Sterile tubes

Human milk collection has been described previously [48 (link)]. Briefly, this involved mothers washing their hands thoroughly with soap and water and cleaning the nipple and areola of the expressing breast with alcohol and chlorhexidine prep pads (70% isopropyl alcohol and 2% chlorhexidine digluconate, Reynard Health Supplies, Artarmon, NSW, Australia), followed by rinsing with sterile saline solution (Livingstone, Mascot, NSW, Australia) and drying with sterile gauze swabs (Livingstone, Mascot, NSW, Australia). 10–20 mL of human milk was hand-expressed directly into sterile tubes (Greiner Bio-One, Kremsmünster, Austria). Human milk samples were stored at 4 °C in the fridge at the participant’s home before being collected within 24 h and transported on ice to the laboratory, where they were immediately aliquoted into sterile tubes (Sarstedt, Numbrecht, Germany) and stored at −80 °C until further analysis.

HM samples were collected by mothers at 2 months postpartum. Mothers elected one breast from which to donate the HM sample. Mothers were asked not to breastfeed or express milk from the elected breast for at least two hours prior to sample collection. Mothers washed their hands thoroughly with soap and water and wore disposable nitrile powder-free gloves (Complete Office Supplies, NSW, Australia) during sample collection. The nipple and areola of the expressing breast were cleaned with alcohol and chlorhexidine prep pads (70% isopropyl alcohol and 2% chlorhexidine digluconate, Reynard Health Supplies, NSW, Australia), followed by rinsing with sterile saline solution (Livingstone, NSW, Australia) and drying with sterile gauze swabs (Livingstone, NSW, Australia). Up to 20 mL (otherwise as much as possible) of HM was hand-expressed directly into sterile tubes (Greiner Bio-One, Kremsmünster, Austria). HM samples were stored at 4 °C in the fridge at the home of the participant before being collected within 6–24 h and transported to the laboratory on ice, where they were immediately aliquoted into sterile tubes (Sarstedt, Numbrecht, Germany) and stored at −80 °C until further analysis.
Mothers answered a background questionnaire at the time of recruitment, and an infant and maternal questionnaire on sample collection day.

Deep cough swabs were collected after obligate coughs using sterile cotton swabs and placed in DNA LoBind Tubes (Eppendorf; no. 30120094) (40 (link)). The swabs were trimmed with sterile scissors and immediately stored at −80°C until further processing. Sputum was collected according to the CFFT Therapeutics Development Network standard operating procedure 530.00 (70 ) for sputum induction after inhalation of 3% NaCl in sterile tubes (Sarstedt; no. 62.547.004) and immediately stored at −80°C until further processing.

Deep cough swabs were collected after obligate coughs using sterile cotton swabs and placed in DNA LoBind Tubes (Eppendorf; no. 30120094) (40 (link)). The swabs were trimmed with sterile scissors and immediately stored at −80°C until further processing. Sputum was collected according to the CFFT Therapeutics Development Network standard operating procedure 530.00 (70 ) for sputum induction after inhalation of 3% NaCl in sterile tubes (Sarstedt; no. 62.547.004) and immediately stored at −80°C until further processing.

All patients were resident at altitudes less than 600 m and had not been to heights greater than 1,500 m in the 3 months preceding the study. Patients flew to La Paz, Bolivia (3,600 m), and spent 4 or 5 days there before ascending for 90 minutes by road to the Chacaltaya Laboratory (5,200 m).
Fig. 1Ashows the ascent profile of the expeditions and the timing of blood sampling. Sea-level samples were taken before the expedition on Apex 4 but after the expedition on Apex 2. Blood was drawn using a 21-gauge needle, with minimal tourniquet pressure and collected in sterile tubes (Sarstedt, Nümbrecht, Germany) containing sodium citrate or EDTA. Peripheral arterial oxygen saturation (SpO
₂) was measured for each patient prior to departure and on each day of investigation using a pulse oximeter (Masimo Rad 5, Irvine, United States). Patients self-recorded symptoms of acute mountain sickness (AMS) using the Lake Louise self-assessment score (LLS)
17
and a visual analogue scale symptom score.
18 (link)

Identical protocols and consumables were used in both settings. Cord blood samples (20–50 ml) were collected in sterile tubes (Sarstedt AG, Nümbrecht, Germany) containing 20 ml of RPMI‐1640 (Invitrogen‐Life Technologies, Melbourne, Australia) and 20 IU/ml of preservative‐free heparin (Pfizer, West Ryde, NSW, Australia). Samples were processed within 18 h from the time of collection to isolate cord blood mononuclear cells (CBMC) by centrifuging twice over a Ficoll‐Hypaque gradient (Lymphoprep, Alexis‐Shield, Oslo, Norway). Cells were cryopreserved at concentrations of 20–50 × 10⁶ cells/ml in 50% heat‐inactivated (HI) fetal calf serum (FCS) (JRH BioSciences, Lenexa, KS, USA) and 7·5% dimethyl sulphoxide. Cells from PNG newborns were transported in a dry‐shipper to Perth for further experiments.

1 tube of 2.6 ml of blood on EDTA was centrifuged (FrontierTM, Ohaus, FC5718R, Germany) for 20 min at 340 xg at room temperature, then the supernatant was collected with a pasteur pipette (Sarstedt, 86.1171.010) and pipetted 0.5 ml into 2 sterile tubes (Sarstedt, 72.694.006).