3Automated system precalibrated column and gradient HPLCAutomated system precalibrated column and gradientfully automated cation exchange HPLC system which uses buffers increasing ionic strength to pass through a column and other conditions specifically designed to separate and quantitate HbA2 and HbF, Specific haemoglobin variants to elute within certain retention time frames. within 6-5 minute program.Direction of flowDetector3
4Hemoglobin is eluted in a stepped manner by Buffers of Increasing Ionic strength 4
11HbA2 concentration in Bthalassaemia carriers of diverse ethnic backgrounds is reported to be 4-9%, but HbA2 concentrations in some 5' fl-gene deletion thalassaemias may range from 7-12%. HbE concentrations in heterozygotes are typically in excess of 20%. Therefore, HbA2 concentrations in excess of perhaps 10% should suggest coelution of an abnormal haemoglobin such as HbE.HbE do not cause clinically important anaemia, compound heterozygotes for HbE and B-thalassaemia (Beta-E/beta-thal) may express a phenotype indistinguishable from homozygous B-thalassaemia.
12Heterozygous Beta thalassemia Mother of 8 mnth old childHbF can be elevated in thal trait. Usually A2 4-8/10= BTT, A %, HbF normal, cbc normal, asymptomatic= Hb lepore trait RT before min. A2 between 25-35% HbE trait (normal cbc)A2 more than 60% with HbF 2-10%= HbE homozygous, microcytic anemia asymptomaticA2 more than 50% with HbF more than 10%= double heterozygous for HbE & B thal, cbc microcytic anemia with severe symptoms.A %, HbF normal, cbc normal, asymptomatic= HbD-IranHeterozygous Beta thalassemia
13HbA2- Normal RBC indices- Normal RBC indices s/o thal Silent β thalassaemiaRBC indices s/o thalCo existing IDACo inheritance of α thalassaemiaδβ thalassaemiaHbA2 LOW in α thalassaemia
28Tests may not be accurate if… Patient had a blood transfusion within the past four months.Patient has polycythemia (increased red blood cell production) or underlying anemiaIf the patient is on certain medicationsAged/ degenerated sample28