The validation cohort comprised a subset of Gambian participants of the Grand Challenges 6–74 (GC6–74) project, as previously described [4 (link),5 (link),15 (link)]. Briefly, between March 5, 2007 and October 21, 2010, household contacts of TB cases were longitudinally followed for up to 2 years, with assessments at baseline, at 6 months, and at 18 months. Immunological sensitization to M. tuberculosis was performed by TST. TB progressors who developed microbiologically confirmed pulmomary TB during follow-up were retrospectively identified and matched 1:4 to healthy nonprogressors. Individuals in whom TB disease developed within 3 months of baseline were excluded. Blood and plasma were collected in lithium heparin tubes (Becton Dickinson) at enrollment, month 6, and month 18 of the GC6–74 project, and 254 plasma samples from 34 progressors and 115 nonprogressors were included for validation. Further details about clinical and epidemiological attributes and the selection of progressors and nonprogressors are provided in S2 Text and S2 Table . Participants provided written, informed consent, and the protocols were approved by the Joint Medical Research Council and Gambian Government ethics review committee, Banjul, The Gambia (SCC.1141vs2).
Lithium heparin tube
Manufactured by BD
Sourced in United States, Australia, United Kingdom
Lithium heparin tubes are laboratory equipment used for the collection and storage of blood samples. They contain lithium heparin, an anticoagulant that prevents the blood from clotting, allowing for further analysis and testing.
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Lab products found in correlation
Lymphoprep, by Axis-Shield (6 mentions)
Edta tube, by BD (3 mentions)
Macs cell separation column, by Miltenyi Biotec (2 mentions)
Lymphoprep, by STEMCELL (2 mentions)
Lithium heparin tubes, by Merck Group (2 mentions)
Dipyridamole, by Merck Group (2 mentions)
Erythro 9 2 hydroxy 3 nonyl adenine hydrochloride, by Merck Group (2 mentions)
K2edta tubes, by BD (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Fgf 21 quantikine elisa kit, by R&D Systems (2 mentions)
Vetscan hm5 hematology analyzer, by Abaxis (1 mentions)
Piccolo xpress chemistry analyzer, by Abaxis (1 mentions)
Luer lok tip, by BD (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Midimacs system, by Miltenyi Biotec (1 mentions)
Six well culture plate, by Corning (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Histopaque, by GE Healthcare (1 mentions)
Egm 2 plus bullet kit, by Lonza (1 mentions)
71 protocols using lithium heparin tube
1
Validation of Gambian TB Progression Cohort
2
Isolation and Characterization of PBMCs
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PBMC experiments were carried out with ethical approval. Demographics of healthy donors are given in Table 2 . Peripheral blood sample (approx. 50 mL) was drawn into lithium heparin tubes (Becton Dickinson). Samples were maintained at room temperature and processed immediately following venepuncture. PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque. Proliferation and cultured ELISpot assay of PBMCs were performed immediately after PBMC isolation. The median number of PBMCs routinely derived from healthy donor samples was 1.04 × 106 PBMC/mL whole blood (range: 0.6 × 106 – 1.48 × 106 / mL). The median viability as assessed by trypan blue exclusion was 93% (range 90–95%).
Healthy donor details.
| Donor | 1 | 2 | 3 | 4 | 5 | 6 |
|---|---|---|---|---|---|---|
| Age (years) | 20–40 | 40–60 | 20–40 | 40–60 | 20–40 | 20–40 |
| Sex | Male | Male | Female | Male | Male | Female |
| Smoking Status | Ex-smoker | Non-smoker | Non-smoker | Ex-smoker | Non-smoker | Non-smoker |
| HLA type | A2 | A3, 32 | A3, 24 | A1, 2 | A2, 29 | A2, 29 |
| B7, 41 | B14, 35 | B15, 27 | B8, 44 | B44, 51 | B15, 44 | |
| C7, 17 | C4, 8 | C2, 3 | C5, 7 | DR7, 11 | C3, 16 | |
| DR7, 52 a, 13 | DR2, 11 | DR4, 52 a | DR3, 51 a, 15, 52 a | DQ2, 0301 | DR4, 7, 53 a | |
| DQ2, 3 | DQ1, 7 | DQ3 | DQ2, 6 | DP4, 5 | DQ2, 3 | |
| DP1, 4 | DP3, 4 | DP4, 9 | DP1, 4 | DP2 |
3
Biomarker Analysis of Murine Blood
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Whole blood was obtained by retro-orbital bleeding of isoflurane-anesthetized mice immediately prior to euthanasia by an experienced lab animal technician, with part of the sample placed in lithium heparin tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for clinical chemistry analysis. Clinical chemistry was analyzed with a Piccolo Xpress Chemistry Analyzer (Abaxis, Union City, CA, USA). Heparinized blood was centrifuged at 8,000 × g for 5 min to collect plasma to measure cytokine levels. Retro-orbital blood was also collected in EDTA tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for obtaining a CBC with automated cell differential count. The assay was performed with a VetScan HM5 Hematology Analyzer (Abaxis).
4
Quantifying Osteopontin in Tumor Microenvironment
5
Fentanyl Pharmacokinetics in Horses
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Each horse was weighed and a baseline physical exam was performed prior to each treatment. Horses were instrumented with a 14 gauge intravenous catheters (Mila International, Inc., KY, USA) in one jugular vein for blood collection during the first 24 h of treatment. Subsequent samples were collected via direct venipuncture. Blood samples were obtained at baseline and again at 2, 4, 8, 12, 16, 24, 32, 40, 48, 56, 64, 72, and 96 h after placement. Following removal of a 10 ml waste sample, a total of 6 ml of whole blood was obtained at each timepoint, and stored in lithium heparin tubes (Becton, Dickinson, and Company, NJ, USA) for no longer than 1 h prior to processing. Blood was centrifuged at 1,300 g for 10 min and plasma was harvested and placed in cryovials (VWR, International, PA, USA) prior to storage at −80°C until analysis. All fentanyl patches were labeled and stored at −80°C until analysis to determine the amount of residual fentanyl in the patches.
6
Plasma Preparation for NMR Analysis
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Additional blood was collected into vacutainer lithium-heparin tubes [Becton Dickinson, product number 367375] and stored at room temperature for at least 30 min before centrifugation at 2200 × g for 10 min. Plasma was immediately aliquoted and stored at −80°C. For NMR analysis, the plasma samples were defrosted at room temperature and centrifuged at 100000 × g for 30 min at 4°C. Then, 150 μL of the plasma supernatant was diluted with 450 μL of 75 mM sodium phosphate buffer prepared in D2O [pH 7.4]. Samples were then centrifuged at 16000 × g for 3 min to remove any precipitate before transferring to a 5-mm NMR tube.
7
Isolation and Differentiation of Primary Human Monocytes
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Peripheral venous blood was collected in lithium heparin tubes (Becton Dickinson) from healthy volunteers with informed consent (study approved by Nepean Blue Mountains Local Health Service Human Ethics committee). Mononuclear cells were isolated by Ficoll-Paque (GE Healthcare) gradient centrifugation and monocytes were isolated using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotech, Germany) as per manufacturer's instructions. The purity for CD14 monocyte isolations was routinely >90% by flow cytometry.
For macrophage experiments, peripheral blood mononuclear cells were plated in complete RPMI 1640 medium at a density of 2.5 × 106 cells per well. Cells were incubated for 2 hours at 37°C in order to adhere to plastic, nonadherent cells removed, and adherent PBMCs were cultured overnight in 1 mL complete media, washed once the following day, and cultured for 6 more days.
For macrophage experiments, peripheral blood mononuclear cells were plated in complete RPMI 1640 medium at a density of 2.5 × 106 cells per well. Cells were incubated for 2 hours at 37°C in order to adhere to plastic, nonadherent cells removed, and adherent PBMCs were cultured overnight in 1 mL complete media, washed once the following day, and cultured for 6 more days.
8
Isolation and Culturing of BOECs
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BOECs were isolated as previously described with some modifications [24 (link)]. Peripheral blood samples were collected from healthy adult Europeans (30 mL; n = 5) and Ghanaian children (2–8 mL; n = 34) in lithium heparin tubes (Becton Dickson). Blood samples were diluted 1:1 with PBS, and peripheral blood mononuclear cells (PBMCs) isolated by density-gradient centrifugation over Histopaque (GE) or Lymphoprep (StemCell Technologies). European PBMCs were washed twice with PBS, then seeded in six-well culture plates (Corning) coated with 50 μg/mL rat tail collagen type 1 (BD Biosciences). Medium was changed every second day once colonies (from day 6 post-seeding) started to appear. Following isolation, PBMCs from the Ghanaian children were washed twice with PBS, and suspended in 350 μL EGM-2 plus Bullet Kit (Lonza) supplemented with 10% FBS, 50 μL DMSO, and 100 μl FBS. PBMC from the Ghanaian children were frozen using a Mr. FrostyTM freezing container and stored overnight at -80˚C, then transferred to liquid nitrogen. Following transfer to Denmark, the frozen PBMC were thawed, washed in pre-warmed EGM-2 and processed as above. All experiments described in this paper were carried out with BOECs at passage 2 to 8.
9
Infant Blood Processing for cAMP Measurement
10
PBMC Isolation and Characterization
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PBMC experiments were carried out with ethical approval. Demographics of healthy donors are shown in Table 1 . Peripheral blood samples (approx. 50 ml) were drawn into lithium heparin tubes (Becton Dickinson). Samples were maintained at room temperature and processed immediately the following venepuncture. PBMCs were isolated by density gradient centrifugation using Ficoll-Hypaque. Proliferation assays were performed immediately after PBMC isolation. The median number of PBMCs routinely derived from healthy donors was 1.36 × 106 PBMC/ml whole blood (range 0.6 × 106–1.8 x 106/ml). The median viability as assessed by trypan blue exclusion was 90.6% (range 80–97%). For CD25 depletion PBMCs were processed as above and then immediately enriched with anti-CD25 microbeads and MACS cell separation columns (Miltenyi).
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