Presentation is loading. Please wait.

Presentation is loading. Please wait.


Similar presentations

Presentation on theme: "HAEMOGLOBINOPATHY CASES on HPLC"— Presentation transcript:

Dr. Archana Vazifdar Head Pathologist Hindlabs, HLL Lifecare Ltd.


3 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

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

5 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: % RT: HbA2: % (2-3.6%) RT: 5

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

7 8 mnth/M, pallor, failure to thrive


9 Homozygous beta thalassemia

10 22/Female

11 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.

12 Heterozygous Beta thalassemia
Mother of 8 mnth old child HbF 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 asymptomatic A2 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-Iran Heterozygous Beta thalassemia

13 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

14 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

15 Hb Lepore Trait

16 34/M, Kolkata HbE elutes in A2 window.
A2 between 25-35%, HbF will be normal, cbc normal. PS- target cells.

17 Heterozygous E thalassemia

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

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

20 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.

21 Recently transfused c/o homozygous beta thalassemia

22 Case 2: 28/F, Severe anemia

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

24 Borderline elevated HbA2 due to macrocytosis

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

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

27 Double heterozygous for HbE and β thalassaemia

28 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

29 29


Similar presentations

Ads by Google