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Cold Agglutinin Titer

Cold agglutinins are IgM antibodies which agglutinate red blood cells at cold temperatures between 0 and 30oC. They usually bind to polysaccharides on the RBC surface. The most common targets on the RBC membrane are the I and i blood group antigens. Cold agglutinins can cause acrocyanosis and hemolytic anemia.

Cold agglutinin disease accounts for 20 to 30% of all autoimmune hemolytic anemia cases. Primary cold agglutinin disease is a low-grade monoclonal B-cell disorder without evidence of lymphoma or other malignancy. Secondary cold agglutinin syndrome is associated with lymphoma or infection. Cold agglutinin syndrome can be triggered by infections with Mycoplasma pneumoniae, Epstein Barr Virus, HIV, SARS-CoV-2, legionella, legionella, Citrobacter, influenza and varicella. Cold autoantibodies often appear one to two weeks after infection.

Hemolysis is mediated by complement fixation on RBC membranes and phagocytosis. Proximal complement components become bound to the RBC membrane by interaction with the IgM autoantibody. IgM often dissociates from the membrane at warmer temperatures. Bound C3b leads to opsonization and phagocytosis by reticuloendothelial cells located primarily in the liver. Bound C3b is converted to C3d which is detectable by the direct antiglobulin test. The incidence of both venous and arterial thrombosis is increased in patients with cold agglutinin disease.

Most cases have extravascular hemolysis, but 15 to 20% of cases have intravascular hemolysis. Acute infections and cardiopulmonary bypass are common triggers of intravascular hemolysis. The latter cases exhibit increased lactate dehydrogenase, decreased haptoglobin and hemoglobinuria.

The titer of cold agglutinins is the highest dilution of plasma that agglutinates RBCs. Cold agglutinins are present in 95% of healthy patients at titers of 16 or less. Titers less than 32 are considered negative. Titers above 512 are usually clinically relevant, but the best prediction of the biological activity of cold autoantibodies is thermal amplitude, which is the highest temperature at which antibodies agglutinate RBCs. Harmless cold autoantibodies react with RBCs up to a temperature of 10 to 15 degrees C in vitro, while antibodies reacting at 30 degrees C or higher can cause RBC agglutination in the acral circulation. The latter can cause acrocyanosis, Raynaud’s phenomenon, ulcerations and gangrene.

In the past, cold agglutinin titers were often used as a surrogate test for Mycoplasma pneumonia, since mycoplasma infections are often associated with elevated anti-I titers.   More specific mycoplasma IgG and IgM immunoassays are preferred to diagnose Mycoplasma pneumonia.

Corticosteroids and splenectomy are ineffective treatments in most cases of cold agglutin disease. Splenectomy can exacerbate the risk of thromboembolism. Severe anemia can be managed with transfusions of phenotypically matched RBCs. Therapeutic plasma exchange can be used in severe cases to rapidly remove cold agglutinins. The American Society for Apheresis considers plasma exchange a Category 2 indication. Medications include rituximab in combination with fludarabine or ibrutinib.

Specimen requirement is one 10 mL SST tube of blood.  The tube should be immediately placed in a 37 C water bath and allowed to clot.  After 10 minutes, the tube can be centrifuged, and the serum transported to the laboratory at ambient temperature.  If a water bath is not available, the tube should be transported at ambient temperature without centrifugation.


Patriquin CJ and Pevenski K. Transfusion 2022; 62:2-10.

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