Xp 300 automated haematology analyzer

Whole blood was collected in EDTA Vacutainer^® tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for differential cell count and C-reactive protein measurements (Clinical chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden), as well as PBMC isolation, as previously described [18 (link)]. Isolated cells were counted using a Sysmex XP-300 Automated Haematology Analyzer (Sysmex Europe, Norderstedt, Germany) and resuspended at a concentration of 2 × 10⁶ cells/mL. Approximately 2 million cells were seeded per well and grown in RPMI complete medium (RPMI 1640, 1% [v/v] L-Glutamine and 1% penicillin-streptomycin, all from Hyclone, Logan, UT, USA) and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO₂. Cells were treated for 48 h with 0.2 µg/mL αCD3 (Purified NA/LE Mouse anti-Human CD3, clone UCHT1 (BD Pharmingen™, BD Biosciences, Franklin Lakes, NJ, USA)) and 0.33 µg/mL αCD28 (Purified NA/LE Mouse anti-Human CD28, clone CD28.2 (BD Pharmingen, BD Biosciences)), 2.5 µM dexamethasone (Merck, Branchburg, NJ, USA), either both treatments concurrently or medium only as control. Cells were collected and used in subsequent RNA or flow cytometry analyses.

Using sterile needles and syringes, qualified phlebotomists obtained 3 mL of venous blood from each participant. The blood samples were dispensed into labelled EDTA tubes to prevent clotting and were later used for haematological analyses. Complete blood counts were run on the blood using the Sysmex XP-300 Automated Haematology Analyzer. The analyser provided cell count data on red blood cells (RBC), white blood cells (WBC), lymphocytes, neutrophils and platelets. Other parameters measured included haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean platelet volume (MPV), plateletcrit and red blood cell distribution width (RDW).

From each participant, 2 ml of venous blood was drawn by a trained phlebotomist in the hospital laboratory. This was transferred into an EDTA tube with a unique participant identifier. The blood sample was used to prepare thin and thick films for microscopic examination and automated complete blood counts (CBCs). Blood parameters were estimated using the Sysmex XP-300 Automated Haematology Analyzer. The cell counter provided data on red blood cell (RBC) count, haemoglobin (Hb) level, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) level, WBC, lymphocyte, neutrophil, platelet counts, mean platelet volume (MPV), plateletcrit and the red blood cell distribution width (RDW).