Cold Agglutinin Disease (CAD)

Cold agglutinin disease (CAD) is a rare type of autoimmune hemolytic anemia (AIHA) in which cold agglutinins, or autoantibodies, bind to the surface of erythrocytes, promoting hemolysis. Most cold agglutinins target the carbohydrate I/i antigens that are ubiquitous on the surface of red blood cells (RBCs), forming weak hydrogen and van der Waals bonds sensitive to temperature.1,2 These weak bonds between antigen and cold agglutinin are strengthened by lower ambient temperatures via the reduction of spontaneous brownian motion within the bonds.1


The I antigen is inactive until after birth, when an enzyme that catalyzes the addition of lactosamine disaccharides to galactose becomes activated, progressively converting the little i antigen into the big I antigen.1,2 Therefore, anti-i autoantibodies typically agglutinate neonatal RBCs, whereas anti-I autoantibodies typically agglutinate adult RBCs.2 The big I antigen is found on the erythrocytes of approximately 99% of adults; little i is found in fewer than 1% of adults, who are termed “big I negative.”1 

In particular, cold agglutinins target erythrocyte surface antigens that contain polysaccharide epitopes within glycoproteins or glycolipids that include glycophorins or ceramide. Complex polysaccharides form the precursors of the ABO and Lewis blood group antigens. Specific anti-I or anti-i antibodies, labeled anti-IA, anti-IB, and so forth, contain components of the ABO or Lewis antigens as well. In rare cases, some unique antibodies (called anti-j antibodies) react equally well with both the I and i antigens.1

Cold Agglutinin Antibodies

CAD is caused predominantly by immunoglobulin M (IgM) antibodies in approximately 90% of cases.3 Immunoglobulin G (IgG) and immunoglobulin A (IgA) have been reported as antibodies that may contribute to the remaining 10% of cases of CAD, although it has been observed that IgA antibodies may not cause the typical hemolysis found in CAD.3-6

The pentameric structure of IgM antibodies allows them to bind to multiple erythrocytes, a feature that accounts for the notable degree of agglutination associated with them.1  

Cold Agglutinin Clonality

Cold agglutinins may be monoclonal or polyclonal. Monoclonal cold agglutinins develop from a single clone of lymphocytes that have one epitope specificity. Polyclonal cold agglutinins develop from multiple independent lymphocytes that have a variety of epitope specificities. Monoclonal cold agglutinins are more pathogenic than polyclonal agglutinins and are correlated with the lymphoproliferative disorders that are believed to be the basis of primary CAD.1 


It is believed that most cases of primary CAD originate from a low-grade lymphoproliferative disorder. Somatic mutations in the KMT2D (MLL2) and CARD11 genes have been observed in primary CAD.7     

Thermal Amplitude

Natural cold agglutinins have an optimum temperature of 3°C to 4°C and occur at low titers (<1:64) when measured at 4°C.1,2 They are inactive at higher temperatures.2 Pathologic cold agglutinins can cause erythrocytes to agglutinate at approximately 28°C to 31°C (sometimes even at approximately 37°C).1,2 

Location of Symptoms

Agglutination reactions occur especially in acral body parts, such as the nose, ears, fingers, and toes, which tend to be cooler than the more central regions of the body.2,8 The cold-induced symptoms that develop include Raynaud phenomenon, acrocyanosis, livedo reticularis, pain on swallowing cold food or liquids, and in more severe cases necrosis or ulceration.1  

Mechanism of Hemolysis via Complement Activation

Once cold agglutinins bind to the surface of RBCs, they activate the complement system by recruiting components of the classical complement pathway, including C1, C2, and C4.1,9 The enzyme C1 esterase activates C2 and C4 to yield C3 convertase. C3 convertase separates C3 into C3a and C3b.1 

Erythrocytes covered by C3b complement are targeted by macrophages of the immune system and typically undergo phagocytosis in Kupffer cells within the liver; thus, CAD hemolysis is usually extravascular, although it may be intravascular in some cases.1 

Intravascular hemolysis may occur when an individual with CAD is in an increased inflammatory state because of concurrent infection or a surgical procedure.1 These cases are classified as secondary CAD, which is CAD caused by an underlying condition, such as malignancy (eg lymphoplasmacytic lymphoma, marginal zone lymphoma, aggressive non-Hodgkin lymphoma, Waldenström macroglobulinemia) or infection (eg, Epstein Barr virus or cytomegalovirus infection, hepatitis B or C, influenza A, adenovirus or Mycoplasma pneumoniae infection, listeriosis, Escherichia coli lung infection, Legionella pneumophila pneumonia, Chlamydia pneumonia).8 

“Mixed” AIHA, in which both  IgG and IgM antibodies are involved, may result in relatively severe hemolysis that is in part intravascular.1 A key differentiator for determining whether hemolysis is intravascular or extravascular is the presence of hemoglobinuria, which typically does not occur in cases of extravascular hemolysis.1 

Phagocytosis mediated by IgM affects the entire antigen-antibody RBC complex, whereas phagocytosis mediated by IgG occurs only around a section of the cell membrane. When an individual with CAD is exposed to warmer temperatures, the IgM-antigen bonds weaken and IgM dissociates from the erythrocytes; however, the C3b remains attached to the erythrocytes and undergoes cleavage to form C3c and C3d.1,8 C3d is the complement detected by the Coombs test, one of the primary methods of diagnosing CAD.1   


  1. Brugnara C, Berentsen S. Cold agglutinin disease. UpToDate. Accessed September 9, 2021.
  2. Aljubran SA. What is the pathophysiology of cold agglutinin disease? Medscape. Updated December 2, 2020. Accessed September 9, 2021.
  3. Swiecicki PL, Hegerova LT, Gertz MA. Cold agglutinin disease. Blood. 2013;122(7):1114-1121. doi:10.1182/blood-2013-02-474437
  4. Silberstein LE, Berkman EM, Schreiber AD. Cold hemagglutinin disease associated with IgG cold-reactive antibody. Ann Intern Med. 1987;106(2):238-242. doi:10.7326/0003-4819-106-2-238
  5. Pereira A, Mazzara R, Escoda L, Alcorta I, Nomdedeu B, Roelcke D. Anti-Sa cold agglutinin of IgA class requiring plasma-exchange therapy as early manifestation of multiple myeloma. Ann Hematol. 1993;66(6):315-318. doi:10.1007/BF01695974
  6. Römer W, Rother U, Roelcke D. Failure of IgA cold agglutinin to activate C. Immunobiology. 1980;157(1):41-46. doi:10.1016/S0171-2985(80)80060-X
  7. Małecka A, Trøen G, Tierens A, et al. Frequent somatic mutations of KMT2D (MLL2) and CARD11 genes in primary cold agglutinin disease. Br J Haematol. 2018;183(5):838-842. doi:10.1111/bjh.15063 
  8. Gabbard AP, Booth GS. Cold agglutinin disease. Clinical Hematology International. 2020;2(3):95-100. doi:10.2991/chi.k.200706.001
  9. Shi J, Rose EL, Singh A, et al. TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins. Blood. 2014;123(26):4015-4022. doi:10.1182/blood-2014-02-556027

Reviewed by Harshi Dhingra, MD, on 9/10/2021.