Cell dyn 3500

EDTA-anticoagulated peripheral blood (PB) samples (two 2.7 mL specimens) were drawn within 24 hours of stroke onset, as well as on days 3 and 7, from stroke patients, and once from control subjects. The absolute numbers of leukocytes and lymphocytes in PB were determined using an automatic cell counter (Cell-Dyn 3500, Abbott Diagnostics, Santa Clara, CA, USA). High-sensitivity CRP (hsCRP) was assayed using a latex-enhanced immunonephelometric assay on a BN II analyzer (Dade Behring, Newark, DE), whereas fibrinogen was measured on this device by immunoassay. The full population of peripheral blood nucleated cells (PBNCs) was obtained after the lysis of red blood cells using 1 × BD Pharm Lyse Buffer (Pharmingen, BD Biosciences, San Diego, CA, USA).

The following blood samples were collected from all cattle: 5 ml of EDTA blood for haematological analysis, 10 ml of whole blood for serum biochemistry, 2 ml of whole blood mixed with 0.2 ml heparin for venous blood gas analysis and 5 ml of EDTA blood for the glutaraldehyde test. Haematological analysis included the determination of PCV, total leukocyte count and the concentrations of fibrinogen and total protein using an automated blood analyzer (CELL-Dyn 3500, Abbott Diagnostics Division, Baar). A differential leukocyte count was done in cattle with leukopenia (< 5000 leukocytes/μl blood) or leukocytosis (> 10,000 leukocytes/μl blood). The concentrations of serum urea nitrogen and bilirubin and the activities of the enzymes aspartate aminotransferase (ASAT), γ-glutamyltransferase (γ-GT) and glutamate dehydrogenase (GLDH) were determined at 37 °C using an automated analyser (Cobas-Integra-800-Analyser, Roche Diagnostics, Basel) and the manufacturer’s reagents (Roche-Reagents) according to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Venous blood gas analysis was done using an automated analyser (RapidLab 248, (Siemens Schweiz AG, Zurich). A glutaraldehyde test (Glutaltest^®, Graeub AG, Bern) was performed according to the manufacturer’s instructions. Results were compared to reference intervals recently reported [18 (link)].

PB samples will be collected in sterile evacuated blood collection tubes with anticoagulant ethylenediaminetetraacetic acid (EDTA; 3 ml) at the following time points:

preoperatively (T0), at 24 h (T1), 72 h (T2), 7 (T3), and 10 (T4) postoperative days in groups S and C

T0 in group H

The absolute numbers of leukocytes and lymphocytes in PB will be determined at the same time with an automatic cell counter (Cell-Dyn 3500, Abbott Diagnostics, Santa Clara, CA, USA). Peripheral whole blood samples (100 μl) will be used for fluorescence-activated cell sorting (FACS) analysis, and the plasma samples will be collected and stored at −80 °C.
Biochemical tests will be determined daily, according to the routine procedure in the surgical ward and ICU, and performed in the laboratories at University Hospital Foggia, according to the institutional guidelines.
Demographic, anamnestic, clinical, and laboratory data will be recorded.

On day 37, blood (3 ml) was collected from non-fasted birds (10 birds/treatment; 2 birds/pen), via the brachial wing vein, in ethylenediaminetetraacetic acid-coated tubes and immediately placed on ice. The hematologic profile of 1 ml of whole blood samples was measured using the Cell-Dyn 3500 automated hematology analyzer calibrated for chicken blood (Abbott Diagnostics, Abbott Park, IL) within 3 h of sampling. Data collected included the percent of heterophils, lymphocytes, monocytes, eosinophils, and basophils and the calculated heterophil to lymphocyte (H/L) ratio. For gene expression analysis, 250 μl of blood was added to tubes containing 750 μl of TRIzol LS reagent according to manufacturer's recommendations (Life Technologies Corporation, CA, US). The birds' bodyweights were 2.61 ± 0.07, 2.60 ± 0.07, and 2.72 ± 0.08 kg for SCP, LCP, and SP-LCP, respectively.

For each test subject (30/30), platelet count (PLT/μL) leucocyte count (WBC/μL) and erythrocyte count (RBC/μL), were calculated on WB and L-PRP by an automatic analyzer using optical and volumetric impedance measurements (Cell-Dyn 3500 analyzer, Abbott Diagnostics Europe), while neutrophils (cells/μL), monocytes (cells/μL) and lymphocytes (cells/μL) counts were calculated with the same instrument and checked with a manual differential count of blood smears in only 19/30 subjects, for technical reasons. In the same 19 subjects the smears were also used for assessment of possible platelet clumping. All sample were stored at room temperature on a laboratory blood rocker for a minimum of 5 min before counts were performed.

Serum levels of CK, LDH, U, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were performed by enzymatic methods using a Targa bt 3000® biochemical autoanalyzer. The biochemical reagents used were of the brand Wienner lab® (Wiener Lab, Rosario, Argentina). Concentrations of hemoglobin (Hb) and hematocrit (Ht) were measured by the spectrophotometric method using Cell-Dyn 3500 (System Operators Manual, Abbott Laboratories, Abbott Park, Illinois, USA). Mean corpuscular hemoglobin concentration (MCHC) was also calculated. Quantification of FT and cortisol (C) in the serum were done using kits (Elecsys 2010, Roche diagnostics, Germany) and analyzed by electrochemiluminescence and chemiluminescence, respectively, using an Hitachi Modular equipment (Hitachi, Tokyo, Japan). All analyses followed the protocol of their manufacturers and were performed in duplicates. Between and within coefficients of variation for all assays were less than 10% for all biochemical analyses.