Venipuncture

Due to the limited quantity of P. vivax IRBC healthy donor blood was used for the initial side-by-side comparisons of leukocytes and platelet removal methods. Ten ml of whole blood were collected onto Lithium heparin from five healthy donors by venepuncture. The whole blood was centrifuged at 500 g for 5 min at room temperature. The plasma supernatant was removed, but the buffy coat fraction containing white blood cells (WBC) was added back to the red blood cell (RBC) pellet. The RBC+WBC mix from each donor was divided into five two ml portions to which an equal volume of RPMI was added with gentle mixing. The first tube was used as the control for this study. The 2^nd tube was centrifuged again as above and the PBS supernatant and buffy coat carefully removed from the packed RBC, this tube was the 'Buffy Coat Removal' treatment. The 3^rd tube was loaded into a five ml syringe (the plunger removed) and mounted onto a Plasmodipur™ filter (Euro-Diagnostica^®) that was pre-rinsed with a sterile PBS solution. Then gentle pressure was applied to the syringe attached to the Plamodipur unit and the filtered RBC/PBS (50% haematocrit) mix was collected as the 'Plasmodipur' treatment. The 4^th and 5^th tubes were added to PBS-wetted CF11 columns. The filtered 4^th sample was then collected as the 'CF11' treatment. The filtered 5^th sample was then added to another unused CF11 column (pre-wetted with 5 ml of PBS). The double filtered 5^th sample was the 'CF11x2' treatment. In addition to the above samples, three more healthy volunteers were recruited, and 8 ml of whole blood were collected onto Lithium heparin. This blood was processed as above and divided into four 2 ml samples. The first and 2^nd tubes were processed as described above for the control and the 'CF11' treatments, respectively. The contents of the 3^rd tube were layered over 2 ml of Lymphoprep™ (Greiner Bio-One^®) and centrifuged at 600 g for 20 min at 20°C, the leukocytes that banded at the interface were removed using a pipette, and the supernatant RBC fraction was the 'Lymphoprep' treatment. The 4^th tube was processed as above for the 'Lymphoprep' treatment, but then subsequently processed using the 'Plasmodipur' treatment protocol described above. This final sample was called the 'Lymphoprep and Plasmodipur' treatment.
Please note; unless specifically stated, the term 'CF11 filtration' involved only a single CF11 filter, as opposed to 'CF11x2' which involved processing the sample using two CF11 columns.
Complete blood counts of the control samples and the suspensions obtained from the six treatments were conducted using an Automated Hematology Analyzer (Model pocH-100i, Sysmex Company) and by microscopic examination (x100 oil immersion) of Giemsa-stained thick (250 fields) and thin smears (450 fields). The data from the thin smears were used for differential lymphocyte counts.

Cholinesterase activities were measured in all the whole blood samples drawn from arterial lines or venepuncture at three different points in time: preoperatively (on the evening before surgery or right before anaesthesia induction), 15 min after surgery, and the next morning after surgery at 8 a.m. (± 1 h). We analysed the samples with the portable point-of-care photometry test kit and machine “ChE check mobile” according to the instructions of the manufacture (SECURETEC, Neubiberg, Germany). This test kit applies a modified Ellmann reaction according to Worek et al. to determine the activity of AChE (normalized to haemoglobin U/g Hb) and BChE (U/l). The testing was performed immediately after collecting the blood sample.
In order to compare cholinesterase activities between delirium and no delirium, we first adjusted the delirium incidence for our observation period. Patients were classified as delirium positive if they were positively detected with the CAM/CAM-ICU at least once until the first postoperative day.

The blood samples were collected via antecubital venipuncture during Week 1 and 6 visits. A phlebotomist drew 10 ml of blood from each participant’s arm into a K2 EDTA tube and then 2.5 ml of blood into a PAXgene RNA tube. To separate plasma from red blood cells, the whole blood from the K2 EDTA tubes was centrifuged at the speed of 1500 RPM for 15 min at room temperature (15 °C). Plasma was then aliquoted in cryovials and stored at −80 °C. The frozen plasma samples were transferred and assayed at University of California, Irvine for Aβ42, Aβ40, tTau, and pTau-181. The PAXgene RNA tubes were gently inverted ten times right after sample collection and kept at room temperature for between 2.0 and 70.2 h (mean = 6.95 h) to comply with the PAXgene RNA tube requirement of 2- to 72-h stabilization time. The samples were then temporarily stored in a −20 °C freezer before being stored at −80 °C. They were transported for analysis to the Social Genomics Core Lab at University of California, Los Angeles.