Vacutainer serum collection tube

Blood samples were collected from 146 consenting patients using an 18–20 gauge syringe needle and placed in BD Vacutainer serum collection tubes before being centrifuged at 2500× g for 20 min at room temperature within 60 min. The serum supernatant was extracted and frozen as 500 uL aliquots and stored at −80 °C.

Blood samples were collected into four collection tubes [two Vacutainer serum collection tubes containing no additives (Becton Dickinson, Franklin Lakes, NJ), one Monoject plasma collection tube containing 0.10 mL of 15% K₃EDTA (Covidien, Mansfield, MA), and one Vacutainer plasma collection tube containing 15 mg of sodium fluoride and 12 mg of potassium oxalate (Becton Dickinson) for glucose determination]. Following collection, all tubes were inverted several times. Plasma tubes were immediately placed on ice, while serum tubes were allowed to clot before being placed on ice. Between 1 and 8 h after collection, all samples were centrifuged at 1,500 × g at 4°C for 30 min. Plasma and serum were transferred into multiple 2-mL microcentrifuge tubes and stored at −20°C until analysis.
Maternal plasma glucose, serum urea N, and serum non-esterified fatty acids (NEFA) were analyzed as described in Niederecker et al. (2018) (link), and plasma triglycerides were analyzed as described in Larson-Peine et al. (2022) (link). For each assay, samples were analyzed in duplicate, and pooled control samples were used. The intraassay and interassay CV were 2.7% and 2.2% for plasma glucose, 3.2% and 3.7% for serum urea N, 3.4% and 5.1% for serum NEFA, and 2.8% and 2.3% for plasma triglycerides, respectively.

After each calf stood but before it suckled, both cow and calf were removed from the pen, and the cow was directed into a nearby chute. Cow jugular blood samples were collected (76.3 ± 77.5 min post-calving) into the same 4 tubes and processed as previously described for gestational blood sampling. The most full rear quarter was selected based upon visual inspection and palpation and was hand-milked completely prior to the calf suckling (57.5 ± 17.5 min post-calving) at described in Rathert-Williams et al. (2023) (link). Colostrum volume and weight were recorded, and subsamples were aliquoted and stored at −20°C for later analysis.
Calf jugular blood samples were collected at 0 h of age (post-standing but pre-suckling; 34.3 ± 20.2 min of age), and at 48 h of age (48.2 ± 0.5 h of age). Blood was collected into 4 tubes at each time point, including 2 Vacutainer serum collection tubes containing no additives, 1 Monoject plasma collection tube containing 0.10 mL of 15% K₃EDTA, and 1 Vacutainer plasma collection containing 10.8 mg of K₂EDTA (6 mL draw; Becton Dickinson, Franklin Lakes, NJ) for mineral determination, and processed as previously described for cow samples.

Whole blood was obtained in Vacutainer Serum Collection Tubes (Becton Dickinson) and serum was collected following centrifugation at 1500 rcf/g  for 10 min and stored at −80C until analysis. Neutralizing antibody screening assays were carried out as previously described^{53 (link)}. Briefly, assays were performed in 96 well format with 5 × 10⁴ CHO-Lec2 cells per well. Serial dilutions of study participant serum were pre-incubated with 1 × 10⁹ genome copies of AAV2 reporter virus for 1 hour at 37 °C and then added to cells that were infected with Adeno Helper virus. After 48 hours, Promega Bright-Glo substrate was added to the cells and luciferase expression was quantified using the Biotek Synergy Mx luminometer. Both positive and negative monkey sera controls were included with each assay. All animals selected for the study had less than 50% inhibition of transduction when serum was diluted to 1:20.