The workup begins when evidence of hemolysis is found together with red cells coated with antibody or complement. The laboratory pattern of autoimmune hemolytic anemia (AIHA) combines accelerated red-cell destruction with immune sensitization. Cold-reactive AIHA includes two mechanisms. In cold agglutinin disease (CAD) and cold agglutinin syndrome (CAS), an IgM cold agglutinin activates complement. In paroxysmal cold hemoglobinuria (PCH), a biphasic IgG hemolysin binds in the cold and produces complement lysis after the cells rewarm.1,2
Establishing immune hemolysis
The laboratory first establishes evidence of hemolysis, then determines whether antibody or complement is coating the red cells. Interpret a direct antiglobulin test (DAT) result within that sequence. Mild or compensated hemolysis may preserve the hemoglobin concentration. The reticulocyte response may be inadequate early in an episode or during severe hemolysis.2-4
Red-cell autoantibodies often recognize a self-antigen carried by most people. The same antibody can therefore react with the patient's red cells, reagent cells, and many donor units, producing broad serologic reactivity that can conceal an alloantibody.1,3
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| Question | Supporting findings | Important limits |
|---|---|---|
| Is red-cell survival shortened? | Falling hemoglobin or hematocrit; increased absolute reticulocyte count; polychromasia; indirect bilirubin and lactate dehydrogenase (LDH) increase; haptoglobin decrease | Reticulocytes can be low early or when marrow compensation fails. Liver disease, inflammation, and specimen hemolysis can alter individual markers. |
| Is destruction intravascular? | Plasma free hemoglobin; hemoglobinuria; urine dipstick positive for heme with few or no red cells by microscopy; later urinary hemosiderin | Hemoglobinemia and hemoglobinuria follow active lysis. Urinary hemosiderin usually appears about 1 week after onset and can reveal a recent episode. |
| Is immune sensitization present? | DAT with polyspecific antihuman globulin, followed by monospecific anti-IgG and anti-C3d | Recent transfusion, a drug, passive antibody, and incidental coating can produce DAT reactivity. The complete pattern determines its significance. |
Use EDTA-anticoagulated blood for the DAT because EDTA limits complement attachment after collection and preserves complement that bound in vivo. Resolve a reactive polyspecific DAT with monospecific anti-IgG and anti-C3d. The monospecific DAT pattern helps classify AIHA and directs the next serologic studies.1-3
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| Pattern | Hemolysis and serology | Main interpretation |
|---|---|---|
| IgG positive, with or without C3d | Usually extravascular hemolysis; warm-reactive IgG in plasma or eluate | Warm AIHA, a drug-related pattern, an alloantibody coating transfused cells, or another cause of IgG sensitization |
| C3d strongly positive; IgG absent or weak | Cold agglutinin detectable in serum; chronic hemolysis or an acute exacerbation | Cold agglutinin disease or cold agglutinin syndrome |
| C3d positive; IgG usually absent by routine DAT | Acute intravascular hemolysis; cold agglutinin absent or too weak to explain the episode | Paroxysmal cold hemoglobinuria; confirm with a Donath-Landsteiner test |
| IgG and C3d strongly positive | Warm-reactive IgG plus a cold agglutinin with clinically important thermal amplitude | Mixed AIHA |
| Routine DAT nonreactive | Convincing hemolysis after other causes are assessed | Reference-laboratory testing for low-density or low-affinity IgG, IgA, or IgM may demonstrate immune sensitization |
C3d predominance directs cold-reactive studies. The hemolysis pattern, cold agglutinin findings, and Donath-Landsteiner result separate CAD or CAS from PCH.
Cold autoantibody significance
Most healthy adults have a low-level cold autoantibody that reacts only at low temperature. Two measurements help assess whether cold reactivity may be pathogenic. Titer measures how far the serum can be diluted while preserving reactivity at a stated temperature. Thermal amplitude is the highest temperature at which the antibody reacts in the test system. Reactivity extending toward physiologic temperatures is more clinically significant than a high titer confined to 4 °C.1,5,6
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| Feature | Incidental cold autoantibody | Clinically relevant cold antibody |
|---|---|---|
| Clinical setting | No hemolysis or cold-induced symptoms | Hemolysis, anemia, cold-induced symptoms, or a compatible associated disease |
| Titer | Often low, but overlap occurs | May be high; no single titer establishes pathogenicity |
| Thermal behavior | Usually limited to colder temperatures | Reactivity at higher temperatures raises concern |
| DAT | May be nonreactive or weakly C3d positive | Often strongly C3d positive; reaction strength is not diagnostic |
| Specificity | Can include anti-I, anti-i, or anti-IH | Supports interpretation but does not establish severity |
No titer or thermal-amplitude cutoff separates these groups in every method and patient. Interpret both measurements with hemolysis evidence, the monospecific DAT, symptoms, and the associated condition.5,6
Anti-I is the usual cold agglutinin in cold agglutinin disease and in many cases associated with Mycoplasma pneumoniae. Adult red cells express I strongly, and cord cells express mainly the i precursor. Anti-i can occur after Epstein-Barr virus infection and reacts more strongly with cord cells. Anti-IH requires both I and H and commonly reacts most strongly with group O and A2 cells, which have more H antigen than A1 and A1B cells. These associations and reaction patterns support interpretation. A complete interpretation also needs evidence of hemolysis and serologic correlation.1,6
Preanalytic control and interference
Cold agglutination can continue after collection. For cold agglutinin titration and monoclonal immunoglobulin studies, keep the specimen at 37 to 38 °C from collection until serum or plasma has been separated from the cells. The performing laboratory supplies the tube, transport, separation, and storage requirements. A separately collected EDTA specimen supports the DAT and CBC.5,6
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| Test affected | Cold-antibody effect | Laboratory response |
|---|---|---|
| Automated CBC | Red-cell aggregates are counted as fewer, larger particles, producing a falsely low RBC count and hematocrit with high MCV, MCH, and especially MCHC | Inspect the film and analyzer flags; warm the specimen to 37 °C and repeat according to the validated analyzer procedure |
| ABO and D typing | Spontaneous agglutination can add false reactions to forward typing or weak-D testing | Warm and wash patient cells under the validated method; use appropriate controls and monospecific anti-IgG when an antiglobulin phase is required |
| Antibody detection and compatibility testing | Broad cold reactivity can obscure a clinically significant alloantibody | Keep testing above the antibody's thermal range and use validated prewarming or adsorption methods; preserve detection of 37 °C-reactive alloantibodies |
| DAT | Complement can attach in vitro when a clotted sample cools | Test EDTA-anticoagulated red cells and resolve a reactive DAT with monospecific reagents |
Detailed prewarming, adsorption, elution, and compatibility procedures belong in the transfusion service's validated method. Recent transfusion can place donor cells in the patient's sample, so the transfusion history determines whether autologous cells are suitable for adsorption. The Blood Group Immunology lesson explains thermal range, antiglobulin detection, and complement deposition.
Cold agglutinin disease and syndrome
Cold agglutinin disease (CAD) usually presents in middle-aged or older adults. It is a clonal B-cell disorder of the bone marrow that produces a monoclonal cold agglutinin, usually IgM kappa with anti-I specificity. Cold agglutinin syndrome (CAS) describes the same cold-antibody hemolytic mechanism secondary to another condition, such as an acute infection, an overt B-cell malignancy, or an autoimmune disease. Infection-associated CAS is often transient.5,6
In the cooler peripheral circulation, IgM binds red cells, agglutinates them, and activates the classical complement pathway. As the blood warms, IgM dissociates and C3 fragments remain bound. Macrophages in the liver remove C3b-coated cells, producing predominantly extravascular hemolysis. Terminal complement activation can also cause intravascular hemolysis during an acute exacerbation. Agglutination in acral vessels can produce acrocyanosis and other cold-induced circulatory symptoms.1,6
The diagnostic pattern for CAD includes all of the following:
- chronic hemolysis shown by bilirubin, LDH, haptoglobin, reticulocytes, and the blood film
- a monospecific DAT strongly positive for C3d, with absent or weak IgG reactivity
- a cold agglutinin titer of at least 1:64 at 4 °C as a conventional supporting criterion, interpreted with thermal amplitude and the clinical pattern
- evaluation for a recent relevant infection or overt malignant disease so CAD can be separated from CAS
Serum protein electrophoresis with immunofixation and immunoglobulin quantification can demonstrate the monoclonal IgM. Bone-marrow morphology and flow cytometry characterize the underlying clone. A small clone may fall below the detection limit of one or more studies, so the core hemolysis and immunohematologic criteria remain the basis of the diagnosis.2,5,6
Transfusion support and clinical handoff
Early communication between the clinical team and transfusion service allows serologic work to continue while urgent support is prepared. Perform compatibility testing at 37 °C, which avoids most cold autoantibody reactivity, so that clinically significant alloantibodies stay detectable. Select ABO-compatible red cells that lack the antigen for any identified alloantibody. The clinical team keeps the patient and infusion extremity warm, and an approved in-line blood warmer is used when the laboratory and transfusion policy call for it.1,3,5
In life-threatening anemia, transfusion proceeds when the risk of delay exceeds the benefit of completing the serologic investigation first. Historical antibodies, current alloantibody assessment, phenotype or genotype information, and urgency guide unit selection. Broad incompatibility caused by the autoantibody can persist after clinically significant alloantibodies have been assessed.3,4
The laboratory distinguishes CAD from secondary cold agglutinin syndrome, documents the thermal range and complement pattern, and reports evidence of hemolysis and an underlying clonal process. These findings guide hematology in deciding whether to address a clonal or complement-mediated process and whether a secondary disorder needs clinical evaluation.2,6
Paroxysmal cold hemoglobinuria
Paroxysmal cold hemoglobinuria (PCH) is usually an acute, postinfectious AIHA in young children, but it can occur at any age. The Donath-Landsteiner antibody is classically a biphasic IgG anti-P (GLOB1) hemolysin. It binds P-positive red cells and fixes early complement components at a cool temperature. Rewarming to 37 °C allows complement activation to continue, causing intravascular lysis. The DAT therefore commonly shows C3d with little or no detectable IgG.1,7
PCH can produce abrupt fever, pallor, jaundice, back or abdominal pain, and red-brown urine. The laboratory pattern is acute intravascular hemolysis: a rapid hemoglobin fall, increased LDH and indirect bilirubin, depleted haptoglobin, hemoglobinemia, and hemoglobinuria. Reticulocytopenia can occur early in children. When a child shows this pattern, a C3d-positive DAT, and little cold-agglutinin activity, arrange Donath-Landsteiner testing immediately, because the specimen must stay at 37 °C from collection.4,7
Donath-Landsteiner testing
Collect the serum specimen into the tube required by the performing laboratory and keep it at 37 °C from collection until clotting and serum separation are complete. Cooling the specimen before separation can adsorb the antibody onto the patient's red cells and cause a false-negative result. Notify the testing laboratory before collection because the warm transport and processing steps must be ready.7,8
The test combines the patient's serum, P-positive group O reagent red cells, and a fresh complement source when the patient's complement is depleted. Parallel conditions establish the biphasic reaction:
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| Incubation sequence | Expected result in PCH |
|---|---|
| Cool phase followed by 37 °C | Hemolysis |
| 37 °C throughout | No hemolysis |
| Cool phase without rewarming | No hemolysis |
Hemolysis confined to the cool-then-warm condition demonstrates a biphasic hemolysin. A low antibody titer, complement depletion, recovery from the acute episode, or loss of antibody during specimen handling can produce a false-negative result. The controls and exact procedure follow the reference laboratory's validated method.7,8
Postinfectious childhood PCH is usually transient. The laboratory reports the abrupt intravascular hemolysis pattern and Donath-Landsteiner result promptly to pediatrics or hematology. If red-cell transfusion is ordered, the transfusion service supplies compatible units and follows the local warming policy. P-negative units are generally impractical because P is nearly universal and the antibody is usually transient. Pediatrics or hematology directs the remaining supportive care and treatment.4,7,9
PCH and paroxysmal nocturnal hemoglobinuria (PNH) have similar names. PNH arises from an acquired hematopoietic clone with deficient glycosylphosphatidylinositol-anchored complement regulators, including CD55 and CD59. Flow cytometry demonstrates the deficient populations. PCH is identified by the postinfectious pattern and a Donath-Landsteiner antibody.1
Marker timing in intravascular hemolysis
Free hemoglobin first consumes circulating haptoglobin. Once binding capacity is exceeded, hemoglobinemia and hemoglobinuria appear. These findings can fade quickly after lysis stops. Methemalbumin can remain detectable over the following days. Renal tubular cells retain iron from filtered hemoglobin, and urinary hemosiderin usually appears about 1 week after onset and can persist after plasma and urine free hemoglobin have cleared. Interpret each marker with the time since the episode began, because plasma free hemoglobin can be normal when urinary hemosiderin is positive.4,7
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| Feature | CAD | CAS | PCH |
|---|---|---|---|
| Usual setting | Primary clonal marrow B-cell disorder | Infection, overt lymphoid malignancy, or autoimmune disease | Acute postinfectious illness, usually in a young child |
| Antibody | Usually monoclonal IgM anti-I | Usually IgM; anti-I or anti-i depends on the association | Usually biphasic IgG anti-P |
| DAT | Strong C3d; IgG absent or weak | Strong C3d; IgG absent or weak | C3d; IgG often absent by routine DAT |
| Hemolysis | Chronic extravascular pattern with possible acute intravascular exacerbations | Follows the underlying condition | Acute intravascular pattern |
| Confirming study | Cold agglutinin titer, thermal amplitude when needed, and clonal evaluation | Cold agglutinin studies plus evaluation of the associated condition | Donath-Landsteiner test |
Practice
References
- Bloch EM, Campbell-Lee S, McKenna DH Jr, Montemayor-Garcia C, Schwartz J, Shaz B, Storry J, eds. Technical Manual. 22nd ed. AABB; 2026. Back to text
- Jäger U, Barcellini W, Broome CM, et al. Diagnosis and treatment of autoimmune hemolytic anemia in adults: recommendations from the First International Consensus Meeting. Blood Rev. 2020;41:100648. doi:10.1016/j.blre.2019.100648 Back to text
- 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 Back to text
- Hill QA, Stamps R, Massey E, Grainger JD, Provan D, Hill A. The diagnosis and management of primary autoimmune haemolytic anaemia. Br J Haematol. 2017;176(3):395-411. doi:10.1111/bjh.14478 Back to text
- Ubezio G, Barcellini W, Coluzzi S, et al. Standardized diagnostic approach for cold agglutinin disease: results from a Delphi-based expert consensus. Blood Transfus. Published online July 15, 2026. doi:10.2450/BloodTransfus.1314 Back to text
- 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 Back to text
- 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 Back to text
- Michigan Medicine MLabs. Donath-Landsteiner Test. Updated February 18, 2026. Accessed August 28, 2026. Back to text
- Cooling LL. Kids, colds, and complement: paroxysmal cold hemoglobinuria. Transfusion. 2017;57(6):1332-1335. doi:10.1111/trf.14128 Back to text