Diagnosis of CAD

Cold agglutinin disease (CAD) is a condition in which the body’s own immune system attacks and destroys red blood cells — the cells that carry oxygen through the blood to all tissues in the body. Specifically, in CAD, the immune system makes antibodies, called cold agglutinins, which stick to red blood cells at cold temperatures, causing them to clump together (agglutinate) and ultimately be destroyed.

A CAD diagnosis is generally made through a combination of physical exams and laboratory tests.

Additional tests may be required to identify the underlying cause of the disease in the case of secondary CAD, a form of CAD that is associated with other illnesses, including infections, cancers, or other autoimmune disorders. Further tests may also be required to differentiate CAD from other diseases that can also lead to the premature destruction of red blood cells.

Physical examination

CAD is often first suspected when a person experiences certain symptoms in response to cold. For example, unexplained pain and discomfort when ingesting cold food or drinks, or a bluish discoloration (acrocyanosis) in the hands and feet when exposed to cold temperatures.

A physical exam can help identify these and other symptoms that may be indicative of CAD, such as a patchy, net-like pattern of purplish veins on the skin (livedo reticularis), jaundice (yellowed skin), or spleen enlargement.

Nailfold Capillaroscopy

A clinician may also perform a nailfold capillaroscopy, a non-invasive diagnostic test to examine blood vessels in fingernail beds under a microscope or other magnification device. This can help detect Raynaud’s phenomenon, a symptom of CAD and other diseases.

Normally, blood vessels are regularly shaped and evenly spaced across nail beds. In people with Raynaud’s phenomenon, these vessels can have an abnormal shape or be haphazardly spaced, with some areas of the nail bed not having any blood vessels at all.

Laboratory tests

Blood smear

A blood smear, as its name suggests, is a procedure in which a small amount of blood is “smeared” on a glass slide and then examined for abnormalities under a microscope. In CAD, blood smears may reveal decreased levels of red blood cells and/or agglutination, when cells clump together at cold temperatures.

Agglutination at cold temperatures is also sometimes noticed incidentally when blood samples are taken for other reasons, which may also be helpful in diagnosing CAD.

Complete blood count

A complete blood count is a test that measures the total number of cells and other components in a blood sample. In people with CAD, a complete blood count will typically indicate low red blood cell levels.

CAD patients can also have elevated levels of immature red blood cells, called reticulocytes. This condition, called reticulocytosis, occurs in CAD because the body is attempting to compensate for the lack of mature red blood cells due to agglutination and the cells’ premature destruction.

Coombs Test

The Coombs test, which looks for antibodies that may bind to red blood cells, can also be used to detect the presence of cold agglutinins in people with CAD. There are two variations of the Coombs test: direct, which looks for cold agglutinins or other antibodies that are bound to red blood cells; and indirect, which looks for free-floating antibodies in the blood.

Cold agglutinin titer

In this test, a sample of plasma (the liquid portion of blood) is serially diluted, and then incubated with red blood cells. The test then measures which dilutions are able to trigger red blood cell agglutination at low temperatures, which is indicative of CAD.

Everyone has small amounts of cold agglutinins in their blood. But people with CAD have so many of these self-reactive antibodies that even greater dilutions still have enough cold agglutinins to make red blood cells clump. The titer is the highest dilution of plasma at which red blood cell clumping occurs at cold temperatures. In general, titer levels of 1:512 or higher are considered clinically significant. In CAD patients, titers may be as high as 1:65,536.

Thermal amplitude test

In this test, a patient’s blood sample is incubated with red blood cells at different temperatures, allowing clinicians to determine the range of temperatures at which agglutination is induced. In general, the closer to body temperature that clumping occurs, the more harmful are a patient’s cold agglutinins.

Other blood tests

There are several substances in the blood that can be measured to check whether red blood cells are being destroyed. For example, the destruction of red blood cells commonly leads to increased levels of lactate dehydrogenase (LDH) and bilirubin, whereas it causes the levels of another protein, called haptoglobin, to drop. Measuring the levels of these molecules may be useful in diagnosing CAD and other forms of anemia.

Liver function may be indirectly assessed by measuring the levels of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in the blood, since high levels of these liver enzymes are indicative of liver damage. This is important in CAD because the liver plays a critical role in clearing damaged red blood cells, which are much more abundant in people with this disease.


Last updated: Oct. 14, 2021


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