1. Introduction
Congenital hypothyroidism (CH) is one of the most common pathologies of the thyroid present at birth. It is also one of the most common causes of mental retardation, which is preventable if treated early. It occurs in approximately 1:2000 to 1:4000 newborns and presents almost no symptoms until weeks after birth, when most of the damage in the brain is irreversible [
1]. For this reason, neonatal screening was started in the 1970s, including the determination of either thyroxine (T
4) or thyroid stimulating hormone (TSH), to detect children with such a condition as early as possible [
2].
For a deeper understanding of the present work and the values discussed in it, it is important to recall the physiology of the thyroid hormone system after birth in term and preterm infants. In the first 30 min after birth, TSH rises abruptly, as a consequence of both exposure to a colder environment and the clamping of the umbilical cord. After this initial peak, the serum TSH concentration decreases rapidly over the first 24 h post-partum. The concentration continues to fall in the following week, but at a much slower pace.
This initial surge in TSH stimulates the production of T
4, which presents a peak a little later at approximately 24–36 h after birth. The same is true for triiodothyronine (T
3), which rises because of both the TSH and T
4 peaks. Newborn screening (NBS) is usually performed after these initial changes. In Switzerland, this screening occurs between 72–96 h after birth. In the subsequent week, the concentrations of these hormones fall and then effectively stabilize a level that is slightly higher than that of adults [
3].
Premature born infants undergo these same transitional changes, but their hypothalamic–pituitary–thyroid axis (HPT-axis) is still immature. Consequently, these changes are quantitatively much smaller and serum concentrations of thyroxin-binding globulin (TBG), tT
4, fT
4, and T
3 are lower than in term-born infants [
4]. As mentioned in the previous paragraph, T
4 concentrations drop after the first peak at 72–96 h. In premature-born infants, this drop is greater due to a higher T
4 clearance. Serums T
4 and T
3 concentrations then rise again after the first week, reaching concentrations comparable to those of term-born infants by three to six weeks of life. This difference appears to be more marked in very preterm infants (<33 weeks), while more mature healthy preterm infants (34–36 weeks) have T
4 values comparable to those of term-born infants after the first week of life [
4]. Other factors that could affect tT
4 concentrations are sex, ethnicity, seasonality, age at sampling, and other factors, such as TBG and albumin.
It is important to distinguish the two main types of CH: primary CH (T-CH) and central CH (C-CH). Primary congenital hypothyroidism, also called thyroidal hypothyroidism, affects approximately 1:2000–1:4000 newborns [
5]. It is caused by a lack of thyroid hormone production, namely T
4 and T
3, mostly due to a dysfunctional thyroid gland (thyroid dysgenesis or dyshormonogenesis).
Secondary CH, also called hypopituitary CH or central CH, is not of thyroidal origin, but rather of pituitary origin. This means that the problem lies within the production of TSH. C-CH is a much rarer cause of hypothyroidism, with a prevalence of 1:20,000 to 1:80,000 in the general population [
5,
6]. Among infants with C-CH, the TSH values are more U-shaped, which means that TSH measurements can result in being either low, normal, or increased. Also, fT
4 levels are usually lower in C-CH than in T-CH with comparable TSH values [
7].
Understanding this distinction between T-CH and C-CH is important for understanding the rationale behind the various screening programs. The primary T
4-follow up TSH screening effectively detects T-CH, some cases of C-CH, and a delayed TSH rise, but it misses cases of subclinical T-CH. The primary TSH screening detects T-CH and subclinical T-CH effectively but misses most cases of C-CH and a delayed TSH rise if just one specimen is tested [
8].
Like most neonatal screening programs worldwide, Switzerland has TSH-based NBS. If the values measured in the NBS are abnormal or if a particular infant needs its values monitored, they get recalled for a second blood draw, in which TSH and total T4 gets checked. To date, there are no reference values for T4 concentration in DBSs in term- or in premature-born infants.
The main aim of this study was to determine a reference range of total T4 in DBSs for term- and premature-born infants. If we can define a reference range of total T4 at birth, we could potentially spare children with abnormal or borderline TSH values at the NBS a second visit at a clinic for blood drawing. Instead, we could directly measure this value from the DBSs taken for NBS and interpret it with the reference values according to the gestational age (GA) of the newborn.
To get a good idea of the reference ranges of tT4 in term and preterm newborns, there is a need for a big sample size due to the big variance of tT4 values depending on birth weight, gestational age, etc. For this, we initiated a miniature study to determine the stability of tT4 in our stored DBSs.
2. Materials and Methods
This was a cross-sectional, experimental study carried out at the newborn screening laboratory at the University Children’s Hospital Zurich. The experiment was divided into two main parts. For the first part of the study, we used dried blood samples (DBSs) where tT4 was already measured and remeasured these samples after 0–11 weeks to determine its stability. The second part was subdivided into two additional parts, preterm and term-born infants, where we used the data from the first part to determine which samples were still indicative of the T4 concentration at the NBS.
2.1. tT4 Stability in Dried Blood Samples
To determine the stability of tT4 in the DBSs, we compared values, which were measured soon after the blood was drawn from the infant, i.e., reliable tT4 concentrations, with the results of our measurements taken in the same week and up to multiple weeks after storage of the DBSs. The cards were stored at room temperature in a dark and dry room inside the newborn screening laboratory.
For every storage time span we wanted to check the stability for, we used 10 specimens. For the first batch of 10 cards, the first measurement was within the same week; for the second batch, the first measurement dated back 1 week, and so on. We used a time span interval of 1 week for up to 7 weeks of storage. After 7 weeks, we increased the time span interval to approximately 2 weeks, and then increased it again to 3 weeks between measurements (
Table 1, see Results).
For some weeks, the desired minimum number of 10 DBSs was not available. The reason for this was, as mentioned earlier, that in Switzerland, we have a TSH-based screening; therefore, thyroxine was only measured after a first pathological or borderline value of TSH, resulting in relatively few measurements per week.
As seen in
Table 1 (see Results), we measured 10–11 samples for each of the storage times considered. For the storage times of 5, 19, and 21 weeks, we removed one outlier, leaving those groups consisting of only nine samples.
For this first part of the study, we measured the thyroxine values of a total of 255 specimens belonging to newborns with (borderline) pathological TSH measurements at the newborn screening.
2.2. Determination of tT4 Ranges
2.2.1. tT4 Values of Preterm-Born Infants
For the main part of the study, we selected all premature-born infants who were born in the preceding 21 weeks. This time span was determined through the results of the first part, in which we found that thyroxine could be considered stable, with some correction, over a maximum of 21 weeks. The correction was applied to all DBSs with a storage time of 36 to 146 days and amounted to 10%, i.e., a 10-week-old DBS with a measured thyroxine value of 95 nmol/L was corrected by adding 10%, resulting in an effective value of 104.5 nmol/L (see Results).
In total, we measured 1245 dried blood samples of premature newborns at different gestational ages. We also included further 127 tT
4 measurements, which were assessed outside of this experiment as part of a CH-screening. After excluding the measurements that were taken at >7 days of age, the duplicate values between the two data sets and the outlying values, our data set consisted of 944 DBSs of infants born at a gestational age between 24 and 36 weeks (
Figure 1).
We adopted the World Health Organization (WHO) definition of premature, i.e., infants born before completion of the 37th pregnancy week. The most premature infants mentioned in this paper where born in the 24th pregnancy week.
The specimens were measured using the GSP® Neonatal Thyroxine (T4) kit by PerkinElmer (Turku, Finland).
2.2.2. tT4 Concentrations of Term-Born Infants
The thyroxine values for the term-born infants were obtained the same way as the values of the premature-born infants. Because full-term-born infants were more prevalent than premature-born infants, we were able to measure the thyroxine values of 973 infants over the time it took to collect the measurements for the premature-born infants. These values were taken together with the regular newborn screening measurements; therefore, no correction for storage time had to be added.