Cold Agglutinin Disease (CAD)

Cold agglutinin disease (CAD) is a rare condition that is found in association with 15% of cases of autoimmune hemolytic anemia (AIHA). Hemolysis mediated by cold agglutinins results in mild to moderate chronic anemia. Besides hemolysis, clinical features of CAD include cold-induced circulatory conditions such as Raynaud disease, acrocyanosis, and livedo reticularis.1 CAD is diagnosed mainly with various laboratory tests.2 

CAD is characterized by the presence of hemolytic anemia; increased serum bilirubin, reticulocyte, and lactate dehydrogenase levels; and Coombs test results that are positive for anti-C3 antibodies and typically negative for anti-IgG antibodies.1

Handling of Specimens in Cases of CAD

The proper handling of specimens is crucial during testing for cold agglutinins. Drawn blood should be stored in prewarmed tubes and kept warm until tested. If this precaution is not taken, the red blood cells stick together, so that the values obtained for the hemoglobin level and hematocrit are inaccurate. The cold agglutinin titers used to determine the number of antibodies in the plasma may also be inaccurate if the antibodies sticj together and sink to the bottom of the tube. As a consequence, the diagnosis of CAD is usually delayed. It is recommended that water baths be used to maintain the temperature of samples at 98.6°F.2

Complete Blood Cell (CBC) Count and Peripheral Blood Smear

Red blood cell agglutination is the characteristic finding in the peripheral blood smear, usually in combination with polychromasia and anisocytosis, features suggestive of compensatory erythropoiesis. The changes in the CBC count are due to agglutination, which leads to falsely high values for the mean corpuscular volume (MCV) and falsely low values for the red blood cell count. The estimated values for the hematocrit are therefore inaccurate. In rare cases, agglutination of white blood cells (WBCs), including monocytes and granulocytes, may also be seen because of the presence of I antigen on the surface of WBCs.This may occasionally result in spurious values for the total or differential WBC count.3

Reticulocyte Count

Reticulocytosis is usually observed in cases of chronic or ongoing hemolysis. However, the reticulocyte count may be normal if hemolysis has taken place recently. It may also be normal in cases of underlying bone marrow disease, which interferes with compensatory erythropoiesis, or if the autoantibodies bind to both precursor and mature erythrocytes.4

Biochemical Tests

Increased levels of indirect bilirubin suggest extravascular hemolysis. Elevated levels of lactate dehydrogenase indicate severe intravascular destruction of red blood cells. Decreased haptoglobin levels are also a sensitive marker of hemolysis.5

The Coombs test or direct antiglobulin test is central to the diagnosis of AIHA. Monospecific antisera (anti-IgG, anti-IgA, anti-IgM, anti-complement [C]) are used. The test helps to distinguish between warm AIHA due to an IgG autoantibody (direct antiglobulin test [DAT] result positive with IgG or IgG+C at low titer) and cold AIHA due to an IgM antibody (DAT result positive with anti-C and high titer of cold agglutinins).5 The diagnostic criteria for CAD include chronic hemolysis, positive polyspecific DAT result, and strongly positive monospecific DAT result for C3d.3,6 However, the DAT result can be weakly positive for IgG in addition to C3d in up to 20% of cases.3

Measurement of the cold agglutinin titers is required for all patients with suspected CAD. The thermal amplitude is measured only in the management of complex cases.4 The cold agglutinin titer indicates the strength (concentration and avidity) of the antibody. The titer is measured by testing serial dilutions of the patient’s serum to determine its ability to agglutinate red blood cells. The highest dilution at which the cold-induced agglutination of red blood cells takes place is the titer.4 Patients with CAD have high cold agglutinin titers. The threshold titer for a diagnosis of CAD is 1:64. A titer of 1:512 is considered clinically significant. In many cases of CAD, the titer is higher than 1:2048.4

Serum Protein Electrophoresis and Immunoelectrophoresis

Serum protein electrophoresis and serum immunoelectrophoresis or immunofixation are preliminary tests used to diagnose dysproteinemia. Further, the serum levels of IgG, IgA, and IgM must be quantified when the results of initial tests are abnormal. The results of these tests may be normal or abnormal, showing an increase in levels of IgM with κ or λ light chains.7

Urine Tests

Urinalysis is important to detect hemoglobinuria, hemosiderinuria, and raised urobilinogen levels. The urine sample should be fresh; otherwise, in vitro hemolysis of red blood cells in the urine can lead to a false finding of hemoglobinuria. Urine immunoelectrophoresis must be done if abnormal serum globulins are present. A 24-hour urine sample is required to exclude the presence of light chains.7

Tests for Hematolymphoid Malignancies

Bone marrow aspiration and biopsy are recommended to exclude underlying hematolymphoid diseases. Flow cytometry studies of bone marrow samples are useful to confirm the presence of an abnormal monoclonal lymphoid population. A lymph node biopsy is also required in cases of unexplained lymphadenopathy. This is preferred to fine-needle aspiration because preservation of the nodal architecture is crucial for an accurate diagnosis.5

Testing for Infections

For patients who have a fever or any respiratory symptoms, particularly of recent onset, it becomes important to search for an underlying infection. The infections mainly implicated in secondary CAD are Mycoplasma pneumonia and infectious mononucleosis.Tests for Mycoplasma infection are required in cases of atypical pneumonia. Specific tests for infectious mononucleosis are particularly required for a child or young adult presenting with symptoms.4 

Imaging Studies

A chest radiograph is required in cases of Mycoplasma pneumoniae infection to look for pulmonary infiltrates. Computed tomography of the chest and abdomen is needed to assess for splenomegaly and lymphadenopathy if lymphoma is suspected.7

Workup for Underlying Autoimmune Disease

Testing for an autoimmune disease is required in cases with features of malar rash, cytopenias, arthralgias, or arthritis.4


  1. Swiecicki PL, Hegerova LT, Gertz MA; Cold agglutinin disease. Blood. 2013;122(7):1114-1121.
  2. Broome CM.  Diagnosis and treatment of cold agglutinin disease. Clin Adv Hematol Oncol. 2019;17(3).
  3. Berentsen S. (2018), How I manage patients with cold agglutinin disease. Br J Haematol. 2018;181:320-330.
  4. Brugnara C, Berentsen S. Cold agglutinin disease. Updated April 22, 2021. Accessed September 8, 2021.
  5. Barcellini W, Giannotta J, Fattizzo B. Autoimmune hemolytic anemia in adults: primary risk factors and diagnostic procedures. Expert Rev Hematol. 2020;13(6):585-597. doi:10.1080/17474086.2020.1754791
  6. Berentsen S. Cold agglutinin disease. Hematology Am Soc Hematol Educ Progam. 2016;2016(1):226-231. doi:10.1182/asheducation-2016.1.226
  7. Aljubran SA. Cold agglutinin disease workup. MedScape. Updated August 23, 2021. Accessed September 8, 2021.

Reviewed by Kyle Habet, MD, on 9/9/2021.