Cobas 6000 c501

A total of 16 serological markers were used in the study. The standard laboratory protocol was followed for the assessment of each marker. Low-density lipoprotein (LDL) (Direct), high-density lipoprotein (HDL), Triglyceride, Lipoprotein-Associated Phospholipase (Lp-PLA2) Test (PLAC), Homocysteine, and small dense low-density lipoprotein-cholesterol (sdLDL) were measured by the enzymatic-colorimetric method using Beckman Coulter AU680 clinical analyzer. Myeloperoxidase (MPO) was measured by latex enhanced Immunoturbidimetric method and total cholesterol was measured by the cholesterol dehydrogenase method using Beckman Coulter AU680 clinical analyzer. Oxidized low-density lipoprotein (ox-LDL) was measured by ELISA using Hamilton Microlab STAR. Apolipoprotein A1 (Apo A-1) and Apolipoprotein B (Apo B) were measured by the immunoturbidometric method, while high-sensitivity C-reactive protein (hs-CRP) and Lipoprotein(a) (Lp(a)) were measured by the particle-enhanced immunoturbidimetric assay using Roche Cobas 6000 c 501. N-terminal pro b-type natriuretic peptide (NT-proBNP) was measured by the electrochemiluminescence Immunoassay using Roche Cobas 6000 c 501. The concentration of LDL cholesterol (denoted as LDL-C) was calculated using triglycerides and HDL concentration using Friedewald’s formula (LDL cholesterol = total cholesterol − HDL cholesterol − (triglycerides/5).

Blood samples were collected on a weekly basis before the morning feeding from the jugular vein; serum was obtained using 9-mL vacutainer tubes (Vacuette; Greiner Bio-One, Kremsmünster, Austria). Acute phase proteins concentration analyses including Hp and SAA were determined using a Tridelta phase range Multispecies SAA ELISA kit (Tridelta Development Ltd., Greystones, Co., Wicklow, Ireland), SAA serum samples were diluted 1:500 and samples with optical density values above the standard curve were diluted again (1:400 or 1:250) and analyzed once more. No dilution of serum was needed for Hp measurement. Liver enzymes including alkaline phosphatase (ALP), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and gamma-glutamyl transferase (GGT) were measured with a conventional large-scale analyzer for clinical chemistry at the laboratory of the Central Clinical Pathology Unit, University of Veterinary Medicine, Vienna. The standard enzymatic colorimetric analyses with a fully automated autoanalyzer for clinical chemistry (Cobas 6000/c501; Roche Diagnostics GmbH, Vienna, Austria) was used. The intra-assay variation was controlled by limiting the coefficient of variation to ≤10% for SAA and <5% for other blood variables.

Seven animals were hospitalized for a period of 19 to 164 days at the animal clinic. Initial examination (including weighing) was performed upon admission. Weight records and blood samples from three animals were collected once or twice a week. Red and white blood cell count was determined using an automatic hematology analyzer (ADVIA 2120i, Siemens Healthcare, Vienna, Austria). Glucose, total protein, albumin, cholesterol, NEFA, total bilirubin, creatinine, potassium, sodium, AST, GLDH, LDH were measured in blood plasma using an automatic analyzer (Cobas 6000/c501, Roche Diagnostics GmbH, Rotkreuz, Switzerland).

Bodyweight, food intake and fasting glucose level were recorded every month. Blood was collected from tail vein, and plasma was isolated by centrifugation. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), low‐density lipoprotein cholesterol (LDL‐C) and HDL‐cholesterol (HDL‐C) levels were measured with an auto‐analyser (Cobas 6000 c501; Roche Diagnostics). Fasting blood glucose was measured with a One‐Touch Accu‐Chek Glucometer (Roche Diagnostics), and fasting serum insulin was determined using a rat‐specific insulin ELISA kit (Ultrasensitive Rat Insulin ELISA; Mercodia) following the kit instruction.

Blood smears were stained using the May–Grünwald–Giemsa stain (Hemacolor Rapid staining of blood smear kit; Merck KGaA, Darmstadt, Germany) [4 (link)], following which 100 leukocytes, including granular (heterophils, eosinophils, and basophils) and nongranular (lymphocytes and monocytes), were counted per slide using light microscopy (Leitz Orthoplan, Leitz, Wetzlar, Germany) at 100-times magnification. The heterophil:lymphocyte ratio was calculated [10 (link)]. Serum glucose, uric acid, triglycerides, cholesterol, and non-esterified fatty acids (NEFA) were determined using standard enzymatic colorimetric analysis using an autoanalyzer for clinical chemistry (Cobas 6000/c501; Roche Diagnostics GmbH, Vienna, Austria).

Blood samples were collected with anticoagulant (K3EDTA) and tested with an automated haematology analyser, Celltac Alpha (Nihon Kohden, Tokio, Japan). The following parameters were measured: white blood cell count, eosinophil count and percentage, red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, lymphocyte count and percentage, monocyte count and percentage, granulocyte count and percentage, red cell distribution width, procalcitonin test, mean platelet volume, platelet distribution width, and platelet count.
Sera prepared from the blood samples were analysed with an automated biochemistry analyser (Cobas 6000 C501; Roche, Basel, Switzerland). The following biochemical parameters were measured: total protein, albumin, gamma-glutamyl transferase, bilirubin, cholesterol, alanine aminotransferase, glucose, calcium, phosphorus, creatinine, and blood urea nitrogen.