1. HAEMOGLOBINOPATHY CASES on HPLC
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 strength4

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

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