Blood count and hemoglobin (Hb) analysis are mandatory steps in the laboratory testing of a patient belonging to a community at high risk for hemoglobinopathies, especially during pregnancy, the aim being to detect possible transmission of a harmful variant and, eventually, to propose a prenatal diagnosis. When evidence for an unexpected peak is found, a thorough Hb analysis is required. We describe a patient in whom thorough molecular studies and Hb analysis unmasked a rare complex variant and demonstrated pitfalls of Hb analysis. 1,2 The 23-year-old patient born in Yogoupon (Ivory Coast) was followed up for a twin pregnancy. She had to be hospitalized briefly because of gestational hypertension, hepatic cytolysis and renal dysfunction. She was not known to suffer from any red cell abnormality, but because of her origin, hemoglobinopathies investigations were proposed to confirm the absence of any such abnormality. Laboratory tests during hospitalization revealed hematologic data in the normal range for her pregnancy, except for an additional Hb peak detected using capillary electrophoresis (eCap) (Capillarys, Sebia, Lisses, France) in the D-zone which was suspected to be Hb F (Figure 1A). Since this Hb fraction was estimated at 8%, which is higher than the slightly increased Hb F value expected during pregnancy (below 5%), genetic counseling was offered and informed consent was obtained to perform the full identification of the variant with a complete Hb study and globin gene analysis, in order to ensure there was no rare variant harmful for the twins. Biochemical analysis during hospitalization and 4 months after delivery revealed no constitutive abnormality (all laboratory are presented in Table 1). After delivery, the twins were included in a scheme for neonatal detection of hemoglobinopathies and dried blood spots were collected on cardboard for analysis. To the full Hb study we added cation exchange HPLC (CE-HPLC) (Variant 2, Bio-Rad, Hercules CA) and isoelectric focusing (Figure 1B), which showed that the peak was not, in fact, Hb F, but an unknown variant amounting to around 8% according to E cap. We thus studied the isolated globin chains by reverse-phase HPLC (RP-HPLC) 2 and mass spectrometry analysis using the Maldi-8020 (Shimadzu, Kyoto, Japan), combined with Neohemog ® , 3 a dedicated solution for hemoglobinopathy screening (Biomaneo, Dijon, France). This demonstrated that:(1) the variant was a beta variant and not Hb F, since no γ-chains could be resolved using RP-HPLC, and (2) the variant was expressed in the same quantity as the normal chain (RP-HPLC and MALDI TOF mass spectrometry analysis Figure 1C,D). RP-HPLC analysis demonstrated that the twins were free of the variant. We then performed molecular identification of this variant using Sanger analysis of the β-globin gene and multiplex ligation-dependent probe amplifica-
