Hemoglobinopathies and

Chromatogram interpretation
 Whom to screen ?

• Screen parents of Thalassemia Major / Sickle cell

disease

• Extended Family screening for Index case

• Screen all Pregnant women- if positive screen the

spouse

• Screen high risk communities

• Any patient having low MCV , Low MCH , High RBC

count , Normal RDW- should raise suspicion of Thal
Minor (Hb may be normal or reduced)

• MCV < 80 fl, MCH < 27 pg, RBC count which looks
higher as compared to Hb.
 D10 Hemoglobin Testing system

Cation Exchange high pressure liquid

chromatography (CE-HPLC)
It is a fully automated, high-throughput hemoglobin analyzer
providing an integrated method for sample preparation,
separation and determination of the relative percent of specific
hemoglobins in whole blood.

Gold Standard Method & Reference Technology

TYPICAL A2/F/A1c
CHROMATOGRAM
Hemoglobin A0
Hemoglobin A2
Hemoglobin A1c

Fetal Hemoglobin

HbA1a & HbA1b

Minor components of
HbA
Total area 1M-4M
A2/F/A1c Result
 Dual Cartridge Insert
Peak Name RT (min.) Window (min.)
A1a 0.21 0.16-0.26
A1b 0.29 0.23-0.35
F 0.485 0.41-0.56
LA1c/CHb-1 0.715 0.56 - 0.87
LA1c/CHb-2 0.735 0.60 - 0.87
A1c 0.83 0.66 - 1.00
P3 1.43 1.23 - 1.63
A0 1.70 1.55 - 1.85
A2 3.01 2.59 - 3.43
S 4.16 4.02 - 4.30
C 4.80 4.70 - 4.90
 Chromatogram review

• Base line – properly constructed, straight line

• Peak shape – sharp & symmetrical
• Total area – between 1 to 4 million
1 to 5 millions for A1c

If TAC less – increase the sample ratio

If TAC high – decrease the sample ratio
Calibration Review

• Base line – properly

constructed, straight line
• Peak shape – sharp &
symmetrical
• Total area – between 22L
to 28L
• Check if Cal 1 and Cal 2 is
proper
• Check A2 RT
• New Slope – 0.8 to 1.25
• New Intercept -2 to +0.3
(not more than 0.3)
 Control Review

• Base line – properly

constructed, straight line
• Peak shape – sharp &
symmetrical
• Total area
• Check if Control 1 and
Control 2 is proper
• Check for Control OK
• Check for low control A2
• Keep it as close to mean
as possible (+0.2)
 NORMAL
Look for the following :
A1a –– normal up-to 2.5%
A1b – normal up-to 2.5%

LA1c/CHb – 1 – Labile A1c –

normal upto 4%
LA1c/CHb – 2 – Carbamylated
Hb – normal up-to 4%

A1c – changes with the

glycemic status
P3 – up-to 6 % acceptable,
A0 – non glycated fraction of
Adult hemoglobin
A2 –normal range 2 to 4 %
β - THALASSEMIA TRAIT

Look for the following :

Hb – Normal to low

MCV <80 fl

MCH <27 pg

RBC which looks higher in

comparison to the Hb

Hb A2 > 4.0 %

Hb F - 0 - 2%
(in some cases this may also be
elevated up-to 8%)
 Anemia

• Iron deficiency – A2 found to be slightly lower

• Megaloblastic anemia – A2 found to be higher

Pre-analytical subject-related factors
that might modify HbA2 levels
Some comments for reporting purposes;

For normal indices and normal HbA2 (2-4%)

Suggestive of absence of beta thalassemia trait and absence of common
abnormal hemoglobin. Kindly repeat the test after correction of anemia and
confirm by doing parental screening and DNA analysis. Alpha thalassemia
cannot be ruled out.

For HbA2 between 4-8%.

MCV < 80, MCH < 27, RBC which looks higher as compared to Hb
Suggestive of beta thalassemia trait. Kindly confirm by doing parental
screening and DNA analysis. Husband's or wife's screening is necessary (if in
reproducible age).

For micro hypo indices but borderline HbA2 between 3.6 to 4.0%.
HbA2 is slightly raised (borderline). This can be due to beta thalassemia trait.
However, in view of anemia and macrocytosis (if present), it is advisable to
repeat after correction of anemia.
False elevation in HbA2 can be due to megaloblastic anemia, hyperthyroidism,
antiretroviral therapy, hypertrophic osteoarthropathy, alpha triplication,
Pseudoxanthoma elasticum. Husband's screening is necessary.
Hb F levels from birth to 1 year

•1 day: 69.7% to 84.3%

•5 days: 71.0% to 82.6%
•3 weeks: 62.7% to 77.3%
•6-9 weeks: 41.9% to 63.9%
•3-4 months: 7.2% to 39.2%
•6 months: 2.5% to 6.9%
•8-12 months: 0.6% to 2.6%
•>1 year: 0.0% to 2.0%
 β - THALASSEMIA MAJOR
First evaluate age and transfusion history
If transfusion is involved
report with parental screening

If no transfusion is involved
Look for the following :
Increased NRBC count
Marked variation in shape and size

Hb F elevated upto 90%

Reduced Hb A
Normal or elevated Hb A2

HbF can be high due to;

Beta thalassemia homozygous or
Double heterozygous for beta thalassemia
or Double heterozygous for beta
thalassemia and delta beta thalassemia or
double heterozygous for beta thalassemia
and HPFH or HPFH homozygous or delta
beta homozygous (Give these differentials
depending on the CBC)
Hb E TRAIT

Look for the following :

Hb E elutes in the A2 window

For a Hb E trait :
Hb A2 - 22 to 35%
Hb F - 0 to 6%

NO NEED TO SEPARATE HbA2 from

HbE

How to know it is HbE trait from

HPLC report?
1. A0 should be more than 50%
2. A2 should be between 22 to 35%
3. HbF should be between 0 to 6%
4. Hb and indices will be normal or
microcytic/hypochromic
5. Patient will be asymptomatic
 Hb E HOMOZYGOUS

Look for the following :

Hb E elutes in the A2 window

For a Hb E homozygous:
Hb A2 will be between 70 to 80%
Hb F will be between 2-10%
Hb normal to low and indices will
be microcytic/hypochromatic
Clinically the patient will be
asymptomatic or mildly anemic.

These values will be variable from

patient to patient and will also
vary if there is a history of blood
transfusion as in this case.
 Hb E- β-THALASSEMIA

Look for the following :

Reduced to very low indices
Hb E elutes in the A2 window
Hb A2 will be > 50%
Hb F will be > 12%

Clinically the patient will be

moderately anemic or on regular
transfusion

These values will be variable from

patient to patient and
will also vary if a history of blood
transfusion is involved as in this
case.
 Hb S TRAIT

Look for the following :

Normal to microcytic/hypochromic
indices
Hb S elutes in the S window

For HbS trait :

Hb A2 will be normal
(however due to the elution of
some glycated sickle products the
A2 may be falsely elevated in some
cases)

Hb F – 0 to 6%
Hb S – 22 to 40%
 Hb S HOMOZYGOUS

Hb S elutes in the S window

Look for the following :

Normal to very low indices
For a Hb S homozygous:
Hb A2 will be normal
Hb F will be elevated (5 – 35%)
Hb S will be > 50%

Clinically the patient will be mild

or moderate or severe.

These values will be variable from

patient to patient and will vary if a
history of blood transfusion is
involved
 Hb S-β-THALASSEMIA

Hb S elutes in the S window

If transfusion is involved
report with parental screening

Look for the following:

Reduced indices

Hb A2 will be elevated > 5%

Hb F will be elevated (5 – 35%)
Hb S will be > 50%

Clinically the patient will be mild

or moderate or severe.

These values will be variable from

patient to patient and will vary if
there is a history of blood
transfusion
 Hb D TRAIT

Look for the following:

Normal indices
Hb D elutes in an unknown
window of retention time of
3.8 + 0.1mins

For Hb D trait :
Hb A2 will be normal
Hb F will be normal
Hb D (unknown) will be
between 25-40%

Clinically the patient will

be asymptomatic.
Hb D-β-THALASSEMIA

Look for the following :

Normal or reduced indices
Hb D elutes in an unknown
window of retention time of
3.8 + 0.1mins

Hb A2 will be normal or elevated

Hb F will be mildly elevated
Hb D will be > 50%

Clinically the patient will be

normal or mildly anemic
HbD-Iran Heterozygous

Look for the following :

Normal or reduced indices

For a Hb D Iran trait :

Hb A2 will be 40-48%
The retention time of Hb A2
will be slightly earlier than
normal

Hb F will be normal

Clinically the patient will be

asymptomatic.
HPFH δβ thalassemia
Heterozygous Heterozygous

Look for the following :

Look for the following :
Reduced indices
Normal indices
MCV < 80fl
MCH < 27pg
For HPFH trait :
Hb A2 will be normal
For δβ (delta beta) thalassemia
Hb F will be 10-35%
trait:
Hb A2 will be normal
Clinically the patient will be
Hb F will be 4-20%
asymptomatic
Clinically the patient will be
asymptomatic
 Hb Q India TRAIT Hb LEPORE TRAIT

Micro hypo
indices like
β thalassemia
trait

HbQ India elutes in an unknown

window of RT of 4.4 + 0.1mins
HbJ Heterozygous

Look for the following :

Normal indices

P3 or Unknown between 15-25%

Clinically the patient will be

asymptomatic
 Hb A2
0-2 % : New born
0-2 % : Alpha Thal (indicative only)
2-3.5 % : Normal
3.5 – 4.0 % : Indeterminate zone
4.0 – 9 % : Beta thalassemia trait
10 – 20 % : Hb Lepore (rare)
22 - 37 % : Hb E trait
40 – 48 % : Hb D-Iran
(the A2 peak may be broken)
70 – 90 % : Hb E homozygous
HbA2 and HbF values, together with some relevant red cell indices, in
normal infants and β thalassemia carriers during the 1st year of life
 Peak table

• P3 – upto 6 % acceptable ,
- 6 to 12 % may indicate sample deterioration
- 15 to 25 % indicate Hb J

Unknown – may appear anywhere in the peak

table/chromatogram
 Beta gene variants

• Hb E – elutes with A2
• Hb D – Unknown peak at 3.8 +/- 0.1 min
• Hb S – S- window
• Hb C – C-window

• 22-40 % - heterozygous
• 70 –90 % - homozygous
• Compound conditions also present (S+ Beta
thal , Hb SD , E+ Beta thal etc)
 Alpha gene variants

• Hb J Meerut – P3 between 15 to 25 %
• Hb Q India – Unknown peak at 4.4 +/- 0.1 min
• Usually between 15 to 25 %

• Being single gene mutation, these conditions are

clinically silent.
D10 Online Library
Online Chromatogram Library
Other considerations for
interpretation

• Blood transfusion affects the Hb

pattern
• Pregnancy – may elevate the Hb F
• Ethnic back ground
• Family history
• Age
• RBC indices