Hplc interpretation

Hemoglobin Disorders
Interpretation By
HPLC
Presentor: Dr.Manisha Raj JR2
Moderator: Dr.Geeta Yadav
Dr.Nishant Taur

Normal Hemoglobin Structure
 Hemoglobin A is a tetramer composed of 4 subunits: –
 2α and 2β.
 Each subunit has a porphyrin ring which holds an iron molecule.
 This is the binding site of oxygen site of oxygen Hemoglobin tetramer.

The structure of hemoglobin is highly complex and can be viewed at four levels.
1 The primary structure : sequence of the amino acids in the polypeptide chain
which constitutes the globin chain.
2 The secondary structure : arrangement of the polypeptide globin chains into α
helices separated by non-helical turns;
70–80%of the amino acid residues of hemoglobin form part of the helices.

3. The tertiary structure :
 Arrangement of the coiled globin chain into a three-dimensional
structure.
 Surface haem-containing pocket between the E and F helices;
 Binding of haem between two specific histidine residues in the E and
F helices respectively
4 .The quaternary structure :
 Relationship between the four globin chains, which is not fixed.
 Strong α 1 β 1 and α 2 β 2 bonds (dimeric bonds) hold the molecule
together in a stable form, while the α β 2 and α 2 β 1 bonds
(tetrameric bonds) both contribute to stability, permit the chains to
slide on each other and rotate.
 Alteration in the quaternary structure of hemoglobin is responsible
for the sigmoid oxygen dissociation curve.

 Alteration in the quaternary structure of hemoglobin is responsible for the sigmoid
oxygen dissociation curve.
 the Bohr effect and the variation of oxygen affinity consequent on interaction
with2,3-DPG
 Contacts between like chains, α 1 α 2 and β 1 β2, are also of physiological
significance.
 The interaction between the four globin chains is such : oxygenation of one haem
group alters the shape of the molecule in such a way that oxygenation of other
haem groups becomes more likely.
 This is known as cooperativity and is reflected in the shape of the oxygen
dissociation curve.
 It is consequent on the fact in the deoxygenated state, the Fe2+ atom is out of the
plane of the porphyrin ring of haem.

 Oxygenation of Fe2+ causes it to move into the plane of the porphyrin
ring .
 Because of the link between haem and the histidine residues of globin, there is
an alteration in the tertiary structure of that haemoglobin Monomer
 This, in turn, causes the oxygenated monomer to alter its position in relation to
other haemoglobin monomers, i.e. the quaternary structure of the haemoglobin
molecule is altered

Other Hemoglobins in normal adults
HbA2: –
 Decreased in iron deficiency,
 alpha-thalassemia
 Elevated in megaloblastic anemia,
 hyperthyroidism
 Beta-thalassemia
HbF: –
 Elevated in HPF: Sickle cell anemia (preferential survival of RBCs because
HgF inhibits sickling),
 Beta thalassemia major
 Normal levels in Beta-thalassemia minor
 Normal or mildly elevated in congenital hemolytic Anemia
 Marked elevation in juvenile CML (up to 70%)

Hemoglobin Abnormalities
 There are 3 main categories of inherited Hemoglobin
abnormalities
 – Structural or qualitative: The amino acid sequence is altered
because of incorrect DNA code(Hemoglobinopathy).
 Quantitative: Production of one or more globin chains is reduced
or absent (Thalassemia).
 Hereditary persistence of Fetal Hemoglobin(HPFH): Complete or
partial failure of γγ globin to switch to β globin.

Abnormal Hemoglobin
 Reasons to suspect a hemoglobin disorder: –
 Patient presents with suspicious history or physical exam
 Laboratory tests: Microcytic hypochromic RBCs,
hemolytic anemia
 Screening test abnormality (primarily in neonates)

Laboratory Methods to evaluate
Hemoglobin
 Red cell morphologies: –
 HbS: Sickle cells
 HbC: Target cells, crystals after splenectomy
 Thalassemias: Microcystosis,
 target cells,
 basophilic stippling


Laboratory Methods to evaluate Hemoglobin
Solubility test : Test to identify HbS.
Solubility test : – Test to identify HbS.
 HbS is relatively insoluble compared to other Hemoglobins.
 Add reducing agent – It will precipitate forming an opaque solution and
compared with the clear pink solution seen in HbS is not present.

Electrophoresis:
 Alkaline (Cellulose Acetate) pH 8.6:
 All Hemoglobin molecules have a negative charge, and migrate
towards the anode proportional to their net negative charge.
 Amino acid substitutions in hemoglobin variants alter net
charge and mobility.
 Acid (Citrate Agar ) pH 6.2
 Hemoglobin molecules separate based on charge differences
and their ability to combine with the agar.
 Used to differentiate Hemoglobin variants that migrate together
on the cellulose gel (i.e. HbS from HbD and HbG,
HbC from HbE)

High-Performance Liquid
Chromatography( HPLC)–
 Weak cation exchange column.
 The ionic strength of the eluting solution is gradually increased solution is
and causes the various Hemoglobin molecules to have a particular
retention .
 Amino acid substitutions will alter the retention time relative to HbA.
 There is some analogy between retention time and pattern on alkaline
electrophoresis.

INTRODUCTION
 HPLC is a form of liquid chromatography used to separate components that are
dissolved in solution.
 HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a
separation column, and a director.
 Compounds are separated by injecting a sample mixture onto the column.
 The different components in the mixture pass through the column at differentiates
due to differences in their partition behavior between the mobile phase and the
stationary phase.

What is HPLC?
HPLC is a separation technique that involves:
 the injection of a small volume of liquid sample into a tube packed with tiny particles (3
to 5 micron ( μm ) in diameter called the (stationary phase)
 where individual components of the sample are moved down the packed tube (column)
with a liquid (mobile phase) forced through the column by high pressure delivered by a
pump.
 These components are separated from one another by the column packing that involves
various chemical and/or physical interactions between their molecules and the packing
particles.
 These separated components are detected at the exit of this tube (column) by a flow-
through device (detector) that measures their amount. An output from this detector is
called a “liquid chromatogram”.

1 - Flat baseline –
The baseline should be properly
constructed
2 - Peak profile & shape –
Peaks should appear sharp and
symmetrical
3 – Order of peaks –
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
should be – 0.7 to 1.3
6 - Hb A2 retention time –
The retention time of Hb A2
for the calibrator should be 3.65 + 0.1
Checkpoints in the calibrator
1
4
3
2
6
5

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
hemoglobin
A2 –normal range 2 to 4 %

Most common Hemoglobin
abnormalities
• Thalassemias –
• Alpha
• Beta
• Hemoglobinopathies –
• HbS trait; disease
• HbC trait; disease –
• HbE
• Hereditary Persistence of Hemoglobin F(HPHF)

Approach for Reporting
Reporting Protocol for Abnormal Hemoglobin study by HPLC
(Beta2 variant Programme-Biorad)
 Although a cut off of >4.0% of HbA2 is recommended for identifying b-
thalassemia carriers, each laboratory needs to establish their own normal
ranges.
 Also some cases of silent b-thalassemia can have HbA2 between 3.6 to 3.9%
 Blood transfusion within 3 months will have dilutional effect & false low
HbA2 & Hb F
 Coexisting Iron deficiency tends to decrease Hb A2 & cause microcytosis
 Coexisting Folate & B12 deficiency tend to falsely elevate Hb A2 levels
 Some hemoglobins co-elute in the Hb A2 window like Hb E, Hb D Iran, Hb G
Copenhagen, Hb Lepore and Hb Osu Christianbourg making it impossible to
quantitate HbA2 in the presence of these hemoglobins

Typical thalassemia carriers
Hypochromic, Microcytic blood film
Hb A2 > 4.0 %
Hb F < 2.0%
( in some cases Hb F may also be elevated )

BETA THALASSEMIA TRAIT
Phenotype: Normal or mildly Anemic.
Ethnicity: Prevalence in Indian population is around is 3%.
Much higher in Gujrat, Hryana and Eastern U.P.
Pathophysiology: molecular defect cause absence or reduced
synthesis of β globin chain leading to
Ineffective erythropoiesis
Mild reduction in Hemoglobin(9-11 g /dl )
 Lab findings: Marked Anisopoikiliocytosis
 Target cell, basophilic stippling
 polychromasia.
 MCV<80 fl
 MCHC<27 pg
 Reduced MCHC

First evaluate age and transfusion history
If transfusion is involved
report with parental screening
If transfusion interval is greater than 30- 60
days
Variable degree of anemia
Marked red cell changes
Hb F elevated upto 90%
Reduced Hb A
Normal or elevated Hb A2

BETA THALASSEMIA INTERMEDIA
Phenotype: Similar.to to Beta Thalassemia Major
Patient may not dependent on regular blood transfusion
for survival.
Ethnicity: Same as Beta Thalassemia Major
Pathophysiology: Variable degree of anemia.
Ineffective erythropoiesis.
Extra medullary hematopoiesis.
Usually appear later than 2 years of age.
 Lab findings: Marked Anisopoikiliocytosis
 Hypochromia with mild reticulocytosis.
. MCV,MCH :Markedly Reduced.
 Marked Increased Fetal Hemoglobin
 Variable Reduction in Hemoglobin A(10-35%)

First evaluate age and transfusion history
If no transfusion is involved
Increased NRBC count
Marked variation in shape and size
Hb F elevated upto 90%
Reduced Hb A
Normal or elevated Hb A2

BETA THALASSEMIA MAJOR
Phenotype: Severe Anemia
Pathophysiology: Ineffective erythropoiesis.
Iron overload resulting from transfusion.
Increased iron absorption.
Clinical manifestation seen after 6 month of life.
 Lab findings: Variable degree of Anemia
 Marked Anisopoikilocytosis
 Marked Increase of Fetal Hemoglobin (>85%)
 Marked Reduction in Hemoglobin A (<0.3%)
 Major band at HbF region on Electrophoresis

Hb
(g/dl)
MCV
(fl)
Hb
F
Hb E SEVERITY
Normal 80 -
90
<1% 25-35% Asymptomatic
Hb E elutes in the A2 window
For a Hb E trait :
Hb A2 will be between 25-35%
Hb F will be normal

HbE Heterozygous
Phenotype: Normal
Ethnicity : Most common in South East Asia.
Genotype: B chain mutation in26th position of Glutamic acid with Lysine.
Pathophysiology: HbE mutation partially activates a cryptic splice site in
Exon 1,resulting in a proportion of abnormally spliced mRNA.
Thus less Beta globin chain is synthesized.
 Lab findings: Microcytosis ( Low MCV)
 Hypochromia(Low MCH ).
 HbE around 30%
 HbE elutes in HbA2 window.

Hb
(g/dl)
MCV
(fl)
Hb F Hb E SEVERITY
10-12 65-75 >2% >60% Mild
For a Hb E homozygous:
Hb A2 will be between >60%
Hb F will be between 2-10%
These values will be variable from
patient to patient and will also vary if
there is a history of blood transfusion

HbE homozygous
Phenotype: Normal or features of mild hemolytic anemia with
Jaundic and splenomegaly.
Ethnicity : Most common in South East Asia.
Pathophysiology: HbE mutation partially activates a cryptic splice site in Exon
1,resulting in a proportion of abnormally spliced mRNA.
Thus less Beta globin chain is synthesized.
 Lab findings: Prominent Microcytosis ( Low MCV)
 Hypochromia(Low MCH ) with Target cells and leptocytes.
 Normal or reduced Hb
 Normal reticulocyte count.
 HbE around 85-95%
 HbE elutes in HbA2 window
 HbF : Mildely increased or Normal.
 OFT: reduced

Hb
(g/dl)
MCV
(fl)
Hb F Hb E SEVERITY
<10 <70 >10% >50% Severe
Reduced indices
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

Double Hetrozygous For HbE and
Beta Thalassemia
Phenotype: Severest forms of Beta
Pathophysiology: Ineffective erythropoiesis.
Iron overload resulting from transfusion.
Increased iron absorption.
Clinical manifestation seen after 6 month of life.
 Lab findings: Variable degree of Anemia
 Marked Anisopoikilocytosis
 Marked Increase of Fetal Hemoglobin (>85%)
 Marked Reduction in Hemoglobin A (<0.3%)
 Major band at HbF region on Electrophoresis

Hb
(g/dl)
MCV
(fl)
Hb A2 Hb F Hb S SEVERITY
Normal 80 - 90 < 4.0 % <1% 30- 40% Asymptomatic
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
elevated in some cases , do not
consider it as a compound
heterozygous case)
Hb F will be normal
Hb S will be between 30-40%

SICKLE CELL HETEROZYGOUS
Phenotype: Clinically asymptomatic except when exposed to low
oxygen tension. Ex: high altitude.
Genotype: Glutamic acid is replaced by Valine at 6th position of Beta
chain.
Pathophysiology: When exposed to low oxygen tension, it may lead
to vaso-occulusive crisis in the patient.
Hematuria and deficient urine concentration ability are most common
associated with renal abnormalities in trait.
Lab findings: NCNC Usuaally..
 HbS : Reduced (<30%)
 HbA2 may be raised.

Hb
(g/dl)
MCV
(fl)
<12 70-75 < 5 % > 5% >50% Mild
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
transfusion is involved

HbS HOMOZYGOUS
Phenotype: Chronic hemolytic anemia.
Genotype: Glutamic acid is replaced by Valine at 6th position of Beta chain.
Pathophysiology: Tendency to detoxify Hbs . Polymerization Innumerable
Clinical expression of sickling syndromes.
Clinical symptoms: Mild and Severe forms of sickle cell trait
Asymptomatic disorders include HbS trait, double hetrerozygous of HbS with
HPFH.
 Lab findings: Anemia (4-8 g/dl )
 NCNC
 Sickle cell with Target cells
 Reticulocyte count :Raised
 In vasso-occular crisis :Polymorphonuclear leukocytosis
 Thrombocytopenia.
 HbA2 : slightly raised.
 HbF :15-25%
 HbS: >50%

Hb
(g/dl)
MCV
(fl)
<10 <70 > 5.0 % > 5% >50% Severe
Reduced indices
For a Hb S thalassemia:
Hb A2 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

Double Heterozygous for HbS and
Beta Thalassemia
Phenotype: Clinically symptomatic..
Pathophysiology: Clinical resemble Sickle cell Anemia and splenomegaly
persists through adult life.
Lab findings: Microcytic hypochromic
Target cells predominates.
MCV, MCH, MCHC : Decreased with increased RBC count.
HPLC: HbA2 :Elevetaed
HbF :Elevated
HbS >50%

Hb
(g/dl)
MCV
(fl)
Hb A2 Hb F Hb D Hb S SEVERITY
</= 14 80-90 <4% <20% <50% >50% Variable
between
mild to
severe
Normal or reduced indices
For a Hb S- Hb D disease :
Hb D will be < 50%
Hb S will be < 50%

Double Heterozygous for HbS and HbD
Phenotype: Moderately severe clinical presentation of sickle cell trait.
Ethnicity :Uncommon condition encounter in Punjabis
Higher In Muslims in consanguineous marriage.
Pathophysiology: The Beta 121 glutamine residue increases the polymerization of
HbS.
Lab findings: Mild to moderate Anemia (4-8 g/dl )
Increased Reticulocytosis.
 HPLC: Separate peaks of HbD in the “D window” and
 HbS in the “S window”.
 HbD (<50%)and HbS (<50%)
 HbF peak (10-20%)
 Electrophoresis: Single bed on SD region.


Hb
(g/dl)
MCV
(fl)
Hb A2 Hb F SEVERITY
12-14 80-90 10-18% <10% Asymptomatic
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
earlier than normal - 3.45 to 3.6mins

HbE LEPORE TRAIT
Phenotype: Features indistinguishable
Genotype: Unequal cross-over during meiosis with deletion of 3rd
part and 5th part of Beta gene leading to formation of Delta Beta
fusion.
Pathophysiology: Features Of Delta Beta Thalassemia.
Three different types of Lepore Hemoglobins have identified.:
1)Hemoglobin Lepore Boston
2)Hemoglobin Lepore Hollandia
3)Hemoglobin Lepore Baltimore.
Lab findings: Anemia and Reticulocytosis.
MCV,MCH : Reduced.
 Abnormal HbA2:10-18%
 Retention time of HbA2 will be earlier than normal
( 3.45-3.6 mins)

Hb
(g/dl)
MCV
(fl)
Hb A2 Hb F SEVERITY
<12 <75 <4% <3-20 % Asymptomatic
Reduced indices
For HPFH trait :
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
 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.

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