Qiacube automated isolator

The primary biological material for DNA testing was 9 mL of peripheral blood, which was collected in EDTA tubes. DNA was isolated from whole blood using the desalting method, which provides high quality DNA suitable for long-term storage. Alternatively, we performed isolation using the QIAcube automated isolator (www.qiagen.com accessed on 12 February 2022).

All 337 blood samples from RhD-negative pregnant women were collected into two parallel 9 mL tubes (“A” and “B”) containing ethylenediaminetetraacetic acid (EDTA). Anticoagulated blood was placed on ice immediately after collection and was processed within 4 h after sampling. Plasma was separated from the cellular fraction of blood using double centrifugation (2700× g for 10 min and 3500× g for 20 min). The plasma samples were frozen until further processing at −28 °C. Plasma-cell-free (cf) DNA was isolated in each of the two parallel tubes (“A” and “B”). The DNA isolation of 1 mL of plasma was performed using the QIAamp DNA Mini Kit (Qiagen, Venlo, The Netherlands). The incubation step for the isolation took place at 56 °C, with an elution volume of 65 µL. The isolation of maternal DNA from peripheral blood leukocytes was performed with a Qiacube automated isolator (Qiagen) using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s instructions. The isolation of the control DNA from newborn buccal swabs was performed using the QIAamp DNA Mini Kit (Qiagen), according to the manufacturer’s instructions.