The use of reactivity testing and genotyping is necessary to differentiate cold agglutinin disease (CAD) from other forms of autoimmune hemolytic anemia (AIHA) to provide optimal care for patients, according to a new study published in Hematology/Oncology Clinics of North America.

“In the presence of autoantibodies, the failure to identify an underlying alloantibody may lead to a (hemolytic) transfusion reaction. Therefore, the aim of pretransfusion testing for patients with AIHA, just as it is for all patients who require transfusion, is to identify (clinically significant) underlying alloantibodies,” the authors said.

CAD may result in patients with lymphomas or chronic lymphocytic leukemia. It may also occur as a result of infections such as Mycoplasma pneumoniae or mononucleosis. AIHA, including CAD, is becoming increasingly reported following treatment with anticancer drugs or transplantation, especially allogeneic hematopoietic stem cell transplantation.

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Most cold-reactive autoagglutinins found in patients with CAD have specific reactivity to I antigens and much less commonly to i antigens, the authors said. The I antigen is primarily found in red blood cells (RBCs) of adult patients and is less common in cord blood samples while the opposite is true of the i antigen.

Read more about CAD differential diagnosis

Patients with CAD often have autoanti-I titers of 1000 or above in contrast to levels of 64 or lower in most healthy people. These autoanti-I agglutinins are usually immunoglobulin M (IgM) and only rarely immunoglobulin G. 

In CAD, the IgM binds below temperatures of 37 °C and maximally at temperatures between 0 °C and 5 °C. When collecting blood samples, it is important to keep the blood at 37 °C until the RBCs are separated from the serum, or else spontaneous agglutination may occur and cause problems with ABO/RhD determination and typing for other antigens. In rare occurrences, the autoantibody specificity may include epitopes of glycophorin A such as anti-Pr or anti-M.

“Autoantibodies, whatever their etiology, interfere with the pretransfusion testing of patients requiring RBCs transfusion making compatibility testing complex and labor-intensive,” the authors said. “Transfusion of RBCs that are selected based on the patient’s extended phenotype can provide a significant measure of safety as they avoid the patient being immunized to antigens absent from their RBCs.”


Lomas-Francis C, Westhoff CM. Red cell antigens and antibodies. Hematol Oncol Clin North Am. 2022;36(2):283-291. doi:10.1016/j.hoc.2021.12.002