affect the triage decision-making process for those patients.

NSE & S100B protein blood level assessment during a long-distance trail race.

The acute and chronic consequences of long-distance running on brain function have received little attention. The impact of such a hard-physical burden associated with sleep privation during such events such has never been explored in terms of neuropsychological function and brain damage. METHODS: Blood samples were collected from 4 athletes before, during and at the end of one of two races: Grand Raid de la Réunion 2017 (GRR: 165 km, elevation gain: 9529 m, 2 runners) and Trail de la Bourbon 2017 (TB: 111 km, elevation gain: 6433 m, 2 runners). Serum S100B and NSE levels were measured for each runner before, during and after the race. RESULTS: Serum S100B levels (normal range: < 0.15 µg/L) increased early during the race and remained high up to the end of the race in all 4 runners (range: 0.17-0.59 µg/L). NSE level (normal range: < 15 µg/L) increased in 3 of the 4 runners (range: 16.8-39.2 µg/L). CONCLUSIONS: This preliminary study shows the potential interest of S100B and NSE serum assessment during long-distance races. Further studies are needed to confirm these results and to investigate the origins and significance of this increase in brain injury markers.

NADPH oxidase is the major source of placental superoxide in early pregnancy: association with MAPK pathway activation.

First-trimester placenta (<10 gestational weeks (GW)) develops in a low oxygen environment (≈2%). Early oxygen exposure can cause oxidative damage leading to pregnancy disorders. The aim of this work was to determine the major sources of placental superoxide during early pregnancy - more specifically before 10 GW - and to study redox adaptation to increased oxygen pressure after 12 GW. Our results show that NADPH oxidase (Nox) is the main source of superoxide in first-trimester chorionic villi. Its activity is higher before 10 GW and concomitant with the location on the syncytiotrophoblast apical pole of p47phox, the Nox organizer subunit. After the increase in pO2 pressure (12-14 GW), the activities of the antioxidant enzymes SOD1, catalase and GPX1 are increased. The redox-sensitive MAPK pathways show increased phosphorylated-p38 expression, but no variation in the phosphorylation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) during first trimester, suggesting a physiological redox adaptation, whilst ERK1/2 phosphorylation is higher after 12 GW. Nox is the major superoxide source in early pregnancy (<10 GW). Increased superoxide production at 7-9 GW is associated with p38 MAPK pathway activation, suggesting that it is involved in physiological placental function and healthy early development of the placenta, through MAPK pathways.

Is Capillary Electrophoresis a New Tool to Monitor Acute Lithium Poisoning in Human?†.

A 38-year-old man was admitted in the intensive care unit (ICU) after supposed ingestion of 504 sustained-release tablets of Theralithe™ corresponding ~200 g of lithium carbonate. At the admission, ~19.5 h after ingestion, the patient was conscious with trembling limbs, intense thirst, profuse sweats and vomiting and lithium serum concentration was 14.2 mmol/L. Toxicological screenings performed in urine and serum, were negative. Patient was treated with continuous extrarenal epuration by continue veno-venous hemodiafiltration starting (CCVHDF) 24 h post-admission and was carried on until 64 h. After 11 days in ICU, the patient was dismissed to the service without sequelae, and transferred to a psychiatric unit. To follow lithium concentrations in serum, urines and dialysates, we developed a simple, rapid and reliable method by capillary zone electrophoresis (CZE). Separation was achieved in 7 min. The method was linear between 0.14 and 1.44 mmol/L for serum samples, and between 0.07 and to 1.44 mmol/L for urines and dialysates. Limits of quantification were 0.15 mmol/L and 0.07 mmol/L for serum and others fluids, respectively. Intra- and inter-day precisions expressed as CV were systematically inferior to 12.1% for serum and 8.2% for other fluids. Results obtained regarding precision, accuracy, recovery and stability were satisfying, with recoveries ranging from 91.0 to 102.0%. Serum, urine and dialysate samples were measured using CZE and flame photometry. We observed a strong correlation between both methods as assessed by linear regression and Bland-Altman analysis. For the intoxicated patient, the assay was successfully applied to serum, urine and dialysates to determine the amount of lithium present in circulation and excreted. Lithium amounts in dialysates were estimated to correspond to 89% of total lithium excreted during CCVHF session while urine excretion account only for 11%.

Sickle SCAN™ (BioMedomics) fulfills analytical conditions for neonatal screening of sickle cell disease.

Sickle SCAN™ is a rapid, qualitative, point-of-care lateral flow immunoassay for the identification of AS, AC, SS/Sß0thal, SC and CC/Cß0thal phenotype. We evaluated this test under the conditions encountered in the French newborn screening (NBS) program for sickle cell disease: a total of 104 dried blood spots (DBSs) were tested with an HPLC reference method and then with the Sickle SCAN™ device. Sickle SCAN™ identified the hemoglobin (Hb) phenotype correctly on 96% of cases. In the four non-concordant cases, the antibody anti-HbS cross-reacted with HbE (n=2), HbD (n=1) or HbX (n=1). There were no false negative. In order to test Sickle SCAN™'s sensitivity to low levels of HbA and HbS in the presence of high HbF levels, we selected another 21 DBS cards with low percentages of HbA (0.6-4.2%) and HbS (2.0-6.9%). HbA and HbS were always detected when present at levels of more than 1% and 2%, respectively. Sickle SCAN™ appears to be an accurate point-of-care method for the identification of newborns with SCD trait. The device meets the criteria for sickle cell disease NBS programs in endemic countries with poor access to laboratory equipment.

[Evaluation of procalcitonin assay on the Abbott Architect i2000 SR® analyzer]. / Évaluation du dosage de la procalcitonine plasmatique sur l'analyseur Abbott Architect i2000-SR®.

Procalcitonin (PCT) is a useful biomarker for the diagnosis of bacterial infection. Its measurement is used routinely as a valuable tool for antibiotic treatment decision. The aim of this study is to assess the analytical performance of the new Architect Brahms PCT® reagents on the Abbott Architect i2000-SR® immuno-analyzer. The Architect PCT® assay was evaluated according to the modified SFBC protocol, in accordance with the NF EN ISO 15189 standard. Sixty two samples from patients hospitalized in Necker Hospital (Paris) have been tested with the evaluated method, and results were compared to those of the PCT Kryptor Brahms® method. Analytical performances tested complied with those announced by the manufacturer. Linear regression showed a strong correlation between the two methods (r >0.99), despite a tendency for overestimation by the new method (y=1.10 x +0.05). Bland-Altman plots confirmed this strong correlation by showing only two points outside the acceptable limits without clinical incidence, given the high PCT concentrations (over 10 ng/mL) in those samples. In conclusion, the new PCT assay on the Abbott Architect i2000-SR® shows excellent analytical performances, even at low concentrations. A slight positive bias compared to the Brahms Kryptor® was observed, but did not lead to inappropriate clinical decisions.