Anaemia with Hyperbilirubinaemia

A 49-year-old female presented with dyspnoea on exertion of 1 month duration. Examination reviled pallor and icterus. There was no lymphadenopathy, clubbing, koilonychia, platonychia, petechiae or purpura. There was no oedema of feet. The pulse was 90/min and the blood pressure 130/70 mm of Hg. Examination of the respiratory, cardiac and nervous systems did not show any abnormality. There was no organomegaly.

The haemoglobin was 4.9 g/dL with an erythrocyte count 1.37 x 1012/L, haematocrit of 16%, MCV of 116.78 fL, MCH of 35.77 pg and MCHC 30.63 of g/L.  The leucocytes count was 2800 with 35% neutrophils and 65% lymphocytes. The platelet count was 90 x 109/L. The peripheral smear showed macrocytosis and anisocytosis. Hypersegmented neutrophils were seen. The reticulocyte count was 3%.

The bilirubin was 2.1 mg/dL with a direct bilirubin of 1.8mg/dL and an indirect bilirubin of 0.3mg/dL. The Lactate dehydrogenase was 1417IU (normal 105 – 333 IU/L).

Anaemia and unconjugated hyperbilirubinaemia are characteristic of haemolysis. Does this patient have haemolytic anaemia?

Haemolysis shortens erythrocyte lifespan and results in increases haemoglobin breakdown. Haemoglobin is made of heme and globin. Heme consists of porphyrin ring at the centre of which is iron in the ferrous state. Iron released from catabolism of heme is reused. The porphyrin ring is catabolised to bilirubin. The bilirubin is transported to the liver for conjugation and excretion (see haemoglobin catabolism). Patients of haemolytic anaemia have unconjugated hyperbilirubinaemia because the increased bilirubin production overwhelms the hepatic bilirubin conjugation capacity.

One of the characteristics of megaloblastic anaemia is ineffective erythropoiesis. Ineffective erythropoiesis is defined as a sub-optimal (fewer) production of mature erythrocytes from a proliferating pool of immature erythroblasts. Each immature erythroblast produces less than the optimal number of erythrocytes because of premature death of erythroid precursors including haemoglobinized precursors. The haemoglobin released from haemoglobinized erythroid precursors is catabolised in the same manner as haemoglobin released from lysed erythrocytes (see haemoglobin catabolism). Megaloblastic anaemias are associated with unconjugated hyperbilirubinaemia because of death of haemoglobinized erythroid precursors.

The treatment of haemolytic anaemia and megaloblastic anaemia are different? How does one differentiate megaloblastic anaemia from that because of haemolytic anaemia? Does this patients have a haemolytic anaemia or megaloblastic anaemia?

Haemolytic anaemia is characterised by shortened erythrocyte survival. Erythrocytes survival is estimated by the use of radionucleotides something that is not possible at most centres. In clinical practice, a shortened erythrocyte survival is inferred from a high reticulocyte count. Reticulocytes are erythrocytes that have been produced in the preceding 24 hours. The erythrocytes survival is about 120 days and about 1% of erythrocytes are produced every day. Consistent with this the normal reticulocyte count is 0.5-1.5%.In patients of haemolytic anaemia, ddestruction of erythrocytes is matched by an increased production by the bone marrow. This manifests as reticulocytosis (see reticulocyte count). Megaloblastic anaemia occurs because of decreased production of erythrocytes and this manifests as reticulocytopenia. The difference between haemolytic anaemia and megaloblastic anaemia is the reticulocytosis in the former reticulocytopenia in the latter. This patient had a high reticulcoyte count but after correction both the reticulocyte production index [0.43] and corrected reticulocyte count [1.07%] were low excluding haemolysis. This patient was evaluated for megaloblastic anaemia.

The haemogram has clues to differentiate between haemolytic anaemia and megaloblastic anaemia. These include

  1. A very high MCV: The MCV is very high. Patients with haemolytic anaemia have a mild elevation in MCV. An MCV value >110fL is almost exclusively found in megaloblastic anaemias because of folate and/or B12 deficiency.
  2. Pancytopenia: B12 and folate deficiency impair DNA synthesis impairing erythrpoieis, myelopoiesis and megakaryopoiesis. Nutritional megaloblastic anaemias because of vitamin B12 and/or folate deficiency may show pancytopenia.
  3. Hypersegmented neutrophils (>5% neutrophils with >5lobes) is a feature of megaloblastic anaemia

Other features of megaloblastic anaemia include rise serum transferrin receptor, increased serum iron, serum ferritin and methemalbumin levels. Like haemolytic anaemia the serum haptoglobin is low and the LDH high. LDH levels in megaloblastic anaemia can ve very high.

This patients had a low serum B12 and was treated with parental B12 (1mg alternate day for 5 doses) and was evaluated for cause of vitamin B12 deficiency. As Schilling’s test was not available a diagnosis of pernicious anaemia was made by documenting gastric atrophy and anti-parietal cell antibodies.


The Erythrocyte Membrane

Compared to other cells of the body the erythrocyte, consistent with its simple functional requirements, has a simple structure. The erythrocyte delivers oxygen to tissue and aids in carriage of carbon dioxide back to the lungs. Oxygen is carried by haemoglobin. Haemoglobin is a very reactive molecule. Extracellular haemoglobin is toxic because of it’s ability to scavenge nitric oxide and cause oxidative injury to the vessels and kidney (Cold Spring Harb Perspect Med 2013 Jun; 3(6): a013433). The erythrocyte is a packet of concentrate haemoglobin solution enclosed by a membrane. It has no organelles. All the biochemical pathways are geared to maintain iron in a ferrous state and prevent oxidative damage.

Oxygen delivery involves protecting tissue from the toxicity of haemoglobin and delivering oxygen to the narrowest parts of circulation. The erythrocyte membrane participates in both the functions. The former is achieved by a selective permeability. The erythrocyte membrane reduces the NO scavenging 1000 fold (J Biol Chem 2005 Nov 25;280(47):39024-32 ) and protects tissue from oxidative injury. The latter is achieved by extreme deformability the erythrocyte membrane provides.

The erythrocyte has a cell with a diameter of 7-8µm. It needs to squeeze past capillaries (3µm) and sinusoids (1-2µm). Extreme deformability of erythrocytes allows a smooth passage through the narrowest parts of the circulation. A sphere is has the smallest surface area for a given volume and as a result the least deformability. Erythrocyte has a biconcave shape that gives it more membrane for volume and allow extreme deformability.

Membrane dysfunction result in erythrocyte change in shape and deformability. Such cells are not able to pass thought splenic sinusoids and are haemolysed. The haemolysis is usually extravascular. Extravascular haemolysis has few clinical consequences other than those related to increased bilirubin production. Membrane damage due to physical, chemical and immune mediated injury results in intravascular haemolysis that has a potential to cause serious injury.

Organization of the Erythrocyte Membrane

The red cell membrane consists of a lipid bilayer, traversed by proteins and anchored to a cytoskeletal scaffolding made of spectrin. The cytoskeleton provides the extreme deformability and the lipid layer provides the selective permeability. Erythrocyte membrane defects may be due to defects in the lipid bilayer, defect in the cytoskeleton or defects in the attachments of the lipid bilayer to the cytoskeleton. Defects in cytoskeletal and anchoring proteins are inherited (see table below) and those in the lipid layer are usually acquired.

The figure below is a schematic relationship between erythrocyte membrane proteins and lipid membrane bilayer. The main component of cytoskeleton is spectrin. Spectrin is a tetrameter made of two α- and two β- subunits. Spectrin is tethered to the cell membrane by vertical interactions with band 3 proteins via ankyrin and to protein 4.2. Spectrin also has horizontal interactions with protein 4.1, actin, tropomodulin, tropomyosin and adducin. Protein 4.1 interacts with glycophorin C, a trans-membrane protein.

Red Cell Membrane-600px

Erythrocyte Membrane Proteins


Spectrin is made up of two intertwining chains the α and the β chain. The two chains associate to form a dimer and two dimers associate to form a tetramer. The α chain is encoded by the gene SPTA1 at 1q23 (Entrez 6708) and the β-spectrin is encoded by the gene SPTB at 14q23 (Entrez 6710). Spectrin three functions

  1. Supporting the lipid layer
  2. Maintaining cell shape
  3. Regulating the lateral movement of integral membrane proteins.

Spectrin is a tetramer made of two chains α and β. The α and β that wind around each other to form dimers. Two dimers associate head to head to form a tetramer. The α-chain is encoded by the gene SPTA1 (OMIM 182860). The β-chain is encoded by the gene SPTB (OMIM 182870). Defects is either of the spectrin genes cause hereditary spherocytosis but with a different inheritance pattern. The rate of synthesis of the β-spectrin limits the formation of the spectrin tetramer. Spectrin deficiency may occur even with slight impairment of β-spectrin synthesis. This may be seen in a patient who is heterozygous for a β-spectrin defect. Autosomal dominantly inherited hereditary spherocytosis is a consequence of defects in the β-spectrin gene ((type 2 hereditary spherocytosis).

Unlike β-spectrin α-spectrin is synthesised in a excess. Both the alleles need to carry mutation for levels to fall to a level that hampers spectrin tetramer assembly. Hereditary spherocytosis (type 3 hereditary spherocytosis) due to α-spectrin is inherited in an autosomal recessive manner.



Ankyrins are adaptor proteins.The erythrocyte ankyrin, ankyrin-R is encoded by the gene ANK1 (Entrez 286) located at 8p11.2. Ankyrin binds spectrin to the red cell membrane. Ankyrin deficiency leads to decreased incorporation of spectrin on the membrane despite normal spectrin synthesis. Ankyrin gene mutations lead to autosomal dominant hereditary spherocytosis (type 1 hereditary spherocytosis).

Band 3

Band 3 glycoprotein of the erythrocyte membrane that is coded by the gene is SLC4A at 17q21.31 (Entrez 6521). The membrane domain of band 3 transports anions across the cell membrane. The cytoplasmic domain binds the lipid membrane to spectrin via ankyrin. Mutations in the band 3 glycoprotein result in hereditary spherocytosis (type 4 hereditary spherocytosis), hereditary acanthocytosis, southeast asian ovalocytosis and hereditary stomatocytosis.

Protein 4.2

Protein 4.2 regulates the interaction of band 3 with ankyrin. It is encoded by the gene EPB42 at 1p33-p34.2 (Entrez 2038). Deficiency causes an autosomal recessive form of hereditary spherocytiosis (type 5 hereditary spherocitosis)

Protein 4.1

Protein 4.1 stabilized the spectrin-actin interactions. It is encoded by the EPB41 gene at 1p35.3 (Entrez 2035). Deficiency causes hereditary elliptocytosis.


Erythrocyte Membrane Defects
Protein Gene Chromosome Disorders
α-Spectrin SPTA1 (Entrez 6708) 1q23.1 Hereditary Elliptocytosis, Hereditary Pyropoikilocytosis, Hereditary Spherocytosis
β-Spectrin SPTB (Entrez 6710) 14q23 Hereditary Elliptocytosis, Hereditary Pyropoikilocytosis, Hereditary Spherocytosis
Ankyrin-1 ANK1 (Entrez 286) 8p11.2 Hereditary Spherocytosis
Band 3 SLC4A1 (Entrez 6521) 17q21.31 Hereditary Spherocytosis, Hereditary Acanthocytosis, Southeast Asian Ovalocytosis, Hereditary Stomatocytosis
Protein 4.1 EPB41 (Entrez 2035) 1p35.3 Hereditary Spherocytosis
Protein 4.2 EPB42 (Entrez 2038) 15q15.2 Hereditary Spherocytosis
Rhesus Asociated Polypeptide RHAG (Entrez 6005) 6p12.3 Overhydrated Hereditary Stomatocytocytosis
Glycophorin C GYPC (Entrez 2995) 2q14.3 Hereditary Elliptocytosis (seen only with Leach phenotype)

 Disorders of Erythrocyte shape

Disruption in the cytoskeleton is the basis of in a viariey erythrocyte disorders charecterzid by alterations in erythrcoyte shape (see tabel and figure above). Disruption in vertical interactions results in instability of lipid layer resulting in loss of lipid layer and spherocytosis. Disruptions in horizontal interactions results in hereditary elliptocytosis.

Changes in the lipid layes also results in changes in erythrocyte shape. Unlike disorders ofthe cytskeleton, most of these disorders are accquired.

  1. Target Cells: Traget cells or codocytes are cells that have an appearance of a shooting target with a central bulls eye. Reletive increase in the membrane lipids results in the formation of target cells. This is seen in severe microcytis anaemias like severe iron deficiency, thalassaemia, haemoglobin C disease and haemoglobin E disease where the intracellular contents decrease. It may aslo bee seen in obstructive liver disease where the lipid and cholesterol content of the membrena increase.
  2. Stomatocytes: Stomatocytes are erythrocytes with a central elongated mouth-like area of pallor. Expansion of the inner layer results in stomatocytosis. This may be seen in alcoholism and with the use of vinca alkaloids.
  3. Echinocytes Ecchinocytes are cells that are no longer disc shaped and are covered by 10-30 short projections. The change is because of expansion of outer lipid layer. Ecchinocytes are seen in uraemia, pyruvate kinase deficiency or may be a fixing/staining artefact.
  4. Acanthocytes: Acanthocytes are cells with a few spiny projections on the surface (from acanthus, The Greek word for thorn). The result from accumulation of cholesterol (liver disease) or sphingomyelin (abetalipoproteinaemia) in the outer lipid layer results in acanthocytosis.

Sickle Cells

Sickle Cell - 100X - IMG_0542

Sickle Cells 40X

Sickle Cell 100X - IMG_0540

Sickle Cells – 100X

Sickle Cell 100X - IMG_0538

Sickle Cells – 100X

The three photomicrographs above show sickle cells from a patients with sickle cell anemia. Sickle cell anaemia occurs because an A→T substitution in codon 6 of the β globin chain of haemoglobin. The single nucleotide polymorphism results in valine substituting for glutamic acid resulting in the formation of haemoglobin S (HbS). HbS crystallizes in hypoxic conditions resulting in sickling of erythrocytes.

Related articles
Sickle Haemoglobin and Variants

Erythrocyte Metabolism

GlucoseMetaboloisRBC As erythrocytes lack mitochondria they are not able to use fats or generate energy from Krebs cycle. Though they have enzymes to synthesize glycogen the balance between synthesis and breakdown favours breakdown. Normal erythrocytes do not have glycogen and depend on a continuous supply of glucose to meet their energy requirements.  Glucose enters the erythrocyte freely by facilitated diffusion. Insulin does not have any effect on the entry of glucose into the erythrocyte.

They erythrocyte metabolism needs ATP as a source of energy and NADH and NADPH cofactors. The erythrocyte does not synthesize nucleic acids but it has a small requirement for ribose to synthesize nucleosides for energy transfer The metabolic needs of erythrocytes are met by metabolism of glucose through three pathways glycolysis, the hexose monophosphate shunt and Rapport-Luebering glycolytic shunt.

Glycolysis in Erythrocytes
Glycolysis is a process in which one molecule of glucose is converted to two molecules of pyruvate with the a net formation of two ATP and two NADH molecules. The energy released in the process stored as ATP. The electron released in the conversion of glyceraldehyde-3-phosophate to 1,3-bisphosphoglycerate is accepted by NAD+. Glycolysis can not proceed in the absence of an electron acceptor. NADH produced by glycolysis is transported to the mitochondria where it generates a net of 2 ATPs (3 ATP are generated but one is consumed to transport NADH into the mitochondria). Erythrocytes do not have mitochondria. Methaemoglobin reductase is the only sink for NADH. Glycolysis can proceed only if NAD+ is regenerated. Conversion of pyruvate to lactate by lactate dehydrogenase regenerates NAD+ allowing glycolysis. The end product of glycolysis in erythrocytes is lactate.

The Rappaport-Luebering Glycolytic Shunt
The main functions of the erythrocyte to deliver oxygen to peripheral tissue. Anaemia decreases the oxygen carrying capacity of blood. Oxygen delivery is maintained in anaemic patients by increasing blood flow and increasing the oxygen extraction (see pathophysiology of anaemia). Patients with anaemia have a decrease in the oxygen affinity of haemoglobin allowing a greater amount to be offloaded for a given PO2. 2,3-Bisphosphoglycerate (2,3-BPG) and H+ ions decrease the oxygen affinity of haemoglobin. The Rappaport-Luebering glycolytic shunt synthesizes 2,3-BPG from 1,2-BPG. The shunt bypasses an ATP producing. The erythrocyte pays for increased oxygen affinity as ATP.

The Hexose Monophosphate Shunt
Erythrocytes are subjected to a high degree of oxidative stress from exposure to drugs and chemicals and from oxygen transport. The reactive oxygen species thus generated can affect cell visibility and function by conversion of ferrous iron of haemoglobin to ferric iron giving methaemoglobin. It can also damage lipid membranes shortening the erythrocyte lifespan. Glutathione is a tripeptide that scavenges reactive oxygen species and is oxidized in the process. Glutathione reductase regenerates glutathione by using NADPH as an electron donor. The only non-mitochondrial source of NADPH is the hexose monophosphate shunt. One molecule of glucose passing through the shunt gives two molecules of NADPH and is converted to ribulose-5-phosphate. The ribulose-5-phosphate can be converted to ribose-5-phosphate for nucleoside synthesis. It is also possible to generate 5 molecules of glucose-6-phosphate out of 6 molecules of ribulose-5-phosphate. This involves two enzymes transaldolase and transketolase both of which use thymine pyrophosphate as co-enzyme and are able to transfer carbon from one sugar to another. As the erythrocyte has only a small need of ribose it reconvert most of the ribulose-5-phosphate to glucose-6-phosphate. Normally about 10% of the glucose is metabolized through this shunt. When the erythrocyte is faced by an oxidizing stress almost 90% of the glucose may be metabolized through the shunt. The first and the rate limiting enzyme of the pathway is glucose-6-phosphate dehydrogenase (G6PD). G6PD deficiency is the commonest enzymatic deficiency causing haemolytic anaemia. It manifests as haemolysis in response to oxidative stress like exposure to drugs, chemicals of infection. The hexose monophosphate shunt is critical to the erythrocyte survival.

Evaluating Anaemia

Anaemia is not disease but a manifestation of an underlying disease. The list of diseases causing anaemia is exhaustive. The list includes disease as innocuous as a nutritional iron deficiency or as serious as a leukaemia or iron deficiency from a colonic cancer. Every patient of anaemia must have a complete evaluation. Failure to do so may may delay the diagnosis of serious diseases like cancer. Figure 1 gives the outline for evaluation of a patients with anaemia. The details of evaluation are given below.


Approach to Anaemia

Approach to anaemia: Given above is a flowchart for the approach to a patients with anaemia. The diagnosis depends on whether the anaemia occurs with changes in platelets and/or leucocytes and whether the bone marrow is responding normally to anaemia. Bone marrow response to anaemia manifest as reticulocytosis. Abbreviations used AA: Aplastic anaemia, BM: Bone Marrow, CML: Chronic Myeloid Leukaemia, MDS: Myelodysplastic syndrome, MDS/MPN: Myelodysplastic/myeloproliferative neoplasms, MPD: Myeloproliferative disease (click for a larger image)

Definition of Anaemia

Anaemia is defined and severity classified as per the WHO guidelines given in table 1. These definitions are for non-smokers individuals living at sea level (<1000 meters above seas level). Definitions for smokers and those living ≥1000 meters above sea level can be found at Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity. World Health Organization (WHO/NMH/NHD/MNM/11.1)

Population Normal haemoglobin Mild Anaemia Moderate Anaemia Severe Anaemia
6-59 months ≥11 g/dL 10-10.9 g/dL 7-9.9 g/dL 7 g/dL
5-11 years  ≥11.5 g/dL 11-11.4 g/dL  8-10.9 g/dL  <8 g/dL
12-14 years  ≥12 g/dL 11-11.9 g/dL  8-10.9 g/dL  <8 g/dL
Pregnant Woman  ≥12 g/dL 11-11.9 g/dL 8-10.9 g/dL  <8 g/dL
Non-Pregnant Woman  ≥11 g/dL 10-10.9 g/dL 7-9.9 g/dL  <7 g/dL
Men (≥15 years)  ≥13 g/dL 10-12.9 g/dL 8.8-10.9 g/dL  <8 g/dL

Initial Evaluation of a Patient of Anaemia

  1. The diagnostic workup of a patient of anaemia should establish the
    1. type of anaemia,
    2. the cause of anaemia and
    3. complications caused by anaemia.
  2. Red cell transfusions in anaemic patients are for relieving symptoms due to a low haemoglobin. Patients without symptoms should not be transfused. A pre-transfusion serum sample should be stored to perform tests likely to be affected by blood transfusion (e.g. serum ferritin, serum B12, serum folic acid and haemoglobin electrophoresis). Apart from risks associated with transfusion the relief of symptoms following transfusion reduces the patient’s and the doctor’s zeal to pursue a complete diagnosis.
  3. The history, examination, haemogram and red cell indices are available in all anaemic patients. This information gives a reasonable idea about the cause of anaemia. A diagnosis can be made with a few additional investigations. No patient should be treated without a complete diagnosis.
  4. Iron deficiency is the commonest cause of anaemia. It may be a result of an innocuous cause like nutritional iron deficiency or may be a manifestation of a serious underlying disease like a cancer of the gastrointestinal tract. Most patient of anaemia will respond to iron supplementation. Iron supplementation without complete investigation of anaemia is a common but dangerous practice as it may delay the diagnosis of dangerous underlying illnesses like colorectal carcinoma.

Clinical manifestations of anaemia result from manifestations of  impaired oxygen deliver, manifestations of the underlying disorder and manifestations due to compensation to anaemia.

Manifestations of of anaemia: The symptoms of anaemia depend on the

  1. Degree of anaemia
  2. Rate of fall of haemoglobin
  3. Conditions of the vessels.

The body adapts to anaemia by increasing cardiac output, redistribution of blood from non-critical to the critical circulations and the and increased oxygen extraction (see Pathophysiology of anaemia). These adaptation occurs slowly. Patients with a gradual fall in haemoglobin can tolerate a more severe anaemia than a patient who become  anaemic rapidly because they get time to adapt to anaemia. Vascular disease results in the patient becoming symptomatic at a higher haemoglobin level. These patients manifest with ischaemic symptoms of the region supplied by the vessel. A patient with a gradually developing anaemia and without coronary heart disease becomes symptomatic at a lower haemoglobin than a patient who develops anaemia rapidly or one having a coronary artery disease or one having both.  Nutritional anaemias have a slow onset and thus present with a lower haemoglobin than anaemia or blood loss or acute haemolysis. If one sees a patients who is asymptomatic with severe anaemia one can be fairly certain that the patients suffers from nutritional deficiency. If the haemogram shows microcytosis the anaemia is likely to be due to iron deficiency. If there is macrocytosis the anaemia is likely to be due to B12 or folate deficiency.
Manifestations of anaemia are a result of impaired oxygen delivery. In patients without a significant coronary compromise the symptoms of anaemia include fatigue, lightheadedness and breathlessness. Frank cardiac failure sets in as the severity increases. Ischaemic symptoms, typically angina pectoris, may predominate in patients with significant coronary narrowing. Pallor, the most prominent sign of anaemia is of less diagnostic use than it is percieved to be. It has been reported to have a sensitivity of 19% to 70% and a specificity of 70% to 100%. There is inter-observer variation and clinical examination can not exclude mild anaemia. There have been suggestions that the tongue is the best site for diagnosing anaemia others have differed (PLoS ONE 5(1): e8545. doi:10.1371/journal.pone.0008545). Lack of pallor does not exclude mild anaemia and a haemoglobin assessment must be done is every patient with suspected anaemia.

Clinical features may give a clue to the nature of the disease causing anaemia:

The table below gives clinical features that may give a clue to the nature of the disease causing anaemia.

Clinical Finding Significance
Painful Crisis Sudden onset of pain is a feature of sickle cell disease. Painful crisis commonly affects bone, chest, abdominal and joints
  1. Achloruric Januduce: Achloruric jaundice is a result of increased haemoglobin catabolism (see haemoglobin catabolism) It is seen in patients with haemolytic anaemia and megaloblastic anaemia. In haemolytic anaemia the source of haemoglobin is erythrocytes and in megaloblastic anaemia the it the haemoglobinized erythroid precursors that are destroyed as a part ineffective erythropoiesis. The presence of reticulocytosis in haemolytic anaemias (see diagnosis of haemolytic anaemia) as opposed to reticulocytopenia megaloblastic anaemia differentiates the two conditions. Every anaemia with achloruric jaundice may not be a haemolytic anaemia or megaloblastic anaemia. Gilbert’s syndrome is a common asymptomatic defect of bilirubin metabolism that may co-exist of anemia of another cause. Rarely achloruric jaundice with reticulocytosis may be seen in large occult haematomas. Haematomas of the retroperitoneal region may present in this manner.
  2. Chloruric Jaundice: Patinets with haemolytic anaemia may develop chloruric jaundice because of biliary obstruction from pigment stones.
Nail Changes Platonychia (flat nails) and koilonychia (spoon shaped nails) are features of iron deficiency. These changes may be congenital and the duration of the changes must be enquired into.
Clubbing Clubbing is seen in infectious endocarditis, chronic liver diseases, bronchiectasis, inflammatory bowel disease and lung cancer
Leg ulcers Leg ulcers are a complication typically seen in patients with sickle cell anaemia. They are not specific to sickle cell disease. They may be seen in severe α and β thalassaemia, hereditary spherocytosis and pyruvate kinase deficiency. The incidence of leg ulcers increases with age. They are more common in the tropics where footwear is not worn. The are less common in Sβ0 and not seen in Sβ+ and SC disease. Co-inheritance of α thalassaemia with sickle cell disease decreases the incidence of ulcers. They are typically seen on the medial aspect of the tibia of behind the medial malleolus, are persistent and are a major cause of morbidity in sickle cell disease
Bleeding Manifestations Bleeding due to thrombocytopenia is characterized by petichiae and purpura. Anaemia with thrombocytopenia may be seen in

  1. Bone marrow pathology: Aplastic anaemia, acute leukaemia, bone marrow infiltrations and myelodysplastic syndromes
  2. Increased periphreal distruction/sequestration of platelets: Thrombotic thrombocytopenic purpura/Haemolytic ureaemic syndrome, Evan’s syndrome and hypersplenism
  3. Anaemia caused by bleeding associated with thrombocytopenia
Mouth ulcers Mouth ulcers are a feature of aplastic anaemia
Lymphadenopathy Generalized lymphadenopathy is seen in patients with acute or chronic lymphoid leukaemia and sometimes in autoimmune diseases
Splenomegaly Splenomegaly may be seen in patients with lymphoid neoplasia, chronic myeloid leukemia, idiopathic myelofibrosis, extra-hepatic portal hypertension and hairy cell leukemia
Neurological changes
  1. Subacute Combined degeneration of the Spinal Cord: Subacute combined degeneration is a myelopathy caused by B12 deficiency. It presents with parasthesiae of the upper and lower limbs that progress to weakness and ataxia in untreated patients. Vibratory and joint position sense is lost early in disease and progresses to impaired pinprick, light touch and temperature sensations. Upper motor neuron involvement results in hyperreflexia of the knees but the ankle reflexes are depressed due to a co-existing peripheral neuropathy. Plantars reflexes give a extensor response. Rarely features of autonomic involvements may be seen including orthostatic hypotension and bladder and bowel incontinence.
  2. Neuropathy: Chronic lead poisoning is characterized by a peripheral neuropathy that manifests with motor disturbances with few sensory symptoms.
  3. Other Neurological Manifestations: B12 deficiency can rarely cause cognitive or psychiatric manifestations (3%) or bilateral optic neuropathy and blindness (0.5%). Exposure to lead can cause encephalopathy.

Manifestations due to compensatory changes:

  1. Cardiovascular: Tachycardia, wide pulse pressure and decreased exercise tolerance result from cardiovascular compensation to anaemia.
  2. Musculoskeletal: Chronic haemolytic anaemias result in marrow hyperplasia. Marrow hyperplasia causes typical facies and increased risk of fracture. Facial features include bossing of the skull hypertrophy of the maxilla resulting in exposure of upper teeth, prominent malar eminences with depression of the bridge of the nose, puffiness of the eyelids and a mongoloid slant of the eyes.

Initial Investigations in a Patient of Anaemia

The starting point in evaluation of an anaemic patient is a haemogram and a reticulocyte count. The aim of initial investigation is determine

  1. If the pathology involves only the red cells or is there involvement of the white cells and /or platelets.
  2. If the bone marrow responding to anaemia by producing erythrocytes

Alterations in more than one cell lines of blood cells occurs because blood cells share precursors, developmental microenvironment and antigens. Diseases involving more than one series of blood cell include.

  1. Diseases of haemopoietic precursors: Aplastic anaemia, Acute leukaemia, myeloproliferative diseases, Myelodysplastic syndrome, Myelodysplastic syndrome/myeloproliferative neoplasm.
  2. Pathology of bone marrow microenviorment: Bone marrow infiltration, myelofibrosis
  3. Antibodies mediates diseases
    1. Antibody to antigen of precursors: Aplastic Anaemia
    2. Shared antigen Aplastic anaemia: Autoimmune pancytopenia, Evan’s syndrome
  4. Complement Mediated Damage: Paroxysmal nocturnal haemoglobinuria
  5. Other diseases: Thrombotic thrombocytopenia purpura/haemolytic uraemic syndrome (TTP/HUS)

All the above diseases other the TTP/HUS need a bone marrow examination for diagnosis. TTP/HUS is characterized by microangiopthic haemolytic anaemia in a patients with characteristic clinical presentation.

Anaemia induces erythropoietin production. Erythropoietin stimulated red cell production. Increased erythrocyte production manifests as reticulocytosis (see reticulocyte count). Reticulocytosis in an anaemic patient indicates that the anaemia is because of erythrocyte loss and the bone marrow is normally responding to anaemia. Patients with erythrocyte loss may fall into three catagories.

  1. Acute blood loss: These patients give history of blood loss and present with anemia with reticulocytosis without evidence of haemolysis.
  2. Chronic Blood loss: Blood loss is a alarming symptom that prompts patients to immediately seek medical advise. Patients with occult blood loss, those with altered blood loss or those with not capable of recognizing a blood loss may present with manifestations of chronic blood loss. Chronic blood loss causes iron deficiency. Unlike acute blood loss the manifestations of chronic blood loss are those of iron deficiency anaemia (microcytic anaemia with reticulocytopenia).
  3. Haemolytic anaemia: Patinets who have anaemia with reticulocytosis but have no demonstrable blood loss should be considered to have a haemolytic anaemia.

Evaluation of Microcytic Anaemia

Evaluation of Microcytic Anaemia

As haemoglobin forms about 97% of the erythrocyte proteins. Disorders impairing haemoglobin synthesis cause microcytic anaemia. Haemoglobin synthesis needs an functional genetic code, normal heme synthesis, adequate supply of iron and proper incorporation of heme in haemoglobin. If any of these are defective a microcytic anaemia develops. The commonest cause of microcytic anaemia is iron deficiency. The first investigation in a patients of microcytic anaemia is assessment of serum iron, total iron binding capacity and serum ferritin. The results of iron studies microcytic anaemia are depicted in figure 3. Further evaluation of patients depends on the result of iron studies. If the irons studies are normal then an study for abnormal haemoglobin should be performed either by HPLC or cellulose acetate electrophoresis in alkaline pH. Patients with HbA2 between 3.5-7% have heterozygous β-thalassaemia. Higher HbA2 values should be considered to be due to an abnormal haemoglobin that separates with HbA(e.g. HbE). Patients with homozygous and heterozygous HbE disease may be diagnosed in this manner. Those with a normal hemoglobin electrophoresis are likely to have α-thalassaemia trait. α-Thalassaemia needs estimation of globin chains synthesis. This investigation may not be readily available. If HbH disease is prevalent in the ethnic group the patients belongs to, it is essential to compete evaluation for α-thalassaemia to prevent Bart’s hydrops fetalis. Patients with transferrin saturation more than 50% need to have  bone marrow aspiration for diagnosis of sideroblastic anaemia.



Evaluation of Normocytic Anaemia

Evaluation of Normocytic Anaemia

Patinets with normocytic anaemia need to be evaluated for liver disease, renal failure or endocrine disease. The endocrine disease associated with anaemia include hypothyroidism, hyperthyroidism, adrenal insufficiency and hypopituitarism. If these disorders are not found the iron status should be assessed. Patinets may have iron deficiency (transferrin saturation <16%), anaemia of chronic disease (transferrin saturation ≥16% with a low total iron binding capacity). A bone marrow aspiration should be performed in whom diagnosis can not be made by the above investigations.



Evaluation of Microcytic Anaemia

Evaluation of Macrocytic Anaemias

Macrocytic anaemias may be megaloblastic or non-megaloblastic. Features of megaloblastic anaemia include hypersegmentation of neutrophils, macroovalocytosis, indirect hyperbilirubinaemia and markedly elevated lactate dehydrogenase levels. Macrocytosis is pronounced and almost all patients with an MCV > 110fl have nutritional megaloblastic anaemia, are of antiviral therapy or on chemotherapy. The converse is not true. Patients of megaloblastic anaemia may have a MCV <110fl in early disease or in the presence of co-existing iron deficiency. Serum iron levels are increase in nutritional megaloblastic anaemia. They  fall rapidly within 24-48 hours after therapy. Iron studies should be performed after a few days after administration of vitamins when the iron levels have stabilized. Nonmegaloblastic macrocytic anaemias are seen with alcoholism, hypothyroidism, liver disease and myelodysplastic syndromes.

Sickle Haemoglobin and Varients

Substitution of the amino acid glutamic acid by valine at position 6 of the beta globin gene (HBB glu6val) results in a mutant haemoglobin that polymerizes at low oxygen pressure. This mutation results in sickle shaped cells under hypoxic conditions. The haemoglobin gets its name, sickle haemoglobin, from the phenomena. Sickling is responsible for symptoms of sickle cell anaemia. Shown above are sickle cells from the smear of a patient with sickle cell anaemia.

HbS is an autosomal co-dominant trait. Homozygous individuals suffer from sickle cell anaemia (SS). The clinical profile of compound heterozygous depends on the non-HbS allele.

  1. HbA: HbA does not participate in sickling. Patients with AS have about 35% HbS and 65% HbA there is little sickliness and symptoms. These patients said to have sickle cell trait. A patient with as with an apparent AS pattern can have see sickling (see below).
  2. β-Thalassaemia: The severity of interaction between HbS and β-thalassaemia depends on the severity of the co-thalassaemia. β0-thalassaemia is identical to SS and β+-thalassaemia is a milder disease with severity depending on the degree of impairment of the β-chain synthesis.
  3. Other Sickling haemoglobinopathies: Co-inheritnce of Hb-O Arab and Hb D Punjab produces a sickle cell anaemia like disease. Co-inheritance of Hb E produces a sickling disease milder than SS.
  4. Haemoglobinopathies than mimic AS on electrophoresis but produce sickling: There are two categories in this class. First, is a disease that resulting from co-inheritance of an abnormal haemoglobin, Hb Quebec Chori, migrates like HbA but promotes sickling. Patients compound heterozygous for Hb S and and Hb Quebec Chori show and electrophoretic pattern of AS but show sickling. Second are variants of HbS where a second mutation has resulted in an increased tendency to precipitate making a individual heterozygous for these variants symptomatic. These include Hb South End, Hb Jamican Plain Hb S Antilles and Hb S-Oman. The first three have mutations reducing the affinity of the haemoglobin increasing polymerization and sickling.

Diagnosis of Haemolytic Anaemia

Haemolysis is decreased erythrocytes lifespan (normal about 100-120 days). The bone marrow responds to haemolysis by increasing erythrocyte production. Anaemia occurs only when the erythrocyte life span is reduced to about 15-20 days. A compensated haemolytic state is when a patient has a shorted erythrocyte lifespan that is adequately compensated by increased erythrocyte production. These patients may have features of haemolysis except anaemia.

Figure 1. Manifestations of Haemolysis

Clinical Manifestations of Haemolysis

The site of haemolysis, intravascular or extravascular, determines the clinical manifestations of haemolysis (figure 1). Extravascular haemolysis is an exacerbation of a physiological catabolic process. The end product of extravascular haemolysis, like that of normal erythrocyte breakdown, is unconjugated bilirubin. Unconjugated bilirubin can cause brain damage in the forms of kernicterus. The conjugation of bilirubin is so efficient that the serum bilirubin rarely increase to more than 5mg/dL only due to bilirubin overproduction. The blood brain barrier prevents entry of bilirubin in the brain. Both the mechanisms are compromised in a neonate making them prone to kernicterus in case of haemolysis as is seen in haemolytic disease of the newborn. The only consequences of extravascular haemolysis occurring in patients beyond the neonatal period are anaemia, unconjugated hyperbilirubinaemia, mild to moderate splenomegaly and pigment gallstones.

Intravascular haemolysis is lysis of cells within the blood vessels. Cell free haemoglobin is oxidizing and pro-inflammatory. Haptoglobin binds and detoxifies cell free haemoglobin but it has a limited capacity. Haemoglobinaemia resulting from intravascular haemolysis causes serious clinical manifestations including renal failure, hypertension, smooth muscle spasm and a prothrombotic state. Many of these result from the nitric oxide scavenging capacity of haemoglobin (Rother et al JAMA 293:1653;2005). Persistent haemoglobinuria is a feature of chronic intravascular haemolysis, e.g. those with haemolysis due to prosthetic valves and paroxysmal nocturnal haemoglobinuria. Iron deficiency may accompany chronic intravascular haemolysis because of haemoglobinuria. Extravascular haemolysis is not associated with haemoglobin loss and does not cause iron deficiency.

Intravascular haemolysis often presents with acute severe haemolysis as may be seen with drug induced immune haemolysis, haemolysis in G6PD deficient patients, severe malaria and mismatched transfusion reactions. The diseases causing extravascular haemolysis have a less acute presentation.

The Diagnosis of Haemolytic Anaemia

Estimation of erythrocyte lifespan is too cumbersome to be used routinely in clinical practice. The diagnosis of haemolytic anaemia instead relies on indirect evidence of increased erythrocyte destruction. This includes:

  1. Increased production of erythrocytes in an anaemic patient with no evidence of blood loss
  2. Increase in products of erythrocyte destruction – Lactate dehydrogenase (LDH) and haemoglobin
  3. Consequences of haemoglobinaemia – low haptoglobin, haemoglobinuria, renal failure and hemosiderinuria

Increased erythrocyte production manifests as reticulocytosis (see The Reticulocyte Count for performing and interpreting reticulocyte count). If polycythaemia is not seen despite reticulocytosis, an equivalent erythrocyte loss is presumed to be occurring. Erythrocytes loss may internal, e.g., haemolysis, or external, e.g., bleeding. Reticulocytosis is seen when a large amount of blood is lost over a short period. Such losses are rarely occult. If there is no obvious blood loss, reticulocytosis in a patient whose haemoglobin is not increasing indicates haemolysis. Large occult haematomas, typically occurring retroperitoneally, may mimic haemolysis.

Lactate dehydrogenase (LDH) is abundant in brain, erythrocyte, liver and lung. Injury to any of these organs increases LDH. LDH is also increased in diseases characterized by increased cell proliferation e.g. acute leukaemia, lymphomas (particularly high grade non-Hodgkin lymphoma), myeloproliferative diseases and myelodysplastic syndrome. The clinical picture, radiology and laboratory investigations can exclude non-erythrocyte sources of LDH.

Haemolytic anaemias are characterized by an increase in LDH. The increase in LDH is more pronounced in patients with intravascular haemolysis and is matched by that seen in patients with nutritional megaloblastic anaemia. Unlike megaloblastic anaemia, intravascular haemolysis is characterized by reticulocytosis, haemoglobinaemia and haemoglobinuria.

The alterations caused by catabolism of haemoglobin released during haemolysis depend on the site of haemolysis (see haemoglobin catabolism). Haemoglobin released during extravascular haemolysis is metabolized to unconjugated bilirubin. The liver responds by increasing conjugation and excretion of bilirubin which increases urinary urobilinogen. Unconjugated hyperbilirubinaemia (unconjugated bilirubin >85% of total bilirubin) with increased urobilinogen is a characteristic of haemolytic anaemia. It differentiates haemolytic anaemia from Gilbert’s Syndrome, a common disorder of bilirubin conjugation that is seen in 3-7% of population. The urobilinogen levels in Gilbert’s syndrome are low. Crigler-Najjar syndrome type I and II are present with a more pronounced increase in bilirubin (>5mg/dL and >20mg/dL respectively) and are rarely considered as a differential diagnosis of an uncomplicated haemolytic anaemia. Bilirubin congugation defects do not show reticulocytosis. The bilirubin levels in patients with haemolytic anaemia rarely exceed 5mg/dL. Higher levels indicate a co-existing illness. If the bilirubin is unconjugated then an incidental co-inheritance of Gilbert’s syndrome should be suspected. Haemolytic anaemia increases the risk of pigment stones. These may cause obstructive jaundice. These patients, unlike those with haemolysis have a higher proportion of conjugated bilirubin with an elevated alkaline phosphatase. Donor screening for hepatitis B and C have made transfusion induced hepatitis a thing of the past.

Haemolysis releases haemoglobin. Cell free haemoglobin has oxidizing and pro-inflammatory properties. Haptoglobin is a haemoglobin scavenger that protects the body from the toxic effects of cell free haemoglobin. Haemolysis decreases serum haptoglobin (see haemoglobin catabolism). Extravascular haemolysis does not cause haemoglobinaemia, but contrary to what may be expected, it does cause low haptoglobin levels.  Low haptoglobin is of no value in differentiating intravascular and extravascular haemolysis. It appears that there is some haemoglobinaemia during extravascular haemolysis. It is possible that there is some intravascular component to extravascular haemolysis or there is regurgitation of haemoglobin from the macrophage during phagocytosis. Haptoglobin is synthesized by the liver. Decrease synthesis limits the usefulness of haptoglobin in the diagnosis of haemolysis in the presence eof chronic liver disease. Haptoglobin is an acute phase reactant that in increased by corticosteroid administration. Patients with acute inflammation and those on corticosteroid therapy may have a normal haptoglobin despite haemolysis.

Haemoglobin filtered into the glomerular fluid results in haemoglobinuria when the absorptive capacity of renal tubules (5g/day, Turgeon, ML. Clinical Hematology: Theory and procedures 4th ed. Lippincott Williams and Wilkins, 2005:89) is exceeded. Haemoglobin can precipitate in the tubules and causes renal failure. The haemoglobin absorbed by the renal tubules in metabolized by haemoglobin oxygenase to iron, bilirubin and amino acids. The iron is stored in the renal tubular cells as hemosiderin. These cells desquamate and cause hemosiderinuria. Hemosiderinuria appears a few days after hemolysis and may persist for a week or more after hemolysis has abated. Iron deficiency anaemia caused by hemosiderinuria may complicate chronic low grade intravascular haemolysis as is seen in prosthetic valve haemolysis, paroxysmal nocturnal haemoglobinuria or march haemoglobinuria.

Diagnosis of haemolysis by transfusion requirements

Even with complete cessation of erythropoiesis only about one unit of blood is needed every week in an adult to maintain haemoglobin of about 10g/dL. A greater requirement in the absence of evidence of blood loss suggests haemolysis. Transfused blood will only be lysed if there is an extrinsic defect e.g. autoimmune haemolytic anaemia. Cells transfused to patients with an intrinsic defect e.g. hereditary spherocytosis will have a normal life span. A patient who needs more than one unit of blood per week to maintain haemoglobin of about 10g/dL is likely to have a haemolytic anaemia if blood loss can be excluded. This haemolysis is likely to be due to an extrinsic cause.

Differential Diagnosis of Haemolysis

Haemolysis may be confused with conditions associated with unconjugated hyperbilirubinaemia, anaemia and reticulocytosis. These include bilirubin conjugation defects, acute blood loss and megaloblastic anaemia.

Bilirubin Conjugation Defects Bilirubin conjugation defects include Gilbert’s syndrome and Crigler-Najjar syndrome types I and II. The bilirubin levels in Crigler-Najjar syndrome type I and II are more than 5mg/dL so these entities are rarely confused with haemolysis. Gilbert’s syndrome is characterized by bilirubin levels which can be seen in haemolysis but there is no associated reticulocytosis. Bilirubin conjugation defects are characterized by low urobilinogen levels.

Acute blood loss: Acute blood loss, like haemolysis, causes anaemia and reticulocytosis but these are not the presenting complains. Blood loss needed to cause reticulocytosis is rarely ignored by the patient and is in fact the reason for seeing the doctor. When severe, it may be accompanied by signs of hypovolemia. The presenting complains of patients with haemolytic anaemia are related to anaemia and hyperbilirubinaemia. Chronic occult blood loss has a different presentation that acute blood loss or haemolysis. The gradual fall in haemoglobin associated with occult blood loss makes the patients tolerant to anaemia unless a cardiovascular or respiratory co-morbidity exist. These patients present with a clinical picture of iron deficiency namely, fatigue, malaise and hypochromic microcytic anaemia with anisocytosis. There is no jaundice and there is reticulocytopenia. Large occult haematomas cause anaemia and reticulocytosis. As the haematoma resolves jaundice may be seen.

Megaloblastic anaemia Megaloblastic anaemia, like haemolytic anaemia, presents with macrocytic anaemia, indirect hyperbilirubinaemia, and a low serum haptoglobin. These patients have reticulocytopenia. The macrocytosis is more pronounced. An MCV of >110 fl usually indicates a megaloblastic anaemia. Patients with nutritional megaloblastic anaemia respond to treatment with pronounced reticulocytosis, usually seen by 5-7 days after initiating therapy and lasting for up to 2 weeks.  Reticulocyte counts may be as high as 20-25%. A history of treatment with vitamin B12 and/or folic acid must be sought in every patient of haemolytic anaemia to avoid confusion with megaloblastic anaemia.


Related Pages

  1. Reticulocyte Count
  2. Evaluating Anaemia
  3. Haemoglobin Catabolism
  4. Sickle Haemoglobins and Variants
  5. Red Cell Indices

Haematology Atlas Pages

  1. Sickle Cells
  2. Pappenheimer Bodies