1. Overview of HemoglobinOverview of Hemoglobin
ElectrophoresisElectrophoresis

2. Normal Hemoglobin StructureNormal Hemoglobin Structure
 Hemoglobin A is a tetramer composed ofHemoglobin A is a tetramer composed of
4 subunits:4 subunits:
– 22αα and 2and 2ββ
 Each subunit has a ring (porphyrin ring)Each subunit has a ring (porphyrin ring)
which holds an iron molecule.which holds an iron molecule.
– This is the binding site of oxygenThis is the binding site of oxygen

3. Normal Hemoglobin StructureNormal Hemoglobin Structure
Hemoglobin tetramer

4. Normal Hemoglobin StructureNormal Hemoglobin Structure
Fe
O
O
The oxygen atom binds to the Fe atom
perpendicular to the porphyrin ring
Porphyrin ring O2 binding site

5. Hemoglobin FunctionHemoglobin Function
 The function of the Hemoglobin moleculeThe function of the Hemoglobin molecule
is to pick up oxygen in the lung and deliveris to pick up oxygen in the lung and deliver
it to the tissues utilizing none of theit to the tissues utilizing none of the
oxygen along the way.oxygen along the way.

6. Hemoglobin FunctionHemoglobin Function
 The normal hemoglobin molecule is wellThe normal hemoglobin molecule is well
suited for its functionsuited for its function
– Allows for OAllows for O22 to be picked up at high Oto be picked up at high O22 tension intension in
the lung and delivered to the tissues at low Othe lung and delivered to the tissues at low O22
tension.tension.
– The oxygen binding is cooperative:The oxygen binding is cooperative:
 As each OAs each O22 binds to hemoglobin, the moleculebinds to hemoglobin, the molecule
undergoes a conformational change increasing the Oundergoes a conformational change increasing the O22
affinity for the remaining subunits.affinity for the remaining subunits.
 This creates the sigmoidal oxygen dissociation curveThis creates the sigmoidal oxygen dissociation curve

7. Normal Hemoglobin FunctionNormal Hemoglobin Function
The hemoglobin dissociation curveThe hemoglobin dissociation curve

8. Normal Hemoglobin FunctionNormal Hemoglobin Function
 Many variables influence the dissociation curve:Many variables influence the dissociation curve:
– pH:pH:
 An increase in pH (dec. COAn increase in pH (dec. CO22) shifts the curve to the left (increased) shifts the curve to the left (increased
OO22) affinity) affinity
 A decrease in pH (inc. COA decrease in pH (inc. CO22) shifts the curve to the right (decreased) shifts the curve to the right (decreased
OO22 ) affinity) affinity
– Temperature:Temperature:
 Increased temp with increased metabolic demands causesIncreased temp with increased metabolic demands causes
decreased Odecreased O22 affinity (right shift) and increased Oaffinity (right shift) and increased O22 deliverydelivery
– 2,3 DPG (2,3-diphosphoglycerate):2,3 DPG (2,3-diphosphoglycerate):
 Lowers OLowers O22 affinity by preferentially binding toaffinity by preferentially binding to BetaBeta chain ofchain of
deoxyhemoglobin, stabilizing it and reduces the intracellular pHdeoxyhemoglobin, stabilizing it and reduces the intracellular pH
– As hemoglobin concentration decreases, 2,3 DPG increases, allowingAs hemoglobin concentration decreases, 2,3 DPG increases, allowing
more Omore O22 to be unloadedto be unloaded

9. Hemoglobin variantHemoglobin variant
 Hemoglobin variants are mutant forms ofHemoglobin variants are mutant forms of
hemoglobin in a population (usually ofhemoglobin in a population (usually of
humans), caused by variations inhumans), caused by variations in
genetics.genetics.
 Some hemoglobin variants such as sickle-Some hemoglobin variants such as sickle-
cell anemia causes diseases, hence theycell anemia causes diseases, hence they
are hemoglobinopathies. Other variantsare hemoglobinopathies. Other variants
cause no detectable disease, thuscause no detectable disease, thus
considered non-pathological variants.considered non-pathological variants.

10. Other Hemoglobins in normalOther Hemoglobins in normal
adults (non pathological)adults (non pathological)
HemoglobinHemoglobin StructureStructure %%
AA αα22 ββ22 92%92%
AA22 αα22 δδ22 2.5%2.5%
AA1C1C αα22 ((ββ-N-glucose)-N-glucose) 3%3%
FF αα22 γγ22 <1%<1%
Gower-1Gower-1 ζζ22 εε22 0*0*
Gower-2Gower-2 αα22 εε22 0*0*
PortlandPortland ζζ22 γγ22 0*0*
* Indicates early embryonic form not seen in adults

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

12. Hemoglobinopathy?Hemoglobinopathy?
 Hemoglobinopathy is a genetic defect thatHemoglobinopathy is a genetic defect that
results in abnormalresults in abnormal structurestructure of one of theof one of the
globin chains of the hemoglobin molecule.globin chains of the hemoglobin molecule.
 Causes theCauses the depletion in the synthesisdepletion in the synthesis ofof
sufficient normal hemoglobinssufficient normal hemoglobins

13. Pathological variantsPathological variants
 1.1. Hemoglobin H (Hemoglobin H (β4) -β4) - A variant form ofA variant form of
hemoglobin, formed by a tetramer ofhemoglobin, formed by a tetramer of ββ
chains, which may be present in variantschains, which may be present in variants
ofof αα thalassemia.thalassemia.
 2.2. Hemoglobin Barts (Hemoglobin Barts (γ4) -γ4) - A variant formA variant form
of hemoglobin, formed by a tetramer ofof hemoglobin, formed by a tetramer of γγ
chains, which may be present in variantschains, which may be present in variants
ofof αα thalassemia.thalassemia.

14. Pathological variantsPathological variants
 3.3. Hemoglobin S (Hemoglobin S (α2βα2βS2) - A variant form ofS2) - A variant form of
hemoglobin found in people with sickle cellhemoglobin found in people with sickle cell
disease. There is a variation in thedisease. There is a variation in the β-β-chainchain
gene, causing a change in the properties ofgene, causing a change in the properties of
hemoglobin, which results in sickling of redhemoglobin, which results in sickling of red
blood cells.blood cells.
 4.4. Hemoglobin C (Hemoglobin C (α2βα2βC2) - Another variantC2) - Another variant
due to a variation in thedue to a variation in the β-β-chain gene. Thischain gene. This
variant causes a mild chronic hemolyticvariant causes a mild chronic hemolytic
anemia.anemia.

15. Pathological variantsPathological variants
 5.5. Hemoglobin E (Hemoglobin E (α2βα2βE2) - Another variantE2) - Another variant
due to a variation in thedue to a variation in the β-β-chain gene. Thischain gene. This
variant causes a mild chronic hemolyticvariant causes a mild chronic hemolytic
anemia.anemia.
 6.6. Hemoglobin AS - A heterozygous formHemoglobin AS - A heterozygous form
causing Sickle cell trait with one adult genecausing Sickle cell trait with one adult gene
and one sickle cell disease geneand one sickle cell disease gene
 7.7. Hemoglobin SC disease - A compoundHemoglobin SC disease - A compound
heterozygous form with one sickle gene andheterozygous form with one sickle gene and
another encoding Hemoglobin C.another encoding Hemoglobin C.

16. Sickle cell diseaseSickle cell disease
 Sickle cell disease is aSickle cell disease is a recessive genetic disorderrecessive genetic disorder of theof the
blood caused by ablood caused by a single nucleotide alteration in the β-globinsingle nucleotide alteration in the β-globin
genegene
 This condition is inherited in an autosomal recessive pattern,This condition is inherited in an autosomal recessive pattern,
which means both copies of the gene in each cell havewhich means both copies of the gene in each cell have
mutations. The parents of an individual with an autosomalmutations. The parents of an individual with an autosomal
recessive condition each carry one copy of the mutated generecessive condition each carry one copy of the mutated gene
 People with this disorder have atypical hemoglobinPeople with this disorder have atypical hemoglobin
molecules calledmolecules called hemoglobin Shemoglobin S that is distorted.that is distorted.
 A molecule ofA molecule of HbS contains two normal α-globin chains andHbS contains two normal α-globin chains and
two mutant β-globin chainstwo mutant β-globin chains, in which, in which glutamateglutamate at position 6at position 6
has been replaced withhas been replaced with valinevaline..

17. Sickle cell diseaseSickle cell disease
 UnderUnder low oxygenlow oxygen conditions, theconditions, the absenceabsence of aof a polarpolar
amino acidamino acid of the β-globin chain promotes theof the β-globin chain promotes the
polymerisation of haemoglobinpolymerisation of haemoglobin, which, which distorts reddistorts red
blood cells into a sickle shapeblood cells into a sickle shape andand decreasesdecreases theirtheir
elasticity.elasticity.
 Sickle cellsSickle cells block the flow of bloodblock the flow of blood in the narrowin the narrow
capillaries.capillaries.
 This interruption in the supply of oxygen leads toThis interruption in the supply of oxygen leads to
localizedlocalized anoxiaanoxia (oxygen deprivation) in the tissue,(oxygen deprivation) in the tissue,
causing pain and eventually death (causing pain and eventually death (infarctioninfarction) of cells) of cells
in the vicinity of the blockage.in the vicinity of the blockage.
 (sickle cell disease video)(sickle cell disease video)

18. Abnormal HemoglobinAbnormal Hemoglobin
 Reasons to suspect a hemoglobinReasons to suspect a hemoglobin
disorder:disorder:
– Patient presents with suspicious history orPatient presents with suspicious history or
physical examphysical exam
– Laboratory tests: Microcytic hypochromicLaboratory tests: Microcytic hypochromic
RBCs, hemolytic anemiaRBCs, hemolytic anemia
– Screening test abnormality (primarily inScreening test abnormality (primarily in
neonates)neonates)

19. Most common HemoglobinMost common Hemoglobin
abnormalitiesabnormalities
 ThalassemiasThalassemias
– AlphaAlpha
– BetaBeta
 HemoglobinopathiesHemoglobinopathies
– HbS trait; diseaseHbS trait; disease
– HbC trait; diseaseHbC trait; disease
– HbEHbE
– Hereditary Persistence of Hemoglobin FHereditary Persistence of Hemoglobin F
(HPFH)(HPFH)

20. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Red cell morphologies:Red cell morphologies:
– HbS: Sickle cellsHbS: Sickle cells

21. Sickle cells on peripheral smearSickle cells on peripheral smear

22. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Red cell morphologies:Red cell morphologies:
– HbS: Sickle cellsHbS: Sickle cells
– HbC: Target cells, crystals after splenectomyHbC: Target cells, crystals after splenectomy

23. HbC crystals with Target cellsHbC crystals with Target cells

24. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Red cell morphologies:Red cell morphologies:
– HbS: Sickle cellsHbS: Sickle cells
– HbC: Target cells, crystals after splenectomyHbC: Target cells, crystals after splenectomy
– Thalassemias: Microcystosis, target cells,Thalassemias: Microcystosis, target cells,
basophilic stipplingbasophilic stippling

25. Alpha Thalassemia withAlpha Thalassemia with
basophilic stipplingbasophilic stippling

26. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 High-Performance LiquidHigh-Performance Liquid
Chromatography (HPLC):Chromatography (HPLC):
– Weak cation exchange column. The ionicWeak cation exchange column. The ionic
strength of the eluting solution is graduallystrength of the eluting solution is gradually
increased and causes the variousincreased and causes the various
Hemoglobin molecules to have a particularHemoglobin molecules to have a particular
retention time.retention time.
 Amino acid substitutions will alter the retentionAmino acid substitutions will alter the retention
time relative to HbA.time relative to HbA.
 There is some analogy between retention time andThere is some analogy between retention time and
pattern on alkaline electrophoresis.pattern on alkaline electrophoresis.

27. Normal HPLC patternNormal HPLC pattern

28. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Solubility testSolubility test
(Sickledex):(Sickledex):
– Test to identify HbS. HbSTest to identify HbS. HbS
is relatively insolubleis relatively insoluble
compared to othercompared to other
Hemoglobins.Hemoglobins.
– Add reducing agentAdd reducing agent
– HbS will precipitate formingHbS will precipitate forming
and opaque solutionand opaque solution
compared with the clearcompared with the clear
pink solution seen in HbSpink solution seen in HbS
is not present.is not present.

29. Laboratory Methods to evaluateLaboratory Methods to evaluate
HemoglobinHemoglobin
 Electrophoresis:Electrophoresis:
– Alkaline (Cellulose Acetate) pH 8.6:Alkaline (Cellulose Acetate) pH 8.6:
 All Hemoglobin molecules have a negative charge, andAll Hemoglobin molecules have a negative charge, and
migrate towards the anode proportional to their net negativemigrate towards the anode proportional to their net negative
charge.charge.
– Amino acid substitutions in hemoglobin variants alter netAmino acid substitutions in hemoglobin variants alter net
charge and mobility.charge and mobility.
– Acid (Citrate agar) pH 6.2:Acid (Citrate agar) pH 6.2:
 Hemoglobin molecules separate based on chargeHemoglobin molecules separate based on charge
differencesdifferences andand their ability to combine with the agar.their ability to combine with the agar.
– Used to differentiate Hemoglobin variants that migrate togetherUsed to differentiate Hemoglobin variants that migrate together
on the cellulose gel (i.e. HbS from HbD and HbG, HbC fromon the cellulose gel (i.e. HbS from HbD and HbG, HbC from
HbE).HbE).

30. Hemoglobin ElectrophoresisHemoglobin Electrophoresis
PatternsPatterns

31. Hemoglobin VariantsHemoglobin Variants
 Hb A : normal individualHb A : normal individual
 Hb A + Hb F: normal neonateHb A + Hb F: normal neonate
 Hb F : beta thalassemia major individualHb F : beta thalassemia major individual
 Hb S : Homozygous HbSHb S : Homozygous HbS
individual(individual(α2βα2βS2)S2)
 Hb A + Hb S: Heterozygous SickleHb A + Hb S: Heterozygous Sickle
 Hb S + Hb C: sickle cell disease individualHb S + Hb C: sickle cell disease individual
((α2βα2βC2)C2)

32. THE ENDTHE END