The biochemical parameters of severity were serum amylase, pancreatic trypsin, pancreatic myeloperoxidase (MPO) activity, and lung MPO activity. Amylase levels were tested using a kinetic method with a Roche automated clinical chemistry analyzer (Burgess Hill, United Kingdom). Trypsin activity was measured by a fluorogenic assay, using Boc-Gln-Ala-Arg-AMC substrate converted by trypsin to a fluorescent product (excitation 380 nm, emission 440 nm).21 (link) For MPO activity, 20 μL of extract was added to 200 μL phosphate buffer (100 mM, pH 5.4, with 0.5% HETAB) and 20 μL 3,3′,5,5′-tetramethylbenzidine (20 mM) in dimethyl sulfoxide. This mixture was incubated at 37°C for 3 minutes, followed by addition of 50 μL of H2O2 (0.01%) for further incubation over 3 minutes. The difference of absorbance between 0 and 3 minutes at 650 nm was calculated using a standard curve compiled using human MPO.22 (link)
Automated clinical chemistry analyzer
Manufactured by Roche
Sourced in United Kingdom, Switzerland
The Roche automated clinical chemistry analyzer is a laboratory instrument designed to perform a wide range of automated clinical chemistry tests. It is capable of analyzing various biological samples such as blood, urine, and body fluids to measure the levels of specific substances, including enzymes, proteins, and metabolites.
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6 protocols using automated clinical chemistry analyzer
1
Biochemical Markers of Pancreatitis Severity
2
Longitudinal Biomarker Measurement Protocol
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Venous blood samples were drawn from each subject at baseline and at 6 months. The patients did not have specific appointment to the laboratory, but due to the operating hours of the regional laboratory services, the samples were obtained in the morning. Serum was separated by centrifugation and stored at − 80 °C prior to analysis. Serum IL-1Ra concentration was measured by Quantikine high sensitivity enzyme-linked immunosorbent assay (ELISA) (R&D Systems Europe, Abingdon, UK). The detection limit and the interassay coefficient of variation were 15.6 pg/ml and 7.4%, respectively. High Sensitivity CRP (hs-CRP) was measured using particle-enhanced immunoturbidimetric method on an automated clinical chemistry analyzer (Roche Diagnostics, Basel, Switzerland). Concentration of serum IL-6 was determined using Quantikine high sensitivity ELISA kit according to the instructions of the manufacturer (R&D Systems, Abington Science Park, UK).
3
Serum Biomarker Measurement in Patients
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Each blood sample was allowed to clot and was centrifuged at 1,500 ×g for 10 minutes. Sera were collected, aliquoted, and kept frozen at ‒70°C until analysis. This molecular study was approved by the Ethics Committee of the study institute. The SAA levels were measured in serum samples using commercial enzyme-linked immunosorbent assay (ELISA) kits from Invitrogen (Camarillo, CA) according to the manufacturer’s instructions. All of the ELISAs were performed in duplicate. The results were obtained by a standard curve prepared according to the manufacturer’s instructions and with a coefficient of variation < 10%. The plasma CRP concentrations were immunoturbidimetrically quantified using a Roche automated clinical chemistry analyzer (Roche Diagnostics, Belleville, NJ). As described in previous study [15 (link)], patient plasma EBV DNA concentrations were routinely measured by quantitative polymerase chain reaction before treatment.
4
Clinical Laboratory Biomarkers in COVID-19
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All medical laboratory data including the numbers of leukocytes, lymphocytes, and eosinophils; percentages of lymphocyte and eosinophils; concentrations of D-dimer, high-sensitivity C-reactive protein (hsCRP), procalcitonin (PCT), and serum creatine kinase were generated by the clinical laboratory of Tongji Hospital and the Second Xiangya Hospital. The samples for laboratory tests were collected on admission and during the hospital stay. Peripheral venous blood was collected for routine blood test using automatic hematology analyzer. The biochemical parameters such as liver and renal function were measured by Roche automated clinical chemistry analyzer (Roche Diagnostics, Mannheim, Germany). Serum PCT was measured by the luminescence immunoassay, and hsCRP was measured with a latex particle-enhanced immunoturbidimetric assay (Roche Diagnostics). It is worth mentioning that coagulation tests were detected using a STA-R MAX coagulation analyzer and original reagents (Diagnostica Stago, Saint-Denis, France) in both hospitals. The laboratory data for some patients were missing due to the absence of types of tests or delayed results.
5
SARS-CoV-2 Biomarker Monitoring Protocol
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Real-time RT–PCR tests were performed to detect SARS-CoV-2 nucleic acid; the detailed protocols were described previously [32 (link)]. During hospitalization, serial monitoring of 30 laboratory biomarkers (Table S1 ) was conducted for each COVID-19 patient, and blood samples were collected at different time points when laboratory biomarkers were requested by physicians to monitor disease progression. Laboratory biomarkers, such as routine peripheral blood biomarkers, inflammatory biomarkers, coagulation biomarkers, serum biochemical biomarkers, and anti-SARS-CoV-2 IgG and IgM antibodies, were assessed during hospitalization. Peripheral blood was collected for routine blood tests using an automated hematology analyzer. Biochemical biomarkers such as albumin, alanine aminotransferase, aspartate aminotransferase, and creatinine were measured using a Roche automated clinical chemistry analyzer (Roche Diagnostics). Coagulation tests were conducted with a new coagulation analyzer, STAR Max® (Diagnostica Stago). Serum quantitative measurements of anti-SARS-CoV-2 IgM and IgG antibodies were conducted using commercial chemiluminescence kits of iFlash-SARS-CoV-2 IgM (Cat. No. C86095M) and iFlash-SARS-CoV-2 IgG (Cat. No. C86095G) from Shenzhen YHLO Biotech. Seroconversion was defined by the cutoff of IgG ≥ 10 AU/mL or IgM ≥ 10 AU/mL.
6
Surgical Resection Outcomes and CRP Levels
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Available data at baseline (surgery date) included age, gender, body mass index (BMI), percent predicted forced expiratory volume in the first second of expiration (FEV 1 ) and plasma level of CRP at baseline (CRP 0 ). In addition, CRP level was evaluated 3 days (CRP 3 ) after surgery, and the corresponding maximum value (CRP max ) after surgery was recorded. CRP was quantified by immunoturbidimetry using a Roche automated clinical chemistry analyzer (Roche Diagnostics, Belleville, NJ) from the same laboratory through the entire study period.
Of 2183 consecutive resections from 2001 patients, we excluded resections that did not have: (i) TNM stage of tumour (16 resections), (ii) CRP 0 , and/or CRP 3 , and/or CRP max (272 resections) and (iii) follow-up information (15 resections). Of the remaining 1880 evaluable resections, we selected the cohort formed by 1750 patients who underwent their first resection during the considered period (Fig. A.1) .
Each member of the study population accumulated person-years of follow-up from baseline until the date of death (all causes mortality being the outcome of interest for the current study) or till 26th June 2016 for survivors.
Of 2183 consecutive resections from 2001 patients, we excluded resections that did not have: (i) TNM stage of tumour (16 resections), (ii) CRP 0 , and/or CRP 3 , and/or CRP max (272 resections) and (iii) follow-up information (15 resections). Of the remaining 1880 evaluable resections, we selected the cohort formed by 1750 patients who underwent their first resection during the considered period (Fig. A.1) .
Each member of the study population accumulated person-years of follow-up from baseline until the date of death (all causes mortality being the outcome of interest for the current study) or till 26th June 2016 for survivors.
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