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PORPHYRIAS:

PORPHYRIAS,
HEMOGLOBINOPATHIES AND
THALASSEMIAS

Impaired production of heme

Primary cause of porphyrias specific enzyme
deficiency
Acquired or hereditary

ACQUIRED PROPHYRIAS:

LEAD POISONING:

Lead poisoning / plumbism

Porphyria cutanea tarda

Lead inhibits pyrimidine 5-nucleotidase

Basophilic stippling
Lab profile: hypochromic RBCs with
basophilic stippling, toxic granulation in
neutrophils

PORPHYRIA CUTANEA TARDA:

HEREDITARY PORPHYRIAS:

Acquired or inherited
Clinical feature: photosensitivity
Exacerbating factors: Alcohol, iron
overload, hepatic injury

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 and
erythropoietic protoporphyria, Xlinked Erythropoietic
Protoporphyria

CONGENITAL ERYTHROPOIETIC
PORPHYRIA

Associated with severe photosensitivity,

scarring and excessive hair growth,
deformity of the fingers and fingernails,
reddish discoloration of the teeth and
chronic hemolysis
Werewolf

HEMOGLOBINOPATHIES:
Hemoglobin variants
Due to the differences in the arrangement of
amino acid in the peptide chain
Differentiated from one another through:

Electrophoresis

Solubility
Mobility in an electrophoretic field (cellulose actetate
buffer)

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MAJOR GROUPS OF
HEMOGLOBINOPATHIES:

DISEASE VS. TRAIT

Disease

Trait

either the homozygous

occurrence of the gene
for the abnormality or the
possession of a
heterozygous, dominant
gene that produces a
hemolytic condition.

heterozygous and
normally
asymptomatic state
must be inherited
from both parents

Alpha-hemoglobinopathies: 2nd most

common
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: Hb S trait (Hb AS) and Hb C trait (Hb AC)

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SOME ABNORMAL HEMOGLOBIN:

1. S
2. C
3. E
4. M-Saskatoon
5. M- Milwaukee 1
6. M- Milwaukee 2 (Hyde
Park)

7. Hiroshima
8. Rainier
9. Bethesda
10.Agenogi
11.Beth-Irael
12.Yoshizuka

Hiroshima
Rainier
Bethesda

Associated with increased oxygen affinity

M-Saskatoon
M- Milwaukee 1
M- Milwaukee 2 (Hyde Park)

Methemoglobinemia and cyanosis

Hb M

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:

226GluVal

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 forms
sickle
Precipitate consists of tactoids (liquid crystals)
Tactoids reflect and deflect light TURBID solution (+ result is
turbidity)

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SODIUM METABISULFITE TEST:

Na metabisulfite: deoxygenates Hb
Under deoxy state, Hb S causes the formation of sickle
cells
Procedure:
1 drop of blood (slide) + Na Metabisulfite (2 drops if
normal 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 to
stand 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:

226GluLys
2 crystals related to Hb C
Hb SC crystals: Washington Monument in appearance; crystals protruding RBC
membrane
Hb CC crystals: hexagonal, elongated crystals; crystals are formed within the RBC
membrane

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THALASSEMIA:
Hb S

226GluVal

Hb C

226GluLys

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, basophilic
stippling, Target cells
Alpha thalassemia: alpha globin chains are affected
Beta thalassemia: beta globin chains are affected

COOLEYS ANEMIA

GENETIC DEFECTS OF THALASSEMIA

Described by Cooley and Lee

Reduced or absent transcription of mRNA

mRNA processing errors
Translation errors
Deletion of one or more globin genes

Anemia, splenomegaly, mild hepatomegaly and mongoloid facies

Untreated beta-thalassemia

PATHOPHYSIOLOGY
1. Reduced or absent production of globin chain
(decreased Hb synthesis)
2. Unequal production of alpha and beta globin
chains (decreased RBC survival)

Alpha thalassemia:
-/ = single gene deletionsilent carrier state
--/ or /- = two gene deletionalpha
thalassemia minor
--/- = three gene deletionHb H disease (4)
--/-- = four gene deletionHb Bart (hydrops
fetalis); increased oxygen affinity; (4)

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MECHANISM OF ALPHA THALASSEMIA

Hb H = 4 beta chains
pitted golf ball / raspberry shaped when stained
with Brilliant Cresyl Blue
Hb H vs. Heinz bodies:
Hb H inclusionsin multiples and cover the cell surface
Heinz bodieseccentrically located and there are very
few per cell

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

alpha chain = gamma chain (fetus) do not precipitate but forms tetramers (4)

or Hb Bart
After 6 months after birth, gamma will be replaced by beta
It will also for tetramers (4) or Hb H
Patients with alpha thalassemia do not have severe ineffective RBC
production
As the RBC matures, Hb H will form inclusion bodies mild hemolytic
anemia
Tissue hypoxia: Hb Bart and Hb H have high affinity to oxygen

Hydrops fetalis: tissue hypoxia

causing heart failure and
massive edema to the baby

Beta thalassemia:
Generally: increased Hb A2 and Increased Hb F
Some clinical syndromes:

Silent carrier state

Beta thalassemia minor
Beta thalassemia majortransfusion 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 erythropoiesispremature death of the RBCs in the marrow
Severe beta thalassemia: asymptomatic at fetal life up to 6 months (Hb F) and
manifests 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 with

Hematopietic stem cell transplantation
Hb F induction agents: hydroxyl urea, 5azacytidine, thalidomine derivatives (gamma
chains)
Lentiviral Beta globin chain transfer (2010)

DIAGNOSIS

OTHER PROCEDURES:

History and physical exam

Alkali denaturation

Laboratory methods:

CBC with PBS

Retics
Supravital staining
Electrophoresis
HPLC
Capillary zone electrophoresis (CZE)
Molecular/ Genetic Testing

Most 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 test

Also used for estimation of fetal maternal hemorrhage
Make PBS, fix it with ethanol
Immerse in citrate acid buffer (pH 3.3)

FETAL-MATERNAL HEMORRHAGE:
leakage of fetal cells into the maternal
circulation is the mechanism through
which Rh sensitization arises

Adult RBCs are eluted (will appear as ghost cells)

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

ELUTION:
The process of extracting one material from
another by washing with a solvent to remove
adsorbed material from an adsorbent

OTHER PROCEDURES:

Osmotic fragility test

Hypochromic RBCs have decreased osmotic fragility
0.375% saline for 5 mins.
Normal cellslysis
Hypochromic cellsno lysis

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