Hemolytic Anemia

Definition & Types of Hemolysis — What is hemolysis, and what are the two main mechanisms by which red cells are destroyed?

There are two main types:
(1) Intravascular hemolysis — red cells undergo lysis directly within the circulation, releasing their contents (haemoglobin, enzymes) into the plasma; this produces haemoglobinaemia and haemoglobinuria.
(2) Extravascular hemolysis — red cells are taken up by cells of the reticuloendothelial (RE) system (mainly in the spleen and liver), where they are destroyed and digested; this produces elevated unconjugated bilirubin and splenomegaly.

Most hereditary haemolytic anaemias involve extravascular haemolysis, while complement- or toxin-mediated conditions tend to cause intravascular haemolysis.

Hereditary Spherocytosis — What is the pathogenesis of hereditary spherocytosis, and how do the molecular defects lead to spherocyte formation?

Hereditary spherocytosis (HS) is a congenital haemolytic disorder caused by an inherited defect in the red cell membrane cytoskeleton.
Normally, the lipid bilayer is anchored to the underlying protein skeleton by two major linkages:
(1) Ankyrin interacts with spectrin in the skeleton and band 3 in the bilayer;
(2) Glycophorin C interacts with protein 4.1.
A deficiency in any of these proteins (ankyrin, spectrin, band 3, or protein 4.1) weakens the contact between the lipid bilayer and the skeleton.
This causes a decrease in the surface area of the red cell relative to its volume, resulting in the formation of spherocytes.
Spherocytes are less deformable than normal red cells and are therefore trapped and destroyed in the spleen.

The most common mode of inheritance is autosomal dominant, though autosomal recessive transmission occurs in some cases.

Hereditary Spherocytosis — What are the clinical features, laboratory findings, and the osmotic fragility test?

Clinically, the majority of patients present in childhood with: mild to moderate anaemia, intermittent jaundice, splenomegaly, gallstones (from chronic haemolysis and bilirubin accumulation), and chronic leg ulcers.
Laboratory findings include: mild to moderate anaemia, reticulocytosis, and the characteristic finding on blood smear — spherocytes (small, round, dense red cells with no central pallor).
Bone marrow examination shows erythroid hyperplasia.
The osmotic fragility test is a key diagnostic test:
red cells are suspended in decreasing concentrations of hypotonic saline solutions, and the amount of haemolysis is measured. Spherocytes are more susceptible to osmotic stress and lyse at higher saline concentrations than normal red cells, giving an increased osmotic fragility result.
Treatment: splenectomy for severe HS. Folate supplementation is necessary in moderate-to-severe disease to prevent megaloblastic anaemia secondary to increased erythrocyte turnover.

β Thalassaemia Major — What is the pathogenesis of anaemia in β thalassaemia major, and what are the systemic complications?

In β thalassaemia major (also called Cooley's anaemia), the genetic defect prevents adequate synthesis of β globin chains.
This causes:
(1) Excess free α chains that precipitate within erythroblasts and red cells → lysis of erythroblasts in the bone marrow (ineffective erythropoiesis);
(2) Red cells containing α chain aggregates have reduced flexibility and are trapped in the spleen;
(3) Reduced haemoglobin synthesis → microcytic hypochromic red cells;
(4) Excessive peripheral destruction → progressive splenomegaly;
(5) HbF (α₂γ₂) becomes the predominant haemoglobin, which has high oxygen affinity and exacerbates tissue hypoxia.

Systemic complications include:
Skeletal changes — severe anaemia drives extreme bone marrow hyperplasia, causing expansion and deformity of skull and facial bones ("hair-on-end" appearance on X-ray) and possible pathological fractures.
Iron overload — from increased intestinal iron absorption due to ineffective erythropoiesis, and from regular blood transfusions; this damages the pancreas (diabetes), liver (cirrhosis), gonads (infertility), parathyroid (hypoparathyroidism), thyroid (hypothyroidism), and heart (arrhythmias and heart failure).
Hypersplenism and extramedullary haematopoiesis compressing vital structures (e.g., spinal cord) can also occur.
Untreated patients typically die from anaemia or infections before 5 years of age.

β Thalassaemia Major — What are the laboratory features and the principles of treatment?

Laboratory features:
Haemoglobin is severely reduced (2–6 g/dl).
Anaemia is microcytic and hypochromic.
Peripheral blood smear shows marked anisopoikilocytosis, severe hypochromia, target cells, basophilic stippling, and nucleated red cells; reticulocytosis is modest. Haemoglobin electrophoresis characteristically shows elevated HbF; HbA2 may be normal or elevated; HbA is absent or markedly reduced.
Bone marrow shows severe erythroid hyperplasia. Serum ferritin is elevated (increased storage iron). Unconjugated serum bilirubin is elevated.

Treatment:
(1) Regular red cell transfusions to maintain Hb constantly above 9.5–10 g/dl — prevents anaemia, hypoxia, and suppresses endogenous (ineffective) erythropoiesis;
(2) Iron chelation therapy (usually started at age 3 years) using desferrioxamine (DF) 25–60 mg/kg SC over 12 hours for 5–6 days/week; alternatives include deferiprone (risk of agranulocytosis, arthropathy, hepatic fibrosis) and deferasirox (oral, 20–30 mg/kg/day; side effects: GI troubles, renal impairment);
(3) Splenectomy for hypersplenism (avoided before age 5–6 years due to sepsis risk);
(4) Folic acid supplementation and hormone replacement therapy for endocrine failure;
(5) Haematopoietic stem cell transplantation — the only curative therapy.

β Thalassaemia Minor — What are the clinical and laboratory features of β thalassaemia minor?

β thalassaemia minor is the heterozygous carrier state for β thalassaemia.
Clinically, patients are usually asymptomatic, but may develop anaemia during physiologically demanding situations such as infections or pregnancy.
Laboratory features: Haemoglobin level is either normal or mildly decreased, generally not less than 9 g/dl.
Red blood cells characteristically show reduced MCV (microcytic) and reduced MCH (hypochromic).
Red cell count is increased (more numerous but smaller cells). Reticulocyte count and serum bilirubin are slightly elevated. Haemoglobin electrophoresis: the hallmark finding is elevated HbA2 (>3.5%), which is the diagnostic criterion for β thalassaemia minor. This distinguishes it from iron deficiency anaemia, which also presents with microcytic hypochromic red cells.

α Thalassaemias — What are the clinical forms of α thalassaemia based on the number of deleted α globin genes?

Humans have four α globin genes (two on each chromosome 16). The severity of α thalassaemia depends on the number of genes deleted:
(1) Silent α⁺ thalassaemia carrier (−α/αα) — 3 functional genes; red cell morphology and indices are normal or only slightly reduced MCV/MCH; clinically silent.
(2) α Thalassaemia trait — 2 functional genes; either homozygous α⁺ (−α/−α) or heterozygous α⁰ (−−/αα); usually detected incidentally; very mild anaemia, microcytic and hypochromic red cells, decreased MCV and MCH; asymptomatic.
(3) HbH disease (−−/−α) — 1 functional gene; marked α chain deficiency causes excess β chains to form β₄ tetramers (HbH); these precipitate in older red cells forming inclusions, which are destroyed in the spleen; presents with anaemia (Hb 7–10 g/dl), icterus, and hepatosplenomegaly; transfusions usually not needed; blood film shows anisopoikilocytosis, hypochromia, microcytes, and target cells.
(4) Hb Bart's Hydrops Foetalis syndrome (−−/−−) — 0 functional genes; absolute deficiency of α chains in foetal life leads to excess γ chains forming γ₄ tetramers (Hb Bart's); infants are either stillborn or die shortly after birth with severe anaemia, massive anasarca (generalised oedema), and hepatosplenomegaly; this is the most lethal form.

Sickle Cell Disease — What is the molecular basis of HbS and how does sickling occur?

Sickle cell disease results from a point mutation: a single base change (A→T) in the sixth codon of the β globin gene causes substitution of valine for glutamic acid at position 6 of the β polypeptide chain (6 Glu→Val).
The resulting abnormal haemoglobin is HbS.
Under deoxygenated conditions, HbS molecules polymerize inside the red cells, forming rigid, insoluble fibres that distort the cell into the characteristic sickle shape.
Sickled cells have rigid membranes and are trapped and destroyed in the spleen (extravascular haemolysis) and also obstruct microvasculature (vaso-occlusion). Sickle cell anaemia (SCA) is the homozygous state (HbSS) — both parents contribute the sickle gene. Sickle cell trait is the heterozygous state (HbAS) — one normal and one sickle gene; these individuals are usually asymptomatic under normal conditions.

Factors Influencing Sickling — What factors promote or worsen sickling, and why is each clinically important?

Five main factors promote HbS polymerization and sickling:
(1) High intracellular concentration of HbS — the higher the HbS relative to HbF or HbA, the greater the tendency to sickle; this is why HbSS (sickle cell anaemia) is severe while HbAS (sickle trait) is mild;
(2) Cellular dehydration — increases the intracellular concentration of HbS, promoting polymerization; treatment with adequate hydration is important;
(3) Decreased oxygen tension (deoxygenation) — the primary trigger for sickling; deoxy-HbS is far more prone to polymerize than oxy-HbS; maintaining oxygenation is critical during crises;
(4) Cold — induces vasoconstriction which reduces blood flow, prolongs deoxygenation, and increases sickling; patients should keep warm;
(5) Low pH/acidosis — promotes the deoxy state of haemoglobin, thereby increasing polymerization; maintaining acid-base balance is important during management.

Sickle Cell Disease — What are the haematological and vaso-occlusive crises, and how do they differ?

Crises in sickle cell disease are divided into:
Vaso-occlusive (painful) crises — the most common type; sickled cells obstruct small blood vessels causing tissue ischaemia and infarction; presents as:
sudden onset of severe pain in bones, joints, chest, or abdomen.

Haematological crises:
(1) Aplastic crisis — caused by parvovirus B19 infection, which selectively infects erythroblasts and suppresses erythropoiesis; usually transient (7–10 days); transfusion support is essential during this phase;
(2) Haemolytic crisis — sudden fall in haemoglobin with increased icterus and rising reticulocyte count;
(3) Splenic sequestration crisis — sudden and massive pooling of blood in the spleen over several hours causing rapid splenomegaly, progressive anaemia, and circulatory failure; this can be life-threatening.

Sickle Cell Disease — What are the multi-organ complications of sickle cell disease?

Multi-organ complications arise from chronic vaso-occlusion and tissue hypoxia:
(1) Spleen — splenomegaly in infants; in later life, repeated splenic infarctions cause the spleen to become small and fibrotic (autosplenectomy), leading to functional asplenia and increased susceptibility to encapsulated bacteria;
(2) Infections — children (especially <5 years) are susceptible to fulminant infections due to impaired splenic phagocytic function; pneumococcal prophylaxis is essential;
(3) CNS — ischaemic strokes;
(4) Genitourinary — ischaemia of renal medulla with papillary necrosis, manifesting as haematuria; priapism (due to obstruction of venous return from the corpora cavernosa);
(5) Skeletal — dactylitis (painful swelling of hands and feet in children); impaired growth and development;
(6) Respiratory — pulmonary hypertension and cor pulmonale;
(7) Hepatobiliary — hepatic damage from infarctions and transfusion-transmitted hepatitis;
(8) Skin — chronic leg ulcers.

Sickle Cell Disease — What are the laboratory features and treatment strategies?

Laboratory features:
Haemoglobin is moderately reduced (6–9 g/dl); anaemia is normocytic and normochromic.
Peripheral blood smear shows sickle cells. Reticulocyte count is elevated. Unconjugated serum bilirubin is elevated.
HbS is identified and confirmed by haemoglobin electrophoresis.

Treatment:
(1) Prevention of crises — early detection and treatment of infections; pneumococcal vaccine, influenza vaccine, and penicillin prophylaxis in early childhood;
(2) Vaso-occlusive crisis management — analgesia, keeping patient warm, adequate hydration, oxygenation, and treatment of infections;
(3) Exchange transfusion — prior to surgery to reduce HbS percentage and lower risk of vaso-occlusion;
(4) Packed red cell transfusions — during symptomatic anaemia to improve oxygen-carrying capacity;
(5) Hydroxyurea — increases HbF production; since HbF does not participate in sickling, it retards HbS polymerization and reduces the frequency and severity of crises;
(6) Haematopoietic stem cell transplantation — the only curative treatment.

G6PD Deficiency — What is the pathogenesis of haemolysis in G6PD deficiency?

G6PD (glucose-6-phosphate dehydrogenase) deficiency is characterised by reduced G6PD enzyme activity in red cells, leading to haemolysis usually triggered by oxidant stress.

G6PD catalyses the first step of the hexose monophosphate (HMP) shunt, generating NADPH from NADP. NADPH is required for the continuous production of reduced glutathione (GSH). GSH detoxifies harmful hydrogen peroxide (H₂O₂) to water.

In G6PD deficiency, H₂O₂ accumulates and causes oxidation of haemoglobin, leading to denaturation and precipitation of globin chains. The precipitated globin chains form inclusions called Heinz bodies, which cause membrane rigidity and extravascular destruction in the spleen.
Additionally, intravascular haemolysis occurs.
G6PD deficiency is X-linked recessive and therefore occurs almost exclusively in males. Females are usually carriers.

G6PD Deficiency — What are the clinical manifestations, triggers, and laboratory features of G6PD deficiency?

Clinical manifestations include:
(1) Drug-induced haemolytic anaemia — triggered by oxidant drugs (e.g., primaquine, dapsone, sulphonamides);
(2) Favism — a unique form seen in Mediterranean and Arab populations, precipitated by ingestion of fava beans, which contain oxidants; haemolysis begins hours to days after ingestion and can be fatal;
(3) Haemolysis following infection;
(4) Neonatal jaundice;
(5) Chronic haemolytic anaemia (in some variants).
Clinical presentation during a haemolytic episode: sudden development of pallor, jaundice, and dark-coloured urine (haemoglobinuria); acute renal failure may develop in severe cases.
Laboratory features during haemolysis: general features of haemolytic anaemia; peripheral blood smear shows fragmented red cells and bite cells; unconjugated hyperbilirubinaemia; haemoglobinuria.

Diagnostic tests: qualitative fluorescent spot test (screening); quantitative G6PD enzyme assay for confirmation.

Treatment: avoid oxidant drugs and triggers; prompt treatment of infections; supportive care during haemolytic episodes; blood transfusion in severe cases; maintain adequate urinary output to prevent renal damage from haemoglobinuria.

Classification of Immune Haemolytic Anaemias — What are the three categories of immune haemolytic anaemia and their subtypes?

Immune haemolytic anaemias are caused by antibodies directed against red cell antigens. They are classified into three major categories:
(1) Autoimmune haemolytic anaemia (AIHA) — the body produces antibodies against its own red cells;
two subtypes: Warm-reactive antibody type (IgG antibodies maximally active at 37°C) and Cold-reactive antibody type (IgM antibodies maximally active at 0–4°C); each can be primary (idiopathic) or secondary.
(2) Alloimmune haemolytic anaemia — antibodies arise from exposure to foreign red cell antigens; includes haemolytic disease of the newborn (Rh or ABO incompatibility) and haemolytic transfusion reactions.
(3) Drug-induced haemolytic anaemia — drugs act as haptens or trigger autoantibody production against red cells.

Warm Antibody AIHA — What is the pathogenesis, clinical features, laboratory findings, and treatment?

Warm antibody AIHA is the most common form of AIHA.

IgG antibodies (active at 37°C) or complement (C3b) bind to the red cell membrane and are recognised by specific receptors on macrophages in the spleen, causing extravascular haemolysis.
It can be primary (idiopathic) or
secondary to: autoimmune disorders (e.g., SLE), neoplastic disorders (CLL, malignant lymphoma, ovarian teratoma).

Clinical features: anaemia, icterus (jaundice), and splenomegaly.
In secondary AIHA, clinical features of the underlying disease predominate. Laboratory features: peripheral blood smear shows anaemia, microspherocytosis, and reticulocytosis; nucleated red cells may be present; elevated unconjugated serum bilirubin.

The key diagnostic test is the Coombs' (antiglobulin) test:
Direct Antiglobulin Test (DAT) — demonstrates antibodies or complement attached to red cells in vivo (positive in AIHA);
Indirect Antiglobulin Test (IAT) — demonstrates antibodies or complement in the serum.

Treatment: (1) Identify and treat underlying disease; (2) Corticosteroids (1 mg/kg/day) — majority of patients respond; (3) Splenectomy — when steroids fail; (4) Immunosuppressive therapy (azathioprine or cyclophosphamide) — for cases unresponsive to steroids and splenectomy; (5) Rituximab (anti-CD20) — targets B lymphocytes; (6) Blood transfusion — only when absolutely essential.

Cold Antibody AIHA — What is cold antibody AIHA, how does it differ from warm AIHA, and how is it treated?

Cold antibody AIHA is caused by autoantibodies (characteristically IgM) that react with red cells maximally at cold temperatures (0–4°C).

At low temperatures, IgM antibodies agglutinate red cells and activate complement, causing haemolysis predominantly when blood circulates through cold peripheral extremities.
It can be primary (idiopathic cold haemagglutinin disease) or
secondary to infections (Mycoplasma pneumoniae, EBV, CMV, malaria) or lymphoproliferative disorders.
A separate subtype involves IgG cold antibodies secondary to Treponema pallidum or viruses (Donath-Landsteiner antibody).

Laboratory features: anaemia (usually mild to moderate, occasionally severe); characteristic autoagglutination of red cells visible on the peripheral blood smear; positive DAT.

Treatment differs markedly from warm AIHA: (1) Identify and treat the underlying cause (e.g., lymphoma treatment); (2) Avoid exposure to cold — critical in management; (3) Corticosteroids and splenectomy are NOT helpful (unlike warm AIHA); (4) Cytotoxic therapy (e.g., chlorambucil) to reduce immunoglobulin production and decrease red cell destruction; (5) Plasmapheresis to reduce circulating antibody levels — a temporary measure; (6) Blood transfusions only when absolutely essential, with warming of blood prior to administration.

General Laboratory Features of Haemolytic Anaemia — What laboratory findings indicate haemolysis regardless of the underlying cause?

The general laboratory findings that indicate haemolysis include:
Evidence of increased RBC destruction: (1) Elevated unconjugated (indirect) serum bilirubin — from haem catabolism; (2) Elevated LDH (lactate dehydrogenase) — released from lysed red cells; (3) Low or absent serum haptoglobin — haptoglobin binds free haemoglobin and is rapidly cleared; (4) Haemoglobinaemia and haemoglobinuria — present in intravascular haemolysis specifically; (5) Haemosiderinuria — in chronic intravascular haemolysis.

Evidence of compensatory increased RBC production:
(1) Reticulocytosis — the most consistent finding;
(2) Erythroid hyperplasia on bone marrow examination;
(3) Elevated MCV (if reticulocytosis is pronounced).
Blood film abnormalities depend on the cause: spherocytes (HS or warm AIHA), sickle cells (SCD), target cells and hypochromia (thalassaemia), bite cells and fragments (G6PD deficiency), autoagglutination (cold AIHA).

Overview of Inherited vs. Acquired Haemolytic Anaemias — How are haemolytic anaemias broadly classified, with key examples?

Haemolytic anaemias are broadly divided into inherited and acquired types.
Inherited haemolytic anaemias result from intrinsic defects in the red cell and include: (1) Membrane defects — Hereditary Spherocytosis (defects in ankyrin, spectrin, band 3, or protein 4.1);
(2) Haemoglobin defects — Thalassaemias (reduced/absent globin chain synthesis: α and β types) and Sickle Cell Disease (structural HbS mutation causing polymerization and vaso-occlusion);
(3) Enzyme defects — G6PD deficiency (X-linked; oxidant-triggered haemolysis due to inadequate NADPH and GSH production).

Acquired haemolytic anaemias result from extrinsic factors acting on otherwise normal (or previously normal) red cells and include:
(1) Immune causes — Autoimmune (warm IgG or cold IgM antibodies), alloimmune (transfusion reactions, haemolytic disease of the newborn), and drug-induced;

(2) Non-immune causes — microangiopathic haemolytic anaemia, infections (e.g., malaria), hypersplenism, and mechanical destruction.

The distinction between intravascular and extravascular haemolysis helps narrow the differential: intravascular haemolysis produces haemoglobinaemia and haemoglobinuria; extravascular haemolysis primarily produces jaundice and splenomegaly