Laboratory Tests to Diagnose Cold Agglutinin Disease

Cold agglutinin disease (CAD) is a rare autoimmune disorder in which exposure to cold temperatures (32o to 50o Fahrenheit) triggers the immune system to mistakenly attack red blood cells (RBCs), causing them to lyse (disintegrate). Blood samples taken at a hospital or clinic may be used to diagnose CAD. The blood is analyzed using multiple laboratory tests, which may include a complete blood count, a blood smear, biochemical tests, the Coombs test, a cold agglutinin titer, or a thermal amplitude test.

Complete blood count

A complete blood count (CBC) is a blood test in which the numbers of each type of blood cells within a blood sample are counted. Blood cell types include red blood cells, white blood cells, platelets, and others.

In patients with CAD, RBC counts are lower than normal, while white blood cell and platelet cell counts remain at normal levels. Occasionally, patients with CAD will have an increase in reticulocytes (immature RBCs) as the body tries to compensate for the low numbers of RBCs. This is called reticulocytosis.

Blood smear

A blood smear is an analysis in which a drop of blood is placed on a glass slide and examined under a microscope. For patients with CAD, the RBCs tend to visibly clump, especially in cold conditions, which is easily detectable with  this test.

Biochemical tests

There are several biochemical assays that can be performed on blood samples to test for CAD. The activity of an enzyme called lactate dehydrogenase (LDH) can be elevated in patients with CAD. LDH activity in the blood is normally quite low, but damage to tissues or to cells may dramatically increase the levels of LDH in the blood.

As the liver clears up the RBCs that have been destroyed by the immune system, a protein called bilirubin is produced. Normally produced at low levels by the recycling of RBCs, bilirubin levels in the blood can increase dramatically in CAD. In extreme cases, bilirubin levels increase to the point that it can cause jaundice (yellowing of the skin and eyes).

Haptoglobin is a protein that normally binds to free hemoglobin (the protein in RBCs that carries oxygen to tissues). In CAD, free hemoglobin is produced in large quantities, which means haptoglobin levels in the blood decrease.

Coombs test

A Coombs test, or direct antiglobulin test, is performed to detect immune system proteins such as C3d, and antibodies like IgM, that lyse RBCs. In patients with CAD, these proteins will be attached to the surface of the RBCs to target them for destruction.

Cold agglutinin titer

To confirm a diagnosis of CAD, a cold agglutinin titer may be performed. In this test, serum (the liquid portion of blood that does not contain cells) is diluted in series and added to normal blood samples. Serum from CAD patients contains immune proteins such as antibodies, which attack the RBCs of the normal blood sample, causing clumping. The highest dilution at which cold-induced clumping of RBCs occurs is determined; if clumping occurs at greater than 1 to 64 serum dilution, it is considered indicative of CAD.

Thermal amplitude test

While not necessary to confirm a CAD diagnosis, the temperature at which cold agglutinin binds to RBCs can be determined. The thermal amplitude test determines the highest temperature at which antibodies bind to RBCs.


Last updated: Aug. 8, 2019


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