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)



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.

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.

Laboratory diagnosis of Iron Deficiency

The investigation to diagnose iron deficiency include:

  1. Haemoglobin and red cell parameters
  2. Bone marrow iron staining
  3. Serum Iron, total iron binding capacity and transferrin saturation
  4. Serum ferritin
  5. Zinc Protoporphyrin
  6. Soluble transferrin receptor

Haemoglobin and Red cell Parameters

Patients undergo evaluation for iron deficiency because they are anaemic. Latent iron deficiency is characterized by a progressive decrease in bone marrow iron stores in patients who have yet not developed symptoms. These patients are asymptomatic and latent iron deficiency is not a clinical problem. Iron deficiency causes microcytic hypochromic anaemia. There are no reliable test to differentiate iron deficiency from other causes of microcytic hypochromic anaermia.  The red cell indices that have been proposed to be useful include

  1. Red cell distribution width (RDW): RDW is a measure of anisocytosis. Iron deficiency anaemia shows more anisocytosis than β-thalassaemia and is associated with a higher RDW. The promise held out by RDW to differentiate iron deficiency and thalassaemia has not fulfilled.
  2. Reticulocyte haemoglobn content: Changes in erythropoiesis are reflected earliest in the reticulocyte. Reticulocytes form a small fraction of erythrocytes. Change in reticulocyte indices do not change erythrocyte indices. Some automated counters are able to measure reticulocyte indices. Reticulocyte haemoglobin content falls with iron deficiency anaemia but the finding is not specific. It has been found to asses iron deficiency in patient of chronic renal failure being treated with erythropoietin accurately. It has not been found to be useful in diagnosing iron deficiency in patients with thalassaemia.

Bone Marrow Iron Staining

Bone marrow is stained for iron content by the Prussian blue reaction and graded in a semiquantative method. Bone marrow iron is the gold standard for diagnosis of iron deficiency. The test is invasive and suffers from an inter-observer variation. Bone marrow iron staining is resorted to only when diagnosis can not be reached by other methods.

Serum iron, total iron binding capacity and transferrin saturation

Iron deficiency is diagnosed by a transferrin saturation of less than 16%. The serum iron is low and the total iron binding capacity is usually increased. Patients with low total iron binding capacity have anaemia of chronic disease if the transferrin saturation is ≥16%  or iron deficiency along with anaemia of chronic disease if the transferrin saturation is <16%.

Serum ferritin

Ferritin is one of the iron storage proteins. Serum ferritin levels co-relates with body iron content. A ferritin level less than 12ng/mL is diagnostic of iron deficiency. Inflammation increases ferritin. Chronic inflammatory diseases like rheumatoid arthritis and ulcerative colitis have anaemia of chronic disease and may also have iron deficiency. A patient with iron deficiency in the setting of an inflammatory disease may not have a low ferritin. There is no consensus for diagnosing iron deficiency in patients with anaemia of chronic disease. Inflammation rarely increases the serum ferritin values more than 60-100ng/mL. Iron deficiency can be excluded in patients with ferritin above this cutoff.

Zinc Portoporphyrin

Iron is added to propoporphyrin in the final step of heme synthesis. Zinc takes the place of iron in patients with iron deficiency. A rise in the concentration of zinc protoporphyrin is the earliest manifestations of iron deficiency. Zinc protoporphyrin levels rise in about 2 weeks from the onset of iron deficiency and need more than a month to normalize after restoration of normal iron levels.

Soluble transferrin Receptor

Iron deficiency results in an increase in soluble transferrin receptor (sTfR). Inflammation impacts serum ferritin but not sTfR making it a potentially useful investigation for differentiating anaemia of chronic disease and iron deficiency. The assay has been difficult to standardize. A ratio of sTfR/log ferritin is more useful.

The sTfR levels reflect the density of transferrin receptors cells and number of cells. sTfR increases when there is erythroid hyperplasia due to any cause like haemolytic anaemia and may not reflect iron deficiency in these disorders.


Development of erythrocytes involves coordinated changes in the nucleus and the cytoplasm of erythroid processors (see Morphology of Erythroid Precursors) . The proerythroblast is a large cell with a fine chromatin, the earliest forms not being different from other blasts. As the cell matures the chromatin becomes more clumped and the nucleus reduces in size and the cytoplasm becomes acidophilic. Finally a dark pyknotic nucleus is extruded from the orthchromatophilic normoblast to give a reticulocyte.

Figure 1. Basophilic Normoblast

A group of basophilic normoblast are shown above. The cytoplasm is basophilic and the chromatin more clumped than a proerythroblast. The two cells on the right are less mature than the two on the left.

Figure 2. A group of orthochromatophilic normoblasts

The figure above show a group of  orthochromatophilic normoblasts. The cells of the left are more mature. The nucleus is reduced to a dense body in the more mature forms. The cytoplasm still has a blue tinge. which contrasts from the megaloblasts shown below.

Figure 3. Basophilic, polychromatophilic and orthochromatophilic normoblasts

The figure above shows three stages of erythroid maturation. The cell on the top right is a basophilic normoblast, bottom right is a polychromatophilic normoblast and the bottom left is an orthochromatophlic normoblast. Note the evolution of nuclear and cytoplasmic changes.

Figure 4. Megaloblasts

The figure above shows a group of megaloblasts. Cells in the right lower corner have a cytoplasm which is fully haemoglobinized and resembles the mature erythrocyte. These cell still have a nucleus. The cell on the left upper corner has a cytoplasm resembling a orthochromatophilc normoblast (see figure 2 and 3) but nuclear features resembling a basophilic normoblast (figures 1 and 2). Megaloblastic anaemia results from conditions that hamper DNA synthesis (B12 deficiency, folate deficiency, Chemotherapy drugs). The nucleus of a megaloblast thus matures slower than the cytoplasm resulting in cells having a nuclear morphology resembling a previous stage. This, known as nucleo-cytoplasmic dissociation, is the characteristic feature of megaloblastic anaemia.

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.

Morphology of Erythroid Precursors

Figure 1. Maturation of erythroid cells in the bone marrow

Pleuripotent stem cells give rise to erythrocytes by the process of erythropoiesis. The stem cell looks like a small lymphocyte and lacks the functional capabilities of the erythrocyte. The stem cells have the capacity of infinite division something the mature cells lack. Some of the daughter cells arising from the stem cell acquire erythroid characters over generations and time. Most of the erythroid cells in the bone marrow have a distinct morphology but commitment to erythroid maturation is seen even in cells that have not acquired morphological features distinctive of the erythroid lineage. These cells are recognized by the type of colonies they form in vitro. Two such cells are recognized. Burst-forming unit erythroid (BFU-E) arise from the stem cell and gives rise to colony-forming unit erythroid (CFU-E). CFU-E gives rise to pronormoblast, the most immature of erythroid cells with a distinct morphology (figure 1). BFU-E and CFU-E form a very small fraction of bone marrow cells and are not important in diagnosis. Examination of Romanovsky stained (Giemsa, Wright’s) bone marrow smears is central to the haematological diagnosis. Morphologically five erythroid precursors are identifiable in the bone marrow stained with Romanovsky stains. The five stages from the most immature to the most mature are the proerythroblast, the basophilic normoblast (early erythroblast), polychromatophilic normoblast (intermediate erythroblast), orthochromatophilic normoblast (late erythroblast) and reticulocyte (figure 1). As the cell matures the following morphological changes take place

  1. Cell becomes smaller
  2. Nucleus becomes smaller, chromatin more clumped and the nucleoli disappear
  3. Cytoplasm shrinks
  4. The cytoplasmic basophilia decreases: Haemoglobin is a major constituent of the red cell takes a pink to red colour on staining with Romanovsky stains. The machinery to synthesize haemoglobin (ribosomes) must appear before haemoglobin. Ribosomes make the cytoplasm basophilic (blue) because of their RNA content. As the haemoglobin content approaches the desired levels the number of ribosomes decreases. The cytoplasm of the maturing erythroid cell captures these changes and changes from deep blue (mainly ribosomes) in basophilic normoblast to polychromatophilic (ribosomes and haemoglobin) in polychromatophilic normoblast and resembling that of a erythrocyte (mainly haemoglobin) in orthochromatophilic normoblast.
  5. The earliest nucleated stages are least numerous and the later stages the most numerous

Figure 2. Proerythroblasts. The nucleus has multiple nucleoli a feature that distinguishes this stage.

Proerythroblast: Proerythroblast (figure 2) is a large cell (12-20μm in size or about 1.5-3 times a normal erythrocyte) with a large nucleus that occupies almost 80% of the cell and a blue cytoplasm that forms a thin rim around the nucleus. The chromatin is finely granular and stripped. The nucleus shows multiple nucleoli (the multiple pale staining areas in the nucleus). The cytoplasm may show a small pale area that corresponds to the Golgi apparatus and may have a pale perinuclear halo. While it is usually possible to tell a proerythroblast from other blasts (myeloblasts, lymphoblasts and monoblasts) by it’s more homgenous of cytoplasm, larger size ,a chromatin that coarser and the perinuclear halo in very immature cells this distinction may be impossible.

Figure 3. Basophilic Normoblasts. All four cells have basophilic cytoplasm but lack nucleoli. Nucleoli are a feature of proerythroblasts. Out of the four cells seen the two on the left are larger, have a less clumped chromatin. The cytoplasm of all four cells shows no polychromasia.

Basophilic Normoblast: The basophilic normoblast (figure 3, 4 and 5) is a smaller (12-17μm) cell. The nucleus is round like that of a proerythroblast but lacks nucleoli. Condensation of chromatin with the appearance of heterochromatin begins at this stage giving the nucleus a coarse and granular appearance. The number of ribosomes peak at in basophilic normoblasts and this reflects in the cytoplasmic colour. The basophilic normoblast has one of the bluest cytoplasm amongst the bone marrow cells. It may have a perinuclear halo. The nucleus may assume a wheel spoke arrangement like a plasma cell. The spoke wheel arrangement, the blue cytoplasm and similar size makes the basophilic normoblast resemble a plasma cell (figure 4). The plasma cell is elliptical with an eccentric nucleus while the basophilic normoblast is round with a central nucleus.

Figure 4. A basophilic normoblast (1) with a polychromatophilic normoblast (2) which is smaller with a cytoplasm more like a mature erythrocyte and a nuclear chromatin that is more clumped

Figure 5. A basophilic normoblast with two plasm cells. The plasma cells have an eccentric nucleus and are elliptical

Polychoromatophilic Normoblast: The polychromatophilic normoblast (figure 4) is a smaller (12-15μm). The distinguishing feature of this stage is the appearance of haemoglobin which reduces the basophilia of the cytoplasm. The chromatin shows a greater degree of clumping and irregular dense areas of staining are seen in the nucleus. Nucleoli are nor seen. Figure 4 shows adjacent basophilic and polychromatophilic normoblasts to contrast the size, the clumping of the chromatin and cytoplasmic staining (see normoblast maturation for more images of maturing polychromatophilic normoblasts).

Figure 6. Orthochromatophilic Normoblast. The cell slightly larger than an erythrocyte, nucleus is condensed and the cytoplasm is almost the colour of the erythrocyte

Orthochromaphilic Normoblast: The process of haemoglobinization is almost complete by the stage of orthochromatophilic normoblast (figure 6). “Ortho-” in Greek means straight, upright or correct. Though orthochromatophilic suggests that the colour of the cytoplasm is the same as mature erythrocyte, this is not the case. The orthochromatophilic normoblast is the nucleated erythroid precursor that is closest to a mature erythrocyte in terms of size (8-12μm) and cytoplasmic staining. The cytoplasm however retains a blue tinge much like a reticulocyte. The nucleus is greatly condensed, shrunk and assumes a variety of bizarre shapes (buds, clover leaves, double spheres). The chromatin is greatly condensed and almost completely homogenous.

Figure 7. The Reticulocyte. Reticulocytes (blue arrows) are larger than the normal erythrocytes. Some

Reticulocyte: The Orthochromatophilic normoblasts finally extrudes the nucleus and reticulocyte (figure 7)is formed. The reticulocyte is about 20% larger (7-9μm) than the mature erythrocyte. The lifespan of the reticulocyte is about 3 days. It spends 2 of these in the bone marrow. A day after appearing in the peripheral blood the reticulocyte looses the blue colour and becomes a erythrocyte.

Table 1. The summary of morphological features of erythroid cells
Cell Size Nucleus Cytoplasm
BFU-E, CFU-E The two cells are indistingushible from blasts of other series. They posses no morphological characters that indicates their erythroid origin
Pronormoblast 12-20 μ Fine chromatin, many nucleoli Blue, a perinuclear halo and a small pale area (Golgi apparatus) may be seen
Basophilic Normoblast 12-17 μ Granular chromatin, no nucleoli Very deep blue, perinuclear halo may be seen.
Polychromatophilic Normoblast 12-15 μ Cromatin is visibly clumped with dark staining areas Basophilia reduced but still not as pink as an erythrocyte
Orthochromatophilic normoblast 8-12 μ A featureless nucleus with dense chromatin Almost the colour of a reticulocyte
Reticulocyte 7-9μ No Nucleus Slightly blue compared to an erythrocyte
The nucleus of the small lymphocyte and the normoblast are about 7.5μ

Relates posts in this site

  1. Morphology of Myeloid Precursors
  2. Megakaryocyte Morphology