Endemic Autoimmunity: Cold Auto-agglutinins in Melanesia

Sera from two Melanesian populations, one living at sea level and the other at 7000 feet, were examined for the incidence, strength and specificity of cold agglutinins. The cold auto-agglutination incidence ranged from 48 to 90 per cent for the sea-level group to 94 per cent for the highlanders. Titres of the sera ranged from 10 to 640. The specificity of the antibodies was in the main anti-I, although four probable examples of anti-AI, four examples of anti-I^T and two 'non-specific'cold agglutinins were found. Blood group A subjects in both populations had a higher incidence and titre of cold agglutinins than O subjects.
The light chain types of 160 of 200 cold agglutinins tested were both k and λ, 24 were K only and 16 were λ only. Fingerprints of the H chains of six New Guinea cold agglutinins showed some peptide spots missing compared with normal IgM, but more spots were present than in a lymphoma cold agglutinin, suggesting that the cold agglutinins represented a heterogeneous population of antibodies.
No obvious relationship was found between environmental factors and the occurrence of the cold agglutinins.     

Chemical analyses of variable regions of heavy and light chains of cold agglutinins

Abstract. The variable regions of cold agglutinins with anti-I and anti-Pr specificities were investigated by N-terminal amino acid sequencing and by analyses of pyrrolidone-carboxylic acid-blocked peptides after digestion of polypeptide chains with Nargase. Results showed that the heavy chains of four IgM anti-I cold agglutinins are exclusively VHI subgroups and their light chains are exclusively VKII subgroup. In contrast, the light chains of two cold agglutinins with anti-Pr specificity are not VKII, while their heavy chains are not restricted to a single subgroup. The amino acid sequences at the first hypervariable region of light chains (positions 25–35) are similar in two of the four anti-I cold agglutinins. These sequences are different from that of the light chain of another cold agglutinin with anti-Pr specificity. These results support the concept that only antibodies with the same specificity can share similar primary structure at their antigen combining sites.     

Clinical and immunologic features of transient cold agglutinin hemolytic anemia

Atypical pneumonitis, often induced by infection with Mycoplasma pneumoniae, is frequently associated with elevated cold agglutinin titers but only rarely with significant hemolytic anemia. An example and documentation of the clinical and immunologic course of such transient cold agglutinin hemolytic anemia is presented, and the immunochemical characteristics of cold agglutinins in this syndrome are assessed in regard to their biologic implications and diagnostic significance. Transient cold agglutinins, such as observed in this case, commonly appear to be of a restricted polyclonal character and as first demonstrated in this case may possess structural determinants usually considered unique for monoclonal cold agglutinins. Although the immunopathogenetic mechanisms and certain clinical manifestations of the various forms of cold agglutinin hemolytic anemia appear similar, the immunogenic basis of cold agglutinin production and the molecular structure of transient cold agglutinins are quite distinctive and provide reasonably reliable guidelines for the differential diagnosis of the cold agglutinin syndromes and for consideration of appropriate clinical management.     

An Unusual Sialoglycoprotein Associated with the Webb-Positive Phenotype

A 65-year-old white man had severe hemolytic anemia due to a mixture of low-titer IgG lambda and IgM lambda agglutinins showing optimum reactivity at 22 degrees C. The IgG agglutinins were detected by manual indirect antiglobulin test (IAT) using anti-IgG, and had a titer of 1 at 37 degrees C, 128 at 22 degrees C and 16 at 4 degrees C against adult type O red blood cells (RBC). The corresponding titers with cord RBC were 1 (37 degrees C), 64 (22 degrees C) and 8 (4 degrees C). Proteolytic enzyme and neuraminidase treatment of RBC did not decrease these titers. No known specificity could be assigned to these agglutinins. The isolated agglutinins (recovered by cold adsorption, warm elution) were shown by immunoelectrophoresis to be IgG lambda antibodies. They did not bind complement in vitro, consistent with the finding that the patient had negative manual direct antiglobulin test (DAT) by anti-C3d. It could be shown only by automated IAT that patient's serum also contained IgM cold agglutinins which also reacted best at 22 degrees C and appeared to be of lambda light-chain type. The patient responded to corticosteroid therapy and remains well without treatment 14 months after the hemolytic episode. The presence of IgG cold agglutinins may be predictive of a favorable response to corticosteroid therapy.     

A High Frequency of Cold Agglutinins of Anti-IA Specificity in a New Guinea Highland Population

Summary. A high incidence (37 out of 188) of anti-IA cold agglutinins was found in the sera of indigenous New Guineans living at Minj (alt. 1,600-1,800 m) in the Western Highlands District of New Guinea. Anti-IA did not occur in three other populations tested, although these possessed a high incidence of anti-I antibodies. The earlier findings concerning the incidence of specificity of cold haemagglutinins in New Guinea are reviewed and it is suggested that they may be induced by different agents, probably of a parasitic nature, in different localities. It is predicted that cold agglutinins should be found in other populations which are subject to high rates of infection similar to those in New Guinea.     

Reaction of Anti-Gd, Anti-Fl and Anti-Sa Cold Agglutinins with p Erythrocytes

Among four anti-Gd cold agglutinins two showed a slight preferential reaction with p erythrocytes, two did not. Anti-Fl and anti-Sa cold agglutinins, sharing some serologic characteristics with Gd antibodies, showed no preferential reactions with p cells. The findings point to different fine specificities of monoclonal anti-Gd cold agglutinins.     

Cold agglutinin syndrome: nursing management

Although cold agglutinin syndrome rarely results in an acute hemolytic episode, consideration should be given to the potential problems that exist when cold agglutinins are present. This autoimmune response may be present in an acute or chronic form. Viral and bacterial infections, especially mycoplasma pneumonia, may precipitate acute CAS, whereas, chronic CAS may be idiopathic or associated with malignant lymphoma. Both acute and chronic CAS may result in RBC hemolysis as the IgM antibody reacts with the I-antigen on the RBC, setting the complement cascade into action. Therefore, screening for cold agglutinins should be done before any procedure that would introduce cold fluids into the body, e.g., blood transfusions and cold cardioplegia during coronary artery bypass surgery.     

B-Cell neoplasms with homogeneous cold-reacting antibodies (cold agglutinins)

Among 78 patients with persistent cold agglutinins, 31 had lymphoma, 13 had macroglobulinemia of Waldenström, six had chronic lymphocytic leukemia and 28 had chronic cold agglutinin disease. The average age was over 60 years. Patients with chronic cold agglutinin disease had more hemolytic crises, bleeding and Raynaud's phenomena, and less frequently lymphadenopathy or hepatosplenomegaly. The frequency of anemia, positive Coombs test results, cryogtobulinemia and Bence Jones proteinuria was similar in the various groups. Survival time from diagnosis was on average two years in lymphoma, two and a half years in Waldenström's macroglobulinemia, more than six years in chronic lymphocytic leukemia and more than five years in chronic cold agglutinin disease. Anti-I were common in chronic cold agglutinin disease (74 percent) and rare in other groups (32 to 33 percent). Anti-I and other cold agglutinins were rare in chronic cold agglutinin disease and common in lymphoma and Waldenström's macroglobulinemia. In chronic cold agglutinin disease, and in Waldenström's macroglobulinemia, cold agglutinins usually had κ light chains—92 percent and 71 percent, respectively—whereas in lymphoma, 71 percent of cold agglutinins had λ light chains. The type of light chains related to the specificity of cold agglutinins: 58 percent of IgM/κ were anti-i, 75 percent of IgM/λ had other specificities. Cold agglutinins were cytotoxic to autologous and allogeneic lymphocytes. Occasionally, more autologous than allogeneic cells were killed implying that the former may be precoated in vivo with the antibodies. In conclusion, conditions with persistent cold agglutinins are a spectrum that varies from “benign” autoimmune-like chronic cold agglutinin disease to malignant lymphoma. Marked differences in the light chain type of cold agglutinins, specificity toward membranous antigens and severity of clinical manifestations were noted in benign and malignant varieties.     

The Discovery of the Gc System

The second example of anti-F1 cold agglutinins recognizing a developmentally regulated antigen like I is described. The antibody occurred transiently and simultaneously together with an anti-I cold agglutinin. Although anti-F1 and anti-I specificities are entirely different, the data on the biochemistry of F1 and I antigenic determinants provide an explanation for the simultaneous occurrence of anti-F1 and anti-I cold agglutinins in connection with a poly- or oligoclonal autoimmune response.     

Chronic hemolytic anemia due to a monoclonal IgG cold agglutinin with anti-Pr specificity

Immunologic studies performed in a case of autoimmune chronic hemolytic anemia with low titer cold agglutinins (1/16) demonstrated that the cold agglutinins corresponded to monoclonal IgG with anti-Pr specificity. This antibody had a large thermal amplitude, being active at 37 degrees C. These unusual characteristics may define a distinct subset of chronic cold agglutinin disease.     

IgG Cold Agglutinins and First Trimester Abortion

Abstract. Two patients with first trimester abortion and idiopathic cold agglutinin syndrome due to cold auto-antibodies of the IgM and IgG immunoglobulin classes are reported.
Sucrose gradient ultracentrifugation and 2-mercapto-ethanol treatment indicated the presence of IgM as well as IgG cold agglutinins.
The possible relationship between the abortions and these cold agglutinins is discussed.     

Restriction in the repertoire of the immunoglobulin light chain subgroup in pathological cold agglutinins with anti-Pr specificity

BACKGROUND AND OBJECTIVES: In cold agglutinin disease, monoclonal red blood cell autoantibodies, termed cold agglutinins, induce haemolysis in patients exposed to the cold. Commonly, these autoantibodies are directed against the developmentally regulated I/i blood groups. A second blood group system, the Pr system (located on glycophorins), is involved less frequently. Anti-Pr cold agglutinins recognize either alpha 2,3- or alpha 2,6-linked N-acetylneuraminic acid as the immunodominant group. Cold agglutinins of anti-I/i specificity show a remarkable restriction in their genomic repertoire of the immunoglobulin heavy and light-chain immunoglobulin-variable domain (i.e. exclusive use of VH4-34 in heavy chains). For anti-Pr cold agglutinins, preliminary data on the repertoire of the light-chain variable domain indicate a preference for the subgroup Vkappa IV. To elucidate restrictions in the light-chain variable-domain subgroup repertoire of anti-Pr cold agglutinins systematically, and to discuss these results in the context of their anti-Pr(1-3) subclassification and immunodominant sialic acid, light chains in 13 anti-Pr cold agglutinins were investigated. MATERIALS AND METHODS: The anti-Pr light chains were isolated using temperature-dependent absorption/elution techniques. Subsequently, they were subjected to N-terminal Edman degradation, and the light chain Vkappa subgroup was affiliated using the Kabat database. RESULTS: Five of 13 (38%) light chains belonged to Vkappa IV, five of 13 (38%) to Vkappa I and three of 13 (23%) to Vkappa III. Anti-Pr with Vkappa IV subgroup light chains exclusively recognized alpha 2,3-linked N-acetylneuraminic acid. CONCLUSIONS: Including data from the literature, the repertoire of the light-chain variable domain in pathological anti-Pr cold agglutinins exhibits a clear bias towards the use of the single germline gene-derived subgroup, Vkappa IV (eight of 17 or 47%). The association of Vkappa IV subgroup light chain-containing anti-Pr cold agglutinins with binding to alpha 2,3-, but not alpha 2,6-linked N-acetyneuraminic acid raises speculations about a possible role of subgroup-derived determinants in anti-Pr binding.     

Demonstration of Low-Titer Anti-Pr Cold Agglutinins

Abstract. Hemagglutination tests with protease- and neuraminidase(RDE)-treated red cells are essential to demonstrate anti-Pr cold agglutinins, since RDE and proteases inactivate the Pr antigens in contrast to the I/i antigens. RDE treatment of red cells, however, reveals the T antigen which reacts with anti-T present in anti-Pr sera. Furthermore, anti-I, also present in anti-Pr sera, shows increased reactions with RDE- and protease-treated red cells. Therefore, T-anti-T and I-anti-I reactions mask the loss of anti-Pr activity against RDE- and protease-treated red cells when low-titer anti-Pr sera are studied. Absorption of anti-Pr sera with RDE-treated red cells removes anti-T and anti-I, leaving anti-Pr cold agglutinins which only react with untreated red cells, not with RDE- or protease-treated red cells. Anti-I contaminating anti-Pr in warm eluates from untreated red cells or stroma can be also removed by absorption with enzyme-treated red cells. By eliminating T-anti-T interference from sera, it was possible to show that all Pr determinants known are determined by N-acetylneuraminic acid.     

Cold hemagglutination in acute and chronic hemolytic syndromes

1. Three cases have been described of hemolytic anemia associated withautoagglutination of erythrocytes in the cold. These cases were characterizedclinically by pallor, slight icterus and lack of splenomegaly. Raynaud’s phenomenon was present in two of the three patients. The first patient had anacute self-limited course. The second and possibly the third were chronic andapparently persistent.

2. A hemolysin was demonstrated in the chronic variety of hemolytic anemia.This antibody was active at body temperature, required complement and appeared to be independent of the cold agglutinin. It disappeared following therapywith cortisone. In the third case, hemolysin was demonstrated directly attachedto the surface of the patient’s erythrocytes by the addition of complement tothe washed red cells. The hemolytic reaction was enhanced by the use of trypsinized test erythrocytes.

3. "Complete and incomplete" cold agglutinins were demonstrated by theuse of saline, albumin and trypsin technics as well as by the Coombs’ antiglobulin reactions. Erythrocyte coating antibodies were not neutralized with humangamma globulin. Cold agglutinins and erythrocyte coating antibodies wereunaffected by cortisone therapy.

4. Survival of transfused erythrocytes was increased in one patient duringthe period of cortisone treatment, despite the fact that cold agglutinins persisted.

5. Cold agglutinated erythrocytes have increased mechanical fragility whenthe cold agglutinin is present in high titer. Incubation at 37 C. for 24 hourscauses a marked shift to the left in the osmotic fragility curve.

6. Study of the serum in one patient for antibodies to Newcastle virus disease and influenza failed to disclose these antibodies in significant titer.

Submitted on May 25, 1954 Accepted on August 4, 1954     

Four cases of pseudothrombocytopenia due to platelet cold agglutinins

We report 4 cases of pseudothrombocytopenia due to platelet cold agglutinins. Case 1 was a 57 y.o. female whose platelet count was 97 x 10(3)/microl. Case 2 was a 37 y.o. male with a platelet count of 96 x 10(3)/microl. Case 3 was a 74 y.o. male with a platelet count of 28 x 10(3)/microl. Case 4 was a 62 y.o. female whose platelet count was 34 x 10(3)/microl. The platelet counts in these 4 cases were decreased and blood smears showed platelet clumping in blood drawn in a tube without anticoagulant just after withdrawal, as well as in blood drawn in a tube with anticoagulant. The platelets from these patients agglutinated at a temperature below 10 degrees C (case 1 and 4) and 24 degrees C (case 2). The immunoglobulin class of the platelet cold agglutinins in cases 1, 2 and 4 was IgM. Agglutinated platelets showed no activation marker, such as CD62P, CD63 or CD40L, on the surface of the platelets. The target antigen of cold agglutinins was GPIIb-IIIa in cases 1 and 2. We considered that the detection of platelet agglutination in blood without anticoagulant is important to diagnose pseudothrombocytopenia due to platelet cold agglutinins. Although this disease is considered to be very rare, we suspect that this disease may be misdiagnosed as pseudothrombocytopenia due to the presence of an anticoagulant, and overlooked.     

Cold agglutinin autoimmune hemolytic anemia in nonhematologic malignancies.

Four patients with nonhematologic malignancies had the simultaneous finding of hemolytic anemia due to high-titer cold agglutinins. In each patient, the cold agglutinin had "anti-I" specificity and was of the IgM kappa immunoglobulin class. Although patients with hematologic malignancies not uncommonly have cold agglutinins, the association between these antibodies and nonhematologic malignancies is unusual.     

Severe hemolytic anemia and excessive leukocytosis masking mycoplasma pneumonia

The formation of cold agglutinins is frequently observed during Mycoplasma pneumoniae infections. Nevertheless, severe hemolysis is exceptional. We report a case of life-threatening hemolytic anemia caused by M. pneumoniae. As the leucocyte count was excessively elevated, the differential diagnosis primarily comprised hematological malignancies. The presence of cold agglutinins indicated the correct diagnosis, which was confirmed by highly elevated levels of both IgG and IgM antibodies to M. pneumoniae and a chest X-ray suggestive of atypical pneumonia. The patient was treated with roxithromycin and showed a favorable recovery within ten days after admission. This case demonstrates that, even in patients with clinically mild pneumonia, M. pneumoniae may be the cause of severe anemia.     

Cold agglutinins in a case of chronic lymphatic leukaemia. A study of the lymphocyte surface.

A case of chronic lymphatic leukaemia with cold agglutinins is presented in which peripheral blood lymphocytes of varying maturity appeared to possess T- and B-cell markers. A cold agglutinin IgM in type is identified in the serum and at the cell surface. This seems to be responsible for the spontaneous rosetting of sheep red blood cells (SRBC); A single clonal origin of these cells is suggested with maturation arrest. It is also suggested that with configurational change in lymphocyte surface cryoglobulins may sometimes produce spontaneous rosetting with SRBC.     

Cold Autoagglutinins with Anti-Pr Specificity Associated with Fresh Varicella Infection

Cold agglutinins with the rare anti-Pr specificity were identified in an adult patient with fresh varicella infection. The antibody was of the IgM ? type with subspecificity anti-Pr_3d and caused a haemolytic episode in the patient. In the only previously reported case in which cold agglutinins were associated with varicella infection, the antibody specificity was also anti-Pr.     

Essential monoclonal gammopathy with an IgM paraprotein that is a cryoglobulin with cold agglutinin and EDTA-dependent platelet antibody properties

A patient with apparent anaemia and thrombocytopenia caused by a monoclonal paraprotein is described. The patient's serum contained a monoclonal IgM kappa, a cryoglobulin and a cold agglutinin. The cryoglobulin, similar to the serum paraprotein, was a monoclonal IgM kappa. Serum was studied to determine the relationship of the cryoglobulin with the cold agglutinin. The cryoglobulin and cold agglutinin were found to be the same paraprotein. Moreover, with absorption and elution techniques the reactivity of the autoantibody with both erythrocytes and platelets was demonstrated.
Reports of cryoprecipitable cold agglutinins are rare and therefore this case is exceptional given that the IgM kappa paraprotein was found to be a cold agglutinin which was also reactive with platelets.