Other macrocytic considerations listed in lecture include oval RBCs, erythroblasts in bone marrow with abnormal maturation, reticulocytosis, myeloma, myelodysplasia, aplastic anaemia.
Megaloblastic Anaemia
Megaloblastic anaemia is characterised by abnormal erythroblasts in the bone marrow with asynchronous maturation of cytoplasm and nucleus (delayed maturation).
Common causes:
Vitamin B12 (cobalamin) deficiency.
Folate deficiency.
Metabolic abnormalities of B12 or folate (rare).
Clinical features include jaundice, oral/skin manifestations, and weight changes (see below).
Dietary folate absorbed in duodenum/jejunum; absorbed as methyl-tetrahydrofolate (methyl-THF) in plasma; inside cells converted to folate polyglutamates for use.
Absorption/activation diagram note: polysaccharide forms are converted to active polyglutamates for intracellular use.
B12 / Folate Absorption and Interplay
Folate polyglutamates are absorbed after dietary folate is converted to methyl-THF in plasma.
B12 indirectly supports folate utilization by regenerating THF from methyl-THF, enabling dTMP synthesis.
Effect on haematology markers (example data from figure): isolation of reticulocytes rise after treatment; other counts (WBC, Hb, platelets) show improvement over time.
Other macrocytic anaemias (>97 fL) not due to megaloblastic processes include:
Alcohol use.
Liver disease.
Hypothyroidism.
Reticulocytosis.
Cytotoxic drugs.
Myelodysplastic syndromes.
Pregnancy.
Smoking.
Myeloma and paraproteinaemia.
Neonatal states.
Haemoglobin Basics
Hb is composed of two pairs of globin chains: \text{Hb} = (\alpha1\beta1)(\alpha2\beta2) attached to haem.
Quaternary structure enabling transport of O2 and CO2; also carries nitric oxide (NO).
~640\times 10^6 Hb molecules per RBC.
Fully loaded Hb can carry up to 4 O2 molecules.
In deoxygenated Hb, the β chains undergo conformational changes allowing 2,3-DPG to enter and decrease Hb affinity for O2 (rightward shift).
Normal Hb-O2 dissociation curve is modulated by 2,3-DPG, H+, CO2, and Hb structure.
Right shift (P50 up) indicates decreased affinity; left shift (P50 down) indicates increased affinity. Examples: Hb S (right shift) vs Hb F (left shift).
Hb F: small amounts postnatally; gamma chain declines after birth (3–6 months transition to beta).
Other developmental Hb types include Gower and Portland during fetal development.
Haemoglobinopathies
Definition: disorders due to abnormal Hb structure or reduced synthesis of normal globin chains (alpha or beta) (i.e., thalassaemias).
Major Hb variants include: Hb S (sickle), Hb C, Hb D, Hb E, and many others (>1000 globin gene mutations reported).
Classification summary:
Unstable or crystalline Hb (e.g., Hb S, C, D, E).
Thalassaemias (reduced globin chain synthesis).
Sickle cell disease/trait.
Methaemoglobinaemia (Hb M).
Other variants and clinical syndromes.
Geographical distribution of thalassaemias and Hb abnormalities (as per lecture illustration).
Investigations of Hb Variants
Full blood count (FBC) with Hb and RBC indices to screen for thalassaemias and structural variants.
Blood film may show characteristic changes:
Target cells in HbC trait.
Sickle cells in HbS disease.
Irregularly contracted RBCs in HbC or unstable Hb.
Reticulocyte count (indicator of marrow response).
Diagnostic tests to confirm Hb variants include:
High-performance liquid chromatography (HPLC).
Capillary electrophoresis (CE).
Cellulose acetate electrophoresis (CAE).
Isoelectric focusing (IEF).
Sickle solubility test.
Sickle Solubility Test
Used to differentiate HbS from HbD or HbG on cellulose acetate electrophoresis;
HbS is insoluble in deoxygenated state; crystals form and refract light, giving turbidity.
A positive sickle solubility test supports presence of HbS; confirmation by HPLC or IEF.
HPLC for Hb Diagnosis
HPLC is used as a first-line method to diagnose Hb disorders.
Different Hb types elute at characteristic times on the column; their concentrations are measured.
Example: a patient carrier of sickle cell disease shows distinct Hb A, Hb S peaks.
Hb Electrophoresis Patterns
Pattern interpretation examples:
Normal or α-thalassaemia trait: Hb A with possibly Hb A2 and Hb F patterns.
Sickle cell trait: Hb A and Hb S bands.
Sickle cell disease: predominantly Hb S with reduced Hb A; presence of Hb F may vary.
β-thalassaemia trait: elevated Hb A2 and sometimes Hb F.
β-thalassemia major: little or no HbA, predominantly HbF, with possible Hb A2.
Sickle cell/β-thalassemia major; Sickle cell/Hb C disease; Hb H disease (rare, in α-thalassaemia contexts).
Methaemoglobinaemia
Methaemoglobinaemia: Hb iron in the oxidised Fe^{3+} state rather than Fe^{2+}.
Causes: deficiency of methaemoglobin reductase, Hb M variants, exposure to oxidising substances.
Clinical feature: cyanosis due to reduced oxygen delivery.
α-Thalassaemia
Cause: deletion of α-globin gene(s), reducing α-chain synthesis.
Normal α-globin gene dosage: 4 copies (αα/αα).
Clinical severity depends on number of missing/inactive genes:
Loss of all 4 genes (--/--) → hydrops fetalis (incompatible with life).
Loss of 3 genes (--/-α) → HbH disease (adult) or Hb Bart’s (fetal) → severe microcytic, hypochromic anaemia with splenomegaly.
Loss of 1 or 2 genes → α-thalassaemia trait; usually no severe anaemia but reduced MCV/MCH; DNA analysis may be needed for diagnosis.
Figures show HbH disease and supravital stain (golf ball cells).
β-Thalassaemia
Cause: deletion or mutation in β-globin gene leading to reduced β-chain synthesis.
β-Thalassaemia major: severe microcytic, hypochromic anaemia; little or no HbA; predominantly HbF; clinical features include growth issues, bone expansion, splenomegaly, hepatomegaly.
β-Thalassaemia trait: asymptomatic; common in certain populations; lab features include microcytosis and hypochromasia; elevated HbA2 and variable HbF by HPLC.
Hb S (Sickle Cell Disease) – Overview
HbS results from a mutation in the β-globin chain causing structural abnormality.
In oxygenated form, HbS is soluble; upon deoxygenation, HbS polymerises, distorting RBCs to a sickle shape.
Sickle cell trait (heterozygous): usually asymptomatic but may show sickling under extreme conditions.
Sickle cell disease (homozygous HbS): severe haemolytic anaemia with vaso-occlusive crises.
Sickle Cell Disease – Lab and clinical features
At birth: normal blood count; in first year, HbF is replaced by HbS; decreased Hb; increased reticulocytes (5–20%).
Adult: Hb typically 60–100 g/L; marked fall during crises.
Blood film features: sickled cells, target cells, Howell-Jolly bodies, polychromasia, irregularly contracted RBCs, and sometimes blister cells.
Sickle cell trait: MCV and MCH usually normal; may be reduced if coexisting α-thalassemia trait.
Sickle Cell Crises and Complications
Vaso-occlusive crisis: triggered by infection, dehydration; infarcts cause severe pain in bones/soft tissues (bones, lungs, spleen, brain).
Visceral sequestration crisis: sickling within organs with pooling of blood → anaemia, possible acute chest syndrome, splenic sequestration.
Aplastic crisis: transient cessation of erythropoiesis.
Haemolytic crisis: rapid RBC destruction.
Other complications: leg ulcers, pulmonary hypertension, hepatic dysfunction, gallstones, kidney infarctions/dysfunction, osteomyelitis; may require exchange transfusion and risk iron overload.
Growth retardation and osteonecrosis are noted.
Sickle Cell Disease – Pattern and Imagery
The sickle polymer formation underlies the pathophysiology of crises and organ damage.
Visuals depict presentation in pediatric and young adult patients (painful dactylitis, facial changes, etc.).
Review / Practice Prompts
Macrocytic anaemias are characterised by increased RBC size (MCV > 97 fL). In B12 or folate deficiency, the bone marrow contains megaloblastic erythroblasts with asynchronous cytoplasmic/nuclear maturation and delayed nuclear maturation due to impaired DNA synthesis.
Major differential diagnoses for macrocytosis include liver disease, alcoholism, hypothyroidism, reticulocytosis, cytotoxic drugs, myelodysplastic syndromes, pregnancy, and smoking.
B12 deficiency is usually caused by B12 malabsorption due to pernicious anaemia, with antibodies directed against gastric parietal cells or intrinsic factor; resulting in deficiency of intrinsic factor, which is essential for B12 absorption in the gut.
Dietary folates are converted to methyl-THF; B12 is required to convert methyl-THF back to THF, enabling DNA synthesis.
Hb is composed of two α/β globin pairs and a heme group; Hb has a quaternary structure and transports O2/CO2; a fully loaded Hb can carry 4 O2 molecules.
Hb types: Foetal Hb (Hb F) is mainly α2γ2 with higher O2 affinity to support placental transfer; Adult Hb A is α2β2; Hb A2 is α2δ2; Hb F declines after birth as γ is replaced by β.
Haemoglobinopathies include thalassaemias (reduced globin synthesis) and Hb variants such as Hb S; diagnosis relies on FBC, smear, HPLC, CE, CAE, IEF, and sickle solubility tests; Hb electrophoresis patterns help distinguish conditions (HbS, HbC, HbD, HbE, β-thal traits/full disease, Hb H disease, etc.).
Sickle cell disease features a spectrum of crises: vaso-occlusive, splenic sequestration, aplastic crisis, and hemolytic crises; other complications include growth abnormalities, osteonecrosis, infections, and organ dysfunction.
A Question to Ponder
What are these? Clue: a type of body fluid, collected over the recovery period of a condition we will learn about next week.