IT 1_LIN Folic Acid & B12 Deficiency Anemia , Thalassemia & Hemoglobinopathia (BIOKIMIA)

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

NN

CH3CH3

H2NCOCH2CH2

H3C

H2NCOCH2

CH2CONH2

H2NCOCH2

CH3

H2C

CH2CH2CONH2

CH3

CH3

CH2

NH

O

CH2CONH2

O

H3C

P

O

O

O

OH

HO

N

N

CH3

CH3

Co

CN

CH3

H3CH

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