Laboratory analyses
LYSIS, BETA THALASSEMIA, AMPLIFICATION, SEPARATION, POINT MUTATION
To assess beta thalassemia and structural hemoglobin variants affecting the beta globin gene, separation and quantification of hemoglobin in the stored red blood cells were performed by high-performance liquid-chromatography (HPLC) with commercial reagents and equipment (Hemoglobin Variants—HPLC, Chromsystems GmbH, Germany). The elucidation of atypical hemoglobin in the HPLC was performed by PCR amplification of the beta globin gene using specific primers and subsequent Sanger sequencing (Beckman Coulter CEQ 8800).DNA extraction was done with the ISOLATE II Genomic DNA Kit (BIOLINE, UK) according to manufacturer instructions by LGC Lab in Berlin, Germany. Sample lysis was conducted with chaotropic salt ions and Proteinase K. Following this, genomic DNA was bound to a DNA mini spin column to wash off contaminants and impurities. The purified DNA was then eluted using an elution buffer. DNA samples were aliquoted with each aliquot normalized to a concentration of 50 µg/µl. The extracted DNA was examined for abnormalities in the alpha globin gene using Multiplex Ligation-dependent Probe Amplification (MLPA; probe mix P140-C1 HBA; MRC Holland, Amsterdam, The Netherlands). This test allowed the identification of copy number variations in the alpha globin locus, and the Hb Constant Spring point mutation in the alpha-2-globin gene.