Plasma metabolites were analyzed at 37 °C by an automated clinical analyzer (ILAB 650, Instrumentation Laboratory, Lexington, MA), using the methodologies previously reported (Calamari et al., 2016) . Commercial kits were used to measure glucose, total cholesterol, urea, inorganic phosphorus, total protein, albumin, total bilirubin, aspartate aminotransferase (GOT), γglutamyltransferase (GGT), and creatinine (Instrumentation Laboratory SpA, Werfen, Monza, Milan, Italy), NEFA (Wako, Chemicals GmbH, Neuss, Germany), β-OH-butyric acid (BHBA, kit Ranbut, Randox Laboratories Limited, Crumlin, County Antrim, UK), thiol groups (SHp, Kit from Diacron srl, Grosseto, Italy). The ferric reducing antioxidant power (FRAP) assay was assessed by adapting the colorimetric method of Benzie and Strain (Benzie and Strain, 1996) to the clinical auto-analyzer ILAB 650 (Instrumentation Laboratory, Lexington, MA).
Ilab 650
Manufactured by Werfen
Sourced in United States, Italy, United Kingdom
The ILAB 650 is a fully automated clinical chemistry analyzer designed for high-volume testing in clinical laboratories. It features a modular design and provides a range of analytical capabilities to perform a variety of routine and specialized clinical chemistry tests.
Automatically generated - may contain errors
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32 protocols using ilab 650
1
Automated Plasma Metabolite Analysis
2
Comprehensive Toxicological Analysis of Biological Samples
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For each case, a comprehensive toxicological analysis, including general screening and quantification of drugs of abuse and medicinal drugs, was performed. Particularly, analyses for alcohol were done by gas chromatography coupled to a Flame Ionization Detector (Shimadzu QP 2010 Plus, Kyoto, Japan). Blood samples were initially screened for illicit drugs (cannabinoids, cocaine, opiates, methadone, and amphetamines/methamphetamines/MDMA/MDA) by immunoassay (ILab 650, Werfen, Barcelona, Spain) [19 (link)]. Confirmation analyses for cannabinoids were performed with a Shimadzu GC-2010 Plus gas chromatograph equipped with a model AOC-6000 auto-sampler system and interfaced with a QP 2010 Ultra mass spectrometer (Shimadzu, Kyoto, Japan) using a previously validated method [20 (link)]. Confirmation analyses for other illicit drugs and screening/confirmation for 68 psychoactive medications (benzodiazepines, Z-drugs, antipsychotics, antidepressants, and medical opioids) were performed with an ACQUITY UPLC® System (Waters Corporation, Milford, MA, USA) equipped with an Acquity UPLC® HSS C18 column (2.1 × 150 mm, 1.8 μm; Waters) using a previously validated method [21 (link),22 (link)].
3
Serum VEGF Levels in Pregnancy
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After obtaining informed written consent from the study participants, detailed history including that of chief complaint with onset, duration, and progress of current condition and obstetric history were elicited. Five mL of venous blood was drawn from anterior cubital vein by venepuncture after overnight fasting in a red vacutainer® (BD™ Biosciences) and was allowed to clot and then centrifuged for ten minutes at 4,000 rpm to obtain the serum. Biochemical analysis including renal function tests, liver function tests, and other routine biochemical parameters were analysed immediately on I Lab 650® clinical chemistry analyser (Werfen®, Germany) using manufacturer guidelines and around 1–1.5 mL of serum was stored in aliquots at –70°C for further estimation of serum VEGF by ELISA. Urinary protein was assayed by dipstick method (Mission® Urinalysis reagent strips, ACCH Biotech Hangzhou China Cat. number U031-021). Serum VEGF levels were assessed by double sandwich ELISA method using Novex® Human VEGF ELISA kit (Life Technology® Cat. number KHG0111).
4
Hair Analysis for Drug Addiction Treatment
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Head hair samples from 150 patients with a history of drug addiction and undergoing treatment at the substance abuse service were collected from January 2022 to March 2023 in the metropolitan area of Bologna, Italy. After performing the analysis for clinical purposes, an aliquot was taken and stored according to the 2022 SoHT Consensus on general recommendations for hair testing. For the purpose of this study, the samples were treated completely anonymously. 10 The samples consisted of head hair, ranging from 3 to 6 cm in length, and were collected by cutting as close to the skin as possible. The hair strands were maintained in alignment until analysis. Toxicological analyses were performed within 72 h of sampling. Two analyses were performed on each sample, an IA analysis using ILab 650 (Werfen, Milan, Italy) and a UPLC-MS/MS analysis using an ACQUITY UPLC® System (Waters Corporation, Milford, USA). The 1 st IA test was utilized as the screening test to be optimized, and the 2 nd test (UPLC-MS/MS) as the confirmatory test.
5
Quantification of Intracellular Lipids
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TAG, lactate and 3‐OHB were measured in collected media. For intracellular TAG, cells were lysed and heated to 95°C before being centrifuged at 15,000g for 10 min and supernatant removed for analysis on the ILab 650 (Instrumentation Laboratory, Werfen; Warrington, UK).
All assays were carried out according to manufacturer's instructions and used an appropriate calibrator or standard curve for quantification, as well as quality control samples. Assays were previously optimized for low concentrations found in vitro (Green et al.2015a ), with complete media used for a background measurement and results normalized to protein concentration.
All assays were carried out according to manufacturer's instructions and used an appropriate calibrator or standard curve for quantification, as well as quality control samples. Assays were previously optimized for low concentrations found in vitro (Green et al.
6
Oxidative Stress Biomarkers Determination
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Parameters of oxidative stress were determined as described previously on an ILAB 650 analyzer (Instrumentation Laboratory, Milan, Italy). In brief, paraoxonase 1 (PON1) activity was determined kinetically with a substrate-paraoxon (Chem Service Inc., West Chester, Pennsylvania, USA) using the method of Richter and Furlong [18 (link)]. The total sulfhydryl groups' (tSHG) levels were determined using dinitrodithiobenzoic acid as a reagent in alkaline buffer referred to Ellman's method [19 (link)]. Advanced oxidation protein products (AOPP) were measured according to the method of Witko-Sarsat, by a reaction with potassium iodide and glacial acetic acid [20 (link)]. Lipid hydroperoxides (LOOH) were determined by the method of Gay and Gebicki, using the ferric-xylenol orange method, following the precipitation in perchloric acid [21 (link)].
7
Plasma Metabolite and Hormone Analysis
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Blood samples were drawn into heparinized syringes, and plasma was prepared rapidly at 4 °C and immediately frozen at − 80 °C before analysis. Plasma glucose and NEFA concentrations were measured enzymatically using commercially available kits on an ILAB600 or ILAB650 clinical analyzer (Instrumentation Laboratory UK, Warrington, UK). Insulin and C-peptide were measured by ELISA (Invitron, Monmouth, UK) at a reference laboratory (Diabetes Research Unit Cymru, Swansea University, UK). Cortisol was measured by a colorimetric assay (R&D Systems, Abingdon, UK).
8
Biomarker Assessment in Plasma and Milk
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Plasma was assessed for biomarkers associated with metabolism (glucose, cholesterol, β-hydroxy-butyrate (BHB), nonesterified-fatty-acids (NEFA), creatinine and urea), innate immune response (haptoglobin, ceruloplasmin and zinc) and liver function (aspartate transaminase (AST/GOT), γ-glutamyl-transferase (GGT), total bilirubin, albumin, globulin, total protein and paraoxonase). Blood assays were performed as previously described [36 (link)] using the Instrumentation Laboratory Spa Tests (Werfen Co., Milan, Italy) on an ILAB 650 clinical auto-analyser (Instrumentation Laboratory, Lexington, MA, USA).
Milk samples were analysed for protein, fat, lactose, urea and somatic cell count (SCC) using a FT-NIR FOSS 6000 (FOSS Analytics, Hilleroed, Denmark). BHB was analysed in milk using the same test as for plasma. Cortisol, a biomarker of chronic stress, was assayed in milk and in hair, both following the procedure described by Sgorlon et al. [37 (link)].
Milk samples were analysed for protein, fat, lactose, urea and somatic cell count (SCC) using a FT-NIR FOSS 6000 (FOSS Analytics, Hilleroed, Denmark). BHB was analysed in milk using the same test as for plasma. Cortisol, a biomarker of chronic stress, was assayed in milk and in hair, both following the procedure described by Sgorlon et al. [37 (link)].
9
Standardized Assessment of Metabolic and Functional Parameters
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At enrollment and at each time-point, metabolic and functional parameters (i.e. glucose, insulin, lipid profile, liver and renal function) were assessed by a standardized validated protocol, using an automatic biochemical analyzer (ILAB 650, Instrumentation Laboratory, Lexington, MA). Low density lipoprotein cholesterol (LDL-C) concentration was estimated using the Friedewald formula [33 (link)], while non-high density lipoprotein cholesterol (non-high density lipoprotein-cholesterol, HDL-C) was calculated by subtracting HDL-C from total cholesterol (TC). The HOMA-Index and Cockroft-Gault index were calculated according to the relevant formula [34 (link), 35 (link)].
10
Plasma Biomarker Measurement Procedure
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The blood samples were drawn into heparinized syringes, and plasma was prepared
rapidly at 4°C and immediately frozen at −80°C before analysis.
The plasma glucose, NEFA, and glycerol concentrations were measured enzymatically
using commercially available kits on an ILAB600 or ILAB650 clinical analyzer
(Instrumentation Laboratory UK, Warrington, UK). Cortisol was measured using a
colorimetric assay (R&D Systems, Abingdon, UK). Insulin and C-peptide were
measured using an enzyme-linked immunosorbent assay (Invitron, Monmouth, UK) in an
accredited reference laboratory (Diabetes Research Unit, Swansea University, Swansea,
UK). Interleukin (IL)-6 was measured using an enzyme-linked immunosorbent assay
(Thermo Fisher Scientific, Loughborough, UK).
rapidly at 4°C and immediately frozen at −80°C before analysis.
The plasma glucose, NEFA, and glycerol concentrations were measured enzymatically
using commercially available kits on an ILAB600 or ILAB650 clinical analyzer
(Instrumentation Laboratory UK, Warrington, UK). Cortisol was measured using a
colorimetric assay (R&D Systems, Abingdon, UK). Insulin and C-peptide were
measured using an enzyme-linked immunosorbent assay (Invitron, Monmouth, UK) in an
accredited reference laboratory (Diabetes Research Unit, Swansea University, Swansea,
UK). Interleukin (IL)-6 was measured using an enzyme-linked immunosorbent assay
(Thermo Fisher Scientific, Loughborough, UK).
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