In this program the samples were collected from a heelprick onto filter paper when the child was 7-10 days old. A dried blood spot of 0.3 cm diameter was obtained from which Hb was eluted to be analyzed by isoelectric focusing (IEF) and by cation exchange high performance liquid chromatography (HPLC) (VARIANT I; Bio-Rad Laboratories, Hercules, CA, USA). When an abnormal Hb is observed in a baby, venous blood from his parents is collected into EDTA vacutainers for further analysis.

Hematological parameters were obtained by routine methods (Coulter STKS; Beckman Coulter, Miami, FL, USA). Electrophoretic and chromatographic studies were done as previously described [4]. Procedures for separation of globin chains, aminoethylation, and digestion with trypsin are detailed elsewhere [4]. Electrospray mass spectrometry (ES-MS) studies on the globin and by tandem mass spectrometry (MS/MS) on the peptides were done as previously described [4].