Epigenomic newborn screening for conditions with intellectual disability and autistic features in Australian newborns.

This study describes a protocol to assess a novel workflow called Epi-Genomic Newborn Screening (EpiGNs) on 100,000 infants from the state of Victoria, Australia. The workflow uses a first-tier screening approach called methylation-specific quantitative melt analysis (MS-QMA), followed by second and third tier testing including targeted methylation and copy number variation analyzes with droplet digital PCR, EpiTYPER system and low-coverage whole genome sequencing. EpiGNs utilizes only two 3.2 mm newborn blood spot punches to screen for genetic conditions, including fragile X syndrome, Prader-Willi syndrome, Angelman syndrome, Dup15q syndrome and sex chromosome aneuploidies. The program aims to: identify clinically actionable methylation screening thresholds for the first-tier screen and estimate prevalence for the conditions screened.

[Box: see text].

Estimating the sensitivity of genomic newborn screening for treatable inherited metabolic disorders.

INTRODUCTION: Over 30 research groups and companies are exploring newborn screening using genomic sequencing (NBSeq), but the sensitivity of this approach is not well understood. METHODS: We identified individuals with treatable inherited metabolic disorders (IMDs) and ascertained the proportion whose DNA analysis revealed explanatory deleterious variants (EDVs). We examined variables associated with EDV detection and estimated the sensitivity of "DNA-first" NBSeq. We further predicted the annual rate of true positive and false negative NBSeq results in the United States for several conditions on the Recommended Uniform Screening Panel (RUSP). RESULTS: We identified 635 individuals with 80 unique IMDs. In univariate analyses, Black race (OR = 0.37, 95% CI: 0.16-0.89, p = 0.02) and public insurance (OR = 0.60, 95% CI: 0.39-0.91, p = 0.02) were less likely to be associated with finding EDVs. Had all individuals been screened with NBSeq, the sensitivity would have been 80.3%. We estimated that between 0 and 649.9 cases of RUSP IMDs would be missed annually by NBSeq in the United States. CONCLUSIONS: The overall sensitivity of NBSeq for treatable IMDs is estimated at 80.3%. That sensitivity will likely be lower for Black infants and those who are on public insurance.

Diagnostic options, physiopathology, risk factors and genetic causes of permanent congenital hypothyroidism: A narrative review.

Background: In Permanent congenital hypothyroidism (PCH) is a lifelong condition characterized by a deficiency in thyroid hormone, leading to various neurodevelopmental complications. Early clinical signs are often nonspecific and easily overlooked, but newborn screening programs have improved early detection. Methods: This narrative review aims to provide insights comparatively transient and permanent PCH and also the diagnosis, risk factors, underlying pathophysiology, and genetic causes associated with PCH. Relevant studies were identified through a comprehensive search using the term 'Permanent congenital hypothyroidism' (Mesh) across scientific databases of electronic databases such as PubMed, Scopus, and Web of Science. Results: Prompt initiation of thyroid hormone replacement therapy, particularly within the initial two weeks postpartum, crucially enhances neurocognitive development outcomes. Multiple predictive approaches, encompassing screening TSH levels, maternal thyroid history, and levothyroxine dosage per kilogram assessment, aid in identifying PCH. Recent studies have demonstrated a mounting prevalence of PCH, contributing significantly to the overall rise in CH incidence. Genetic factors, primarily DUOX2 and DUOXA2 mutations, alongside environmental influences such as post-term birth, low birth weight, and macrosomia, may induce PCH. Nonetheless, reliable markers for early PCH prediction upon diagnosis remain elusive, leading to delayed recognition post-ceasing levothyroxine treatment around age 3. Conclusions: Recent studies have observed an increased incidence of PCH, contributing substantially to the overall rise in cases of congenital hypothyroidism. Understanding the diagnostic options and genetic etiologies associated with PCH is crucial for the early identification and appropriate management.

Outcomes of early-treated infants with spinal muscular atrophy: A multicenter, retrospective cohort study.

INTRODUCTION/AIMS: While prompt identification and treatment of infants with spinal muscular atrophy (SMA) can ameliorate outcomes, variability persists. This study assessed management and outcomes of early-treated infants with SMA. METHODS: We analyzed retrospective data at 12 centers on infants with SMA treated at age ≤6 weeks from August 2018 to December 2023. RESULTS: Sixty-six patients, 35 with two SMN2 copies and 31 with ≥3 SMN2 copies, were included. Twenty-five (38%, 22 with two SMN2 copies), had SMA findings before initial treatment which was onasemnogene abeparvovec in 47 (71%) and nusinersen in 19 (29%). Thirty-two received sequential or combination treatments, including 16 adding nusinersen or risdiplam due to SMA findings following onasemnogene abeparvovec. All sat independently. Compared to children with ≥3 SMN2 copies, those with two SMN2 copies were less likely to walk (23/34 [68%] vs. 31/31 [100%], p < .001) and less likely to walk on time (9/34 [26%] vs. 29/31 [94%], p < .001); one non-ambulatory child was <18 months old and was excluded from this analysis. No patients required permanent ventilation or exclusively enteral nutrition; six required nocturnal non-invasive ventilation and four utilized supplemental enteral nutrition, all with two SMN2 copies. DISCUSSION: Early treatment of infants with SMA can improve outcomes as indicated by our cohort, all of whom sat independently and are without permanent ventilation. However, our study demonstrates ongoing disability in most children with two SMN2 copies despite early monotherapy and emphasizes the need for additional research, including earlier monotherapy, initial combination therapy, prenatal treatment, and non-SMN modifying treatments.

Newborn screening algorithm distinguishing potential symptomatic isovaleric acidemia from asymptomatic newborns.

Newborn screening (NBS) for isovaleric acidemia (IVA) reduces mortality and morbidity; however, it has also resulted in the detection of individuals with an asymptomatic or mild presentation for which early detection via newborn screening has not been proven to alter neurological outcome. We reevaluated biochemical and molecular data for newborns flagged positive for IVA in aim of developing a new screening algorithm to exclude the latter from positive screening. Among 2 794 365 newborns underwent routine newborn screening in Israel, 412 flagged positive for IVA, of which, 371 were false positives on recall sample testing and 41 positive newborns were referred to the clinic. 38/41 have biochemical and molecular confirmation in keeping with IVA. Among the 38 patients, 32% (12/38) were classified as symptomatic while, 68% (26/38) were classified as asymptomatic. 69% of the latter group harbor the known variant associated with mild potentially asymptomatic phenotype, c.932C>T; p. Ala311Val. Among asymptomatic patients, only 46% (12/26) are currently treated. Two novel variants have been detected in the IVD gene: c.487G>A; p. Ala163Thr and c.985A>G; p. Met329Val. Cut-off recalculation, of referred newborns' initial biochemical results, after classifying the referred patients to two binary groups of symptomatic and asymptomatic, resulted in an improved NBS algorithm comprising of C5 >5 µM and C5/C2>0.2 and C5/C3>4 flagging only those likely to have the classic symptomatic phenotype.

A Qualitative Study on Engaged Families' Experiences with Long-Term Follow-Up Care in the Colorado/Wyoming Newborn Screening System.

Understanding whether the long-term follow-up (LTFU) system is working for families is critical to measuring the success of newborn screening (NBS) and understanding why some families are lost to follow-up. Caregivers were recruited from six pediatric specialty care clinics. Data were gathered from caregivers via five focus groups and one individual interview (n = 24). Caregiver participants represented a wide range of children's ages and conditions identified through NBS. While this is not the first study to gather caregivers' input on LTFU, it provides a wide breadth of perspectives (e.g., metabolic, endocrine, hemoglobinopathy, etc.). When asked about goals for their children, caregivers identified health-related goals (i.e., children able to care for themselves, not hindered by diagnosis) and non-health related goals (i.e., defining themselves outside of disease, participating in sports, making friends). In describing the LTFU care they want and need for their child and the key factors that influence access and engagement, caregivers identified three themes: communication and relationships with providers; care team roles and factors; and care access and utilization factors. The themes identified are not disjointed; they are intertwined and illustrate the lived experiences of a sample of families engaged in LTFU care.

Wilson and Jungner Revisited: Are Screening Criteria Fit for the 21st Century?

Driven by technological innovations, newborn screening (NBS) panels have been expanded and the development of genomic NBS pilot programs is rapidly progressing. Decisions on disease selection for NBS are still based on the Wilson and Jungner (WJ) criteria published in 1968. Despite this uniform reference, interpretation of the WJ criteria and actual disease selection for NBS programs are highly variable. A systematic literature search [PubMED search "Wilson" AND "Jungner"; last search 16.07.22] was performed to evaluate the applicability of the WJ criteria for current and future NBS programs and the need for adaptation. By at least two reviewers, 105 publications (systematic literature search, N = 77; manual search, N = 28) were screened for relevant content and, finally, 38 publications were evaluated. Limited by the study design of qualitative text analysis, no statistical evaluation was performed, but a structured collection of reported aspects of criticism and proposed improvements was instead collated. This revealed a set of general limitations of the WJ criteria, such as imprecise terminology, lack of measurability and objectivity, missing pediatric focus, and absent guidance on program management. Furthermore, it unraveled specific aspects of criticism on clinical, diagnostic, therapeutic, and economical aspects. A major obstacle was found to be the incompletely understood natural history and phenotypic diversity of rare diseases prior to NBS implementation, resulting in uncertainty about case definition, risk stratification, and indications for treatment. This gap could be closed through the systematic collection and evaluation of real-world evidence on the quality, safety, and (cost-)effectiveness of NBS, as well as the long-term benefits experienced by screened individuals. An integrated NBS public health program that is designed to continuously learn would fulfil these requirements, and a multi-dimensional framework for future NBS programs integrating medical, ethical, legal, and societal perspectives is overdue.

Charting the Ethical Frontier in Newborn Screening Research: Insights from the NBSTRN ELSI Researcher Needs Survey.

From 2008 to 2024, the Newborn Screening Translational Research Network (NBSTRN), part of the National Institute of Child Health and Human Development (NICHD) Hunter Kelly Newborn Screening Program, served as a robust infrastructure to facilitate groundbreaking research in newborn screening (NBS), public health, rare disease, and genomics. Over its sixteen years, NBSTRN developed into a significant international network, supporting innovative research on novel technologies to screen, diagnose, treat, manage, and understand the natural history of more than 280 rare diseases. The NBSTRN tools and resources were used by a variety of stakeholders including researchers, clinicians, state NBS programs, parents, families, and policy makers. Resources and expertise for the newborn screening community in ethical, legal, and social issues (ELSI) has been an important area of focus for the NBSTRN and this includes efforts across the NBS system from pilot studies of candidate conditions to public health implementation of screening for new conditions, and the longitudinal follow-up of NBS-identified individuals to inform health outcomes and disease understanding. In 2023, the NBSTRN conducted a survey to explore ELSI issues in NBS research, specifically those encountered by the NBS community. Since NBS research involves collaboration among researchers, state NBS programs, clinicians, and families, the survey was broadly designed and disseminated to engage all key stakeholders. With responses from 88 members of the NBS community, including researchers and state NBS programs, the survey found that individuals rely most on institutional and collegial resources when they encounter ELSI questions. Most survey responses ranked privacy as extremely or very important in NBS research and identified the need for policies that address informed consent in NBS research. The survey results highlight the need for improved collaborative resources and educational programs focused on ELSI for the NBS community. The survey results inform future efforts in ELSI and NBS research in the United States (U.S.) and the rest of the world, including the development of policies and expanded ELSI initiatives and tools that address the needs of all NBS stakeholders.

Clinical, Biochemical, and Molecular Characteristics of Filipino Patients with Tyrosinemia Type 1.

Hereditary tyrosinemia type I (HT1), or hepatorenal tyrosinemia, is an amino acid disorder which may cause hepatic failure as well as renal and neurologic comorbidities. Early detection of this disorder is possible with newborn screening (NBS). The objective of this study is to describe the clinical, biochemical, and molecular characteristics of Filipino patients diagnosed with HT1 through the expansion of the Philippine NBS program in 2014. There were a total of 16 patients with confirmed HT1 from then until September 2022. Clinical and biochemical data during confirmation and initial evaluation, as well as molecular data, were obtained from the patients' medical records. The cohort included children between the ages of 18 and 54 months at the time of data collection. The mean age at treatment initiation was 26.8 days. The mean succinylacetone level from dried blood spot sampling using tandem mass spectrometry (MS) was 11.1 µmol/L. Biochemical confirmatory tests via plasma amino acid analysis showed mean levels of tyrosine, phenylalanine, and methionine of 506.1 µmol/L, 111.5 µmol/L, and 125.4 µmol/L, respectively. Upon urine organic acid (UOA) analysis, succinylacetone was detected in all except for one patient, who was managed prior to UOA analysis. The most common clinical characteristics were abnormal clotting times (62.5%), elevated alpha fetoprotein (37.5%), anemia (31.3%), and metabolic acidosis (31.3%). The most common genotype was homozygous c.122T>C p.Leu41Pro in 64.3% of patients. The allelic frequency of this pathogenic variant is 71.4%. The inclusion of HT1 in the Philippine NBS program allowed early diagnosis and management of HT1 patients.

Diagnostic Transitions of Cystic Fibrosis and CRMS Compared After 12 Years of Newborn Screening in California.

OBJECTIVE: To compare the long-term diagnostic transitions for cystic fibrosis (CF) and cystic fibrosis transmembrane conductance regulator (CFTR)- related metabolic syndrome (CRMS) side-by-side since the onset of newborn screening in California. STUDY DESIGN: Using real-world data, we conducted a retrospective cohort study to compare long-term observations of CRMS and CF in California and the diagnostic transitions from one to the other using clinical and diagnostic metrics. The California Genetic Disease Screening Program newborn screening for CF employs an immunoreactive trypsinogen tier-1 laboratory test, followed by molecular testing. This approach captures CF and CRMS, a diagnosis of "watchful waiting" among infants at risk for CF but with signs and symptoms that may emerge outside the screening window. Waiting entails periodic diagnostic reviews that can continue for many years; the California Genetic Disease Screening Program routinely conducts 5 years of follow-up for each child identified with a disorder. We used categorial logistic regression to compare the transitions with CRMS. RESULTS: After screening 5 944 700 newborns between July 2007 and July 2019, 694 CF cases and 1257 CRMS cases were identified. Of the 1257 CRMS cases, 66 (5.2%, 95% CI 3.9%-6.4%) transitioned from CRMS to CF (CRMS2CF) at a mean age of 3.3 years (median = 2.9 years). CRMS2CF cases had longer follow-up periods and were more likely later to develop positive sweat chloride and fecal elastase test results after 6 months of life than other CRMS cases. CONCLUSIONS: These results suggest that children who have a CRMS2CF transition are more likely to develop positive biochemical markers than other patients who are diagnosed as CRMS and have few clinical indications during the first 5 years of follow-up.

Comparison between Liquid and Tablet Formulations in the Treatment of Congenital Hypothyroidism up to 3 Years of Age: The First Italian Study.

Background/Objectives: Levothyroxine (L-T4) is available for use in congenital hypothyroidism (CH) in three formulations: tablets, drops, and oral solution. This study aims to compare the efficacy and safety of all three L-T4 formulations. Methods: We enrolled 63 children born between January 2019 and April 2023 in the Emilia-Romagna Region (Italy) and diagnosed with CH by newborn screening. They were divided according to the L-T4 formulation used: drops (Group D), oral solution (Group S), and tablets (Group T). Clinical and laboratory data were collected up to 3 years after the start of replacement therapy. Results: Serum-free thyroxine (sFT4) and thyroid stimulating hormone (sTSH) normalization occurred within the first month of treatment in most patients of all groups. No negative effects on growth and cognitive development were observed. At 7-15 days we found higher median sTSH levels (p = 0.031) and a greater percentage of patients with sTSH > 5 µU/mL (p = 0.011) in Group S than in Group T, but comparable sFT4 levels. At 12 months, a greater percentage of patients of Group D showed sFT4 values below the normal range than Group S (p = 0.011) and Group T (p = 0.038); Conclusions: Overall, our study reported an equal efficacy of the L-T4 oral solution compared to drops and tablets in CH treatment. A larger series of patients and a long-term follow-up are needed.

Incorporating Next-Generation Sequencing as a Second-Tier Test for Primary Carnitine Deficiency.

BACKGROUND: Newborn screening (NBS) for primary carnitine deficiency (PCD) has poor performance. This study aimed to evaluate the feasibility of incorporating next-generation sequencing (NGS) as a second-tier PCD test. METHODS: Between March and December 2020, 60,070 newborns were screened for inherited metabolic disorders. Newborns with free carnitine (C0) levels below 8.5 µmol/L were selected for second-tier genetic testing. RESULTS: In total, 130 (0.22%) newborns with low C0 levels underwent second-tier genetic testing, 87 (66.92%) had positive genetic testing results, and 30 (23.08%) carried pathogenic variants of the SLC22A5 gene. Six newborns were diagnosed with PCD. The incidence of PCD was approximately 1 in 1:10,012 newborns. The PPV reached 20% after combining with second-tier NGS. Of the eight variants identified in patients with PCD, the three most common variants were c.760C>T (p.Arg254*), c.51C>G (p.Phe17Leu), and c.1400C>G (p.Ser467Cys). The C0 levels of patients with PCD were significantly lower than those of PCD carriers (p = 0.0026) and PCD-negative individuals (p = 0.0005). CONCLUSIONS: Our results showed that the PPV reached 20% after combining with second-tier NGS. The MS/MS-based NBS and second-tier NGS combination can effectively reduce the false-positive rate and detect PCD in patients.

Perspectives and Insights Into Phenylketonuria: Patient Narratives About the Early Years Following Newborn Screening.

Newborn screening for Phenylketonuria (PKU) began in 1963, and since then knowledge and treatment recommendations have evolved. In the decades following newborn screening for PKU, individual and family experiences varied widely. We present narratives by people living with PKU during these years, including individuals actively following in PKU clinic and those who have been out of PKU clinic for many years. These stories describe different individual experiences, including diet discontinuation in childhood, changing treatment guidelines, and new treatments that have become available.

Perspectives and Insights Into Phenylketonuria: Provider Narratives About the Early Years Following Newborn Screening.

The understanding of phenylketonuria (PKU), guidelines, and treatment landscape have evolved dramatically over the decades since newborn screen implementation. We capture this rich history from the stories and experiences of a multidisciplinary provider team from Boston Children's Hospital's PKU Clinic, who treated PKU from the early years of newborn screening and who worked together for over 40 years.

Phenylketonuria - newborn screening as a health protection in society.

OBJECTIVE: Aim: Phenylketonuria is the most prevalent inherited metabolic disorder. Early detection and prompt treatment can prevent serious neurological consequences. This has become possible thanks to the implementation of newborn screening programmes. The objective of this review is to provide readers with a comprehensive understanding of the phenylketonuria and the role that neonatal screening plays in the protection of public health. PATIENTS AND METHODS: Materials and Methods: A review of the literature was conducted using the PubMed database, with the search period encompassing the most recently published scientific sources. Analysis of the literature. This article presents phenylketonuria as an example of an inherited metabolic disorder, outlines the treatment options, and discusses the potential implications of hyperphenylalaninemia. Furthermore, it also delineates the various aspects of health that are influenced by newborn screening. CONCLUSION: Conclusions: Phenylketonuria represents a significant health problem in the population. The development of screening tests has transformed healthcare, including improvements in quality of life, prognosis, and reductions in the number of comorbidities in patients. It is essential to disseminate knowledge among the society about the importance of newborn screening tests in order to enhance awareness and prevent refusal to participate.

Lysosomal storage diseases.

Lysosomal storage disorders (LSDs) are a group of inherited metabolic diseases caused by dysfunction of the lysosomal system, with subsequent progressive accumulation of macromolecules, activation of inflammatory response, and cell death. Neurologic damage is almost always present, and it is usually degenerative. White matter (WM) involvement may be primary or secondary. Diseases with primary WM involvement are leukodystrophies, demyelinating (Krabbe disease and metachromatic leukodystrophy), and hypomyelinating leukodystrophies (free sialic acid storage disease, fucosidosis, and mucolipidosis type IV). LSDs with secondary WM involvement are classified as leukoencephalopathies and include gangliosidosis, mucopolysaccharidosis (MPS), ceroid neuronal lipofuscinosis, multiple sulfatase deficiency, alpha-mannosidosis, Pompe disease, and Fabry disease. Neurologic manifestations may overlap among LSDs and include developmental delays, motor, cognitive and speech impairments, seizures, visual failure, ataxia, and extrapyramidal signs. Most of LSDs are typically present in early or late infancy, but juvenile and adult forms also exist and are associated with predominantly neuropsychiatric and behavioral symptoms. The outcome of these disorders is generally poor and specific treatments (enzyme replacement therapy, hematopoietic stem cell transplantation, or gene therapy) are only available in a small number of them.

Clinical Course of a Child With Cystic Fibrosis and the Genotype F508del/CFTRdup1_11: A Case Report.

Cystic fibrosis (CF) is a hereditary disease with great genetic complexity as not all mutations are disease-causing and genotype doesn't always predict phenotype. This case involves a child with CF and genotype F508del/CFTRdup1_11. The CFTRdup1_11 duplication was not reported previously, and genetic counseling was based on reports describing the clinical course of people carrying smaller duplications of the same area combined with F508del. The predicted clinical presentation was CF with pancreatic insufficiency. However, the case presented has so far shown no clinical symptoms and has borderline sweat chloride concentrations.

Optimizing clinical outcomes: The journey of twins with CRIM-negative infantile-onset Pompe disease on high-dose enzyme replacement therapy and immunomodulation.

Infantile-onset Pompe disease (IOPD) is caused by a deficiency in the enzyme acid alpha-glucosidase (GAA). It is characterized by severe and progressive hypertrophic cardiomyopathy and muscle weakness with death in the first 2 years of life if left untreated. Enzyme replacement therapy (ERT) with alglucosidase-alfa is lifesaving, but its effectiveness is influenced by the patient's cross-reactive immunologic material (CRIM) status, dose of ERT, and the development of high antibody titers, which can reduce the therapy's efficacy. The inability of CRIM-negative IOPD patients to produce native GAA exposes them to a high risk of development of anti-rhGAA IgG antibody titers, leading to treatment failure. We present the case of CRIM-negative dizygotic twins treated with high-dose alglucosidase-alfa (40 mg/kg/week), initiated at 28 days (Twin A) and 44 days (Twin B). Both twins received immune tolerance induction (ITI) with rituximab, methotrexate, and IVIG to mitigate antibody response. Initial evaluations revealed elevated left ventricular mass index (LVMI) and elevated biomarkers (urine glucose tetrasaccharide (Glc4), creatine kinase (CK), and aspartate aminotransferase (AST)) in both twins. Following treatment, cardiac function and biomarkers normalized within several months, with a slight delay in Twin B compared to Twin A, likely attributed to the later initiation of ERT. Both twins safely tolerated ITI, achieving immune tolerance with low antibody titers. At 28 months, the twins transitioned to avalglucosidase-alfa (40 mg/kg every other week (EOW)), which was well tolerated without an increase in antibody titers. At 39 months, both twins exhibited normal cardiac function, LVMI, and biomarkers. Motor skills continued to improve, though some kinematic concerns persisted. These cases underscore the importance of early, high-dose ERT combined with ITI in managing CRIM-negative IOPD. While transitioning to avalglucosidase-alfa at 40 mg/kg/EOW was beneficial and well-tolerated in our patients, further studies are needed to confirm its long-term efficacy compared to the high-dose weekly 40 mg/kg alglucosidase-alfa.

Outcome of Expanded Newborn Screening Among 194 000 Neonates at Northeast México.

Objectives. To describe the results of a 16-year experience of a state-coverage expanded newborn screening program (NBSP) in Northeast México. Methods. Between 2002 and 2017, dried blood spots of newborns were screened for congenital hypothyroidism (CH), congenital adrenal hyperplasia (CAH), biotinidase deficiency, galactosemia, cystic fibrosis, and glucose-6-phosphate dehydrogenase (G6PD) deficiency via immunofluorescence and amino and fatty acid disorders and organic acidemias using tandem mass spectrometry. Frequency rates were determined. Results. Overall, 192 487 samples were processed; 99.4% had negative results, and 598 were diagnosed. The frequency was 3.01/1000 newborns. G6PD deficiency, CH, amino acidemia, organic acidemia, cystic fibrosis, CAH, fatty acid oxidation disorder, galactosemia, and biotinidase deficiency cases were 1:773, 1:962, 1:4277, 1:4476, 1:11,322, 1:10,693, 1:10,693, 1:38,497, and 1:64,162, respectively. Conclusion. Using different technologies in NBSP increased the number of conditions detected, facilitating infant morbidity and mortality prevention. The frequency of disorders depends on the population's genetic background and diagnostic capacity.

Total Cholesterol Determination Accuracy in Dried Blood Spots.

Background Detecting total cholesterol in dried blood spots could aid in identifying individuals with a high likelihood of familial hypercholesterolemia and could be used as a screening measure. This study aims to assess the diagnostic accuracy of dried blood spots on Whatman 903 paper cards using a manual enzymatic technique. Methods: A total of 394 samples were collected as serum and dried blood spots were compared. Cholesterol was determined in serum using the automated reference method, while cholesterol on paper was measured using a manual enzymatic method. Within- and between-day diagnostic variability were analyzed. The correlation between both methods was assessed using Passing-Bablok regression and Bland-Altman plot. Internal validation of our correlation formula was performed on 149 samples, along with external validation of the formula proposed by Corso et al. Results: The within- and between-day coefficient of variation was found to be lower than 10.14% and 14.09%, respectively. Passing-Bablok regression indicated a precision of 0.803 and an accuracy of 0.96. Internal validation precision was measured at 0.716. The resulting positive and negative predicted values were 0.77 and 0.92, respectively, vs. 0.46 and 0.96 from the external formula. Conclusions: Total cholesterol analysis in dried blood spots demonstrates high precision and reproducibility. This method reliably enables the incorporation of this biological marker into neonatal screening for familial hypercholesterolemia detection.