Lithium heparin

Human peripheral blood was obtained by venipuncture using coated with lithium heparin (Greiner Bio-One, Austria) vacuum tubes CAT serum separator clot activator (Greiner Bio-One, Austria) or coated with lithium heparin (Greiner Bio-One, Austria) and then transferred in 2 ml tubes. In both the procedures the samples were allowed to clot for at least 30 min at room temperature in an upright position and were then centrifuged at 1800xg for 10 min. Serum was recovered and stored in fresh 1.5 ml tubes at −20°C. Serum levels of total of IgG1, IgG4, and IgE were determined on a BNII nephelometer (Siemens Healthcare Diagnostic Products, Vienna, Austria). The measured concentrations of total sera IgG1, IgG4, and IgE were normalized using a z-normalization formula for each class separately.
z=x−xˉσ $\bar{x}$ = mean of total sera concentration for the specific class; σ = standard deviation of total sera concentration for the specific class.
We obtained three values: z-normalized IgG1 (zIgG1) values, z-normalized IgG4 (zIgG4) values, and z-normalized IgE (zIgE) values.
Then, we hypothesized that the zIgG4 values could correlate positively with the metastatic stage of CRC and zIgE values could correlate negatively with the metastatic stage of CRC. To demonstrated that we created the zIgG4/-zIgE ratio that could include both the characteristics. As controls, zIgG1/-zIgE ratios were used.

A trained phlebotomist collected venous blood samples into a plastic serum vacuette tube (9 mL, cat no: Lithium Heparin; Greiner, Kremsmünster, Austria), which we had shown to be free of endotoxin. All samples were allowed to clot for 2 h at room temperature and subsequently centrifuged at 3500 g at 4 °C for 15 min (Hettich Universal 320R, Geldermalsen, Netherlands). Raw plasma was decanted into pyrogen free tubes (cat: 80-507 USP Type 1 flint borosilicate glass tubes with caps; Lonza, Walkersville, MD, USA) using pyrogen free pipette tips (Cat; BE10051, Lonza, Walkersville, MD, USA). Aliquots of raw serum were stored at −80 °C [32 (link)] pending further analysis.

Blood was collected by venipuncture in vacuum tubes containing separating gel and lithium heparin (4 mL; Greiner Bio-One, Frickenhausen, Germany) or separating gel and silica coagulation activator (4 mL; Greiner Bio-One). After resting for 30–60 min, the tubes were centrifuged at 2,400×g for 5 min. Aliquots of serum and plasma were stored initially at −20°C for less than 1 month, after which the samples were transferred to −70°C and kept frozen until analysis.

Animals were immunized twice, two months apart, with the ANRS recombinant MVA-HIV B vaccine (Transgene) at a dose of 4 x 10e⁸ plaque forming units (PFU) by ID injections distributed over 10 injection points (150 μL/point of injections, all along the back, in two columns). Recombinant HIV-1 antigens consisted of the complete sequence of gag, fused to fragments from pol (residues 172-219, 325-383 and 461-519) and nef (residues 66-147 and 182-206) of the Bru/Lai isolate. Blood samples were taken longitudinally before and after immunizations in either lithium-heparin (Greiner Bio-One) for mass cytometry analysis, or in ethylenediaminetetraacetic acid (EDTA) (Greiner Bio-One) for complete blood counts (CBCs) and plasma preparation. The same batch and dose of vaccine, and vaccine schedule, but a different route of administration, were used relative to a previous preclinical study that analyzed innate responses after SC immunization (9 (link)), and for which the mass cytometry dataset was reused here.

Blood samples were collected in lithium heparin (Greiner Bio-one) and processed fresh without storage. Peripheral blood mononuclear cells (PBMCs) were extracted by Lymphoprep™ (Axis-Shield) density gradient separation. Monocytes were isolated from PBMCs by positive selection using anti CD14 microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD14⁺ monocyte purity was assessed by flow cytometry (CD3, CD19, CD15, CD16, and CD14). Only samples with purity of greater than 98% were used.

Venous peripheral blood was obtained from healthy donors with known HLA types. Each blood donor had signed a written consent form, allowing for the use of his or her blood for research purposes. The procedure was approved by local ethics legislation. The blood was collected in heparinized tubes (Vacuette, Lithium Heparin, Greiner Bio One, Frickenhausen, Germany). Isolation of peripheral blood mononuclear cells (PBMCs) was accomplished by using Lymphoprep ™ gradient centrifugation (Axis-Shield, Oslo, Norway). The PBMCs were either used directly after the isolation or stored at −140 °C in a storage medium (RPMI 1640 (Gibco, Life Technologies, Carlsbad, CA, USA), 40% heat-inactivated fetal calf serum (FCS) (Gibco), 10% dimethyl sulfoxide (Merck Millipore, Darmstadt, Germany), 100 U/mL penicillin/10 µg/mL streptomycin (Ampliqon, Odense, Denmark)). The CD4+ T cells were isolated from PBMCs using either a Dynabeads^® CD4 Positive Selection kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) or a Dynabeads^® Regulatory CD4+ CD25+ T Cell Kit (Invitrogen), using only the CD4+ isolation part. The entire procedure was carried out according to the manufacturer’s guidelines. The purity of the isolated cells was evaluated by flow cytometry.