A Brief Overview of A Brief Overview of Hemoglobin Hemoglobin

Electrophoresis Electrophoresis Sarah Walter, M.D.Sarah Walter, M.D.

Normal Hemoglobin Normal Hemoglobin StructureStructure

Hemoglobin A is a tetramer Hemoglobin A is a tetramer composed of 4 subunits:composed of 4 subunits:– 22αα and 2 and 2ββ

Each subunit has a porphyrin ring Each subunit has a 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

Normal Hemoglobin Normal Hemoglobin StructureStructure

Hemoglobin tetramer

Normal Hemoglobin Normal Hemoglobin StructureStructure

FeO

O

The oxygen atom binds to the Fe atomperpendicular to the porphyrin ring

Porphyrin ring O2 binding site

Hemoglobin FunctionHemoglobin Function The function of the Hemoglobin The function of the Hemoglobin

molecule is to pick up oxygen in the molecule is to pick up oxygen in the lung and deliver it to the tissues lung and deliver it to the tissues utilizing none of the oxygen along utilizing none of the oxygen along the way. the way.

Hemoglobin FunctionHemoglobin Function The normal hemoglobin molecule is The normal hemoglobin molecule is

well suited for its functionwell suited for its function– Allows for OAllows for O2 2 to be picked up at high Oto be picked up at high O22

tension in the lung and delivered to the tension in the lung and delivered to the tissues at low Otissues at low O2 2 tension.tension.

– The oxygen binding is cooperative:The oxygen binding is cooperative: As each OAs each O22 binds to hemoglobin, the molecule binds to hemoglobin, the molecule

undergoes a conformational change increasing undergoes a conformational change increasing the Othe O22 affinity for the remaining subunits. affinity for the remaining subunits.

This creates the sigmoidal oxygen dissociation This creates the sigmoidal oxygen dissociation curvecurve

Normal Hemoglobin Normal Hemoglobin FunctionFunction

The hemoglobin dissociation curveThe hemoglobin dissociation curve

Normal Hemoglobin Normal Hemoglobin FunctionFunction

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 ) shifts the curve to the left (increased O(increased O22) affinity) affinity

A decrease in pH (inc. COA decrease in pH (inc. CO22) shifts the curve to the right ) shifts the curve to the right (decreased O(decreased O22 ) affinity ) affinity

– Temperature:Temperature: Increased temp with increased metabolic demands causes Increased temp with increased metabolic demands causes

decreased Odecreased O2 2 affinity (right shift) and increased Oaffinity (right shift) and increased O22 delivery delivery– 2,3 DPG:2,3 DPG:

Lowers OLowers O2 2 affinity by preferentially binding to affinity by preferentially binding to BetaBeta chain of chain of deoxyhemoglobin, stabilizing it and reduces the intracellular pHdeoxyhemoglobin, stabilizing it and reduces the intracellular pH

– As hemoglobin concentration decreases, 2,3 DPG increases, As hemoglobin concentration decreases, 2,3 DPG increases, allowing more Oallowing more O22 to be unloaded to be unloaded

Other Hemoglobins in normal Other Hemoglobins in normal adultsadults

HemoglobiHemoglobinn

StructureStructure %%

AA αα22 ββ22 92%92%AA22 αα22 δδ22 2.5%2.5%AA1C1C αα22 ( (ββ-N--N-

glucose)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

Other Hemoglobins in Other Hemoglobins in normal adultsnormal adults

HbAHbA22: : – Decreased in iron deficiency, alpha-thalassemiaDecreased in iron deficiency, alpha-thalassemia– Elevated in megaloblastic anemia, Elevated in megaloblastic anemia,

hyperthyroidism, Beta-thalessemiahyperthyroidism, Beta-thalessemia HbF:HbF:

– Elevated in HPFH, Sickle cell anemia (preferential Elevated in HPFH, Sickle cell anemia (preferential survival of RBCs because HgF inhibits sickling), survival of RBCs because HgF inhibits sickling), Beta thalessemia majorBeta thalessemia major

– Normal levels in Beta-thalassemia minorNormal levels in Beta-thalassemia minor– Normal or mildly elevated in congenital hemolytic Normal or mildly elevated in congenital hemolytic

anemiaanemia– Marked elevation in juvenile CML (up to 70%)Marked elevation in juvenile CML (up to 70%)

Hemoglobin AbnormalitiesHemoglobin Abnormalities There are 3 main categories of inherited There are 3 main categories of inherited

Hemoglobin abnormalities:Hemoglobin abnormalities:– Structural or qualitative: The amino acid Structural or qualitative: The amino acid

sequence is altered because of incorrect DNA sequence is altered because of incorrect DNA code (Hemoglobinopathy).code (Hemoglobinopathy).

– Quantitative: Production of one or more globin Quantitative: Production of one or more globin chains is reduced or absent (Thalassemia).chains is reduced or absent (Thalassemia).

– Hereditary persistence of Fetal Hemoglobin Hereditary persistence of Fetal Hemoglobin (HPFH): Complete or partial failure of (HPFH): Complete or partial failure of γγ globin globin to switch to to switch to ββ globin. globin.

Abnormal HemoglobinAbnormal Hemoglobin Reasons to suspect a hemoglobin Reasons to suspect a hemoglobin

disorder:disorder:– Patient presents with suspicious history Patient presents with suspicious history

or physical examor physical exam– Laboratory tests: Microcytic Laboratory tests: Microcytic

hypochromic RBCs, hemolytic anemiahypochromic RBCs, hemolytic anemia– Screening test abnormality (primarily in Screening test abnormality (primarily in

neonates)neonates)

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

Red cell morphologies:Red cell morphologies: – HbS: Sickle cellsHbS: Sickle cells

Sickle cells on peripheral Sickle cells on peripheral smearsmear

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

Red cell morphologies:Red cell morphologies:– HbS: Sickle cellsHbS: Sickle cells– HbC: Target cells, crystals after HbC: Target cells, crystals after

splenectomysplenectomy

HbC crystals with Target HbC crystals with Target cellscells

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

Red cell morphologies:Red cell morphologies:– HbS: Sickle cellsHbS: Sickle cells– HbC: Target cells, crystals after HbC: Target cells, crystals after

splenectomysplenectomy– Thalassemias: Microcystosis, target Thalassemias: Microcystosis, target

cells, basophilic stipplingcells, basophilic stippling

Alpha Thalassemia with Alpha Thalassemia with basophilic stipplingbasophilic stippling

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

Electrophoresis:Electrophoresis:– Alkaline (Cellulose Acetate) pH 8.6:Alkaline (Cellulose Acetate) pH 8.6:

All Hemoglobin molecules have a negative charge, All Hemoglobin molecules have a negative charge, and migrate towards the anode proportional to their and migrate towards the anode proportional to their net negative charge.net negative charge.

– Amino acid substitutions in hemoglobin variants alter Amino acid substitutions in hemoglobin variants alter net charge and mobility.net charge and mobility.

– Acid (Citrate agar) pH 6.2:Acid (Citrate agar) pH 6.2: Hemoglobin molecules separate based on charge Hemoglobin molecules separate based on charge

differences differences andand their ability to combine with the agar. their ability to combine with the agar.– Used to differentiate Hemoglobin variants that migrate Used to differentiate Hemoglobin variants that migrate

together on the cellulose gel (i.e. HbS from HbD and together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).HbG, HbC from HbE).

Hemoglobin Electrophoresis Hemoglobin Electrophoresis PatternsPatterns

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

High-Performance Liquid High-Performance Liquid Chromatography (HPLC):Chromatography (HPLC):– Weak cation exchange column. The Weak cation exchange column. The

ionic strength of the eluting solution is ionic strength of the eluting solution is gradually increased and causes the gradually increased and causes the various Hemoglobin molecules to have a various Hemoglobin molecules to have a particular retention time.particular retention time. Amino acid substitutions will alter the Amino acid substitutions will alter the

retention time relative to HbA.retention time relative to HbA. There is some analogy between retention There is some analogy between retention

time and pattern on alkaline electrophoresis.time and pattern on alkaline electrophoresis.

Normal HPLC patternNormal HPLC pattern

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

Solubility test Solubility test (Sickledex):(Sickledex):– Test to identify HbS. Test to identify HbS.

HbS is relatively HbS is relatively insoluble compared to insoluble compared to other Hemoglobins.other Hemoglobins.

– Add reducing agentAdd reducing agent– HbS will precipitate HbS will precipitate

forming and opaque forming and opaque solution compared with solution compared with the clear pink solution the clear pink solution seen in HbS is not seen in HbS is not present.present.

Most common Hemoglobin Most common Hemoglobin abnormalitiesabnormalities

ThalassemiasThalassemias– AlphaAlpha– BetaBeta

HemoglobinopathiesHemoglobinopathies– HbS trait; diseaseHbS trait; disease– HbC trait; diseaseHbC trait; disease– HbEHbE– Hereditary Persistence of Hemoglobin F Hereditary Persistence of Hemoglobin F

(HPHF)(HPHF)

Case 1Case 1 47 year old female 47 year old female

presents with a presents with a history of peptic history of peptic ulcer disease, H. ulcer disease, H. Pylori an anemia.Pylori an anemia.

Labs:Labs: Hgb: 10.2Hgb: 10.2 Hct: 30.9Hct: 30.9 MCV: 96.4MCV: 96.4 B12: 338B12: 338 Iron: 122Iron: 122 Ferritin: 304.5Ferritin: 304.5 IBC: 226IBC: 226

Case 1Case 1

HbF: 1.3%HbA2: 4.1%Sickledex test POSITIVE

Case 1Case 1

Case 1Case 1 Hemoglobin S/C disease:Hemoglobin S/C disease:

– Second most common hemoglobin variant Second most common hemoglobin variant in Africans; 1 in 1000 births of African in Africans; 1 in 1000 births of African AmericansAmericans

– Relatively benign condition; Milder disease Relatively benign condition; Milder disease than Sickle cell disease. Patients have than Sickle cell disease. Patients have normal growth and developmentnormal growth and development

– Do not see the classic sickle cellsDo not see the classic sickle cells– Peripheral smear reveals anisocytosis, Peripheral smear reveals anisocytosis,

target cells, poikilocytosis, polychromasiatarget cells, poikilocytosis, polychromasia

Case 1Case 1 Hemoglobin S/C disease:Hemoglobin S/C disease:

– Most patients have moderate Most patients have moderate splenomegaly with many having splenomegaly with many having autosplenectomy, usually older age than autosplenectomy, usually older age than with Sickle cell diseasewith Sickle cell disease

– May have veno-occlusive disease, but less May have veno-occlusive disease, but less common and less severe than in sickle cell common and less severe than in sickle cell diseasedisease

– May have aseptic necrosis of bone with May have aseptic necrosis of bone with osteomyelitisosteomyelitis

– ~50% HbS: 50% HbC; rarely is HbF >2%~50% HbS: 50% HbC; rarely is HbF >2%

Case 2Case 2 A 45 year old A 45 year old

German man who is German man who is asymptomatic is asymptomatic is seen for seen for microcytosis.microcytosis.

Peripheral smear Peripheral smear shows microcytosis, shows microcytosis, hypochromia, target hypochromia, target cells, basophilic cells, basophilic stippling, stippling, polychromasiapolychromasia

Labs:Labs: Hgb: 11.8Hgb: 11.8 Hct: 37.5Hct: 37.5 MCV: 65.9MCV: 65.9 Iron: 119Iron: 119 Ferritin: 506Ferritin: 506 IBC: 275IBC: 275 Fe Sat: 43%Fe Sat: 43%

Case 2Case 2

HbF: 1.6%* HbA2: 5.1%

Case 2Case 2Cellulose acetate gel performed

HbS

HbS

Case 2Case 2 Beta Thalassemia Minor:Beta Thalassemia Minor:

– The thalassemia seen most commonly is caucasians The thalassemia seen most commonly is caucasians (primarily Mediterranean descent)(primarily Mediterranean descent)

– Beta thalassemia minor is loss of one of two genes for Beta thalassemia minor is loss of one of two genes for Beta globin on chromosome 11Beta globin on chromosome 11

– Patients generally asymptomaticPatients generally asymptomatic– May have mild microcytic anemia (MCV: 60-70; Hgb: 10-May have mild microcytic anemia (MCV: 60-70; Hgb: 10-

13) with a normal or slightly increased RBC count13) with a normal or slightly increased RBC count– The peripheral smear will show target cells and basophilic The peripheral smear will show target cells and basophilic

stipplingstippling– See increased HbASee increased HbA22 in the range of 5-9% with normal HbF in the range of 5-9% with normal HbF– Thalassemia found most commonly in caucasiansThalassemia found most commonly in caucasians– See mild microcytosisSee mild microcytosis

Case 2Case 2 Beta Thalassemia Minor:Beta Thalassemia Minor:

– Primary indication is a slightly elevated HbAPrimary indication is a slightly elevated HbA22 detected by HPLC (usually around 4-7%, up to detected by HPLC (usually around 4-7%, up to 10%) typically without elevation of HbF10%) typically without elevation of HbF

– Diagnosis may be obscured in concomitant iron Diagnosis may be obscured in concomitant iron deficiency present because Beta-thalassemia deficiency present because Beta-thalassemia causes an increase in HbAcauses an increase in HbA22 while iron deficiency while iron deficiency causes a decrease in HbAcauses a decrease in HbA22. Both create a . Both create a microcytosis.microcytosis. May see a anemia that partially responds to iron therapyMay see a anemia that partially responds to iron therapy Always want to look at iron studies when interpreting Always want to look at iron studies when interpreting

hemoglobin electrophoresis; usually wait to diagnose hemoglobin electrophoresis; usually wait to diagnose until nutritional deficiencies have first been corrected.until nutritional deficiencies have first been corrected.

Case 2Case 2 Beta Thalassemia Major:Beta Thalassemia Major:

– Homozygous double gene deletion with no Beta Homozygous double gene deletion with no Beta globin productionglobin production

– Presents with lethal anemia, jaundice, Presents with lethal anemia, jaundice, splenomegaly, growth retardation, bone splenomegaly, growth retardation, bone malformations, deathmalformations, death

– Severe hypochromic, microcytic anemia with Severe hypochromic, microcytic anemia with very bizarre cells very bizarre cells

– HbAHbA22 is not increased is not increased– HgF is at nearly 100%HgF is at nearly 100%– Abundant intra-erythrocyte precipitation of alpha Abundant intra-erythrocyte precipitation of alpha

monomers that are insolublemonomers that are insoluble

Case 3Case 3 47 year old African 47 year old African

American female American female presents to the ER presents to the ER with drug with drug intoxication and intoxication and marked anemia. marked anemia. She is unable to She is unable to provide any provide any adequate history to adequate history to the clinicians.the clinicians.

Labs:Labs: Hgb: 5.9Hgb: 5.9 Hct: 17.8MCV: 97.1Hct: 17.8MCV: 97.1 RDW: 20.9RDW: 20.9 Iron: 83Iron: 83 Ferritin: 394.3Ferritin: 394.3 IBC: 144IBC: 144 Fe Sat: 58%Fe Sat: 58%

Case 3Case 3

Sickledex is POSITIVE; Peripheral smear with 2+ sickle cellsHbF: 1.0%; HbA: 38.7%; HbA2: 4.4%; HbS: 56.1%

Case 3Case 3

Case 3Case 3 Sickle cell anemia:Sickle cell anemia:

– In sickle cell trait, usually see HbS In sickle cell trait, usually see HbS concentrations of 35 to 45% of total concentrations of 35 to 45% of total Hemoglobin because the HbS has a Hemoglobin because the HbS has a slower rate of synthesis than HbAslower rate of synthesis than HbA If HbS is less than 33%, start thinking about If HbS is less than 33%, start thinking about

S-alpha-thalassemiaS-alpha-thalassemia If HbS is greater than 50%, worry about S-If HbS is greater than 50%, worry about S-

Beta-thalassemia or Sickle cell disease with Beta-thalassemia or Sickle cell disease with transfusiontransfusion

Case 3Case 3 Sickle cell anemia:Sickle cell anemia:

– This patient was transfused with two units This patient was transfused with two units of RBCs before the HPLC was performed.of RBCs before the HPLC was performed.

– It is important to know the appropriate It is important to know the appropriate ratios of HbS: HbA expected. If the ratios of HbS: HbA expected. If the patient does not fit, patient does not fit, alwaysalways look at the look at the transfusion history.transfusion history. If concerned about overlying Beta-If concerned about overlying Beta-

thalassemia, repeat HPLC after four months of thalassemia, repeat HPLC after four months of most recent transfusionmost recent transfusion

Case 3Case 3

HbAHbA HbSHbS HbAHbA22 HbFHbFHb ASHb AS 55-55-

606040-4540-45 2-32-3 <1<1

Hb SSHb SS 00 90-9590-95 2-32-3 5-105-10Hb S-Hb S-αα-thal-thal 7575 2525 2-32-3 <1<1Hb S- Hb S- ββ thal thal majormajor

00 90-9590-95 Inc.Inc. 5-105-10

Hb S- Hb S- ββ thal thal minorminor

5-305-30 60-9060-90 Inc.Inc. 5-105-10

Hb S HPFHHb S HPFH 00 70-8070-80 2-32-3 20-3020-30Hb SCHb SC 00 5050 2-32-3 <1<1

Expected ratios

Case 4Case 4 31 year old healthy 31 year old healthy

female, pregnant female, pregnant with moderate with moderate target cells target cells detected on detected on routine peripheral routine peripheral smearsmear

Labs:Labs: Hgb: 15.0Hgb: 15.0 Hct: 42.5Hct: 42.5 MCV: 87.8MCV: 87.8 MCH: 31.0MCH: 31.0 RDW: 12.6RDW: 12.6

Case 4Case 4

HbF: 0.6%; HbA2: 2.9%; HbA: 56.3%

Case 4Case 4

Case 4Case 4 Hemoglobin C trait:Hemoglobin C trait:

– Hemoglobin C trait (Heterozygotes) are Hemoglobin C trait (Heterozygotes) are clinically and hematologically wellclinically and hematologically well

– Moderate target cells seen on peripheral smearModerate target cells seen on peripheral smear– HbA and HbC in a 60:40 ratio on HPLCHbA and HbC in a 60:40 ratio on HPLC– 2% of African Americans have HbC trait2% of African Americans have HbC trait– Homozygotes have mild hemolytic disease, Homozygotes have mild hemolytic disease,

cholelithiasis and occasional aplastic crisis.cholelithiasis and occasional aplastic crisis. See reduced MCV with increased MCHCSee reduced MCV with increased MCHC

– Intracellular HbC crystals, block-like structures Intracellular HbC crystals, block-like structures may be seen and are pathognomonic of HbC.may be seen and are pathognomonic of HbC.

THE END!!!THE END!!!