Haemolytic anaemias are those anaemias which result from an increase in the rate of destruction of circulating red cells, with a compensatory increase in production from bone marrow. ^[1]

Why this definition matters: In uncomplicated haemolysis the marrow is capable of increasing output, so reticulocytosis is expected. It is not guaranteed: reticulocytosis may be blunted by marrow failure, haematinic deficiency, renal failure, infection (especially parvovirus B19), or very early sampling. That caveat prevents the unsafe shortcut “no reticulocytosis = no haemolysis.” Aplastic anaemia differs because the production apparatus itself has failed.

Two classification axes: (1) Hereditary vs Acquired; (2) Intrinsic vs Extrinsic ^[1]

Why two axes? Because "intrinsic" (defect is within the RBC itself) and "extrinsic" (something outside the RBC damages it) don't perfectly map onto inherited vs acquired. The important exception is PNH — it is an acquired intrinsic defect (a somatic mutation in the PIGA gene).

History: Family history, Ethnicity, Infection, Drugs, Transfusion history, Co-existing illnesses (autoimmune diseases, lymphoproliferative neoplasm), History of gallstones ^[1]

Examination: Pallor, Jaundice (no tea-coloured urine as in obstructive jaundice), Haemoglobinuria in intravascular haemolysis (e.g. PNH), Splenomegaly ^[1]

Why each matters — explained from first principles:

Anaemia (mildly macrocytic usually) with reticulocytosis ^[1]

Increased unconjugated bilirubin, LDH ^[1]

Reduced serum haptoglobin ^[1]

Increased methaemalbumin ^[1]

Blood film: Polychromasia, Spherocytes (HS, immune HA), RBC fragmentation (MAHA), RBC agglutination (cold agglutinin disease) ^[1]

Direct antiglobulin test (DAT/Coombs') — positive in immune haemolytic anaemia ^[1]

Why each lab finding occurs:

Due to autoimmune antibodies against self erythrocytes. Warm or cold auto-antibodies depending on the temperature at which the antibody binds better with erythrocytes (37°C vs 4°C). ^[1]

Blood count — increased reticulocyte. Blood film — microspherocytes. Increased bilirubin (unconjugated), LDH. Decreased haptoglobin, increased methaemalbumin. Specific test — Direct Antiglobulin Test (Direct Coombs' test). ^[1]

Why microspherocytes in AIHA? Antibody-coated RBCs pass through the spleen → macrophages nibble away at the membrane (partial phagocytosis) → RBC loses surface area but retains volume → spherical shape. These are microspherocytes because the membrane loss is progressive.

Reading the direct antiglobulin test diagram

The slide depicts the test as a sequence rather than simply a “positive/negative” result:

1. The patient's RBCs are already coated in vivo with IgG and/or complement if immune haemolysis is present.
2. The laboratory washes the RBCs to remove unbound plasma immunoglobulin.
3. Antihuman globulin (Coombs reagent) is added.
4. The reagent bridges adjacent coated RBCs; visible agglutination is a positive DAT.

This is the direct test because it detects material already attached to the patient's RBCs. The indirect antiglobulin test instead detects free antibodies in serum after incubation with reagent RBCs; it is used in antibody screening and compatibility testing, not as the primary test for AIHA.

1. Transfusion if necessary ^[1] 2. Folate supplement ^[1] 3. Decrease further haemolysis: ^[1]

• Steroid or other immunosuppressants in autoimmune haemolytic anaemia
• Avoidance of certain drugs in drug-induced haemolytic anaemia
• Keep warm in cold haemagglutinin disease
• Splenectomy 4. Treat the underlying cause (e.g. AIHA due to underlying lymphoproliferative disorder) if necessary ^[1]

The slide combines warm and cold disease into one historical treatment list. Current management separates them because the mechanisms and effective therapies differ: ^[16][17][18]

Folate is most relevant when ongoing brisk haemolysis, dietary insufficiency, or deficiency creates increased need; it is not a substitute for disease-directed treatment.

Pancytopenia resulting from bone marrow hypoplasia or aplasia ^[1]

Congenital vs acquired. Acquired: idiopathic vs secondary. ^[1]

Why is it called "aplastic"? The bone marrow becomes hypocellular ("aplastic" = without growth). Unlike haemolytic anaemia where the marrow is working overtime, here the marrow itself fails → all three lineages drop → pancytopenia (low RBCs, WBCs, and platelets).

Isolated aplasia of the erythroid series. Congenital: Diamond-Blackfan syndrome. Acquired: lymphoproliferative disease, thymoma, parvovirus B19. ^[1]

Why parvovirus B19? Parvovirus B19 has tropism for erythroid progenitor cells (it enters via the P antigen/globoside receptor on erythroid precursors). In a healthy person, this causes a transient drop in RBC production that goes unnoticed. But in patients with underlying chronic haemolysis (e.g. sickle cell, HS), the temporary halt in erythropoiesis precipitates a dramatic aplastic crisis because they depend on high marrow output to compensate for ongoing haemolysis.

Idiopathic — 70-80% ^[1]

Congenital: Fanconi anaemia, Shwachman-Diamond syndrome, Dyskeratosis congenita ^[1]

Drugs: Gold, Alcohol, Diclofenac acid, Indomethacin, Chloramphenicol, anti-convulsants ^[1]

Infection: non A,B,C hepatitis (sero-negative hepatitis) ^[1]

Environmental exposure: benzene, pesticide ^[1]

Expanded drug list from senior notes: ^[2][3]

• Cytotoxic drugs (anticipated, nadir day 7-10)
• Antibiotics: chloramphenicol, sulphonamide
• DMARDs: penicillamine, gold
• NSAIDs: phenylbutazone, indomethacin, diclofenac
• Thionamides: carbimazole, propylthiouracil
• Anticonvulsants: carbamazepine, phenytoin
• Alcohol
• Toxins: benzene, toluene (glue-sniffing)

Immune (T-cell) mediated suppression of marrow stem cells ^[1]

Why this matters for treatment: If the disease is driven by autoreactive T-cells attacking haematopoietic stem cells, then immunosuppressive therapy (ATG + cyclosporine) makes mechanistic sense — you're suppressing the T-cells that are destroying the marrow. This also explains why some patients respond to cyclosporine alone.

Symptoms of pancytopenia (anaemia, neutropenia, thrombocytopenia) ^[1]

NO lymphadenopathy. NO hepatosplenomegaly. ^[1]

In young patients, watch out for the presence of congenital physical abnormalities (inherited bone marrow failure syndromes) ^[1]

Why is organomegaly usually absent? Aplastic anaemia does not create a proliferating infiltrative cell population. Therefore, substantial lymphadenopathy or hepatosplenomegaly should prompt a search for leukaemia/lymphoma, infection, another marrow disorder, or an alternative diagnosis rather than being treated as an absolute exclusion rule.

Symptoms by lineage:

Blood count: pancytopenia; macrocytic anaemia; low reticulocyte count ^[1]

Blood film ^[1]

Autoimmune markers ^[1]

Vit B12 and folate levels ^[1]

Bone marrow: trephine biopsy is required for the assessment of cellularity ^[1]

Specialized tests: ^[1]

• Flow cytometry for CD55 and CD59 deficient RBC (to rule out PNH)
• Chromosome breakage with diepoxybutane (to screen for Fanconi anaemia)

Why each investigation:

Reading the marrow histology slide

The side-by-side trephine images are a cellularity comparison. The aplastic marrow is dominated by large clear adipocyte spaces with only sparse residual haematopoietic cells, whereas normal marrow contains abundant dark-staining haematopoietic tissue between fat spaces and bony trabeculae. The visual lesson is why a trephine core, not an aspirate alone, is needed: cellularity and patchiness are architectural findings. Hypocellularity is not specific, so hypoplastic MDS, inherited marrow-failure syndromes, drugs/toxins, infection, and sampling error must be excluded. ^[19]

Severe aplastic anaemia (SAA): ^[1]

• BM cellularity < 25% (or 25–50% with < 30% residual haematopoietic cells)
• 2 out of 3:
  • Neutrophils < 0.5 × 10⁹/L
  • Platelets < 20 × 10⁹/L
  • Reticulocytes < 20 × 10⁹/L

Very severe aplastic anaemia: As for SAA but neutrophils < 0.2 × 10⁹/L ^[1]

The slide uses the traditional manual absolute-reticulocyte cut-off of < 20 × 10⁹/L. Current BSH criteria use < 60 × 10⁹/L when the reticulocyte count is automated; always state the method with the threshold. ^[19]

Why these numbers matter: They determine treatment urgency. Very severe AA has the worst prognosis and requires the most aggressive therapy. The neutrophil count is the strongest prognostic factor — lower neutrophils = higher infection risk = higher mortality.

First line — allogeneic haematopoietic stem cell transplantation (for young patients with matched sibling donors) ^[1]

Anti-thymocyte globulin (ATG) + cyclosporine ^[1]

Cyclosporine A ^[1]

Supportive (blood products, iron chelation) ^[1]

Eltrombopag (high dose) + ATG and cyclosporine ^[1]

Current treatment framing: The slide's age cut-off is a useful memory aid but not a stand-alone rule. Current BSH guidance makes the decision from severity, age/fitness, comorbidity, donor availability, and transplant risk. For newly diagnosed severe/very severe acquired AA, standard first-line options are: ^[19]

1. Matched-sibling allogeneic HSCT for suitable patients; it offers definitive replacement of the failed marrow.
2. Horse ATG + ciclosporin + eltrombopag when transplant is not the preferred initial option. Eltrombopag supports multilineage haematopoiesis; ciclosporin alone is not equivalent therapy for newly diagnosed SAA.
3. Specialist transplant pathways may use alternative donors in selected patients; the lecture's simple “under 40 = sibling transplant, otherwise IST” rule should not replace individual assessment.
4. Supportive care throughout: irradiated/leucocyte-reduced blood products as locally indicated, prompt antimicrobial management/prophylaxis, and avoidance of unnecessary transfusion before potential transplant. Iron chelation is considered for established clinically significant transfusional iron overload, not automatically with the first transfusions.

Four pathophysiological categories: ^[1]

1. Inherited bone marrow failure syndromes → pancytopenia
2. Disorders of red cell membrane
3. Disorders of haemoglobin (haemoglobinopathy and thalassaemia)
4. Disorders of RBC metabolism

Think of the RBC as a bag of haemoglobin wrapped in a membrane powered by metabolism. Any component can be defective:

• Membrane defects → cells are abnormally shaped → trapped and destroyed in spleen
• Haemoglobin defects → abnormal or reduced globin chains → haemolysis or ineffective erythropoiesis
• Enzyme defects → inability to generate energy or handle oxidative stress → haemolysis

Family history – pattern of inheritance, Age of onset, Drug history, Symptoms of haemolysis, Transfusion requirement, Complications (including those due to long-term red cell transfusion) ^[1][2]

Pallor, Jaundice, Splenomegaly, Skin complexion, Skeletal deformities ^[1][2]

Why skeletal deformities? In severe congenital anaemias (e.g. thalassaemia major, Fanconi anaemia), marrow expansion to compensate for ineffective erythropoiesis causes bony changes — frontal bossing, maxillary hyperplasia ("chipmunk face"), thinning of cortical bone. In Fanconi, the deformities are developmental (thumb/radius anomalies).

^[1]

Understanding vertical vs horizontal interactions:

• Vertical interactions (ankyrin, band 3, protein 4.2) anchor the cytoskeleton to the lipid bilayer. Defects → membrane loss (vesiculation) → spherocytosis
• Horizontal interactions (spectrin-spectrin, spectrin-actin via protein 4.1) maintain the structural integrity of the cytoskeleton lattice. Defects → membrane fragmentation → elliptocytosis or poikilocytosis

In the slide diagram, the lipid bilayer is the roof, band 3/glycophorins span it, and the spectrin–actin lattice runs beneath it. Read vertical as “bilayer-to-skeleton anchoring” and horizontal as “side-to-side integrity within the lattice.” That spatial distinction explains why vertical failure sheds membrane and rounds the cell, while horizontal failure destabilises shape.

Presence of spherocytes, Haemolytic anaemia — mild to severe, commonly autosomal dominant ^[1]

60% result from a defect in the ankyrin-spectrin complex or vertical interaction ^[1]

Clinical features: Jaundice (indirect hyperbilirubinaemia), Splenomegaly, Gallstones, Aplastic crisis — primary parvovirus B19 infection ^[1]

Extravascular haemolysis, Blood film — spherocytes, MCHC is often raised ^[1]

Osmotic fragility test, Eosin-5-maleimide (EMA) binding (decreased binding in HS) ^[1]

Pathophysiology explained step by step:

1. Mutation in vertical interaction proteins (ankyrin, spectrin, band 3) → weakened anchoring of lipid bilayer to cytoskeleton
2. Membrane undergoes microvesiculation — small blebs of lipid bilayer pinch off
3. RBC loses surface area but retains intracellular volume → surface-area-to-volume ratio decreases → cell becomes spherical
4. Spherocytes are less deformable → cannot squeeze through the narrow splenic sinusoids (3μm slits)
5. Trapped spherocytes → phagocytosed by splenic macrophages → extravascular haemolysis

Why MCHC is raised: The cell has lost membrane (volume stays same or slightly less, Hb content stays same) → same Hb concentrated in a smaller package → MCHC goes up. This is one of the few conditions where MCHC > 36 g/dL. ^[4]

Why EMA binding is decreased: EMA binds to band 3 protein on the RBC surface. In HS, there is reduced band 3 (or reduced membrane in general) → less EMA binding → detectable by flow cytometry. This is now the preferred diagnostic test over osmotic fragility. ^[1][4]

Why osmotic fragility is increased: Spherocytes have reduced surface-area-to-volume ratio → less room to swell before bursting. When placed in hypotonic solution, they lyse earlier than normal biconcave RBCs.

How to read the osmotic-fragility graph: Moving left means progressively more hypotonic saline. Normal discocytes can take up more water before bursting; HS cells begin lysing at a higher NaCl concentration, so the HS curve is shifted to the right. A right shift therefore means increased osmotic fragility. Osmotic fragility is supportive but less specific than a validated EMA-binding assay and can be normal in mild HS unless incubated. ^[20]

Treatment: Folic acid supplement, Compensated haemolysis — no specific treatment, Cholecystectomy if symptomatic gallstones, Splenectomy in severe haemolysis ^[1]

Why splenectomy works in HS: The spleen is the main site of destruction. Spherocytes persist after splenectomy (the membrane defect remains), but RBC survival improves. Splenectomy is reserved for appropriately selected severe or symptomatic disease, not mild compensated HS, because infection and thrombotic risks are lifelong. Folate supplementation is generally aimed at moderate/severe haemolysis or documented increased need, not every mild case. Before elective splenectomy, follow current local pneumococcal, MenACWY, MenB, and Hib vaccination guidance (ideally completed at least 2 weeks beforehand), provide fever/OPSI education, and use antibiotic prophylaxis according to age and local protocol. ^[20][21]

G6PD deficiency, Pyruvate kinase deficiency, Deficiency of other glycolytic pathway enzymes → Congenital non-spherocytic haemolytic anaemia ^[1]

The lecture slide shows the glycolytic pathway and pentose phosphate shunt diagram. ^[1]

Why these pathways matter for RBCs: Mature RBCs have no mitochondria → they rely entirely on anaerobic glycolysis (Embden-Meyerhof pathway) for ATP and on the pentose phosphate shunt (hexose monophosphate shunt) for NADPH to combat oxidative stress.

• Glycolysis → ATP (maintains cation pumps, membrane integrity) + 2,3-DPG (regulates O₂ affinity)
• Pentose phosphate shunt → NADPH (reduces glutathione via glutathione reductase → reduced glutathione protects Hb and membrane from oxidative damage)

Reading the metabolic pathway diagram

Follow the glucose-6-phosphate branch point:

• Straight down glycolysis, pyruvate kinase generates ATP; deficiency therefore produces an energy-starved, poorly deformable RBC and chronic non-spherocytic haemolysis.
• Across the pentose-phosphate shunt, G6PD generates NADPH. NADPH allows glutathione reductase to regenerate GSH, and glutathione peroxidase uses GSH to convert hydrogen peroxide to water.
• The diagram's NADH–methaemoglobin reductase loop keeps iron in functional haemoglobin reduced, while the Rapoport–Luebering side shunt generates 2,3-DPG to tune oxygen affinity.

Thus, pyruvate kinase deficiency is chiefly an ATP problem, while G6PD deficiency is chiefly an oxidant-defence problem.

X-linked. The slide quotes historical Hong Kong prevalence estimates of 4.5% in males and 0.5% in females ^[1]

The slide's “G6PD B” means a historical biochemical wild-type variant name; do not confuse this with WHO's newer variant class B below ^[1][22]

Why X-linked matters clinically: Hemizygous males usually have a fairly uniform deficient RBC population. Heterozygous females are mosaics because of lyonisation (random X-inactivation), so their enzyme activity and haemolytic risk can range from deficient to normal; “female = unaffected carrier” is unsafe. Quantitative testing is therefore important when a haemolytic-risk medicine is being considered.

The lecture shows the old WHO I–IV table. It is useful for recognising older exam language, but its statement that class III has “no” clinical effects is incorrect: these variants may cause acute trigger-related haemolysis. WHO revised the variant classification because residual activity overlaps substantially between the former classes II and III. ^[22][23]

This classifies genetic variants, not the severity of every individual patient. Phenotype also depends on sex, X-inactivation, age of circulating RBCs, dose/exposure, and intercurrent illness.

Intravascular haemolysis with haemoglobinuria — acute or chronic (not common) ^[1]

Precipitated by infection or acute medical illness, drugs and Fava beans ^[1]

Drugs: sulphonamides, rasburicase, primaquine ^[1]

Why G6PD-deficient RBCs are vulnerable to oxidative stress:

1. G6PD is the rate-limiting enzyme of the pentose phosphate shunt
2. G6PD generates NADPH → needed to regenerate reduced glutathione (GSH) via glutathione reductase
3. GSH protects Hb and RBC membranes from reactive oxygen species (ROS)
4. Without adequate G6PD → ↓NADPH → ↓GSH → Hb precipitates as Heinz bodies → membrane damage → intravascular lysis and splenic removal of damaged cells
5. Triggers (infection, drugs, fava beans) increase ROS production → overwhelm the already impaired antioxidant system

Triggers: infection/acute illness, fava beans, naphthalene (mothballs), and particular oxidant medicines. Avoid a single memorised blanket list: risk depends on the drug and dose, and recommendations change. Rasburicase is contraindicated in G6PD deficiency; 8-aminoquinoline malaria drugs such as primaquine/tafenoquine require guideline-directed quantitative G6PD assessment and regimen selection. Check the current product label, local formulary, and specialist guidance. Hong Kong newborn counselling also specifically warns about fava beans and mothballs. ^[24][25]

Blood film — polychromasia, ghost or hemi-ghost cells ^[1]

G6PD assay — may be normal during the acute phase as young red cells and reticulocytes may have normal G6PD levels ^[1]

Blood film findings explained:

• Polychromasia: Reticulocytosis (compensatory)
• Blister/hemighost cells: Haemoglobin is displaced to one side of the cell, leaving a pale vacuole-like or almost empty segment; this reflects severe oxidative membrane damage
• Heinz bodies (seen with supravital staining): Denatured, precipitated Hb within RBCs
• Bite cells: Splenic macrophages remove Heinz bodies by "pitting" → creates indentations in the RBC

In the slide montage, scan for the pale “empty” half of hemighost/blister cells and for sharply indented bite cells. Heinz bodies themselves are generally not visible on a routine Romanowsky film; they require a supravital stain.

Supportive. Blood transfusion. Folic acid. Stop and avoid offending agents. ^[1]

The most important management is PREVENTION: stop the trigger, treat infection, monitor Hb/haemolysis and renal function, maintain hydration when appropriate, and transfuse only for clinically significant anaemia. Give patients locally maintained medicine/food-exposure advice rather than an uncurated internet list. Routine folate is not required for every asymptomatic person, but may be used when sustained haemolysis or deficiency increases need.