porphyrias, hemoglobinopathies and thalassemias

8/19/2019 Porphyrias, Hemoglobinopathies and Thalassemias

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PORPHYRIAS,HEMOGLOBINOPATHIES ANDTHALASSEMIAS

PORPHYRIAS:

Impaired production of hemePrimary cause of porphyrias specific enzyme

deficiency

Acquired or hereditary

ACQUIRED PROPHYRIAS:

Lead poisoning / plumbism

Porphyria cutanea tarda

LEAD POISONING:

Lead inhibits pyrimidine 5’-nucleotidase

Basophilic stippling

Lab profile: hypochromic RBCs withbasophilic stippling, toxic granulation inneutrophils

PORPHYRIA CUTANEATARDA:

Acquired or inherited

Clinical feature: photosensitivity

Exacerbating factors: Alcohol, ironoverload, hepatic injury

HEREDITARY PORPHYRIAS:

1. Acute intermittent porphyria

2. Congenital erythropoietic porphyria

3. Hereditary coproporphyria

4. Variegate porphyria

5. Erythropoietic protoporphyria

6. Porphyria cutanea tarda

7. X-linked erythropoietic protoporphyria


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3 hematologically significant:congenital erythropoietic anderythropoietic protoporphyria, X-linked ErythropoieticProtoporphyria

CONGENITAL ERYTHROPOIETICPORPHYRIA

Associated with severe photosensitivity,scarring and excessive hair growth,deformity of the fingers and fingernails,reddish discoloration of the teeth andchronic hemolysis

Werewolf

HEMOGLOBINOPATHIES:

Hemoglobin variants

Due to the differences in the arrangement ofamino acid in the peptide chain

Differentiated from one another through:Solubility

Mobility in an electrophoretic field (cellulose actetatebuffer)

Electrophoresis


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DISEASE VS. TRAIT

Disease

either the homozygousoccurrence of the genefor the abnormality or thepossession of aheterozygous, dominantgene that produces ahemolytic condition.

Trait

heterozygous andnormallyasymptomatic state

must be inheritedfrom both parents

MAJOR GROUPS OFHEMOGLOBINOPATHIES:

Alpha-hemoglobinopathies: 2nd mostcommon

Beta hemoglobinopathies: most common

Gamma hemoglobinopathies

Zeta hemoglobinopathies

BETA HBPATHIES

2 major types:

Homozygous

Both beta genes are mutated

Abnormal Hb becomes the dominant Hb type

Hb A is absent

Ex: sickle cell disease (HbSS) and HbC disease (HbCC)

Heterozygous

1 beta gene is mutated, other is normal

Abnormal < Hb A

Ex: HbS trait (HbAS) and HbC trait (HbAC)


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SOME ABNORMAL HEMOGLOBIN:

1. S2. C3. E4. M-Saskatoon5. M- Milwaukee 16. M- Milwaukee 2 (Hyde

Park)

7. Hiroshima8. Rainier9. Bethesda10.Agenogi11.Beth-Irael12.Yoshizuka

M-SaskatoonM- Milwaukee 1

M- Milwaukee 2 (Hyde Park)

Methemoglobinemia and cyanosis

Hb M

Hiroshima

Rainier

Bethesda

Associated with increased oxygen affinity

Agenogi

Beth-Israel

Yoshizuka

Associated with decreased oxygen affinity


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HB S

Glutamic acid on the 6th position of the Beta- chain is replaced by valine

Defined by structural formula:

α2β26GluVal

Reduced oxygen conditions sickling of RBCs

Homozygous state causes sickle cell anemia

Heterozygous state has sickle cell trait

Patients with sickle cell trait seem resistant to P. falciparum

DITHIONITE TEST FOR HB S

Principle:

RBCs lysed by saponin

Na Dithionite removes oxygen from the test environment

Not confirmatory, just screening test

Hb S polymerizes in the resulting deoxygenated state and formssickle

Precipitate consists of tactoids(liquid crystals)

Tactoidsreflect and deflect lightTURBID solution (+ result isturbidity)


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SODIUM METABISULFITETEST:

Na metabisulfite: deoxygenates Hb

Under deoxy state, Hb S causes the formation of sicklecells

Procedure:

1 drop of blood (slide) + Na Metabisulfite (2 drops ifnormal Hb; 1 drop if decreased Hb)

Cover with glass slip and seal with petroleum jelly

Examine after 1 hour

* Sometimes RBCs may take on a “holly-leaf” form found in sickle cell trait and reported as positive

* No sickling after 1 hour allow the prep tostand at room temp for 24 hrs reexamine

* Sickle cells / holly leaf = Positive result

* Normal RBCs / slightly crenated = Negative result

HB C

Glutamic acid on the 6th position of BETA chain is replaced by lysine

Structural formula:

α2β26GluLys

2 crystals related to Hb C

Hb SC crystals: “Washington Monument” in appearance; crystals protruding RBCmembrane

Hb CC crystals: hexagonal, elongated crystals; crystals are formed within the RBCmembrane


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HbS

α2β26GluVal

HbC

α2β26GluLys

THALASSEMIA:

Greek – “thalassic” anemia (great sea)

Quantitative defects of globin synthesis

Reduction or total absence of synthesis of one or more of the globin

Microcytic and hypochromic anemia

Other abnormalities: NRBCs ( metarubricytes), polychromasia, basophilicstippling, Target cells

Alpha thalassemia: alpha globin chains are affected

Beta thalassemia: beta globin chains are affected

COOLEY’S ANEMIA

Described by Cooley and Lee

Anemia, splenomegaly, mild hepatomegaly and mongoloid facies

Untreated beta-thalassemia

GENETIC DEFECTS OF THALASSEMIA

Reduced or absent transcription of mRNA

mRNA processing errors

Translation errors

Deletion of one or more globin genes

PATHOPHYSIOLOGY

1. Reduced or absent production of globin chain(decreased Hb synthesis)

2. Unequal production of alpha and beta globinchains (decreased RBC survival)

Alpha thalassemia:

-α/αα = single gene deletion—silent carrier state

--/αα or –α/-α = two gene deletion—alphathalassemia minor

--/-α = three gene deletion—Hb H disease (β4)

--/-- = four gene deletion—Hb Bart (hydropsfetalis); increased oxygen affinity; (γ4)


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Hb H = 4 beta chains“pitted golf ball” / raspberry shaped when stained

with Brilliant Cresyl Blue

Hb H vs. Heinz bodies:Hb H inclusions—in multiples and cover the cell surface

Heinz bodies—eccentrically located and there are veryfew per cell

MECHANISM OF ALPHA THALASSEMIA

Can manifest in utero (component of fetal and adult Hb)

↓ alpha chain = ↑ gamma chain (fetus) do not precipitate but forms tetramers (γ4)or Hb Bart

After 6 months after birth, gamma will be replaced by beta

It will also for tetramers (β4) or Hb H

Patients with alpha thalassemia do not have severe ineffective RBCproduction

As the RBC matures, Hb H will form inclusion bodiesmild hemolyticanemia

Tissue hypoxia: Hb Bart and Hb H have high affinity to oxygen

Hydrops fetalis: tissue hypoxiacausing heart failure andmassive edema to the baby

Beta thalassemia:

Generally: increased HbA2 and Increased Hb F

Some clinical syndromes: Silent carrier state Beta thalassemia minor Beta thalassemia major—transfusion dependent Hb < 7g/dL, usually every 2-5 weeks Iron accumulation = chelation [deferoxamine(standard),

deferasirox and deferiprone(both are oral)] Beta thalassemia intermedia


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MECHANISMS IN BETA THALASSEMIA

The unpaired excess alpha chains will precipitate = inclusion bodies, oxidative

stress, apoptosis of RBC Ineffective erythropoiesis—premature death of the RBCs in the mar row

Severe beta thalassemia: asymptomatic at fetal life up to 6 months (Hb F) andmanifests after 6 – 24 months.

Erythroid hyperplasia (inc. cell number)

Bone deformities

Children: lacy/lucent appearance of the bone, “hair on end” skull appearance,frontal bossing, hepatosplenomegaly, jaundice

Thalassemia can be treated withHematopietic stem cell transplantation

Hb F induction agents: hydroxyl urea, 5-azacytidine, thalidomine derivatives (gammachains)

Lentiviral Beta globin chain transfer (2010)

DIAGNOSIS

History and physical exam

Laboratory methods: CBC with PBS

Retics

Supravital staining

Electrophoresis

HPLC

Capillary zone electrophoresis (CZE)

Molecular/ Genetic Testing

OTHER PROCEDURES:

Alkali denaturationMost human hemoglobins are denatured on

exposure to a strong alkali

Hb F resists alkaline denaturation


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OTHER PROCEDURES:

Kleihauer-betke acid elution slide testAlso used for estimation of fetal maternal hemorrhage

Make PBS, fix it with ethanol

Immerse in citrate acid buffer (pH 3.3)

Adult RBCs are eluted (will appear as ghost cells)

Hb F resists elution (it will take up the stain)

FETAL-MATERNAL HEMORRHAGE:leakage of fetal cells into the maternalcirculation is the mechanism throughwhich Rh sensitization arises

ELUTION:

The process of extracting one material fromanother by washing with a solvent to removeadsorbed material from an adsorbent

OTHER PROCEDURES:

Osmotic fragility testHypochromic RBCs have decreased osmotic fragility

0.375% saline for 5 mins.

Normal cells—lysis

Hypochromic cells—no lysis


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porphyrias, hemoglobinopathies and thalassemias

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