Checkpoints in the calibrator

1 - Flat baseline –
The baseline should be properly
constructed
5
3

2 - Peak profile & shape –

Peaks should appear sharp and
symmetrical
6
3 – Order of peaks – 4
The peaks should follow the
order F, P2, P3, Ao, A2

4 - Total peak area

Should be 1 to 3 million

5 – Hb A2 and Hb F response-
The new calibration factor
2
should be – 0.7 to 1.3

6 - Hb A2 retention time –
The retention time of Hb A2 1
for the calibrator should be 3.65 + 0.1
 Interpretation of chromatograms
• Flat baseline

• Total peak area

• Hb A2 retention time

• Peak profile & shape

• Review the CBC data and Interpret result in conjunction with CBC

• Consider ethnic origin

• Related clinical information

• Examine the relative percentages of the hemoglobin fractions found

• Determine whether a variant is present

• Consider the possibility of more than one hemoglobinopathy being present

Look for the following :
F – Less than 2%

P2 – changes with the glycemic status,

upto 6 % acceptable

P3 – upto 6 % acceptable ,

A0 – non glycated fraction of Adult hemoglo-

bin

A2 –normal range 2 to 4 %
Look for the following :
Typical thalassemia carriers

Hypochromic, Microcytic blood film

Hb A2 > 4.0 %

Hb F < 2.0%
( in some cases Hb F may also be elevated )
First evaluate age and transfusion history
If transfusion is involved
report with parental screening

If transfusion interval is greater than 30- 60

days

Look for the following :

Variable degree of anemia

Marked red cell changes

Hb F elevated upto 90%

Reduced Hb A
Normal or elevated Hb A2
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
 Hb MCV Hb F Hb E SEVERITY
(g/dl) (fl)

Normal 80 - <1% 25-35% Asymptomatic

90

Look for the following :

Hb E elutes in the A2 window

For a Hb E trait :
Hb A2 will be between 25-35%
Hb F will be normal
 Hb MCV Hb F Hb E SEVERITY
(g/dl) (fl)

10-12 65-75 >2% >60% Mild

Look for the following :

Hb E elutes in the A2 window

For a Hb E homozygous:
Hb A2 will be between >60%
Hb F will be between 2-10%

These values will be variable from pa-

tient to patient and will also vary if
there is a history of blood transfusion
 Hb MCV Hb F Hb E SEVERITY
(g/dl) (fl)
<10 <70 >10% >50% Severe

Look for the following :

Reduced indices
Hb E elutes in the A2 window
For a Hb E homozygous:
Hb A2 will be between >50%
Hb F will be between > 10%

These values will be variable from patient to

patient and
will also vary if a history of blood transfusion
is involved
 Hb MCV Hb A2 Hb F Hb S SEVERITY
(g/dl) (fl)

Normal 80 - 90 < 4.0 % <1% 30- 40% Asymptomatic

Look for the following :

Normal indices
Hb S elutes in the S window

For a S trait :
Hb A2 will be normal (however due
to the elution of some glycated
Sickle products the A2 may be ele-
vated in some cases , do not con-
sider it as a compound heterozygous
case)

Hb F will be normal
Hb S will be between 30-40%
 Hb MCV Hb A2 Hb F Hb S SEVERITY
(g/dl) (fl)
<12 70-75 <5% > 5% >50% Mild

Hb S elutes in the S window

Look for the following :

For a Hb S homozygous:
Hb A2 will be normal
Hb F will be elevated
Hb S will be > 50%

These values will be variable from patient to

patient and will vary if a history of blood trans
fusion is involved
 Hb MCV Hb A2 Hb F Hb S SEVERITY
(g/dl) (fl)

<10 <70 > 5.0 % > 5% >50% Severe

Hb S elutes in the S window

If transfusion is involved
report with parental screening

Look for the following :

Reduced indices

For a Hb S thalassemia:
Hb A2 will be elevated
Hb F will be elevated
Hb S will be > 50%

These values will be variable from patient

to patient and will vary if there is a history
of blood transfusion
 Hb MCV Hb A2 Hb F Hb D SEVERITY
(g/dl) (fl)
Normal 80 - 90 < 2% <1% 30-45% Asymptomatic

Look for the following :

Normal indices
Hb D elutes in D window

For Hb D trait :
Hb A2 will be normal
Hb F will be normal
Hb D will be between 30-40%
 Hb MCV Hb A2 Hb F Hb D SEVERITY
(g/dl) (fl)

<12 <75 <4% 3-6% >50% Variable be-

tween mild
to severe

Look for the following :

Normal or reduced indices
Hb D elutes in D window

For a Hb D beta thalassemia :

Hb A2 will be normal or elevated
Hb F will be mildly elevated
Hb D will be between >50%
 Hb MCV Hb A2 Hb F Hb D Hb S SEVERITY
(g/dl) (fl)
</= 14 80-90 <4% <20% <50% >50% Variable be-
tween mild
to severe

Look for the following :

Normal or reduced indices

For a Hb S- Hb D disease :
Hb A2 will be normal
Hb F will be elevated
Hb D will be < 50%
Hb S will be < 50%
 Hb MCV Hb A2 Hb F SEVERITY
(g/dl) (fl)

Normal Normal 40-48% <2% Asymptomatic

Look for the following :

Normal or reduced indices

For a Hb D Iran trait :

Hb A2 will be 40-48%
Hb F will be normal
 Hb MCV Hb A2 Hb F SEVERITY
(g/dl) (fl)
12-14 80-90 10-18% <10% Asymptomatic

Look for the following :

Normal indices

For Hb Lepore trait :

Hb A2 will be 10-18%
Hb F will be normal
The retention time of Hb A2 will be ear-
lier than normal - 3.45 to 3.6mins
 Hb MCV Hb A2 Hb F Hb Q SEVERITY
(g/dl) (fl)

12-14 80-90 <4% <2% 8-25% Asymptomatic

Look for the following :

Normal indices

For Hb Q trait :
Hb A2 will be normal
Hb F will be normal
Hb Q will elute at retention time 4.7 +
0.1 mins constituting 8-25%
 Hb MCV Hb A2 Hb F SEVERITY
(g/dl) (fl)

12-14 80-90 <4% <5-30 % Asymptomatic

Look for the following :

Normal indices

For HPFH trait :

Hb A2 will be normal
Hb F will be 5-30%
 Hb MCV Hb A2 Hb F SEVERITY
(g/dl) (fl)
<12 <75 <4% <3-20 % Asymptomatic

Look for the following :

Reduced indices

For HPFH trait :

Hb A2 will be normal
Hb F will be 3-20%
Additional points
• P3 – upto 6 % acceptable ,
– 6 to 12 % may indicate sample deterioration
– 15 to 25 % indicate Hb J (refer to Dr Nardi’s
algorithm)

• Iron deficiency – Hb A2 found to be slightly lower

• Megaloblastic anemia – Hb A2 found to be higher

Unknown peak
• May appear any where in the peak table
• More than 1 unknown peak may be seen
• Upto 6 % not significant
• If, above 6% - look for the RT for Hb identification.