Dogs were initially classified as cases or controls based on clinical diagnoses from expert veterinarians certified in veterinary internal medicine. The clinical diagnoses of cases were subsequently supported by thyroid typical serological measurements (thyroid-stimulating hormone (TSH) and free thyroxine (fT4)) using Siemens IMMULITE Immunoassay System [31 (link), 37 (link)]. In order to be classified as cases, dogs had to present with increased levels of TSH (> 40 mU/L) and reduced levels of fT4 (< 7 pmol/L), whereas controls had to be older than seven years of age. A subsequent careful survey of clinical records and/or questionnaires completed by dog owners was performed to exclude cases with another condition potentially influencing the dog’s wellbeing as well as controls with a history of immune-mediated conditions.
Immulite immunoassay system
Manufactured by Siemens
Sourced in Germany
The Immulite Immunoassay System is a diagnostic instrument designed for the detection and quantification of various analytes in biological samples. It utilizes an automated, random-access chemiluminescent immunoassay technology to perform a wide range of immunoassay testing. The system is capable of processing multiple sample types and providing accurate and reproducible results.
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8 protocols using immulite immunoassay system
1
Diagnosing Canine Hypothyroidism
2
Canine Serum Testosterone Assay
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At the T0 and T1 time points, blood was collected from the saphenous vein of each dog between 8:00 a.m. and 8:30 a.m. A portion of blood was allowed to stand for 10‐15 min at 4°C, and then centrifuged at 1.500 × g for 10 min at 4°C prior to aspiration of serum. Serum samples were stored at −20°C until thawed and assayed for testosterone concentrations by a chemiluminescence technique (Immulite Immunoassay System, Siemens Healthcare, Munich, Germany).
3
Testosterone Measurement in Blood
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Blood was sampled by vein-puncture at T0 and T3. Testosterone was measured considering the physiological secretory patterns. All collections were made between 9:30 and 11:30 a.m. The blood collected was immediately centrifuged (1500g, 15 min at room temperature) so the serum could be separated and stored at −20°C until assay. Testosterone was measured by a chemiluminescence technique (Immulite Immunoassay System, Siemens, Healthcare Diagnostics S.r.l., Milano, Italy).
4
Multiplexed Biomarker Measurement Protocol
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Biomarker measurements were performed in duplicate on stored frozen plasma samples from the day of study enrollment. Plasma levels of IL-6, LBP, and IL-2R were measured using the IMMULITE immunoassay system (Siemens Medical Solutions Diagnostics, Germany). Additionally, plasma levels of ELA2, HGF, IL-6, IL-8, LTF, NGAL, resistin, sFas, TSP-1, and TRAIL were measured with the MILLIPLEX® MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 and the MILLIPLEX® MAP Human Sepsis Magnetic Bead Panel 3 (Merck Millipore, Germany) on a Luminex MAGPIX® following the manufacturer’s instructions (Luminex Multiplexing Instrument, Merck Millipore). Biomarker levels below the lowest standard were extrapolated corresponding to the equation of the standard curve.
5
Serum Vitamin D and IL-6 in COVID-19
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To assess whole-body VitD status of patients, levels of total 25(OH)D were measured retrospectively in cryopreserved (−80 °C) serum samples collected in gel tubes at the time of admission and SARS-CoV-2 testing. Serum levels of total 25(OH)D were quantified using a commercially available immunoassay (ADVIA Centaur Vitamin D Total Assay®, Siemens Healthcare GmbH, Erlangen, Germany).
In inpatients, serum levels of Interleukin-6 (IL-6) were measured prospectively at the time of hospitalization. IL-6 was quantified using a commercially available immunoassay (IMMULITE® Immunoassay System, Siemens Healthcare GmbH, Erlangen, Germany).
All measurements were carried out at the Department of Clinical Chemistry of the Heidelberg University Hospital using accredited laboratory methods (certified according to ISO 15189 by Germany’s national accreditation body).
In inpatients, serum levels of Interleukin-6 (IL-6) were measured prospectively at the time of hospitalization. IL-6 was quantified using a commercially available immunoassay (IMMULITE® Immunoassay System, Siemens Healthcare GmbH, Erlangen, Germany).
All measurements were carried out at the Department of Clinical Chemistry of the Heidelberg University Hospital using accredited laboratory methods (certified according to ISO 15189 by Germany’s national accreditation body).
6
Testosterone Levels in Dogs After hCG Injection
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To determine testosterone levels at time intervals T0 to T3, dogs received subcutaneous (SC) injections of 1,000 international units (I.U.) of human chorionic gonadotropin (hCG) (Creative Biomart, CD, Inc.) [2 ,17 (link)]. At 120 min after the hCG injections, blood was collected as previously described [2 ]. Testosterone was measured by a chemiluminescence technique (Immulite Immunoassay System, Siemens).
7
Biomarker Changes in Exercise Response
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Biochemical analysis was conducted in the serum from the participants collected before and after each exercise bout. Systemic leptin (Peprotech Inc, USA) and insulin growth factor-1 (IGF-1) (BOSTER, USA) levels were evaluated by enzyme-linked immunosorbent assay (ELISA). Cortisol concentrations were measured using an Immulite immunoassay system (Siemens, USA). Blood glucose, lactate, and uric acid levels were evaluated through automated biochemical assay using Bioclin BS120 (BioClin, Brazil).
8
Canine hCG-Induced Testosterone Assay
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Between 8:00 a.m. and 8:30 a.m. at T0 to T12 dogs received subcutaneous injections of 1,000 international units of human chorionic gonadotrophin (hCG) (Creative Biomart, CD Inc.) in order to stimulate gonadal testosterone levels [20 (link)-22 (link)]. Blood was collected from the saphenous vein of each dog 120 min after the hCG injections. A portion of blood was allowed to stand for 10-15 min at 4°C, then centrifuged at 1,500 g for 10 min at 4°C prior to aspiration of serum. Serum samples were stored at -20°C until thawed and assayed for testosterone concentrations by a chemiluminescence technique (Immulite Immunoassay System, Siemens).
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