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Recognizing hemolysis and its mechanism

15 min

Skills you'll build4 skills
  • Recognize hemolysis from haptoglobin, indirect bilirubin, LDH, and reticulocyte results
  • Tell immune, fragmentation, and intrinsic hemolysis apart using the DAT and film
  • Recognize when a normal G6PD result cannot exclude deficiency
  • Recognize a spherocyte in the monolayer of a blood film

At the bench: Relate red cell indices and morphology to the anemia workup

Try first

Try first

A hospitalized patient's hemoglobin has fallen over 2 days, and the lactate dehydrogenase (LDH) comes back markedly increased. Nothing else has been checked yet. Does the LDH result by itself establish hemolysis?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Laboratory markers of hemolysis

No single result proves that red cells are being destroyed faster than the marrow can replace them.1 Hemolysis raises the reticulocyte count once the marrow responds, raises indirect bilirubin and LDH as cells break down, and lowers haptoglobin as it binds the released hemoglobin.1 LDH also rises with liver disease, tissue injury, ineffective erythropoiesis, and a hemolyzed collection, so a raised LDH alone does not establish hemolysis. A low haptoglobin, a raised indirect bilirubin, and a rising reticulocyte count on a properly collected specimen, interpreted together, support red-cell destruction.

Extravascular and intravascular hemolysis

PatternKey findings
ExtravascularReticulocytosis, indirect bilirubin and LDH increase, haptoglobin decrease, spherocytes in some disorders
IntravascularMarked haptoglobin depletion, plasma free hemoglobin, hemoglobinuria, schistocytes in fragmentation disorders

Identifying the mechanism

The direct antiglobulin test (DAT) and the film help identify the mechanism. Spherocytes form in warm autoimmune hemolysis and in hereditary spherocytosis, so a reactive DAT for IgG or C3d, interpreted with objective hemolysis and the transfusion and drug history, supports an immune cause.2 A nonreactive DAT with a family pattern points toward membrane studies such as eosin-5'-maleimide (EMA) binding.4 Schistocytes point toward fragmentation.

A normal G6PD result during an episode

Reticulocytes have more glucose-6-phosphate dehydrogenase (G6PD) activity than older red cells, and the most deficient cells are lost first during hemolysis, so an assay run during or soon after an episode can be normal in a deficient patient. Recent transfusion can mask deficiency in the same way.3 State the limitation in the report, and repeat the quantitative assay at steady state, commonly 2 to 3 months later.3

Follow the bench demonstration

Sources for this step
References
  1. Keohane EM, Preston MM, Mirza KM, Walenga JM, eds. Rodak's Hematology: Clinical Principles and Applications. 7th ed. Elsevier; 2025. Accessed September 26, 2026. https://www.us.elsevierhealth.com/rodaks-hematology-9780323936507.html
  2. Michel M, Crickx E, Fattizzo B, Barcellini W. Autoimmune haemolytic anaemias. Nat Rev Dis Primers. 2024;10(1):82. doi:10.1038/s41572-024-00566-2
  3. Luzzatto L, Ally M, Notaro R. Glucose-6-phosphate dehydrogenase deficiency. Blood. 2020;136(11):1225-1240. doi:10.1182/blood.2019000944
  4. Wu Y, Liao L, Lin F. The diagnostic protocol for hereditary spherocytosis: 2021 update. J Clin Lab Anal. 2021;35(12):e24034. doi:10.1002/jcla.24034

Watch one

Read each decision and its explanation.

A hemoglobin of 8.9 g/dL is falling further over 2 days. The reticulocyte count is increased, indirect bilirubin and LDH are increased, and haptoglobin is undetectable. The film shows spherocytes in the monolayer and no schistocytes. The DAT is positive for IgG. The patient has no personal or family history of anemia and no recent transfusion. What mechanism does the pattern support?

  1. Check the markers together. Reticulocytes, bilirubin, and LDH are increased, and haptoglobin is undetectable, so hemolysis is present.

    Hemolysis is confirmed before its type is identified.

  2. Review the film. Spherocytes are present and no schistocytes are seen, which argues against a fragmentation process.

    Red-cell morphology narrows the mechanism before serologic results are reviewed.

  3. Check the DAT. It is positive for IgG, which supports an immune cause when interpreted with objective hemolysis.

    Spherocytes cannot distinguish an immune cause from a hereditary one.

  4. Check the history. There is no personal or family history of anemia and no recent transfusion, so nothing argues against an acquired immune cause.

    A family pattern or long-standing mild anemia would point toward hereditary spherocytosis.

The pattern supports immune-mediated hemolysis. Spherocytes are present with a positive DAT for IgG, no schistocytes are seen, and no family or transfusion history suggests another cause.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

Results show decreased haptoglobin, hemoglobinemia, and hemoglobinuria. Which clearance route does this pattern reflect?

Correct. Red cells rupturing in the circulation release hemoglobin into plasma. Once haptoglobin and hemopexin are saturated, free hemoglobin appears in plasma and urine, and hemosiderinuria can follow with ongoing loss.

Incorrect. Haptoglobin can fall in either route, but macrophage clearance releases no free hemoglobin into plasma and urine. Extravascular hemolysis raises indirect bilirubin and lactate dehydrogenase (LDH) with reticulocytosis.

Incorrect. Pitting removes inclusions such as Howell-Jolly bodies and returns the red cell to the circulation, so it releases no hemoglobin.

Hint
  1. Haptoglobin binds free hemoglobin released directly into the plasma.
  2. Macrophage clearance in the spleen and liver does not put hemoglobin into the plasma or urine.

Review Erythrocyte clearance and iron recycling

Problem 2 of 3 · Hints available

A film shows numerous schistocytes with thrombocytopenia, and lactate dehydrogenase (LDH) is markedly elevated; PT and activated partial thromboplastin time (APTT) are near normal. What is the laboratory's action?

Correct. Schistocytes, thrombocytopenia, and a high LDH with relatively preserved clotting times suggest thrombotic thrombocytopenic purpura (TTP) or another thrombotic microangiopathy. Urgent notification comes first, and an ADAMTS13 specimen is collected before treatment when feasible. Activity below 10% supports TTP.

Incorrect. In TTP the PT and APTT are often less disturbed than in overt disseminated intravascular coagulation (DIC), so near-normal clotting times fit the urgent pattern.

Incorrect. Acute DIC shows prolonged clotting times, low fibrinogen, and a positive D-dimer. Schistocytes and thrombocytopenia with near-normal clotting times fit a thrombotic microangiopathy.

Hint
  1. Look at the platelet count and the clotting times together with the film.
  2. Compare the low platelet count and fragmented red cells with the near-normal PT and APTT.

Review Urgent laboratory patterns

Problem 3 of 3 · On your own

Near the feather edge of a film, several red cells appear dense with no central pallor. In the monolayer, red cells show normal central pallor. What do you report?

Outside the monolayer, overlapping or flattened red cells can lose their central pallor and look dense. Spherocytes are judged where cells lie singly.

Called cells outside the monolayer spherocytes

Outside the monolayer, overlapping or flattened red cells can lose their central pallor and look dense. Calling them spherocytes suggests hereditary spherocytosis or immune hemolysis the specimen does not show. Spherocytes are judged where cells lie singly and normal cells keep their pallor.

In the monolayer, where cells lie singly, the red cells keep their normal central pallor. The dense appearance at the edge is a preparation effect and does not represent true spherocytes.

Central pallor is judged where cells lie singly and are not overlapping or flattened. The monolayer is the correct area for this assessment.

Review Red-cell morphology on the stained film

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • A woman, MRN 0271946, is admitted with fatigue and yellowing of the eyes.
  • Her hemoglobin has fallen since admission 2 days ago.
  • She has no history of transfusion, recent travel, or new medications.
  • The film shows spherocytes in the monolayer and no schistocytes.
  • The blood bank reports the DAT positive for IgG.
  • A quantitative G6PD assay drawn this admission is within the reference interval.
TestResultPreviousReference intervalFlag
Hemoglobin8.9 g/dL LowAdmission: 11.4 g/dL11.4 g/dLAdmission12.0–15.5 g/dL12.0–15.5Low
Reticulocytes (absolute)185 × 10³/µL HighAdmission: 62 × 10³/µL62 × 10³/µLAdmission25–100 × 10³/µL25–100High
Indirect bilirubin3.8 mg/dL HighAdmission: 0.6 mg/dL0.6 mg/dLAdmission0.1–1.0 mg/dL0.1–1.0High
Lactate dehydrogenase410 U/L HighAdmission: 160 U/L160 U/LAdmission100–220 U/L100–220High
HaptoglobinUndetectable LowAdmission: 90 mg/dL90 mg/dLAdmission30–200 mg/dL30–200Low

Specimen: H 3, L 8, I 2. Serum and EDTA whole blood, drawn on admission day 2

Decision 1 of 3

Does this panel show hemolysis, or a production problem alone?

Haptoglobin is undetectable, indirect bilirubin and LDH are increased, and the reticulocyte count has risen. Together these show accelerated red-cell destruction with a marrow response.

A low reticulocyte count would suggest reduced production. A rising reticulocyte count here is the marrow's response to destruction.

The reticulocyte count, LDH, indirect bilirubin, and haptoglobin already indicate hemolysis with a marrow response. A biopsy is not needed.

Review Evidence of increased hemoglobin catabolism

Decision 2 of 3

Which mechanism does the pattern support?

Fragmentation hemolysis shows schistocytes. None are seen here, and the film shows spherocytes.

Spherocytes with a positive DAT for IgG, interpreted with objective hemolysis and no family or transfusion history, support an antibody-mediated cause cleared by the spleen.

The film and DAT already point to an immune cause. A single G6PD result drawn during this episode cannot confirm or exclude enzyme deficiency.

Review Extrinsic and immune hemolysis

Decision 3 of 3

What do you do with the normal G6PD result drawn during this admission?

Acute hemolysis removes the most deficient cells and leaves younger, higher-activity cells behind, so a deficient patient can test normal during an episode. This result cannot exclude deficiency.

Excluded G6PD deficiency from one result during recovery

Reticulocytes carry more G6PD than older cells, and the most deficient cells are lost during hemolysis, so activity measured during or soon after an episode can look normal. Recent transfusion masks it the same way. State the limitation in the report, and repeat the quantitative assay at steady state, commonly 2 to 3 months later.

The result was drawn during active hemolysis, when activity can look falsely normal. The report states that limitation, and a repeat 2 to 3 months later at steady state would be needed if deficiency remains a concern.

A same-day repeat is still within the same hemolytic episode and has the same limitation. Only a repeat at steady state avoids it.

Review G6PD deficiency testing

Spherocytes with no schistocytes and a DAT positive for IgG, with no family or transfusion history, point to immune-mediated hemolysis. The G6PD was drawn during the episode, so it cannot be reported as excluding deficiency.

For reference

Sources checked