© Springer International Publishing Switzerland 2015
Gianni Bona, Filippo De Luca and Alice Monzani (eds.)Thyroid Diseases in Childhood10.1007/978-3-319-19213-0_55. Laboratory Aspects in Neonatal Screening of Congenital Hypothyroidism
(1)
Dipartimento della Donna, della Mamma e del Neonato, Laboratorio di Riferimento Regionale per lo Screening Neonatale, Ospedale dei Bambini “V. Buzzi”, AO Istituti Clinici di perfezionamento, Via L.Castelvetro n° 32, Milan, I-20154, Italy
Keywords
Newborn screeningCongenital hypothyroidismScreening testQuality assurance policy5.1 Introduction
The aim of a newborn screening program is to identify affected babies in the newborn population. This implies that screening tests and laboratory methodologies are characterized by performance parameters (accuracy, analytical range, analytical specificity, blank reading, detection limit, interferences, precision, reagent stability, etc.) able to meet the medical usefulness of a newborn screening program and specific criteria such as sensitivity (positivity in disease), specificity (absence of disease), predictive value of positive test (percent of patients with positive test results who are diseased), predictive value of negative test (percent of patients with negative test results who are nondiseased) [1].
For what strictly concerns newborn screening for congenital hypothyroidism (CH), many aspects should be taken into consideration. A crucial aspect is to define the spectrum of pathologies to be screened: primary CH, secondary (central) CH, severe forms of CH, mild forms of CH. Moreover, the complex regulation of thyroid hormone biosynthesis, as well as the rapid modification of reference intervals of thyroid and pituitary hormones in the first weeks of life, may affect many laboratory aspects. In addition, there are features concerning the biological matrix universally used for newborn screening, the dried blood spot (DBS). DBS is collected on a special filter paper with a high degree of uniformity and designed to absorb a specific volume of blood. The quantitative nature of DBS allows the quantitative assessment of biomarkers of many congenital diseases and the definition of cutoff values to differentiate asymptomatic newborns that may have a disease from those who may not [2]. These features meet the needs of the analytical process and guarantee efficiency and effectiveness of the newborn screening program.
It is universally accepted that only high-technology laboratories which use standardized operating procedures and have appropriately trained staff with large experience in automated immunoassay procedures, quality assurance policy, and information technology [3] can successfully process the high workload requested by a newborn screening program for CH. In fact, it is recommended that the number of babies screened yearly by a screening center is at least 35,000-50,000 [4].
The criteria on the basis of which a laboratory test may be applicable to a newborn screening program have been clearly defined [5, 6]. A screening test must be applicable to DBS samples and feasible for high-throughput platforms. Furthermore, it should have high sensitivity and specificity in order to identify the highest number of affected babies in the newborn population. It should also be inexpensive. Finally, in the USA, the Centers for Disease Control and Prevention (CDC) classified the biochemical genetic testing and newborn screening as essential laboratory services for the screening, detection, diagnosis, and monitoring of inherited metabolic diseases, endocrine and hemoglobin disorders, and other rare diseases [7]. Biochemical genetic tests and newborn screening tests are considered high-complexity tests. Laboratories that perform these tests must meet more severe regulations for the total testing process in comparison with general laboratories. Moreover, the qualification of laboratory personnel, including training and experience, is a critical factor for ensuring the quality of laboratory test results.
5.2 Biomarkers and Screening Strategies Used in Newborn Screening for Congenital Hypothyroidism
In general terms, the laboratory tests [8] most commonly used to evaluate thyroid hormone dysfunction are classified into
Hormone concentration tests:
Total thyroxine (T4)
Total triiodothyronine (T3)
Free thyroxine (fT4)
Free triiodothyronine (fT3)
Thyrotropin (Thyroid-stimulating hormone) (TSH)
Reverse Triiodothyronine (rT3)
Serum-binding protein tests:
Thyroxine-binding globulin (TBG)
Thyroxine-binding prealbumin (Tranthyretin) (TBPA)
Autoimmune Thyroid Disease tests:
Antithyroglobulin antibodies (TgAb)
Antithyroid peroxidase antibodies (TPO Ab)
TSH receptor antibodies (TRAb)
Other Hormones and Thyroid-Related Protein tests:
Thyrotropin-releasing hormone (TRH)
Thyroglobulin (Tg)
Calcitonin (CT)
Some of these analytes (TSH, T4, fT4, fT3, TBG) can be detected in DBS samples (http://www.cdc.gov/labstandards/pdf/nsqap_analyte_list.pdf).
According to the clinical target of a newborn screening program for CH, i.e., detection of babies with primary CH and/or central CH, the screening protocol should use appropriate biomarkers in order to obtain a highly effective screening program [11, 12]. Generally, an analyte expressing the effect of a genetic and/or functional defect is an optimal biomarker. For example, phenylalanine is the best analyte in newborn screening for phenylketonuria (PKU). This rare inherited disorder is caused by a genetic defect causing the lack of Phe-hydroxylase, an enzyme needed to process phenylalanine. Without the enzyme, phenylalanine increases in blood. This vision explains why T4 was identified as an optimal biomarker when newborn screening for primary CH was introduced. However, it was realized that T4 is not an ideal indicator of thyroid status, in part because of the effects of variations in serum-binding protein levels and also because the relationship between T4 and T3 (the primary active thyroid hormone) is not always predictable [8]. Differently, TSH serum (or blood) concentration reflects an integrative action of all thyroid hormones in one of its target tissues, the pituitary cells that secrete TSH. Moreover, TSH serum or blood levels are inversely proportional to T4 concentration, and a twofold change in FT4 causes an approximate 100-fold change in serum (or blood) TSH concentration [13]. This log-linear ralationship explains why little changes in T4 level are reflected in great variations in TSH levels. This feature, which allows the identification of subclinical thyroid diseases, together with the increasing accuracy of TSH measurement which has been achieved over the years, are the main factors on the basis of which TSH is considered the best biomarker to identify primary CH in the first days of life. Even though the measurement of TSH alone is not appropriate to identify babies with central CH, which requires the combined use of other biomarkers (mostly T4 or FT4), the TSH-centered strategy for screening evaluation of thyroid function in newborns is both cost effective and medically efficient.
Although TSH as primary screening test is the most used screening strategy for CH worldwide, other strategies are possible:
1.
T4-backup TSH
2.
Tandem T4 and TSH (in all samples)
3.
T4, TSH, TBG (combined method)
4.
Tandem fT4 and TSH
In the T4-backup TSH strategy, T4 is the primary screening test and TSH is measured only in samples whose T4 concentrations are below the 10th centile. In the 1990s, this strategy was the most used worldwide. Over the years, the improvement of the analytical and functional sensitivity of TSH assays, as well as the increasing use of the retest at 2–4 weeks of life (serial testing) in preterm or sick newborns [14], has increased the use of TSH as primary screening test for CH. At present, T4 is used as primary screening test only in some states of the USA and in Israel, whereas the strategy with simultaneous (tandem) T4 and TSH testing is applied only in a limited number of screening programs [15]. The widespread use of TSH as primary screening test for CH is also confirmed by annual reports of the most common international program of quality assurance for newborn screening, the Newborn Screening Quality Assurance Program (NSQAP) of the Centers for Disease Control, Atlanta [16]. In 2013, 578 newborn screening laboratories in 73 different countries took part in the Program. Among the 492 laboratories participating in the Proficiency Testing Program, 310 participated for TSH and only 84 for T4. Similarly, among the 445 laboratories participating in the Quality Control Program, 294 participated for TSH and only 72 for T4.
It is important to understand which strategy provides the best performance metrics in relation to the aims of a newborn screening program. In a recent study, performance metrics of four screening strategies used from 1994 to 2010 were compared: T4-backup TSH, tandem T4 and TSH, TSH (no serial testing), TSH plus serial testing [17]. In terms of effectiveness, the TSH plus serial testing strategy resulted to have the best performances, although the tandem T4 and TSH strategy allows the identification of cases with central CH. It has been reported that the measurement of fT4 can be used instead of T4 in tandem strategy. The use of fT4 avoids the effects of variations in serum-binding protein levels on the T4 measure. In Japan, some NBS laboratories are using the fT4 measure as primary marker with the simultaneous measure of TSH in all DBS samples: the fT4 measurement enables the detection of CH of central origin, with an estimated incidence in Japan’s regions of 1:30.833 live births [18, 19]. In the Netherlands, the Dutch NBS program is using a different strategy: primary T4 test with sequential TSH measured in the lowest 20 % and TBG measured in samples with the lowest 5 % of T4 values. Also, this strategy allows the detection of both primary and secondary CH, the latter with an incidence of 1:15.000 live borns [20].