Subject(s)
COVID-19 , Virus Diseases , Humans , SARS-CoV-2 , Seroepidemiologic Studies , COVID-19 Testing , Antibodies, ViralABSTRACT
BACKGROUND: The International Standard ISO 15189 based on the ISO 9001:2008 emphasizes specific requirements for quality and ability of medical laboratories. The accreditation of medical laboratories according to ISO 15189 includes the validation of biological methods, which depends on collection of bibliographic data and experimental proofs. Moreover, these results must be compared to provider data sheets and independent scientific data. In the immunodiagnostic field, independent published data are deeply lacking. The aim of our work was to share experience of method validation for virological immune markers on the widely used Architect i2000sr. METHODS: After risk analysis, intra- and inter-assay variability, and inter-sample contamination were evaluated for each method, and sensitivity was investigated for antigen detection tests. A comparison between the two Architect i2000sr available in our laboratory was also performed. RESULTS: All tested methods were consistent with the manufacturer data (from the data sheet). No inter-sample contamination was observed. Both devices are broadly equivalent and can be used indifferently or as a backup solution of the other. CONCLUSIONS: To our knowledge, those results are the first independent complete data on the reliability of the Architect i2000sr in real-life experience. These data are needed to the accreditation of our platform and potentially useful for the accreditation of other laboratories using the same equipment.
Subject(s)
Immunoenzyme Techniques/standards , Laboratories, Hospital/standards , Accreditation , HIV Core Protein p24/blood , Hepatitis B Surface Antigens/blood , Hospitals, University , HumansABSTRACT
Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbßG83C. We investigate the potential side effects of rHbßG83C on endothelial cells. The rHbßG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbßG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb ßG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.
Subject(s)
Blood Substitutes/pharmacology , Hemoglobins/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Biomarkers , Blood Substitutes/toxicity , Cell Survival/drug effects , Dextrans/pharmacology , Dextrans/toxicity , Down-Regulation/drug effects , Drug Evaluation, Preclinical , E-Selectin/biosynthesis , E-Selectin/genetics , Gene Expression Regulation/drug effects , Heme Oxygenase-1/biosynthesis , Heme Oxygenase-1/genetics , Hemoglobins/analysis , Hemoglobins/chemistry , Hemoglobins/toxicity , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Hydroxyethyl Starch Derivatives/pharmacology , Hydroxyethyl Starch Derivatives/toxicity , Inflammation/chemically induced , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/genetics , Methemoglobin/analysis , Models, Molecular , Nitric Oxide Synthase Type III/biosynthesis , Nitric Oxide Synthase Type III/genetics , Oxidative Stress/drug effects , Plasma Substitutes/pharmacology , Plasma Substitutes/toxicity , Protein Conformation , Real-Time Polymerase Chain Reaction , Vasoconstriction/drug effectsABSTRACT
BACKGROUND: Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium. METHODOLOGY/PRINCIPAL FINDINGS: Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2) or normoxic (21% O2) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3(rd) passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability. CONCLUSION/SIGNIFICANCE: We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells.
Subject(s)
Cell Differentiation , Endothelium, Vascular/cytology , Hematopoietic Stem Cells/cytology , Muscle, Smooth, Vascular/cytology , Oxygen/metabolism , Animals , Endothelial Cells/cytology , Endothelial Cells/metabolism , Extracellular Matrix/metabolism , Hematopoietic Stem Cells/metabolism , Male , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Phenotype , RabbitsABSTRACT
The lack of blood donations and the threat of infections from blood and blood products have led to extensive research into the development of blood substitutes. The latest generation of hemoglobin based oxygen carriers (HBOC) has been shown to induce side effects like hypertension, vasoconstriction, inflammation and oxidative stress. HBOC are able to restore volemia and transport oxygen after a hemorrhagic shock, the reperfusion leading to the restoration of the blood flow in vessels. We propose an innovative approach, more closely emulating clinical situations, to assess the impact of HBOC perfusion on endothelial cells (EC) in vitro. Through this approach we quantified levels of oxidative stress, vasoactive factors and inflammation. EC were cultivated under a laminar flow to reproduce the return of shear stress (SS) during the reperfusion. We showed that heme oxygenase I transcription correlated with changes in oxidatively modified heme and methemoglobin; all were lower under SS. SS induced increased nitric oxide production, which may have implications for the mechanism of in vivo vasoconstriction and hypertension. E-selectin changes under SS were greater than those of ICAM-1. Our results demonstrate how it is essential to include SS in assays attempting to understand the potential vascular side effects of HBOC perfusion.
