Hereditary spherocytosis and G6PD deficiency

Overview

The red cell needs a strong, flexible membrane and working enzymes to survive 120 days.

• In hereditary spherocytosis (HS) the membrane skeleton is faulty. Cells lose membrane, become round and are trapped in the spleen: a chronic extravascular haemolysis.
• In G6PD deficiency the cell cannot protect itself from oxidants. Haemoglobin denatures and the cell bursts: an acute, usually intravascular haemolysis after a trigger.

G6PD deficiency is common in Pakistan and is particularly important because primaquine is used for vivax malaria.

Hereditary spherocytosis

Genetics

• The commonest inherited haemolytic anaemia in people of Northern European descent (about 1 in 5000), but seen worldwide.
• Usually autosomal dominant (about three-quarters have a family history); some cases are recessive or new mutations.

Role of spectrin and the membrane skeleton

• The red cell membrane is a lipid bilayer held up by a mesh of proteins underneath: spectrin, ankyrin, band 3, protein 4.1 and protein 4.2.
• In HS, the defect is in the vertical links that tie the bilayer to the skeleton. Commonest: ankyrin (about 45%), then band 3 and spectrin.
• The poorly supported bilayer buds off as small vesicles. The cell loses surface area but keeps its volume, so the surface-to-volume ratio falls and the cell becomes a sphere.
• Spherocytes are rigid; they cannot squeeze through the splenic cords and are eaten by splenic macrophages.
• The membrane also leaks sodium, so the cell uses more ATP to pump it out.

Clinical findings

• Variable: from no symptoms to severe anaemia.
• Neonatal jaundice may be the first sign.
• Triad: anaemia, jaundice (fluctuating, acholuric) and splenomegaly.
• Pigment gallstones, even in children.
• Leg ulcers (rare).
• Family history of anaemia, jaundice, gallstones or splenectomy.
• Crises:
  • Aplastic crisis with parvovirus B19, which infects red cell precursors and stops production for a short time; Hb falls sharply with low reticulocytes.
  • Haemolytic crisis with infections.
  • Megaloblastic crisis from folate exhaustion.

Investigations

• FBC: anaemia (mild to moderate), raised reticulocytes, MCHC raised, MCV normal or slightly low.
• Blood film: spherocytes (small, dense cells with no central pallor) and polychromasia.
• Haemolysis screen: raised unconjugated bilirubin, LDH, urobilinogen; low haptoglobin.
• Direct Coombs test: negative (excludes AIHA, which also shows spherocytes).
• Osmotic fragility test: spherocytes lyse more readily in hypotonic saline (they have no spare room to swell). The incubated test is more sensitive.
• EMA (eosin-5-maleimide) binding test by flow cytometry: reduced dye binding; the preferred screening test in many laboratories today.
• SDS-PAGE of membrane proteins or genetic tests in difficult cases.
• Family screening.

Management

• Folic acid for moderate or severe haemolysis.
• Transfusion during aplastic crises or severe anaemia.
• Splenectomy:
  • Indicated for symptomatic anaemia, growth failure, massive splenomegaly or recurrent gallstones.
  • Delay until after early childhood (ideally after about 6 years) because of the risk of overwhelming infection with encapsulated bacteria.
  • Vaccinate against pneumococcus, Haemophilus influenzae type b and meningococcus at least 2 weeks before; give lifelong penicillin prophylaxis afterwards.
  • Remove the gallbladder at the same operation if there are stones.
  • Partial splenectomy may be used in young children.
• After splenectomy: anaemia and haemolysis resolve, but spherocytes persist; Howell–Jolly bodies and target cells appear; platelets rise; there is an increased risk of thrombosis as well as infection.

G6PD deficiency

Biochemistry

• G6PD is the first enzyme of the hexose monophosphate (pentose phosphate) shunt.
• It makes NADPH, which keeps glutathione in its reduced form. Reduced glutathione mops up hydrogen peroxide and other oxidants.
• Red cells have no mitochondria, so G6PD is their only source of NADPH.
• Without it, oxidants damage haemoglobin, which precipitates as Heinz bodies on the inside of the membrane, and cross-link membrane proteins. Cells become rigid and lyse.

Genetics

• X-linked recessive (gene at Xq28). Males are affected; heterozygous females are usually mild but can haemolyse because of random X-inactivation (lyonisation).
• Very common in Africa, the Mediterranean, the Middle East, South Asia and South-East Asia. Carriers have some protection against falciparum malaria.
• Variants:
  • African (A−): mild; only old cells are deficient, so haemolysis is self-limiting even if the drug continues.
  • Mediterranean: severe; all cells are deficient; haemolysis can be life-threatening; associated with favism.

Precipitating factors

Clinical findings

1. Acute haemolytic episode 1–3 days after a trigger:
   • Sudden pallor, jaundice, dark (cola-coloured) urine from haemoglobinuria, back or abdominal pain, fever.
   • Can cause acute kidney injury and shock.
2. Neonatal jaundice, which can be severe enough to cause kernicterus.
3. Chronic non-spherocytic haemolytic anaemia in rare severe variants.
4. Between attacks the patient is usually well with a normal blood count.

Lab findings

• Falling Hb; reticulocytosis after a few days.
• Intravascular haemolysis: haemoglobinaemia, haemoglobinuria, very low haptoglobin, raised LDH and unconjugated bilirubin; later haemosiderinuria.
• DAT negative.

Microscopic picture

• Heinz bodies: purple inclusions of denatured haemoglobin. They are only seen with a supravital stain (methyl violet or crystal violet), not on a routine Romanowsky film.
• Bite cells (degmacytes): a "bite" taken out where splenic macrophages removed the Heinz body.
• Blister cells: haemoglobin pulled away to one side, leaving a clear blister.
• Irregularly contracted cells and polychromasia.

Confirming the diagnosis

• G6PD screening test (fluorescent spot or dye reduction test) or quantitative enzyme assay.
• Pitfall: just after a crisis the oldest, most deficient cells have been destroyed and young reticulocytes have higher enzyme levels, so the test may be falsely normal. Repeat 2–3 months later (or use a quantitative assay with a reticulocyte comparison).
• Heterozygous females may give borderline results.

Management

• Stop and avoid the trigger drug or food; give a written list of drugs to avoid.
• Treat the infection.
• Hydration and keep a good urine output to protect the kidneys.
• Blood transfusion for severe anaemia; it can be life-saving in the Mediterranean type.
• Folic acid if haemolysis is chronic.
• Neonatal jaundice: phototherapy or exchange transfusion.
• Splenectomy is not usually helpful (destruction is mainly intravascular).
• Prevention: newborn screening in high-prevalence areas, and testing before primaquine.

Comparison

Pyruvate kinase deficiency (brief)

• The commonest glycolytic enzyme defect; autosomal recessive.
• Low ATP makes cells rigid; chronic haemolysis with splenomegaly; "prickle cells" on the film.
• Raised 2,3-BPG shifts the oxygen curve to the right, so the anaemia is well tolerated.
• Splenectomy helps patients who need frequent transfusion.

Quick revision

• HS: autosomal dominant; ankyrin > band 3 > spectrin; spherocytes destroyed in spleen.
• HS labs: high MCHC, reticulocytosis, DAT negative, increased osmotic fragility, low EMA binding.
• HS: parvovirus B19 aplastic crisis; pigment gallstones.
• Splenectomy after about age 6, vaccinate first, penicillin after; spherocytes persist.
• G6PD: X-linked; NADPH and glutathione fail; oxidant haemolysis.
• Triggers: infection, primaquine, dapsone, sulphonamides, nitrofurantoin, quinolones, rasburicase, methylene blue, fava beans, mothballs.
• Film: bite and blister cells; Heinz bodies with supravital stain.
• Test may be normal straight after a crisis; repeat later.

Written by The House Job from standard references (Kumar and Clark's Clinical Medicine; Pathoma: Fundamentals of Pathology).

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