Newborn Screening

Specimen

Currently, almost all disorders on the RUSP are screened by laboratory analysis of the DBS specimen of the newborn. The two exceptions are a point-of-care hearing test and pulse oximetry that are performed directly on the newborns to screen for hearing loss and critical congenital heart disease (CCHD), respectively.

Specimen Collection Procedure

The blood specimen is generally obtained from the heel of the infant. This simple sampling method conceived and introduced by Guthrie and Susi, has made NBS feasible since blood is easily obtained and can be easily and inexpensively delivered to a central testing facility by mail or courier. There are no serious complications from obtaining these newborn specimens, contrary to early fears that their collection would lead to infection or result in excessive bleeding.

The blood specimen should be obtained from the lateral or the medial side of the heel ( Fig. 18.1 ). Blood should be applied to only one side of the filter paper card, but it should saturate each circle on the card. Contamination of the filter paper specimen with iodine, alcohol, petroleum jelly, stool, urine, milk, or a substance such as oil from the fingers can adversely affect the results of the screening tests. In addition, exposure to heat and humidity can inactivate enzymes and produce false results. The specimen should be dried in air at room temperature for at least 3 hours before being placed in an envelope.

Specimens are sometimes collected in capillary tubes, by venipuncture of a dorsal vein or from a central line, and then spotted on filter paper. There is little or no substantial difference in analyte levels between blood collected directly from the heel and that collected by any of these other methods. However, there is the danger of introducing amino acids into the specimen in infants receiving total parenteral nutrition (TPN) if the blood is collected from a central line, resulting in a false-positive increase in the levels of amino acids or interference in some molecular assays by the heparin within the line. In general, it is preferable that a blood screening be spotted on filter paper directly from the heel.

Timing of the Collection

Specimen collection timings differ around the world. In the United States, most specimens are collected between 24 and 48 hours after birth. In Europe and Australia, screening specimens are collected within 48 to 72 hours, and in the United Kingdom the specimen is not collected until the newborn is 5 to 8 days old. The specimen should be obtained from every newborn before nursery discharge or by the third day of life, whichever is first. In newborns whose initial specimen was obtained within the first 24 hours after birth, as may happen with the practice of early nursery discharge, a second blood specimen should be obtained at no later than 7 days old to be certain that a diagnosis is not missed.

NBS encompasses a gamut of conditions, each with its own ideal screening period during which there is the greatest chance of diagnosing the disorder before the onset of symptoms. As a result, it is worth noting that recommendations on the timing of specimen collection, although appropriate for most conditions, may not be ideal for all conditions on the screening panel. For instance, for CAH, in which the symptoms can manifest themselves within the first week of life, the optimal time for collection of the specimen is within 24 to 48 hours after birth. Conversely, there remain concerns that with the NBS specimen collected early, often during the first day of life, some newborns with metabolic disorders or with CH might not have a sufficient degree of abnormality for identification. The MS/MS method with its increased sensitivity and specificity has considerably increased the reliability of screening for metabolic conditions in early specimens. Furthermore, use of thyroid-stimulating hormone (TSH) as the primary marker for CH, or as a second-tier test when T4 is the primary marker, has similarly allowed early screening for CH to be more acceptable.

Special circumstances require specific attention to newborn blood specimen collection. Premature newborns or those with low birthweight (LBW), and newborns who are sick and those in neonatal intensive care units (NICU), are at risk of unreliable screening owing to factors such as the unique physiology of the newborn, therapeutic interventions, and a focus on critical activities in caring for the very sick neonate. Consequently, a single specimen is inadequate for screening newborns in this subpopulation, and additional specimens should be collected for retesting. Serial screening with collection of three specimens, (1) on admission to the NICU prior to interventions regardless of age, (2) between 24 and 48 hours after birth, if a prior specimen was collected at less than 24 hours of birth and, (3) at discharge or at 28 days old, whichever is sooner, has been proposed as an adequate and efficient protocol for this population. In addition, some programs recommend that screening be performed every month until discharge for babies who continue to remain in the NICU.

A blood specimen should be collected from any infant who is being transferred to a different hospital or to an NICU, regardless of age. The first specimen should be collected before transfer, and a second specimen should be collected at the receiving hospital by 4 days of age. This dual collection policy covers the child from whom a newborn specimen might not have been obtained in the turmoil that frequently accompanies the transfer of neonates.

In a newborn who is to receive a blood transfusion within 24 hours of birth precluding collection of an ideal routine specimen, a screening specimen should ideally be collected before transfusion, and a second specimen should be collected 2 days after the transfusion. If a pretransfusion specimen has not been obtained, a screening specimen should be obtained 2 months after the last transfusion, when most of the donor red blood cells have been replaced, to ensure reliable testing for analytes present in red blood cells.

Screening Tests

NBS tests are usually performed in a centralized state, provincial, or regional laboratory. In a regional program, the specimens may be received by the state program and then delivered to the regional state or private laboratory, or they may be sent directly to the regional laboratory. In either case, the individual state programs serve as the state data and follow-up centers.

The testing procedure begins with punching small discs (each usually 3 mm in diameter) from the screening specimen. These small discs are then analyzed by various methods for the individual markers being sought. Amino acids (AA) and acylcarnitines (AC), the markers for a large proportion of the screened metabolic conditions, are simultaneously measured by MS/MS. MS/MS is superior in terms of accurate measurement of the individual analytes when compared with alternative methods originally used for screening the AA, such as bacterial assays or fluorometric techniques. Immunoassays, including fluoroimmunoassay and enzyme-linked immunosorbent assays, are used to test for endocrinopathies such as CH and CAH, for infectious diseases such as congenital toxoplasmosis, and for CF. Hemoglobin analysis of blood eluted from the filter paper can be performed by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), or isoelectric focusing (IEF) to identify abnormal hemoglobin variants (Hb) associated with the sickling hemoglobinopathies. An enzyme-based assay is often used to screen newborns for galactosemia and is always used to screen newborns for biotinidase deficiency and the lysosomal storage disorders (LSD).

A molecular assay, quantitative polymerase chain reaction (qPCR), is employed to identify SCID by quantifying TRECs, a marker of newly formed, antigenically naïve thymic emigrant T cells, and another qPCR-based assay is used to identify homozygous absence of Exon7 of SMN1 , expected in 95% of individuals with SMA. TREC analysis was the first NBS test to use DNA as the primary analyte. Before implementation of screening for SCID, molecular assays were used only as second-tier tests to detect targeted mutations in disorders such as CF following an out-of-range biomarker. Several platforms, DNA microarrays, and microsphere-based assays can multiplex several molecular and immunologic assays for high-throughput screening and are used by screening programs. NGS with the high-throughput and massively parallel DNA sequencing technologies has substantially reduced the cost and time required for sequencing and made it possible to sequence the whole exome and entire coding regions of a gene/genes. NGS has already been adopted by a few screening laboratories to provide supplemental genotyping information as a second-tier test, and offers the prospect of becoming a first-tier test to screen newborns for genetic disorders that do not have a biomarker identifiable by current screening.

Secondary Tests

An abnormal finding on an NBS test is not diagnostic. Abnormalities in the newborn specimen can be transient or artifactual. Accordingly, when an abnormality is identified, the original specimen is retested for the primary analyte that was abnormal. In addition, secondary tests, biochemical or molecular, can be performed by the screening laboratory to substantiate the finding and increase the specificity of screening. However, it should be noted that the primary analyte(s), the second-tier tests, and even the testing algorithms applied vary across screening programs.

In screening for CH, many programs have adopted protocols in which the primary analysis is for TSH, and T4 is measured as a second-tier test in specimens with high concentrations of TSH to improve specificity. Some programs initially measure T4. Specimens in which a low T4 level is found are further tested for an increased level of TSH, which would indicate CH. Similarly, in screening for galactosemia, in some laboratories an elevated galactose measurement in a specimen can trigger the analysis of GALT activity as a second-tier test, while in others, a decreased GALT activity prompts galactose measurement as the second-tier test.

An out-of-range IRT, the primary marker for CF, could prompt second-tier molecular testing to identify pathogenic mutations on the CFTR gene. However, the IRT concentration that prompts the molecular analysis, methods used for mutation selection and detection, and the number of mutations in the screening panel vary greatly, with some regions testing only for the most common mutation (F508del) and others for more than 400 mutations. Sensitivity and specificity consequently vary across regions, but screening programs following this two-tier IRT-DNA approach can identify up to 99% of patients with CF and report a positive predictive (PPV) value ranging between 1/9.5 and 1/25. Molecular assays to detect disease-causing mutations are currently used as second-tier tests for several other disorders, and their use continues to expand with advances in DNA technology. Some examples include testing for a panel of several GALT mutations in galactosemia screening and sequencing the relevant genes in screening for X-ALD and the LSD.

The final interpretation of the screening results is based on the primary analysis and, if available, the results of second-tier testing. The second-tier tests are used to either reduce the number of positive screens reported, or to provide supplemental information and categorize results based on probability of the disorder. However, it is important to realize that screening is not intended to be diagnostic; abnormal screening results must be supported by confirmatory investigations. These studies require additional specimens and are performed by clinical laboratories or sometimes by the screening laboratory.

Physician Contact for Abnormal Results

Tables 18.3 and 18.4 indicate disorders or other reasons for abnormal screening results, sorted according to the primary analyte usually used to screen the newborn for the condition. For example, a low T4 level together with an elevated TSH concentration indicates CH, and a marked elevation of 17-hydroxyprogesterone (17-OHP) level indicates the likelihood of CAH. An elevation of an acylcarnitine could indicate an organic acid or fatty acid oxidation disorder.

Any infant for whom an abnormal screening result is reported should be evaluated by the primary healthcare provider as soon as possible to facilitate the next steps toward the confirmation and management of the disorder. However, several conditions screened are extremely rare, and primary healthcare professionals might not have sufficient information available to direct appropriate intervention in screen-positive infants. To overcome the challenge, one-to-two-page explanations of the possible disorders, suggested follow-up action and confirmatory diagnostic investigations for the screening abnormality, known as ACTion (ACT) Sheets and Algorithms, are readily available on the website of the American College of Medical Genetics and Genomics (ACT Sheets and Algorithms [ https://acmg.net ]). Similar explanations are often included with the screening report.

Although all screening results with a metabolite concentration that crosses its threshold are considered screen positive, all screen-positive results are not associated with the same likelihood of being associated with a disorder. Most infants with a positive screening result that is only mildly abnormal are less likely to have a disorder (see the later discussion of false-positive results) than are infants with analyte concentrations that are severalfold above the cutoff. Applying a uniform approach for all screen-positive results because of urgency of intervention or battery of tests suggested can result in unnecessary parental anxiety and medical costs. However, if recommendations for further action and workup are customized in accordance with the potential significance of the abnormality, both parental anxiety and the costs associated with false-positive results can be reduced. To achieve this goal, some programs subcategorize positive screening results and the primary care providers are supplied with category-based, customized fact sheets when a positive screening result is reported (personal communication). These sheets include information on disorders associated with the marker, the estimated likelihood of being affected, clinical presentations of likely disorders, factors contributing to false positives, and recommendations for further management. The follow-up recommendations can range from immediate admission to a hospital to simply repeating the analysis on a DBS specimen collected a few days later. Other programs approach this problem differently, but with the same goal of providing the primary care providers with the information needed to put the result in the appropriate context for the family.

When specific guidelines based on the individual results are not provided by the screening program, and the infant is ill or the likely disorder is among those that manifest acutely within the first few days of life (indicated in Tables 18.1 and 18.2 ), a specialist should be contacted. The infant may need to be admitted to the hospital, where further evaluation and therapy for the illness can be initiated without delay.

If the infant is clinically well on initial evaluation and the suspected disorder does not require immediate attention, a second DBS can be obtained and sent to the screening laboratory for repeat testing, or confirmatory testing can be performed on a less urgent basis. In many cases, confirmatory testing or referral to a specialist is required only if the second specimen also indicates the presence of the disorder. However, the follow-up of an initial positive screening result can differ. In some cases, specific confirmatory testing is the appropriate first response to a positive newborn screen, with a less intense time frame for individuals in whom the level of suspicion is lower.

The physician should contact the screening laboratory when an infant whose screen has been reported as normal or whose screening results have not yet been reported has symptoms that suggest a disorder on the NBS panel. The screening laboratory can check the results in the infant’s newborn specimen. If the testing has been completed and the newborn specimen is retained in storage, the laboratory may wish to recover the specimen and repeat the tests. The physician should also contact the screening laboratory for the repeated test results and inform the family of the results as soon as possible. If the second result is normal, the family’s anxiety may be shortened.

Metabolic Disorders