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8/19/2019 Porphyrias, Hemoglobinopathies and Thalassemias
1/11
29/02/20
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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