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

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.

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.

 

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


The erythrocyte membrane is subject a great deal of physical stress in circulation. It needs to withstand the high sheering stress in the arteries, it needs to squeeze past capillaries that ma be as small as 7.5µm and need to withstand the ionic changes. It has a well-developed network of proteins known as the erythrocyte cytoskeleton below the lipid bilayer of the plasma membrane to meet these needs. Inherited defects in these proteins have been associated with disorders of erythrocyte shape including hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis, Southeast Asian stomatocytosis and hereditary acanthocytosis (see table below).

Protein Gene Chromosome Disorders
α-Spectrin SPTA1 1q22-q23 Hereditary ElliptocytosisHereditary PyropoikilocytosisHereditary Spherocytosis
β-Spectrin SPTB 14q23-q24.1 Hereditary ElliptocytosisHereditary PyropoikilocytosisHereditary Spherocytosis
Ankyrin-1 ANK1 8p11.2 Hereditary Spherocytosis
Band 3 SLC4A1 17q21 Hereditary SpherocytosisHereditary AcanthocytosisSoutheast Asian OvalocytosisHereditary Stomatocytosis
Protein 4.1R EPB41 1p33-p34.2 Hereditary Spherocytosis
Protein 4.2 EPB42 15q15-q21 Hereditary Spherocytosis
Stomatin STOM 9q33.1 Hereditary Stomatocytocytosis
Glycophorin C GYPC 2q14-q21 Hereditary Elliptocytosis
Glycophorin D GYPD 2q14-q21 Hereditary Elliptocytosis

Organization of the Erythrocyte Membrane

The erythrocyte membrane consists of a lipid layer on protein scaffolding known as the cytoskeleton. The relationship between erythrocyte membrane proteins and lipid membrane bilayer is shown in the figure below. The main component of cytoskeleton is spectrin. Spectrin is tethered to the cell membrane by vertical interactions with band 3 proteins via ankyrin and protein 4.2. Spectrin also has horizontal interactions with protein 4.1R, actin, tropomodulin, tropomyosin and adducin. Protein 4.1R interacts with glycophorin C, a trans membrane protein.

Red Cell Membrane-600px

Erythrocyte Membrane Proteins

Spectrin

Spectrin has three functions

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

It is made of two chains α and β that interwine to form dimers. Two dimers associate to form a tetramer which is the functional subunit. The α-chain is encoded by the gene SPTA1 at 1q22-q23 and the β-spectrin is encoded by the gene SPTB at 14q23-q24.1.

Ankyrin

Ankyrins are ubiquitous adapter proteins thattarget diverse proteins to specialized membrane domains of smooth muscles and endoplasmic reticulum. The erythrocyte ankyrin, ankyrin-R is encoded by the gene ANK1 located at 8p11.

Band 3

Band 3 glycoprotein of the erythrocyte membrane that is coded by the gene is SLC4A1 at 17q21. The membrane domain transports anions across the cell membrane and the cytoplasmic domain provide binds the lipid membrane to spectrin via ankyrin.  major integal

Protein 4.2

Protein 4.2 regulates the interactiom of band 3 with ankyrin. It is encoded by the gene EPB42 at 1p33-p34.2.

Protein 4.1R

Protein 4.1R stabilized the spectrin-actin interactions. It is encoded by the EPB42 gene at 1p33-p34.2.

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.

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