[POCT-management during the first wave of Covid-19 in France. Results of a national survey leaded by the SFBC-POCT Working Group]. / Gestion de la biologie délocalisée lors de la première vague de Covid-19 en France. Résultats de l'enquête nationale menée par le groupe de travail des EBMD de la SFBC.

During the first wave of Covid-19 in France, in spring 2020, healthcare institution's laboratory had to adapt itself quickly to the growing demand for emergency biology, in particular by reorganizing their POCT analyzers: redeployment of analyzers and/or new installations. In order to analyze this management, a subgroup of 15 hospital biologists from the SFBC Working Group "Biochemical markers of Covid-19" sent, in fall 2020, an on-line survey to French hospital laboratories using POCT. Answers analysis (n = 86) shows a territorial disparity related to the severity of the first wave: increased activity essentially in red zones, management of unexpected situations, training of additional nursing staff for 40 % of the laboratories... The survey also showed simplification of aspects related to accreditation those periods of health crisis. An additional survey, carried out in the spring of 2021, showed good overall satisfaction of the healthcare services (n = 139) concerning the services provided by biology in the POCT sector. Because of their great adaptation capacity, the laboratories and their POCT-teams have played a key role in the management of the first wave of Covid-19 in France. However, the success of these organizations requires an essential collaboration between laboratories and healthcare services. The results of this survey are fundamental in the context of the prolongation of the pandemia throughout the world with a POCT sector appearing to be growing.

Advanced mask aligner lithography: new illumination system.

A new illumination system for mask aligner lithography is presented. The illumination system uses two subsequent microlens-based Köhler integrators. The second Köhler integrator is located in the Fourier plane of the first. The new illumination system uncouples the illumination light from the light source and provides excellent uniformity of the light irradiance and the angular spectrum. Spatial filtering allows to freely shape the angular spectrum to minimize diffraction effects in contact and proximity lithography. Telecentric illumination and ability to precisely control the illumination light allows to introduce resolution enhancement technologies (RET) like customized illumination, optical proximity correction (OPC) and source-mask optimization (SMO) in mask aligner lithography.

False elevation of blood lactate reveals ethylene glycol poisoning.

Ethylene glycol poisoning is a medical emergency. Making a definitive diagnosis is challenging because few institutions have timely access to direct measurement of ethylene glycol. After ingestion, primary metabolism of ethylene glycol takes place in the liver, leading to glycolic acid and glyoxylic acid. These compounds may cross-react with L-lactate oxidase used in blood gas analyzers lactate electrodes to induce false elevation of blood lactate. We present the case of a 47-year-old male patient initially admitted to the intensive care unit for severe lactate acidosis of unknown cause (pH 6.96, lactate, 30 mmol/L). Knowledge of potent artifactual lactate results was the key to the diagnosis of ethylene glycol poisoning, and false lactate measurements were found at the central laboratory on our 3 different blood gas analyzers.

Point-of-care testing: false elevation of cardiac troponin I assayed in the emergency department.

In October 2007, an 18-year-old woman with no cardiac history was admitted to the emergency department for a major epigastric pain. The chest pain led to assay cardiac troponin I (cTnI) with the emergency department point-of-care testing analyzer (Stratus CS), which disclosed a value of 0.70 ng/mL, discordant with the atypical clinical presentation and the noncontributive electrocardiography. The biologist contacted for biological advice controlled cTnI with an Access II, which disclosed a value less than 0.04 ng/mL. These findings were confirmed with the discordant results of a new sample assayed on both analyzers. The interference of heterophilic antibodies (HAs) was suspected because these antianimal antibodies may lead to analytical errors in sandwich immunoassays using animal sources of immunoglobulins and can cause false-positive results. In this case, the HAs bind to the capture antibody and the conjugate antibody, simulating cTnI. The diagnosis of HA involvement was confirmed using Heterophilic Blocking Tube, a device that contains a blocking reagent composed of specific binders that attach HA. After treatment in Heterophilic Blocking Tube, the cTnI concentration measured by the Stratus CS decreased from 0.62 to 0.05 ng/mL. Finally, potentially invasive test or this patient's unnecessary hospitalization in cardiology was avoided. Our experience supports that collaboration between staffs of laboratories and medical departments owning point-of-care testing analyzers is essential to avoid mistaken diagnosis linked to analytical interferences and to ensure quality of results assayed outside the laboratory.

Evaluation of the GEM Premier 4000: a compact blood gas CO-Oximeter and electrolyte analyzer for point-of-care and laboratory testing.

BACKGROUND: GEM Premier 4000 (Instrumentation Laboratory), a new blood gas/CO-Oximeter/hematocrit/electrolyte/glucose/lactate analyzer was evaluated. The only reagents required were disposable cartridges (GEM Premier 4000 PAK comprise all components necessary for analysis, including quality controls). METHODS: The evaluation was performed in two laboratories according to the Valtec protocol guidelines. Analytical performances (imprecision at three levels, linearity, inaccuracy by method comparison using patient samples, interferences) and instrument practicability were compared with three routinely used laboratory blood gas analyzers (ABL 835 and 725, Radiometer; OMNI S, Roche Diagnostics) and the centrifuged microhematocrit method. RESULTS: Within-run and between-run imprecision yielded good coefficients of variation values for all parameters. Linearity was satisfactory and within the expected ranges provided by the manufacturer. Method comparisons demonstrated close agreement (Deming's regression correlation coefficients 0.92-1.00). No interferences of lipemia, hyperbilirubinemia and hemolysis were found on CO-Oximetry parameters. Fetal hemoglobin >35% overestimated carboxyhemoglobin measurements, but the magnitude of error was reduced with the fetal hemoglobin correction analysis mode. CONCLUSION: The GEM Premier 4000 analytical performance was validated for all parameters with the accuracy and the reliability of traditional systems. This blood gas analyzer fulfills most of the requirements for both point-of-care and laboratory use.

Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification.

BACKGROUND: Detecting and quantifying circulating free DNA in patient serum has become a major challenge. New methods using conventional or automated DNA amplification have been developed. As quantitative real-time PCR (QPCR) remains expensive and requires dedicated automated instrumentation, we questioned whether simple quantification using fluorescent dyes is efficient for determination of free DNA levels in serum. METHODS: Serum samples from 180 cancer patients and 58 healthy volunteers were used for DNA quantification according to three methods: (i) using an exonic part of the beta-globin gene as the amplifying target; (ii) amplifying a 105-bp intron 1 part of the housekeeping cyclophilin A gene, both referring to specific standard curves; and (iii) using a PicoGreen DNA quantification kit without amplification. RESULTS: The 58 samples from healthy controls showed a reference limit of (95th percentile) <160 cyclophilin gene copies/mL. The 180 cancer samples displayed values ranging between 300 and 215,000 copies/mL. The cyclophilin method showed a high level of correlation with both the beta-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods. CONCLUSIONS: Aside from the disadvantage that the QPCR assays can only be used in clinical biochemistry laboratories that possess QPCR apparatus, the use of direct PicoGreen quantification displays major advantages in a routine context: it is less time-consuming and is quite inexpensive, but is still correlated with QPCR.

PHProteomicDB: a module for two-dimensional gel electrophoresis database creation on personal web sites.

PHProteomicDB is a PHP-written module to help researchers in proteomics to share two-dimensional electrophoresis gel data using personal web sites. No technical or PHP knowledge is necessary except a few basics about web site management. PHProteomicDB has a user-friendly administration interface to enter and update data. It creates web pages on the fly displaying gel characteristics, gel pictures, and numbered gel spots with their related identifications pointing to their reference pages in protein databanks. The module is freely available at http://www.huvec.com/index.php3?rub=Download.

Sonic Hedgehog-dependent proliferation in a series of patients with colorectal cancer.

BACKGROUND: The Hedgehog (Hh) gene family is known to regulate development of stem cells. In addition, activation is responsible for the induction of GLI1 proto-oncogene and subsequent cellular proliferation. Sonic Hedgehog (SHh), one of the Hh family members promotes carcinogenesis in airway and pancreatic epithelia, is expressed in colonic stem cells. As differentiated colonic cells arise from constant renewal of Hedgehog-expressing colonic stem cells, SHh could be involved in human colonic carcinogenesis. METHODS: Tissue samples of colorectal adenocarcinoma (T) and adjacent normal colon tissue (NT) were sampled from each of 44 consecutive patients with colorectal cancer. Specific transcription of SHh, GLI1, and the GLI1 downstream target FOXM1 were evaluated using semiquantitative reverse transcriptase polymerase chain reaction. Similar in vitro measurements of mRNA of GLI1 and FOXM1 transcription levels after specific induction by SHh-Np were performed in the HT-29 colorectal tumor cell line to confirm the in vivo results. RESULTS: SHh mRNA was overexpressed in colorectal adenocarcinomas in 38 of 44 (86%) patients. Expression of transcription levels of GLI1 and FOXM1 correlated with SHh expression (SHh vs GLI1, r = 0.77, P < .0001; GLI1 vs FOXM1, r = 0.68, P < .0001; SHh vs FOXM1, r = 0.79, P < .0001). SHh overexpression did not appear to correlate with the patient characteristics evaluated. Similarly, when studied in the HT-29 colorectal cell line, exogenous SHh promoted cell proliferation, while inhibition of SHh expression decreased proliferation. Expression of GLI1 and FOXM1 mRNA increased with exogenous exposure to SHh. CONCLUSIONS: We demonstrated increased expression of SHh mRNA in human colonic adenocarcinomas and in a colorectal cell line with downstream increased expression of GLI1 and FOXM1 mRNA known to promote cell proliferation. This upregulation within human colorectal adenocarcinoma tissue confirms the potential role of the Hh pathway in colorectal carcinogenesis and suggests a potential therapeutic target of Hh blockade in colorectal cancer.

Plasma homocysteine measurement with ion exchange chromatography (Jeol Aminotac 500): a comparison with the Abbott IMx assay.

OBJECTIVE: We evaluated ion exchange chromatography (IEC) on the Jeol Aminotac 500 analyzer for total homocysteine (tHcy) determination and compared it with an immunoassay method using fluorescence polarization on an Abbott IMx analyzer. METHODS: IEC method validation (linearity, limit of detection, precision, interference) was made according to the French Biology Society guidelines (Société Française de Biologie Clinique). Moreover, during a 2-month period, 55 plasma samples from patients scheduled for routine tHCy measurement were assayed by both methods for determining correlation. RESULTS: The IEC method was found linear up to at least 190 micromol/l, and the limit of detection was 1.6 micromol/l. Precision was studied with 3 controls at 6, 15 and 30 micromol/l. Intra-assay coefficients of variation (n = 14) were 8.3, 3.1 and 2.3%, respectively, and inter-assay coefficients of variation (n = 15) were 9.6, 5.1 and 4.9%, respectively. No interference was found with other sulfur-containing amino acids (methionine, cysteine). An excellent agreement was found between IEC and fluorescence polarization (Deming regression; y = 0.99x - 1.23; r = 0.97; p < 0.001). CONCLUSION: The IEC method for tHcy measurement shows adequate precision and correlates highly with the IMx assay. The IEC method is more time-consuming but less expensive in reagent cost and allows simultaneous determination of plasma methionine concentration which may help to explain the underlying mechanism responsible for hyperhomocysteinemia.

Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis.

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

Development of an internal dynamic web site to promote quality assurance in a clinical laboratory.

In clinical laboratories, one challenging quality assurance objective is to maintain standardized practices. Meeting this objective entails ensuring information flow, which is necessary to smooth running of the laboratory. To facilitate information flow, we developed an internal quality Web site on our local network. The dynamic generated pages of the site were constructed with EasyPHP v.1.6, a complete freeware package providing PHP dynamic language and databases. The site comprises various sections: general news, specific laboratory units news, documents (quality manual, guidelines, emergency processes), schedules, National Quality Control results, forum, etc. Five to 10 pages are updated each week. This work was facilitated by the use of PHP-written pages and data tables, which enable us to record in real time the operation of our assurance quality project and to improve traceability. This approach could be extended to other aspects of quality management and could help meet the future IS015189 standard requirements.

Dose dependency of the effect of ornithine alpha-ketoglutarate on tissue glutamine concentrations and hypercatabolic response in endotoxaemic rats.

The optimal dosage of ornithine alpha-ketoglutarate (OKG) for repleting tissue glutamine (Gln) concentrations and maintaining N homeostasis after injury is unknown. We set out to perform 'dose-ranging' of OKG supplementation after an endotoxaemic challenge. Sixty-one male Wistar rats were injected with 3 mg lipopolysaccharide (LPS) from Escherichia coli/kg (n 50) or saline vehicle (9 g NaCl/l; controls n 11). After a 24 h fast, survivors were fed by gavage for 48 h with a polymeric standard diet (879 kJ/kg per d and 1.18 g N/kg per d) supplemented with non-essential amino acids (control, n 11; LPS-OKG-0.0, n 9), or with 0.5 g OKG/kg per d (LPS-OKG-0.5, n 12), 1.5 OKG/kg per d (LPS-OKG-1.5, n 11) or 4.5 g OKG/kg per d (LPS-OKG-4.5, n 10). The diets for all groups were made isonitrogenous with the LPS-OKG-4.5 diet by adding an appropriate amount of non-essential amino acids. Rats were killed on day 3 for blood and tissue sampling (muscle, jejunum mucosa, liver). Urine was collected daily for 3-methylhistidine and total N assays. The OKG dose was correlated with Gln concentrations in every tissue and with cumulative N balance (Spearman test, P<0.01). 3-Methylhistidine excretion was increased in endotoxaemic groups compared with controls (ANOVA, P<0.05) except in the LPS-OKG-4.5 group. Only the LPS-OKG-4.5 group achieved a positive post-injury N balance (t test, P<0.05). In conclusion, OKG exerted a dose-dependent effect on tissue Gln concentration and N balance, but only the highest dosage counteracted myofibrillar hypercatabolism and caused a positive N balance.

Enhanced saquinavir exposure in human immunodeficiency virus type 1-infected patients with diarrhea and/or wasting syndrome.

The protease inhibitor saquinavir was administered to 100 human immunodeficiency virus type 1 (HIV-1)-infected patients as a single 600-mg oral dose (hard gelatin capsules) with a standard breakfast, including 200 ml of grapefruit juice, during an open-label trial to assess whether diarrhea and/or wasting syndrome has consequences on its pharmacokinetics. Three groups of patients were enrolled: group 1, asymptomatic patients (n = 30); group 2, AIDS symptomatic patients without body weight loss or diarrhea (n = 37); and group 3, AIDS symptomatic patients with severe body weight loss and/or diarrhea (n = 33). Clinical and biological data (covariates) were collected. A population approach was performed with three blood samples per patient to estimate the mean population pharmacokinetic parameters (clearance [CL]/oral bioavailability [F], V/F, k(a), and lag time) and the derived ones (k(el), C(max), T(max), and area under the curve [AUC]). The relationships between groups, exposure (i.e., estimated individual post hoc AUCs), and covariates were explored by using multiple linear regressions. A significant increase in median AUCs (165, 349, and 705 ng. h. ml(-1) for groups 1, 2, and 3, respectively [P < 0.0001]) was observed. The enhancement in saquinavir exposure could be due to the destruction of the transporters in enterocytes and/or to the enlargement of their tight junctions, allowing a paracellular crossing of saquinavir as the illness spreads. Because of grapefruit juice intake by every patient, no implication of CYP3A4 could be assessed. These results strongly suggest that, despite its low intrinsic oral bioavailability, saquinavir can be considered as a relevant treatment for HIV-1-infected patients with diarrhea and/or wasting syndrome. This must be evaluated in a long-term period.

Osmolar gap hyponatremia in critically ill patients: evidence for the sick cell syndrome?

OBJECTIVE: Accumulation of nondiffusible solutes in plasma leads to redistribution hyponatremia with an increased osmolar gap (i.e., the difference between measured and calculated osmolality). In critically ill patients, intracellular solutes may leak out of the cell because of an increased membrane permeability and may lead to redistribution hyponatremia with increased osmolar gap, a concept called the "sick cell syndrome." The aims of this prospective study were to determine whether an increased osmolar gap related to endogenous solutes accumulation was present in intensive care patients with true hyponatremia and to identify the solutes accounting for this increased osmolar gap. SETTING: A 14-bed medical intensive care unit in an 821-bed university hospital. DESIGN: A 20-wk prospective observational study. PATIENTS: Fifty-five consecutive patients with a measured plasma sodium concentration 10 mosm/kg. Total plasma amino acid concentration also was measured. Organ dysfunctions were assessed with the Sequential Organ Failure Assessment. Thirty of the 55 patients (54%) had an osmolar gap >10 mosm/kg (17.2 +/- 7.1 mosm/kg). Sequential Organ Failure Assessment score was significantly higher in the osmolar gap patients (6.4 +/- 3.2 vs. 4.5 +/- 2.0; p =.015). No difference of amino acids concentration was observed between osmolar gap and non-osmolar gap patients, and no correlation was observed between osmolar gap and amino acid concentration. Accumulation of ketone bodies and lactic acid was also unlikely. During correction of hyponatremia in osmolar gap patients, a significant decrease of plasma osmolar gap was observed and a statistically significant inverse relationship was demonstrated between osmolar gap decrease and plasma sodium concentration increase. CONCLUSION: Hyponatremia with increased osmolar gap related to endogenous solutes accumulation is observed frequently in hyponatremic intensive care patients, especially in patients with the most severe organ dysfunctions. The nature of the endogenous solutes accounting for the increased osmolar gap remains to be determined. Simultaneous correction of sodium and osmolar gap suggests a causal link between increased osmolar gap and hyponatremia and may support the concept of sick cell syndrome.