Haemolytic Anaemias: Classification and Causes

• Haemolytic anaemias result from an increase in the rate of red cell destruction.
• Red cell destruction may increase before a patient becomes anaemic due to erythropoietic hyperplasia and bone marrow extension, resulting in compensated haemolytic disease.

Classification of Haemolytic Anaemias

• Broadly classified as intracorpuscular (mainly hereditary) or extracorpuscular (mainly acquired).
• Can also be classified as hereditary or acquired.
• Hereditary causes include membrane disorders (HS, HE), enzyme disorders/enzymopathies (G6PD deficiency, PK deficiency), and haemoglobin disorders/haemoglobinopathies (Hb S, Hb C).

Acquired Causes of Haemolytic Anaemia

• Immune-related: auto immune, allo immune, and drug-induced.
• Non-immune-related: red cell fragmentation syndrome, march haemoglobinuria, infections, chemical and physical agents, secondary causes, and paroxysmal nocturnal haemoglobinuria.

Enzymopathies

• These involve defective red cell metabolism that can be quantitative or qualitative, and are mostly inherited.
• Red cell metabolic processes: glycolysis, redox reaction, and nucleoside metabolism.

Glycolysis

• Red cells feed on plasma glucose and excrete lactate and pyruvate.
• The conversion of glucose to pyruvate yields a net of 2 ATP molecules per glucose molecule.

Redox Reaction

• Maintaining normal red cell shape requires normal ion pump operation, dependent on ATP.
• Haemoglobin must be in a functional state with reductive potentials provided by glycolysis in the form of NADH.

Haemoglobin Oxidation

• Circulating red cell haemoglobin undergoes gradual oxidation from divalent ferrohemoglobin to trivalent ferrihaemoglobin (methemoglobin).

Methemoglobin Reduction

• A small amount of methemoglobin is constantly formed and converted back to haemoglobin via methemoglobin reductases like NADH Diaphorase, NADPH Diaphorase, GSH, ascorbic acids, and Methylene Blue.
• NADH Diaphorase is the primary enzyme for reducing methaemoglobin back to haemoglobin.

NADH Role in Glycolysis

• NADH generated in the PPP pathway of glycolysis leads to methemoglobinemia and tissue anoxia if methemoglobin cannot be reduced.
• Methaemoglobin production generates highly reactive and toxic O₂ radicals (OH-, O2-), which produce H2O2, toxic to RBC membrane and haemoglobin.

Glutathione Importance

• Key for preserving sulfhydryl groups in proteins and preventing oxidative damage.
• Crucial in red cells due to their role as O2 carriers, which creates a risk of damage from generated oxygen radicals.

Reactive Oxygen Species

• Highly reactive oxygen radicals can decay spontaneously or be converted by superoxide dismutase to hydrogen peroxide (H2O2).
• H2O2 must be quickly detoxified into water by glutathione-dependent enzymes, specifically glutathione peroxidase (GSHPx).
• Reduced Glutathione (GSSH) is constantly regenerated by glutathione reductase, an NADPH-linked flavoenzyme.
• NADPH is produced by G6PD and 6PGD reactions in the HMS shunt or PPP.

Glutathione Redox Cycle

• GSH + H2O2 becomes GSSG + H2O.
• One molecule of GSH is oxidized to GSSG for every molecule of H2O2 detoxified.
• Glutathione functions if it is continuously and stoichiometrically regenerated to GSH by glutathione reductase.

Nucleoside Metabolism

• While glucose is the main metabolic fuel, purine nucleosides can enter red cells and convert to pentose phosphate via nucleoside phosphorylase.

G6PD Deficiency

• Intracorpuscular RBC disorder under enzymopathies leads to haemolytic anaemia, primarily intravascular but also extravascular.
• G6PD is an enzyme in the first step of PPP, reducing NADP to NADPH while oxidizing G6P.
• G6PD is the only source of NADPH in RBCs, and since NADPH is needed for reduced glutathione, G6PD deficiency makes RBCs prone to oxidant stress.

G6PD Enzyme Structure

• G6PD enzyme is dimeric or tetrameric.
• The gene is X-linked (Xq28).
• A monomer has 514 amino acids with a molecular weight of 59 kDa.
• The gene is near those for factors viii/ix and colour blindness.
• It has high substrate specificity.

G6PD Inheritance

• Inherited via simple Mendelian X-linked inheritance.
• Inherited as a codominant trait; female heterozygotes show somatic cell mosaicism.
• Half of cells will be Gd+ and half will be Gd-.

G6PD Prevalence

• The X-linked disorder is more common in males.
• Female carriers may have mild G6PD deficiency.
• Affected (homozygous) females (carriers) are also known.
• G6PD can be associated with chronic granulomatous disease (CGD).

G6PD Genetics/Epidemiology

• Defects (deficiency and carrier states) arise from mutations/deletions at the gene locus.
• There are over 400 types of mutations, named after the city of discovery (e.g., G6PD Ilesha, G6PD Sunderland, G6PD Minnesota).
• Adenine replaced by Guanine at codon 376 of the G6PD gene resulting into Arsenic acid replaced by Asparaginase at position 126 of G6PD.
• The gene is designated Gd; normal is Gd+, deficient is Gd-.
• Gd+/Gd+ is normal homozygote, Gd-/Gd- is deficient homozygote, Gd+/Gd- is heterozygote.
• Males are either Gd+ or Gd- due to X-linkage.
• G6PD deficiency is the most common genetically determined enzyme deficiency worldwide, affecting over 400 million people.

G6PD Types

• Normal G6PD is designated G6PD B.
• G6PD A is the normal variant in Africa.
• The four main variants are G6PD B-, G6PD A-, G6PD Mediterranean (associated with Favism), and G6PD Santiado de Cuba.
• G6PD A occurs in about 10% of Africans and African-Americans.
• G6PD Mediterranean is prevalent in the middle.

G6PD Deficiency Definition

• Reduced normal enzyme activity is a G6PD deficiency.
• Can be quantitative (low concentration) or qualitative (normal quantity, abnormal activity).

G6PD Distribution

• Found in Africa, the Mediterranean, the Middle East, South East Asia, and the Indian subcontinent.
• Distribution overlaps with Plasmodium falciparum malaria areas.
• Protects against lethal falciparum malaria.

G6PD Pathophysiology

• G6PD deficient cells are more susceptible to oxidative damage.
• NADPH from G6PD regenerates glutathione (GSH), which detoxifies H2O2 via GSH peroxidase.
• Sources of oxidative damage: H2O2 producing bacteria, chemicals, and divicine in fava beans.

Haemoglobin Denaturation

• Oxidative agents cause haemoglobin denaturation, forming Heinz bodies, or lipid peroxidation on the RBC membrane.
• Heinz bodies lead to red cell destruction in the RES (extravascular haemolysis).
• Lipid peroxidation affects red cell structure and may lead to destruction in peripheral circulation (intravascular haemolysis).

Intravascular vs Extravascular Haemolysis

• Intravascular haemolysis is dominant in acute haemolytic anaemias.
• Extravascular destruction dominates in chronic non-spherocytic haemolytic anaemia.

G6PD Deficiency: Clinical Features

• Most patients are asymptomatic until an acute episode.
• Manifestations can be acute or chronic.
• Includes acute haemolytic anaemia, neonatal jaundice, and chronic non-spherocytic haemolytic anaemia.

Acute Haemolytic Anaemia

• As low as 3% G6PD enzyme activity is sufficient for normal RBC functions.
• Haemolysis occurs when exogenous factors impose extra stress and the NADPH supply is low.
• Triggers: fava beans, infections, and drugs.

Fava Beans

• Offending agents are divicine and isouramil, with concentrations varying in fava beans; highly concentrated in fresh and raw beans.
• Oxidative damage is based on the amount ingested.
• Cultivated mainly in the Mediterranean (Spaniards, Italians, Greeks, Armenians, and Jews).

Infections

• Infections such as pneumococcus and streptococcus produce large amounts of H2O2, trigger haemolysis.

Drugs

• Certain drugs can also trigger haemolysis: Primaquine, Pamaquine, Sulphonamides, Sulphones, Dapsone, Septrin, Acetaphenetidin(phenacetin), Acetylsalicyclic acid (asprin), -naphtol, stibophan, Niridazole, Naphthalene(mouth balls), Probenecid, and Methylene blue.

Severity of Haemolysis

• Ranges from mild anaemia/jaundice to severe anaemia.
• Massive haemoglobinuria is more common in children with favism.
• Renal failure is rare despite haemoglobinuria.
• Haemolysis is commonly intravascular.

Neonatal Jaundice (NNJ)

• G6PD deficient babies are more prone to NNJ than normal babies.
• G6PD deficiency and HDN are common causes of NNJ.
• Develops late (2-3 days after birth) and is deeper in G6PD deficiency cases.
• Offending agents, such as drugs given to the mother before or to the babies during delivery, are sometimes the cause.

Chronic Haemolytic Anaemia

• Residual enzyme activity is less than 2%.
• Patients suffer life-long haemolysis and anaemia due to severe qualitative or quantitative enzyme abnormalities.
• This is the rarest presentation with normocytic normochromic anaemia.
• Reticulocytosis and bone marrow erythroid hyperplasia.
• Onset is frequently at birth with recurring anaemia and jaundice.

Chronic Haemolytic Anaemia Signs

• There is chronic hyperbilirubinemia, decreased haptoglobin, and increased lactate dehydrogenase (LDH).
• Haemoglobinuria is rare.
• Haemosiderinuria may be detected.

Laboratory Diagnosis of G6PD Deficiency

Full Blood Count

• Reveals anaemia and reticulocytosis.

Peripheral Blood Film

Normocytic normochromic anaemia accompanied by:

• Marked anisocytosis and poikilocytosis
• Regularly and irregularly contracted red cells
• Red cells with uneven haemoglobin distribution, called hemighost.

Specific Test Types

• Screening and confirmatory tests.

Screening Tests

• Brilliant cresyl blue decolorization test
• Methaemoglobin reduction test
• Ultra violet spot test

Special Staining

• Special staining of reticulocytes reveals inclusion bodies, unlike those normally seen.
• Heinz bodies, denatured haemoglobin, are 1-3µm in diameter and appear leaning from the interior against the cell membrane.

Confirmatory Tests

• A G6PD assay with a threshold of enzyme activity of about 20% of normal.

Management of G6PD Deficiency

For AHA

• Bed rest.
• Administer urgent red cell transfusion.
• Perform haemodialysis in case of renal failure.
• Treat underlying infections as detected.
• Remove any offending agents.

For NNJ

• Implement phototherapy.
• Perform exchange blood transfusion.

For Chronic Haemolytic Anaemia

• Administer recurrent blood transfusions.
• Perform splenectomy after age of 6 years.

Pyruvate Kinase Deficiency

• The second commonest enzyme deficiency after G6PD, though very rare.
• Prevalence of 1:10,000 population.

Pyruvate Kinase Deficiency: Genetics/Epidemiology

• Caused by mutations in the PKLR gene on chromosome 15q22.
• The PKLR gene is active in the liver and in red blood cells.
• Inheritance is autosomal recessive, although autosomal dominant inheritance can also occur.
• Affects males and females equally.
• About 55 mutations and 2 deletions on chromosome 1q22.
• Has no worldwide distribution.

Pyruvate Kinase Deficiency: Pathophysiology

• Pyruvate kinase is the last enzyme in the glycolytic pathway, transferring the phosphate group from phosphenol pyruvate to ADP, generating ATP and pyruvate.
• Represents the second ATP-producing step and the third regulatory reaction.
• Results in a 50% reduction in ATP generated per molecule of glucose and a complete absence of pyruvate, with precursor products accumulating.

Pyruvate Kinase Deficiency: Clinical Features

• Symptoms of anaemia are less than expected due to high concentrations of 2,3DPG.
• Jaundice.
• Anaemia occurs in infancy/childhood with Hb levels of 4-10g/dl.
• Risk of gallstones from hyperbilirubinaemia.
• Biliary Colic.
• Cholecystitis.
• Splenomegaly.
• Impaired growth.
• Frontal bossing.

Pyruvate Kinase Deficiency: Lab Diagnosis

Blood Film

• Macrocytosis
• The presence of bizzare prickle cells
• Moderate reticulocytosis
• Auto haemolysis is abnormal with poor correction via glucose.

Specific Test

• PK assay.

Pyruvate Kinase Deficiency Treatment

• Severely affected fetus dies in utero.

Treatment

• Regular folate replacement (5mg daily).
• Iron supplementation.
• Repeated/occasional blood transfusions.
• Splenectomy.