Newborn screening for severe combined immunodeficiency; the Wisconsin experience (2008-2011).

Severe combined immunodeficiency is a life-threatening primary immune deficiency characterized by low numbers of naïve T cells. Early diagnosis and treatment of this disease decreases mortality. In 2008, Wisconsin began newborn screening of infants for severe combined immunodeficiency and other forms of T-cell lymphopenia by the T-cell receptor excision circle assay. In total, 207,696 infants were screened. Seventy-two infants had an abnormal assay. T-cell numbers were normal in 38 infants, abnormal in 33 infants, and not performed in one infant, giving a positive predictive value for T-cell lymphopenia of any cause of 45.83% and a specificity of 99.98%. Five infants with severe combined immunodeficiency/severe T-cell lymphopenia requiring hematopoietic stem cell transplantation or other therapy were detected. In summary, the T-cell receptor excision circle assay is a sensitive and specific test to identify infants with severe combined immunodeficiency and severe T-cell lymphopenia that leads to life-saving therapies such as hematopoietic stem cell transplantation prior to the acquisition of severe infections.

Development of a routine newborn screening protocol for severe combined immunodeficiency.

BACKGROUND: Severe combined immunodeficiency (SCID) is characterized by the absence of functional T cells and B cells. Without early diagnosis and treatment, infants with SCID die from severe infections within the first year of life. OBJECTIVE: To determined the feasibility of detecting SCID in newborns by quantitating T-cell receptor excision circles (TRECs) from dried blood spots (DBSs) on newborn screening (NBS) cards. METHODS: DNA was extracted from DBSs on deidentified NBS cards, and real-time quantitative PCR (RT-qPCR) was used to determine the number of TRECs. Positive controls consisted of DBS from a 1-week-old T(-)B(-)NK(+) patient with SCID and whole blood specimens selectively depleted of naive T cells. RESULTS: The mean and median numbers of TRECs from 5766 deidentified DBSs were 827 and 708, respectively, per 3.2-mm punch ( approximately 3 muL whole blood). Ten samples failed to amplify TRECs on initial analysis; all but 1 demonstrated normal TRECs and beta-actin amplification on retesting. No TRECs were detected in either the SCID or naive T-cell-depleted samples, despite the presence of normal levels of beta-actin. CONCLUSIONS: The use of RT-qPCR to quantitate TRECs from DNA extracted from newborn DBSs is a highly sensitive and specific screening test for SCID. This assay is currently being used in Wisconsin for routine screening infants for SCID.

Statewide newborn screening for severe T-cell lymphopenia.

CONTEXT: A newborn blood screening (NBS) test that could identify infants with a profound deficiency of T cells may result in a reduction in mortality. OBJECTIVE: To determine if quantitating T-cell receptor excision circles (TRECs) using real-time quantitative polymerase chain reaction on DNA extracted from dried blood spots on NBS cards can detect infants with T-cell lymphopenia in a statewide program. DESIGN, SETTING, AND PARTICIPANTS: Between January 1 and December 31, 2008, the Wisconsin State Laboratory of Hygiene screened all infants born in Wisconsin for T-cell lymphopenia by quantitating the number of TRECs contained in a 3.2-mm punch (approximately 3 microL of whole blood) of the NBS card. Flow cytometry to enumerate the number of T cells was performed on full-term infants and preterm infants when they reached the equivalent of at least 37 weeks' gestation with TREC values of less than 25/microL. Infants with T-cell lymphopenia were evaluated by a clinical immunologist. MAIN OUTCOME MEASURES: The number of infants with TREC values of less than 25/microL with T-cell lymphopenia confirmed by flow cytometry. RESULTS: Exactly 71,000 infants were screened by the TREC assay. Seventeen infants aged at least 37 weeks' gestation had at least 1 abnormal TREC assay (TREC values < 25/microL), 11 of whom had samples analyzed by flow cytometry to enumerate T cells. Eight infants demonstrated T-cell lymphopenia. The causes of the T-cell lymphopenia included DiGeorge syndrome (n = 2), idiopathic T-cell lymphopenia (n = 2), extravascular extravasation of lymphocytes (n = 3), and a Rac2 mutation (n = 1). The infant with the Rac2 mutation underwent successful cord blood transplantation. CONCLUSION: In a statewide screening program, use of the TREC assay performed on NBS cards was able to identify infants with T-cell lymphopenia.

Screening newborns for congenital disorders.

The Newborn Screening Laboratory at the Wisconsin State Laboratory of Hygiene (WSLH) tests all newborn babies in the state of Wisconsin for 26 congenital disorders. The screening is mandated by state statute (253.13) and attempts to identify those babies at highest risk for any of the screened-for congenital disorders. The Newborn Screening Laboratory at the WSLH is part of the state's newborn screening program, a cooperative effort between the Wisconsin Department of Health and Family Services, state birthing hospital staff, several public and private specialty clinics, primary care providers, and parents. Screening occurs within the first hours (median 38 hours) of life when a few drops of blood from the baby's heel is collected, applied to a special paper, dried, and sent to the WSLH for analysis. Those babies determined to have at-risk test results get repeat testing to confirm the initial test results and, if warranted, the baby is referred to a specialty clinic for a diagnostic work-up and treatment if necessary. Since its inception in the early 1960s, newborn screening in Wisconsin has saved approximately 1300 children from serious mental or other medical problems.

Newborn screening for congenital adrenal hyperplasia has reduced sensitivity in girls.

OBJECTIVES: To characterize Wisconsin-born infants with 21-hydroxylase deficiency-congenital adrenal hyperplasia (21-OH-D-CAH) who were not identified by the newborn screening for 21-OH-D-CAH, and to examine male and female screening 17-hydroxyprogesterone (17-OHP) levels. STUDY DESIGN: Information on infants with false-negative results was gathered. Results of the Wisconsin newborn screening for 21-OH-D-CAH from January 1, 2000, to June 30, 2003, were analyzed to detect possible differences between male (n=119,842) and female (n=114,951) infants. RESULTS: Six of 7 female infants with false-negative results had genital masculinization, and 4 of 8 infants with false-negative results had laboratory evidence of salt-wasting. None died, had a salt-wasting crisis, or was assigned the wrong sex. A significant difference in the mean 17-OHP levels between male (17.5 ng/mL) and female (15.4 ng/mL) infants (P <.0001) was detected. The sensitivity of newborn screening for female infants was 60%, compared with 80% for male infants. CONCLUSIONS: Male and female infants have significantly different mean 17-OHP levels on newborn screening, and female infants comprise most of the infants with false-negative results. Although health professionals should not assume that newborn screening for 21-OH-D-CAH is a means of identifying all affected infants, the primary goals of newborn screening for CAH (prevention of salt-wasting crises and sex misassignment) are fulfilled.

Newborn screening with tandem mass spectrometry: examining its cost-effectiveness in the Wisconsin Newborn Screening Panel.

OBJECTIVE: To examine the cost-effectiveness of tandem mass spectrometry (MS/MS) in a neonatal screening panel for 14 fatty acid oxidation and organic acidemia disorders in the Wisconsin Newborn Screening Program. STUDY DESIGN: An incremental cost-effectiveness analysis with a hypothetical cohort of 100,000 infants was performed. A threshold of $50,000/QALY (quality-adjusted life-year) was used to determine whether screening for medium-chain acyl-CoA dehydrogenase deficiency (MCAD) alone is cost-effective or whether additional disorders would need to be incorporated into the analysis to arrive at a conclusion regarding the overall cost-effectiveness of MS/MS. RESULTS: Under conservative assumptions, screening for MCAD alone yields an incremental cost-effectiveness ratio of $41,862/QALY. With the use of more realistic assumptions, screening becomes more cost-effective ($6008/QALY) and remains cost-effective so long as the incremental cost of screening remains under $13.05 per test. Adding the incremental costs of detecting the 13 other disorders on the screening panel still yields a result well within accepted norms for cost-effectiveness ($15,252/QALY). CONCLUSIONS: In Wisconsin, MS/MS screening for MCAD alone appears to be cost-effective. Future analyses should examine the cost-effectiveness of alternative follow-up and treatment regimens for MCAD and other panel disorders.

Multiplexed genetic analysis using an expanded genetic alphabet.

BACKGROUND: All states require some kind of testing for newborns, but the policies are far from standardized. In some states, newborn screening may include genetic tests for a wide range of targets, but the costs and complexities of the newer genetic tests inhibit expansion of newborn screening. We describe the development and technical evaluation of a multiplex platform that may foster increased newborn genetic screening. METHODS: MultiCode PLx involves three major steps: PCR, target-specific extension, and liquid chip decoding. Each step is performed in the same reaction vessel, and the test is completed in approximately 3 h. For site-specific labeling and room-temperature decoding, we use an additional base pair constructed from isoguanosine and isocytidine. We used the method to test for mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The developed test was performed manually and by automated liquid handling. Initially, 225 samples with a range of genotypes were tested retrospectively with the method. A prospective study used samples from >400 newborns. RESULTS: In the retrospective study, 99.1% of samples were correctly genotyped with no incorrect calls made. In the perspective study, 95% of the samples were correctly genotyped for all targets, and there were no incorrect calls. CONCLUSIONS: The unique genetic multiplexing platform was successfully able to test for 31 targets within the CFTR gene and provides accurate genotype assignments in a clinical setting.