Newborn Specimens Research Articles

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

Capture of a new-born shortfin mako shark Isurus oxyrinchus (Lamniformes: Lamnidae), with updated records from the Turkish marine waters

The study reports on the capture of a new-born specimen of shortfin mako shark Isurus oxyrinchus Rafinesque, 1810 off Karantina islet coast, Urla, Izmir (north Aegean Sea). This specimen measured 761 mm in total length and weighed 2900 g. The historical and recent captures of the species in the area shows the presence of new-born, juvenile and adult males and females. The distribution of these specimens suggests the potential occurrence of a nursery ground in the same area. To preserve the extirpation of the species throughout the area where it finds favourable environmental parameters to live, a management plan should be conducted to ensure the establishment of a viable population in the Turkish marine waters

Management of refined and personalized newborn blood specimen collection

BackgroundIatrogenic blood loss is an important cause of neonatal anemia. In this study, a spreadsheet tool was developed to reduce blood collection, providing a new idea for the prevention of iatrogenic blood loss in newborns. MethodsBased on hematocrit, minimum test volume and dead volume, a new tool was to calculate the minimum blood collection volume and the number of containers required for the test portfolio. We collected data from October 2022 to October 2023 from Xiamen Maternal and Child Health Hospital for analysis and validation. ResultsDuring this year, there were 16,434 patients and 13,696 plasma/serological samples in the neonatology department. Among them, there were 8 test combinations of greater than 1%, and 9490 samples in total. According to the hospital manual, the recommended amount of blood collection is 27,534 ml and 9490 containers. Through the analysis of this tool, total blood collection was 8864.77 ml, marked qnantity of upward containers (closest level to the calculated blood collection volume) was 10301 ml, and the amount of containers was 8835, which decreased by 67.8%, 62.58% and 6.9% respectively. Besides, if the hematocrit information cannot be obtained in advance and the high hematocrit is calculated as 0.8, the recommended amount of blood collection is 14334.3 ml, and the marked amount of the upward container markering is 17340 ml, decreasing by 47.9% and 37.02% respectively. ConclusionWe have developed an auxiliary tool that can manage neonatal blood specimen collection in a fine and personalized way and can be applied among different laboratory instruments by parameters modification.

Biological Testing and Interpretation of Laboratory Results Associated with Detecting Newborns with Substance Exposure.

Substance use during pregnancy is common, as is biological testing that is intended to help identify prenatal exposures. However, there is no standardized requirement for biological testing with either maternal or newborn specimens, nor is there standardization related to when testing occurs, how frequently testing occurs, what specimen(s) to test, what substances to test for, or how to perform testing. We review common specimen types tested to detect maternal and newborn substance exposure with a focus on urine, meconium, and umbilical cord tissue. We also review common analytical methods used to perform testing, including immunoassay, and mass spectrometry platforms. Considerations regarding the utilization of testing relative to the purpose of testing, the drug analyte(s) of interest, the specific testing employed, and the interpretation of results are emphasized to help guide decisions about clinical utilization of testing. We also highlight specific examples of unexpected results that can be used to guide interpretation and appropriate next steps. There are strengths and limitations associated with all approaches to detecting substance exposure in pregnant persons as well as biological testing to evaluate a newborn with possible substance exposure. Standardization is needed to better inform decisions surrounding evaluation of substance exposures in pregnant people and newborns. If biological sampling is pursued, testing options and results must be reviewed in clinical context, acknowledging that false-positive and -negative results can and do occur.

Cardiac Fibrosis and Innervation State in Uncorrected and Corrected Transposition of the Great Arteries: A Postmortem Histological Analysis and Systematic Review

In the transposition of the great arteries (TGA), alterations in hemodynamics and oxygen saturation could result in fibrotic remodeling, but histological studies are scarce. We aimed to investigate fibrosis and innervation state in the full spectrum of TGA and correlate findings to clinical literature. Twenty-two human postmortem TGA hearts, including TGA without surgical correction (n = 8), after Mustard/Senning (n = 6), and arterial switch operation (ASO, n = 8), were studied. In newborn uncorrected TGA specimens (1 day-1.5 months), significantly more interstitial fibrosis (8.6% ± 3.0) was observed compared to control hearts (5.4% ± 0.8, p = 0.016). After the Mustard/Senning procedure, the amount of interstitial fibrosis was significantly higher (19.8% ± 5.1, p = 0.002), remarkably more in the subpulmonary left ventricle (LV) than in the systemic right ventricle (RV). In TGA-ASO, an increased amount of fibrosis was found in one adult specimen. The amount of innervation was diminished from 3 days after ASO (0.034% ± 0.017) compared to uncorrected TGA (0.082% ± 0.026, p = 0.036). In conclusion, in these selected postmortem TGA specimens, diffuse interstitial fibrosis was already present in newborn hearts, suggesting that altered oxygen saturations may already impact myocardial structure in the fetal phase. TGA-Mustard/Senning specimens showed diffuse myocardial fibrosis in the systemic RV and, remarkably, in the LV. Post-ASO, decreased uptake of nerve staining was observed, implicating (partial) myocardial denervation after ASO.

Seno cavernoso: anatomía, histología y terminología.

Although the cavernous sinus (CS) has been studied since 1695, its anatomy and name are still under discussion. Anatomy and histology of 40 CS from human cadavers were studied, included both from a newborn specimen. Two walls limit the CS, an inferior medial one composed only of the dura's outer layer and a superior lateral one consisting of both dura's layers. Sinusoidal veins pass through the lateral wall of the CS as a transition between venous tributaries and the CS. An endothelial layer covers the inner surface of the CS and the outer surface of the internal carotid artery. The space within the CS shows trabeculae, which are rarer in adults compared to the newborn. The loss of trabeculae in the CS may be a natural process along with life. In conclusion, the CS is a real sinus, and the term "cavernous sinus" is appropriately applied.

Abstract 11554: Biochemical and Molecular Newborn Screening for Familial Hypercholesterolemia

Introduction: Familial Hypercholesterolemia (FH) is a common genetic disorder resulting in elevated low-density lipoprotein cholesterol (LDL-C) causing premature atherosclerosis. Despite guidelines, universal screening rates remain low. Newborn screening could increase FH diagnostic rates. Hypothesis: Biochemical newborn screening for LDL-C, total cholesterol (TC), and apolipoprotein B (apoB) followed by selective molecular testing identifies pathogenic and likely pathogenic variants causing FH. Methods: De-identified, residual newborn screening bloodspots collected for routine screening at 24-48 hours of life were analyzed for LDL-C, TC, and apoB. Values are expressed as multiples of median (MoM). Mahalanobis distance, measuring how far MoM values differed positively from 1, was computed. Samples with the most extreme Mahalanobis distance were selected for molecular testing (panel includes LDLR , APOB , PCSK9 , LDLRAP1 , APOE , STAP1 , LIPA , ABCG5 , and ABCG8 ). Results: Distributions of apoB, TC, and LDL-C from 5,248 samples are shown below (figure). ApoB followed a Gamma distribution; TC and LDL-C followed non-normal but symmetric distributions. ApoB and LDL-C accounted for 93% of variance among biomarkers. Of the first 2500 samples, 192 samples were selected for molecular testing; a pathogenic variant for monogenic FH was detected in 1 sample and risk factors for polygenic FH in 4 samples (table below). Conclusions: Pathogenic or likely pathogenic variants for FH were identified with an incidence of 1 in 500 in the first 2500 newborn specimens tested. Further study will determine if this two-tiered screening strategy adequately detects FH in newborns.

Trends in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Seroprevalence in Massachusetts Estimated from Newborn Screening Specimens.

BackgroundEstimating the cumulative incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for setting public health policies. We leveraged deidentified Massachusetts newborn screening specimens as an accessible, retrospective source of maternal antibodies for estimating statewide seroprevalence in a nontest-seeking population.MethodsWe analyzed 72 117 newborn specimens collected from November 2019 through December 2020, representing 337 towns and cities across Massachusetts. Seroprevalence was estimated for the Massachusetts population after correcting for imperfect test specificity and nonrepresentative sampling using Bayesian multilevel regression and poststratification.ResultsStatewide seroprevalence was estimated to be 0.03% (90% credible interval [CI], 0.00–0.11) in November 2019 and rose to 1.47% (90% CI: 1.00–2.13) by May 2020, following sustained SARS-CoV-2 transmission in the spring. Seroprevalence plateaued from May onward, reaching 2.15% (90% CI: 1.56–2.98) in December 2020. Seroprevalence varied substantially by community and was particularly associated with community percent non-Hispanic Black (β = .024; 90% CI: 0.004–0.044); i.e., a 10% increase in community percent non-Hispanic Black was associated with 27% higher odds of seropositivity. Seroprevalence estimates had good concordance with reported case counts and wastewater surveillance for most of 2020, prior to the resurgence of transmission in winter.ConclusionsCumulative incidence of SARS-CoV-2 protective antibody in Massachusetts was low as of December 2020, indicating that a substantial fraction of the population was still susceptible. Maternal seroprevalence data from newborn screening can inform longitudinal trends and identify cities and towns at highest risk, particularly in settings where widespread diagnostic testing is unavailable.

The Dorsal Integument of the Southern Long-Nosed Armadillo Dasypus hybridus (Cingulata, Xenarthra), and a Possible Neural Crest Origin of the Osteoderms. Discussing Evolutive Consequences for Amniota

Osteoderms are present in most tetrapod lineages with considerable lineage-specific variation. It has been hypothesized that osteoderms are a case of “deep homology” in craniates, but the embryonic origin of osteoderms -and other related postcranial exoskeletal elements- is still under debate. Most authors support its mesodermal origin, while others suggest that osteoderms are derived from neural crest scleroblastic cells in sauropsids. The armadillos (Xenarthra, Cingulata) are the only living mammals and the only extant synapsids with osteoderms. Here, we aim to identify skeletogenic neural crest cells in the dorsal skin of armadillos in order to assess if osteoderms have a neuroectodermal origin in mammals, similar to what is observed in sauropsids. For this purpose, skin samples from fetuses and newborn specimens of Dasypus hybridus were processed and the embryological development of osteoderms was characterized using different immunohistochemical markers (HNK-1, PDGFR α, S-100, and C5). For the first time, we report cell populations that were reactive to skeletogenic neural crest markers, indicating an ectomesenchymal origin of the mammalian osteoderms. Our results demonstrate similar molecular expression for mammals as in sauropsids and, therefore, this strongly suggests that osteoderms in both groups would have a homologous embryonic origin.

Electronic informed consent information for residual newborn specimen research: findings from focus groups with diverse populations.

We developed a video and an app for obtaining consent about allowing newborn blood spots (NBS) to be used as biospecimen resources for biobanking. Newborn screening programs test for treatable diseases and leave residual biospecimens that can be used in future research activities. We conducted focus groups and interviews with three diverse communities to determine (a) how well the consent tools worked and (b) participant familiarity with NBS. Participants preferred the video and noted that they were unaware that NBS could be used for future research. Providing information about how biospecimens could be used was a key issue.

Improved Outcomes From Newborn Screening Timeliness and Transcutaneous Hyperbilirubinemia Screening

Our staff was routinely obtaining a heelstick specimen for both New York state (NYS) newborn screening as well as a serum bilirubin after midnight on the day of discharge. This was painful for the newborns and relatively time intensive for nursing. In addition, national recommendations, and eventually NYS regulations, for newborn screening specimen timing had decreased to Age 24–36 hours. Many of our specimens, particularly after cesarean births, were delayed. We wanted to implement a less painful, more efficient, hyperbilirubinemia screening and, by eliminating the need for a potential second heelstick, to improve our timing on newborn screening specimens. This project was granted an exemption by Montefiore Nyack Hospital’s Institutional Review Board. A grant was procured from our Ladies Auxiliary to purchase a transcutaneous bilimeter. Two units were trialed by nursing staff, results correlated, and one was chosen. Our policy and protocol were updated, and fields were created in the electronic medical record for documentation and review. Training was developed and administered both via the learning management system and live return demonstrations. NYS’s newborn screening report card statistics as well as number of serum bilirubin tests ordered were monitored. The timing of our newborn screening specimens improved immediately and by 3 months after the transcutaneous bilimeter go-live, we were ranked first in NYS. Serum bilirubins declined, which represented cost savings to the organization. There were several cases where the specimen was drawn just short of 24 hours, necessitating a repeat screen for the neonate. Individual employees were coached and reinforcement offered at staff meetings and huddles. Staff expressed great satisfaction with the new protocol, verbalizing that it was easy and could be used at anytime for a quick screen. We found an interdisciplinary transcutaneous hyperbilirubinemia screening protocol to be efficacious, efficient, and cost effective. Nurses are responsible for overseeing and, in many cases collecting, newborn blood specimens. Device selection by shared decision making increased adoption. As their patients’ advocates, nurses appreciate the improvement to patient comfort, ease of use, facilitation of better compliance with state regulations, and improved quality measures.

Validation and Implementation of a Highly Sensitive and Efficient Newborn Screening Assay for Mucopolysaccharidosis Type II.

Mucopolysaccharidosis Type II (MPS II), also known as Hunter syndrome, is a lysosomal storage disorder (LSD) caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). MPS II satisfies all criteria defined by the Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC) for inclusion in the Recommended Uniform Screening Panel (RUSP) for newborn screening, apart from the fact that only minimal prospective population screening data are available. This report details the analytical validation, clinical validation, and implementation of a fluorometric assay for measurement of IDS activity in newborn dried blood spot (DBS) specimens at the Missouri State Public Health Laboratory (MSPHL). The assay is performed in a microwell plate format requiring approximately 15 min of hands-on time per plate and an incubation time of two hours. The analytical validation of this assay included linearity, analytical sensitivity, precision, and carry-over testing. Clinical validation was completed using more than 5000 deidentified presumptive normal newborn DBS specimens as well as seven specimens from patients known to be affected with MPS II. Following validation, MSPHL began prospective screening using the IDS assay on 1 November 2018. In the first 18 months of screening (to 30 June 2020), 146,954 specimens were prospectively screened using the method. Two newborns were identified with severe Hunter syndrome and the assay had a presumptive positive rate of 0.022%.

Anatomical study of dorsalis pedis artery

Background: The dorsalis pedis artery is the dorsal artery of the foot. The knowledge of any variation in the course and distribution of the artery is clinically important and it is used to record peripheral pulsation. The aim of this study was to study origin, course, relation and branches of the artery. The length and diameter of surgically important branch were recorded.
 Methods: An observational cross-sectional study was conducted at dissection hall of anatomy department at Chitwan medical college. Thirty lower limbs from fifteen cadavers were dissected for the study. Photographs were taken at different site of dissection and on finding anomalies. Newborn specimens and Conical enlargements if present were excluded.
 Results: The diameter of the artery ranged from 3.0 to 4.6mm and its length ranged between 5.6cm to 8.4cm.The length of deep plantar artery was between 5.6cm to 8.4cm. Double lateral tarsal artery was found in (24)80% of the studied specimen and (6)20% were with single lateral tarsal artery. In 90% of the specimen medial tarsal artery originated as proximal and distal branch form medial aspect of dorsalis pedis. The arcuate artery emerged directly from dorsalis pedis artery in (100%) all studied specimen. The 2nd ,3rd and 4th metatarsal artery were placed on their corresponding intermetatarsal space.
 Conclusions: Awareness of anatomical variation of vasculature of foot is important for the vascular surgeons, angiographers and reconstruction surgeons. Dorsalis pedis artery is excellent for the revascularization since it is the largest artery below the ankle joint.

ANATOMICAL STUDY OF DORSALIS PEDIS ARTERY

Background: The dorsalis pedis artery is the dorsal artery of the foot. The knowledge of any variation in the course and distribution of the artery is clinically important and it is used to record peripheral pulsation. The aim of this study was to study origin, course, relation and branches of the artery. The length and diameter of surgically important branch were recorded.
 Methods: An observational cross-sectional study was conducted at dissection hall of anatomy department at Chitwan medical college. Thirty lower limbs from fifteen cadavers were dissected for the study. Photographs were taken at different site of dissection and on finding anomalies. Newborn specimens and Conical enlargements if present were excluded.
 Results: The diameter of the artery ranged from 3.0 to 4.6mm and its length ranged between 5.6cm to 8.4cm.The length of deep plantar artery was between 5.6cm to 8.4cm. Double lateral tarsal artery was found in (24)80% of the studied specimen and (6)20% were with single lateral tarsal artery. In 90% of the specimen medial tarsal artery originated as proximal and distal branch form medial aspect of dorsalis pedis. The arcuate artery emerged directly from dorsalis pedis artery in (100%) all studied specimen. The 2nd ,3rd and 4th metatarsal artery were placed on their corresponding intermetatarsal space.
 Conclusions: Awareness of anatomical variation of vasculature of foot is important for the vascular surgeons, angiographers and reconstruction surgeons. Dorsalis pedis artery is excellent for the revascularization since it is the largest artery below the ankle joint.

Biomechanical evaluation of femoral anteversion in developmental dysplasia of the hip and potential implications for closed reduction

BackgroundEarlier clinical reports have identified femoral anteversion as a factor associated with developmental dysplasia of the hip. This study investigates the biomechanical influence of femoral anteversion on severe dislocations and its effect on hip reduction using the Pavlik harness. MethodsA computational model of an infant lower-extremity, representing a ten-week old female was used to analyze the biomechanics of anteversion angles ranging from 30° to 70° when severe dislocation was being treated with the Pavlik harness. Specifically, the effects and relationships between muscle passive response and femoral anteversion angle were investigated over a range of hip abduction and external rotation. FindingsResults of this study suggest that increased femoral anteversion may decrease the success rate for treatment of high-grade developmental dysplasia of the hip when using the Pavlik harness. However, hip external rotation and decreased abduction in the harness may facilitate initial reduction in these cases. InterpretationThis biomechanical study may help explain why dissections of newborn specimen with developmental dysplasia of the hip have shown normal distribution of femoral anteversion in contrast to studies of patients requiring surgery where greater frequency of increased femoral anteversion has been reported. This study also suggests that adjusting the Pavlik harness to increase external hip rotation and decrease hip abduction may facilitate initial reduction for severe dislocations with increased femoral anteversion.

Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens

BackgroundDecreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens. MethodsGALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples. ResultsWe present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 μmol/l/h with a limit of detection of 0.066 μmol/l/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples. ConclusionsA fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from β-galactosidase.

Chronic maternal cortisol excess during late gestation leads to metabolic alterations in the newborn heart.

Our laboratory has previously shown in an ovine model of pregnancy that abnormal elevations in maternal cortisol during late gestation lead to increased fetal cardiac arrhythmias and mortality during peripartum. Furthermore, transcriptomic analysis of the fetal heart suggested alterations in TCA cycle intermediates and lipid metabolites in animals exposed to excess cortisol in utero. Therefore, we utilized a sheep model of pregnancy to determine how chronic increases in maternal cortisol alter maternal and fetal serum before birth and neonatal cardiac metabolites and lipids at term. Ewes were either infused with 1 mg·kg-1·day-1 of cortisol starting at gestational day 115 ( n = 9) or untreated ( n = 6). Serum was collected from the mother and fetus (gestational day 125), and hearts were collected following birth. Proton nuclear magnetic resonance (1H-NMR) spectroscopy was conducted to measure metabolic profiles of newborn heart specimens as well as fetal and maternal serum specimens. Mass spectrometry was conducted to measure lipid profiles of newborn heart specimens. We observed alterations in amino acid and TCA cycle metabolism as well as lipid and glycerophospholipid metabolism in newborn hearts after excess maternal cortisol in late gestation. In addition, we observed alterations in amino acid and TCA cycle metabolites in fetal but not in maternal serum during late gestation. These results suggest that fetal exposure to excess maternal cortisol alters placental and fetal metabolism before birth and limits normal cardiac metabolic maturation, which may contribute to increased risk of peripartum cardiac arrhythmias observed in these animals or later life cardiomyopathies.

Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS/MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders

Background and aimsIncreased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.MethodsThe assay was developed using liquid chromatography–tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.ResultsReliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.ConclusionsA liquid chromatography–tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening panel (‟Newborn screening 2020”; Newborn Screening Center, University Hospital Heidelberg).

Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach.

Methanobrevibacter smithii is the main human methanogen almost always found in the digestive tract of adults. Yet, the age at which M. smithii establishes itself as part of the developing intestinal microflora remains unknown. In order to gain insight into this, we developed a polyphasic approach, including microscopic observation by fluorescence in situ hybridisation, polymerase chain reaction (PCR) sequencing detection, identification and culture, to isolate and genotype M. smithii in one-day-old newborns' gastric juice specimens. In the presence of negative controls, 50/50 (100%) prospectively analysed newborn gastric juice specimens were PCR-positive for methanogens, all identified as M. smithii by sequencing. We succeeded in cultivating M. smithii in 35/50 (70%) newborn gastric juice specimens, while 15/50 specimens remained sterile. Further, M. smithii was observed by direct microscopic investigation using fluorescence in situ hybridisation. Multispacer sequence typing found one of seven different genotypes per specimen, these genotypes having all been previously described in adult human stools. Methanobrevibacter smithii is an early inhabitant of the human stomach, colonising the gastric mucosa just after birth, and the mother's gut microbiota is a probable source of colonisation.

Limitations and opportunities of whole blood bilirubin measurements by GEM premier 4000\xae

BackgroundNeonatal hyperbilirubinemia has traditionally been screened by either total serum bilirubin or transcutaneous bilirubin. Whole blood bilirubin (TwB) by the GEM Premier 4000® blood gas analyzer (GEM) is a relatively new technology and it provides fast bilirubin results with a small sample volume and can measure co-oximetry and other analytes. Our clinical study was to evaluate the reliability of TwB measured by the GEM and identify analytical and clinical factors that may contribute to possible bias.Methods440 consecutive healthy newborn samples that had plasma bilirubin ordered for neonatal hyperbilirubinemia screening were included. TwB was first measured using the GEM, after which the remainder of the blood was spun and plasma neonatal bilirubin was measured using the VITROS 5600® (VITROS).Results62 samples (14%) were excluded from analysis due to failure in obtaining GEM results. Passing-Bablok regression suggested that the GEM results were negatively biased at low concentrations of bilirubin and positively biased at higher concentrations relative to the VITROS results (y = 1.43x-61.13). Bland-Altman plots showed an overall negative bias of the GEM bilirubin with a wide range of differences compared to VITROS. Both hemoglobin concentration and hemolysis affected the accuracy of the GEM results. Clinically, male infants had higher mean bilirubin levels, and infants delivered by caesarean section had lower hemoglobin levels. When comparing the number of results below the 40th percentile and above the 95th percentile cut-offs in the Bhutani nomogram which would trigger discharge or treatment, GEM bilirubin exhibited poor sensitivity and poor specificity in contrast to VITROS bilirubin.ConclusionsAn imperfect correlation was observed between whole blood bilirubin measured on the GEM4000® and plasma bilirubin on the VITROS 5600®. The contributors to the observed differences between the two instruments were specimen hemolysis and the accuracy of hemoglobin measurements, the latter of which affects the calculation of plasma-equivalent bilirubin. Additionally, the lack of standardization of total bilirubin calibration particularly in newborn specimens, may also account for some of the disagreement in results.