Diagnosis of Cold Agglutinin Disease

Cold agglutinin disease (CAD) is a form of autoimmune disease, characterized by hemolytic anemia, caused by the attack and destruction of red blood cells (RBCs) by autoantibodies.

The diagnosis of CAD is a multi-step process that includes a physical examination to check for signs and symptoms and laboratory tests to establish hemolytic anemia induced by autoantibodies also called cold agglutinins.

Further tests may be required to identify the underlying cause of the disease, such as infections, lymphoid cancers, or other autoimmune diseases in the case of secondary CAD.

Moreover, tests may be required to differentiate CAD from other autoimmune diseases that can also lead to hemolytic anemia.

Physical examination

The main reason to suspect CAD includes the fact that signs and symptoms are cold-induced. For example, patients may have pain and discomfort when swallowing cold food or drink, develop hemolytic anemia upon cold exposure, or the RBCs may clump together in a cooled blood collection tube or blood smear.

Physical examination can also identify typical symptoms of cold-induced hemolytic anemia such as pale bluish tinge on fingertips, toes, and nose (acrocyanosis), patchy, reticulated vascular pattern on the skin with a red-blue or violet coloration (livida reticularis), weakness, weight loss, dizziness, headaches, pain in the back or legs, vomiting or diarrhea, chest pain, and jaundice.

Sometimes, physical examination can reveal a spleen enlargement (splenomegaly).

Laboratory tests

Complete blood count

A routine blood test analyzes the relative numbers of different types of blood cells including RBCs, white blood cells (WBCs), platelets, and others. In general, RBC counts are significantly lower, but WBC and platelet counts are normal in people with CAD.

Moreover, a complete blood count can also reveal reticulocytosis, a condition generally seen in anemia wherein immature RBCs called reticulocytes are found in the blood to compensate for the reduced numbers of mature RBCs.

Blood smear

The analysis of a blood smear can reveal RBC clumping, especially under cold conditions, which is characteristic of CAD.

Biochemical tests

Biochemical assays for lactate dehydrogenase (LDH) enzyme activity and amounts of bilirubin and haptoglobin protein are generally performed to confirm hemolytic anemia in CAD. In general, bilirubin and LDH levels are high, whereas haptoglobin protein levels are low in CAD samples compared to normal ones.

Coombs test

A Coombs or direct antiglobulin test is performed to detect complement proteins such as C3d, and antibodies such as IgM, which are attached to the surface of RBCs in the case of autoimmune diseases such as CAD, targeting them for destruction and causing hemolytic anemia.

Cold agglutinin titer

In many instances, to confirm CAD, the titer of cold agglutinins is determined. In this case, the serum, or liquid, portion of the blood of the patient containing antibodies is serially diluted and the highest dilution at which cold-induced clumping of RBCs occurs is determined. Generally, the accepted titer for diagnosis is above 1:64.

Thermal amplitude test

While not necessary for confirming CAD, the temperature at which the cold agglutinin binds to RBCs can be determined. Thermal amplitude is generally the highest temperature at which the autoantibody will bind to the RBCs.

In general, a positive diagnosis for CAD is reached if:

  • There is evidence of hemolysis based on high reticulocyte count, high LDH, high indirect bilirubin, and low haptoglobin;
  • The Coombs test for C3d and IgM is positive;
  • Cold agglutinin titer equal to or above 64 at 4°C.

Other details

Once CAD is confirmed, other tests may be necessary to identify the underlying cause of the disease such as infections, lymphoid malignancies or cancers, or other autoimmune disorders.

Also, a differential diagnosis may be required to rule out other causes of cold-induced symptoms such as primary Raynaud’s phenomenon, Raynaud’s phenomenon associated with other underlying disorders, and cryoglobulinemia.

Finally, the differential diagnosis of CAD against other causes of hemolytic anemias such as warm antibody hemolytic anemia, drug-induced immune hemolytic anemia, hemolytic transfusion reaction, and paroxysmal cold hemoglobinuria may also be performed.


Last updated: July 28, 2019


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