Haemolytic Anaemia - Strictly According to Robbins Pathology

Robbins & Kumar Basic Pathology (Robbins Pathology), 10th ed., ISBN 9780323790185

Definition and Overview

• Marrow erythroid hyperplasia
• Peripheral blood reticulocytosis (hallmarks of haemolytic anaemia)
• In severe cases: extramedullary haematopoiesis in liver, spleen, and lymph nodes

Classification

1. By Origin of Defect

2. By Site of Haemolysis (Clinically more useful)

Extravascular Haemolysis

• Hyperbilirubinaemia and jaundice (from Hb degradation in macrophages)
• Splenomegaly (work hyperplasia of phagocytes)
• Cholelithiasis with pigment (bilirubin-rich) gallstones if long-standing
• Low serum haptoglobin (macrophages regurgitate enough Hb to deplete it)
• No iron deficiency (iron recycling by phagocytes is efficient)

Intravascular Haemolysis

• Haemoglobinaemia - free Hb in plasma
• Haemoglobinuria - Hb passes into urine (Hb molecule is small enough)
• Haemosiderinuria - iron accumulation in renal tubular cells shed into urine
• Iron deficiency - iron is lost via urine rather than recycled
• Low serum haptoglobin (also seen in extravascular)

Types of Haemolytic Anaemia

1. Hereditary Spherocytosis

• Spectrin (major protein) - long, flexible heterodimer
• Actin filaments
• Ankyrin and band 4.1 - linker proteins
• Connected to intrinsic membrane proteins band 3 and glycophorin

• Peripheral smear: spherocytes are dark red and lack central pallor
• Compensatory marrow erythroid hyperplasia + reticulocytosis
• Splenomegaly (most prominent of all haemolytic anaemias) - splenic weight 500-1000 g (normal 150-200 g)
• Marked congestion of splenic cords, increased macrophages
• Cholelithiasis in 40-50% of patients

2. Sickle Cell Anaemia

• HbS differs from HbA: valine instead of glutamate at β-globin position 6
• On deoxygenation, HbS molecules undergo conformational change → polymers form via intermolecular contacts involving the abnormal valine → red cells assume elongated crescentic (sickle) shape
• Sickling is initially reversible on reoxygenation; with repeated episodes → membrane damage (calcium influx, K⁺ and water loss) → irreversibly sickled cells → haemolysis

1. Intracellular levels of non-HbS Hb - HbA retards HbS polymerization greatly → HbS heterozygotes (sickle cell trait) have little sickling in vivo. HbF also retards polymerization → newborns asymptomatic until HbF falls (~5-6 months). HbC (lysine instead of glutamate) interacts with HbS only moderately → compound heterozygotes have milder disease
2. Intracellular Hb concentration - high MCHC favours polymerization; dehydration worsens sickling
3. Length of time in deoxygenated state - slow blood flow prolongs deoxygenation

• Peripheral smear: sickle-shaped red cells, target cells, nucleated red cells
• Vaso-occlusive crises - hallmark; microinfarcts in bones, spleen, liver, brain, lungs, penis
• Spleen: initially enlarged by red pulp congestion with sickled cells; progressive splenic infarction leads to autosplenectomy (fibrotic, shrunken spleen by adulthood)
• Severe splenomegaly can occur in compound heterozygotes (HbSC)
• Bone marrow hyperplasia → cortical bone thinning, "crew cut" appearance on skull X-ray
• Increased risk of aplastic crisis (Parvovirus B19), sequestration crisis, infections by encapsulated bacteria (functional asplenia)

1. Increasing HbF levels
2. Anti-inflammatory effect (inhibits WBC production)
3. Increases red cell size, lowering intracellular Hb concentration
4. Metabolizes to NO (vasodilator, inhibits platelet aggregation)

3. Thalassemia

β-Thalassemia

• Point mutations impair transcription, splicing, or translation of β-globin mRNA
• Excess α-globin chains form insoluble precipitates → damage red cell membranes → ineffective erythropoiesis (destruction of erythroid precursors in marrow) + haemolysis
• Both extravascular haemolysis and ineffective erythropoiesis contribute to anaemia

• Marked microcytosis, hypochromia, poikilocytosis, anisocytosis, nucleated red cells (normoblasts)
• Target cells (increased surface area-to-volume ratio)
• Striking erythroid hyperplasia filling intramedullary space → cortical bone thinning, impaired growth, skeletal deformities
• Extramedullary haematopoiesis → prominent splenomegaly, hepatomegaly, lymphadenopathy
• Growth retardation and cachexia
• Severe haemosiderosis from iron overload (transfusions + increased gut iron absorption due to suppressed hepcidin from expanded erythropoiesis)

• β-Thalassemia minor/trait: asymptomatic, normal life expectancy; mild microcytic hypochromic anaemia
• β-Thalassemia major: manifests postnatally as HbF diminishes; growth retardation from infancy; survival into 2nd-3rd decade with transfusions, but iron overload develops; chelation therapy (deferoxamine) + bone marrow transplant can be curative

α-Thalassemia

• Caused by deletion of α-globin genes (usually entire genes deleted)
• Severity depends on number of genes deleted (0-4):
  • 1 gene deleted: silent carrier state
  • 2 genes deleted: α-thalassemia trait - mild microcytic anaemia
  • 3 genes deleted: HbH disease - excess β-chains form β₄ tetramers (HbH); relatively stable but high O₂ affinity (poor O₂ delivery)
  • 4 genes deleted: Hydrops fetalis - lethal in utero; excess γ-chains form γ₄ (Hb Bart); near-zero O₂ delivery capacity

4. Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency

• Oxidant stress (from drugs, infections, foods) overwhelms deficient GSH
• Oxidants attack globin chains → oxidized haemoglobin denatures → precipitates as Heinz bodies (intracellular inclusions)
• Heinz bodies damage the red cell membrane → intravascular haemolysis
• Cells with less damage lose deformability; splenic macrophages "pluck out" Heinz bodies → bite cells (Fig. 10.6) → trapped and destroyed in spleen

• Antimalarials (primaquine), sulfonamides, nitrofurantoin, phenacetin, high-dose aspirin, vitamin K derivatives
• Fava beans (favism)
• Infections (most common trigger - phagocytes generate oxidants as host response)

• Haemolysis 2-3 days after drug exposure; variable severity
• Males uniformly affected (X-linked)
• Heterozygous females: two RBC populations due to lyonization; most unaffected unless "unfavorable lyonization" (large proportion of deficient cells)
• G6PD A- variant (Africa): only older red cells lysed (modest enzyme decrease); haemolysis self-limited as marrow replaces with new cells with adequate G6PD
• G6PD Mediterranean (Middle East): more marked deficiency; more severe haemolysis

5. Paroxysmal Nocturnal Haemoglobinuria (PNH)

• Classic (but uncommon) presentation: nocturnal haemolysis (sleep-related CO₂ retention → decreased pH → enhanced complement fixation)
• Most present with chronic anaemia and iron deficiency from chronic intravascular haemolysis
• Association with aplastic anaemia (may precede or follow PNH)
• Most feared complication: thrombosis in abdominal vessels (portal vein, hepatic vein) - related to excessive complement activity

• Does NOT affect early complement fixation → C3b continues to deposit → continuing extravascular haemolysis
• Blocks C5b-C9 → risk of Neisseria (meningococcal) infections → all patients must be vaccinated against N. meningococcus

6. Immunohemolytic Anaemia

Warm Antibody Type

• IgG (rarely IgA) antibodies active at 37°C

• 60% idiopathic; 25% in immunologic disorders (SLE) or drug-induced

• Mechanism: IgG-coated cells phagocytosed in spleen; also "nibbling" by macrophages removes membrane → spherocytes → rapid splenic destruction (same as hereditary spherocytosis)
• Drug mechanisms:
  • α-methyldopa: induces autoantibodies against Rh blood group antigens
  • Penicillin: binds covalently to red cell membrane proteins → neoantigens → antibody response
  • Some drugs form immune complexes that deposit on red cells → fix complement or act as opsonins

Cold Antibody Type

• Low-affinity IgM antibodies binding at temperatures <30°C (e.g., distal extremities in cold)
• Transient forms: Mycoplasma pneumonia, infectious mononucleosis (mild, clinically unimportant)
• Chronic forms: B-cell neoplasms or idiopathic
• Pathogenesis: IgM initiates complement fixation; later steps inefficient at <37°C → cells coated with C3b and C3d but not lysed. In warm areas, IgM released but C3b/C3d remain → phagocytosis by macrophages (mainly spleen and liver) → mostly extravascular haemolysis
• IgM pentavalency → crosslinks red cells → agglutination → sludging in capillaries → Raynaud phenomenon

7. Mechanical Haemolysis

1. Traumatic (macroangiopathic) haemolysis:
   • Dysfunctional cardiac valve prostheses - turbulence creates "blender effect"
   • Repeated physical pounding (marathon racing, karate, bongo drumming)
2. Microangiopathic haemolytic anaemia (MAHA):
   • Small vessels partially obstructed by fibrin strands, platelet thrombi, or other lesions → passing red cells are sheared into schistocytes (helmet cells, triangular cells)
   • Causes: DIC (most common - fibrin deposition), severe hypertension, thrombotic thrombocytopenic purpura (TTP), haemolytic-uraemic syndrome (HUS)

8. Malaria

• Haemolysis caused by Plasmodium parasites invading and destroying red cells
• P. falciparum is the most severe form; protective pressure explains why HbS, G6PD deficiency, and thalassemia mutations are prevalent in malaria-endemic areas
• Haemolysis is both intravascular (rupture of parasitized cells) and extravascular (phagocytosis of parasitized cells)

Summary Table

Megaloblastic Anaemia - Strictly According to Robbins Pathology

Primary source: Robbins & Kumar Basic Pathology (Robbins Pathology), 10th ed. (ISBN 9780323790185); supplemented by Robbins, Cotran & Kumar Pathologic Basis of Disease (ISBN 9780443264528)

Definition

Pathogenesis (Common to All Megaloblastic Anaemias)

• Thymidine deficiency causes abnormalities in rapidly dividing cells throughout the body, but the haematopoietic marrow is most severely affected
• Synthesis of RNA and cytoplasmic elements proceeds at a relatively normal rate and outpaces that of DNA
• This creates nuclear-cytoplasmic asynchrony in haematopoietic precursors

1. DNA synthesis is so defective in many red cell progenitors that a DNA damage response is triggered → apoptosis (ineffective haematopoiesis)
2. Other progenitors mature into red cells but do so after fewer cell divisions, further diminishing output
3. Granulocyte and platelet precursors are also affected (though less severely)
4. Most patients present with pancytopenia (anaemia + thrombocytopenia + granulocytopenia)

Morphology (Common Features)

Bone Marrow

• Markedly hypercellular with numerous megaloblastic erythroid progenitors
• Megaloblasts are larger than normal normoblasts and have delicate, finely reticulated nuclear chromatin (indicative of nuclear immaturity)
• As megaloblasts acquire haemoglobin, the nucleus retains its finely distributed chromatin and fails to undergo chromatin clumping typical of normoblasts - classic nuclear-cytoplasmic asynchrony
• Granulocytic precursors show asynchrony → giant metamyelocytes and giant band forms
• Megakaryocytes may be abnormally large with bizarre multilobed nuclei

Peripheral Blood

• Earliest change: Appearance of hypersegmented neutrophils (before onset of anaemia)
  • Normal neutrophils: 3-4 nuclear lobes
  • Megaloblastic anaemia: 5 or more lobes (hypersegmentation)
• Red cells: large oval-shaped macroovalocytes
• MCV often >110 fL (normal 78-98 fL)
• Though macroovalocytes appear hyperchromic, their haemoglobin concentration is actually normal (apparent hyperchromia due to larger cell size)
• Marked anisocytosis and poikilocytosis
• Reticulocyte count is LOW (despite marrow hyperplasia - due to ineffective erythropoiesis)
• Large, misshapen platelets may be seen
• Nucleated red cell progenitors may appear in circulation when anaemia is severe

Causes of Megaloblastic Anaemia (Robbins PBD Table 14.5)

Type 1: Folate (Folic Acid) Deficiency Anaemia

Aetiology

• Folate is present in nearly all foods but is destroyed by 10-15 minutes of cooking → stores are marginal in many healthy persons
• Highest risk groups: food insecurity, elderly, pregnant women, patients with chronic haemolytic anaemias (e.g., sickle cell disease)
• Absorption problems: Food folates are predominantly in polyglutamate form and must be split into monoglutamates for absorption; this conversion is inhibited by:
  • Acidic foods
  • Substances in beans and other legumes
  • Phenytoin (interferes with folate absorption)
  • Methotrexate (inhibits folate metabolism)
• Malabsorptive disorders affecting the upper third of the small intestine (e.g., coeliac disease, environmental enteropathy) impair folate uptake

Pathogenesis

Clinical Features

• Onset is insidious with nonspecific symptoms: weakness, easy fatigability
• May be complicated by coexistent deficiency of other vitamins (especially in alcohol use disorder)
• GI epithelium (rapidly turning over cells) also affected → sore tongue (glossitis)
• NO neurologic abnormalities (key distinction from B12 deficiency)

Type 2: Vitamin B12 (Cobalamin) Deficiency Anaemia

Normal Vitamin B12 Metabolism

• B12 is widely present in animal products (meat, fish, milk, eggs); daily requirement 2-3 μg; resistant to cooking
• Body handles B12 efficiently - stored in the liver with reserves for 5-20 years → clinical presentation typically follows years of unrecognised malabsorption

1. Pepsin in stomach frees B12 from food binding proteins
2. B12 binds to salivary protein haptocorrin
3. In duodenum, pancreatic proteases release B12 from haptocorrin
4. B12 associates with intrinsic factor (IF) secreted by parietal cells of fundic mucosa
5. IF-B12 complex travels to ileum → endocytosed by ileal enterocytes via cubilin receptor
6. Within ileal cells, B12 associates with transcobalamin II → secreted into plasma
7. Transcobalamin II delivers B12 to liver and other tissues

Biochemical Functions of Vitamin B12 (Robbins PBD)

Methylcobalamin (methyl-Cbl) serves as cofactor for methionine synthase in the conversion of homocysteine to methionine. In the process, methylcobalamin recovers a methyl group from N⁵-methyltetrahydrofolic acid (N⁵-methyl FH₄), which is then converted to tetrahydrofolic acid (FH₄).

• B12 deficiency → methionine synthase impaired → folate remains trapped as N⁵-methyl FH₄ → cannot be converted to FH₄
• Deprives thymidylate synthetase of its folate coenzyme → impaired DNA synthesis
• This is why folate administration reverses the anaemia of B12 deficiency (it bypasses the trap)

Adenosylcobalamin serves as cofactor for methylmalonyl-CoA mutase, which converts methylmalonyl-CoA to succinyl-CoA.

• B12 deficiency → methylmalonate and propionate accumulate in plasma and urine
• Elevated methylmalonic acid in urine = specific marker for B12 deficiency (vs folate)
• Abnormal fatty acids may be incorporated into neuronal lipids → myelin breakdown → subacute combined degeneration of the spinal cord
• The neurologic complications are not improved by folate (and may be worsened)

Pernicious Anaemia (Most Important Cause)

• Autoreactive T-cell response initiates gastric mucosal injury → triggers autoantibody formation
• Autoantibodies are diagnostically useful but are not the primary cause of gastric pathology
• Associated with other autoimmune disorders: autoimmune thyroiditis and adrenalitis (shared predisposition to autoimmunity)

Other Causes of Vitamin B12 Malabsorption

• Gastrectomy → loss of intrinsic factor-producing parietal cells
• Ileal resection → loss of IF-B12 complex-absorbing cells
• Crohn disease, Whipple disease, environmental enteropathy → disruption of distal ileum
• Achlorhydria/gastric atrophy (especially in older persons) → cannot release B12 from food
• Exocrine pancreatic insufficiency → cannot release B12 from haptocorrin
• Fish tapeworm (Diphyllobothrium latum) - competitive uptake
• Bacterial overgrowth in blind loops
• Nitrous oxide (recreational use) → oxidises and inactivates B12
• Increased demand: pregnancy, hyperthyroidism, disseminated cancer

Morphology of Pernicious Anaemia

• Diffuse chronic gastritis with fundic gland atrophy affecting both chief cells and parietal cells (parietal cells virtually absent)
• Glandular epithelium replaced by mucus-secreting goblet cells resembling large intestine → intestinalization (intestinal metaplasia)
• Affected cells and their nuclei may be twice normal size ("megaloblastic" change in mucosal epithelium - analogous to marrow changes)
• These gastric/metaplastic changes are due to autoimmunity and persist after parenteral B12 treatment (in contrast to the marrow and gut "megaloblastic" changes which are readily reversible)

• Central nervous system lesions found in ~75% of florid pernicious anaemia cases; can occur even without overt haematologic findings
• Demyelination of dorsal and lateral spinal tracts (posterolateral columns)
• Begins: symmetric numbness, tingling, burning in feet or hands
• Progresses to: unsteadiness of gait, loss of position sense (particularly in toes)
• Anaemia responds dramatically to parenteral B12, but neurologic manifestations often fail to resolve
• Mechanism: abnormal fatty acid incorporation into neuronal lipids from methylmalonyl-CoA accumulation → myelin breakdown

Diagnosis

• MCV >110 fL
• Hypersegmented neutrophils (≥5 lobes)
• Macroovalocytes
• Low reticulocyte count
• Bone marrow hypercellularity with megaloblasts and giant metamyelocytes
• Pancytopenia

Key Summary Points

"Although the anemia responds dramatically to parenteral vitamin B12, the neurologic manifestations often fail to resolve." - Robbins & Kumar Basic Pathology, p. 397