Georgia state spinal muscular atrophy newborn screening experience: Screening assay performance and early clinical outcomes.

The purpose of this study is to provide the results of the newborn screening (NBS) program for Spinal Muscular Atrophy (SMA) in the state of Georgia to determine disease incidence, time to diagnosis and treatment, and early outcomes. NBS for SMA was performed using real time PCR assays from February 2019 through February 2020 in a pilot phase of screening. This method continued as part of our official state panel, and here we describe the pilot period as well as the first year of standard screening through February 2021. Medical records of infants with a positive NBS were reviewed for time to confirmation and neurologic evaluation, SMN2 copy number, clinical information, and treatment. Descriptive statistics were applied. Of the 301,418 samples screened, there were 15 true positive (eight males) and 24 false positive cases. One patient was missed due to human error early in the pilot phase and presented after symptom onset. The incidence of SMA in Georgia is approximately 1 in 18,840 births per year. After the pilot phase, the false positive rate was found to be so low that all patients who test positive were immediately referred to neurology for further care. Four patients died prior to intervention. Ten patients received intervention. Gene therapy was the preferred treatment. One patient was lost to follow-up; another was clinically followed. In conclusion, trends for treated patients show improved or stable motor function. Long-term follow-up will help determine the durability of treatment.

A Novel Approach to Improve Newborn Screening for Congenital Hypothyroidism by Integrating Covariate-Adjusted Results of Different Tests into CLIR Customized Interpretive Tools.

Newborn screening for congenital hypothyroidism remains challenging decades after broad implementation worldwide. Testing protocols are not uniform in terms of targets (TSH and/or T4) and protocols (parallel vs. sequential testing; one or two specimen collection times), and specificity (with or without collection of a second specimen) is overall poor. The purpose of this retrospective study is to investigate the potential impact of multivariate pattern recognition software (CLIR) to improve the post-analytical interpretation of screening results. Seven programs contributed reference data (N = 1,970,536) and two sets of true (TP, N = 1369 combined) and false (FP, N = 15,201) positive cases for validation and verification purposes, respectively. Data were adjusted for age at collection, birth weight, and location using polynomial regression models of the fifth degree to create three-dimensional regression surfaces. Customized Single Condition Tools and Dual Scatter Plots were created using CLIR to optimize the differential diagnosis between TP and FP cases in the validation set. Verification testing correctly identified 446/454 (98%) of the TP cases, and could have prevented 1931/5447 (35%) of the FP cases, with variable impact among locations (range 4% to 50%). CLIR tools either as made here or preferably standardized to the recommended uniform screening panel could improve performance of newborn screening for congenital hypothyroidism.

Newborn Screening for X-Linked Adrenoleukodystrophy in Georgia: Experiences from a Pilot Study Screening of 51,081 Newborns.

We screened 51,081 newborns for X-linked adrenoleukodystrophy (ALD) using a two-tiered strategy quantifying very long chain lysophosphatadylcholines (LPC). Our testing strategy used flow injection tandem mass spectrometry for the first-tier analysis of LPCs, and second-tier quantification of C26:0 LPC using liquid chromatography tandem mass spectrometry. There were 364 specimens considered abnormal using our first-tier algorithm that relied on the four LPC measurements and post-analytical tools. Second-tier test results were reported as normal or abnormal based on a cutoff for the single analyte, C26:0 LPC. Eleven cases were reported as abnormal based on second-tier test results. One male with ALD was identified, and two females with peroxisomal biogenesis disorders were also identified. A single female case remains unresolved, due to a loss to follow up after a negative molecular test result for ABCD1 gene sequencing. The positive predictive value for confirmed, clinically relevant disorders during this pilot study was 27.3%. Challenges identified during the study period were based around coverage for confirmatory testing, particularly if family members needed molecular testing, which is an ongoing issue with newborn screening in Georgia. We also encountered issues with the follow up for a patient who remained asymptomatic. Due to the different timelines involved with clinical findings in ALD, follow-up coordination may be more difficult, particularly if the child identified by newborn screening (NBS) is the only member of the family affected, or able to be tested.

Post-Analytical Tools for the Triage of Newborn Screening Results in Follow-up Can Reduce Confirmatory Testing and Guide Performance Improvement.

Georgia uses post-analytical tools through Collaborative Laboratory Integrated Reports (CLIR) to triage abnormal newborn screening (NBS) results for follow-up. Condition specific tools are used to assign each case a risk level, which is used to guide follow-up recommendations. Follow-up recommendations include assessment by the child's primary care provider as well as testing, either a repeat NBS or confirmatory testing. Triaging abnormal cases using these tools has been advantageous in managing the workflow for the follow-up team, as well as prioritizing cases that appropriately require more attention and resources. The initial goal in utilizing these tools was to reduce the amount of confirmatory testing, particularly for disorders where there are many false positives. We assessed the performance of these tools retrospectively for three of the most commonly detected conditions by tandem mass spectrometry in Georgia: phenylketonuria, medium chain acyl-CoA dehydrogenase deficiency and very long chain dehydrogenase deficiency. The post-analytical tools appropriately assigned all true positive cases to the higher levels of follow-up testing and reduced the level of intervention for a significant number of cases as well. Based on the experience gained from our utilization of the tools in the follow-up program, we are well situated to move forward with using the tools in a more prospective manner, and reduce the number of cases that will be reported, rather than just assigning resources appropriately at follow-up. Post-analytical tools are an improvement over trying to capture the variation in the newborn population using multiple cutoffs. It also easily identifies significant abnormalities that are unrelated to inherited disease, such as large amino acid elevations due to total parenteral nutrition.

Two-Tiered Newborn Screening with Post-Analytical Tools for Pompe Disease and Mucopolysaccharidosis Type I Results in Performance Improvement and Future Direction.

We conducted a pilot newborn screening (NBS) study for Pompe disease (PD) and mucopolysaccharidosis type I (MPS I) in the multiethnic population of Georgia. We screened 59,332 infants using a two-tier strategy of flow injection tandem mass spectrometry (FIA-MSMS) enzyme assays. The first tier of testing was a 2-plex assay measuring PD and MPS I enzyme activity, followed by a second-tier test with additional enzymes to improve specificity. Interpretation of results was performed using post-analytical tools created using Collaborative Laboratory Integrated Reports (CLIR). We identified a single case of infantile onset PD, two cases of late onset PD, and one pseudodeficiency. The positive predictive value (PPV) for PD screening during the study was 66.7%. No cases of MPS I were identified during the study period, but there were 2 confirmed cases of pseudodeficiency and 6 cases lost to follow up. The two-tier screening strategy was successful in reducing false positive results and allowed for the identification and early treatment of a case of infantile PD but the frequency of pseudodeficiency in MPS I is problematic. Molecular testing is required and should be covered by the screening program to avoid delays in case resolution.

Increased parental anxiety and a benign clinical course: Infants identified with short-chain acyl-CoA dehydrogenase deficiency and isobutyryl-CoA dehydrogenase deficiency through newborn screening in Georgia.

The long-term consequences and need for therapy in children with short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBDD) identified via newborn screening (NBS) remains controversial. Initial clinical descriptions were severe; however, while most cases identified through NBS have remained asymptomatic, clinical concerns have been raised in these populations. It is not clear whether these children are asymptomatic because of the success of NBS, or because the normal clinical course of these disorders is relatively benign. To evaluate these possibilities in our program, we evaluated the clinical outcomes of children with SCADD or IBDD identified by the Georgia NBS compared to the health status of a healthy age-matched control group. We also assessed parental anxiety during a phone interview both subjectively and objectively using the Pediatric Inventory for Parents (PIP), a validated measure of illness-related parental stress. The general health of 52 SCADD and nine IBDD cases from 2007 to 2016 were compared to the general health of unaffected control children obtained through the Centers for Disease Control and Prevention (CDC) parent listserv. We also collected statements from parents who participated in a phone survey regarding events they experienced during and after their diagnostic process. Overall, the children with SCADD and IBDD had no major health problems. There was no significant difference in cognitive development (p = .207). We identified a slightly higher incidence of reported neonatal hypoglycemia in the SCADD group; two of these occurred in the context of maternal diabetes. All interviewed parents reported extreme anxiety during the diagnostic period and current feelings of uncertainty about their child's future. PIP scores for all six caregivers who responded to that portion of the survey were consistent with some degree of parental stress. The greatest reported stressor was the unknown long-term impact of the illness. All children with SCADD and IBDD had no significant long-term sequelae. The phone interviews revealed substantial parental anxiety about the identification and follow-up of SCADD and IBDD. Based on our findings, the anxiety parents experience may be unwarranted given that we see no disease-associated morbidity or mortality in these children. Consideration should be given to the removal of these conditions from NBS panels, or if that is not possible, clinicians could educate parents on the benign nature of these diagnoses and release them from follow-up without treatment.

Newborn screening for medium chain acyl-CoA dehydrogenase deficiency: performance improvement by monitoring a new ratio.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder included on newborn screening (NBS) panels in many regions that have expanded to using tandem mass spectrometry for acylcarnitine screening. False positive (FP) screening results for MCAD deficiency have previously been linked to very low birth weight (VLBW) infants and those who are heterozygous for the common mutation, p.K324E. Previous studies have identified these causes of FP screens by sequencing residual dried blood spots. From our cohort of FP screens in Georgia, we identified an elevation at the same mass as octenoylcarnitine (C8:1) causing elevations of octanoylcarnitine (C8) not due to MCAD deficiency. We reviewed biochemical results from 2011 to 2013 for all newborn screens positive for MCAD deficiency in Georgia to identify screening criteria to allow these cases to be identified prospectively, thus saving families the stress of additional testing on their newborn and reducing healthcare costs while improving screening performance for the screening program. We identified the C8/C8:1 ratio as an effective marker, and developed criteria that will reduce FP screening results due to this interfering substance.

Comparative evaluation of newborn bloodspot specimen cards by experienced laboratory personnel and by an optical scanning instrument.

A major factor in determining the suitability of a dried blood spot (DBS) specimen is the subjective nature of evaluation by laboratory personnel. Using newborn screening DBS specimen cards as they were submitted to a public health NBS program, we conducted a systematic pilot study of DBS evaluation by multiple experienced laboratory personnel (ELP) and by an automated optical scanning instrument (OSI) (CardScan (tm), BSD Robotics). OSI confirmed the satisfactory status of all newborn DBS specimen cards that passed initial review by the first ELP. Among the questionable cards selected for further review, 58% passed multiple ELP consensus assessment, and 62% passed OSI evaluation. The overall agreement between ELP and OSI was 86%. Among questionable specimen cards, ELP and OSI were more strongly correlated when multiple ELP assessment was unanimous. We conclude that subjective assessment by ELP is essential and that OSI evaluation is a useful adjunct when ELP assessment does not reach consensus. OSI further allows the selection of optimal locations for punching DBS from unsatisfactory or questionable specimens, optimizing the quality of interim analyses that may be conducted while repeat specimens are being collected. Instrument evaluation of specimen cards would also be valuable as an independent reference method for training laboratory and specimen collection personnel. OSI technology merits further studies to confirm and extend our findings.

Impact of Hurricane Katrina on newborn screening in Louisiana.

OBJECTIVE: The Louisiana Office of Public Health and the Centers for Disease Control and Prevention assessed the extent to which newborn screening was disrupted from August 15 to September 21, 2005, the immediate period before and after Hurricane Katrina. METHODS: A list of hospitals with labor and delivery services was obtained from the Louisiana Hospital Association. A survey sent to hospitals on October 17, 2005, asked about the number of live births during the assessment period, disruption in hospital services, the number of specimens sent to alternative laboratories, and the number of children without screening results. RESULTS: Among 64 Louisiana hospitals with labor and delivery units, 6 remained closed at the time of the survey. Of the 58 open hospitals, 53 (91.4%) completed the questionnaire. Twenty-one (36.2%) of 58 hospitals experienced disruption of newborn screening services. Respondents from 31 (58.5%) of the 53 open hospitals acknowledged receiving the advisory from the Office of Public Health regarding resumption of newborn screening laboratory services. Hospitals stated that of 5958 specimens submitted, reports had not been received for 1207 (20.3%) newborns. The Office of Public Health laboratory reviewed the names of 2828 newborns received from hospitals and determined that no specimen was received within 14 days of collection for 631 (22.3%). Thirty percent of the specimens received from infants who were born between August 15 and September 21 were rejected as a result of having been received >14 days after collection. Ten children had confirmed positive screening results during the assessment period; all were located, and treatment was initiated. CONCLUSIONS: Collaboration between the Office of Public Health and the Centers for Disease Control and Prevention was essential to increase awareness of changes in laboratory procedures after the hurricane and to help identify infants who might be in need of screening or rescreening.

Diagnosis and characterization of Hb C/Hb Iowa: a rare but easily misidentified compound heterozygous condition.

Hb Iowa is a rare hemoglobin (Hb) variant with a Gly --> Ala substitution at position 119 of beta-globin. It was previously reported only in an African American infant who was also heterozygous for Hb S [beta6(A3)Glu --> Val] and her mother (Hb A/Iowa). Here we describe the second report of Hb Iowa, the first in conjunction with Hb C [beta6(A3)Glu --> Lys]. The patient was an African American girl, originally diagnosed as homozygous Hb C during neonatal screening. When seen in our clinic, hematological data for both the child and her mother (Hb C trait) indicated mild anemia with slightly low mean corpuscular volume (MCV) but normal red blood cell (RBC) count. Analysis of blood from the child by capillary isoelectric focusing (cIEF) identified Hb C and an unknown Hb variant with an isoelectric point (pI) intermediate to that of Hbs F and A. The unknown variant was identified as Hb Iowa by DNA sequence analysis of the beta-globin gene (GGC --> GCC). Both reported cases of Hb Iowa indicated that there are no abnormal hematological manifestations associated with this rare Hb variant. In both cases, however, Hb Iowa was mistaken for Hb F during routine neonatal screening by high performance liquid chromatography (HPLC) and/or gel IEF. Neonatal misidentification of Hb Iowa led to misdiagnosis of sickle cell disease and Hb C disease, respectively. In our patient, Hb Iowa was also misidentified as Hb A at 2 years of age by a commercial reference laboratory using cellulose acetate and citrate agar gel electrophoresis. This led to an incorrect diagnosis of Hb C trait. These results show that commonly used analytical methods can easily misidentify Hb Iowa as Hbs F or A in neonates, or older individuals, resulting in incorrect identification of the Hb phenotype. We conclude that the presence of Hb Iowa, or other variants with similar pIs, should be considered in cases where the results of follow-up testing conflict with neonatal diagnosis of sickle cell or Hb C disease, or where clinical presentation does not agree with diagnosis of either homozygous or heterozygous Hb S or Hb C.