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The diagnosis of the main hemoglobinopathies (HbS, HbC, HbE, heterozygous beta-thalassemia) is easy for laboratories using Hb electrophoresis and/or cation-exchange high performance liquid chromatography (CE-HPLC) methods. However, the diagnosis of alpha-thalassemias and of rare Hb variants is much more complex. Six French laboratories, forming together the "Pathologie héréditaire de l'érythrocyte" network, routinely practice the molecular diagnosis of hemoglobinopathies. These laboratories have shared their experiences to propose flowcharts for the phenotypical diagnosis and the molecular characterization of the main hereditary Hb pathologies. These flowcharts are applicable to any single patient with an adult erythropïesis (more than two-years-old), that is to say after the fetal to adult (gamma>beta) Hb commutation, when the HbF level is stabilized.

Hemoglobinopathies have become a significant national health problem in France. The biologists have a pivotal role in the genetic diagnoses. Although sickle cell disease (SCD) is the most frequent abnormality found: not less than 200 new cases are observed each year at birth, many other globin gene variations are found in the various ethnic groups. Since 1995 a neonatal sickle cell screening program has been established for at risk newborns. This programme is supported by the "Association française de dépistage et prévention des handicaps de l'enfant" (AFDPHE). The characterization of hemoglobin genetic variations requires a comprehensive set of laboratory techniques for which we specify here main clinical and technical recommendations.

Unlabelled: We report a five-year experience of targeted neonatal screening for sickle cell disease in the northern part of the Paris area as well as the follow-up procedure of screened patients., Population: This geographic area in France is characterized by a high frequency of populations at risk for sickle cell disease., Results: Among 115,480 tested newborns, 250 patients were diagnosed (frequency: 1/462). The quality of the screening was attested by the high frequency (5.34%) of newborn carriers for a hemoglobin abnormality (n = 6168). We developed an optimized strategy which avoids the majority of pitfalls (false positive and false negative responses), except for S/HPFH. More than 95% of sickle cell disease was followed, the majority by medical sickle cell disease experts from hospitals. Only two deaths were recorded during this time period., Conclusion: We demonstrate the efficiency of targeting the proposed methodological strategy and the follow-up of affected newborns, a major argument demonstrating the importance of newborn screening.

Separation of hemoglobins F, Fac, S, C and A1c was performed using high performance liquid chromatography with a cation exchange Polycat A column in a 15-minute assay. HbF titration results were well correlated with those of the reference alkali denaturation technique for values below 12% (r = 0.95; P < 0.001). This technique may be used as a confirmation test for neonatal screening of sickle cell disease.

A nine-year-old boy from Béjà (North-Western Tunisia) was found to have both HbD Iran and beta-thalassemia. This patient presented with anemia and slight enlargement of the spleen and had a history of acute episodes of hemolysis. Structural studies on this hemoglobin variant used several miniaturized techniques, mainly carboxy-methyl-cellulose chromatography, reverse-phase high performance liquid chromatography and manual peptide sequencing using Chang's technique. The glutamic acid in position 22 on the beta chain was found to be replaced by a glutamine, establishing the diagnosis of HbD Iran. Concomitant presence of a thalassemia trait was suggested by the finding in the index patient of microcytosis, hypochromia and increased HbA2. The family study confirmed this patient's combined heterozygous anomalies, showing the D trait in the father and the beta thalassemia trait in the mother. The same combination was found in the index patient's sister who was, however, free of clinical symptoms. The explanation of this difference in clinical expression was provided by the ADN study which disclosed deletion of an alpha gene in the girl. The resulting alpha chain deficiency counterbalanced the beta chain deficiency.

Hb D Punjab is a hemoglobin abnormality due to the substitution of a glutamic acid by a glutamic on the hemoglobin beta chain: beta 121 Glu----Gln. Authors report here the first case of this rare variant in a tunisian family.