Hemoglobin Electrophoresis PDF

ORGANIZATION OF THE GLOBIN GENES  Hemoglobin Abnormalities Qualitative: The amino acid sequence is altered because of incorrect DNA code (Structurally abnormal Hb e.g HbS and HbC). Quantitative: Production of one or more globin c...

ORGANIZATION OF THE GLOBIN GENES  Hemoglobin Abnormalities Qualitative: The amino acid sequence is altered because of incorrect DNA code (Structurally abnormal Hb e.g HbS and HbC). Quantitative: Production of one or more globin chains is reduced or absent (e.g.Thalassemia). Hereditary persistence of Fetal Hemoglobin (HPFH): Complete or partial failure of γ globin to switch to β globin.  HEMOGLOBIN ELECTROPHORESIS Hb electrophoresis is used as screening test to detect normal and abnormal hemoglobins. It uses an elecric current to separate normal and abnormal types of Hemoglobin Hemoglobin types have different electrical charges and move at different speeds CELLULOSE ACETATE HEMOGLOBIN ELECTROPHORESIS  Principle: Separation is largely determined by electrical charge. At this alkaline pH, all hemoglobins are negatively charged and moves toward the positively charged anode. These hemoglobins move at different rates depending on their net negative charge, which in turn is controlled by the composition (amino acids) of the Hb molecule (globin chain). A.A. substitutions in Hb variants alter net charge and mobility  Procedure  Supporting medium: Cellulose acetate membrane  Buffer: Tris-EDTA borate (TEB) which provides alkaline pH (8.5)  The sample is placed in a cellulose acetate membrane, which is positioned in an electrophoresis tray near the cathode (-).  The cellulose acetate membrane is then stained in order to color the proteins (Hbs).  By noting the distance each Hb has migrated and comparing this distance with the migration In HbA (normal Hb): Glutamate A.A. is present in the 6th position on the Beta globin chain. Glutamate is negatively charged. Thus,it produces a band closer to the Anode In HbS (sickle cell anemia): Glutamate A.A. in the 6th position on the Beta globin chain is replaced by valine. This Hb loses its negative charge. Thus, it produces a band in the middle between the cathode and anode. In HbC (HbC disease): Glutamate A.A. in the 6th position on the Beta globin chain is replaced by Lysine which is positively charged. This Hb acquire positive charge. Thus, it produces a band closer to the cathode. Catho Anode de + - Increased HbF > (2%) (wider band) as no HbA Increased HbF > (2%) and HbA2 (wider band) as decrease