HAEMOGLOBINOPATHY CASES on HPLC Dr. Archana Vazifdar Head Pathologist Hindlabs, HLL Lifecare Ltd.

Automated system precalibrated column and gradient HPLC Automated system precalibrated column and gradient fully 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 flow Detector 3

Hemoglobin is eluted in a stepped manner by Buffers of Increasing Ionic strength 4

HbF: 1-2% RT: 1.03-1.13 HbA2: 1.75-3.25% (2-3.6%) RT: 3.63-3.64 CHROMATOGRAMS Peak Output Time Area Flat baseline Total peak area HbA2 / HbF RT Peak profile & shape (sharp, well defined, symmetrical) RT (min) Total Area Count: 1-3 million HbF: 1-2% RT: 1.03-1.13 HbA2: 1.75-3.25% (2-3.6%) RT: 3.63-3.64 5

P2- Glycemic status, upto 6% acceptable P3- 6% acceptable, 6-15% deterioration 15-25% HbJ

8 mnth/M, pallor, failure to thrive

Homozygous beta thalassemia

22/Female

HbA2 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.

Heterozygous Beta thalassemia Mother of 8 mnth old child HbF can be elevated in thal trait. Usually A2 4-8/10= BTT, A2 10-18%, HbF normal, cbc normal, asymptomatic= Hb lepore trait RT before 3.45-3.6 min. A2 between 25-35% HbE trait (normal cbc) A2 more than 60% with HbF 2-10%= HbE homozygous, microcytic anemia asymptomatic A2 more than 50% with HbF more than 10%= double heterozygous for HbE & B thal, cbc microcytic anemia with severe symptoms. A2 40-50%, HbF normal, cbc normal, asymptomatic= HbD-Iran Heterozygous Beta thalassemia

HbA2- Normal RBC indices- Normal RBC indices s/o thal Silent β thalassaemia RBC indices s/o thal Co existing IDA Co inheritance of α thalassaemia δβ thalassaemia HbA2 LOW in α thalassaemia

HbE/Hbβ double heterozygous HbA2% 2-3.6 HbF% ≤ 2 Hb N MCV Symptoms - FINAL NORMAL 4-10 ≤ 2/ m Borderline Asymptomatic β THAL TRAIT 10-18 2- 10 Hb LEPORE TRAIT 25-35 ≤ 1 HbE TRAIT 40-48 HbD IRAN Heterozygous 50-60 ≥ 10 Severe HbE/Hbβ double heterozygous ≥ 60 2-10 m HbE homozygous

Hb Lepore Trait

34/M, Kolkata HbE elutes in A2 window. A2 between 25-35%, HbF will be normal, cbc normal. PS- target cells.

Heterozygous E thalassemia

All criteria fit provided there is NO history of recent blood transfusion ALWAYS CORRELATE with clinical history with CBC & peripheral blood picture Degenerated sample

Case 1: 2 yr/M, anemia, hepatosplenomegaly Hb 7 MCV 84 MCH 28 MCHC 32.4 RDW 20.2

HPFH (HbF 5-30%, CBC N, asymp) Heterozygous δβ thal (HbF 3-20%, asymp) Elevated HbF: HPFH (HbF 5-30%, CBC N, asymp) Heterozygous δβ thal (HbF 3-20%, asymp) Homozygous beta thal Pregnancy Gap between Transfusion& analysis should be atleast 1-2 mnths. Ideally 3 mnths.

Recently transfused c/o homozygous beta thalassemia

Case 2: 28/F, Severe anemia

Borderline HbA2 levels: Silent carrier of thalassemia β thal with superadded IDA Macrocytosis αβ thalassemia

Borderline elevated HbA2 due to macrocytosis

P3: 6% acceptable, 6-12% deterioration 15-25% HbJ

Hb: 6.5 MCV: 63 MCH: 23 MCHC: 26 RDW: 19.6

Double heterozygous for HbE and β thalassaemia

Tests 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 anemia If the patient is on certain medications Aged/ degenerated sample 28

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