MALDI-MS in first-line screening of newborns for sickle cell disease: results from a prospective study in comparison to HPLC.

OBJECTIVES: Newborn screening (NBS) for sickle cell disease (SCD) requires a robust, high-throughput method to detect hemoglobin S (HbS). Screening for SCD is performed by qualitative methods, such as isoelectric focusing (IEF), and both qualitative and quantitative methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and tandem mass spectrometry (MS/MS). All these methods detect HbS, as well as low-level or absent HbA, and also other variants of hemoglobin. HPLC is considered as a reference method for NBS, because of its high sensitivity and specificity in detecting HbS. NeoSickle®, a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, combined with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation, has increased the throughput of SCD testing. The purpose of this study was to compare the performances of NeoSickle® and HPLC. METHODS: A prospective study was conducted including 9,571 samples from the NBS program to compare MALDI-MS using NeoSickle® with an HPLC method. Correlation between the two methods was studied. For the MALDI-MS method, sensitivity, specificity, NPV, and PPV were calculated. RESULTS: We found over 99.4 % correlation between the HPLC and MALDI-MS results. NeoSickle® showed 100 % of sensitivity and specificity in detecting SCD syndrome, leading to positive and negative predictive values of 100 %. CONCLUSIONS: NeoSickle® is adapted to NBS for SCD, and can be used in first-line high-throughput screening to detect HbS, and beta-thalassemia major warning. When HbS is detected, second-line use of another specific method as HPLC is necessary.

[Newborn screening for sickle cell disease in France]. / Dépistage néonatal de la drépanocytose en France.

Newborn screening (NBS) for sickle cell disease (SCD) in France has allowed the identification of 9,260 children with SCD since 1989, including 583 in 2019. In mainland France, however, SCD screening is targeted to newborns identified at risk of SCD, i.e born from parents originating from countries with a high SCD prevalence. This screening program, combined to prophylactic measures and a well-organized social and health network in France, has demonstrated considerable efficacy in reducing childhood mortality as well as severe infectious, anemic and neurovascular complications in childhood. SCD NBS has additionally allowed the identification of 180,687 heterozygous (AS) children since 1989. The increasing incidence of SCD (1/1,303 new-borns identified in 2019 versus 1/2,089 in 2009) now pleads for universal NBS and strong advocacy regarding SCD, the most frequent disease identified by NBS in France, and a major public health issue.

TITLE: Dépistage néonatal de la drépanocytose en France. ABSTRACT: Le dépistage néonatal de la drépanocytose, la plus fréquente des maladies rares en France, a permis, entre 1984 et 2019, l'identification de 9 260 nouveau-nés atteints de drépanocytose (dont 586 en 2019) et de 180 687 hétérozygotes AS. Ce dépistage a permis la mise en œuvre précoce de mesures prophylactiques chez ces enfants, grâce à un tissu sanitaire et social structuré. Depuis que ce dépistage est organisé, on a pu observer, dès l'âge pédiatrique, une diminution majeure de la mortalité et de la morbidité de la drépanocytose, qui concerne notamment les complications infectieuses invasives, anémiques et neuro-vasculaires. En métropole, ce dépistage garde la particularité d'être ciblé vers les nouveau-nés dont les parents sont originaires de régions à risque. La fréquence croissante de la drépanocytose (1/1 303 nouveau-nés identifiés en 2019 contre 1/2 089 en 2009) et l'augmentation de la fréquence des hétérozygotes plaident aujourd'hui pour un dépistage systématique étendu à tous les nouveau-nés et pour une meilleure information sur cette maladie, devenue un enjeu majeur de santé publique.

Prenatal biochemical diagnosis of two forms of congenital diarrheal disorders (congenital chloride diarrhea and congenital sodium diarrhea): A series of 12 cases.

OBJECTIVE: Congenital diarrheal disorders (CDDs) are a group of rare diseases among which some present as inherited disorders of intestinal electrolyte transportation: congenital chloride diarrhea (CCD) and congenital sodium diarrhea (CSD) with prenatal manifestations, mainly polyhydramnios, leading to premature delivery. Affected neonates present with watery stools, sometimes mistaken as urine, leading to a misdiagnosis of Bartter syndrome. The aim of this study was to study the value of a prenatal biochemical pattern in the case of suspected CDD. METHODS: We retrospectively studied 12 amniotic fluids of CDD-affected fetuses prenatally suspected and confirmed after birth. Digestive enzymes, proteins, and electrolytes were assayed and showed abnormal biochemical patterns. RESULTS: The 12 infants (eight CCD- and four CSD-affected) were born prematurely with a normal birth weight. Electrolytes and the Bartter index were normal for all cases. Amniotic fluid enzyme patterns were abnormal: anal leakage for nine, as expected, but vomiting of bile was observed for three infants, for whom an occlusive syndrome required surgery, and thereafter severe complications appeared with a poor prognosis. CONCLUSION: Amniotic fluid biochemical patterns differentiate CDD from Bartter syndrome. If a vomiting bile pattern is observed, postnatal management should take into account the hypothesis of a most severe complication.

Appropriate thresholds for accurate screening for ß-thalassemias in the newborn period: results from a French center for newborn screening.

Objectives: Newborn screening (NBS) for ß-thalassemia is based on measuring the expression of the hemoglobin A (HbA) fraction. An absence or very low level of HbA at birth may indicate ß-thalassemia. The difficulty is that the HbA fraction at birth is correlated with gestational age (GA) and highly variable between individuals. We used HbA expressed in multiples of the normal (MoM) to evaluate relevant thresholds for NBS of ß-thalassemia. Methods: The chosen threshold (HbA≤0.25 MoM) was prospectively applied for 32 months in our regional NBS program for sickle cell disease, for all tests performed, to identify patients at risk of ß-thalassemia. Reliability of this threshold was evaluated at the end of the study. Results: In all, 343,036 newborns were tested, and 84 suspected cases of ß-thalassemia were detected by applying the threshold of HbA≤0.25 MoM. Among the n=64 cases with confirmatory tests, 14 were confirmed using molecular analysis as ß-thalassemia diseases, 37 were confirmed as ß-thalassemia trait and 13 were false-positive. Determination of the optimum threshold for ß-thalassemia screening showed that HbA≤0.16 MoM had a sensitivity of 100% and a specificity of 95.3%, whatever the GA. Conclusions: NBS for ß-thalassemia diseases is effective, regardless of the birth term, using the single robust threshold of HbA≤0.16 MoM. A higher threshold would also allow screening for carriers, which could be interesting when ß-thalassemia constitutes a public health problem.

Finding vacuolated lymphocytes in fetal effusions improves the prenatal diagnosis of lysosomal storage diseases.

OBJECTIVES: There are many causes of fetal effusions, including the rare lysosomal storage diseases (LSDs). Vacuolated lymphocytes (VLs) are found in the blood of infants with LSDs, and their presence in fetal effusion could increase the risk of underlying LSD. METHODS: Between 2006 and 2018, all fetal effusions samples from 43 fetal multidisciplinary centers were referred to a single laboratory. Cells were counted, and, if observed, VLs were categorized and counted. Screening for LSDs was performed by metabolite analyses on amniotic fluid supernatant. The diagnosis of an LSD was confirmed by measuring the activity of the corresponding enzyme and/or mutation analysis. RESULTS: Our laboratory received 614 ascitic fluids and 280 pleural fluids sampled between 22 and 33 weeks of gestation. The final diagnosis was LSD in 16 cases (1.8%). VLs were reported in all these 16 cases, in a mix of lymphocytes with and without vacuoles. Vacuoles in VLs varied in size and number. In most cases, VLs were easy to recognize, with numerous, large, round, well-defined vacuoles, but in three cases of LSDs, VLs were atypical. CONCLUSION: The finding of VLs in fetal effusions is an inexpensive first-line test that may help to prioritize biochemical and genetic tests for LSDs.

[Newborn screening of sickle cell disease and management of care.] / Dépistage néonatal de la drépanocytose et filières d'organisation des soins.

Newborn screening of sickle cell disease -NBS- is systematic in the French West Indies and targeted in Metropolitan France to babies from "at risk" countries. The percentages of targeted babies range from 9.1% in Brittany to 73.6% in the Parisian area. NBS is performed on a drop of blood collected by heel stick and put on a blotting paper. Electrophoretic and chromatographic techniques are used to separate and quantify hemoglobin fractions. When a test finds abnormal results, confirmatory tests using a different technique are needed. In case of heterozygote baby, results are disclosed by mail or during a consultation according to local protocols. For children with major sickle cell syndromes -SCD-, results are disclosed by a specialist of SCD. All infants have to be included in a network of physicians including proximity health care providers and expert centers. Parents must be taught how to be the first line actors and must learn the signs prompting for going to Emergency department. In 2016, 431 children with SCD have been diagnosed in France, which represents 1/771 screened babies.

DÉPISTAGE NÉONATAL DE LA DRÉPANOCYTOSE ET FILIÈRES D'ORGANISATION DES SOINS Le dépistage néonatal de la drépanocytose est systématique chez tous les nouveau-nés des départements, régions et collectivités d'outre-mer français, et il est ciblé en métropole chez les nouveau-nés dont les parents appartiennent à un groupe à risque pour la drépanocytose. Le taux de ciblage va de 9,1 % en Bretagne à 73,6 % en Île-de-France. Le dépistage néonatal est réalisé à partir d'un échantillon de sang total obtenu par piqûre au talon du nouveau-né et déposé sur un buvard. L'analyse est fondée sur des techniques séparatives, électrophorèse et chromatographie, permettant d'une part de séparer les hémoglobines en fonction de leurs caractéristiques physicochimiques et d'autre part de quantifier les différentes fractions d'hémoglobine. Tout résultat positif doit être systématiquement vérifié à l'aide d'une technique différente de celle utilisée en première intention. Les résultats des nouveau-nés hétérozygotes, qui ne sont pas malades, sont rendus aux parents par simple courrier ou au décours d'une consultation selon les organisations locales. Les résultats des nouveau-nés possiblement atteints sont adressés au pédiatre référent. Celui-ci organise une consultation d'annonce avec les parents et le nouveau-né. La première consultation doit être faite par un médecin expert. Tout enfant doit être au coeur d'un réseau de soins maillant des acteurs de proximité et des équipes soignantes expertes. Les parents doivent être formés à devenir les premiers acteurs de soin, et notamment à reconnaître les signes exigeant une consultation dans un service hospitalier d'accueil des urgences. En 2016, 431 enfants ont été dépistés, soit une incidence de la maladie à la naissance de 1/771 dans la population testée.

Newborn Screening for Sickle Cell Disease in Europe.

The history of newborn screening (NBS) for sickle cell disease (SCD) in Europe goes back almost 40 years. However, most European countries have not established it to date. The European screening map is surprisingly heterogenous. The first countries to introduce sickle cell screening on a national scale were France and England. The French West Indies started to screen their newborns for SCD as early as 1983/84. To this day, all countries of the United Kingdom of Great Britain and Northern Ireland have added SCD as a target disease to their NBS programs. The Netherlands, Spain and Malta also have national programs. Belgium screens regionally in the Brussels and Liège regions, Ireland has been running a pilot for many years that has become quasi-official. However, the Belgian and Irish programs are not publicly funded. Italy and Germany have completed several pilot studies but are still in the preparatory phase of national NBS programs for SCD, although both countries have well-established concepts for metabolic and endocrine disorders. This article will give a brief overview of the situation in Europe and put a focus on the programs of the two pioneers of the continent, England and France.

Newborn screening for sickle cell disease in Europe: recommendations from a Pan-European Consensus Conference.

Sickle Cell Disease (SCD) is an increasing global health problem and presents significant challenges to European health care systems. Newborn screening (NBS) for SCD enables early initiation of preventive measures and has contributed to a reduction in childhood mortality from SCD. Policies and methodologies for NBS vary in different countries, and this might have consequences for the quality of care and clinical outcomes for SCD across Europe. A two-day Pan-European consensus conference was held in Berlin in April 2017 in order to appraise the current status of NBS for SCD and to develop consensus-based statements on indications and methodology for NBS for SCD in Europe. More than 50 SCD experts from 13 European countries participated in the conference. This paper aims to summarise the discussions and present consensus recommendations which can be used to support the development of NBS programmes in European countries where they do not yet exist, and to review existing programmes.

Sickle SCAN™ (BioMedomics) fulfills analytical conditions for neonatal screening of sickle cell disease.

Sickle SCAN™ is a rapid, qualitative, point-of-care lateral flow immunoassay for the identification of AS, AC, SS/Sß0thal, SC and CC/Cß0thal phenotype. We evaluated this test under the conditions encountered in the French newborn screening (NBS) program for sickle cell disease: a total of 104 dried blood spots (DBSs) were tested with an HPLC reference method and then with the Sickle SCAN™ device. Sickle SCAN™ identified the hemoglobin (Hb) phenotype correctly on 96% of cases. In the four non-concordant cases, the antibody anti-HbS cross-reacted with HbE (n=2), HbD (n=1) or HbX (n=1). There were no false negative. In order to test Sickle SCAN™'s sensitivity to low levels of HbA and HbS in the presence of high HbF levels, we selected another 21 DBS cards with low percentages of HbA (0.6-4.2%) and HbS (2.0-6.9%). HbA and HbS were always detected when present at levels of more than 1% and 2%, respectively. Sickle SCAN™ appears to be an accurate point-of-care method for the identification of newborns with SCD trait. The device meets the criteria for sickle cell disease NBS programs in endemic countries with poor access to laboratory equipment.

Characterization and origin of heme precursors in amniotic fluid: lessons from normal and pathological pregnancies.

BACKGROUND: Heme is the prosthetic group of numerous proteins involved in vital processes such as oxygen transport, oxidative stress, and energetic mitochondrial metabolism. Free heme also plays a significant role at early stages of development and in cell differentiation processes. The metabolism of heme by the fetal placenta unit is not well-established in humans. METHODS: In a retrospective study, we measured heme precursors in the amniotic fluid (AF) of 51 healthy women, and 10 AF samples from pregnancies with either upper or lower intestinal atresia or ileus were also analyzed. RESULTS: We showed that the porphyrin precursors aminolevulinic acid, porphobilinogen, and protoporphyrin IX are present at the limit of detection in the AF. Total porphyrin levels decreased progressively from week 13 to week 33 (p < 0.01). Interestingly, uroporphyrin, initially detected as traces, increased with maturation, in contrast to coproporphyrin. Uro- and coproporphyrins were type I immature isomers (>90%), suggesting a lack of maturity in the fetal compartment of the heme pathway. Finally, the differential analysis of AF from normal and pathological pregnancies demonstrated the predominant hepatic origin of fetal porphyrins excreted in the AF. CONCLUSION: This study gives the first insight into heme metabolism in the AF during normal and pathological pregnancies.

New approach to accurate interpretation of sickle cell disease newborn screening by applying multiple of median cutoffs and ratios.

BACKGROUND: The main goal of newborn screening (NBS) for sickle cell disease (SCD) is to detect affected neonates so that specific preventive care can be implemented. High-performance liquid chromatography (HPLC) used for NBS has high sensitivity and specificity, but we lack guidelines for quantitative hemoglobin (Hb) fraction interpretation. The purpose of this study was to determine cutoff values to standardize quantitative interpretation in SCD NBS for different clinical situation such as, red blood cell transfusion or beta-thalassemia, which can be real screening pitfalls. METHODS: Retrospective study of 75,026 samples from the neonatal screening program analyzed in our laboratory. Precise HbA and HbS percentages at birth were recorded and median values established for each gestational age, allowing percentage results to be expressed in normal gestation-specific multiples of the median (MoM). Three threshold values of clinical interest were determined. RESULTS: High levels of HbA (>2.5 MoM) allowed identification of newborns who received transfusions. Low levels of HbS (≤0.7 MoM) allowed detection of the association between HbS and other mutations of the beta-globin gene (i.e., HbHope, ß0-thalassemia, etc.). An HbA/HbS ratio <0.5 to distinguish healthy carriers from SCD with S/ß+-thalassemia. The screening accuracy for each threshold was established. The screening accuracy of low-level HbA, which is determinant in identifying the subgroup of patients at risk of ß-thalassemia, will be determined prospectively. CONCLUSIONS: This new approach introduces tools for a quantitative interpretation in SCD NBS by HPLC methods and could allow standardization of interpretation between centers.

Prenatal diagnosis of megacystis microcolon intestinal hypoperistalsis syndrome by biochemical analysis of fetal urine.

OBJECTIVES: The objective of the study is to determine a model of fetal urine biochemical markers to differentiate megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) from other megacystis. METHOD: This is a retrospective study of biochemical analysis of fetal urine in patients who presented prenatally with megacystis. We studied ß2-microglobulin, sodium, calcium, and phosphorus. Twenty-six patients subsequently diagnosed with MMIHS were compared with 2 control groups: one of end-stage renal failure (64 fetuses) and the second of "good" postnatal renal function (control group, 64 fetuses). RESULTS: Mean fetal urine ß2-microglobulin was significantly higher (P < .001) in end-stage renal failure (15.7 mg/L) than in MMIHS (2.2 mg/L) and the control group (3.2 mg/L). Fetal urine profiles differed significantly (P < .001) between MMIHS and the control group: median sodium 46.5 and 51 mmol/L, median calcium 1.12 and 0.73 mmol/L, and median phosphorus 0.03 and 0.15 mmol/L respectively. Fetal urinary ionic index [ratio: calcium / (phosphorus × sodium)] gave an area under the ROC curve of 0.86, at 54% sensitivity and 97% specificity, with correct classification in 84% of cases. We defined a nomogram to obtain a probability for MMIHS. CONCLUSION: Fetal urinalysis can be helpful in prenatal differentiation of MMIHS from posterior urethral valves with good postnatal renal function.

Amniotic fluid biochemistry in isolated polyhydramnios: a series of 464 cases.

OBJECTIVE: To report amniotic fluid biochemistry in a large series of 464 cases of isolated polyhydramnios in order to analyze both the outcome and the benefit of amniotic fluid biochemistry. METHODS: This retrospective cohort (2008-2012) included polyhydramnios cases for which amniotic fluid samples were sent to our laboratory for biochemical analysis (total protein, alpha-fetoprotein and gamma-glutamyl transpeptidase) so as to investigate the etiology. A Bartter index and an esophageal atresia index were defined. Final diagnoses were compared between groups to determine the association between these indices and the frequency and type of adverse outcomes. RESULTS: Among 464 cases of polyhydramnios considered isolated at ultrasound examination, severe fetal diseases were diagnosed in 136 (29.3%): 46 (9.9%) chromosomal anomalies, 28 (6%) Bartter syndrome, 23 (4.95%) other genetic syndromes, 22 (4.75%) swallowing disorders and 17 (3.7%) uro-nephrological disorders. Amniotic fluid biochemistry identified esophageal atresia with 66.6% (10/15) sensitivity and 100% specificity and Bartter syndrome with 85.7% (24/28) sensitivity and 84.2% specificity. CONCLUSION: Isolated polyhydramnios is associated with a high risk of severe fetal diseases. Molecular cytogenetics and amniotic fluid biochemistry are helpful tools.