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Folic acid & B12 Deficient

Anemia, Thalassemia &


Hemoglobinopathia
By
Dr.Liniyanti D.Oswari,MNS.,MSc
For block 14
Medical School, University of
Sriwijaya

Learning Objective
Understand of the Hematopoesis
Understand the Metabolism of folic acid
Cyanocobalamine in Erythropoesis.
Understand the etiology and the
management of Anemia.
Understand about Thalassemia
Understand about Hemoglobinopathies
Understand the management of
Thalassemia and Hemoglobinopathies.

Hematopoiesis
Factors affecting erythropoiesis:A)-Oxygen supply of tissues:
Decreased oxygen supply (hypoxia) to tissues stimulates
secretion of erythropoietin (EP) hormone.
Hypoxia stimulates kidney to release renal erythropoietic
factor (REF).
Hypoxia stimulates liver to produce a special type of
globulin.
Both REF & globulin unite in plasma and form EP.
EP then stimulates bone marrow to produce RBCs.
Erythropoietin accelerates nearly all stages of RBCs
formation,
i.e. it stimulates proliferation & differentiation of
progenitor stem cells to produce mature RBCs.

Hematopoiesis
Factors affecting erythropoiesis:B) Dietary factors:
i-Proteins: Proteins of high biological value are
needed in the formation of RBCs.
ii-Metal ions:

Iron Fe: is essential for RBCs formation because it


enters in the formation of the hem part.

Copper Cu: It is carried & transported by plasma


protein ceruloplasmin. It catalyses the oxidation
of Fe++ to Fe+++, a reaction that must occur
before transferrin can combine and transport iron.

Cobalt Co: It stimulates EP release from kidney.


So, excess Co may produce polycythaemia.

Hematopoies
is
Factors affecting erythropoiesis:B) Dietary factors:
iii-Vitamins:

Both vitamins B12 & folic acid are essential for


final maturation of RBCs because they are
needed in DNA synthesis.

Deficiency of either B12 or folic acid results in


failure of nuclear maturation and causing
maturation failure anemia.

Vitamin C is a strong reducing agent which is


important in reducing the ferric form of iron to
ferrous to facilitate its absorption and
transport.

Hematopoiesi
s erythropoiesis:Factors affecting
C) Hormonal factors:
i-Androgens: increase erythropoiesis by
stimulating the production of erythropoietin
from kidney.
ii-Thyroid hormones:
Stimulate the metabolism of all body cells
including the bone marrow cells, thus,
increasing erythropoiesis.
Hypothyroidism is associated with anemia
while hyperthyroidism is associated with
polycythaemia.

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors:
iii-Glucocorticoids:

Stimulate the general metabolism and also


stimulate the bone marrow to produce more
RBCs.

In Addisons disease (hypofunction of adrenal


cortex) anemia present, while in Cushings
disease (hyperfunction of adrenal cortex)
polycythaemia present.

Hematopoiesis
Factors affecting erythropoiesis:C) Hormonal factors:
iv-Pituitary gland: Affects erythropoiesis both
directly and indirectly through the action of
several hormones.
v- Haematopoietic growth factors: Are secreted
by lymphocytes, monocytes & macrophages
to regulate the proliferation and
differentiation of proginator stem cells to
produce blood cells.

Hematopoiesis
Factors affecting erythropoiesis:D)-State of liver & bone marrow:
i-Liver: Healthy liver is essential for
normal erythropoiesis because the
liver is the main site for storage of
vitamin B12 , folic acid, iron & copper.
In chronic liver disease anemia
occurs.
ii-Bone marrow: When bone marrow is
destroyed by ionizing irradiation or
drugs, aplastic anemia occurs.

Anemia
Anemia means a decrease in
hemoglobin content,
or RBCs count,
or both of them below the
normal range.
Anemia leads to a decrease in
blood ability to transport oxygen
to tissue cells.

Anemia
Types & causes of anemia:
I-Blood loss anemia:
A-Acute blood loss anemia:
Due to severe hemorrhage.
Plasma volume is replaced rapidly by the
fluids present in tissue spaces.
This leads to marked dilution of the blood.
RBCs are replaced within 2-3 weeks.
Sufficient iron gives normocytic cells but
insufficient iron will produce microcytic
RBCs.

Anemia
Types & causes of anemia:
I-Blood loss anemia:
B-Chronic blood loss anemia:
Due to repeated loss of small amounts of
blood over a long period e.g.:
-Gastrointestinal bleeding (peptic ulcer)
-Excessive menstruation.
-Hemorrhagic diseases.
Due to depletion in iron stores the newly
formed RBCS are microcytic.

Anemia
Types & causes of anemia:
II-Aplastic anemia:
It results from destructione of bone marrow.
It may result from:
1-Excessive exposure to x-rays or gamma rays.
2-Chemical toxins e.g. cancer therapy & prolonged
exposure to insecticides or benzene.
3-Invasion of bone marrow by cancer cells.
4-Following infection by hepatitis.
Damaged bone marrow dont produce any RBCs, so
in aplastic anemia RBCS are normocytic.
It is associated with decrease in WBCs & platelets.

Anemia
Types & causes of anemia:
III-Hemolytic anemia:
It results from increased rate of destruction of RBCs
inside the cardiovascular system.
Causes of hemolytic anemia:
A-Hereditary:
1-Membrane abnormalities.
2-Enzyme deficiency e.g. G-6-P Dehydrogenase.
3-Hemoglobin abnormalities.
B-Acquired:
1-Incompatible blood transfusion.
2-Parasitic infection e.g. malaria.
3-Toxic agents e.g. snake venom & insect poisons.
4-Thermal e.g. several burns.

Anemia
Types & causes of anemia:
IV-Dyshemopoietic anemia: Which may be due
to:
1-Iron deficiency anemia.
2-Maturation failure (megaloblastic) anemia:a-Vitamin B12 deficiency.
b-Folic acid deficiency.
3-Anemia of endocrine disorders.
4-Nutritional anemia.
5-Anemia of renal failure.

Classification of Anemia
Based on cell size (MCV)
Macrocytic (large) MCV 100+ fl
(femtoliters)
Normocytic (normal) MCV 80-99 fl
Microcytic (small) MCV<80 fl
Based on hemoglobin content (MCH)
Hypochromic (pale color)
Normochromic (normal color)

MEGALOBLASTIC
ANEMIA
By
Liniyanti D.Oswari,
MD,MNS,MSc.
For Block 14

MEGALOBLASTIC (Macrocytic)
ANEMIA
High MCV
High MCH
Normal MCHC
Macrocytic Anemia
Megaloblastic : defective DNA
synthesis
Non-megaloblastic : numerous
mechanisms

Nutritional Requirements for


Hematopoiesis

Metals : iron copper cobalt


B12 and Folate
Other vitamins: B6, A, E, C
Riboflavin, Niacin

Megaloblastic Anemias
A form of anemia characterized by the
presence of large, immature, abnormal
red blood cell progenitors in the bone
marrow
95% of cases are attributable to folic
acid or vitamin B12 deficiency

CH3
CH3

H2NCOCH2CH2

CH2CONH2

H2NCOCH2
N CN

H3 C
H3 C
H

corin nucleus

Co
N

cobalt coordinated
N

H2NCOCH2
H2C

CH2CH2CONH2

CH3

CH3

CH3
CH3
CH2 CH2CONH2

N
O

H3C
O

P
O

OH

CH3

benzylimidazole
N

HO
VITAMIN B 12

CH3

Vitamin B12
Source : food of animal origin
- liver
- muscle
- eggs
- cheese and milk
- Not in plants
- Made by bacteria

B12 Absorption
1. Release from food sources gastric
proteases and acids
2. Binding by salivary cobalophilins
3. Digestion of cobalophilin-B12
complex by pancreatic enzymes
4. Binding to intrinsic factor (IF)
IF is secreted by gastric parietal cells
5. Attachment of B12-IF to receptors
6. Endocytosis and binding to
transcobalamin II

B12 Dependant
Reactions
1. Synthesis of methionine from
homocysteine requires : B12 and
folate
2. Synthesis of succinyl CoA from
methyonyl CoA requires :
methylmalonyl CoA mutase

FOLIC ACID
Sources : synthesized by plants
and microorganisms
Vegetables, fruits, dairy
products
Polyglutamated
Thermo labile

Folic Acid Absorption-Transport


1. Polyglutamates converted to monoglutamates Intestinal carboxypeptidase
2. Binding of monoglutamates to brush
border receptor
3. Conversion to
methyltetrahydrofolate during
absorption
4. Bind to serum protein
5. Receptor mediated cell uptake
6. Polyglutamated in cytoplasm

Pernicious Anemia
An Autoimmune Disease
Antibodies against :parietal cells
intrinsic factor
(IF)
Thyroid - myxedema
Melanosomes - vitiligo

Pernicious Anemia
Hematologic features :

anemia
pancytopenia
megaloblastic hematopoiesis
cellular bone marrow
ineffective hematopoiesis

Folate Deficiency
Hematologic features : same as
Pernicious Anemia.
Clinical Picture : no neurologic
findings

Folate Deficiency :Diagnosis


Dietary history
Clinical conditions:
pregnancy
malabsorption (sprue)
hemolytic anemia
drugs
Laboratory:
serum or red cell folate levels

Pernicious Anemia
Presenting Complaint

Symptoms of anemia : 58%


Sensory paresthesis :13%
GI complaints :11%
Sore tongue or mouth : 7%
Weight loss : 5%
Difficulty walking : 3%
Other :3%

Pernicious Anemia Diagnosis

History and Physical


glossitis
pallor
neurologic exam
Laboratory
blood smear
antibody assays
B12 level
Other
Schilling test

Schilling Test
First stage :
1. Inject B12 IM (1,000 ug) to
saturatetranscobalamin II
2. Administer oral B12 - radiolabeled
3. Collect 24 h urine
4. Measure radioactivity in urine
Second stage :
1. Inject B12 IM (1,000 ug) to saturate
transcobalamin II (Same as 1st stage)
2. Administer oral B12 radiolabeled plus intrinsic
factor (HOG)
3. Collect 24 h urine, (Same as 1st stage)
4. Measure radioactivity in urine,(Same as 1st
stage)

Static Test for Folate/B12


Status
Folate
Measured in whole blood (plasma and
cells) and then in the serum alone
Difference is used to calculate the red
blood cell folate concentration (may
better reflect the whole folate pool)
Can also test serum in fasting patient
B12
Measured in serum

Functional Tests for


Macrocytic Anemias
Homocysteine: Folate and B12 are
needed to convert homocysteine to
methionine; high homocysteine may
mean deficiencies of folate, B12 or B6
Methylmalonic acid measurements can
be used along with homocysteine to
distinguish between B12 and folate
deficiencies ( in B12 deficiency)
Schilling test: radiolabeled cobalamin is
used to test for B12 malabsorption

Pernicious Anemia
A macrocytic, megaloblastic anemia caused by a deficiency of vitamin B 12.

Usually secondary to lack of intrinsic factor (IF)

May be caused by strict vegan diet(vegetarian)

Also can be caused by gastric acid secretion, gastric atrophy, Hpylori, gastrectomy, disorders of the small intestine (celiac disease,
regional enteritis, resections), drugs that inhibit B12 absorption
including neomycin, alcohol, colchicine, metformin, pancreatic disease

Symptoms of
Pernicious Anemia
Paresthesia (especially numbness
and tingling in hands and feet)
Poor muscular coordination
Impaired memory and hallucinations
Damage can be permanent

Vitamin B12 Depletion


Stage Iearly negative vitamin B12
balance
Stage IIvitamin B12 depletion
Stage IIIdamaged metabolism: vitamin
B12 deficient erythropoiesis
Stage IVclinical damage including
vitamin B12 anemia
Pernicious anemianumbness in hands
and feet; poor muscular coordination; poor
memory; hallucinations

Causes of Vitamin B12


Deficiency

Inadequate ingestion
Inadequate absorption
Inadequate utilization
Increased requirement
Increased excretion
Increased destruction by
antioxidants

Treatment of B12 Deficiency


Before 1926 was incurable; until 1948 was
treated with liver extract
Now treatment consists of injection of 100
mcg of vitamin B12 once per week until
resolved, then as often as necessary
Also can use very large oral doses or nasal
gel
MNT: high protein diet (1.5 g/kg) with
meat, liver, eggs, milk, milk products,
green leafy vegetables

Pernicious Anemia
Treatment
B12 by IM injection
Frequent at first
Monthly thereafter life long

Folic Acid Deficiency


Tropical sprue; pregnancy; infants born to
deficient mothers
Alcoholics
People taking medications chronically that
affect folic acid absorption
Malabsorption syndromes

Causes of Folate Deficiency

Inadequate ingestion
Inadequate absorption
Inadequate utilization
Increased requirement
Increased excretion
Increased destruction
Vitamin B12 deficiency can cause
folate deficiency due to the
methylfolate trap

Methylfolate Trap
In the absence of
B12, folate in the
body exists as 5methyltetrahydrofolate (an inactive
form)
B12 allows the
removal of the 5methyl group to
form THFA

Stages of Folate Depletion and


Deficiency
Stage Iearly negative folate balance (serum
depletion)
Stage IInegative folate balance (cell
depletion)
Stage IIIdamaged folate metabolism with
folate-deficient erythropoiesis
Stage IVclinical folate deficiency anemia

Diagnosis of Folate
Deficiency
Folate stores are depleted after 2-4
months on deficient diet
Megaloblastic anemia, low
leukocytes and platelets
To differentiate from B12, measure
serum folate, RBC folate (more
reflective of body stores) serum
B12
High formiminoglutamic acid
(FIGLU) in the urine also diagnostic

Hemolytic Anemia
Oxidative damage to cellslysis occurs
Vitamin E is an antioxidant that seems to
be protective.
This anemia can occur in newborns,
especially preemies.
Deficiency Glucose 6 Phospat dehidrogenase
in baby boy(X linked)- block 5

Nonnutritional Anemias
Sports anemia (hypochromic
microcytic transient anemia)
Anemia of pregnancy: dilutional
Anemia of inflammation, infection, or
malignancy (anemia of chronic
disease)
Sickle cell anemia
Thalassemias

Sports Anemia
Transientusually in athletes who are
runners; from compression of RBCs in feet
until they burst, releasing hemoglobin
Check lab values
Counsel about a proper diet

THALASSEMIAS AND
HEMOGLOBINOPATHIES
By
Liniyanti D.Oswari,MD,MNS,MSc.
For Block 14
b

Thalassemias and
Hemoblobinopathies: Module
Objectives
At the end of this module you should be
able to
Explain the pathophysiology that causes
thalassemia and hemoglobinopathies.
Explain how thalassemias are
categorized.
Correlate the results of laboratory
testing with specific thalassemias and
hemoglobinopathies.

Thalassemia
Severe inherited anemia affecting mostly
people of Mediterranean extraction
Basic defect is reduced production of
selected globin chains
Defective globin formation in hemoglobin
leads to increased blood volume,
splenomegaly, bone marrow expansion,
facial deformities, osteomalacia, bone
changes
Iron buildup due to transfusions requires
chelation therapy to remove excess iron

Demographics:
Thalassemia
Found most
frequently in the
Mediterranean,
Africa, Western and
Southeast Asia,
India and Burma
Distribution
parallels that of
Plasmodium
falciparum

Classification & Terminology


Alpha Thalassemia

Terminology
Silent carrier
Minima
Minor
Intermedia
Major

Symbolism Alpha
Thalassemia
Greek letter used to designate globin
chain:

Indicates division between genes


inherited from both parents:

/
Each chromosome 16 carries 2 genes. Therefore
the total complement of genes in an individual
is 4

- : Indicates a gene deletion:


-/

Classification & Terminology


Alpha Thalassemia

Normal
/
Silent carrier - /
Minor -/-
--/
Hb H disease --/-
Barts hydrops fetalis

--/--

Symbolism Other
Thalassemia
Greek letter used to designate globin
chain:

+:

Indicates diminished, but some


production of globin chain by gene:

:Indicates no production of globin chain

by gene:
Superscript

denotes nonfunctioning gene:

Classification & Terminology


Beta Thalassemia
Normal
Minor
Intermedia
Major

/
/ 0
/ +
0/ +
0/ 0
+/ +

Special Cases
Thalassemia
Hb Lepore: fusion seen in some
types
of thalassemia
Hb Constant Spring
chain with 31 additional amino acids
--/cs

Hereditary persistence of fetal


hemoglobin
(HPFH)

Special Cases:
Thalassemia
Hb Barts & hydrops fetalis
Barts is a 4 tetramer
Associated with --/- Lethal
High concentrations are capable of sickling

Hb H
4 tetramer
Associated with --/- thalassemia

Primary Laboratory Investigation


Thalassemia
Variable hemogram results proportional
to the severity of the thalassemia
Severe cases present with
Microcytosis
Hypochromia
Poikilocytosis
RBC counts higher than expected for the
level of anemia

Primary Laboratory Investigation


Thalassemia
Findings in severe cases can mimic
those seen in other
microcytic/hypochromic anemias
Results of the reticulocyte count are
variable
NRBCs may be present (contrast with
iron deficiency anemia)

Course and Treatment


Thalassemia

Time of presentation

Related to degree of severity


Usually in first few years of life
Untreated severe thalassemia
--/--: Prenatal or perinatal death
--/- & --/cs: Normal life span with chronic
hemolytic anemia

Untreated thalassemia
Major: Death in first or second decade of life
Intermedia: Usually normal life span
Minor/Minima: Normal life span

Hemoglobine in Human
being

Hb A1a : < 1 %
: Increase in Diabetic Mellitus
HbA1b
: 2% : Increase in Diabetic Mellitus
HbA1c (2(-N-Glucose)2): 4%:Increase in Diabetic
Mellitus
HbF ( 2(-N-Acetyl)2) : < 1% : Increase in
Thalassemia &

Sickle Cell Anemia.


HbA2(2 2 ) : < 3% : Increase in Thalassemia &

Sickle Cell Anemia.


Hb Gower 1 :(22) : 0
: Increase in early Embryo
Hb Gower 2 ( 2 2) : 0
: Increase in early Embryo
Hb Portland (22) : 0
: Increase in early Embryo
HbH
( 4) : 0
: Increase in Thalassemia
Hb Barts (4 ) : 0
: Increase in Thalassemia

Characteristics:
Hemoglobinopathies
Hereditary disorders that can result
in moderate to severe anemia
Basic defect is production of an
abnormal globin chain
The demographics of
hemoglobinopathies are varied.

Hemoglobinopathy
Genetics
Homozygous: Inheritance of two genes
from each parent coding for the same
type of abnormal hemoglobin, e.g., Hb
SS
Heterozygous: Inheritance of genes
from each parent which code for a
different type of abnormal hemoglobin
each, e.g., Hb SC

Terminology
Hemoglobinopathy
Abnormal hemoglobins discovered earlier have been
given letter designations:

Hb S: A->T( Glutamat (GAG) Valine


(GTG) at codon 6

More recently discovered hemoglobins have been


named by the city or location of discovery:
Hb C-

Harlem
In South East Asia there is specific
hemoglobinopathies is mutation in codon 26:
Hb E : G- A ( GAG AAG)

In Palembang & Malay


,Hemoglobinopathies is mutation in
codon 19.
Hb Malay ( 2219 (B1) AG( AsnSer)

Amino Acid Substitution


Hemoglobinopathy
Greek letter designates affected globin
chain

Superscript number designates affected


amino acid(s), e.g., 6
Letters and numbers in parentheses designate
the helical segment and amino acid sequence
in that segment affected (sometimes
omitted), e.g.,

6(A3)

Amino Acid Substitution


Hemoglobinopathy
Amino acid substitutions are denoted
by the three letter abbreviation for
the normally occurring amino acid
followed by an arrow followed by the
three letter abbreviation for the
substituted amino acid:

6(A3)Glu Val

Classification:
Hemoglobinopathy
Functional Abnormality
Aggregation
Polymerization
Crystallization

Unstable hemoglobins
Methemoglobin
Oxygen affinity

Primary Laboratory Investigation


Hemoglobinopathy
Variety of hemogram findings
depending on
Type
Severity

of the specific disorder


Only sickle hemoglobinopathies and
Hb C will be described here

Primary Laboratory Investigation


Heterozygous & Other Disorders

AS
S-Thal
Other hemoglobinopathies, e.g., SC
Hb C

Sickle Cell Anemia


Protein-energy malnutrition common; may
have poor intake and increased energy
needs
Be careful not to overdo iron in diet or
supplements; iron stores are often high due
to frequent transfusions; avoid iron rich
foods, alcohol, and ascorbic acid which
enhance iron absorption
Promote foods high in copper, zinc and
folate as needs are increased due to
constant replacement of erythrocytes
Zinc supplements may be useful

Morphologic Findings
Hb SS vs. Hb SC vs. Hb CC
=

+
Hb S

Hb C

Hb SC
=

Where Do Sickle Cells Come


From?
Sheared in
microcirculation

Irreversible
Sickle Cell

Sickle Cells

Secondary Laboratory
Investigation
Hemoglobin electrophoresis
Major test for identifying thalassemia
and hemoglobinopathy
Types
Cellulose acetate: Alkaline pH
Citrate agar: Acid ph

Patterns of mobility (see handout)

Secondary Laboratory
Investigation
Cellulose Acetate Hb
Electrophoresis
- A2/C
S
F
Normal
Hb SS
Hb AS
Hb SC
Hb CC
HB AD

A+

Secondary Laboratory
Investigation
Solubility testing-Dithionite tube test
Alkali denaturation test for
quantification of fetal hemoglobin
Acid elution test for fetal hemoglobin
distribution
Unstable hemoglobin testing for
Heinz bodies

Alkali Denaturation for


Hemoglobin F
Recommended assay for hgb F in the
range of 2-40%
Principle
Other hemoglobins are more susceptible
than hgb F to denaturation at alkaline
pH
Denaturation stopped by addition of
ammonium sulphate
Denatured hemoglobin precipitates

Alkali Denaturation for


Hemoglobin F
Remaining hemoglobin (F) can be
measured spectrophotometrically

Specimen: EDTA anticoagulated


whole blood
QC: Normal and elevated controls
should be used with each batch of
specimens

Alkali Denaturation for


Hemoglobin F
Hgb F, %

Diff. Between Duplicates, %

<5
5-15
>15
Sources of error

0.5
1.0
2.0

Too short or too long an incubation time


Filtrate turbidity
Outdated reagents
Incorrect reagent concentrations
Poor quality filter paper

Acid Elution for Fetal


Hemoglobin
Indication of distribution of fetal
hemoglobin in a population of RBC
Homogeneous distribution: hereditary
persistence of fetal hemoglobin
Heterogeneous distribution: thalassemia

Course and Treatment


Sickle Cell Disease
Sickle cell disease
Asymptomatic at birth
Symptoms appear as percentage of fetal
hemoglobin decreases during first year of life
Untreated crises increase morbidity and early
death
Life span can be significantly increased with
early and effective treatment
Studies of natural populations reveal that
individuals with sickle cell disease are capable
of normal life spans

Course and Treatment


In both thalassemia and
hemoglobinopathy therapy is usually
supportive rather than curative
Blood transfusion is used to
Control severe anemia
Reduce the risk of complications of
sickle hemoglobinopathies
(cerebrovascular accident,
hypersplenism, etc.)

Course and Treatment


Chronic blood transfusion
Results in iron overload of major organs
resulting in increased morbidity
Laboratory monitoring
Necessitates the use of chelating agents
to remove excess iron

Course and Treatment


Excess iron can cause the appearance of
sideroblastic conditions
Transfusion interferes with the typical
laboratory findings for the disorder

Alternative treatment
Activation of fetal hemoglobin genes
Bone marrow transplantation