Z serum sep clot activator tubes

Post morten blood samples for serology were collected using 4 ml Z Serum Sep Clot Activator tubes (Greiner BioOne, AT). The serum was centrifuged, separated and frozen at −20°C until further processing. EBHS serology was performed on 32 hares in 2011 using an established competitive enzyme linked immunosorbent assay (ELISA), highly specific for EBHSV [21 , 22 ]. The serologic screening for Treponema sp. was performed on 41 hares in 2011. For this an indirect immunofluorescent test (IFAT) was set up [23 ].

Maternal serum was routinely collected at time of booking for antenatal care, from the cord immediately at birth and from women postnatally within 72 h of delivery. Serum was collected from infants at 7 weeks (1 week prior to commencing primary immunizations) and 5 months of age (1 month after completion of primary immunizations). Maternal blood collected at the time of booking for antenatal care was taken into Vacutainer^® plastic SSTTM II Advance tubes (Becton Dickinson, Wokingham, UK) and stored samples were obtained following patient consent. All other samples were taken into Z Serum Sep Clot Activator tubes (Greiner Bio‐One, Stonehouse, UK) and processed by the study team. Samples were left for a minimum of 30 min prior to centrifugation at 1900 g for 10 min. Maternal and cord blood were processed within 48 h of collection and infant blood within 1 h of collection. All serum aliquots were stored at –80°C prior to further analysis.

Morning samples were collected between 9:00 and 9:30 am, while afternoon samples were collected between 3:00 and 3:30 pm. Participants presented fed for blood collection on day 1 and after an overnight fast on day 3. The participants were free to consume their regular meals of choice for the duration of the study when not required to be fasted. Eight millilitres of whole blood was collected into Z Serum Sep Clot Activator tubes (Greiner Bio-One, Frickenhausen, Germany) using a 21-gauge Vacuette Safety Blood Collection sealed vacuum device (Greiner Bio-One, Frickenhausen, Germany). To ensure sample quality the device was primed by collecting a 5 mL ‘discard’ tube immediately prior to sample collection. Serum was isolated from whole blood within 60 min of sample collection by two cycles of centrifugation at 2500× g for 15 min at 4 °C.