Neonatal screening for sickle cell disease in France.

BACKGROUND: As a result of population growth in African-Caribbean regions of overseas France, and now immigration essentially from North and sub-Saharan Africa to mainland France, neonatal screening for sickle cell disease (SCD) has been performed in France since 1985 in Guadalupe and dependencies, as a universal test. After several pilot studies, screening was gradually extended to mainland France in 1996. Since 2000, the test has been performed at national level for all newborns defined as being "at risk" for SCD based on ethnic origin. METHODS: A dry blood sample is obtained by heel stick and analysed by isoelectric focusing as a first-line method, followed by either high-performance liquid chromatography or acid agar electrophoresis for confirmation, whenever a variant haemoglobin is observed on isoelectric focusing. RESULTS: In 2007, 28.45% of all newborns in mainland France were screened for SCD. Since 1996, a total of 3,890 newborns have been found to have SCD, and they have been followed up by reference paediatricians. CONCLUSION: Although screening for SCD at birth in France is not universal, it appears that missed babies are relatively infrequent. Despite obvious sociological problems inherent to the at-risk population, the follow-up of SCD babies is rather successful. Due to the birth prevalence of SCD in France, especially in comparison with other common genetic diseases, screening all newborns regardless of ethnic origin is an issue that is being addressed.

Abnormal hemoglobins: laboratory methods.

Laboratory methods allowing the detection and characterization of hemoglobin variants are reviewed. Protein chemistry techniques such as isoelectrofocusing, electrophoreses under various experimental conditions, cation exchange and reversed phase high performance liquid chromatography, are the most frequently used for the detection of variants. When associated with a few additional data they may lead to a presumptive diagnosis. DNA studies are also developed in many laboratories. Final identification of a variant may be achieved either by molecular biology techniques or by protein sequence analysis in which mass spectrometry now occupies a key position.

Characterization of alpha-smooth muscle actin positive cells in mineralized human dental pulp cultures.

In response to injury, pulp precursor cells can differentiate into odontoblast-like cells that produce reparative dentine. In culture, pulp cells form mineralizing nodules, but the characteristics of the cells involved in this process are still not fully known. Human pulp cells for culture were obtained from coronal pulp isolated from non-erupted molars, and were maintained in RPMI 1640 medium supplemented with fetal calf serum. Nodules were forming in all human pulp primary cultures (HPPc) and human pulp subcultures observed until their fifth passage (HPSc<5). Mineralization of the nodules was confirmed by the presence of calcium and phosphate that were quantified by X-ray microanalysis. Specific immunolabeling revealed alpha-smooth muscle actin and vimentin in both HPPc and HPSc<5 cells. Cells positive for alpha-smooth muscle actin were either isolated or gathered together in the nodules. Under transmission electron microscopy, some cells in primary pulp cultures exhibited features typical of myofibroblasts or pericytes, such as stress fibers, fibronexus, indented nuclei and gap-junctions. These cells were frequently in close contact with mineral deposits. This work demonstrates for the first time the presence of pericytes or myofibroblasts in mineralized human pulp cultures, but further investigation is required to determine their origin, role and degree of differentiation.

Two new Ggamma chain variants: Hb F-clamart [gamma17(A14)Lys-->Asn] and Hb F-Ouled Rabah [gamma19(B1)Asn-->Lys].

Two new fetal hemoglobin variants affecting the Ggamma chain are reported. Hb F-Clamart was found during investigation of a French newborn who presented with a mild microcytemia. The second variant was found during neonatal screening for hemoglobinopathies of 30,000 babies from a population-at-risk living in the Paris region. It was named Hb F-Ouled Rabah because its structural modification and ethnic distribution is similar to that of Hb D-Ouled Rabah [beta19(B1)Asn-->Lys]. Hb F-Ouled Rabah is clinically silent and occurs at a frequency of ca. 0.1% in newborns originating from Maghreb. Structural characterization of both variants was done by protein chemistry methods, including amino acids analysis and mass spectrometry.

Cation-exchange HPLC evaluated for presumptive identification of hemoglobin variants.

A battery of relatively simple tests allows the presumptive identification of hemoglobin (Hb) variants, making unnecessary structural analysis by protein chemistry methods or DNA sequencing. The primary step in this strategy involves the use of a matrix of electrophoretic mobilities obtained under various experimental conditions. This leads to an unambiguous result in approximately 90% of the cases. Additional tests are required to characterize with more confidence the remaining 10%. We describe here the use of cation-exchange HPLC on the Bio-Rad Variant automated analyzer with the "beta Thalassemia Short" program. By comparing the elution time of 125 human Hb mutants, we found that some variants with almost identical pI values or produced by the same type of amino acid substitution displayed different elution times. We present several examples in which use of the HPLC profile helped establish the diagnosis.