Fall 2012 13 Hem 1 Chapter 13 Hemoglobinpathies(2)

8/10/2019 Fall 2012 13 Hem 1 Chapter 13 Hemoglobinpathies(2)

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Learning objectives:

1.

Describe the common denominator in hemoglobinopathies & name the3 major categories of classification of hemoglobin defects.

2.

Describe the etiology of sickle cell disease (SCD), discuss its

epidemiology & describe its clinical signs and symptoms

3.

Outline laboratory findings that are typical of SCD and briefly describe

the various approaches used for SCD diagnosis.

4.

Compare the conditions of !- and "-thalassemia and outline the

laboratory findings in the various forms of thalassemia.

5.

Describe the conditions of sickle "-thalassemia, sickle-C (SC), andsickle cell trait.

6.

Describe the general characteristics of hemoglobin (Hb) C disease, HbSC disease, Hb D disease, Hb E disease, Hb H disease,

methemoglobinemia & unstable hemoglobins.

Chapter 13

Hemoglobinopathies


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Introduction to Hemoglobinopathies

A group of genetically determined abnormalities of the structureorsynthesisof the globin chain; the heme group is normal

Most common in people of African, Mediterranean, or SoutheastAsian origin

Majority of hemoglobinopathies result from !-globin chainabnormalities.

They frequently associate with chronic hemolytic anemia and othercomplications

Globin chain abnormalities are either qualitative defects (structuralabnormalities) or a quantitative defect of the globin chain synthesis

Qualitative abnormal hemoglobin molecules result from genetic

mutation involving amino acid deletions or substitutions in theglobin protein chain; most common disorder of this type issickle cell anemia.

Quantitativeglobin disorders result from genetic defects thatlead to reduced synthesis of globin chains. This type of

quantitative disorders is known as thalassemias.


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Race or ethnic

group

Average

prevalence per

100,000 live births

White 1.72

Black 289

Hispanic, total 5.28

Hispanic, eastern

states89.8

Hispanic, westernstates

3.14

Asian 7.61

Native American 36.2

Prevalence of SCD by race

or ethnic group in the US

Prevalence of SCD

worldwide


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Sickle Cell Disease (SCD)

SCD is the most common type of hemoglobinopathy aroundthe world; about 7% of world population carry the mutation.

Greatest prevalence is in Africa (central Africa); but alsocommon in the Middle East, Mediterranean, India & Nepal.

Geographic areas with the highest frequency of sickle cell

gene are also areas where infection withPlasmodiumfalciparumis common!suggesting that individuals withHbS trait are resistant to malarial infections (WHY ???)

Sickle cell disease is mostly inherited as an autosomal

recessive trait with over-dominance(= heterozygotes havea selective advantage over homozygotes). HbS heterozygotes are carriers of the defect with little or no clinical

consequences

HbSS homozygotes suffer from sickle cell disease!significant

clinical consequences


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Mode of inheritance of SCD / SC trait


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Other forms of SCD:

Compound heterozygous states in which the person has only

one copy of the mutation that causes HbS and one copy of

another abnormal hemoglobin allele could also lead to SCD.

Such forms include:

Sickle / hemoglobin C disease (HbSC)

Sickle / beta-plus-thalassaemia (HbS/!+)

Sickle / beta-zero-thalassaemia (HbS/!0)


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Etiology of SCD

HbS is the hemoglobin that isproduced when valine

(hydrophobic) substitutesglutamic acid (negativelycharged) at the sixth position inthe !chain

This substitution is on the surfaceof the molecule!a change of netcharge! Changes electrophoretic mobility

of the molecule

In the deoxygenated form,solubility of HbS is markedly

reduced, producing a tendencyfor deoxyhemoglobin Smolecules topolymerizeintorigid aggregates

Following polymerization, thecell assumes a crescent or sickleshape


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Hemoglobin S polymer formation

Normal

Sickle Cell Disease


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Electron micrograph ofHemoglobin S polymers


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Etiology (cont)

The sickling process is enhanced by: Hypoxia

Acidosis

Extreme temperature (high or low)

Hypertonicity of microenvironment Concentration of HbS within erythrocyte itself

(MCHC)

Presence of other intracellular hemoglobin

variants (the proportion of HbS to HbA &HbF)!presence ofHbA or HbF tends to

dilute (minimize) the sickling process


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Sickle Cell Disease


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Etiology (Cont)

When sickled cells attempt to travel through small vessels,

they get stuck!vessels become obstructed. This initiates a pattern of blood not flowing properly to

tissue and creating a lack of oxygen.

Lack of oxygen (hypoxia) causes more sickling and more

deprivation of oxygen to tissue. This process can produce intense pain.

When sickled cells receive oxygen, they return to their

normal shape

Repeated cycles of sickling & unsickling lead to red celldamage, resulting in hemolysis & anemia

Additionally, sickled cells have high tendency to adhere to

vascular endothelial cells of small vessels, leading to

vaso-occlusion painful crises!ischemic injury


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Sickle cell Disease

Normal RBCs

! flexible, disc

shaped

!

move easilythroughout blood

vessels

! lasts four months

in bloodstream

Abnormal RBCs

!stiff, curved shape

resembling a sickle

(crescent moon shaped)!

clogs blood vessels

! last 10-20 days in

bloodstream which can

lead to anemia


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Clinical signs and Symptoms

Sickling leads to damage & defects in various body organs & tissues!enlarged heart, progressive loss of pulmonary & renal function, strokes,arthritis, liver damage, skeletal damage!leads to crises of differentforms: vaso-occlusive crisis, aplastic crisis, sequestration crisis&hemolytic crisis; most episodes of crises last for 5-7 days.

Symptoms usually appear after the age of 6 months

Symptoms of the disease include:

Severe hemolytic anemia

Vaso-occlusion symptoms: develops between 12 months- 6 years.

Hand-Foot syndrome (resulting from vaso-occlusion crises)

Infection (Streptococcus pneumonia and Haemophilus influenzae) isthe major cause of death among children below age of 5 years.

Leg ulcers

Aplastic crises due to viral infection Lagging growth & development

Bone and joint destruction result from repeated ischemia andinfarctions.

Pulmonary complications

Strokes


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Laboratory Findings

Decreased hemoglobin (10-5 g/dL)

Decreased hematocrit, red cell count, and

increased WBC count.

Blood film: Anisopoikilocytosis, hypochromia,

target cells, microcytes, polychromasia, red cellfragments, and sickled red cells.

Reticulocytosis

Increased unconjugated Bilirubin

Decreased haptoglobin & hemopexin


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Laboratory Investigations of SCD

1. Solubility test

Principle: in the deoxygenated form, HbS becomesinsoluble & precipitates!causes the solution to be turbid.

Procedure: Few drops of whole blood in a test tube are mixed with a

solution containing ) hemolyzing agent (saponin) & areducing agent (sodium dithionite).

The tube is held in front of a white card with narrow blacklines and turbidity is read in comparison of a negative and

positive controls.

If the black lines cannot be seen the solution is turbid & thetest is positive for HbS.

The test gives positive results for HbAS, HbSS, & HbSC

It does not differentiate between heterozygous andhomozygous forms.

Hence, a positive test is further confirmed by electrophoresis.


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Solubility TestRed Cells

+

Saponin

+

Sodium Dithionite

Positive Negative


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2. Sickling test of Whole blood

The sickling phenomenon can also bedemonstrated by making a thin wetfilm of whole blood:

a small drop of blood is added to aslide

mix with a reducing agent likesodium metabisulfite orsodiumdithionite & covered with a coverslip

Cover slip is sealed at the edges

observation of sickled cells underthe microscope indicate a positiveresult for HbS

But test does not differentiatebetween heterozygous andhomozygous states

Hence, Hb electrophoresis shouldfollow


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3. Hemoglobin Electrophoresis

Cellulose Acetate Electrophoresis at Alkaline pH

At alkaline pH (pH=8.6), hemoglobins will be

negatively charged!Migrate from cathode (negative

pole) to anode (positive pole)

Hemoglobin variants with highest negative charge will

move the fastest

At alkaline pH, hemoglobins D & G migrate with HbS

while hemoglobins C & E migrate with HbA2. Therefore, electrophoresis at acidic pH should be done

to differentiate these hemoglobins from each others.


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Hemoglobin ElectrophoresispH 8.6

Cathode (-) Anode (+)

HbAA

HbAS

HbSS

PA A2 S F AD

G

C

E


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Hemoglobin Electrophoresis

pH 8.6

Cathode (-) Anode (+)

AA

AS

SS

SC

AC

CC

A2 S F AC D

E G


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Hemoglobin Electrophoresis (Cont)

Citrate Agar Electrophoresis (Acidic pH, 6.2)

Citrate agar electrophoresis separates

hemoglobin fractions that migrate together on

cellulose acetate.

These fractions are hemoglobins S, D, G,

C, E & A2as shown in diagram below


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AA |

AS |

SS |

SC |

AC |

AE? |

Thal Major |

S-Thal |

C S A F

A2

G

D

E

Cathode Anode

CitrateAgarElectropho

resis(AcidicpH,

6.2

)


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Treatment of Sickle Cell Disease

There is no cure for SCD but symptoms can be treated.

Crises accompanied by extreme pain, common problem, istreated with pain relievers.

Special precautions are taken before any type of surgery;

for major surgery some patients receive transfusionsto boost

[Hb]. Blood transfusions may also be used to treat/preventanemia, spleen enlargement, and recurring strokes.

Infants diagnosed with the disease receive daily doses of

penicillin to prevent infections.

Adults with SCD now take hydroxyurea, an anticancerdrug that causes the body to produce RBCs resistant to

sickling!average life expectancy in the US of SCD patients

increased from 42 years in males & 48 years in females to >50

yrs.


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Thalassemia

Basics of Hb structure & synthesis


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Adult Hb is made of 2 gene loci: !

globin locuson chromosome 11 and

the "locus on chromosome 16"locus contains 2 copies of the same

gene aligned one after the other!

total "genes/cell = 4); each

contributes ~25% of the total "

globin chains made in the cell.The 2 !globin genes are active

during fetal growth and produce HbF.

Very early on during embryonic

development, 2 "("1 & "2) make

chains instead of "globin chains)

Adult gene, !becomes active after

birth.

Each of the four globin genes

contribute to the synthesis of the

HbA protein.

Basics of Hb synthesis


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RBC Hb composition under normal conditions

Hemoglobin Structural formula

Adult Hb-A #2 $2 97%

Hb-A2

#2%

21.5-3.5%

Fetal Hb-F #2 !2 0.5-1%

Hb-Barts !4

Embryonic Hb-Gower 1 "2 &2

Hb-Gower 2 #2 &2

Hb-Portland "2 !2


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Hemoglobinopathies

Thalassemia Thalassemias result due to absence or reduced synthesis of "

or !chain protein.

Inherited as autosomal recessive.

Globin genes are located at chromosomes 11 (beta chain) and

16 (the alpha chain).

Only one gene per chromosome, (two per diploid cell),

specifies the !-chain.

Two genes on each homologous chromosome (4/diploid

cell), specify the inheritance of the "-globin chain.

Deficiency of the "-chain lead to alpha thalassemia

deficiency of the beta chain lead tobeta thalassemia.


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Classification & Terminology

Beta Thalassemia

Normal $/$

Minor (heterozy.) $/$0

$/$+

Intermedia $0/$+

Major (homozy.) $0/$0

$+/$+

Keep in mind that +means that there is some synthesis

within a wide range (from very little to almost normal)


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!-Thalassemias

1. !-thalassemia minor:the heterozygous, characterized by:

mild anemia with microcytosis & abnormal erythrocytesmorphology

splenomegaly

2. Thalassemia intermedia: anemia is moderate with presenceof HbA in addition to HbF

3. !-thalassemia major (Cooleys anemia):the homozygous

form, characterized by:

severe anemia

transfusion dependence

organ damage secondary to iron overload

extramedullary erythropoiesis (bone damage)


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!-Thalassemia minor

Lab Findings:

Beta Thalassemia minor could be mistaken by

mild iron deficiency anemia on peripheral blood

film.

Characterized by increased HbA2 (diagnostictest)and decreased MCV.

Normal range for HbA2 is 1.5-3.5%, but in thalassemiaminor it is 3.5-8.0%


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!-Thalassemia Major

Symptoms appear several months after birth following the switchfrom #to !chain synthesis

Pathophysiology:Decreased synthesis of !chain leads to excessof "-chain!excess free "chains are unstable and precipitatewithin the cell causing membrane damage!contributes todestruction of RBCs and development of anemia

Lab Findings:

Decreased Hb, Hct & RBC count

Significantly reduced MCV, MCH, and MCHC

Anisocytosis, poikilocytosis, hypochromia, target cells,polychromasia, and nRBCs.

Increased RDW, reticulocytes, bilirubin, serum iron & serumferritin.

Electrophoresis reveals increased HbF & decreased/absent HbA


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"-Thalassemia

Major cause of "-thalassemia is deletion of one or more of

the genes coding for "-chain on chromosome 16.

Can be classified into four types according to number ofgene deletion:

1.

Silent carrier(one gene is inactive)!three remaining

genes can synthesize adequate amount of "producingnormal amount of Hb!no anemia

2. "-thalassemia trait(two genes are inactive)!imbalance of"- and !-chain synthesis creates an excess in !chains.

These excess !chains may aggregate forming a tetramer(!4) known as HbH. HbHis an unstable hemoglobin that

precipitates on cell membrane. Affected individuals areclinically normal but frequently have minimal anemia andreduced MCV & MCH; RBC count is usually increased,

typically exceeding 5.5 $1012

/L.


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Classification & Terminology Alpha Thalassemia

Normal ##/## Silent carrier - #/##

Minor -#/-#

--/##

Hb H disease --/-#

Barts (hydrops fetalis) --/--


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3. Hemoglobin H disease(three genes are inactive)!excess!-chains aggregate forming again HbH!precipitate oncell membrane!cell damage & short RBC life span.

HbH inclusions (precipitate) are detected when cellsare stained with brilliant cresyl blue.

HbH associates with chronic, moderately severehemolytic anemia; Hb ranges from 8-10 g/dL & allRBCindices are decreased.

HbH migrates ahead of HbA in electrophoretic gel.Hb electrophoresis reveals HbH 4-30% (ahead ofHbA); may also show a small amount of Hb Barts(#4).

4. Hydrops fetaliswith Hb Barts (four genes are inactive)!incompatible with life. Affected fetuses die either in uteroor shortly after birth. On electrophoresis mainly Hb Bartsis present.


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Other Hemoglobinopathies

Hemoglobin Structural formula

Hb-S #2 $26 glu 'val

Hb-C #2 $26glu 'lys

Hb-E #2 $226 glu 'lys

Hb-D Punjab #2 $2121 glu 'gln


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Other Hemoglobinopathies

1. Hemoglobin C disease (HbC)

Differ from HbA by the substitution of lysine instead ofglutamic acid at position 6 of the beta globin chain.

DeoxyHbC has decreased solubility and forms

intracellular crystals (cigar-shaped crystals).

Homozygous form (HbCC) results in mild chronichemolytic anemia with >50% target cells in blood film.

Hemoglobin C trait (HbAC) is symptomless, with target

cells and mild hypochromia.

At alkaline pH electrophoresis, HbC migrates with A2.

At acidic pH it remains at origin


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2. Hemoglobin SC disease (HbSC)

Result from the inheritance of one S gene and one C

gene; milder than SCD (SS) although HbC tends toenhance sickling. Blood film reveals target cells, foldederythrocytes & intracellular crystals.

3. Hemoglobin D disease (Hb D)

Both homozygous and heterozygous are asymptomatic.HbD migrates at same position as HbS & HbG atalkaline pH but migrates with HbA at acidic pH.

4. Hemoglobin E disease (Hb E) In some areas of Thailand, frequency of HbE trait is

almost 50%; heterozygous (HbAE) is asymptomaticwhile homozygous (HbEE) is mildly anemic.


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Patient with thalassemia major:

Note the prominent target cells, anisopoikilocytosis & 3

nucleated red cells (normoblasts)


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Peripheral blood smear from a patient with !-thalassemia

major showing marked anisopoikilocytosis: target cells,

schistocytes, teardrops, and ovalocytesn, RBCs. (Wright-

Giemsa stain)


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Alpha thalassemia


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Brilliant Cresyl Blue Stain

Incubation with

brilliant cresyl bluestain causes HbH toprecipitate

Results incharacteristic

appearance ofmultiple discreteinclusions -golf ballappearance of RBCs.

Inclusions smaller

than Heinz bodiesand are evenlydistributedthroughout cell.

44


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Acid Elution Stain for Detection of HbF

Based on Kleihauer-Betke procedure.

Acidic pH will dissolveHbA from RBCs but notHbF because HbF is

resistant to denaturation& remains in the cell.

Stain slide with eosin.

Normal adult cells

appear as "ghost" cellswhile cells with HbFstain varying shades of

pink.

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"ghost" cells

Cells with HbF


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Sickle CellsSickle cells (drepanocytes) contain a sickling HbS which

polymerizes into long rigid crystals upon exposure to decreased

oxygen or low pH!sickle shape with decreased ability to pass

through small vessels & increased mechanical fragility.


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Hemoglobin C Crystals


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Hemoglobin H Inclusions (Golf ball-shape)


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Differential Diagnosis of Microcytic, Hypochromic

Anemias

RDW Serum Iron TIBC Serum Ferritin

Iron Deficiency Inc Dec Inc Dec

Alpha Thal Norm Norm Norm Norm

Beta Thal Norm Norm Norm Norm

Hgb E Disease Norm Norm Norm Norm

Anemia of Chronic

Disease

Norm Dec Dec Inc

SideroblasticAnemia

Inc Inc Norm Inc

Lead Poisoning Norm Norm Norm Norm

Documents

Fall 2012 13 Hem 1 Chapter 13 Hemoglobinpathies(2)