Mednotes

L14 Blood Haemolytic anaemias and haemoglobinopathy

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tagsblood, lecture
aliasesL14 Blood Haemolytic anaemias and haemoglobinopathy

21 min read

Overview

This lecture covers anaemias caused by shortened red cell survival. It starts with how normal red cells are cleared at the end of their ~120 day lifespan in the spleen, then follows what happens when destruction is excessive: free haemoglobin is scavenged by haptoglobin and haemopexin, haem is degraded to unconjugated bilirubin, and the marrow responds with reticulocytosis. These processes give the laboratory signature of haemolysis. The causes are then worked through as hereditary (membrane defects, enzyme defects, haemoglobinopathies and thalassaemias) and acquired (immune, fragmentation, infection, chemical or physical, secondary), with hereditary spherocytosis, G6PD deficiency, sickle cell disease and the thalassaemias as the worked examples, and with the recurring theme that several of these inherited disorders persist because they give partial protection against malaria.

Red cell clearance and the splenic filter

  • Average red cell lifespan is about 120 days. In severe haemolytic conditions lifespan can fall to as low as 10 days.
  • As red cells age they undergo physical and molecular changes affecting deformability, shape, volume, metabolism and surface molecules.
  • Senescent red cells express more adhesion proteins (CD44, laminin-α5, HA, Lu/BCAM) and are trapped in the splenic cords by adhesion to the extracellular matrix in the red pulp.
  • Sequence in the spleen: old red cell adheres to the cord extracellular matrix, then the red cell lyses releasing haemoglobin into the circulation, then the “ghost” red cell is engulfed and degraded by a macrophage.

What a haemolytic anaemia is

  • Reduced red cell survival in the circulation, either because the red cells themselves are abnormal or because normal red cells are excessively destroyed. Arguably better named “shortened lifespan” anaemias.
  • The consequence is more pressure on the haptoglobin scavenging system.

Handling of free haemoglobin and haem

Haemoglobin and haem are toxic, so plasma has a scavenging system:

  1. Haptoglobin is an abundant plasma protein (0.3 to 2 g/L) synthesised in the liver.
  2. When red cells lyse, haemoglobin dimers are released and mopped up by haptoglobin.
  3. The haemoglobin-haptoglobin complex is endocytosed and degraded by macrophages or monocytes via CD163.
  4. If haptoglobin is used up, haemoglobin dimers are oxidised to methaemoglobin, releasing oxidised (ferric) haem, which is bound by haemopexin and degraded in the liver. Haemopexin is not measured clinically.
  5. Haem degradation: Hb(Fe2+) to biliverdin to bilirubin, with carbon monoxide released. Bilirubin production is increased in haemolysis.

Bilirubin pathway

Ordered pathway across four compartments:

  1. Reticuloendothelial system: red cells to haem to unconjugated bilirubin.
  2. Bloodstream: unconjugated bilirubin binds albumin, forming the unconjugated bilirubin-albumin complex, which is the indirect bilirubin.
  3. Liver: uridine glucuronyl transferase converts it to conjugated bilirubin (direct bilirubin), excreted in bile.
  4. Gut: gut bacteria convert conjugated bilirubin to urobilinogen, of which 80% is excreted in faeces as stercobilin, 2% is excreted in urine as urobilin, and 18% undergoes enterohepatic circulation back to the liver.

Causes of haemolytic anaemia

Hereditary:

  • Membrane defects (membranopathies): hereditary spherocytosis, hereditary elliptocytosis.
  • Metabolic disorders (enzymopathies): G6PD deficiency, pyruvate kinase deficiency.
  • Haemoglobinopathies: Hb SS, SC, E, D.
  • Thalassaemia: alpha or beta.

Acquired:

  • Autoimmune: warm antibody type, cold antibody type.
  • Alloimmune: haemolytic transfusion reactions (acute or delayed), haemolytic disease of the newborn, allografts, drug-associated.
  • Red cell fragmentation syndromes: microangiopathy, for example DIC; march haemoglobinuria.
  • Infections: malaria, clostridia.
  • Chemical or physical agents: especially drugs, severe burns.
  • Secondary: severe liver and renal disease.

Mechanisms: extravascular versus intravascular

  • Extravascular haemolysis: usually caused by alterations that make red cells less deformable. Principal clinical features are anaemia and splenomegaly. Often benefits from splenectomy.
  • Intravascular haemolysis: caused by mechanical injury, complement fixation, intracellular parasites (for example falciparum malaria) or exogenous toxic factors. Sources of mechanical injury include trauma from cardiac valves, narrowing of the microcirculation by thrombi or tumour cells, and repetitive physical trauma such as marathon running and bongo drum beating.

Laboratory features of haemolysis

Features of increased red cell breakdown:

  • Serum bilirubin raised, unconjugated.
  • Urine urobilinogen increased.
  • Serum haptoglobins reduced or absent (low in all acute or chronic haemolysis).

Features of increased red cell production:

  • Reticulocytosis.
  • Bone marrow erythroid hyperplasia (marrow aspirate shows numerous erythroid precursors).

Damaged red cells on the blood film:

  • Spherocytes, sickled red cells, red cell fragments.

Reticulocytes:

  • Reduced red cell survival leads to increased release of young red cells from the marrow.
  • Reticulocytes are young (1 day old) red cells; they appear bluish on a standard stained film, which is polychromasia, and show punctate reticular material on a reticulocyte stain.
  • Reference interval 20 to 100 x10
  • They are about 25% larger than mature red cells, which is why haemolysis tends to produce macrocytosis.

Because cell turnover is increased, these conditions need an increased supply of folic acid.

Typical results in a patient with haemolytic anaemia (reference intervals in brackets):

  • Hb 110 (130 to 175 g/L)
  • MCV 105 (80 to 98 fL), macrocytic because reticulocytes are large
  • MCH 35 (27 to 33 pg)
  • Reticulocytes 200 (20 to 100 x10
  • Total bilirubin 35 (2 to 20 µmol/L), almost all unconjugated (unprocessed)
  • Conjugated bilirubin 6 (0 to 5 µmol/L)
  • Haptoglobin <0.05 (0.3 to 2.0 g/L), all used up

Hereditary spherocytosis

Membrane structure and defect:

  • The red cell membrane has a lipid bilayer with embedded GPA, band 3, RhAG and protein 4.2, linked via ankyrin to an underlying α-spectrin/β-spectrin cytoskeletal lattice.
  • Mutations of spectrin, ankyrin, band 3 or protein 4.2 cause hereditary spherocytosis.

Mechanism in order:

  1. Mutation involving a membrane skeleton protein (band 3, protein 4.2, ankyrin, spectrin).
  2. Loss of membrane, with small membrane fragments budding off, so the cell becomes progressively spherical and less deformable.
  3. Spherocytes cannot pass through the splenic filter.
  4. Phagocytosis by splenic macrophages, so red cell survival is shortened.

Consequences and features:

  • Anaemia (variable), reticulocyte count increased, elevated bilirubin, low haptoglobin, raised MCHC.
  • Raised MCHC is due to dehydration of the spherocytic red cells caused by loss of K+ and H2O.
  • Characteristic clinical features: variable anaemia, splenomegaly, jaundice.
  • Blood film: small densely staining round red cells lacking central pallor.
  • Frequency about 1:5,000, especially in Northern Europe. Severity varies between families. Why it exists is unknown.

Diagnosis and complications:

  • Spherocytosis plus reticulocytosis plus a negative direct antiglobulin test (Coombs test), meaning no antibody or complement on the red cells, is highly suggestive of hereditary spherocytosis.
  • Incubation of red cells with the fluorescent dye eosin-5′-maleimide, which selectively binds band 3, gives reduced staining.
  • Aplastic crises, usually caused by acute parvovirus infection.
  • Haemolytic crises.
  • Gallstones: excess bilirubin in bile precipitates with calcium, forming calcium bilirubinate stones.
  • Splenectomy is a treatment option.

Red cell enzymopathies and G6PD deficiency

Why the pathway matters:

  • Red cells must protect against oxidative damage to proteins and lipids across their 120 day life, and need NADPH, glutathione (GSH) and G6PD to do so.
  • Glucose-6-phosphate dehydrogenase deficiency is the commonest red cell enzymopathy worldwide.
  • X-linked recessive, so males are mostly affected.
  • It clearly protects humans from malaria. Prevalence is highest in sub-Saharan Africa (over 20% in places), with moderate to high prevalence across the Middle East, parts of the Mediterranean and South/Southeast Asia.

The oxidative pathway:

  1. Hb(Fe2+:O2) is oxidised to Hb(Fe3+), methaemoglobin, releasing superoxide anion (O2•–).
  2. Superoxide is converted to H2O2.
  3. Normally G6PD converts glucose-6-phosphate to 6-phosphogluconate, generating NADPH from NADP.
  4. NADPH is used by glutathione reductase to convert GSSG back to GSH.
  5. GSH is used by glutathione peroxidase to convert H2O2 to water.
  6. In G6PD deficiency this detoxification fails, so H2O2 accumulates, causing haemoglobin denaturation, membrane lipid peroxidation and haemolysis.

Variants:

  • The most clinically significant haemolysis is associated with only two variants, G6PDA– and G6PD Mediterranean.
  • G6PDA– is present in about 10% of Black Americans; G6PD Mediterranean is prevalent in the Middle East.
  • Variants associated with haemolysis cause misfolding of the protein, making it more susceptible to proteolytic degradation.
  • Enzyme activity is moderately reduced in G6PDA– but markedly decreased in G6PD Mediterranean.
  • Older red cells are much more prone to haemolysis than younger ones.

Clinical and film features:

  • Acute episodes of haemolysis precipitated by infections, drugs, and fava (broad) beans.
  • Neonatal jaundice, anaemia, increased reticulocytes.
  • Heinz bodies: masses of oxidised, denatured and aggregated haemoglobin, seen with new methylene blue.
  • Bite cells and blister cells: red cells with irregular membrane defects.
  • Electron microscopy shows a red cell caught in transit from splenic cord to sinus, most of the cell having slipped through a gap in the basement membrane while the bunched Heinz body inclusions collect at its tail and impede passage, illustrating the splenic “pitting” mechanism of inclusion removal.

Agents that may cause haemolytic anaemia in G6PD deficiency:

  • Antimalarials: fansidar, chloroquine, quinine.
  • Sulphonamides and sulphones: co-trimoxazole, dapsone.
  • Other antibacterial agents: quinolones, chloramphenicol.
  • Analgesics: aspirin.
  • Moth balls.

Worked case (4 year old male):

  • Haemoglobin 56 g/L (113 to 145), Hct 0.17 (0.33 to 0.42), MCV 86 fL (74 to 87), MCH 29 pg (24 to 29), platelets 425 x10^9/L (150 to 475), WBC 9.5 x10^9/L (4.5 to 12.0), neutrophils 4.7, lymphocytes 3.3, monocytes 1.1 (0.3 to 1.0), eosinophils 0.3, basophils 0.0, reticulocytes 136 x10^9/L (20 to 100).
  • Total bilirubin 56 µmol/L (2 to 20), conjugated bilirubin 8 µmol/L (<6), haptoglobin <0.05 g/L (0.30 to 2.70), G6PD 0.3 U/g Hb at 37°C (reference >6.9).

Warning

The source slide showing the Heinz body electron micrographs has caption text cut off at the right/bottom edge, so part of that description is incomplete in the slides.

Clinical syndromes from abnormal haemoglobins

  • Thalassaemias: reduced production of α or β globin peptides, caused by defective or deleted α or β globin gene(s), resulting in a microcytic anaemia.
  • Haemoglobinopathies: abnormal haemoglobins arising from point mutations or deletions in one or both β globin genes, giving specific variants such as Hb C, D, E and S. Haemolysis occurs in some haemoglobinopathies.

Sickle cell anaemia (HbS)

  • Caused by a point mutation in β-globin: valine substituted for glutamic acid at position 6.
  • Major health problems occur in homozygous individuals.

Mechanism in order:

  1. Point mutation at DNA level converts HbA to HbS.
  2. Deoxygenation of HbS causes it to polymerise into long rigid crystalline structures that damage the red cell membrane; the cell loses K+ and H2O and takes up calcium.
  3. Repeated cycles of deoxygenation increase inflammation, transit time and expression of adhesive molecules on sickled cells.
  4. The end result is an irreversibly sickled, non-deformable cell causing microvascular occlusion and extravascular haemolysis, with rapid phagocytosis by macrophages.
  5. NO scavenging occurs.

Clinical issues:

  • Anaemia from shortened cell survival, that is haemolysis.
  • Sickling crises: acute obstruction of small blood vessels, causing infarction of spleen, bone and brain.
  • Crisis types: aplastic, haemolytic, sequestration and vaso-occlusive (pain).
  • Infections.
  • Blood film shows sickle-shaped (crescent) red cells.

Why the gene persists:

  • The HbS gene is mainly present in Africa, the Middle East and Asia, that is malarial areas; allele frequency is highest across central and west Africa, with lower frequencies across the Mediterranean, Middle East and parts of India, matching historically malaria-endemic regions.
  • Partial protection against malaria through an inhibitory effect on parasite growth from increased red cell potassium loss, decreased red cell pH, and increased endothelial adherence of parasitised red blood cells.

HbS trait (heterozygous state, Hb AS):

  • Mild condition: no anaemia, normal red cells in the blood film.
  • Sickling crisis may occur if pO2 is very low.
  • Protection against malaria occurs even in heterozygotes: the malaria parasite uses O2, leading to sickling of infected cells, which are then removed in the spleen and elsewhere by macrophages.

Thalassaemias

  • A syndrome of inherited haemoglobin disorders characterised by a quantitative deficiency of functional globin chains; among the commonest single-gene disorders. The name means “sea blood”.
  • Features: microcytic anaemia, and a spectrum of severity.
  • Common in Africa, the Mediterranean, the Middle East and Asia.
  • Blood film: microcytic hypochromic red cells with occasional target cells and basophilic stippling.

α-thalassaemia

  • Reduced production of the α chain of haemoglobin.
  • Severity varies enormously with the number of deleted genes, located on chromosome 16 (4 α genes per diploid genome).
  • Gene dosage: normal (4 genes); 1 gene deletion, silent carrier; 2 gene deletion, alpha trait (two configurations, cis and trans); 3 gene deletion, HbH disease; 4 gene deletion, hydrops fetalis.
  • Deletion of all 4 α genes is lethal for the foetus; avoiding fetal death is the reason α-thalassaemia matters clinically.
  • The silent carrier (1 gene deletion) state is common in NZ Māori and Eastern Polynesians (about 20%) and is clinically unimportant.

β-thalassaemia

  • β-globin locus is on chromosome 11, in the gene order Gγ, Aγ, δ, β.
  • Complete loss of 1 β-gene (β0/β) leads to mild β-thalassaemia, which is common.
  • Complete loss of 2 β-genes (β0/β0) gives severe β-thalassaemia.

β thalassaemia major:

  • Dependent on blood transfusions.
  • Greatly expanded bone marrow, causing bony deformities (frontal bossing, maxillary overgrowth, “hair-on-end” skull X-ray).
  • Hb 30 to 70 g/L, MCV 50 to 60 fL.
  • Iron overload, so chelation therapy is essential.
  • Increased illnesses and shortened survival.

Comparison of the β thalassaemia syndromes:

TypeGenotypeAnaemiaRed cell morphologyHb electrophoresisClinical features
Minorβ/β+, β/β0Absent or mild (Hb > 10 g/dl)Microcytic hypochromic, target cells, basophilic stipplingHb A2 > 3.5%; Hb F < 10%Asymptomatic
Intermediaβ+/β+, δβ/δβ, β0/δβModerate (Hb 7 to 10 g/dl)Similar but less severe changesHb F 10 to 95%; absent or low Hb A; Hb A2 normal or raisedOnset later than major, splenomegaly, not transfusion dependent
Majorβ0/β0, β0/β+, β+/β+Severe (Hb < 7 g/dl)Severe anisopoikilocytosis, microcytosis, hypochromia, target cells, basophilic stippling, numerous nucleated red cellsHb F 10 to 95%; absent or low Hb A; Hb A2 normal or raisedOnset in infancy, massive splenomegaly, marked skeletal changes, transfusion dependent

Acquired haemolytic anaemias

Immune haemolytic anaemias:

  • Autoimmune haemolytic anaemia is caused by antibody production by the body against its own red cells.
  • Characterised by a positive direct antiglobulin test (DAT), also known as the Coombs test.
  • Divided into warm and cold types according to whether the antibody reacts more strongly with red cells at 37°C or 4°C.

Warm autoimmune haemolytic anaemia

  • Red cells are coated with immunoglobulin at 37°C, usually IgG alone or with complement, and are taken up by reticuloendothelial macrophages, which have receptors for the Ig Fc fragment.
  • Part of the coated membrane is lost, leading to spherocytes.
  • When cells are coated with IgG and complement, or complement alone, destruction occurs more generally throughout the reticuloendothelial system.
  • Primary (idiopathic) or secondary to autoimmune disorders (particularly systemic lupus erythematosus), drugs (methyldopa), or lymphoid neoplasms.
  • Clinical feature: splenomegaly.
  • Lab findings: spherocytosis, positive DAT.
  • Treatment: remove the underlying cause, corticosteroids, rituximab, splenectomy, blood transfusion.

Cold autoimmune haemolytic anaemia

  • IgM autoantibodies attach to red cells mainly in the peripheral circulation, where blood is cooled.
  • The autoantibody may be monoclonal, as in primary cold haemagglutinin syndrome or in association with lymphoproliferative disorders, or a transient polyclonal response after infections such as infectious mononucleosis or Mycoplasma pneumonia.
  • IgM antibodies bind red cells optimally at 4°C and are highly efficient at fixing complement, so both intravascular and extravascular haemolysis can occur.
  • Only complement factors can be detected on the red cells in lab tests, because the IgM antibody is eluted off as the cells flow through warmer parts of the circulation.

Important

Spherocytes on a blood film are not specific: they occur in both hereditary spherocytosis and warm autoimmune haemolytic anaemia. The direct antiglobulin test separates them, being negative in hereditary spherocytosis and positive in autoimmune haemolysis.

Self-test

  1. Define a haemolytic anaemia and state why the lecturer argues it should be called a “shortened lifespan” anaemia.
  2. Describe the steps by which a senescent red cell is cleared in the spleen.
  3. Explain what happens to free plasma haemoglobin when haptoglobin has been exhausted.
  4. Describe the fate of bilirubin from haem breakdown through to excretion, including the proportions of urobilinogen taking each route.
  5. List the four hereditary categories of haemolytic anaemia, with an example disorder of each.
  6. List the acquired categories of haemolytic anaemia given in the lecture.
  7. Distinguish extravascular from intravascular haemolysis by mechanism and by clinical features.
  8. List the laboratory features of haemolysis under the headings increased breakdown, increased production, and damaged red cells.
  9. Explain why haemolytic anaemia tends to produce macrocytosis, and give the reticulocyte reference interval.
  10. Explain why patients with chronic haemolysis need an increased supply of folic acid.
  11. Describe the steps by which a mutation in a membrane skeleton protein leads to red cell destruction in hereditary spherocytosis.
  12. What is the triad of findings that is highly suggestive of hereditary spherocytosis, and why is the MCHC raised in this condition?
  13. Explain how gallstones arise in chronic haemolysis, and name the type of stone formed.
  14. Describe the pathway by which G6PD normally protects the red cell from H2O2, and predict what accumulates and what results if the G6PD step is blocked.
  15. Distinguish G6PDA– from G6PD Mediterranean.
  16. A patient with known G6PD deficiency develops acute haemolysis. List the classes of precipitant, with an example agent from each drug class.
  17. Describe the blood film findings in acute G6PD haemolysis, and explain what a Heinz body is.
  18. Distinguish thalassaemias from haemoglobinopathies as clinical syndromes from abnormal haemoglobins.
  19. Describe the molecular defect in HbS and the steps from deoxygenation to microvascular occlusion.
  20. Distinguish homozygous sickle cell anaemia from HbS trait in terms of anaemia, blood film and risk.
  21. Explain the three mechanisms by which HbS gives partial protection against malaria, and the separate mechanism operating in heterozygotes.
  22. List the α-globin gene deletion states from 1 to 4 genes deleted, with the phenotype of each, and state which state is common in NZ Māori and Eastern Polynesians.
  23. What are the haemoglobin and MCV values in β thalassaemia major, and what two treatment requirements follow from the disease?
  24. Distinguish β thalassaemia minor, intermedia and major by haemoglobin level, Hb F, and transfusion dependence.
  25. Describe how red cells are destroyed in warm autoimmune haemolytic anaemia, and list the treatment options.
  26. Distinguish warm from cold autoimmune haemolytic anaemia by antibody class, optimal temperature, site of action and what is detectable on the red cells in the laboratory.
  27. Integrative: a patient has anaemia, spherocytes on the film, reticulocytosis, unconjugated hyperbilirubinaemia and undetectable haptoglobin. Explain which single test discriminates the two main diagnoses this picture suggests, and how each result would be interpreted.

Answers

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