Anemia - tutorial
Hemopoiesis
• pluripotent stem cell
– able to give rise to any
blood cell
– properties
 self-renewal
 proliferation and
differentiation into
progenitor cells
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Hemopoietic growth factors
• glycoproteins
• act on the cytokine-receptor
superfamily
• stimulating factors
– erythropoietin
– IL-3, 6, 7, 11, 12
– thrombopoietin
 produced in the kidney and
liver
 controls platelet production
• inhibiting factors
– TNF-α, TGF-β
• use in treatment
– G-CSF
 accelerate recovery after
chemotherapy and hemopoietic
cell transplantation
– EPO
– thrombopoietin receptor agonist
 treatment of immune
thrombocytopenic purpura
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The formation of blood cells
• hemopoietic system
– bone marrow, liver
– spleen, lymph nodes
– thymus
• huge turnover of cells
– tight regulation according to
the needs of the body
• survival
– RBC 120 days
– platelets 7 days
– granulocytes hours
• hemopoiesis during life
– at birth
 in the marrow of nearly
every bone
– as the child grows
 gradually replaced by fat
– in the adult
 central skeleton
 proximal ends of the long
bones
– extramedullary hemopoiesis
 hemopoietic activity in the
liver and spleen
– pathological processes
interfering with normal
hemopoiesis
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Erythropoiesis
• several stages in the bone
marrow
– earliest recognizable is
pronormoblast
– smaller normoblasts result from
cell division
– precursors at each stage contain
less DNA and more Hb in the
cytoplasm
– nucleus becomes more
condensed and lost from the
late normoblast
 reticulocyte
– residual ribosomal RNA
– synthesize hemoglobin
– in the marrow 1 – 2 days
– into circulation
• loose their RNA
• become RBC after 1-2
days
• normoblasts
– in peripheral blood
 normally not present
 present if there is
extramedullary hemopoiesis
• physiologic erythropoiesis
– 10 % of erythroblast may die in
the bone marrow
• erythropoietin
– polypetide, 175 AA, 30kDa
– produced in the kidney (90 %)
and liver (10 %)
– production regulated by oxygen
tension
 hypoxia – HIF-1
 increases proportion of bone
marrow precursors committed
to erythropoiesis
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Erythropoiesis during ontogenesis
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Hemoglobin synthesis
• performs main function of RBC
• adult Hb molecule (HbA)
– 2 α chains, 2 β chains (α2β2)
– 97 % of the Hb in adults
• other types
– HbA2 (α2δ2): 1.5 – 3.2 %
– HbF (α2γ2): < 1 %
• synthesis in the mitochondria
– production of aminolevulinic acid
 ALA synthase
 rate-limiting step
 coenzyme vitamin B6
 inhibited by heme
 stimulated by EPO
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RBC production and
breakdown
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Anemia
• decrease in Hb in the blood below
the reference level for the age and
sex of the individual
– men 135 – 175 g/l
– women 120 – 160 g/l
• classification by MCV
– hypochromic microcytic with a low MCV
– normochromic normocytic with a normal
MCV
– macrocytic with a high MCV
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Classification of anemia
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Anemia classification
• pathogenetic
– increased RBC loss
 bleeding
 hemolytic anaemia
– corpuscular
• membrane
• hemoglobinopathy
• enzymopathy
– extracorpuscular
• toxic
• autoimmune
– insufficient RBC production
 lack of erythropoietin
 lack of essential factors
 bone marrow disorder
• morphologic
– RBC size
– haemoglobin content
– pathologic morphology
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Abnormalities of RBC morphology
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Symptoms
• symptoms
– fatigue, headaches and
faintness
– breathlessness
– angina
– intermittent claudication
– palpitations
• signs
– pallor
– tachycardia
– systolic flow murmur
– cardiac failure
• investigations
– peripheral blood
 RBC indices
 WBC count
 platelet count
 reticulocyte count
 blood film
– bone marrow
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Microcytic anemia
• causes
– iron deficiency
 most common cause affecting 30 % of world‘s population
– anemia of chronic disease
– sideroblastic anemia
– thalassemia
 defect in globin synthesis, other causes – defect in the synthesis of heme
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Disorders characterized by microcytosis
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Iron metabolism
• 15 – 20 mg/day in the
average diet in the UK
– 10 % is absorbed
– 20 – 30 % in iron deficiency
and pregnancy
• source
– non-haem iron
 main part of dietary iron
 cereals
– commonly fortified with
iron
– haem iron
 haemoglobin and
myoglobin from meat
 better absorbed than nonhaem
iron
• iron stores
– 2/3 in the circulation
 haemoglobin
 2.5 – 3 g in adult man
– stored in cells
 hepatocytes
 reticuloendothelial cells
 skeletal muscle cells
 500 – 1500 mg
 2/3 ferritin
 1/3 hemosiderin
– small amounts
 plasma
 myoglobin
 enzymes
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Regulation of iron absorption
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Iron metabolism
• free iron is toxic
– ferritin
 liver, spleen, bone marrow
 plasma levels correlate with
iron stores
– hemosiderin
 from degraded ferritin
 usually only trace amount
 ↑ in iron overload
• balance ensured by regulation of
absorption
– mechanism of excretion does not
exist
– absorption of haem and non-haem
iron differs
– regulation of absorption
 hepcidine
– produced in the liver
– released when stores are ↑
– effect on hepatocytes
– supresses release of iron from
macrophages
• important in anaemia of
chronic disease
• proinflammatory madiators
increase production of
hedcidin
– ↓ hepcidin in hemochromatosis
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Iron deficiency anemia
• inadequate iron for
haemoglobin synthesis
• cause
– blood loss
– increased demands
 growth, pregnancy
– decreased absorption
 post-gastrectomy
– poor intake
• etiology
– food deficiency
 infants
 developing countries
–  absorption
 absorption is supported by
– ascorbic acid
 supressed by
– oxalates, phosphates,
tanins
 malabsorption syndrome
 diarrhea
 gastrectomy
–  demands
 growth, pregnancy
–  losses
 chronic
– GIT bleeding
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Iron deficiency anemia
• hypochromic microcytic
anemia
– after depletion of stores
  serum iron, ferritin and
transferin saturation
 absence of stainable iron in
the macrophages from bone
marrow
• diagnostics
–  hemoglobin, hematocrit
–  iron, ferritin and transferrin
saturation (< 15 %)
• iron supplementation
–  reticulocyte count after 5 –
7 days
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Sideroblastic anemia
• inherited or acquired disorder
• inadequate use of iron
– accumulation in mitochondria of
erythroblasts
 ring sideroblast
– an erythroblast with stainable iron
granules in its cytoplasm
– defect of ALA-synthase
 inherited form
– excess iron in bone marrow
• acquired
– alcohol, drugs
• mutations
– protoporphyrine production is slow
• variable number of hypochromic
microcytes
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Anemia of chronic disease
• common
•  proliferation of RBC precursors
• deteriorated utilization of iron
• causes
– chronic microbial infection
– autoimmune disease
 rheumatoid arthritis
– cancer
 lung cancer
• systemic inflammation
– hepcidin stimulation (IL-6)
 supression of iron release from
macrophages
–  supplementation of RBC
precursors
 defence from bacteries that utilize
iron (H. influenza)
– structural similarity between
hepcidin and defensins
– production of EPO is supressed
• anemia
– mild
– normochromic and
normocytic or hypochromic
microcytic
–  serum ferritin,
–  iron in macrophages
– treatment
 correction of the cause
 possibly EPO
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Normocytic anemias
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Anemia due to blood loss
• acute
– intravascular volume loss
 amount is important
 cardiovascular collapse,
shock, death
– volume repletion
 water shift, ↓ hematocrite
– ↑ EPO production
– iron loss
– blood pressure decrease
 epinephrine release
 granulocytes mobilization
– leukocytosis
– reticulocytosis
 10 – 15 % after 15 days
– thrombocytosis
• chronic
– less activated erythropoiesis
– losses may exceeed
regeneration capacity of
bone marrow
– iron stores depletion
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Aplastic anemia
• chronic failure of hematopoiesis
• etiology
– idiopathic (65 %)
– chemicals
 benzene
 alkylation agents
 antimetabolites
– viral infection
 hepatitis, CMV, EBV
– radiation
– hereditary defects
– Fanconi anemia
 DNA reparation disorder
 defects of telomerase
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Aplastic anaemia
• pathogenesis
– extrinsic cause
 change of stem cells‘
antigens
 activation of Th1
– cytokines, destruction of
progenitors
 up-regulation of
proapoptotic genes
 immunosupresive therapy
– intrinsic cause
 karyotype changes
 short telomeres
• morphology
– hypocellular bone marrow
– common infection, bleeding
• signs
– pancytopenia
 anaemia, petechia,
infection
 reticulocytopenia
• treatment
– transplantation
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Macrocytic anemias
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Megaloblastic anemia
• erytroblasts with delayed nuclear
maturation in the bone marrow
• defective DNA synthesis
– abnormally large RBC and their
precursors
– immature nuclei
• deficit of vitamin B12 or folic acid
– thymidine synthesis
– defects in nucleus maturation
 delay or blocade of cell
division
• morphology
– macrocytes or macroovalocytes
 central brightening is missing
but MCHC is not increased
 anisocytosis, poikilocytosis
 ↓ reticulocytes
 neutrophils
– larger and hypersegmented
 hypercellular bone marrow
 maturation of cytoplasm and
Hb accumulation is normal
 ↑ growth factors
– apoptosis of precursors in
the bone marrow
 mild hemolysis
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Biochemical basis of megaloblastic anemia
• key biochemical problem
– block in DNA synthesis
 inability to methylate deoxyuridine
monophosphate to deoxythimidine
monophosphate
 folate deficiency
– ↓ supply of methylene
tetrahydrofolate
 vitamin B12 deficiency
– slowing the demethylation of
methyl thetrahydrofolate
• other forms
– interference with purine or
pyrimidine synthesis causing an
inhibition of DNA synthesis
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Vitamin B12 metabolism
• vitamin B12 = cobalamin
• essential
• animal sources
– meat, fish, egg, milk
– not in plants
– usually not destroyed by cooking
• stores in the liver
• alternative resorption
– without IF
– up to 1 % of its content in the diet
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B12 deficit = pernicious anemia
• autoimmune gastritis
– intrinsic factor deficit
• ocurrence
– all races
– older people (median 60 years)
• pathogenesis
– autoimmunity
 chronic atrofic gastritis
– parietal cells loss
– autoantibodies are not
specific
 autoreactive T cells
– achlorhydria, ↓ pepsine
– gastrectomy
– exocrine pankreas dysfunction
– ileum resection
– tapeworm
– ↑ demands on B12
 relative deficit
• diagnostics
– megaloblasts
– leucopenia
 hypersegmented granulocytes
– ↓ level of B12
– ↑ homocysteine and
methylmalonyl acid
– gastritis
 ↑ risk of gastric carcinoma
– homocysteine
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Vitamin B12 functions
• 2 reactions dependent of B12
– methionine production
 methyl group acceptor
 formation of TH4
– generation of sukcinyl CoA from
methylmalonyl CoA
 ↑ methylmalonyl acid in
plasma and urine
 abnormal fatty acids in
neuronal lipids
 neurologic complications
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Folic acid deficiency anemia
• tetrahydrofolate (FH4)
– transfer of 1 C groups
 methyl, formyl
– processes dependent on these
tranfers
 purines synthesis
 homocysteine → methionine
 synthesis of deoxythimidylate
monophosphate
• etiology
– decreased intake
 essential, heat inactivation
 small stores (weeks)
– increased demands
 pregnancy, growth, cancer
– disturbed utilization
 methotrexate
– folic acid antagonist
• dihydrofolate
reductase
• distinction from pernicious
anemia
– ↓ folates in the blood
– ↑ homocysteine but not
methylmalonic acid
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Hemolysis: diagnostic and hematological features
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Fate of Hb following hemolysis
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Hemolytic anemias
• common signs
– premature RBC destruction
 normally in mononuclear
phagocytes
 usually extravascular
– phagocytes hyperplasia
• splenomegalia
– increased EPO and
stimulation of erythropoiesis
– accumulation oof
hemoglobin degradation
products
– iron deficit is not present
• clinical signs
– anaemia
– splenomegalia
– jaundice
• intravascular haemolysis
– hemoglobinemia
– hemoglobinuria
– hemosiderinuria
– splenomegalia is missing
– ↓ haptoglobin
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Classification of hemolytic anemias
• corpuscular
– membrane disorders
 hereditary spherocytosis
 eliptocytosis
paroxysmal nocturnal
hemoglobinuria
– metabolism disorders
 glucose-6-phosphate
dehydrogenase
 pyruvate kinase
– disorders of
hemoglobinisation and
hemoglobinopathies
 thalassemia
• extracorpuscular
– damage due to physical and
toxic insults
 mechanical
 heat
 bacterial toxins
 changed lipid metabolism
and membrane disorders
– damage caused by
autoantibodies
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Hereditary spherocytosis
• most common inherited
hemolytic anemia in Northern
Europeans (1:5000)
• autosomal dominant
– one defect (75 %)
• in 75 % neither parent is affected
– spontaneous mutation
– recessive?
• defect in RBC membrane
– deficit of structural proteins
 most commonly ankyrin
 also band 3 or 4.2, spectrin
– mutations
 reading frame shift or
premature stop codon
– RBC lifespan 10 – 20 days
• changes
– young RBC – normal shape
  membrane stability with
aging
  deformability – trapping in
the spleen
– splenectomy
 spherocytes remain
 correction of anaemia
• clinical features
– possible jaundice at birth
– onset can be delayed for many
years
– some patients may go through
life without symptoms
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Hereditary spherocytosis
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Another cell membrane defects
• hereditary elliptocytosis
– inherited disorder
 autosomal dominant
 1:2500 in Caucasians
– deficiency of
 protein 4.1
 spectrin/actin/4.1 complex
– membrane defect
– similar to HS but milder
– minority of patient have
anemia
• hereditary stomatocytosis
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Glucose-6-phosphate dehydrogenase (G6PD) deficiency
• recessive X-linked disease (Xq28)
– more common in males
– affects millions of people
 Africa, Mediterranean, Middle East (20
%)
 South-East Asia (up to 40 % in certain
areas)
• G6PD function
– oxidation of G6P to 6-phosphoglycerate
 production of NADPH
– the only source of RBC
– regeneration of glutathione
• protection of oxidative damage
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G6PD deficiency
• over 400 mutations identified
– mostly amino acids substitutions
– WHO classification
 normal activity
– B+
• almost all Caucasians, 70 % of black Africans
– A+
• 20 % of black Africans
 reduced activity
– A•
mild deficiency, more marked in older cells, young cells have nearly normal
activity
– Mediterranean type
• both young and old RBC have very low enzyme activity
• after oxidant shock, Hb may fall precipitously, transfusion is needed
– mutations provide protection from malaria
 Plasmodium falciparum
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G6PD deficiency
• episodic hemolysis
–  oxidative stress
 infection
– viral hepatitis, pneumonia
 drugs
– antimalarics, sulfonamids
 ingestion of fava beans
– favism
– denaturation of gobin chains
 binding of sulfhydryl groups
 precipitates bound to membrane
– Heinz bodies
•  deformability
• intravascular haemolysis
– clinical features
 anemia
 jaundice
 hemoglobinuria
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Pyruvate kinase deficiency
• the most common defect of RBC
metabolism after G6PD deficiency
– affects thousands people
• autosomal recessive
– variable severity
– homozygotes have anemia and splenomegaly
• lower PK aktivity
– reduced ATP production
– energy deficit
  resitance of membrane – rigid RBC
• diagnostics
– lower enzymatic aktivity
 5 – 20 % in affected homozygotes
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Hemoglobinopathies
• abnormalities occur in
– globin chain production
 thalassemia
– structure of the globin chain
 sickle cell disease
• change of aminoacid
composition of globin chain
or incorrect proportion of
subunits
• highly variable clinical
manifestations
– mild hypochromic anemia
– moderate hematological
disease
– severe, lifelong, transfusiondependent
anemia with
multiorgan involvement
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Sickle cell anemia
• common hereditary
hemoglobinopathy
• point mutation in the codon 6
– valin instead of glutamic acid
– abnormal HbS
• 2 forms
– homozygous
 sickle cell anemia (HbSS)
– heterozygous
 sickle cell trait (HbAS)
• most common in
– Africa
 up to 25 % in some populations
– India, Middle East, Southern
Europe
• HbF synthesis is normal
– manifestation when hbF decreases to
adult levels
 approx. 6 months of age
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Sickle cell anemia
• deoxygenated HbS
– polymerizes and becomes
insoluble
– flexibility of RBC is decreased
– rigid, sickle appearance
– change of shape
 initially reversible
 after repeated sickening
membrane loses flexibility
– irreversibly sicked cell
• sickling can produce
– shortened RBC survival
– impaired passage through
microcirculation
• sickling precipitated by
– infection, dehydration
– cold, hypoxia
• clinical features
– vaso-occlusive crisis
 pain in the hands and feet
– pulmonary hypertension
 in 30 – 40 %
– NO deficiency?
– anemia
 stable Hb 60 – 80 g/l
– splenic sequestration
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Thalassemias
• normally balanced production of
α and β globin chains (1:1)
• defective synthesis of globin
chains in thalassemia
– imbalance
– precipitation of globin chains
 ineffective erythropoiesis
 Hemolysis
• mutations causing  synthesis of
HbA
• heterogenous group
• endemic
– Middle East, tropic Africa, India,
Asia
– one of the most common
hereditary diseases
 heterozygous forms
– protection from malaria
• α-talassemia
– deficit of α chain synthesis
• β-thalassemia
– deficit of β chain
 chromosome 11
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β-thalassemia
• homozygous β-thalassemia
– β0 mutation
 β-chain is missing
– β+ mutation
 β-chain production is reduced
• heterozygous β-thalassemia
– usually symptomless
– microcytosis with or without
mild anemia
• ↓ lifespan of RBC and their
precursors
• precipitation of α-chains –
membrane damage
• ineffective erythropoiesis
– destruction of some RBC in
bone marrow
– remaining RBC are prone to
extravascular hemolysis
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β-thalassemia
• > 200 genetic defects
– mainly point mutations
– highly unstable β-chain
• clinical classification
– major
 2 alleles
 severe anemia requiring
regular transfusions
– minor (thalasemia trait)
 1 allele, heterozygous carrier
 without symptoms
– intermedia
 genetically heterogenous
 moderate anemia not
requiring regular transfusions
• serious β-thalassemia
– erythroid hyperplasia,
extramedullar hemopoiesis
– bone damage
– increased iron absorption
 supressed hepcidin synthesis
 iron from transfussions
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α-thalassemia
• gene for α-globin chain is
duplicated on both chromosomes
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• normal person has 4 α-globin
genes
– deletion of 1 or both α-chain genes
on each chromosome may occur
 most common is deletion of 1
• decreased α-chain synthesis
– excess of unmatched chains
• less severe than β-thalassemia
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Thalassemias - summary
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Extracorpuscular hemolytic anemias
• RBC damage
– mechanical
– toxins or parasites
– antibodies and complement
– antibodies against blood
group antigens
56
Paroxysmal nocturnal hemoglobinuria
• rare form
– mutation affecting hemopoietic
stem cell
– enzyme PIG-A
 impaired synthesis of GPI
– it anchors many proteins to
the cell surface
 synthesis of surface proteins
– not only RBC
– present in most healthy people
 in small number of cells
– deficit of proteins that regulate
complement activity
 intravascular hemolysis of
deficient cells
• night lysis of cells
– in 25 % of patients
– pH
– complement activity
• hemosiderinuria
• clinical signs
– intravascular hemolysis
– hemoglobinuria
– vein thrombosis
• urinary iron loss
– may cause deficiency
• treatment and prognosis
– chronic
– blood transfusions
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