Cold Agglutinin Disease (CAD)

Cold agglutinin disease (CAD) is a rare disease that impacts 15% of patients diagnosed with autoimmune hemolytic anemia.1 Cold-reacting autoantibodies attach to the membrane of erythrocytes, causing premature destruction and resulting in anemia. Immunoglobulin M (IgM) is the autoantibody responsible for approximately 90% of CAD cases. In rare cases, immunoglobulin G (IgG), immunoglobulin A (IgA), or λ light chain restriction result in CAD.2 


Diagnosis of CAD is a multistep process consisting of a comprehensive physical examination and laboratory tests to determine the underlying cause of the disease and rule out other diagnoses such as infections, other autoimmune disorders, and lymphoid cancers.3 

Physical Examination

Physical examination allows for documentation of the signs and symptoms of CAD, including pain and discomfort when swallowing cold foods or beverages and provocation of symptoms with exposure to cold. Common cold-induced circulatory symptoms include acrocyanosis, Raynaud’s disease, and livedo reticularis. Other clinical manifestations are dizziness, headaches, weakness, weight loss, chest pain, pain in the back or legs, jaundice, emesis, diarrhea, and splenomegaly.1,3 One rare clinical manifestation is cutaneous necrosis.1

Diagnostic criteria for CAD include chronic hemolysis, a positive polyspecific direct antiglobulin test (DAT), monospecific DAT for C3d, a cold agglutinin (CA) titer ≥64 at 4 °C, and the absence of malignancy or relevant infections such as Epstein-Barr virus (EBV) or Mycoplasma.3,4  

Infectious disease testing when determining the etiology of CAD must include influenza, hepatitis, malaria, human immunodeficiency virus (HIV) infection, cytomegalovirus infection, Mycoplasma pneumonia, and infectious EBV.5 Blood tests should be performed to determine the presence of collagen vascular diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, and scleroderma (systemic sclerosis).5 

Laboratory Tests

Blood tests used to diagnose CAD include a routine complete blood cell count (CBC), a peripheral blood smear, a reticulocyte count, the Coombs test, a CA titer, and serum protein electrophoresis and serum immunoelectrophoresis ​​(immunofixation).5

Typically, clinical laboratories are the first to report CAD since blood is commonly refrigerated prior to CBC testing. Agglutination increases as blood is cooled to 4 °C and may be present even in anticoagulated blood at room temperature. Due to erythrocyte destruction, CBC results in CAD include a low red blood cell (RBC) count that indicates a mild to moderate anemia. Rarely, severe anemia is observed, and the patient may require transfusion. Leukocytosis may also occur during hemolytic attacks.5

In cases requiring transfusion, blood typing should be performed to determine the compatibility of the donor blood with that of the individual with CAD. Improvements for compatibility may test the patient’s serum for anti-A and anti-B hemagglutinins, as well as compatibility testing reactions at 37 °C to avoid inaccurate results from cold agglutination.5 

Peripheral blood smears commonly show clumping or agglutination of RBCs in individuals with CAD.5

Individuals with CAD demonstrate increased reticulocyte counts with polychromasia on peripheral blood smears. The agglutination of RBCs and reticulocytosis elevate the mean corpuscular volume (MCV). Spherocytes may also be visible, although these are more predominant in warm autoantibody-induced hemolytic anemias than in CAD.5

The Coombs test is a DAT that detects the presence of specific antibodies attached to the membranes of erythrocytes. The Coombs test is almost always positive for IgM in individuals with CAD, as this accounts for up to 90% of cases.2,5,6 In the remaining 10% to 20% of cases, the Coombs test or another monospecific DAT may weakly indicate IgG or IgA as the causative antibody.2,5 The Coombs test may be either a direct or indirect test. The direct Coombs test indicates antibodies that are attached to the erythrocytes’ membranes, whereas the indirect Coombs test detects free floating antibodies in the blood.7  

The Coombs test or other monospecific DATs are also used to detect the presence of complement proteins, such as C3d, that attach to the RBC membranes like the autoantibodies. Chandesris et al reported the following DAT results in 58 patients with CAD: 74% demonstrated C3d alone, 3.4% showed IgG alone, and 22.4% had a combination of C3d and IgG.8 

A CA titer is measured by serially diluting the patient’s serum and determining the highest dilution at which cold-induced clumping of RBCs occurs. In general, the accepted CA titer to diagnose CAD is above 1:64 at 4 °C.3 The CA titer level is less indicative of CAD clinical manifestations since hemolysis can occur at titer levels as low as 1:32.1 Stone et al performed serum assays of 172 patients with IgM monoclonal proteins. Cold agglutinin activity occurred in 10 of 117 patients (8.5%), with CA titers ranging from 1:512 to 1:65,536.9

Serum protein electrophoresis and serum immunoelectrophoresis (immunofixation) may be performed as initial diagnostic tests to indicate dysproteinemia. The quantitation of serum levels of IgG, IgA, and IgM is subsequently performed when dysproteinemia is identified. These serum levels may be normal or abnormal, as in the case of increased IgM levels with κ or λ light chains.5 

The serum immunoglobulin tests and CA titers must be conducted carefully to ensure that the blood is not exposed to cold and is maintained between 37 and 38 °C before testing. Any cooling of the blood results in a false negative when the CAs are removed from the serum by attaching to RBC membranes.4,5  

Thermal Amplitude Test

While it is not necessary for CAD diagnosis, a thermal amplitude test may be conducted to determine the temperature at which CAs bind to RBC membranes. Thermal amplitude is the highest temperature at which autoantibodies bind to RBCs.3 CAD typically correlates to extremely elevated CA titers of greater than 1:10,000 at 4 °C accompanied by a thermal amplitude of up to 30 to 32 °C.5 Improved correlation of CA titers and thermal amplitude testing with clinical hemolytic anemia occurs with the addition of bovine serum albumin (BSA) when compared with saline-suspended cells that do not contain BSA.10 


Bone marrow aspiration or biopsy is only required to rule out malignancies. Flow cytometry studies of bone marrow may be used to detect abnormal monoclonal lymphocytes. Lymph node biopsies may be required to determine the cause of unexplained lymphadenopathy.5 

Biochemical Tests

Biochemical assays including bilirubin levels, haptoglobin protein levels, and activity of lactate dehydrogenase (LDH) can confirm the presence of hemolytic anemia in individuals with CAD. LDH and bilirubin levels tend to be high, while haptoglobin protein levels are low in individuals with CAD.3


  1. Swiecicki PL, Hegerova LT, Gertz MA. Cold agglutinin disease. Blood. 2013;122(7):1114-1121. doi:10.1182/blood-2013-02-474437
  2. Aljubran SA. Cold agglutinin disease: practice essentials. Medscape. Updated August 23, 2021. Accessed September 1, 2021.
  3. Diagnosis of cold agglutinin disease. Cold Agglutinin Disease News. Updated July 28, 2019. Accessed September 1, 2021.
  4. Berentsen S. Cold agglutinin disease. Hematology Am Soc Hematol Educ Program. 2016;2016(1):226-231. doi:10.1182/asheducation-2016.1.226
  5. Aljubran SA. Cold agglutinin disease workup. Medscape. Updated August 23, 2021. Accessed September 1, 2021.
  6. Cold agglutinin disease. Genetic and Rare Diseases Information Center. Accessed September 1, 2021.
  7. Medical tests: Coombs test. UCSF Health. AccessedSeptember 1, 2021. 
  8. Chandesris MO, Schleinitz N, Ferrera V, et al.  [Cold agglutinins, clinical presentation, and significance; retrospective analysis of 58 patients]. Rev Med Interne. 2004;25(12):856-865. French. doi:10.1016/j.revmed.2004.08.001
  9. Stone MJ, McElroy YG, Pestronk A, Reynolds JL, Newman JT, Tong AW. Human monoclonal macroglobulins with antibody activity. Semin Oncol. 2003;30(2):318-324. doi:10.1053/sonc.2003.50077
  10. Garratty G, Petz LD, Hoops JK. The correlation of cold agglutinin titrations in saline and albumin with haemolytic anaemia. Br J Haematol. 1977;35(4):587-595. doi:10.1111/j.1365-2141.1977.tb00623.x

Reviewed by Debjyoti Talukdar, MD, on 9/7/2021.