Cold Agglutinin Disease Workup

Updated: Feb 07, 2022
  • Author: James Carson Collie, MD; Chief Editor: Michael A Kaliner, MD  more...
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Approach Considerations

Ensure proper handling of specimens when looking for cold agglutinins (ie, keeping the blood warm until it is tested). Blood specimens are commonly placed in a laboratory refrigerator until they are tested, but this practice must be avoided when testing for cold agglutinins. Blood studies used in the diagnosis of cold agglutinin disease include the following:

  • Complete blood cell count (CBC) with differential
  • Peripheral blood smear
  • Reticulocyte count
  • Direct Coombs test (direct antiglobulin test [DAT])
  • Serum protein electrophoresis and serum immunoelectrophoresis (immunofixation)
  • Cold agglutinin titer

Note that for the direct Coombs test (direct antiglobulin test [DAT]), blood should be drawn into prewarmed containers and kept at 37-38°C until serum/plasma has been removed. [48] Testing should be performed using polyspecific and monospecific Coombs sera, including monospecific anti-C3 and IgG antisera.

The following findings are required for the diagnosis of cold agglutinin disease [48] :

  • Chronic hemolysis
  • Positive polyspecific DAT
  • Monospecific DAT strongly positive for C3d
  • Cold agglutinin titer ≥64 at 4º C
  • Absence of overt malignancy or relevant infection ( Mycoplasma or Epstein-Barr virus)

Significant findings on other blood tests include the following:

  • Lactate dehydrogenase (LDH) and total and direct bilirubin values are elevated in cold agglutinin disease, depending on the extent of hemolysis.
  • The haptoglobin level may be reduced when hemolysis is active and ongoing with an intravascular spillover from a massive extravascular process and in the absence of significant liver disease.
  • Cryoglobulin levels should be tested only if vascular purpura or other atypical findings, such as elevated levels of IgM and/or hepatitis virus antibodies, are found. Again, proper handling of the sample, by keeping it warm until the test is run, is essential to avoid premature loss of the cryoglobulin


Perform urinalysis with microscopic assessment for red blood cells (RBCs) and a chemical test for hemoglobin to differentiate between hematuria and hemoglobinuria. Urinalysis can reveal hemoglobinuria, hemosiderinuria, and elevated urobilinogen.

Processing only fresh urine samples is an important means of avoiding in vitro hemolysis of RBCs in the urine, leading to an incorrect diagnosis of hemoglobinuria.

Urine immunoelectrophoresis should be performed if serum globulins are abnormal; only a 24-hour urine sample can be used to conclusively exclude the presence of light chains.

Imaging studies

A chest radiograph is obtained if pneumonia is suggested; pulmonary infiltrates are found in cases of Mycoplasma pneumoniae infection. Findings may also indicate lymphadenopathy. Computed tomography (CT) scans of the chest and abdomen are performed to assess for lymphadenopathy and splenomegaly when lymphoma is suggested.


Complete Blood Cell Count and Peripheral Smear

If the cold agglutinin is operative at room temperature, then a falsely high mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) with a low RBC count are obtained due to agglutination of RBCs in the cold automated counter.

Agglutination may also be seen in anticoagulated blood at room temperature. This agglutination worsens with storage and cooling of blood to 4°C and disappears rapidly upon warming the blood to 37°C, unlike with rouleaux formation. Repeating the complete blood count (CBC) after warming the blood to 37°C avoids this problem. Thus, the clinical laboratory is frequently the first to report the presence of a cold agglutinin. Agglutination in the cold may also interfere with typing and cross-matching of blood.

In patients with chronic cold agglutinin disease, a mild to moderate stable anemia is present; occasionally, the anemia is severe. Leukocytosis may be evident during hemolytic episodes. Peripheral blood smears may reveal clumps of RBCs (see the image below).

Peripheral blood smear showing several clumps of R Peripheral blood smear showing several clumps of RBCs with the largest in the center. These are typical of aggregates seen in persons with cold agglutinin disease.

Reticulocytes and Spherocytes

The results of the reticulocyte count are usually increased in patients with cold agglutinin disease, with polychromasia in the peripheral blood smear. The mean corpuscular volume (MCV) is elevated because of reticulocytosis, as well as agglutination of the RBCs.

However, reticulocytosis may be inadequate for the degree of anemia in the patient. This may be due to decreased erythropoiesis caused by an underlying infection.

Spherocytes may be present, although less prominently than in warm autoantibody–induced hemolytic anemias. (See the image below).

Blood smear showing spherocytic and agglutinated r Blood smear showing spherocytic and agglutinated red blood cells.

Blood Typing

Blood typing is performed in the event that a transfusion is needed. The presence of autoantibodies may interfere with blood typing because they may react with the RBCs of potential donors, making detection of alloantibodies difficult. Several techniques are available to improve compatibility testing. These include testing the patient's serum for anti-A and anti-B hemagglutinins and performing the compatibility testing reactions at 37°C to avoid inaccurate test results due to nonspecific agglutination in the cold.


Electrophoresis and Immunoelectrophoresis

Perform serum protein electrophoresis and serum immunoelectrophoresis (immunofixation) as initial tests to look for a dysproteinemia.

Quantitation of serum levels of IgG, IgA, and IgM should follow when a dysproteinemia is suggested based on results from the first 2 tests. These test results may be normal or abnormal (increased IgM with κ or λ light chains).

Careful sample processing, avoiding exposure of blood to the cold, and maintaining the blood at 37°C before testing are essential. If the blood sample is cooled and not kept warm from the time it is collected to the time it is tested, the cold agglutinin attaches to the RBCs and is removed from the serum, causing a false-negative result.


Cold Agglutinin Titers

With the cold agglutinin titer, a titer of 1:64 or greater is considered abnormal when blood is tested at 4°C. Obtain the cold agglutinin titers also at 30°C and 37°C, when needed. Testing at temperatures higher than 4°C is extremely valuable, particularly if the patient is to undergo hypothermia for surgery.

Cold agglutinin disease is usually associated with very high cold agglutinin titers of greater than 1:10,000 at 4°C, with a thermal amplitude of up to 30-32°C. The addition of bovine serum albumin (BSA) while testing for the cold agglutinin titer and thermal amplitude results in a better correlation with clinical hemolytic anemia than does obtaining data in the absence of BSA, using saline-suspended cells. [8]


Infectious and Vascular Disease Testing

Tests for the following infectious diseases can be performed if these disorders are being considered in the etiology of cold agglutinin disease:

  • Infectious mononucleosis (Epstein-Barr virus [EBV] infection)
  • Influenza
  • Mycoplasma pneumonia
  • HIV infection
  • Hepatitis
  • Cytomegalovirus (CMV) infection
  • Malaria (review of thick and thin blood smears)

If collagen vascular disease is a possibility, blood tests for the following disorders should be performed:

  • Systemic lupus erythematosus (SLE)
  • Rheumatoid arthritis
  • Scleroderma (systemic sclerosis)


Perform bone marrow aspiration and biopsy only when they are needed to exclude certain neoplastic or immunoproliferative diseases. Flow cytometry studies of bone marrow are helpful in defining the presence of an abnormal monoclonal population of lymphocytes.

A lymph node biopsy is necessary when unexplained lymphadenopathy is present. Fine-needle aspiration is not reliable in comparison with excision of the largest lymph node for diagnostic purposes; nodal architecture is important for making an accurate diagnosis and is preserved in a lymph node biopsy. The addition of flow cytometry to define the presence of 1 or more abnormal monoclonal lymphocyte populations is also useful.



Depending on the underlying precipitating illness, changes may be seen in the bone marrow and lymph nodes of patients with cold agglutinin disease. The presence of a malignant lymphoproliferative disorder may also be evident in biopsy samples. Clumps of RBCs may be observed in the peripheral smear. (See the images below.)

Peripheral blood smear showing several clumps of R Peripheral blood smear showing several clumps of RBCs with the largest in the center. These are typical of aggregates seen in persons with cold agglutinin disease.
Blood smear showing spherocytic and agglutinated r Blood smear showing spherocytic and agglutinated red blood cells.