HAEMOGLOBINOPATHY CASES
on HPLC
Dr. Archana Vazifdar
Head Pathologist
Hindlabs, HLL Lifecare Ltd.
HPLC
Automated system precalibrated column and gradient
Direction of flow
Detector
• Hemoglobin is eluted in a stepped
manner by Buffers of Increasing Ionic
strength
CHROMATOGRAMS
Peak
Output
Area
Time
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
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
Mother of 8 mnth
old child
Heterozygous Beta thalassemia
HbA2- Normal
• RBC indices- Normal
– Silent β thalassaemia
• RBC indices s/o thal
– Co existing IDA
– Co inheritance of α thalassaemia
– δβ thalassaemia
HbA2 LOW in α thalassaemia
HbA2%
HbF%
Hb
MCV
Symptoms
FINAL
2-3.6
≤2
N
N
-
NORMAL
4-10
10-18
≤ 2/ m
2- 10
Borderline
N
Asymptoma
tic
N
β THAL
TRAIT
Hb LEPORE
Asymptoma
TRAIT
tic
HbE TRAIT
25-35
40-48
≤1
≤2
50-60
≥ 10
≥ 60
2-10
N
N
N
N
Asymptoma
tic
Asymptoma
tic
Severe
m
Asymptoma
tic
HbD IRAN
Heterozygo
us
HbE/Hbβ
double
heterozygou
s
HbE
homozygou
Hb Lepore Trait
34/M, Kolkata
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
Elevated HbF:
•HPFH (HbF 5-30%, CBC N,
asymp)
•Heterozygous δβ thal
(HbF 3-20%, asymp)
•Homozygous beta thal
•Pregnancy
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