Sensitive and Robust LC-MS/MS Assay to Quantify 25-Hydroxyvitamin D in Leftover Protein Extract from Dried Blood Spots

Neonatal dried blood spots (DBS) provide a remarkable resource for biobanks. These microsamples can provide information related to the genetic correlates of disease and can be used to quantify a range of analytes, such as proteins and small molecules. However, after routine neonatal screening, the amount of DBS sample available is limited. To optimize the use of these samples, there is a need for sensitive assays which are integrated across different analytic platforms. For example, after DNA extraction, protein extracts are available for additional analyses. We describe a sensitive and robust LC-MS/MS method for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 optimized for leftover protein extracts from DBS, which has excellent recovery, precision, and accuracy.

Devastating Salt-wasting Crisis in a Four-month-old Male Child with Congenital Adrenal Hyperplasia, Highlighting the Essence of Neonatal Screening

Classic Congenital Adrenal Hyperplasia in males usually presents late until before puberty, but rarely at birth. In cases of undiagnosed CAH, severe deficiency of cortisol may lead to life threatening situation in an infant with acute salt losing crisis. Screening for CAH at birth can lead to better outcomes.

PECULIARITIES OF EXPANDED NEWBORN SCREENING FOR THE INHERITED METABOLIC DISORDERS IN PREMATURE NEWBORNS

Occurrence of the premature birth in Ukraine is about 6%. Premature newborns are the highest risk group of developing chronic pathology of the nervous system, sensory organs, and respiratory system, causing neonatal mortality and disability; the latter is 22 times higher in premature newborns than in full-term ones. Besides, there is a large group of rare metabolic disorders that significantly disrupt the adaptation and nursing of newborns with signs of morpho-functional and enzymes immaturity. The efficacy of medical care of premature newborns to a great extent relates to prompt diagnosis as common somatic, as rare metabolic disorders.In view of the absence of specific symptoms, it is almost impossible to establish a diagnosis of inherited metabolic disordersduring the clinical examination of a neonate.Expanded newborn screening (ENBS) for inborn errors of metabolism (IEMs) proved to be an effective tool to single out newborns with genetic deficiency of certain metabolic enzymes.The practical experience of performingENBS indicates a problematic issue is the interpretation of results for preterm babies.This article is discussed the key factors affecting the predictive value of ENBS results in premature newborns, like peculiarities of blood sampling based on time intervals from delivery for certain nosologies, repeated sample taking for lab examination considering the basic principles of 2-nd Edition of CLSI "Guideline "Newborn Screening for Preterm, Low Birth Weight, and Sick Newborns, 2019". The CLSI Guideline presents the consensus solutions of a global team of neonatologists, metabolic paediatricians and medical geneticists regarding the accuracy, reliability and timing of laboratory determinations of IEM markers in the blood, as well as a set of factors to consider interpreting ENBS results for premature, low birth weight and newborns with perinatal pathology.The current procedure for neonatal screening for premature babies in Ukraine should be updated to modern requirements of the relevant clinical recommendations of world-recognized medical institutions, including CLSI. The critical issue in improving the quality and reliability of neonatal screening for preterm babies is multiple (repeated) blood sampling for laboratory determination of levels of biochemical IEM markers.A properly established and well- functioning system of expanded neonatal screening proved to be a highly effective tool for reducing early infant mortality and disability associated with inherited metabolic diseases.

Web System for Spatial Processing and Analysis: An Application in a Neonatal Screening Program in Southern Brazil

Background Spatial data analysis refers to the process of finding patterns, detecting anomalies, or testing hypotheses and theories by observing phenomena associated with a specific geographic area or location. The literature in the area presents different studies that seek to understand its phenomena through spatial analysis techniques and methods. However, these studies have several problems, such as the frequent use of only one type of analysis, area or punctual. Furthermore, the studies do not formally describe the process of treatment and organization applied to the data to replicate the spatial analyzes in other research areas. Thus, this work proposes a web system for generating, organizing, and processing data compatible with geographic information systems to construct spatial analysis of area and points. Methods The proposed method was developed with the JavaScript programming language and structured in four sequential steps: data acquisition, processing and organization, data validation, and spatial analysis. Data from three diseases (cystic fibrosis, congenital adrenal hyperplasia and hemoglobinopathies) from a neonatal screening program in southern Brazil were used to validate the proposed method and construct the spatial analyses. The choropleth mapping and kernel density estimation methods were used to build the analyses. Results The results obtained made it possible to georeference the data, validate it to its area of study, associate it with its micro and mesoregions, and cross it with public databases. In addition, the results enabled the construction of scientific maps of area and points to visualize the primary evidence from the spatial distribution of disease cases. Conclusions The developed method showed high replication potential for other study contexts. Also, it proved to be relevant in the context of spatial analysis, enabling speed in processing, data organization and, consequently, in the construction of significant results that can be used in public policies that directly impact people's quality of life and health challenges.

Factors Influencing Change in MCV and Age at Transplantation in the Belgian Sickle Cell Disease Registry

The Belgian sickle cell disease registry (BCR) was initiated in 2008 and aims to evaluate mortality, morbidity as well as clinicals practices in participating centers. The current analysis focuses on criteria influencing age at transplantation (HSCT) and on the management of Hydroxyurea (HU) therapy across centers. The methodology of the registry has already been published (Le PQ et al., Pediatric Blood and Cancer, 2015) . Data are recorded prospectively from neonatal screening or first contact until last annual follow-up (FU) or death. The data collected included diagnosis, demography, treatment and outcome data as well as a minimal set of biological values. Data were extracted from the database in May 2021. There are 1029 patients registered by 14 different centers (2 centers exclusively treating adult patients). The median FU is 9 y (1-53 y). Median age at last FU is 13 y (0-61 y). 890 patients (86,5%) have a severe phenotype (SS or Sβ°) and 52% are female. Among them, 561 (55%) are born in Belgium of whom 379 (68%) are diagnosed by neonatal screening. In the absence of neonatal screening, median age at diagnosis is 1 year (range 0-18). 131 patients have been transplanted (126 successfully), 68 HSCT were performed before 2005. At last FU, 646 patients (76%) received at least 1 disease-modifying treatment (DMT) : 598 patients receive HU, 65 are chronically transfused, 8 participate in a study with crizanlizumab. The prescribed HU dose is known for 572 patients. 179 patients (31.3%) receive less than 20 mg/kg/day, 217 (37.9%) less than 25 mg/kg/day, 148 (25.9%) less than 30 mg/kg/day and 28 (4.9%) were prescribed more than 30 mg/kg/day. The majority of HSCT were performed in two centers (68 and 59, respectively). Median age at HSCT was significantly different between both centers (8y (2-15) versus 5y (0-19); p=0.002) (figure 1). Variables associated with a lower age at HSCT are detailed in table 1. In a linear multivariate regression model, birth in Belgium (p=0.002), no treatment with HU (p=0.009) and shorter duration of FU (p<0.001) but not the center are independent factors correlated with younger age at transplantation. This linear model explains 90.60% of the variance (adjusted R²) of age at HSCT. Among not transplanted patients, the proportion of those receiving HU is different between centers (50% to 91%; p=0.050). The median age at which HU was initiated was also significantly different between centers (4y to 21y; p<0.001) as was the management of HU treatment in a multiple comparison model measured by ΔMCV (difference in MCV before start of HU versus at last FU). In a linear univariate regression model, other variables are significant predictors for the variance of ΔMCV (table 2). In the linear multivariate regression model, the variance of ΔMCV between centers is controlled by the duration of FU (p<0.001), neonatal screening (p=0.046), HU dose between 25-30mg/kg/day (p<0.001), all resulting in a higher ΔMCV, while patients not born in Belgium (p=0.033) have a lower ΔMCV. Age at diagnosis, severity of the disease (assessed by the number of VOC/FU year) and HU dose <20 mg/kg/day are not correlated with the variance in ΔMCV. Twenty-seven (2.6%) patients died which accounted for a mortality rate of 0.24/100 patients-years (PY) which increases significantly with age (0.18/100PY <18 years, 0.35/100PY 18-40 years and 1.43/100PY >40 years; p=0.001). Conclusions: BSR has an excellent registration activity from participating centers and represents a reliable tool to evaluate the Belgian SCD population. Mortality remains low with a significant trend to increase with age. Regarding treatment practices, the age at start of HU is significantly different between centers as the approach to further HU treatment, evaluated by ΔMCV. A higher dose of HU resulted in a higher ΔMCV. However, the policy to increase HU to maximal tolerated dose seems not implemented in most centers, as 2/3 of the patients are prescribed less than 25 mg/kg/day. Being born in Belgium and no treatment with HU are associated with younger age at HSCT. Nevertheless since 2005, almost all patients were treated with HU prior HSCT, reflecting the wider implementation of HU in SCD patients living in Belgium. Figure 1 Figure 1. Disclosures Benghiat: Novartis: Consultancy; BMS: Consultancy. Labarque: Bayer: Consultancy; Sobi: Consultancy; NovoNordisk: Consultancy; Octapharma: Consultancy; Novartis: Consultancy.

MODELS AND METHODS OF INFORMATION TECHNOLOGY OF ADVANCED NEONOTAL SCREENING

The paper considers the problems of informational implementation of neonatal screening of newborns in order to improve the overall picture of the nation's health and prevent the development of hereditary diseases. The methodology for solving the problems of complete neonatal screening is based on the methods and mathematical apparatus of discrete mathematics, web technologies, data warehouses, and data mining methods. An information model of the dynamic processes of neonatal screening is proposed, based on the specific processing of data presented by a tuple, which contains coherent sequential processes for obtaining the results of tests for blood analysis of newborns, conducting genetic studies and determining pathologies and deviations from an expanded list (currently up to 44 indicators for the purpose of exiting for more than 60). The block diagram of information support of information technology in the decision support system for carrying out neonatal screening of hereditary metabolic diseases is presented. On the basis of LLC «CDC «PHARMBIOTEST», the research of the algorithm for performing sequential procedures of neonatal screening was carried out. The described algorithm of actions has been tested and fully tested for the continuity of information flows, the stability of the information model graph. As a result of the research, the sufficiency and completeness of the chronological indicators of the processing of information flows have been proved. The criteria for confirming the authenticity of methods for obtaining a diagnosis have been developed.

Prevalence of the five newborn screening tests

Neonatal screening is essential for child health and has the following purposes: (1) pulse oximetry screening to evaluate congenital heart diseases; (2) red reflex examination to investigate eye diseases; (3) newborn hearing screening test to evaluate congenital hearing diseases; (4) tongue test to evaluate the lingual frenulum and identify communication and feeding problems; (5) the Guthrie test to screen for metabolic diseases. This study investigated the prevalence of the five neonatal screening tests and its associated institutional and socio-cultural factors using a cross-sectional study with 415 mother and baby binomials from public maternity hospitals in Natal, RN, Brazil in 2019. Pearson’s chi-squared, Mann-Whitney and Poisson regression tests were used, with a significance of p ≤ 0.05 and a 95% confidence interval. The sample loss was 71 mothers (17%). The prevalence in the first week and at the end of 28 days was 93% and 99.5% (pulse oximetry screening), 60% and 97.6% (red reflex examination), 71.9% and 93.6% (Guthrie test), 35.5% and 68.2% (hearing screening test), and 19% and 48.9% (tongue test). Only 152 newborns (36.6%) underwent all five tests. The performance of the tests was associated in the final model (p ≤ 0.05) with the residence of the mothers in the state capital (PR = 1.36; 95% CI = 1.18–1.56) and the provision of guidance for mothers about the five tests in maternity hospitals (PR = 1.30; 95% CI = 1.08–1.67). None of the tests met full coverage, and regional inequities were identified indicating the need to restructure the institutions, training and qualification procedures to improve of the work processes and longitudinal care.

The Assessment of Risk Factors for Development of Disability in Children with Congenital Hypothyroidism in Uzbekistan within a Neonatal Screening

The aim of this study was to detect the most significant risk factors leading to disability in children with congenital hypothyroidism (CH) in the autonomous Republic of Karakalpakstan (RK) during neonatal screening (NS). Methods and Results: We used data of patients with CH registered within NS in the RK in 1998-2019 by the Center for Screening of Mother and Child. To predict and calculate the most significant risk factors for disability in children with CH, we used the method of normalizing intensive indicators by E. Shigan, based on the Bayes theorem. The study recruited 111 patients with CH aged from 2 months to 20 years. Among the patients, there were 79(71.2%) girls and 32(28.8%) boys. Additionally, 34(30.6%) children with CH had been disabled since childhood. The lack of compensation after the start of treatment had the highest and most significant degree of disability risk (RR=6.39, 95% CI: 7.4-1.2). Among patients diagnosed outside of screening, disability developed 4.1 times more often than with the results of NS (RR=4.0, 95% CI: 1.1-10.6). In CH patients diagnosed outside of screening, “absence of reagents” was a significant factor increasing the risk of disability by 6.1 times (RR=6.1, 95% CI: 1.8-11.2). Such risk factors as “home delivery” and “parental refusal of the primary test” increased the risk of disability by 3.4 times (RR=3.4, 95% CI: 2.5-8.4) and 1.6 times (RR=2.4, 95% CI: 2.93-7.12), respectively. The possible errors or false-negative answers in the “normal” secondary test and the “normal” primary test increased the risk of disability by 3.3 times (RR=4.0, 95% CI: 3.2-10.7) and 2.4 times (RR=2.42, 95% CI: 2.93-7.12), respectively. Factors such as the “late response to retesting” (RR=0.82 95% CI: 0.65-0.54), “late awareness on the part of the medical staff” (RR=0.29, 95% CI: 0.27- 0.08), and "parental refusal of treatment" (RR=1.03, 95% CI: 0.81-0.84) showed less significance in patients' disability. The “starting treatment after 1 month” factor was 4.2 times more likely to result in disability than “starting treatment before 1 month” (RR=4.2, 95% CI: 4.5 -1.1). Cancellation of levothyroxine by parents for children up to 3 years of age and cancellation of treatment by parents after 3 years more likely resulted in disability by 1.4 times (RR=1.43, 95% CI: 1.4 -2.01) and 3.3 times (RR=3.33, 95% CI: 3.3-10.9), respectively. Conclusion: the most significant risk factors for the development of disability in children with CH in the RK were (in descending order): no compensation after starting treatment, no reagents for screening, starting treatment after 1 year, diagnostics outside of screening, cancellation of L-T4 by parents before and after age 3 years, false-negative secondary TSH test, false-negative primary test, parents refusing the primary test, and childbirth at home.