• Hemolytic anaemias are relatively uncommon compared to iron deficiency anaemia, but they carry significant morbidity
• Inherited forms (G6PD deficiency, thalassaemias, sickle cell disease) are among the most common genetic disorders worldwide — concentrated in malaria-endemic regions due to heterozygote advantage (balanced polymorphism)
• Autoimmune hemolytic anaemia (AIHA) has an estimated incidence of 1–3 per 100,000/year

• G6PD deficiency is the most clinically important inherited hemolytic anaemia in Hong Kong
  • Prevalence: ~4.5% of males in Hong Kong (X-linked, so predominantly affects males)
  • Universal neonatal screening programme in Hong Kong since 1984
  • Common variants in Southern Chinese: Canton, Kaiping, Chinese-5
• Thalassaemias are extremely common in Southeast Asia and Southern China
  • α-thalassaemia trait: ~3–5% carrier rate in Hong Kong
  • β-thalassaemia trait: ~1–2% carrier rate
  • HbH disease (--/- α) is encountered in clinical practice
• Sickle cell disease (HbS) is rare in Hong Kong (predominantly African/Mediterranean)
• Hereditary spherocytosis occurs but is less common than in Northern European populations
• Paroxysmal nocturnal haemoglobinuria (PNH): rare, but important acquired intrinsic RBC defect
• Autoimmune hemolytic anaemia: encountered in association with SLE, lymphoproliferative disorders

The adult haemoglobin A molecule consists of 2 alpha (α) globin chains and 2 beta (β) globin chains. Within each globin chain, there is one haem molecule. Within each haem molecule, there is one iron atom in the ferrous (Fe²⁺) state. ^[3]

RBC Components:
├── Membrane (lipid bilayer + skeletal proteins)
│   ├── Spectrin, ankyrin, band 3, protein 4.1, protein 4.2
│   └── GPI-anchored proteins (CD55, CD59) — relevant to PNH
├── Metabolism (enzyme systems)
│   ├── Glycolytic pathway (Embden-Meyerhof) → generates ATP
│   ├── Hexose monophosphate shunt (pentose phosphate pathway) → generates NADPH
│   │   └── G6PD is the rate-limiting enzyme here
│   └── Methemoglobin reductase pathway → keeps iron as Fe²⁺
└── Haemoglobin
    ├── HbA (α₂β₂) — 95–97% of adult Hb
    ├── HbA₂ (α₂δ₂) — 1.5–3.5%
    └── HbF (α₂γ₂) — <1% in adults

• RBCs are anucleate — they cannot synthesize new proteins or repair themselves
• They rely entirely on their existing enzyme stock and membrane integrity for their 120-day lifespan
• Any defect in the membrane skeleton, metabolic enzymes, or haemoglobin structure → premature destruction

• Extravascular haemolysis (majority): occurs in the reticuloendothelial system (RES), primarily the spleen (and liver)
  • Macrophages in the splenic red pulp phagocytose abnormal/damaged/antibody-coated RBCs
  • This is why splenomegaly is a hallmark of chronic extravascular haemolysis
• Intravascular haemolysis: occurs within the blood vessels themselves
  • Free haemoglobin released directly into plasma → binds haptoglobin → when haptoglobin saturated → haemoglobinuria
  • Causes: complement-mediated lysis (PNH, ABO mismatch), mechanical shear (TTP, prosthetic valves), severe infections (C. perfringens)

Two main ways to classify hemolytic anaemias: (1) Hereditary/Inherited vs. Acquired, and (2) Intrinsic vs. Extrinsic. ^[1][2]

There is also an important clinical classification: Extravascular vs. Intravascular haemolysis — this guides your investigation strategy.

If inherited, think in terms of the components of the RBC, and how they can go wrong — either the membrane, the metabolism, or the haemoglobin. ^[1]

If acquired, most cases will be due to something outside that is attacking your red cells → only exception is the intrinsic red cell defect seen in PNH. ^[1][2]

This is the most important pathophysiological distinction because it determines the clinical presentation and lab findings.

Where: Reticuloendothelial system — primarily the spleen (red pulp macrophages)

Why the spleen? The splenic sinusoids have narrow inter-endothelial slits (1–3 μm). Normal RBCs (7–8 μm) must deform to squeeze through. Abnormal RBCs (spherocytes, sickle cells, antibody-coated cells, rigid cells from enzyme deficiency) get trapped and phagocytosed.

Consequences:

• Splenomegaly — the spleen is working overtime
• Unconjugated (indirect) hyperbilirubinaemia — haem is broken down to unconjugated bilirubin within macrophages, overwhelming the liver's conjugation capacity
• Jaundice — but importantly, jaundice WITHOUT tea-coloured urine ^[1]. Why? Because unconjugated bilirubin is water-insoluble and bound to albumin → it cannot be filtered by the kidney. Tea-coloured urine = conjugated (direct) bilirubin in urine = suggests obstructive/hepatocellular jaundice, NOT haemolysis
• Increased urobilinogen — more bilirubin reaching the gut → more urobilinogen reabsorbed enterohepatic circulation → more excreted in urine (detected by dipstick)
• Pigment gallstones — Increase in the amount and concentration of unconjugated bilirubin in hepatic bile → promotion of stone formation within the gallbladder ^[1]. These are black pigment stones (calcium bilirubinate)
• Reticulocytosis — marrow compensatory response
• Raised LDH — released from destroyed RBCs (LDH-1 and LDH-2 isoforms)

Where: Within the blood vessels themselves

When: Complement-mediated lysis (PNH, ABO-incompatible transfusion, cold AIHA), mechanical shear (MAHA, prosthetic valves), severe infections (C. perfringens, malaria with massive parasitaemia)

Consequences:

• Free haemoglobin in plasma (plasma turns pink/red — "haemoglobinaemia")
• Haptoglobin depleted — haptoglobin is an α₂-globulin that binds free Hb in plasma; the Hb-haptoglobin complex is cleared by hepatocytes. In intravascular haemolysis, haptoglobin is rapidly consumed → serum haptoglobin drops to undetectable levels. This is one of the most sensitive markers.
• Haemoglobinuria — once haptoglobin is saturated, free Hb passes the glomerular filter → coca-cola coloured urine ^[1]
• Haemosiderinuria — renal tubular cells absorb filtered Hb, break it down, and store iron as haemosiderin. When these cells are shed into the urine → positive Prussian blue stain on urine sediment. This is a marker of chronic intravascular haemolysis (appears days after the acute event)
• Methaemalbuminaemia — haem released from oxidized Hb binds albumin → methaemalbumin (detected by Schumm's test — rarely done now)
• Renal injury — free Hb is directly nephrotoxic → can cause acute tubular necrosis (ATN). This is why massive intravascular haemolysis (e.g., ABO-incompatible transfusion) can lead to AKI ^[5]

The peripheral blood film is the single most informative investigation in haemolytic anaemia. Always look at it yourself.

• Erythroid hyperplasia with ↓M:E ratio
• In aplastic crisis (parvovirus B19): sudden cessation of erythropoiesis → giant pronormoblasts with nuclear inclusions, absent erythroblasts
• In megaloblastic crisis: folate deficiency (chronic haemolysis uses up folate stores) → megaloblastic changes

• Work hypertrophy → congested red pulp with expanded cords of Billroth
• In chronic haemolysis: haemosiderin-laden macrophages

• Pigment (bilirubin) gallstones — black stones composed of calcium bilirubinate
• Can develop as early as childhood in hereditary spherocytosis or SCD

• In intravascular haemolysis: haemosiderin deposition in renal tubular epithelial cells, ATN in severe cases
• In SCD: papillary necrosis (medullary ischaemia from sickling in the vasa recta — the medullary environment is hypoxic, acidotic, and hypertonic, all promoting sickling)

The Direct Antiglobulin Test (DAT / Direct Coombs Test) is the single most important first-line investigation to separate immune from non-immune haemolysis ^[1][14]. It detects antibodies (IgG) and/or complement (C3d) bound to the surface of the patient's RBCs.

If the DAT is negative but you see spherocytes on the film, the differential is much narrower:

Key learning point: Both hereditary spherocytosis and warm AIHA produce spherocytes on the blood film. The DAT is what separates them. HS = DAT negative; warm AIHA = DAT positive ^[1][14][15].

RBC fragmentation on blood film = microangiopathic haemolysis ^[1][14]. This is a medical emergency in many cases.

The key distinction between G6PD and PK deficiency: G6PD causes episodic haemolysis triggered by oxidant stress, whereas PK deficiency causes chronic haemolysis because ATP depletion is constant, not triggered.

The patient presents with haemoglobinuria. Top 2 differential diagnoses: ^[11]

1. Paroxysmal nocturnal haemoglobinuria (PNH)
2. Paroxysmal cold haemoglobinuria (PCH) — very rare subtype of cold AIHA ^[11]

Other causes of intravascular haemolysis with haemoglobinuria:

• ABO-incompatible transfusion reaction (coca-cola urine; abrupt onset of "sense of impending doom", flushing, fever, rigors, loin/back pain, vomiting, shock) ^[13]
• March haemoglobinuria (mechanical — prolonged marching/running)
• Severe G6PD crisis
• Severe falciparum malaria ("blackwater fever")
• C. perfringens sepsis

PNH — The Classic Acquired Intrinsic Cause

• PIGA gene mutation → GPI-anchor deficiency → loss of CD55/CD59 → complement-mediated intravascular haemolysis ^[1][2]
• Diagnosis: Flow cytometry on peripheral blood — deficiency of GPI-anchored proteins (CD55, CD59) on RBCs and granulocytes; MOST useful and accepted method ^[18]
• Must screen for PNH in all cases of aplastic anaemia and unexplained Coombs-negative haemolysis ^[18]
• Triad: haemolysis + thrombosis + cytopenias (marrow failure)

Bone marrow aspiration and trephine biopsy is NOT routinely needed to confirm haemolysis or diagnose its cause. It is reserved for specific situations:

Folate supplementation is generally recommended for any form of haemolysis — replenish the raw material ^[11]

• Why: Chronic haemolysis → ↑RBC turnover → ↑folate demand for DNA synthesis during compensatory erythropoiesis. If folate stores are depleted, the marrow cannot mount an adequate reticulocyte response → megaloblastic crisis superimposed on haemolysis
• Dose: Folic acid 1–2 mg/day for moderate/severe haemolysis; 5 mg/day if pregnant ^[3][16]
• Exception: Once a patient is on regular transfusion (e.g., thalassaemia major on hypertransfusion), folate supplementation is NOT required — because the marrow is suppressed and not actively producing RBCs ^[19]

Transfusion if necessary ^[11] — indicated for:

• Symptomatic anaemia (dyspnoea, chest pain, haemodynamic compromise)
• Critically low Hb (especially acute drops)
• Aplastic crisis (parvovirus B19) where reticulocytes are absent

Transfusion considerations in haemolytic anaemia:

• In warm AIHA, crossmatching is extremely difficult because the autoantibody reacts with virtually all donor RBCs → the blood bank must find the "least incompatible" unit. Transfusion should NOT be withheld in life-threatening anaemia just because of crossmatch difficulty — transfuse and accept some additional haemolysis
• In thalassaemia major, use pre-filtered products that are leukocyte-depleted and matched for at least D, C, c, E, e and Kell antigens to minimize WBC-induced febrile reaction ^[19]
• In cold AIHA, use pre-warmed IV fluids and a blood warmer during transfusion ^[15]
• Pre-medications for transfusion: IV/PO chlorpheniramine 30 mins before transfusion; IV furosemide at the start of transfusion ^[19]

• AKI from free Hb nephrotoxicity (intravascular haemolysis) → monitor RFT, ensure adequate hydration, aggressive fluid resuscitation ^[13]
• Pigment gallstones → USS abdomen; cholecystectomy if symptomatic
• Aplastic crisis → if sudden severe drop in Hb with absent reticulocytes, test for parvovirus B19
• Iron overload (transfusion-dependent patients) → monitor ferritin, start chelation when indicated

Hereditary Spherocytosis (HS)

No specific treatment can target the underlying membrane defect ^[3][16]

Why splenectomy works in HS: The spleen is where spherocytes are trapped and destroyed. Removing the spleen eliminates the primary site of extravascular haemolysis → Hb normalises (though spherocytes persist on the blood film). The cells are still abnormal — they just have nowhere to be destroyed.

Penicillamine causes worsened neurological symptoms in the first few days — patient is used to copper in brain; once penicillamine is given, they lose copper, the body is not used to it, may exacerbate neurological symptoms ^[7]

For the acute haemolytic crisis in Wilson's: supportive (transfusion, dialysis if AKI), urgent liver transplant assessment.

Mechanism: increase in the amount and concentration of unconjugated bilirubin in hepatic bile → promotion of stone formation within the gallbladder ^[11]

• Type: Black pigment stones (calcium bilirubinate) — distinct from the cholesterol stones of metabolic syndrome
• Incidence: Very common in chronic haemolysis — 50% of adults with hereditary spherocytosis develop pigmented gallstones ^[16]
• Clinical consequence: Biliary colic, acute cholecystitis, choledocholithiasis with obstructive jaundice (now conjugated bilirubin → tea-coloured urine, which can confuse the clinical picture in someone with pre-existing haemolytic jaundice), ascending cholangitis, gallstone pancreatitis
• Management: Cholecystectomy (often performed at the same time as splenectomy in HS)
• Why does this happen from first principles? Chronic haemolysis → ↑haem breakdown → ↑unconjugated bilirubin produced → liver conjugates more bilirubin than normal → higher concentration of conjugated bilirubin excreted in bile → in the gallbladder, bacterial β-glucuronidase deconjugates some of it → the insoluble unconjugated bilirubin precipitates with calcium → black pigment stones

• Pre-hepatic (unconjugated) hyperbilirubinaemia — typically mild, fluctuating, scleral icterus
• Jaundice WITHOUT tea-coloured urine — unconjugated bilirubin is water-insoluble → not filtered by kidneys ^[11]
• Usually well-tolerated in adults but in neonates → unconjugated bilirubin crosses the immature blood-brain barrier → kernicterus (bilirubin encephalopathy)
  • Relevant in neonatal G6PD crisis and HDN
  • Complications of haemolysis in neonates: neonatal jaundice (NNJ) ^[3][16]

Predominantly a complication of intravascular haemolysis (but can occur in severe extravascular haemolysis too):

Free haemoglobin from haemolysed RBCs can cause AKI through multiple mechanisms ^[13]:

• Prevention: Aggressive IV fluid resuscitation, maintain urine output, alkaline diuresis (to prevent Hb precipitation in acidic tubular lumen)
• Classic scenarios: ABO-incompatible transfusion, severe G6PD crisis, PNH crisis, massive falciparum malaria

• Chronic intravascular haemolysis → chronic depletion of NO by free Hb → endothelial dysfunction → vasoconstriction and remodelling of pulmonary vasculature → pulmonary hypertension
• Particularly important in: sickle cell disease, thalassaemia intermedia, PNH
• Screen with echocardiography (tricuspid regurgitant jet velocity)

• PNH: Thrombosis is the leading cause of death — venous thrombosis at unusual sites (hepatic veins → Budd-Chiari syndrome, cerebral venous sinuses, mesenteric veins)
  • Mechanism: free Hb scavenges NO → platelet activation; complement activation on platelets; release of procoagulant microvesicles from lysed RBCs
• Sickle cell disease: Vaso-occlusion is the hallmark complication (see below)
• Post-splenectomy thrombocytosis → risk of portal vein thrombosis and other venous thromboembolism ^[6][16]
  • Splenectomy risks: ↑thromboembolism, ↑life-threatening infection, ↑pulmonary hypertension ^[16]

• Occurs in severe acute intravascular haemolysis (ABO-incompatible transfusion, C. perfringens sepsis)
• Mechanism: activated complement system → platelet activation + cytokine release (TNF, IL-6, IL-8) → activation of coagulation system → DIC ^[13]
• DIC causes consumption of clotting factors and platelets → paradoxical bleeding
• DIC can cause further MAHA (fibrin strands in vessels shear more RBCs) → vicious cycle

Iron complications arise from two sources:

Consequences of iron overload:

Monitoring of iron overload: serum ferritin useful as screening but quantitative liver iron and cardiac iron measurement by MRI is the standard indicator of total-body iron stores ^[16]

Long-term complications of transfusion include haemosiderosis ^[13]

Transient aplastic crisis in patients with underlying haemolytic anaemia — following primary parvovirus B19 infection ^[11]

• Mechanism: Parvovirus B19 has tropism for erythroid progenitor cells (binds to globoside / P antigen on erythroblasts) → directly lyses erythroid precursors → sudden cessation of red cell production
• Normal individuals: Barely notice this because their RBCs survive 120 days — a transient 7–10 day halt in production causes minimal Hb drop
• Patients with chronic haemolysis: Already depend on compensatory reticulocytosis to maintain Hb. When production stops, Hb plummets catastrophically over days (because destruction continues unabated while production has ceased)
• Lab hallmark: Severe anaemia with absent reticulocytes (reticulocyte count near zero) — this is the clue
• Duration: Self-limiting over 7–14 days (once viral clearance occurs, marrow recovers)
• Management: Transfusion support during the nadir; IVIG for immunocompromised patients who cannot clear the virus
• Complications of parvovirus B19 infection ^[11]:
  • Transient aplastic crisis in patients with underlying haemolytic anaemia (e.g., hereditary spherocytosis)
  • Chronic hypoplastic anaemia
  • Pure red cell aplasia in immunocompromised patients
  • Hydrops fetalis in pregnant women

• Megaloblastic crisis due to folate deficiency, usually occurring during pregnancy when there is increased folate need ^[3][16]
• Mechanism: Chronic haemolysis → ↑RBC turnover → ↑folate consumption for DNA synthesis in erythroid precursors → folate stores depleted → megaloblastic change in the marrow → ineffective erythropoiesis
• Risk factors: Pregnancy (↑folate demand), poor dietary intake, alcoholism
• Prevention: Folic acid supplementation 1–2 mg/day for chronic haemolysis; 5 mg/day if pregnant ^[3][16]
• Distinct from aplastic crisis: in megaloblastic crisis, the marrow is present but dysfunctional (megaloblastic precursors); in aplastic crisis, the erythroid precursors are absent

• Haemolytic crisis due to increased severity of haemolysis, uncommon, usually associated with infections ^[3][16]
• An acute exacerbation of the baseline haemolysis → sudden ↓Hb with ↑reticulocytes (unlike aplastic crisis where reticulocytes are absent)
• Triggers: intercurrent infections, drugs (in G6PD), oxidant stress
• Management: supportive, transfusion if needed, treat the trigger

• Chronic compensatory erythroid hyperplasia → marrow cavity expands → cortical bone thinning
• Relevant in: thalassaemia major (if undertransfused), sickle cell disease
• Skeletal complications:
  • Frontal bossing, maxillary hyperplasia ("chipmunk facies") — expansion of the diploic space in the skull
  • "Hair-on-end" appearance on skull X-ray — perpendicular trabecular striations from marrow expansion
  • Pathological fractures — cortical thinning weakens bones
  • Extramedullary haematopoiesis — marrow function "spills over" into spleen, liver, paravertebral masses → hepatosplenomegaly, cord compression

Patients with chronic haemolytic anaemias (especially thalassaemia major, SCD, warm AIHA) require repeated transfusions, exposing them to:

Long-term steroid use (warm AIHA), rituximab, and other immunosuppressants carry their own complications:

• Steroids: Cushingoid features, osteoporosis, diabetes, adrenal suppression, infection susceptibility, avascular necrosis, cataracts
• Rituximab: Hepatitis B reactivation (must screen HBsAg/anti-HBc before starting), progressive multifocal leukoencephalopathy (very rare), late-onset neutropenia
• Splenectomy complications: As detailed above

• Thrombosis (leading cause of death): hepatic vein (Budd-Chiari), cerebral venous sinuses, mesenteric veins, dermal veins
• Bone marrow failure (overlap with aplastic anaemia): PNH clones arise from the aplastic marrow; ~15–20% of aplastic anaemia patients develop PNH
• Chronic kidney disease from recurrent AKI episodes and haemosiderin deposition in renal tubules
• Smooth muscle dystonia (dysphagia, abdominal pain, erectile dysfunction) — due to NO depletion by free Hb

An underappreciated but clinically important complication of haemolytic anaemia:

HbA1c is inaccurate in haemolytic anaemia ^[24]:

• HbA1c measures glycated haemoglobin accumulated over the ~120-day RBC lifespan
• In haemolytic anaemia, RBC lifespan is shortened → less time for glycation → HbA1c is falsely DECREASED
• Conversely, HbA1c is falsely increased in: iron deficiency anaemia, post-splenectomy, HbH disease (older cells with more glycation are preferentially retained) ^[24]
• Clinical implication: Do not rely on HbA1c for diabetes screening or monitoring in patients with haemolytic anaemia — use fructosamine or continuous glucose monitoring instead