Cold Agglutinin Disease

Updated: Sep 12, 2016
  • Author: Salman Abdullah Aljubran, MD; Chief Editor: Michael A Kaliner, MD  more...
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Cold agglutinin disease is a rare form of autoimmune hemolytic anemia caused by cold-reacting autoantibodies. Autoantibodies that bind to the erythrocyte membrane leading to premature erythrocyte destruction (hemolysis) characterize autoimmune hemolytic anemia. (See Pathophysiology and Etiology.) Peripheral blood smears may reveal clumps of RBCs (see the image below.)

Peripheral blood smear showing several clumps of R Peripheral blood smear showing several clumps of RBCs with the largest in the center. These are typical of aggregates seen in persons with cold agglutinin disease.

Autoimmune hemolytic anemia is classified as primary or secondary and is subclassified according to autoantibody type. Primary cold agglutinin disease is characterized by a clonal lymphoproliferative disorder. [1, 2] Secondary cold agglutinin syndrome results from a systemic disease—infection or malignancy. [3]

In 90% of cases, the autoantibody in cold agglutinin disease is immunoglobulin M (IgM); rarely, it may involve monoclonal immunoglobulin G (IgG), immunoglobulin A (IgA), or λ light chain restriction. In contrast, warm autoimmune hemolytic anemia predominantly involves IgG. [1] Donath-Landsteiner hemolytic anemia is also caused by a cold-reacting immunoglobulin, but most cases are due to polyclonal IgG. [1]  

Another autoimmune hemolytic anemia syndrome associated with cold-reacting autoantibodies is paroxysmal cold hemoglobinuria, which involves the IgG Donath-Landsteiner (D-L) antibody. Unlike cold agglutinin disease, in which affected red blood cells (RBCs) are removed via extravascular phagocytosis, paroxysmal cold hemoglobinuria involves intravascular hemolysis. (See DDx.)

Primary and secondary disease

Primary cold agglutinin disease is usually associated with monoclonal cold-reacting autoantibodies. Primary cold agglutinin disease is chronic and occurs after the fifth decade of life, with a peak incidence in the seventh and eighth decades. (See Epidemiology.)

Secondary cold agglutinin disease may be associated with either monoclonal or polyclonal cold-reacting autoantibodies. It predominantly is caused by infection and lymphoproliferative disorders. Monoclonal secondary disease is usually chronic, occurring in adults. Polyclonal secondary cold agglutinin disease, which occurs in children and young adults, is usually transient. (See Etiology and Prognosis.)

Requirements for induction of active hemolytic anemia

Several factors play a role in determining the ability of a cold agglutinin to induce an active hemolytic anemia. [4, 1] These include the following:

  • Ability to initiate
  • Extent of antibody-induced complement activation
  • Concentration of the antibody
  • Range of temperatures, including the highest temperature at which the antibody interacts with the RBC (its thermal amplitude)
  • Qualitative binding of IgM to the RBC
  • Modification of the antibody's ability to fix complement components onto the RBCs


Cold agglutinins, or cold autoantibodies, occur naturally in nearly all individuals. These natural cold autoantibodies occur at low titers, less than 1:64 measured at 4°C, and have no activity at higher temperatures. Pathologic cold agglutinins occur at titers over 1:1000 and react at 28-31°C and sometimes at 37°C.

Cold agglutinin disease usually results from the production of a specific IgM antibody directed against the I/i antigens (precursors of the ABH and Lewis blood group substances) on red blood cells (RBCs). Cold agglutinins commonly have variable heavy-chain regions encoded by VH, with a distinct idiotype identified by the 9G4 rat murine monoclonal antibody. [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20]

Because the I antigen is not activated until after birth, anti-i autoantibodies predominantly agglutinate neonatal RBCs, and anti-I autoantibodies predominantly agglutinate adult RBCs.

VH genes

The 9G4 idiotope is localized to the V4-34 encoded portion of the variable region. [21] It is found on cold agglutinin-producing malignant lymphoid cells in the bone marrow in persons with lymphoproliferative disorders, on a small proportion of normal lymphoid cells, and in the spleen of a 15-week-old fetus. In contrast, the cold agglutinins found in healthy individuals, those with no clinical symptoms, are often derived from a variable segment other than the V4-34 portion. [22, 23]

The VH genes appear to regulate not only the production of cold agglutinins, but also the formation of normal antibodies to other carbohydrate antigens, both sharing the same fundamental mechanism of production. The I/i antigen analogues are present on human lymphocytes, neutrophils, and monocytes and in human saliva, milk, and amniotic fluid. Thus, in disease states, the finding of a clone of B cells producing this antibody may be the result of expansion of a normal clone that is specific for the production of an immunoglobulin with these properties. Autoimmune and lymphoproliferative disorders can also be associated with the production of cold agglutinins.

In vitro studies have shown that human monoclonal antibodies encoded by the V4-34 gene segment not only have cold agglutinin properties but also exhibit multireactivity. This is in contrast to the generally monospecific I/i reactivity of sera from patients with cold agglutinin disease. [18]

Associated infections

The hemolytic anemia associated with monoclonal cold agglutinins is typically more serious than that associated with polyclonal cold agglutinins. The monoclonal form is usually chronic, whereas the polyclonal form is often limited. [24]

Some polyclonal IgM cold agglutinins arise in association with infections with Mycoplasma pneumoniae, infectious mononucleosis, influenza B, and human immunodeficiency virus (HIV), as well as with other infections. (Cold agglutinins develop in more than 60% of patients with infectious mononucleosis, but hemolytic anemia is rare.)

Cytomegalovirus (CMV), rubella virus, varicella-zoster virus (VZV), parvovirus B19, and Chlamydia psittaci have also been implicated. [25]

In the case of infectious mononucleosis, hemolysis tends to develop 1-2 weeks after the onset of illness, but it may occur simultaneously for up to 2 months after onset. [25] Furthermore, increased expression of I/i antigens have been described on hemoglobin SS (HbSS) erythrocytes, which suggests that such patients may have increased susceptibility to cold-mediated hemolysis. [26]

Antigen specificity

In its classic presentation, with hemolytic anemia and Raynaud syndrome, cold agglutinin disease is usually idiopathic. As with most autoimmune diseases of a chronic nature, stimulated B lymphocytes begin to produce pathogenic antibodies against an antigen that is normally present in human tissue. In cold agglutinin disease, the antibody is an IgM, usually monoclonal, with kappa (κ) or lambda (λ) light chains. In chronic cold agglutinin disease, the antibody is usually directed against the I antigen on the membrane of normal adult RBCs.

Uncommonly, the antibody may be directed against only the i antigen found on fetal cord blood RBCs, which lack the mature I antigen; this has been reported in association with infectious mononucleosis. [27]

In a study of 78 patients, κ light-chain specificity was found in the majority of patients with chronic cold agglutinin disease or Waldenström macroglobulinemia, whereas two thirds of cold agglutinins found in patients with lymphomas had λ light-chain specificity. The type of light chain appears to correlate with the antigen specificity of the cold agglutinin. Fifty-eight percent of IgM/κ (usually κIII variable region subgroup) was anti-I, but 75% of IgM/λ had other antigen specificities. [11]

Antigen specificities of cold agglutinins other than the I/i system that have been described include those against Pr, M, P, and Lud and anti-Gd, anti-Fl, and anti-Sa. [7, 10, 13] Exclusive occurrence of κ chains has also been shown with some cold agglutinins. [6] Thus, benign and malignant cold agglutinins exhibit differences in their light chains and their specificities toward membrane antigens.

Association of complement fixation with temperature

In vivo, the IgM antibody attaches to RBCs and causes them to agglutinate at temperatures below 37°C and maximally at 0-5°C, resulting in impaired blood flow to the digits, nose, and ears (ie, areas more likely to have colder temperatures [in the 30°C range]) when exposed to the cold.

Fixation of the C3 component of complement to the RBC by the cold agglutinin usually occurs in vivo at higher temperatures than those required by the IgM cold agglutinin to attach to the RBC, but it is generally less than 31°C. When the IgM/C3b-coated RBC circulates to warmer tissues, the IgM dissociates, leaving complement C3b on the original RBC.

The dissociated IgM cold agglutinin can then bind to another RBC at lower temperatures. Fixation of complement results in C3b and/or C4b components on the RBC membrane, which may lead to phagocytosis by macrophages in the reticuloendothelial system, particularly in the liver, where the macrophages have specific complement receptors. With time, the C3b components are converted enzymatically to C3dg, which is not recognized by macrophage receptors.

Complement levels in hemolysis

In chronic cold agglutinin disease, complement tends to be depleted. Thus, the hemolysis is self-controlled, and anemia may be only mild or moderate, because these C3dg-coated RBCs are no longer capable of reacting with the IgM antibody in the cold, the C3dg-coated RBCs are not recognized by the macrophages, and low complement levels become rate limiting.

Temporary increases in complement levels, as can occur with intercurrent febrile illnesses, can increase hemolysis. Lytic components of complement C5-C9 generally do not form on these cells, and intravascular hemolysis by complement is less likely to occur. [15] Hemolysis develops acutely following M pneumoniae infections and lasts approximately 1-3 weeks. Subclinical mild hemolysis with reticulocytosis may also occur, and the results of a direct Coombs test may be weakly positive, especially with M pneumoniae infections.

Idiopathic disease and lymphoma

Monoclonal cold agglutinin IgM antibodies found in patients with lymphoma are the product of the abnormal clone. Progression of an idiopathic cold agglutinin disease to malignant lymphoma may occur in some cases; thus, affected patients require close, long-term follow-up, with obvious therapeutic implications. [27, 13] One study of 86 patients in Norway showed clonal light-chain predominance in 90% of patients, evidence of lymphoplasmacytic lymphoma in 50% of patients, and lymphoma of any type in 76% of patients overall. [1]

Warm-cold antibody combinations

Hemolysis due to cold agglutinins can sometimes be accompanied by a warm antibody (IgG), resulting in a mixed autoimmune hemolytic anemia, [27, 12] that is, cold agglutinin syndrome and warm antibody autoimmune hemolysis, with the direct antiglobulin (direct Coombs) test results positive for the presence of IgG and complement on the surface of the sensitized RBC.

In mixed antibody syndromes, the IgG and IgM antibody components can be separated. The cold autoantibodies reactive at temperatures of 30°C or higher often show blood group specificity to the adult I antigen, whereas the warm autoantibodies are not directed against this system. A combination of cold agglutinins and cryoglobulins has also been reported with an IgM/κ monoclonal antibody, with specificity to the Pr2 antigen system. [19]

Biphasic hemolysins

The presence of biphasic hemolysins implicates more severe disease. Biphasic hemolysins bind to RBCs at low temperatures and activate complement to produce in vitro hemolysis at warmer temperatures (37°C), whereas monophasic hemolysins bind to RBCs and activate complement at the same temperature. [28]

Modulation of agglutinin production

Data have confirmed an immunomodulatory/immunosuppressive role of the naturally occurring anti-F(ab')2 antibodies in the production of cold agglutinins, with an inverse correlation between the titers of IgG-anti-F(ab')2 and cold agglutinins. [17] This inverse correlation was found only in patients with anti-I/i and in the presence of a monoclonal lymphocyte population.



Cold agglutinins develop in more than 60% of patients with infectious mononucleosis, but hemolytic anemia is rare.

Classic chronic cold agglutinin disease is idiopathic, associated with symptoms and signs in relation to cold exposure. Causes of the monoclonal secondary disease include the following:

Causes of polyclonal secondary cold agglutinin disease include the following:

  • Mycoplasma infections - M pneumoniae [30]
  • Viral infections - Infectious mononucleosis due to Epstein-Barr virus (EBV) or CMV
  • Viral infections, other - Mumps, varicella, rubella, adenovirus, HIV, influenza, hepatitis C
  • Bacterial infections - Legionnaire disease, syphilis, listeriosis, Escherichia coli
  • Parasitic infections - Malaria, trypanosomiasis

Bacterial and viral infections

Transient acute hemolysis may occur secondary to certain infectious diseases, such as M pneumoniae infection and infectious mononucleosis (eg, EBV). Other viral infections, such as influenza, HIV, CMV, rubella, varicella, and mumps, have also been reported to be associated with a hemolytic anemia due to cold agglutinins. Associated illnesses also include subacute bacterial endocarditis, syphilis, and malaria. The development of a febrile illness in a patient with chronic cold agglutinin disease may also accelerate hemolysis.

CANOMAD syndrome

Cold agglutinins are seen in CANOMAD syndrome (chronic ataxic neuropathy ophthalmoplegia M-protein agglutination disialosyl antibodies). This syndrome is described by gait and upper-limb ataxia; cranial nerve involvement with external ophthalmoplegia; and the presence of cold agglutinins, IgM paraprotein, and anti-disialosyl antibodies. [31] The neurologic and hematologic symptoms have been seen to respond to rituximab. [32]


Lymphoproliferative and autoimmune diseases, myeloma, Kaposi sarcoma, and angioimmunoblastic lymphoma may occasionally be associated with the production of cold agglutinins. An idea of associated disease distribution was provided by a study of 78 patients with persistent cold agglutinins. Among these patients, 31 had lymphoma, 13 had Waldenström macroglobulinemia, 6 had chronic lymphoid leukemia, and 28 had chronic idiopathic cold agglutinin disease. [11]

A case of cold agglutinin–induced hemolytic anemia has been described in association with an aggressive natural killer cell (NK-cell) leukemia. [33] Nonhematologic malignancies can occasionally be associated with a high-titer cold agglutinin–induced hemolytic anemia. [9, 34]

Trisomy and translocation

Cytogenetic studies in patients with cold agglutinin disease have revealed the presence of trisomy 3 and trisomy 12. Translocation (8;22) has also been reported in association with cold agglutinin disease. [1, 35, 36]


Cold agglutinin–mediated hemolytic anemia has been described in patients after living-donor liver transplantation treated with tacrolimus and after bone marrow transplantation with cyclosporine treatments. It is postulated that such calcineurin inhibitors, which selectively affect T-cell function and spare B-lymphocytes, may interfere with the deletion of autoreactive T-cell clones, resulting in autoimmune disease. [37, 38, 39]

Systemic sclerosis

Cold agglutinin disease has been described in patients with sclerodermic features, with the degree of anemia being associated with increasing disease activity of the patient’s systemic sclerosis. This may suggest a close association between systemic rheumatic disease and autoimmune hematologic abnormalities. [40]

Hyperreactive malarial splenomegaly

Hyperreactive malarial splenomegaly (HMS) is an immunopathologic complication of recurrent malarial infection. Patients with HMS develop splenomegaly, acquired clinical immunity to malaria, high serum concentrations of anti-Plasmodium antibodies, and high titers of IgM, with a complement-fixing IgM that acts as a cold agglutinin. [41]

DPT vaccination

Diphtheria-pertussis-tetanus (DPT) vaccination has been implicated in the development of autoimmune hemolytic anemia caused by IgM autoantibody with a high thermal range. A total of 6 cases have been reported; 2 followed the initial vaccination and 4 followed the second or third vaccinations. [23, 42, 43, 44, 45]


Equestrian perniosis is a rare cause of persistent elevated titers of cold agglutinins. [20] Also rarely, the first manifestations of cold agglutinin disease can develop when a patient is subjected to hypothermia for cardiopulmonary bypass surgery. [14]



Occurrence in the United States

The development of cold agglutinin syndrome is relatively uncommon, at least in the classic chronic form. Various reports state that 7-25% of cases of autoimmune hemolytic anemia are cold agglutinin mediated. Thus, while the incidence of cold and warm autoimmune hemolytic anemia (combined) is approximately 1 in 80,000, the incidence of cold agglutinin disease is approximately 1 in 300,000. Among autoimmune hemolytic anemias, cold agglutinin disease is the second most common cause, after warm autoantibody–induced immune hemolysis.

International occurrence

Data regarding the incidence of cold agglutinin disease are lacking. Frequency figures listed for the United States probably also apply to Canada and the United Kingdom.

Sex- and age-related demographics

In general, no predilection for either sex is noted, although some report a female predilection in older populations. Autoimmune hemolytic anemia appears to be more common in male children and female adolescents. [5, 27]

Only rarely do Infants and children develop chronic cold agglutinin disease, although Mycoplasma pneumoniae and infectious mononucleosis are diseases of young persons. Chronic cold agglutinin disease typically affects adults who are of middle age and older, with an average age of older than 60 years and peaking in the seventh and eighth decades of life. Although found in persons of all age groups, mixed autoimmune hemolysis is also more frequent in later life.



Cold agglutinin disease may be associated with an excellent long-term prognosis if it is secondary to M pneumonia or viral infections that are, in themselves, self-limited. In children and young adults, acute hemolysis lasts 1-3 weeks; evidence of cold agglutinins disappears within 6 months.

Patients with the mildly to moderately severe primary (idiopathic) variety of cold agglutinin disease are expected to have a good long-term prognosis if excessive exposure to cold is avoided and with close medical surveillance for complications or progression to lymphoma.

The nature of the antigenic specificity of the cold agglutinin, as when it is directed against the Pr antigen system, may be associated with greater severity of disease.

Cold agglutinin disease associated with HIV infection may have a relatively poor prognosis due to the nature of the underlying disease. The same applies to cases associated with lymphoma, with the prognosis dependent on remission of the underlying malignancy.

Morbidity and mortality

Complications of cold agglutinin disease include the following:

  • Brisk hemolysis due to cold exposure
  • Ischemic complications at exposed sites due to prolonged cold exposure
  • Other symptoms related to severe anemia
  • Infrequently, development of malignant disease during follow-up care of a patient with idiopathic chronic cold agglutinin disease
  • Shock or congestive heart failure, resulting from severe hemolysis and anemia
  • Peripheral gangrene and, rarely, fatalities after inadvertent and perhaps prolonged exposure to the cold

Transfusions for life-threatening symptoms due to severe anemia require prewarming and the use of washed RBCs (not cold). In general, autoimmune hemolytic anemia has a mortality rate of 10%.

A study by Kamesaki et al indicated that the clinical characteristics of patients with autoimmune hemolytic anemia who have a positive direct Coombs test (direct antiglobulin test [DAT]) differ from those of patients with this type of anemia and a negative DAT. The report used data from 216 patients with autoimmune hemolytic anemia, including 154 who were DAT negative and 62 who were DAT positive. [46]

The investigators found that patients who were DAT negative tended to have milder anemia and hemolysis than did patients who were DAT positive and that they needed significantly lower steroid doses during maintenance treatment. The 2 groups of patients were found to have an equally good response to steroids. Survival at 1-year follow-up for each group was comparable to that of the other.


Patient Education

It is essential to educate patients with chronic cold agglutinin disease about the importance of keeping all body parts warm at all times and avoiding cooling of body parts. Appropriate clothing is necessary in cold environments, and avoidance of cold foods and working in cold storage areas is also important.

Patients must comprehend the importance of their daily folic acid intake, which supplies a needed hematinic. Folic acid could easily become a rate-limiting hematinic in a patient with a chronic hemolytic process.

Teach patients to watch for signs of anemia, such as dyspnea, palpitations, and pallor, and to observe for signs of hemolysis, such as jaundice and dark urine.

For patient education information, see Anemia.