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Differential Diagnosis of Hypochromia and Iron Overload


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  • Include;
    • Iron deficiency anaemia ü Sideroblastic anaemia
    • Thalassaemia trait (α or β)
    • Chronic inflammation or malignancy

SIDEROBLASTIC ANAEMIA

  • REFRACTORY anaemia with hypochromic cells in the PB[1] and increased marrow iron
  • Characterized by the presence of ring sideroblasts (Erythroblasts with iron granules arranged in a ring around the nucleus)
  • Diagnosed when 15% or more of bone marrow erythroblasts are ring sideroblasts

Ring Sideroblasts

 

  • Acquired

Hereditary

  • X-linked

 

Usually occurs in males

  • Rarely in females

Acquired

  • Primary
    • Myelodysplasia (Refractory anaemia with RS[2])
  • Secondary
    • Other types of myelodysplasia
    • Myelofibrosis
    • Myeloid leukaemia
    • Myeloma
    • Drugs e.g. isoniazid, cycloserine, alcohol and lead
    • Others e.g. haemolytic anaemia, megaloblastic anaemia, malabsorption, rheumatoid arthritis

Pathogenesis

  • Defective haem synthesis is the common end point
  • Mutation of the δ-aminolaevulinic acid synthase (ALA-S) gene on X chromosome
  • So there is reduced level of ALA-S
  • Pyridoxine deficiency or defect in its metabolism may also play a role

Laboratory Findings

  • Hypochromic microcytic anaemia
  • Reduced MCV and MCH
  • There is ineffective erythropoiesis
  • Bone marrow shows erythroid hyperplasia
  • Iron store is normal or increased
  • Serum iron and transferrin saturation are raised

Treatment

  • Pyridoxine therapy is useful in hereditary and in some secondary acquired type but ineffective in primary acquired type
  • Folic acid
  • Red cell transfusion
  • Iron removal by phlebotomy[3]
  • Iron chelation therapy

LEAD POISONING

  • Lead inhibits both haem and globin synthesis at a number of points
  • Red cell lifespan is shortened
  • Mild rise in reticulocytes
  • Basophilic stippling[4] is characteristic
  • Siderotic granules and occasionally cabot rings are found in circulating red cells
  • Bone marrow may show ring sideroblasts
  • Free erythrocyte protoporphyrin is raised

ANAEMIA OF CHRONIC DISORDER

  • This is anaemia that occurs in patients with a variety of chronic inflammatory and malignant diseases
  • It is one of the most common causes of anaemia

Causes of Anaemia of Chronic Disorder

  • Chronic inflammatory diseases ü Infectious

 Pulmonary abscess

 TB

 Osteomyelitis

 Pneumonia

 Bacterial endocarditis

  • Non-infectious RA[5]

 SLE[6]

 Other CT[7] disorders

 Sarcoidosis

 Crohns disease

  • Malignant diseases
    • Carcinomas
    • Lymphomas
    • Sarcomas

Pathogenesis

  • Effect of hepcidin
    • Decreased release of iron from macrophages
    • Decreased absorption of iron
  • Reduced red cell life span

Inadequate erythropoietin response to anaemia caused by effect of IL-1 and TNF on erythropoiesis

Lab Features

  • MCV and MCH are low, normal or mildly reduced
  • ESR is raised
  • C-reactive protein is raised
  • Serum iron and TIBC[8] are reduced
  • Serum transferrin receptor level is normal
  • Serum ferritin is normal or raised
  • Bone marrow storage iron is normal but erythroblast iron is absent or reduced

Treatment

  • The anaemia is corrected by successful treatment of the underlying disease and does not respond to iron therapy

 

 

 

 

 

ERYTHROBLAST

IRON

 Absent

Absent or ↓

Ring forms

N

 

IRON OVERLOAD

  • There is excessive accumulation of iron in;
    • The macrophages in the RES[9]
    • In liver parenchymal cells and
    • Other cells in the body
  • Excess iron accumulation in the RES is relatively harmless
  • Iron accumulation in parenchymal cells causes organ damage

Commonly Affected Organs

  • Liver → liver cirrhosis
  • Heart → cardiac failure and arrhythmias
  • Endocrine glands → failure of pubertal growth and development, hypoparathyroidism, hypothyroidism
  • Some patients can have DM (Diabetes Mellitus) and arthritis

Causes of Iron Overload

  • Excessive iron absorption
    • Hereditary haemochromatosis
    • Massive ineffective erythropoiesis
  • Increased intake
    • Sub-Saharan dietary iron overload (in combination with a genetic determinant of increased absorption)
    • Excessive parenteral intake
  • Repeated red cell transfusions – In transfusion dependent conditions like;
    • β-Thalassaemia major
    • Red cell aplasia
    • Myelodysplasia
    • Aplastic anaemia

Iron overload in an adult occurs after a total loss of about 50 units of blood is transfused over time

HEREDITARY HAEMOCHROMATOSIS

  • Excessive absorption of iron from the GIT

Iron overload of the parenchymal cells of the liver, endocrine organs and in severe cases, the heart

  • Mutation of the HFE[10] gene
  • HFE is involved in Hepcidin synthesis
  • Low hepcidin →↑ iron absorption and ↑release of iron from macrophages Treatment of Iron Overload
  • Regular venesection
  • Iron chelation therapy e.g.
    • Desferrioxamine (DF)
    • Diferiprone (L1)

In transfusion dependent conditions, iron chelation should be commenced after an adult has had a total of 20 units of blood in order to prevent iron overload

Summary

Table of Contents

             

Iron Deficiency Anaemia – PROF. HANNAH O. OLAWUNMI

  • Iron deficiency and iron deficiency anaemia are common nutritional and haematologic disorder worldwide, affecting an estimated 2 billion people
  • In infants and young children, iron deficiency is most commonly due to insufficient dietary iron
  • In young women, it is most often the result of excessive blood loss in menstruation or as a result of pregnancy

PREVALENCE

  • Iron deficiency is the commonest cause of anaemia worldwide and is frequently seen in general practice
  • Iron deficiency is particularly common in infants and pregnant women
  • In infancy, the occurrence of iron deficiency is equal in both sexes
  • It is usually detected between the ages of 6 and 20 months
  • The prevalence of iron deficiency is also higher among people living in chronic poverty

SEQUENCE OF EVENTS OF IRON DEFICIENCY ANAEMIA

  • Depletion of iron stores
  • Iron deficient erythropoiesis
  • Iron deficiency anaemia

Depletion of Iron Stores

  • First event that occurs when the body is in a state of negative iron balance
  • Iron absorption is increased when stores are reduced
  • Serum iron levels may still be normal
  • Serum ferritin has already fallen
  • Anaemia has not developed

Iron Deficient Erythropoiesis (IDE)

  • With further iron depletion, when serum ferritin is below 15µg/L, serum transferrin saturation falls below 15% due to a rise in transferrin concentration and a fall in serum iron
  • This leads to the development of IDE and increasing concentrations of serum transferrin receptor and red cell protoporphyrin
  • At this stage the Hb, MCV and MCH may still be normal

Iron Deficiency Anaemia

If negative balance continues, frank iron deficiency anaemia develops

 TIBC rises and serum iron falls so that percentage saturation of TIBC is usually < 10%  Bone marrow shows total absence of iron

CAUSES OF IRON DEFICIENCY

  • Causes of iron deficiency anaemia include;
    • Poor diet
    • Blood loss
    • Increased demand
    • Malabsorption
  • In infants and children, iron deficiency usually results from poor dietary intake
  • In adults, iron deficiency occurs usually secondary to chronic blood loss particularly from GIT
  • In women of reproductive age group, iron deficiency is usually the result of menstrual disorders and pregnancy

Poor Diet

  • This is a major factor in many developing countries due to poverty especially in infants and children
  • Rarely, the sole factor in developed countries
  • Large vegetarian diet

Blood Loss

 Most common causes in adults include;

  • Hookworm infestation
  • Menorrhagia
  • Peptic ulcer
  • Haemorrhoids
  • Schistosomiasis

Increased Demand

  • Prematurity
  • Pregnancy
  • Growth
  • Erythropoietin therapy

Malabsorption

  • Gastrectomy
  • Gluten-induced enteropathy
  • Autoimmune gastritis

SIGNS AND SYMPTOMS

  • Depend on how rapidly the anaemia develops
  • In cases of chronic, slow blood loss, the body adapts to the increasing anaemia, and patients can often tolerate extremely low Hb concentrations with remarkably few symptoms
  • Impaired growth in infancy, and the growth rate is restored when the deficiency is corrected
  • Irritability, palpitations, dizziness, breathlessness, headache and fatigue
  • Fatigue is particularly a common complaint among patients

TISSUE EFFECTS OF IRON DEFICIENCY

  • Patients with long-standing iron deficiency may develop some symptoms characterized by defective structure or function of epithelial tissue
  • The nails, the tongue and mouth, the hypopharynx and the stomach are commonly affected
  • Koilonychia – ridged or spoon nails
  • Angular stomatitis
  • Pharyngeal webs (Paterson-Kelly syndrome) – dysphagia
  • Glossitis
  • Dietary cravings (Pica)[11]
  • Impaired mental development and function in children

 

 

  • MCV, MCH and MCHC are decreased
  • Red cell distribution width (RDW) is increased
  • Reticulocyte count is low for the degree of anaemia
  • There is usually thrombocytosis
  • Eosinophilia may be present in parasitic infestation

108

  • Microcytic, hypochromic red cells
  • Anisocytosis
  • Pencil shaped poikilocytes
  • Target cells

                                                 

  • FBC – Full Blood Count
  • PBF – Peripheral Blood Film

Iron Deficiency: PBF Showing Microcytic Hypochromic Red Cells

 

BONE MARROW

  • Iron is absent in the macrophages and developing erythroblasts
  • There is mild to moderate erythroid hyperplasia
  • Bone marrow is depleted of stainable iron
  • The erythroblasts are small and have a ragged vacuolated cytoplasm
  • Granulocytic and megakaryocytic lines are normal

Normal Bone Marrow Showing Adequate Iron in Macrophages (Perl’s Stain) with Iron

Granules in Erythroblasts (Insets)

 

Iron Deficiency: BM Showing Absence of Stainable Iron (Perl’s Stain)

 

IRON STUDIES

  • Serum Ferritin is low
  • Serum iron is low
  • Transferrin saturation is low

 

 

 

 

 

BM IRON STORE

Absent

Normal

Normal or↑

N

ERYTHROBLAST IRON

 

Absent

Absent or ↓

Ring forms

N

 

MANAGEMENT OF IDA

  • Identification and Rx[12] of the underlying cause
  • Correction of deficiency by therapy with inorganic iron

Identification of the Underlying Cause

  • Stool for ova and parasites
  • Stool for occult blood[13]
  • Abdominopelvic USS
  • Upper and lower GI endoscopy
  • Test for parietal cell antibodies
  • Duodenal biopsy
  • Test for H. pylori
  • ETC

Treatment

  • Oral iron – 100-200mg/day
    • Children – 3mg/kg/day
    • Rx given for 3-6 months to correct anaemia and to replenish body stores
    • Minimal rate of response is 2 mg/dl rise in Hb every 3 weeks
  • Parenteral iron
    • Ferric hydroxide – sucrose (Venofer) by slow IV injection (the safest)
    • Iron sorbitol (Jectofer) given by slow IM injection
    • Iron dextran (cosmoFer) slow IV infusion

Summary

Table of Contents

             

Vitamin B12 and Folate Metabolism – PROF. HANNAH O. OLAWUNMI

  • Vitamin B12 belongs to a group of compound generally referred to as the cobalamins
  • Like other cobalamins, Vitamin B12 consists of a central cobalt atom surrounded by a corrin ring

 

  • There are 4 main forms of Vitamin B12;
    • Methyl cobalamin – main form found in human plasma
    • Ado (deoxyadenosyl) cobalamin – tissue form of B12
    • Cyanocobalamin – radioactive form for investigation of B12 absorption or metabolism
    • Hydroxocobalamin – main form for treatment of B12 deficiency
  • Cyanocobalamin & hydroxocobalamin are the 2 stable forms pharmacologically

SOURCES OF VIT B12

  • Vit B12 is synthesized in nature by microorganisms
  • Animals acquire it by eating other animal foods, by internal production from intestinal bacteria (not in humans) or by eating bacterially contaminated foods
  • Vitamin B12 is found in foods of animal origin such as liver, meat, fish and dairy products, with highest amount found in Liver and Kidney
  • Fruits, cereals or vegetables do not contain Vit B12
  • Vegetarians usually come down with B12 deficiency
  • Cooking does not usually destroy cobalamin

DAILY REQUIREMENTS AND LOSSES

  • A normal diet contains much in excess of what the body requires
  • A normal Western diet contain between 5 and 30µg of cobalamin daily
  • Adult daily losses (mainly in the urine and feaces) are between 1 and 3µg
  • Body stores are of the order of 2-3 mg and are sufficient for 3-4 years if supplies are completely cut off

ABSORPTION OF VIT B12

  • The bioavailability of food vitamin B12 varies depending on the amount of vitamin B12 in the diet, but normally averages around 50%
  • Vitamin B12 in food is bound to protein and is released in the stomach by the acid environment and by proteolysis of binders by pepsin
  • The released vitamin B12 initially binds to R-binders, which are dietary proteins that have affinity for vitamin B12
  • As the vitamin B12-R-binder complexes pass through the small intestine, the R binder is broken down in the duodenum by the pancreatic enzyme, trypsin, to release B12
  • Which then becomes attached to another glycoprotein called Intrinsic Factor (IF)
  • IF is secreted by the parietal cells of the gastric mucosa
  • IF can also bind B12 in bile forming an entero-hepatic circulation
  • IF-B12 complex is carried to the distal ileum where it gets attached to specific IFreceptor (cubulin) in the distal ileum
  • It is then absorbed into the ileal mucosal (brush border) cells by a receptor-mediated endocytosis
  • In the ileal cells, the IF-B12 complex fuse with lysosomes, then IF is degraded, and the vitamin B12 is released into the cytosol
  • B12 then gets attached to a transport protein, Transcobalamin II (TC II), and is transported to the portal circulation
  • TC II is a polypeptide synthesized by the liver macrophages and the ileal mucosal cells
  • The process of absorption across the gut epithelium takes about 3-4 hours
  • TC II transports B12 in plasma to the bone marrow and other tissues, where it is taken up and utilized for the synthesis of deoxyribonucleic acid (DNA) of developing blood cells
  • The half-life of the TC-II-vitamin B12 complex in plasma is about 6 minutes
  • A deficiency of TC II can also result in megaloblastic anaemia due to failure of B12 to enter the bone marrow
  • Serum B12 level will be normal in TC II deficiency
  • Plasma contains 2 additional vitamin B12-binding glycoproteins or R-binders
    • Haptocorrin (transcobalamin I, TC-I) and
    • Transcobalamin III (TC-III)
  • They are less specific than TC-II and also bind B12 analogs
  • Most of the body store of vitamin B12, estimated at about 2-3 mg, is in the liver
  • 5’-Deoxyadenosylcobalamin, attached to methylmalonylCoA mutase, is the major form of the vitamin in liver
  • Methylcobalamin is the major form in plasma
  • The vitamin is excreted via the urine and via the bile
  • Normally, the enterohepatic circulation results in effective reuptake, via the IF receptor of biliary vitamin B12

 

BIOCHEMICAL FUNCTIONS OF VIT B12

  • The 2 natural forms of B12 are;
    • Methylcobalamin (methyl B12)
    • Deoxyadenosyl cobalamin (ado B12)
  • Methyl B12 is used as a cofactor in the methylation of homocysteine to methionine by methyl tetrahydrofolate
  • Deoxyadenosyl B12 is used during the conversion of methyl malonyl CoA to succinyl CoA

 

FOLATE METABOLISM

  • Folate (folic acid or pteroyglutamic acid) is a yellowish, stable and water soluble compound
  • Various forms of folate include;
    • Folate polyglutamates
    • Dihydrofolates, and
    • Tetrahydrofolates
  • Folate can be synthesized by bacteria but not in humans, so man must obtain it from plant or animal sources;
    • Liver
    • Green vegetables
    • Yeast
  • Folate is heat-labile and easily destroyed by excessive boiling of vegetables and other sources
  • An average daily diet contains between 200-250 µg of folate of which about 150µg is absorbed daily

 

ABSORPTION, TRANSPORT AND BIOAVAILABILITY OF FOLATE

  • Folic acid does not occur in nature, is rarely found in unfortified foods, and is not an active form of the coenzyme
  • However, it is the most common form of folate used in supplements and in fortified food products because it is highly bioavailable, chemically stable, and is readily reduced to tetrahydrofolate, the active coenzyme form of folate
  • The bioavailability of folic acid is close to 100% when consumed on an empty stomach
  • Folate is absorbed mainly in the duodenum and jejunum
  • Dietary folate occurs in the form of polyglutamates which are hydrolyzed to monoglutamates by the enzyme – pteroyl polyglutamates hydrolase
  • The monoglutamates are reduced and then methylated to form methyltetrahydrofolate in the intestinal mucosal cells
  • Methyl THF is absorbed into the blood stream and transported to bone marrow and other body tissues bound to albumin
  • Folate store in the body is about 10-12 mg, which is only sufficient for about 4 months if no folate is taken

BIOCHEMICAL REACTIONS INVOLVING FOLATE

Folates are required in some biochemical reactions in the body;

  • In the synthesis of purine bases
  • In the synthesis of thymidylate which is required for the synthesis of pyrimidine bases

Purine and pyrimidine bases are required in the synthesis of DNA

  • In the interconversion of amino acids e.g. in conversion of homocysteine to methionine and serine to glycine
  • Folate deficiency can cause megaloblastic anaemia by inhibiting the synthesis of thymidylate which is the rate limiting step in the synthesis of DNA
  • There is impairment of chromosomal replication in DNA with formation of chromosomal breaks thereby leading to cell death in the ‘S’ phase of the cell cycle
  • Anti-folate drugs—methotrexate and pyrimethamine—cause megaloblastic anaemia by inhibiting the enzyme DHF reductase which is involved in DNA synthesis
  • Toxicity to methotrexate or pyrimethamine can be reversed by giving Folinic acid (5formyl THF), a more stable and reduced form of folic acid
  • Macrocytosis refers to clinical conditions in which the RBCs are abnormally large with the MCV > 100 femtolitres (fl)
  • Causes of macrocytosis can be divided into;
    • Megaloblastic causes
    • Non-megaloblastic causes

Megaloblastic Causes of Macrocytosis

  • Vitamin B12 deficiency
  • Folate deficiency
  • Pernicious anaemia
  • TC II deficiency
  • Chronic Nitrous oxide administration – during anaesthesia

Non-megaloblastic Causes of Macrocytosis

  • Chronic alcoholism
  • Liver dx
  • Myxedema
  • Smoking
  • Reticulocytosis – from chronic haemolysis/haemorrhage
  • Anti-folate drugs – pyrimethamine, methotrexate
  • Physiological – pregnancy and neonates

 

[1] Peripheral Blood

[2] Ring Sideroblast

[3] Puncture of a vein to remove blood

[4] A stippled pattern – small dots

[5] Rheumatoid Arthritis

[6] Systemic Lupus Erythematosus

[7] Connective Tissue

[8] TIBC – Total Iron Binding Capacity

[9] Reticuloendothelial system

[10] High Iron Gene

[11] Pica – a craving to eat non-food substances such as earth or coal

[12] Treatment

[13] The presence in the feaces of blood that cannot be seen by the naked eye but can be detected by chemical tests