summarizes the programmatic criteria the Wisconsin NBS Program considered prior to adding SCID to the test panel. The responses to these 15 questions made it clear that a screening program for SCID could be successful if effective screening and follow-up protocols were established.
Programmatic criteria considered by the Wisconsin NBS Advisory Committee in evaluating the addition of SCID screening to its NBS test panel
A shows the distribution of the number of TRECs extracted from 5,766 de-identified dried-blood spot samples submitted to the NBS program and analyzed during the proof-of-concept pilot study in 2007. The number of TRECs is shown as TRECs/3.2-mm circular dried-blood spot. A 3.2-mm circular punch from a dried-blood spot contains 3 μL of whole blood. The number of TRECs/3 μL ranged from 0 to 3,900. In this sample, the mean number of TRECs/3 μL was 827, and the median number was 708. The shape of the distribution function, skewed (i.e., exhibiting a “tail” of values) to the right, is typical of many clinical analytes; however, the region of interest when screening for SCID and immunodeficiencies lies with the zero and low values at the left end of the distribution. A newborn exhibiting a significant number of TRECs is, by definition, producing T-cells and, therefore, is negative for SCID and many other T-cell immunodeficiencies. B shows the distribution of TRECs from newborns who had ≤150 TRECs on initial testing. Only about 1.06% of newborns have <75 TRECs/3 μL or the equivalent of 25 TRECs/μL. Using data from this pilot study, we set the internal (intra-laboratory) cutoff at <25 TRECs/μL. Specimens with ≥25 TRECs/μL are considered to be normal (i.e., negative for SCID and other immunodeficiencies). The cutoff of <25 TRECs/μL clears 98.94% of all initial specimens for SCID on the initial tests.
Results of initial RT-qPCR TREC assay conducted by the Wisconsin NBS Program on 5,766 NBS cards during the 2007 SCID-screening pilot studya,b
From these pilot-study results, a screening algorithm was developed to move forward for prospective analyses. Specimens not reported as normal (i.e., ≥25 TRECs/μL) fall into one of two categories: inconclusive or abnormal. If the blood spots are of poor quality for NBS, have low TRECs but are from newborns of very low gestational age, or, in the judgment of the laboratory personnel, cannot be declared negative, results are deemed to be inconclusive. The primary care provider is requested to provide a second filter-paper specimen for retesting of all 47 NBS parameters, including SCID. This category represents approximately 0.17% of all births. The abnormal category represents specimens that on the initial test exhibited <25 TRECs/μL. For these, the original filter paper is re-punched and retested in duplicate. If confirmed to have zero or low numbers of TRECs, the clinical consultant to the NBS program is notified of the results. The consultant (a physician who diagnoses and treats immunodeficiencies in newborns, including those with SCID) contacts the newborn's primary care physician, and they jointly decide to test either a repeat filter-paper specimen or an immediate, micro whole-blood -specimen for immune cell subset levels by flow cytometry. If T-cell markers are absent, an immediate referral to an immunodeficiency clinic and full workup for SCID is recommended. If the repeat filter-paper specimen is again abnormal, a liquid whole-blood specimen is requested. Specimens that are normal on repeat testing (either filter paper or flow) are considered screening false positives.
The intra-laboratory reflexive testing of the initial filter-paper specimen in duplicate enables us to clear 99.98% of full-term newborns from whom we have obtained satisfactory specimens. Similarly, for premature births, the clearance rate is 99.97%. These rates are as good, or better, than the other tests in the current NBS panel; however, the number of inconclusive specimens is much higher in premature newborns, reflecting, in part, on low-birthweight babies in intensive care units, where a final discharge specimen will be forthcoming.
Results from the 2008 prospective study of full-term newborns and premature infants are shown in the . Twelve full-term newborns had abnormal (positive screening) results. Six whole-blood samples and four repeat filter-paper specimens were collected from these full-term newborns and retested. Two of the full-term newborns who initially had abnormal results were not retested (one died, one refused). Of the 10 full-term newborns who were retested, two had normal flow results (false positives), four exhibited non-SCID-related T-cell lymphopenia, and four were found to be normal. None were diagnosed with true SCID. Only 23 of 6,603 premature newborns had abnormal screening results reported during 2008. Five of these infants died before a repeat specimen could be obtained. Of the remaining 18, 12 tested normal on repeat screening, three had normal flow cytometry results (false positives), and three had an abnormal result at the equivalent age of 37 weeks gestation. The follow-up flow cytometry on these three newborns exhibited non-SCID T-cell lymphopenia. Although none of the premature newborns was diagnosed with SCID, eight infants were identified as having clinically significant T-cell lymphopenia, one of whom underwent successful bone-marrow transplantation. These results illustrate that the TREC assay used as part of routine NBS is also a test for T-cell lymphopenia, not just SCID.
Wisconsin Newborn Screening Program severe combined immunodeficiency screening results, 2008 (n=71,000)
Importantly, screening for SCID did not delay reporting of the other 46 NBS tests. Results are available in less than two days from receipt of the initial specimen in the laboratory and are reported as the number of TRECs/μL, along with the interpretation (normal, abnormal, or inconclusive). The addition of SCID screening has been accomplished without significant impact on the existing NBS program.