Folic acid & B12 Deficient Anemia, Thalassemia & Hemoglobinopathia By Dr.Liniyanti D.Oswari,MNS.,MSc For block 14 Medical School, University of Sriwijaya
Jan 12, 2016
Folic acid & B12 Deficient Anemia, Thalassemia & Hemoglobinopathia
ByDr.Liniyanti D.Oswari,MNS.,MSc
For block 14Medical 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.
HematopoiesisFactors 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.
HematopoiesisFactors 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.
Factors affecting erythropoiesis:-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.
HematopoiesiHematopoiesiss
HematopoiesisFactors affecting erythropoiesis:-
C) Hormonal factors:
iii-Glucocorticoids:
Stimulate the general metabolism and also stimulate
the bone marrow to produce more RBCs.
In Addison’s disease (hypofunction of adrenal cortex)
anemia present, while in Cushing’s 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.
HematopoiesisFactors 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.
AnemiaTypes & 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 don’t produce any RBCs, so in
aplastic anemia RBCS are normocytic. It is associated with decrease in WBCs & platelets.
AnemiaTypes & 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.
AnemiaTypes & 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 flBased on hemoglobin content (MCH)• Hypochromic (pale color)• Normochromic (normal color)
MEGALOBLASTIC ANEMIA
ByLiniyanti 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 forHematopoiesis
• 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
N N
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NH
O
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HO
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VITAMIN B 12
corin nucleus
benzylimidazole
cobalt coordinated
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 mono-glutamates 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 AnemiaAn Autoimmune Disease
• Antibodies against :parietal cells intrinsic factor (IF)
• Thyroid - myxedema• Melanosomes - vitiligo
Pernicious AnemiaHematologic 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:
pregnancymalabsorption (sprue)hemolytic anemiadrugs
• Laboratory:serum or red cell folate levels
Pernicious AnemiaPresenting 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 patientB12• 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 AnemiaA macrocytic, megaloblastic anemia caused by a deficiency of vitamin B12.
• 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, H-pylori, 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 I—early negative vitamin B12 balance
• Stage II—vitamin B12 depletion
• Stage III—damaged metabolism: vitamin B12 deficient erythropoiesis
• Stage IV—clinical damage including vitamin B12
anemia• Pernicious anemia—numbness 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 AnemiaTreatment
• 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 TrapMethylfolate Trap• In the absence of B12,
folate in the body exists as 5-methyltetrahydro-folate (an inactive form)
• B12 allows the removal of the 5-methyl group to form THFA
Stages of Folate Depletion and Deficiency
• Stage I—early negative folate balance (serum depletion)• Stage II—negative folate balance (cell depletion)• Stage III—damaged folate metabolism with folate-
deficient erythropoiesis• Stage IV—clinical 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 cells—lysis 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• Transient—usually 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
bByLiniyanti D.Oswari,MD,MNS,MSc.
For Block 14L
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 & TerminologyAlpha 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 & TerminologyAlpha 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: +
0 :Indicates no production of globin chain by gene: 0
Superscript T denotes nonfunctioning gene:
T
Classification & Terminology Beta Thalassemia
• Normal /• Minor /0
/+
• Intermedia 0/+
• Major 0/0
+/+
Special CasesThalassemia
• 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 InvestigationThalassemia
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 InvestigationThalassemia
• 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 (ά2(β-N-Glucose)2): 4%:Increase in Diabetic Mellitus• HbF (ά 2(γ-N-Acetyl)2) : < 1% : Increase in β Thalassemia &• Sickle Cell Anemia.• HbA2(ά2 β2 ) : < 3% : Increase in β Thalassemia &• Sickle Cell Anemia.• Hb Gower 1 :(ζ2€2) : 0 : Increase in early Embryo• Hb Gower 2 (ά 2 €2) : 0 : Increase in early Embryo• Hb Portland (ζ2γ2) : 0 : Increase in early Embryo• HbH (β 4) : 0 : Increase in ά Thalassemia• Hb Barts (γ4 ) : 0 : Increase in ά 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 HemoglobinopathyAbnormal 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 ( ά 2β219 (B1) AG( AsnSer)
Amino Acid SubstitutionHemoglobinopathy
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 SubstitutionHemoglobinopathy
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 InvestigationHemoglobinopathy
• Variety of hemogram findings depending on• Type• Severity
of the specific disorder• Only sickle hemoglobinopathies and Hb C will
be described here
Primary Laboratory InvestigationHeterozygous & 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 inmicrocirculation
IrreversibleSickle 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 A+
NormalHb SSHb ASHb SCHb CCHB AD
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 0.5 5-15 1.0 >15 2.0• Sources of error
• 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 TreatmentSickle 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