Minicollect

As previously described²⁰ , blood was collected and placed in MiniCollect ethylenediaminetetraacetic acid (EDTA) tubes or serum tubes (Greiner Bio One, Monroe, NC). Immediately following sampling, 100 μl of blood was added to 600 μl of AVL viral lysis buffer with carrier RNA (Qiagen) for RNA extraction. For tissue sample processing, approximately 100 mg was stored in 1 ml RNAlater (Qiagen) for 96 hours to stabilize RNA. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer (Qiagen) in a 2-ml cryovial using a tissue lyser (Qiagen) and 1.4 mm ceramic beads (Precellys; Bertin Corp., Saint-Quentin-en-Yvelines Cedex, France). The tissues sampled included right lung upper lobe, right lung middle lobe, right lung lower lobe, left lung upper lobe, left lung middle lobe, left lung lower lobe, liver, spleen, kidney, adrenal gland, pancreas, and brain (frontal cortex). All blood samples were inactivated in AVL viral lysis buffer with carrier RNA, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood using the QIAamp viral RNA kit (Qiagen), from tissues using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit.

Neonatal blood samples were collected regardless of the time in the day or fasting state by heel prick in micro tubes (0.5 mL MiniCollect, Greiner Bio-One GmbH, Kremsmünster, Austria). Other blood samples were collected in fasting state from 7.00 to 10.00 am by venipuncture in test tubes with clot activator (2 or 4 mL Vacuette, Greiner Bio-One GmbH, Kremsmünster, Austria). After clotting, blood samples were centrifuged at 2200 RCF for 10 minutes.
Fresh serum samples were firstly analysed for TPO-Ab and TG-Ab. If antibodies tested negative, samples were further analysed for TSH, TT3, TT4, FT3 and FT4. Neonatal samples were tested for all hormones irrespective of the antibodies test results. Due to limited volume, not all hormones were measured in each individual sample. All measurements were performed on the Abbott Architect i2000 chemiluminescent microparticle immunoassay (Abbott Diagnostics, Abbott Park, USA). Assay precision was evaluated according to CLSI EP15-A2 guidelines using two levels of quality controls measured in triplicate for five consecutive days (Table 1) (21 ). Prior to analysis, authorized personnel verified calibrations and quality controls.