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USMLE Step 1Haematology and oncologyEP 05 of 14

G6PD deficiency vs hereditary spherocytosis, by mechanism

Both cause haemolysis with jaundice, but one is an antioxidant enzyme failure and the other a membrane scaffold failure. The smear, the timing and the antiglobulin test sort them.

  • 5:36 video
  • 3 min read
  • 11 sources
  • Published 15 Nov 2026
Video lesson · 5:36 · Watch on YouTubeSeries: Hematology: Anemia, Coagulation & Oncology Basics · @medservatory
Chapters (13)
By the end of this lesson

Contrast G6PD deficiency (oxidant injury, Heinz bodies, bite cells) with hereditary spherocytosis (membrane defect, spherocytes, EMA binding) by mechanism.

The short version

  1. In both, red cells die early: reticulocytes and indirect bilirubin rise, and haptoglobin falls.
  2. G6PD deficiency means no NADPH, oxidant injury, Heinz bodies and bite cells, in episodes after a trigger.
  3. Hereditary spherocytosis means a weak membrane skeleton, spherocytes, a raised MCHC and trapping in the spleen.
  4. A negative direct antiglobulin test with spherocytes points to the membrane, not antibodies.
  5. EMA binding is the preferred lab test for spherocytosis: fluorescence on flow cytometry is reduced.

What G6PD deficiency and hereditary spherocytosis share

Both conditions are haemolysis: red cells die early. Reticulocytes rise, indirect (unconjugated) bilirubin rises, and haptoglobin falls.[1][7]

So the first lab picture does not separate G6PD deficiency vs hereditary spherocytosis. The mechanism does.

A red cell must survive two threats: oxidant attack on its haemoglobin, and mechanical stress on its membrane. G6PD deficiency is the first defence failing. Hereditary spherocytosis is the second.[2][1]

G6PD deficiency: no NADPH, no shield

G6PD is the first enzyme of the pentose phosphate pathway, and it makes NADPH. NADPH keeps glutathione reduced, and reduced glutathione mops up hydrogen peroxide.[2][3]

  1. G6PD makes NADPHThe first enzyme of the pentose phosphate pathway.
  2. NADPH keeps glutathione reducedReduced glutathione is the red cell's shield.
  3. Glutathione removes hydrogen peroxideWith no shield, oxidant stress goes unchecked.

With no shield, an oxidant stress denatures haemoglobin. Typical triggers are an infection, fava beans, or a drug such as primaquine or dapsone.[2][11]

Heinz bodies and bite cells

Denatured haemoglobin clumps into Heinz bodies, which are seen only with a supravital stain such as crystal violet.[6][2][11]

Splenic macrophages pluck out each Heinz body with a piece of membrane, leaving a bite cell.[6]

Worked example: reading the hook case
  1. A 20-year-old man has dark urine and jaundice two days after an antimalarial tablet.
  2. His smear shows bite cells: Heinz bodies were removed by the spleen.
  3. Bite cells (and Heinz bodies, if a crystal violet stain is done) point to G6PD deficiency.
  4. Ask which oxidant came first: here, the tablet.

Hereditary spherocytosis: a membrane skeleton problem

Hereditary spherocytosis is a membrane problem. Mutations in ankyrin, band 3, spectrin or protein 4.2 weaken the link between the cytoskeleton and the lipid bilayer.[1]

The cell sheds membrane, loses surface area, and shrinks into a sphere with no central pallor. Because the cell is dense, the MCHC is raised.[1][5]

Spheres cannot bend through the splenic cords, so the spleen traps them. This is extravascular haemolysis, with splenomegaly, jaundice and pigment gallstones.[1][11]

Follow the chain: weak link, membrane loss, sphere, trapping in the spleen. Each step explains a finding, from the dense round cell on the smear to the enlarged spleen and the gallstones.[1][5]

Inheritance, the antiglobulin test and EMA binding

  • About three quarters of cases are autosomal dominant, so a family history is common.[1]
  • The direct antiglobulin test is negative. This separates it from autoimmune haemolysis, where spherocytes also appear.[7][8]
  • The preferred lab test is EMA binding: the dye binds band 3 on the membrane, and in spherocytosis the fluorescence on flow cytometry is reduced.[5][6]

Telling them apart: oxidant episode or membrane loss?

G6PD deficiency vs hereditary spherocytosis
FeatureG6PD deficiencyHereditary spherocytosis
DefectNo NADPH, so no glutathione shieldWeak link between cytoskeleton and bilayer
SmearBite cells; Heinz bodies on crystal violetDense spherocytes, no central pallor
MCHCNot a featureRaised
CourseEpisodic, after a triggerChronic, with splenomegaly and gallstones
InheritanceX-linked recessive, mostly malesAbout three quarters autosomal dominant
Direct antiglobulin testNegative too (non-immune)Negative
Confirmatory clueEnzyme assay (repeat weeks to months later if normal during an attack)Reduced EMA fluorescence
  1. Look at the smearBite cells with Heinz bodies on crystal violet point to the enzyme. A dense round cell without central pallor points to the membrane.
  2. Ask about timingEpisodic haemolysis after a trigger suggests G6PD deficiency. Chronic haemolysis with a big spleen and gallstones suggests the membrane.
  3. Use the antiglobulin testA negative direct antiglobulin test with spherocytes points to the membrane, not antibodies.
  4. ConfirmMeasure G6PD activity, repeating it weeks to months later if it was normal during or soon after an attack, or look for reduced EMA fluorescence.

A bite cell, with Heinz bodies on crystal violet, says G6PD deficiency: ask what oxidant came first. A dense round cell, a negative antiglobulin test and a raised MCHC says membrane.

Episodic, after a trigger, in a boy: enzyme. Chronic, with a big spleen and gallstones: membrane.

Practice question

Original · USMLE style
Your turnQB-MS-S1-HEME-013-01

A 7-year-old girl is brought to the clinic with fatigue and yellow eyes that her mother says have been present since early childhood. Her father had a splenectomy as a teenager. She takes no medications and has not had a recent illness. Temperature is 36.9 °C. Examination shows scleral icterus and a spleen palpable below the left costal margin. Laboratory studies show:

Results
Haemoglobin9.4 g/dL
MCV82 fL
MCHC37.2 g/dL
Reticulocytes9%
Bilirubin, indirect3.0 mg/dL
Direct antiglobulin testnegative
A peripheral blood smear shows dense, round red cells without central pallor. Which of the following is the most likely underlying defect?
Pick one option. Your answers stay on this device.

Common questions

How do you tell G6PD deficiency from hereditary spherocytosis?

Use the smear, the timing and the antiglobulin test. Bite cells and Heinz bodies after an oxidant trigger point to G6PD deficiency. Spherocytes, a raised MCHC and a negative antiglobulin test point to the membrane.

What are Heinz bodies and bite cells?

Heinz bodies are clumps of denatured haemoglobin, seen with a supravital stain such as crystal violet. Splenic macrophages remove each one with a piece of membrane, leaving a bite cell.

Why can the G6PD enzyme assay be falsely normal?

During an attack, young red cells carry more enzyme, so the assay can read falsely normal. A normal result is repeated weeks to months later.

What does EMA binding test for?

It supports a diagnosis of hereditary spherocytosis. The dye binds band 3 on the red cell membrane, and in spherocytosis the fluorescence on flow cytometry is reduced.

Why is the direct antiglobulin test negative in hereditary spherocytosis?

The cause is a membrane skeleton defect, not an antibody. A negative test separates it from autoimmune haemolysis, where spherocytes also appear.

Transcript

Read the full narration, by chapter651 words

0:00The case: dark urine after a tablet, and a child with spherocytes

A 20-year-old man takes an antimalarial tablet before a trip. Two days later his urine is dark, he is jaundiced, and his smear shows bite cells. A child on the same ward is jaundiced too, with small round cells and no pale centre. Same word, haemolysis, two faults.

0:20Today: G6PD deficiency vs hereditary spherocytosis, and what both share

Today: contrast G6PD deficiency with hereditary spherocytosis, by mechanism. In both, red cells die early: reticulocytes rise, indirect bilirubin rises, haptoglobin falls. A red cell must survive two threats: oxidant attack on its haemoglobin, and mechanical stress on its membrane. G6PD deficiency is the first defence failing; spherocytosis is the second. G6PD is the first enzyme

0:50The pentose phosphate pathway: G6PD, NADPH, glutathione

of the pentose phosphate pathway, and it makes NADPH. NADPH keeps glutathione reduced, and reduced glutathione mops up hydrogen peroxide. With no shield, an oxidant stress, such as an infection,

1:07Oxidant triggers, Heinz bodies and bite cells

fava beans, or a drug like primaquine or dapsone, denatures haemoglobin. The denatured haemoglobin clumps into Heinz bodies, seen only with a supravital stain like crystal violet. Splenic macrophages pluck out each Heinz body with a piece of membrane, leaving a bite cell.

1:27X-linked and episodic: why the enzyme assay can be falsely normal

The gene is X-linked recessive, so it mostly affects males, and people are usually well until a trigger, so the haemolysis comes in episodes. The enzyme assay can read falsely normal during an attack, because young red cells carry more enzyme, so it is repeated weeks to months later.

1:47Hereditary spherocytosis: the membrane skeleton (band 3, ankyrin, spectrin, protein 4.2)

Hereditary spherocytosis is a membrane problem. Mutations in ankyrin, band three, spectrin or protein 4.2 weaken the link between the cytoskeleton and the lipid bilayer.

2:00Spherocytes, a raised MCHC and trapping in the spleen

The cell sheds membrane, loses surface area, and shrinks into a sphere with no central pallor, so the MCHC is raised. Spheres cannot bend through the splenic cords, so the spleen traps them: extravascular haemolysis, splenomegaly, jaundice, and pigment gallstones. About three quarters of cases are autosomal

2:20Inheritance, family history and the negative direct antiglobulin test

dominant, so a family history is common. The direct antiglobulin test is negative, which separates it from autoimmune haemolysis, where spherocytes also appear.

2:33Confirming it: the EMA binding test

The confirmatory test is EMA binding: the dye binds band three on the membrane, and in spherocytosis the fluorescence on flow cytometry is reduced.

2:44The decision rule: oxidant episode or membrane loss?

Now use it. A bite cell, with Heinz bodies on crystal violet, says G6PD: ask what oxidant came first. A dense round cell, a negative antiglobulin test and a raised MCHC says membrane. Episodic, after a trigger, in a boy: enzyme. Chronic, with a big spleen and gallstones: membrane.

3:11Your turn: an original practice question

Your turn. A 7-year-old girl has fatigue and yellow eyes that have been there since early childhood. Her father had a splenectomy as a teenager. Her spleen is palpable below the left costal margin. Haemoglobin is 9.4, MCV 82, MCHC 37.2 grams per decilitre, reticulocytes 9%, and indirect bilirubin 3.0. The direct antiglobulin test is negative. The smear shows dense round cells without central pallor. Which of the following is the most likely underlying defect? A, reduced NADPH production by the pentose phosphate pathway. B, weakened attachment of the cytoskeleton to the cell membrane. C, an IgG autoantibody against the red cell surface. D, a glutamate-to-valine substitution in the beta-globin chain. E, impaired ATP generation by red cell glycolysis. Pause here and decide.

4:21The answer: weakened cytoskeleton to membrane attachment

A is out: that is G6PD, with episodes after an oxidant, bite cells and Heinz bodies, not chronic spherocytes. C is out: an autoantibody would make the direct antiglobulin test positive, and hers is negative. D is out: that is haemoglobin S, which gives sickle cells, not spheres. E is out: that is pyruvate kinase deficiency, a non-spherocytic haemolysis. So the answer is B. Spherocytes, a raised MCHC, a negative antiglobulin test, splenomegaly and a family history point to a membrane skeleton defect.

5:02High-yield recap, and next lesson: sickle cell disease

Three things to keep. One: G6PD means no NADPH, oxidant injury, Heinz bodies and bite cells. Two: spherocytosis means membrane loss, spherocytes, a raised MCHC, and a reduced EMA signal. Three: a negative antiglobulin test with spherocytes points to the membrane, not antibodies. Next, sickle cell disease: from polymerisation to vaso-occlusion.

Sources

Numbers in the text, e.g. [1], point here
  1. Turpaev K, et al. An overview of hereditary spherocytosis and the curative effects of splenectomy. Front Physiol 2025 (PMC11850534). europepmc.org/article/PMC/PMC11850534
  2. Molecular Mechanisms of Drug-Induced Hemolysis in G6PD Deficiency (PMC12343164). europepmc.org/article/PMC/PMC12343164
  3. Glucose-6-phosphate dehydrogenase deficiency (PMC12872104, 2026). europepmc.org/article/PMC/PMC12872104
  4. Acquired G6PD Deficiency. J Clin Med 2022 (PMC9695330). europepmc.org/article/PMC/PMC9695330
  5. Wu et al. Eosin-5'-maleimide binding test in hereditary spherocytosis. J Clin Lab Anal 2021 (PMC8649336). europepmc.org/article/PMC/PMC8649336
  6. Inherited Anemia review. Healthcare (Basel) 2026 (PMC13704711). europepmc.org/article/PMC/PMC13704711
  7. Hereditary Hemolytic Spherocytosis in the Active-Duty Population (PMC13282025). europepmc.org/article/PMC/PMC13282025
  8. Hereditary spherocytosis at birth (PMC12468025). europepmc.org/article/PMC/PMC12468025
  9. Pyruvate kinase deficiency reviews (PMC13342182, PMC13523542). europepmc.org/article/PMC/PMC13342182 · europepmc.org/article/PMC/PMC13523542
  10. Sickle Cell Disease. Transfus Med Hemother 2024 (PMC11452173); Front Pharmacol 2025 (PMC12411783). europepmc.org/article/PMC/PMC11452173 · europepmc.org/article/PMC/PMC12411783
  11. Wikipedia: Hereditary spherocytosis; Glucose-6-phosphate dehydrogenase deficiency (secondary sources, cross-checked against the papers above). en.wikipedia.org/wiki/Hereditary_spherocytosis · en.wikipedia.org/wiki/Glucose-6-phosphate_dehydrogenase_deficiency

The practice question is original and its patient is synthetic. Every fact is checked against the sources above in independent accuracy reviews. Always follow your local guidelines and your own laboratory’s ranges.

Image credits. Illustrations: Servier Medical Art (smart.servier.com), CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/), adapted (recoloured, outlined, resized): "Capsule", "Urine test", "Spleen" and "Liver and gallbladder". Original diagrams (code-drawn): the pentose phosphate pathway, red cells, Heinz bodies, bite cells, spherocytes, the membrane cross-section, the pedigree, the EMA histogram, the blood count slip and the smear panels (schematic, not real micrographs). No AI-generated teaching images.

Corrections. None so far. Spotted an error? Email info@medservatory.com with the lesson code MS-S1-HEME-013.

For exam study only, not medical advice. Read the medical disclaimer.

In this series

Playlist on YouTube
← Previous · EP 04Intravascular vs extravascular haemolysis: site and labs
  1. EP 01Microcytic anaemia: four causes, four iron fingerprints5:37
  2. EP 02Hepcidin: why iron falls in inflammation5:34
  3. EP 03B12 vs folate deficiency: MMA, homocysteine and the nerves5:23
  4. EP 04Intravascular vs extravascular haemolysis: site and labs5:27
  5. EP 05G6PD deficiency vs hereditary spherocytosis, by mechanism5:36
  6. EP 06Sickle cell disease pathophysiology: HbS to vaso-occlusion5:26
  7. EP 07Primary haemostasis: platelet adhesion, activation and aggregation5:53
  8. EP 08Coagulation cascade: PT vs aPTT, pathway by pathway5:21
  9. EP 09Von Willebrand disease vs haemophilia: the labs that separate them5:32
  10. EP 10Anticoagulants: target, monitoring test and reversal agent5:34
  11. EP 11Heparin-induced thrombocytopenia: why a low count clots5:29
  12. EP 12DIC vs TTP vs HUS: mechanism and the PT/PTT5:33
  13. EP 13AML vs ALL vs CML vs CLL: leukemia classification made simple5:44
  14. EP 14Oncogenes vs tumour suppressor genes: one hit or two5:17