Se9000

During the study period, serum CRP (reference value: <5 mg/L) values as well as albumin (reference value: 35–55 g/L) values were measured through an automated analyzer (Roche Hitachi Cobas 8000, Rotkreuz, Switzerland), and hemoglobin, lymphocyte, platelet, and neutrophil counts were determined through a hematology analyzer (Sysmex SE-9000, Kobe, Japan). Preoperative HS-mGPS of 2, 1, and 0 were assigned to patients identified as having both hypoalbuminemia (<35 g/L) and a high CRP level (>3 mg/L), having either hypoalbuminemia or a high CRP level, and having neither hypoalbuminemia nor a high CRP level, respectively (12 (link)). To calculate the NLR, the neutrophil count/lymphocyte count ratio was derived; to calculate the PLR, the platelet count/lymphocyte count ratio was derived.

To probe the correlation between survival outcomes and systemic inflammatory indices, we performed preoperative blood laboratory tests within 1 week before curative surgery. According to the blood tests and recorded clinical symptoms and signs, severe infection status was excluded in all patients. The medical staff collected the hematological and biochemistrical parameters during the treatment from patient’s charts. Pretreatment biochemistry values of albumin (reference value: 35–55 g/L) and CRP (reference value: <5 mg/L) were measured using biochemistry automated analyzer (Roche Hitachi Cobas 8000, Rotkreuz, Switzerland) during the study period. Hematological results of lymphocyte, neutrophil, hemoglobin, and platelet were measured using the hematology analyzer (Sysmex SE-9000, Kobe, Japan). Preoperative CAR was calculated as follows: CRP level (expressed in mg/L)/albumin level (expressed in g/L). Similarly, we derived the preoperative NLR and PLR as follows: peripheral blood neutrophil count/lymphocyte count and platelet count/lymphocyte count, respectively. Next, mGPS was calculated using previously published methods (McMillan, 2008 (link)). Patients with both hypoalbuminemia (<35 g/L) and increased CRP levels (>10 mg/L), with one of these variables, and with none of these variables were assigned the scores of 2, 1, and 0, respectively.

Blood samples were collected from the antecubital vein within 24 h of the echocardiography. Serum albumin was measured by a dye-binding bromocresol green procedure and C-reactive protein (CRP) was measured by latex agglutination immunoassay using a JCA-BM2250 analyzer (Japan Electron Optics Laboratory, Tokyo, Japan). White blood cells were measured by the direct current detection method using a Sysmex SE 9000 (Sysmex, Kobe, Japan). Blood samples were also analyzed for free T4, free T3, TSH and TRAb by the electrochemiluminescence immunoassay using Modular Analytica E170 (Roche Diagnostics, Rotkreuz, Switzerland).

Single cell suspensions of spleen, lymph nodes and thymus were obtained via 70-µm cell strainers in fluorescence-activated cell sorting (FACS) buffer (PBS containing 5% FCS). After lysis of red blood cells with ACK lysis buffer, cells were separated via 40-µm cell strainers and stained with flurochrome-conjugated antibodies (V500-coupled anti-CD45.2, V450-coupled anti-CD4, fluorescein isothiocyanate-coupled anti-CD8 (BD Biosciences, Heidelberg, Germany); APC-coupled anti-CD3 (Biolegend)) for 30 min at 4 °C in FACS buffer. Flow cytometry was performed using a BD LSR II Flow Cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo, Ashland, OR, USA).
Platelet counts were determined in whole blood drawn from the retroorbital plexus of isoflurane-anesthetized animals into 300 μL acid citrate-dextrose buffer. Platelet numbers were determined with an automated blood analyzer (Sysmex Se 9000, Kobe, Japan).

Leucocyte levels were conducted on a Sysmex SE9000 with a coefficient of variation (CV) of 5%. Neutrophil, eosinophil, basophil, lymphocyte, and monocyte levels on a Sysmex XE-2100 with a CV of 6%, 12%, 6%, 6%, and 15%, respectively. CRP was measured on a Cobas 8000, c702 modul with a CV of 6%, whereas orosomucoid was measured on a Cobas 8000, c502 modul with a CV of 9%. Ferritin was measured on a Modular E-modul with a CV of 7% and PTH was measured on a Cobas 8000 (Roche) with a CV of 7%. Measurements of hemoglobin (CV2%) and thrombocytes were both conducted on a Sysmex XE-2100. The vitamin D measurements of 25(OH)D₃ (CV < 10%) and 1,25(OH)₂D₃ (< 18%) levels were measured with an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS).