Thyroglobulin
Thyroglobulin (Tg) is a large (660kDa molecular weight) glycoprotein that is synthesized in the thyrocyte and secreted into the thyroid colloid. It is the framework for thyroid hormone synthesis and has an important role in intracellular regulation (31-33). Healthy euthyroid glands release Tg into the circulation in small amounts (34-36). Upon hyperstimulation of the thyroid by TSH or thyroid stimulating antibodies, production and release of Tg is elevated. Tg is a sensitive indicator of both low and excess iodine intake, following a U-shaped curve in response to iodine excretion (7), as shown in school-age children (7, 19), pregnant women (5, 6)and infants (4). Research has demonstrated the utility of Tg to confirm improved thyroid function after iodine repletion (19, 21, 37). Due to a high day-to-day variability, however, the utility of Tg as an individual biomarker of iodine status is uncertain (38).
Methods
There are numerous different ways to measure Tg, including immunoassay (including ELISA), radioimmunoassay, dedicated kits, and by mass spectrometry (6, 18, 39).
Note: Blood, as whole blood, plasma, serum or on dried blood spots, is a potential biohazard, and safety precautions should be employed at all times whilst handling or manipulating samples.
LC-MS: For an example method measuring Tg by liquid chromatography-tandem mass spectrometry (LC-MS), see reference (39), available here.
Serum ELISA: Click the following link to download the Serum-ELISA method protocol: Serum Tg ELISA SOP
Kindly provided by the Human Nutrition Laboratory, ETH Zurich, Zurich, Switzerland.
An ELISA technique has been developed using dried blood spots (DBS) (18), which facilitates the collection, processing, storage and transport of blood samples, and is a field-friendly method of Tg assessment, suitable for use in LMIC settings. This method has validated reference ranges for iodine sufficiency in school-age children and pregnant women (6, 7, 19, 21)(see table on page 3).
Click the following link to download the DBS-ELISA method protocol: DBSTg ELISA SOP
Kindly provided by the Human Nutrition Laboratory, ETH Zurich, Zurich, Switzerland.
Notes on the DBS method: Antibodies are ordered from HyTest (see protocol). Orders must be made by email.
When ordering the Anti-Tg HRP-cojugated antibody (antibody 2), ensure to quote “in agreement with ETH Zurich”, since Hytest only manufacture this antibody for laboratories wishing to undertake this assay: “2TG12ccC 5E6cc Anti-Thyroglobulin HRP-conjugated, in agreement with ETH Zurich”.
Quality control
For all methods, in-house quality controls should be prepared from volunteers with low and high Tg concentrations, respectively.
Venous blood samples can be drawn from volunteers and spotted onto filter paper cards using a pipette set to 50 μL. Note: blood should be drawn into the appropriate tube: blood tubes with EDTA as an anticoagulant should not be used to handle blood for Tg analysis, since the EDTA can interfere with sample assessment (40).
Standards should be produced using washed red blood cells and the Biorad Liquicheck Tumor Marker Controls # 547, 548 and 549. Haematocrit and blood spot volume should be carefully controlled when these reference materials are produced. For an example method, please contact OpeN-Global.
VENOUS BLOOD SAMPLES
Venous blood samples should be drawn by appropriately-trained personnel.
Blood should be drawn into the appropriate tube: blood tubes with EDTA as an anticoagulant should not be used to handle blood for Tg analysis, since the EDTA can interfere with sample assessment (38). (This recommendation also applies when collecting blood from volunteers to prepare in-house DBS standards cards.)
Serum or plasma should be separated using a centrifuge according to manufacturers’ instructions, within 60 minutes or less. After centrifuging, check that the serum has properly separated. Discard any haemolysed samples. Transfer into the designated labelled storage vials, and transfer to the freezer without delay.
Plasma/serum samples should be frozen at -20°C or colder until analysis.
Stability: Results from stability testing of Tg in serum is inconsistent. Some reports state that serum Tg is stable when frozen at -20°C and there is no influence of freeze thaw cycles (41), whereas other reports have suggested that serum Tg is vulnerable to freeze thaw cycles (small decrease in Tg with 3 cycles, large decrease after subsequent cycles (40). Repeated freeze-thaw cycles should therefore be minimised to only those necessary.
DRIED BLOOD SPOT (DBS) SAMPLE
It is important to collect dried blood spots correctly. See Common Methods for advice on taking DBS sample cards.
DBS card samples should be frozen at -20°C or colder in airtight bags containing a desiccant, until analysis.
Thyroglobulin is stable on DBS when frozen at -20°C or colder for 15 weeks, and there is minimal effect of freeze-thaw on samples (18, 19, 40), however these should be minimised and care should be taken to ensure the card stays dry by regularly replacing the desiccant in the storage bag and ensuring dry conditions during defrosting.
DATA INTERPRETATION
Assay specific thresholds (serum, DBS) should be applied, and age- and population-specific cut offs should be used. See the table on page 3.
INFLUENCE OF THYROGLOBULIN ANTIBODIES
Thyroglobulin antibody (TgAb) positivity may confound Tg assay measurement (42), however, the relationship is unclear since the association between TgAb and Tg concentration in assays is poor (43), and data to date are inconclusive (39, 44-46). No apparent interference of TgAb with population Tg results measured on DBS from pregnant women was found in data from eleven countries (6), though other studies suggest an interference of TgAb in the metabolic clearance of Tg, and therefore its measurement (47). The influence of TgAb can also method-dependent.
Eventual screening is only indicated in adults as TgAb are generally rare in children (48). It is currently not considered necessary to measure DBS-Tg and DBS-TgAb in parallel for assessment of iodine status in population studies.
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