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Cold autoantibodies and the Donath-Landsteiner test

15 min

Skills you'll build2 skills
  • Judge cold-antibody significance from thermal amplitude and hemolysis
  • Interpret a Donath-Landsteiner test using its temperature sequence and controls

At the bench: Use the antibody workup to select suitable donor units

Try first

Try first

A cold agglutinin titer is ordered. The clotted specimen spent 2 hours in a refrigerator before the serum was separated. What happens to the titer?

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.

Titer and thermal amplitude

Most healthy adults carry a low-level cold autoantibody that reacts only in the cold.1 Two measurements describe it. The titer is how far the serum can be diluted and still react at a stated temperature. The thermal amplitude is the highest temperature at which it reacts. Reactivity near body temperature is more significant than a high titer confined to 4 °C.1,2 No single cutoff separates incidental from pathologic antibodies. Both results are interpreted with the hemolysis markers and the monospecific DAT.2

Keep the specimen warm

Cold agglutination continues in the tube. For a cold agglutinin titer, the specimen stays at 37 to 38 °C from collection until the serum is separated.2 In a cooled EDTA tube, clumps of red cells count as fewer, larger particles. The RBC count and hematocrit read falsely low, and the MCV and MCHC read falsely high. Warming the specimen to 37 °C and repeating it by the validated procedure corrects the count.1,2 In pretransfusion testing, reading at 37 °C avoids most cold autoantibody reactivity and keeps clinically significant alloantibodies detectable.1,3

The Donath-Landsteiner test

Paroxysmal cold hemoglobinuria (PCH) usually follows an infection in a young child. The Donath-Landsteiner antibody is a biphasic IgG anti-P. It binds red cells and fixes early complement in the cold. On rewarming, complement activation is completed and the cells lyse intravascularly. The DAT therefore usually shows C3d with little or no IgG.1,4 The test incubates the patient's serum with P-positive group O cells, with fresh complement added when the patient's is depleted:4,5

IncubationExpected in PCH
Cool, then 37 °CHemolysis
37 °C throughoutNo hemolysis
Cool onlyNo hemolysis

The serum specimen stays at 37 °C from collection until it is separated. Cooling it first lets the antibody bind the patient's own cells and gives a false-negative result. A low titer, depleted complement and testing after the episode can also give a false negative.4,5

Follow the bench demonstration

Sources for this step
References
  1. Bloch EM, Campbell-Lee S, McKenna DH Jr, Montemayor-Garcia C, Schwartz J, Shaz B, Storry J, eds. Technical Manual. 22nd ed. AABB; 2026.
  2. Berentsen S. Diagnosis and management of cold agglutinin disease. Hematology Am Soc Hematol Educ Program. 2025;2025(1):295-304. doi:10.1182/hematology.2025000718
  3. Johnson ST, Puca KE. Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies. Hematology Am Soc Hematol Educ Program. 2022;2022(1):96-104. doi:10.1182/hematology.2022000406
  4. Williams JD, Jayaprakash RK, Kithany H, Tighe MP. How to use Donath-Landsteiner test to diagnose paroxysmal cold haemoglobinuria. Arch Dis Child Educ Pract Ed. 2022;107(3):199-206. doi:10.1136/archdischild-2020-319568
  5. Michigan Medicine MLabs. Donath-Landsteiner Test. Updated February 18, 2026. Accessed September 27, 2026.

Watch one

Read each decision and its explanation.

Jonah Scales, 6, MRN 2285117, had a cold 10 days ago. Today he has pallor and red-brown urine. His EDTA and clotted specimens were collected and kept at 37 °C until separated. The cold agglutinin screen is weak.

The Donath-Landsteiner tubes read:

  • Tube 1, patient serum, cooled then 37 °C: hemolysis.
  • Tube 2, patient serum, 37 °C throughout: no hemolysis.
  • Tube 3, patient serum, cooled only: no hemolysis.
  • Tube 4, normal serum, cooled then 37 °C: no hemolysis.

What does the result show?

TestResultPreviousReference intervalFlag
Hemoglobin7.2 g/dL Low2 days ago: 12.4 g/dL12.4 g/dL2 days ago11.5–14.3 g/dL11.5–14.3Low
Plasma free hemoglobin96 mg/dL High0.0–15.2 mg/dL0.0–15.2High
DAT, anti-IgGNonreactiveNonreactiveNonreactive
DAT, anti-C3dReactive, 2+NonreactiveNonreactive

Specimen: H 96, L 6, I 3. EDTA and clotted specimens kept at 37 °C from collection

  1. Check the hemolysis first. Hemoglobin fell from 12.4 to 7.2 g/dL in 2 days, and plasma free hemoglobin is 96 mg/dL.

    The test is meaningful only with evidence of hemolysis, and free hemoglobin shows the hemolysis is intravascular.

  2. Read the DAT. C3d is 2+ and IgG is nonreactive, with a weak cold agglutinin screen.

    A biphasic IgG hemolysin leaves complement on the cells, and routine anti-IgG often misses the antibody.

  3. Check the handling. The specimens stayed at 37 °C until the serum was separated.

    Cooling before separation lets the antibody bind the patient's own cells and gives a false negative.

  4. Read the control. Tube 4 shows no hemolysis.

    A valid control shows that the cells and complement do not lyse without the patient's antibody.

  5. Read the cool-then-warm tube. Tube 1 shows hemolysis.

    The antibody binds in the cold, and lysis completes at 37 °C.

  6. Read the single-temperature tubes. Tubes 2 and 3 show no hemolysis.

    Lysis confined to the cool-then-warm tube shows that both phases are needed.

The Donath-Landsteiner test is positive, showing a biphasic hemolysin. With acute intravascular hemolysis and a C3d-only DAT, the pattern fits paroxysmal cold hemoglobinuria, and the laboratory reports it promptly to pediatrics or hematology.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A patient's cold autoantibody has a titer of 512 at 4 °C. Which additional finding most increases concern that the antibody is clinically significant?

Incorrect. A higher titer at 4 °C still describes reactivity confined to the cold, and no single titer establishes pathogenicity.

Incorrect. Anti-I occurs in both incidental and pathologic cold agglutinins, so specificity does not show clinical significance.

Correct. Reactivity close to body temperature lets the antibody bind and fix complement in the circulation, so thermal amplitude is more important than a 4 °C titer. Hemolysis findings then show whether the antibody is causing hemolysis.

Hint
  1. Titer and thermal amplitude measure different things.
  2. Ask which finding shows the antibody could bind red cells inside the body.

Review Cold autoantibody significance

Problem 2 of 3 · Hints available

With valid controls, which Donath-Landsteiner pattern demonstrates a biphasic hemolysin?

Correct. The IgG antibody binds and fixes early complement in the cold, and lysis completes only when the cells are rewarmed to 37 °C. The 37 °C-only and cool-only tubes stay unlysed.

Incorrect. Lysis at 37 °C without a cool phase shows no dependence on cold binding, so it does not demonstrate a biphasic hemolysin.

Incorrect. Complement lysis needs the 37 °C phase to finish, so the Donath-Landsteiner antibody leaves cells intact during the cool phase alone.

Hint
  1. Split the test into two phases: antibody binding and complement completing lysis.
  2. Ask which temperature each phase needs.

Review Donath-Landsteiner testing

Problem 3 of 3 · On your own

A 5-year-old has acute intravascular hemolysis after a viral illness and a DAT reactive for C3d only. His serum was kept at 37 °C until separated. The episode has consumed much of his complement, and the Donath-Landsteiner test was set up with his serum alone. Every tube, including the cooled-then-warmed tube, shows no hemolysis. What does the laboratory report?

Lysis needs complement. With his own complement depleted, the antibody can bind in the cold without lysing the cells.

The test is positive only when the cooled-then-warmed tube hemolyzes. Hemolysis in the patient shows the need for the test.

Cold agglutinin disease is a chronic disorder, usually of older adults, with a significant cold agglutinin. This child's weak screen and acute intravascular episode do not fit it.

Depleted complement gives a false negative. Fresh normal serum supplies the complement. A tube of normal serum alone is the control.

Review Paroxysmal cold hemoglobinuria

Use it

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

  • Walter Sampson, 78, MRN 6154820, is scheduled for hip surgery. His CBC and type and screen were drawn at 08:10 and reached the laboratory at room temperature.
  • The blood film shows clumps of red cells.
  • His antibody screen cells and autocontrol react 2+ at immediate spin. All are nonreactive at 37 °C and at the antiglobulin phase.
TestResultReference intervalFlag
RBC count2.60 × 10⁶/µL Low4.35–5.65 × 10⁶/µL4.35–5.65Low
Hemoglobin13.8 g/dL13.2–16.6 g/dL13.2–16.6
Hematocrit30.7 % Low38.3–48.6 %38.3–48.6Low
MCV118 fL High78.2–97.9 fL78.2–97.9High
MCHC45.0 g/dL High32.0–36.0 g/dL32.0–36.0High

Specimen: H 2, L 8, I 1. EDTA specimen at room temperature; red-cell clumps on the film

Decision 1 of 3

What does the laboratory do with the CBC?

Clumped cells count as fewer, larger particles. The RBC count, hematocrit, MCV and MCHC are artifacts, and releasing them would report wrong values.

Warming disperses the clumps. An MCHC of 45.0 g/dL with clumps on the film is the pattern of cold agglutination in the tube.

Cooling makes the agglutination worse. The existing specimen can be warmed and rerun.

Review Cold autoantibody significance

Decision 2 of 3

A cold agglutinin titer, drawn and kept at 37 °C until separated, is 256 at 4 °C. The antibody does not react at 30 °C, and his bilirubin, lactate dehydrogenase and haptoglobin are within their reference intervals. How is the antibody described?

Reactivity stops below 30 °C, and the hemolysis markers are within their intervals. A 4 °C titer alone does not make a cold antibody significant.

Titers at 4 °C overlap between incidental and pathologic antibodies. This one does not react near body temperature and causes no hemolysis.

Called a high 4 °C titer clinically significant

Most adults have a low-level cold autoantibody, and 4 °C titers overlap between incidental and pathologic antibodies. A cold antibody is clinically significant only when it reacts near body temperature or causes hemolysis. Calling a cold-only titer significant can start an unneeded workup for a pathologic cold antibody that is not present.

Cold agglutinin disease needs chronic hemolysis and a strongly C3d-positive DAT. His hemolysis markers are within their intervals.

Review Cold autoantibody significance

Decision 3 of 3

Two units are ordered for surgery. How does the transfusion service crossmatch them?

The cold antibody makes every immediate-spin reading reactive, and an immediate-spin crossmatch cannot show an IgG alloantibody.

Almost every adult donor's red cells carry I. An incidental cold autoantibody does not require antigen-negative units.

Testing above the antibody's thermal range avoids its reactivity and keeps clinically significant alloantibodies detectable.

Review Cold autoantibody significance

The antibody did not react at 30 °C, and there was no evidence of hemolysis, so the titer of 256 represents an incidental cold autoantibody. The CBC was warmed and repeated, and the crossmatch was read at 37 °C.

For reference

Sources checked