Anaemia with Thrombocytosis

A 49 year old woman presented with weakness and fatigability. On examination, other than pallor of the skin and mucosa there was no other finding.  A haemogram was done that showed a haemoglobin: 6.7g/dL, leucocyte count: 9.1 X 109/L and a platelet: 664 X 109/L.

Anaemia and thrombocytopenia is a feature of

  1. Myeloproloferative neoplasm
  2. Chronic inflammation
  3. Underlying malignancy (Paraneoplastic)
  4. Iron deficiency

A haemogram from a automated haematological counter hides a lot of information. Before more investigations are done it is important to assimilate all the information in the haemogram. The haemogram in the above listed conditions shows:

  1. Myeloproliferative Neoplasm: The myeloproliferative neoplasm are diseases characterised by proliferation of bone marrow. They are distinct from acute leukaemia. According to the 2016 WHO classification myeloproliferative neoplasm include chronic myeloid leukaemia (CML), chronic neutrophilic leukaemia (CNL), polycythaemia vera (PV), progressive myelofibrosis (PMF), essential thrombocytosis (ET), chronic myeloproliferative neoplasm unclassified (CMPN-U) and chronic eosinophilic leukaemia (CEL).  Myeloproliferative diseases associated with anaemia and thrombocytosis are CML, prefibrotic phase of PMF and CMPN-U. Haemogram of patients with myeloproliferative diseases shows leucocytosis with presence of immature leucocyte forms. This is most pronounced in patients with CML. In fact leucocytosis with the presence of immature leucocyte forms is the dominant feature of the haemogram of patients with CML. Anaemia of myloproliferative is normocytic and normochromic.
  2. Chronic Inflammation and Paraneoplastic Diseases: Anaemia and thrombocytosis can also be seen in patients with chronic inflammation and as a paraneoplastic finding. An occasional immature leucocyte form may also be seen in these conditions. This picture may be indistinguishable from that myeloproliferative diseases other than CML. The anaemia is normocytic and normochromic. When the chronic disease or neoplasm is associated with blood loss as may be the case in cancers of the gastrointestinal tract or inflammatory bowel disease, microcytosis due to a coexisting iron deficiency may be seen.
  3. Iron Deficiency: Iron deficiency is associated with microcytic hypochromic anaemia. The degree of microcytosis co-relates with the degree of iron deficiency. The leucocyte counts depends on the cause of iron deficiency. Commonly the leucocyte is normal or slightly decreased. Patients who have iron deficiency because of blood loss due to an inflammatory condition may have leucocytosis. Iron deficiency from blood loss due to helmethiasis may cause eosinophilia.

The differential leucocyte count showed 67% polymorphs, 27% lymphocytes, 2% monocytes and 4% basophils. The erythrocyte indices were MCV 61fl, MCH 15.3pg and MCHC 24.9g/dL. The red cell distribution width was 28.5%. The peripheral smear showed hypochromia, microcytosis, anisocytosis and poikilocytosis.

Of the causes of anaemia and thrombocytosis listed above only iron deficiency is characterised by hypochromic microcytic anaemia. Iron deficiency is also characterised by anisocytosis and poikilocytosis. This manifests as increased red cell distribution on the haemogram.

The serum iron was 27.9 µg/dl, the total iron binding capacity 488 µg/dl with a transferrin saturation 5.7%. The serum ferritin was 8.53 ng/ml. The haemoglobin electrophoresis showed a haemoglobin A2 of 2.8%, the HbA 96% and haemoglobin F 1.2%.

Iron deficiency is diagnosed by documenting low body iron stores and/or impaired iron delivery of iron to the erythroid precursors. The gold standard for depletion of iron stores is absence of stainable iron in the bone marrow. Serum ferritin accurately reflects body iron stores. It has become the preferred method to demonstrate depletion of body iron stores because of the invasive nature of bone marrow aspiration. Levels less than 15ng/ml strongly suggest iron deficiency. Serum ferritin is specific but not sensitive for iron deficiency. Its has a sensitivity of 59% if the cutoff is 15ng/mL and 75% if the cutoff is less than 16ng/ml. The low sensitivity makes the test of limited value to exclude iron deficiency. Ferritin in an acute phase reactant. It has a limited value in the presence of inflammation.

Unlike low serum ferritin, low serum iron is of limited value in diagnosis of iron deficiency. Iron delivery to the haemoglobinizing erythroid precursors is a function of transferrin saturation rather than the serum iron levels. One can have a low serum iron and a low total iron binding capacity as may be seen in anaemia of chronic disease and yet have a normal transferrin saturation. Such patients do not benefit from iron supplementation. Patients with iron deficiency have a low transferrin saturation indicated impaired iron delivery to the developing erythroid cells. Lower the iron saturation higher the probability of iron deficiency being present. Patients are considered to be iron deficient if the transferrin saturation is less than 16%. This patient had a transferrin saturation of 5.7% and a serum ferritin of 8.63ng/ml along with microcytic hypochromic anaemia. A diagnosis of iron deficiency anaemia was made.

The diagnosis of iron deficiency is incomplete without diagnosing the cause of anaemia. Iron deficiency in a 49 year old woman frequently is a result of blood loss that is often menstrual.  This woman had attained menopause and is being evaluated for a gastrointestinal blood loss.

Heme Synthesis

Heme, a porphyrin, is a co-factor in haemoglobin, myoglobin, cytochrome, catalase, heme peroxidase, and endothelial nitric oxide synthase. It has a complex structure with four pyrrole rings with a ferrous iron in the centre that allows it to carry oxygen. The synthesis of heme takes place from glycine and succinyl CoA in eight steps and is extensively studied. Mutations in genes encoding for enzymes involved in heme synthesis result in porphyrias.

Steps in Heme synthesis

About 85% of the heme is synthesised in the developing erythroid cells and almost all the remaining is synthesised in the liver. The control of synthesis differs in erythroid and non-erythroid cells reflecting the exceedingly high heme requirement of the former for haemoglobin synthesis. Heme synthesis takes place in the mitochondria as well as cytosol. The first step, formation of δ-aminolevulenic acid, takes place in the mitochondrial matrix. The next few steps take place in the cytosol. The heme precursor, corpoprophyrinogen III, returns to the mitochondria, is converted to protoporphyrin IX and iron incorporated. The steps in heme synthesis are as follows

  1. Synthesis of δ-aminoleuvelinic acid: Synthesis of δ-aminoleuvelinic acid (ALA) from glycine and succnyl CoA catalysed by ALA synthase (ALAS) is the first step in the synthesis of heme. This is a rate limiting step. ALA synthase is encoded by two genes ALAS1 (OMIM 125290) and ALAS2 (OMIM 301300). ALAS2 codes for the erythroid ALAS and ALAS1 for the non-erythroid (housekeeping) ALAS. The gene ALAS1 is located on chromosome 3p21.1. The product has 12 exons and undergoes is alternate splicing to yield two distinct forms, isoform 1 (640 amino acids) and isoform 2 (657 amino acids). The erythroid specific gene (ALAS2) on X chromosome at Xp11.21. It has 12 exons and also undergo alternate splicing to yield two forms, isoform b (587 amino acids), isoform c (574 amino acids). ALAS is synthesised in the cytosol and transported to the mitochondria. It has a short half life. Heme synthesis is consoled by regulating levels and activity of ALAS (discussed below).
  2. Synthesis of prophobilinogen: ALA moves to the cytosol and is dimerised to prophobilinogen by the action of prophobilinogen synthase (ALA dehydratase). The enzyme is a homo-octomer (made of eight similar units) and needs zinc. The gene (gene ALAD, OMIM 125270) encoding the enzyme is located at 9q32. It has 15 exons. Four isoforms from alternate splicing 361 amino acid, 344 amino acid, 321 amino acid and 304 amino acid are known.
  3. Synthesis of hydroxymethylbilane: Prophobilinogen is converted to hydroxymethylbilane by the action of hydroxymethylbilane synthase. This enzyme is also known as propohbilinogen deaminase. The gene (HMBS OMIM 609806) is located at 11q23.3, has 15 exons. Four alternately spliced forms with 361, 344, 321 and 304 amino acids are known.
  4. Synthesis of uroporphyrinogen: Hydroxymethylbilane is converted to enzymatically to uroporphyrinogen III as well as non-enzymatically to uroporphyrinogen I. The enzymatic conversion is catalysed by the enzyme uroporphyrinogen III synthase. Uroporphyrinogen III synthatase is encoded by a gene (UROS, OMIM, 606938) on 10q25.2-q26 that has 16 exons and encodes for a 265 amino acid protein.
  5. Synthesis of corpoporphyrinogen III: Uroporphyrinogen III is decrboxylated to corpoporphyrinogen III by uroporphyrinogen decarboxylase. The gene (UROD, OMIM 613521) for thes enzyme is at 1p34. It has 10 exons and encodes for a protein 367 amino acid long. This is the last step in the cytosol.
  6. Synthesis of protoporphrinogern IX: Coproporphyrinogen III is converted to propoporphyrinogen IX by a reaction catalysed by corpoporphyrinogen oxidase  in the mitochondria in an oxygen dependent reaction. The gene for corpoporphyrinogen oxidase (COPX, OMIM 612732) is at 3q11.2-q12.1 8 exons. The product has 454 amino acids.
  7. Synthesis of protoporphyrin IX: Propoporphyrin is the final product of the pathway into which iron is incorporated. Protoporphyrin IX is synthesised by the action of protoporphyrinogen oxidase. The gene (PPOX, OMIM 600923) for this enzyme is located at 1q22  and has14 exon. It encodes for a 477 amino acid enzyme.
  8. Synthesis of heme: Ferrochelatse (protoporphyrin ferrochelatase) catalysed the incorporation of iron into protoporphyrin IX. The gene (FECH, OMIM 612386) for ferrochelatse is located at 18q21.31 and has 11 exons. It encodes for a 477 amino acid enzyme.

Control of heme sythesis

The rate limiting enzyme of heme synthesis is the synthesis of ALA. ALA synthase has a short half life. Heme synthesis is regulated  by controlling the levels and activity of ALA synthase.

  1. Inhibition of ALA synthase: ALA synthase is subject to feedback inhibition by heme and and it’s oxidation product hemin. ALA synthase is synthesised in the cytosol and transported to the mitochondrial matrix. In addition to being an inhibitor of ALA synthase hemin also inhibits the metochondrial transport of the enzyme.
  2. Promotion of ALA synthase activity: Cellular iron and factors promoting erythroid differentiation increase the synthesis of ALAS-2, the enzyme responsible ALA synthesis in erythroid cells. Erythroid specific factors like GATA-1 promote the transcription of the ALAS-2 gene. Untranslated portions of the ALAS-2 mRNA have iron responsive elements (IRE) that promote translation. The activity of ASLS in iron deficient cells is low.


Porphyrias are inherited diseases resulting from a mutation of genes involved in heme synthesis. With one exception, X-linked porphyria that results from a gain of function mutation of ALAS synthase 2, porphyrias result from a partial deficiency of the enzymes involved in heme synthesis. Enzyme deficiency results in accumulation of substrates for the reaction catalysed by the enzyme encoded by the gene. Symptoms of porphyrias may be intermittant and/or chronic. The symptoms are diverse and include skin changes, photosensitivity, abdominal pain, muscle weakness, CNS disturbance, seizures, hyponatremia, discolouration of urine. Enzyme deficiencies associated with porphyrias as as follows:

  1. ALA synthatase 2: Gains of function mutation in X linked protoporphyria
  2. ALA dehydratase: ALA dehydrate deficient porphyria (ADP). Lead displaces zinc from binding sites inhibiting the function of the  with enzyme. In patients with tyrosinaemia type 1 Succinylacetone (4,6-dioxoheptanoic acid) accumulates in tyrosinaemia type I. It is structurally similar to ALA and a potent inhibitor of ALA dehydratase.
  3. PBG Deaminase deficiency results in acute intermittent porphyria
  4. Uroporphyrin III synthatase deficiency results in congenital erythrocytic porphyria
  5. Uroporphyrin decarboxylase deficiency results in porphyria cutanea tarde. All patients with porphyria cutanea trade do not have a mutation. Only type II has gene mutations. Types I and III are due to mulifactorial effects on the gene.
  6. Coproporphyrin III oxidase deficiency results in hereditary coproporphyria
  7. Protoporphyrin oxidase results in varigate porphyria
  8. Ferrochalase results in erythropoietic porphyria

Further Reading

Porphyrin and Heme Metabolism
Erythroid Heme Biosynthesis and Its Disorders (doi:  10.1101/cshperspect.a011676)


Evaluation of Splenomegaly

The spleen is a secondary lymphoid organ that lies in intraperitoneally in the left hypochondrium, abuting the diaphragm. It spans from the 9th to 11th rib and weighs between 150-200g. Spleen is supplied by the splenic artery and drains into portal circulation via the splenic vein. It is a part of reticuloendothelial system, immune system and is a site of in utero haematopoiesis. The spleen is enlarged in a diverse set of disease of the above mentioned  systems and in portal hypertension.

Normal Functions of the Spleen

The normal functions of the spleen include

  1. Reticuloendothelial functions: The spleen as a component of the reticuloendothelial system is involved in clearing the blood of ageing or damaged erythrocytes, antibody coated cells and opsonised bacteria. It also removes particles from red cells. The spleen ensures that the red cell in circulation have adequate deformability for passage through microcirculation.
  2. Immune Functions: The spleen is a part of the immune system and plays a role in mounting the immune response . Splenectomy increases the risk of infections particularly with capsulated organisms (see Overwhelming Post-Splenectomy Infection (OPSI)).
  3. Haematopoiesis: Spleen is the site for haematopoiesis in utero. In extrauterine life spleen can become a site of haematopoiesis in disease.

Palpating the Spleen

  1. Palpation of the spleen should start from the right iliac fossa. If this is not done there is a risk of missing a massively enlarged spleen.
  2. Move towards the left costal margin in a direction perpendicular to the margin. Move with each breath. At every position ask the patient to take a deep breath. The tip of the spleen will hit your palpating finger.
  3. If the spleen does not hit your finger move your palpating finger to a position closer to coastal margin, ask the patient to take a deep breath and repeat the procedure described above till your finger hits the costal margin.
  4. If the spleen is felt measure the perpendicular distance between the tip and the left coastal margin. Also note the texture and presence of tenderness.
  5. If the spleen is not felt repeat the procedure with patients lying on right side.
  6. Large spleen can rupture with aggressive palpation. The spleen lies directly under the anterior abdominal wall. One does not need to be aggressive.

Causes of Splenomegaly

The spleen enlarges from the left coastal margin in the direction of the umbilicus. It needs to enlarge 2-3 times before it is palpable. Splenomegaly may be caused be increase in portal venous pressure, infiltrative conditions or when the spleen function needs to increase. Clinically it is useful to classify splenomegaly by size. Massive splenomegaly is enlargement of the spleen beyond the umbilicus. The causes of massive splenomegaly include

  1. Malignant: Chronic myeloid leukaemia, Idiopathic myelofibrois, hairy cell leukaemia, splenic marginal zone lymphoma, chronic lymphocytic leukaemia, prolymphocytic leukaemia
  2. Infections: Tropical splenomegaly, AIDS with Mycobacterium avium complex infections, Kala-azar (visceral leishmaniasis)
  3. Others: β-Thalassaemia major and intermedia, Extrahepatic portal venous obstructions,megaloblastic anaemia, diffuse splenic haemagiosis

The causes of splenomegaly include the above and the following

  1. Portal Hypertension: Cirrhosis, Budd-Chairy syndrome, splenic vein thosmbosis, congestive heart failure, hepatic schistosomiasis
  2. Increased splenic function:
    1. Increased functional demands: Haemolytic anaemia commonly hereditary spherocytosis, autoimmune haemolytic anaemia, β-thalassaemia, early sickle cell anaemia, sickle cell β-thalassaemia,
    2. Infections:
      1. Bacterial: Septicaemia, bacterial endocarditis, splenic abscess, brucellosis, tuberculosis, AIDS with Mycobacterium avium complex infections, secondary syphilis
      2. Viral: Viral hepatitis, infectious mononucleosis, cytomegalovirus,
      3. Parasitic: Malaria , Kala-azar (visceral leishmaniasis), Trypanosomiasis,
      4. Fungal: Histoplasmosis
    3. Immune Disorders:
      1. Autoimmune diseases: Rhumatoid arthritis (Felty’s syndrome), systemic lupus erythrmatosis
      2. Other immune disorders: Immune haemolytic anaemia, immune neutropenia, drug reaction, serum sickness, sarcoidosis
      3. Haemophgocytic lymphohistiocytosis
  3. Infiltrations
    1. Haematological Malignancy:
      1. Myeloid: Chronic myeloid leukaemia, myeloproliferative disease, idiopathic myelofibrosis, polycythaemia vera
      2. Lymphoid: Acute lymphoblastic leukaemia, hairy cell leukaemia, chronic lymphocytic leukaemia, prolymphocytic leukaemia, splenic marginal zone lymphoma, angioimmnoblastic T cell lymphoma
      3. Other: Histiocytosis X, eosinophilic granuloma
    2. Storage disorders:Gaucher disease, Niemann-Pick, Tangier disease, mucopolysachroidosis
    3. Other Infiltrations: Amyloid
  4. Others: Iron deficiency anaemia


History and Physical Examination

  1. Fever: Fever is a feature of splenomegaly due to infections, inflammations or malignancy, particularly haematological malignancy. Usually the fever is low grade. High grade fever suggests splenic abscess.
  2. Painful splenemegaly: The nature of pain associated with splenomegaly varies with the cause of splenomegaly.
    1. An enlargement spleen from any cause can cause a dragging pain in the left upper quadrant.
    2. Acute pain left upper quadrant pain is a feature of is a feature of splenic infarct and splenic abscess. Sickle Cell anaemia is associated with small fibrotic spleen because of repeated splenic infarcts. Early in disease the spleen enlarges. Patients may present with acute pain from splenic infarcts. Enlarged spleen from any cause is predisposed to infarction. Acute pain in the left upper quadrant is also a feature of acute splenic abscess.
    3. Splenic vein thrombosis can cause splenomegly and pain in left upper quadrant or epigastric region. It may also cause generalised abdominal pain.
    4. Pancreatitis presents with abdominal pain and can cause painful splenomegaly secondary to splenic vein thrombosis.
    5. Alcohol induced pain is an uncommon but unique feature of Hodgkin lymphoma. Spleen is a common site of involvement by Hodgkin lymphoma. Such patients may have alcohol induced pain in an enlarged spleen.
  3. Pallor: Pallor in a patient with splenomegaly suggests a diagnosis of haemolytic anaemia, haemolymphatic malignancy and infective endocarditis.
  4. Clubbing: Clubbing with splenomegaly is a feature of infective endocarditis and cirrhosis of the liver.
  5. Skin rash: Skin rash in a patient with splenomegaly is seen in systemic lupus erthomatosis, infective endocarditis, lymphoma (angioimmuniblastic T Cell lymphoma, mycosis fungiodes, skin involvement with lymphoma) and drug reaction.  Each of these conditions have a distinct type of rash.
  6. Skin Pigmentation: Hyperpigmantation suggests be seen in hemachromatosis or megaloblastic anaemia. The patients with megaloblastic anaemia may also have knuckle pigmentation.
  7. Jaundice: Jaundice with enlarged spleen is a feature of haemolytic anaemia. The jaundice is usually achloruric. Patients with haemolytic anaemia are predisposed to gallstones. Obstruction of the biliary system from a calculus dislodged from the gall bladder can cause obstructive jaundice with abdominal pain and signs of acute inflammation. Splenomegaly with jaundice is a feature of advanced cirrhosis. Patients with advanced cirrhosis almost always have ascites.
  8. Lymphadenopathy: The enlargement of lymph nodes and spleen is a feature of lymphoid malignancies or diseases that stimulate the lymphoid systems viz. infections and autoimmune diseases and lymphoid malignancy.
  9. Joint symptoms: Arthropathy with splenomegaly suggests the diagnosis of rheumatoid arthritis, systemic lypus erythrmatosis or haematochromatosis.
  10. Oral symptoms: infectious mononucleosis is charecterized by pharyngitis and generalised lymphadenopathy. Bleeding gums and/or gum hypertrophy suggests a diagnosis of leukaemia. Lymphoma can cause tomsillar enlargement. Amyloid is charectetized by macroglossia.
  11. Evidence of Portal Hypertension and Liver Cell Failure: Patients with portal hypertension often have history of haemetemesis. Examination may reveal periumbilical veins (capital medusae), anterior abdominal or flank veins. Patients with evidence liver cell failures with portal hypertension (e.g. jaundice, ascites, spider angiomas, asterxis etc. see Portal Hypertension) have cirrhosis. When the jugular venous pressure is high a diagnosis of congestive cardiac failure should be considered.

Laboratory Evaluation

Haemogram; The haemogram is the most important laboratory test in evaluating a patient with splenomegaly. The significance of findings on haemogram is described in the table below.

Haemogram Finding Conditions
Pancytopenia Hypersplenism, Lymphoma (splenic marginal zone lymphoma), Hairy cell leukaemia, Myelofibrosis, systemic lupus erythrmotosis
Neutrophilic Leucocytosis Acute infections, inflammation
Leucocytosis with premature white cells Chronic myeloid leumaemia, Myeloproliferative disease, Myeloproliferative/Myelodysplastic overlap, Acute lymphoblastic leukaemia
Leucoerythroblastic anaemia Idiopathic myelofibrosis, Bone marrow infiltration
Polycythaemia Polycythaemia vera
Atypical Lymphocytes Infectious mononucleosis
Thrombocytosis Myeloproliferative disease (Chronic myeloid leukaemia, idiopathic myelofibrosis, polycythaemia vera), chronic infections like tuberculosis
Parasites Malaria, bartonelosizs, babesiosis

Other investigations are dictated by the clinical presentations. Commonly performed investigations include biochemistry, microbiology, echocardiography, endoscopy and biopsy of any lymph node or any other mass. Other investigation may be performed as indicated


Imaging is an important aspect of evaluation of the spleen but is beyond the scope of this article. Several good reviews exist e.g Singapore Med J 56(3):133-144.

Oral Iron Therapy

Iron deficiency anaemia is treated by iron supplementation that may be administered orally or parenterally.  Oral iron absorption is inefficient, is interfered by food and is associated with high incidence of gastrointestinal adverse effects. About 17% of the world population is estimated to be suffering from iron deficiency. Iron deficiency is more common the poorer regions of the world. These regions have limited resources allocated for healthcare. Oral iron is inexpensive and convenient to administer making it the modality of choice for initiation of treatment of iron deficiency. The availability of safer parenteral iron preparations that can replete the iron deficit in one dose has reduced the threshold of switching a person intolerant to iron to parenteral iron.

Oral Iron Absorption

Dietary iron may be heme iron or non-heme iron. Heme iron is absorbed after oxidation to hematin. The absorption of heme iron is more efficient. Dietary non-heme iron is present in the ferric state. Ferric iron is insoluble and needs to be reduced to ferrous iron. Gastric acidity aids this conversion. Medicinal iron is most often administered in the ferrous state. Ferrous iron tends to oxidize to ferric iron at physiological pH. Gastric acid lowers the pH and retards oxidation. The absorption of non-heme iron is aided by ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates and retarded by phytates present in grains and vegetables, dietary fibre, polyphemols present in tea, coffee and wine, phosphates and phosphoproteins present in egg yolk, bovine milk, calcium and zinc. For a detailed discussion on iron absorption see intestinal iron absorption. Preparations containing iron in the ferric state have a 3-4 fold lower bioavailability than ferrous iron preparations. Ferric iron is insoluble in the alkaline medium of the duodenum and needs to be converted to ferrous iron (ScientificWorldJournal. 2012; 2012: 846824).

Oral Iron Preperations

Oral iron preparations may be heme or non-heme. Heme iron is available as heme iron polymer. Non-heme iron may be in the ferrous or ferric form. Carbonyl is pure iron prepared from the decomposition of iron pentacarbonyl. The preparations are listed in the table below.


Preparations Iron Content
Ferrous Sulfate, anhydrous 30%
Ferrous Fumarate 33%
Ferrous Sulfate 20%
Ferrous carbonate, anhydrous 48%
Ferrous Gluconate 12%
Ferric Ammonium Citrate 18%
Ferric bisglycinate 20%
Ferric pyrophosphate 12%
Carbonyl iron ~100%
Heme-iron peptide 100%
Polysaccharide iron complex 100%

Indications of Oral Iron Therapy

Oral iron is indicated for prevention and treatment of iron deficiency anaemia. The rate of iron delivery is insufficient to provide iron when erythropoiesis is stimulated by erythrocyte stimulating agents like erythropoietin and darbepoetin. Oral iron should not be used for such patients.

Trial of Oral Iron Therapy

Serum ferritin is an indicator of total body iron. It is also an acute phase reactant. Low ferritin indicates iron deficiency. The traditional cutoff is 12ng/mL. At this cutoff the sensitivity of ferritin for the diagnosis of iron deficiency anaemia is only 25%. The sensitivity can be increased to 92% with a positive predictive value of 83% if a cutoff of 30ng/mL is used. A trial of oral iron may be given if other causes of anaemia are excluded.

Contraindications to Oral Iron Therapy

  1. Primary hemachromatosis: Primary hemochromatosis is a is absolute contraindication
  2. Peptic ulcer, regional enteritis, or ulcerative colitis can be exacerbated by oral iron.
  3. β-Thalassaemia trait is a relative contraindication. Some patients may develop iron overload. Patients should be given iron only if iron deficiency is established by laboratory investigation.

Failure of Oral Iron Therapy

  1. Failure to take prescribed medication: Oral iron therapy causes gastrointestinal adverse effects in a large proportion patients that are severe enough in some to discontinue therapy. A detailed history must be taken to ascertain that the patient has taken the prescribed dose.
  2. Incorrect or incomplete diagnosis: Iron deficiency anaemia is hypochromic microcytic (see Evaluating Anaemia). Other diseases that result in hypochromic microcytic anaemia are thalassaemia and anaemia of chronic disease. Both are common and can both be confused with iron deficiency as well as co-exist with iron deficiency. Thalassemia trait affects about 1.5% of the world population. About 1.4% of the population is estimated to have anaemia due to infection, inflammation or chronic renal disease ( Incidental occurrence of iron deficiency with thalassaemia trait or anaemia of chronic disease will result in an incomplete response to iron supplementation. Some inflammatory conditions e.g. ulcerative colitis cause blood loss. Blood loss may be seen due to use of non-steroidal inflammatory agents in patients with autoimmune arthritis. Anaemia in these patients shows an incomplete response to iron supplementation because part of the anaemia results from chronic inflammation.
  3. Insufficient amount or inappropriately taken oral iron: The ideal dose is 200mg of elemental iron taken 2 hours before or 1 hour after food. Food interferes with iron absorption but also relieves gastrointestinal adverse effects. Gastrointestinal adverse effects are related to dose. Prescriptions of oral iron may have an insufficient dose or may be administered after food to reduce gastrointestinal adverse effects. This results in an inappropriate haemoglobin response.
  4. Iron demand exceeding intake: Iron demand may exceed supply if there is continued blood loss or in patients where erythropoiesis is stimulated with an erythropoiesis stimulating agent like erythropoietin or darbepoietin. In both these situations the rate of oral iron absorption limits iron availability. These patients need intravenous iron.
  5. Malabsorption of iron: Food interferes with oral iron absorption. Ferrous iron is rapidly oxidised to ferric iron at physiological pH. Gastric acid reduces ferric iron to ferrous iron. Proton pump inhibitors, H2 antagonists, antacids and gastrectomy reduce acidity and can interfere with iron absorption. Iron malabsorption may be seen as part of a malabsorption syndrome. Iron deficiency refractory to iron is rare. Iron refractory iron deficiency anaemia is a disorder resulting from mutations in the TMPRSS6. This mutations results in increase hepcidin production which is sensed by the body as an iron repeated state. Iron is not absorbed despite iron deficiency. The patients do not respond to oral iron and shows a partial response to parenteral iron.


  1. Interaction of iron with food: The absorption of non-heme iron is affected by food (See Intestinal Iron Absorption).
    1. Foods enhancing iron absorption: Ascorbate, animal proteins, human milk, keto sugars, organics, amino acids that form soluble chelates with iron enhance absorption of non-heme iron.
    2. Inhibiting iron absorption: `Inhibitors of absorption of non-heme iron include
      1. Phytates present in grains and vegetables
      2. Dietary fibre
      3. Polypohenols present in tea, coffee and wine,
      4. Phosphates and phosphoproteins present in egg yolk, bovine milk
      5. Calcium and zinc.
  2. Drug-Iron interactions
    1. Iron decreases the absorption of ACE inhibitors, bisphosphonates, levodopa, levothyroxine, penicillamine, quinolone and tetracyclines
    2. The absorption of iron is decreased by drugs that reduce gastric pH. These include H2 antagonists(Cimetidine, ranitidine, famotidine), proton pump inhibitors (omeprazole, pantoprqzole, esomeprazole, lansoprazole, rabeprazole, etc), antacids and cholesterol lowering agents (Cholestyramine and Colestipol).


  1. Children: 3–6 mg/kg daily in 3 divided doses.
  2. Adult: Usual therapeutic dosage: 50–100 mg 3 times daily but a dose of 200mg produces the maximal results. A smaller dosages (e.g. 60–120 mg daily) may be given if patients are intolerant of oral iron, but response in such patients takes a longer time.

Side Effects Of Oral Iron

  1. Gastrointestinal: The commonest adverse effects of iron are gastrointestinal symptoms, including heartburn, nausea, abdominal cramps, diarrhoea or constipation. These may be seen in up to a fifth of the patients. They can be reduced by decreasing the dose or taking iron after food. Taking iron with food can reduce the absorption by about 50%. Enteric coated preparation decrease the side effects by delaying the release of iron. Delaying release may bypass the duodenum that is the site of absorption of iron. The decrease in absorption is particularly marked in patients with aclorhydria as they can not dissolve the enteric coating. The stool of patients taking iron supplements may be discoloured black or green.
  2. Discolouration of teeth: Iron syrups may cause staining to teeth.
  3. Iron overload: Iron overload from oral iron therapy is rare. It has been described in patients with hemachromatosis and chronic haemolytic anaemias.
  4. Iron Poisoning: Iron poisoning usually occurs in children, particularly those younger than 5 years of age, because of accidental ingestion of medicinal iron. Children are likely to ingest these believing them to be candies.

Sickle β-Thalassaemia

Sickle cell anaemia and β-thalassaemia are two common haemoglobinopathies. Co-inheritance of the two is called sickle β-thalassaemia. Sickle β-thalassaemia seen in Africa, throughout the  Mediterranean, Arabian Peninsula and sporadically in india. It has heterogeneous clinical presentation. The severity depends on the severity of the thalassaemia allele and the extent to which the impaired haemoglobin synthesis is compensated by foetal haemoglobin synthesis.


With a very few exceptions (Blood 1989; 74: 1817-22) the sickle cell and the thalassaemia gene are arranged in trans i.e on different chromosomes (βsthal). One allele is inherited from the mother and one from the father. One parent carries the a β-thalassaemia trait the other parent has a sickle cell disease that may be sickle cell anaemia, sickle β-thalassaemia or a trait. Sickle β-thalassaemia in Africa and India/Arabia is mild whereas the patients from the Mediterranean region have severe disease. As mentioned above the differences in severity have to do with severity of the β-thalassaemia and the degree to which the impaired haemoglobin A synthesis is compensated by HbF. Weatherall suggested that patients with HbA <15% follow a course similar to severe HbA and those with HbA 20-30% follow a mild course.

  1. African sickle β-thalassaemia: African patients have a mild β-thalassaemia resulting in a relatively higher HbA level and a lower risk of sickling. These patients run a mild clinical course.
  2. Arab/Indian sickle β-thalassaemia: Patients from India and the Arabian peninsula have a sickle cell haplotype that is associated with a high HbF production. The HbF retards sickling. High levels of HbF attenuate symptoms. Patients carrying this haplotype have mild symptoms even when the inherit a severe β- chain defect. Another reason of a mild phenotype in India is the interaction with α thalassaemia.
  3. Mediterranean sickle β-thalassaemia: Mediterranean patients usually inherit a severe form of  β-thalassaemia. These patients have severe sickling because there is very little HbA or HbF to offset inhibit the crystallisation of HbS. Despite only one chromosome carrying HbS the phenotype of these patients resembles sickle cell anaemia.

Clinical Picture of Sickle-β Thalassaemia

The features of sickle-β thalassaemia resemble those of other sickling disease. It is a chronic haemolytic anaemia the course of which is interrupted by acute exacerbations known as crisis. The manifestations include haemolytic anaemia, painful and other crisis, leg ulcers, priapism and complications of pregnancy. The severity of symptoms is variable. One end of the spectrum are patients, usually of origin Mediterranean descent, whose presentation is indistinguishable from sickle cell anaemia. These patients have inherit severe forms of β (β0) chain defects. Those with sickle cell-β+ thalassaemia have milder symptoms. These patients are typically of African ancestory. Unlike patients with sickle cell anaemia patients with sickle-β thalassaemia may have splenomegaly that is more prominent patients with sickle cell-β+ thalassaemia. The spleen is usually moderately enlarged but massive splenomegaly that may be associated hypersplenism neccesisating splenectomy has been reported. The effect of co-inheritance of α-thalassaemia is small. A decrease in the frequency of acute chest syndrome and leg ulcers and a higher persistence of splenomegaly is seen. Co-inheritance of α thalassemia is one of the reasons that sickle-β thalassaemia runs a milder course in India (the other being the high HbF due to the Arab-Indian haplotype of HbS).


The haematological findings vary with severity. More severe phenotypes shows greater anaemia, lower MCHC, higher reticulocytes, HbF and HbA2. A variable number of sickle cells may be found. Unlike sickle cell anaemia both forms of sickle cell-β thalassaemia have an elevated HbA2. The distribution of HbA2 is very similar to heterozygous β thalassaemia. The levels of HbF are variable. High levels are found in patients with the Arab-Indian and Senegal haplotype of HbS.

Sickle cell-β0 thalassaemia needs to be differentiated from sickle cell anaemia. The presentation of both may be identical. However an offspring of a sickle cell-β0 thalassaemia patients and a carrier of β-thalassamia trait has a 25% risk of suffering from β-thalassaemia major. The offspring of a patients with sickle cell anaemia and a carrier of β thalassaemia trait does not carry the risk of β thalassaemia major. Though sickle cell-β0 thalassaemia is characterised by an elevated HbA2 and splenomegaly this can not be relied upon to differentiate between the two conditions. Family and DNA studies are needed. If the studies show one parent to be heterozygous for HbS and the other a carrier of β thalassaemia trait no further studies are needed. If any of the parent has a phenotype of sickle cell anaemia DNA studies may be the only way to make the diagnosis.

Sickle Cell β thalassaemia in cis

Almost all patients with sickle-β thalassaemia have the disorder in trans i.e. the one β globin gene is thalassaemic and the other has a the sickle mutation. Patients with HbS and thalassaemia gene in cis have been described. These patients have a mild hemolysis, HbA2 levels were 6%–7%, HbF approximately 3% and HbS of 10%–11%.


The symptoms of sickle-β thalassaemia are due to sickling need to be treated accordingly.

Clinical Features of Megaloblastic Anaemia

Megaloblastic anaemia is a macrocytic anaemia resulting from the deficiency of vitamin B12 or folic acid characterised by the presence of megaloblasts in the bone marrow. It has haematological and neurological manifestations. The haematological manifestations are seen with folate as well as vitamin B12 deficiency. Folate deficiency in adults does not affect the nervous system.

Cobalamin deficiency is slow and “pure”. Folate deficiency is rapid and “impure”. Deficiecy of vitamin B12 occurs because of loss of intrinsic factor resulting in an isolated defect of B12 absorption. No other nutrients are affected. The body stores of B12 can last months. This results in B12 deficiency being a slow and “pure” deficiency. Symptoms come on slowly, over months. Folate deficiency evolves relatively quickly and is most commonly because of alcoholism or malabsorption. It is associated with other deficiencies and is rapid and “not pure”.


Manifestationf o megaloblastic anaemia

Figure 1. Clinical Manifestations of Megaloblastic Anaemia

Haematological Manifestations

Haematological changes resulting from vitamin B12 deficiency and folate deficiency are indistinguishable. Megaloblastic anaemias are macrocytic anaemia but macrocytosis is not specific to megaloblastic anaemia. It is however exceptional for other diseases characterised by macrocytosis to have an mean capsular volume (MCV) > 110fl.  This value can considered the threshold above which an anaemia is unlikely to be anything other than megaloblastic anaemia.

The earliest change in a megaloblastic anaemia is macrocytosis. This precedes changes in erythrocyte indices. Changes in mean capsular haemoglobin (MCH) follow and then the MCV rises. Haemoglobin usually falls after the MCV increases to >97 fl. As the severity of anaemia increases the peripheral smear shows aniscytosis and poikilocytosis, nucleated cells, Howell-Jolly bodies and Cabot’s ring. Microcytes and erythrocyte fragments that represent dyserythropoiesis may be seen. Polychromasia is absent and this distinguishes megaloblastic anaemia from haemolytic anaemia.

The term megaloblatic anaemia is a misnomer. The disease is actually a panmyelosis.  Erythroid, myeloid and megakaryocytic series are affected. Thrombocytopenia and leucopenia (neutropenia and to a lesser extent lymphopenia) usually occur late in the course. It is uncommon for patients with mild anaemia to have platelets and neutrophils but occasionally changes in leucocytes and/or platelets may dominate.

Iron deficiency or β-thalassaemia trait result in microcytosis and hypochromia and may incidentally co-exist with megaloblastic anaemia. Co-existence of either of these diseases with megaloblastic anaemia may mask macrocytosis of megaloblastic anaemia. Presence of hypersegmented neutrophils in a patients with normocytic normochromic anaemia should raise the suspicion of a megaloblastic anaemia co-existing with Iron deficiency or β-thalassaemia trait.

Neurological Manifestations

Cobalamine deficiceny causes neurological dysfunction. Folate deficiency causes symptoms only in children. Children with inborn errors of folate metabolism may have myelopathy, brain dysfunction and seizures.

The neurological manifestations of B12 deficiency are a result of a combination of upper motor neuron manifestations from subacute combined degeneration of the spinal cord, sensory and lower motor neuron manifestations from peripheral neuropathy and neurophychiatratic manifestations. Subacute combined degeneration of the spinal cord (SACD) is a degerative disease of the spinal cord involving the posterior and lateral column (corticospinal and spinoceribellar tracts) that starts in the cervical and the thoracic region.

The earliest neurological manifestations are impaired sense of vibration and position and symmetric dysesthasia that involve the lower limb. This is frequently associated with sensory ataxia. With progression spastic paraparesis develops. The patients have brisk knee reflexes, reflecting an upper motor neuron involvement and depressed ankle reflex, reflecting a peripheral neuropathy. Bladder involvement is unusual. Some patients may have optic atrophy.

Neuropsychiatric manifestation include memory loss, depression, hypomania, paranoid psychosis with auditory and visual hallucinations.

Other manifestations

Skin and nails can show pigmentations. Mucosa of the villi undergoes megalobkastic change resulting in temporary malabsorption.

Response to therapy

Haematological Recovery

  • Day 1: Feeling better
  • Day2-3: Reticulocytosis appears
  • Day 7-10: Peak retuculocytosis
  • Day 15 onwards: Neutrophilic hypersegmentation disappears
  • Day 56 (8 weeks): Blood counts become fully .normal

Neurological Recovery

Neurologic improvement begins within the first week also and is typically complete in 6 weeks to 3 months. Its course is not as predictable as hematologic response and may not be complete.



Evolution and Spread of HbS

The gene for β globin (OMIM  is present on chromosome 11 (11p15.4) along with other globin genes (ε, γ, γ and δ). This is known as the β-globin cluster . Individuals carrying identical genes on the β-globin gene cluster may not have identical DNA sequences in non-codeing regions of the DNA of the cluster. The non-coding regions include segments of DNA between genes and introns within genes. . Differences in DNA exist between individuals every 1000-2000 bases in the form of single nucleotide polymorphisms (SNPs). Single nucleotide polymorphisms are variations in a single nucleotide that occurs at a specific position in the genome. Many of these differences have no consequences on gene expression because either they do not result in change in amino acid sequence or they occur in regions of DNA that neither code for the gene nor regulate the gene. SNPs evolve by spontaneous mutations over time. The lesser the number of such differences between two individuals closer the individuals are the each other genetically (and in terms of evolution). Fewer differences in SNPs between individuals mean a more recent common ancestor.

One of the meanings of the word haplotype is a pattern of SNPs. A haplotype may be considered as a DNA “environment” in which the gene(s) occurs. This “environment” is created by the sequence of single nucleotide polymorphisms in which the gene(s) exists. As mentioned above differences in SNPs (and hence the “environment” the gene(s) exist in) evolve by spontaneous mutations over period of time. Fewer the differences between the “environments” the genes occurs in the more the likelihood that they come from related individuals.

HbS results from a single base substitution in the codon 6 of the β-globin gene. GAG becomes GTA resulting in substitution of valine for glutamate. This change results in a haemoglobin that crystallizes in hypoxic conditions resulting in a haemolytic anaemia. HbS occurs in diverse population groups including African, Mediterranean, Middle-Eastern and Indian. Is the haplotype of the HbS gene in these regions similar?

The HbS mutation occurs on five different haplotypes four African and one Arab-Indian. The mutation is the same (GAG to GTA on codon 6) but the SNPs are different. The haplotypes are

  1. Senegal: The Senegal HbS haplotype is found in Atlantic West Africa and Portugal
  2. Benin: The Benin HbS haplotype is found Central West Africa, Northern Africa and Mediterranean Europe (Greece, Sicily)
  3. Central African Republic or Bantu: The Central African Republic or Bantu is found in South Central and Eastern Africa
  4. Cameroon: The Cameroon haplotype is found in the Eton ethnic group of eastern Cameroon
  5. Arab-Indian: The Arab-Indian haplotype is the only non-African phenotype of HbS found in the eastern oasis of Saudi Arabia and India.

Origin of Haplotypes

There are two theories about the origin of haplotypes. The first, and the more accepted one, states that the five haplotypes arose from five independent mutations. An alternative hypothesis states that HbS mutation occurred only once and spread to other haplotypes by gene conversion.


Haplotypes and Severity of Symptoms

Symptoms of sickle cell anaemia are a consequence of crystallisation of haemoglobin under hypoxic conditions. HbF inhibits sickling. Patients with high HbF have fewer symptoms. The Arab-Indian and the Senegal haplotype are associated with higher HbF levels (17% and 12.4% respectively). In general patients carrying these haplotypes have milder symptoms than the Bantu or Benin haplotypes (Blood 2014; 123: 481)


Haplotypes and Human Migrations

Trade, conquests and human migrations (voluntary and slave trade) have disseminated the African haplotypes beyond Africa.

  1. The Mediterranean: Most of the Mediterranean (Greece and Scilly) has the Benin haplotype. This reflects pre-historic migrations from Central West Africa along the then fertile Sahara to North Africa. From here it spread to the Mediterranean via the interactions (Trade and wars) between the two regions. The only exception is Portugal. Portugal has the Senegal haplotype which reflects the trading contacts between Portugal and Atlantic West Africa (Angola and Mozambique).
  2. Americas: Neither the native americans nor the original European settlers to the Americas carried the HbS gene. HbS was imported to the Americas with the slaves from Africa. Jamaica was an important slave import hub and records for where tthe slaves arrived from are available. Jamaica has 73% Benin haplotype, 17% Bantu and 10% Senegal haplotypes. These numbers are close to the actual number of slaves who arrived in Jamaica from regions of Africa where these haplotypes are prevalent. Similarly the distribution of haplotype correspond to the origins of slaves in Baltimore and South Carolina (Mariam Bloom. Understanding Sickle Cell Disease, Page 34).
  3. Arab or Indian: It is not clear if the Arab-Indian haplotype originated in India or Saudi Arabia. But considering that all of tribal India has only one haplotype but the East and West Arabian Peninsula have different haplotypes it is possible that the haplotype originated in India.
  4. Spread to Other Parts: As opposed to the era of slave trade modern migration of people in the recent past have been voluntary. These populations have spread across the world as have those form mediterranean but to a lesser extent. These migrations have introduced the HbS gene in areas where it was not indigenous.