Immunochemical characterization of monoclonal protein in the serum of a patient with Waldenstr]om's macroglobulinemia showing both pyroglobulin and cryoglobulin properties.

A patient with Waldenstr]om's macroglobulinemia whose serum demonstrated properties of both pyroglobulin and cryoglobulin was studied. A monoclonal (M) protein in the serum of the patient was identified by immunoelectrophoresis (IEP) and immunofixation electrophoresis (IFE) as IgM-lambda. The M-protein was separated by gel permeation high-performance liquid chromatography (HPLC). Cryoglobulin in the serum was isolated by the method of cold precipitation. The cryoglobulin was identified as IgM-lambda type by IEP and was the same M-protein as that which occurred in this patient's serum. The purified cryoglobulin also had the properties of a pyroglobulin. Neither property disappeared following pretreatment with 2-mercaptoethanol (2-ME), urea, and Triton X-100 detergent, and deglycosylation with N-glycanase (E.C. 3.5.1.52). We suggest that these abnormal properties were caused by the molecular abnormality of the IgM-lambda M-protein.

Human immunoglobulin M paraproteins cross-reactive with Neisseria meningitidis group B polysaccharide and fetal brain.

Three hundred fifty-nine serum samples from patients with immunoglobulin M (IgM) or IgG monoclonal gammopathies were tested for binding to the capsular polysaccharide (PS) of Neisseria meningitidis group B (MenB PS, poly-alpha[2-->8]-N-acetylneuraminic acid). Of 159 IgM paraproteins, 7 (4.4%) were positive, compared with 0 of 200 IgG paraproteins (P < 0.05). Since MenB PS reactivity was limited to the IgM paraproteins, the 159 IgM paraproteins were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with seven other bacterial PSs. None reacted with meningococcal A or C, Haemophilus influenzae type b, or Streptococcus pneumoniae type 3, 6, 14, or 23 PS. The specificity of the MenB PS-reactive antibodies was confirmed by demonstration of binding to N. meningitidis group B cells but not to a capsular PS-deficient mutant and by specific inhibition of binding to solid-phase MenB PS by soluble MenB PS in an ELISA. Five of five antibodies tested protected infant rats from bacteremia caused by Escherichia coli K1, an organism with a PS capsule that also is composed of poly-alpha[2-->8]-N-acetylneuraminic acid. Each of the seven MenB PS-reactive paraproteins had autoantibody activity as defined by binding to homogenates of calf brain in a radioimmunoassay. For six of the seven antibodies, binding to calf brain was inhibited by the addition of soluble MenB PS. Thus, approximately 4% of human IgM paraproteins have autoantibody activity to poly-alpha[2-->8]-N-acetylneuraminic acid, an antigen expressed in fetal brain and cross-reactive with the MenB capsular PS. The reason for this skewing of the IgM paraprotein repertoire toward reactivity with poly-alpha[2-->8]-N-acetylneuraminic acid antigenic determinants is unknown.

Spurious fibrinogen levels secondary to pyroglobulinemia in Waldenström's macroglobulinemia.

We report two cases of pyroglobulinemia detected in the course of routine determination of fibrinogen levels measured by the heat-precipitation method and that led to the diagnosis of Waldenström's macroglobulinemia. The incidence of pyroglobulinemia is briefly discussed, and the potential significance of this laboratory artifact is emphasized.

IgE pyroglobulinemia in multiple myeloma.

A new case of IgE/K myeloma in a 38-year-old male is described. The clinical course, bone marrow picture and serum electrophoretic pattern were typical of multiple myeloma. Heating of the serum at 56 degrees C induced an irreversible gel formation or pyroglobulin that was isolated and proved to be identical to the IgE paraprotein, by biochemical and immunochemical methods. The thermoprecipitability was abolished by treatment with urea, sodium dodecyl sulphate and neutral salts and not inhibited by sugars, alcohols, 2-beta-mercaptoethanol and guanidine HCl. The association of an IgE myeloma protein with a pyroglobulin is a finding not previously reported in a myeloma of this class.

The physicochemical properties and the process of pyrogel formation of IgD pyroglobulin.

The extremely rare IgD pyroglobulin caused opalescence, lost its antigenicity and acquired more negative charge when heated for 30 min at 56 degrees C. Exposure to 60 degrees C for 60 min produced a firm gel. The thermoprecipitability was abolished by the treatment with guanidine, urea and sodium dodecyl sulfate, but not by 2-mercaptoethanol or neuraminidase, and required the presence of both heavy and light chains. However, amino acid analysis showed abnormalities only in the heavy chain. We hypothesize that the irreversible heat-induced aggregation of IgD pyroglobulin is the result of a conformational change, triggered by light chain, which increases the molecular hydrophobicity.

IgD plasma cell leukemia associated with pyroglobulinemia and pyroglobulinuria.

A 70-yr-old man with multiple myeloma IgD developed a plasma cell leukemia producing a serum IgD monoclonal peak and lambda light chains in the urine. When the serum and the urine were heated at 56 degrees C for 30 min both monoclonal bands disappeared. The precipitate failed to redissolve on heating to 100 degrees C. Ion exchange chromatography with a linear gradient of phosphate buffer, pH 8, 0.020-0.300 mol/l and column electrofocusing showed that the serum pyroglobulin was eluted with buffer concentration between 0.040-0.125 mol/l and had an isoelectric point of 5.02, while the pyroglobulin of the urine was eluted with 0.020-0.033 mol/l and had a pI = 7.16. The serum and urine pyroglobulins had a total antigenic community with their correspondent purified proteins. The isolated lambda chains did not change when they were heated at 56 degrees C for 30 min, nevertheless, the heated purified IgD shows some changes in its isoelectric point, molecular mass and antigenicity. These changes in the purified IgD suggest that the pyroprecipitability could be due to conformational features.

Cryoglobulins and pyroglobulins: an overview.

Cryoglobulins are serum proteins with heterogeneous etiopathogenetic and immunochemical properties. What they have in common is temperature-dependent insolubility, in that at temperatures below 37 degrees C (often around 4 degrees C) they precipitate, and then redissolve at 37 degrees C. When the etiopathogenesis of the cryoglobulinemia is unknown, which is true for many patients, the condition is called idiopathic or essential cryoglobulinemia, whereas it is termed secondary whenever it appears to be associated with one of several diseases. Cryoglobulinemia has indeed been found in patients with lymphoproliferative and autoimmune disorders, liver diseases, infectious (viral, bacterial, fungal and parasitic) diseases, and so on. Cryoglobulins are usually classified according to their immunochemical properties as single-type monoclonal, mixtures of a monoclonal Ig with non-immunoglobulin material (DNA, lipoprotein, complement), mixed with one monoclonal Ig or mixed polyclonal, in which constitutive Ig fractions are polyclonal. As compared with normal Ig, cryoimmunoglobulins have sometimes been found to exhibit a peculiar amino acid structure of their heavy chains, less often of their light chains as well, and to have a lower carbohydrate content. Such structural abnormalities may contribute to their loss of solubility at low temperatures, possibly associated to the steric changes induced by the low temperature, causing the precipitate to form. The most common clinical features of cryoglobulins are correlated with vasculitis in the various organs and sometimes with increased viscosity of the plasma. Signs and symptoms include purpura, ulcers of the extremities, arthralgia, proteinuria, hepatic damage, abdominal pain, congestive heart failure, mental confusion, oligo-anuria, hemorrhagic diathesis, and coma. Pyroglobulins are also serum proteins with temperature-dependent insolubility. However, although they precipitate out of serum heated at 56 degrees C for half an hour, they do not resolubilize when the serum is returned to 37 degrees C. Pyroglobulins have been mainly found in patients with lymphoproliferative diseases (especially Waldenström's macroglobulinemia, with or without cryoglobulinemia), systemic lupus erythematosus, and neoplasia. So far, only single monoclonal IgG, IgM or IgA pyroglobulins have been described. Since they precipitate only at 56 degrees C, pyroglobulins do not cause clinical symptoms and they are usually discovered by chance.

'Incomplete' pyroglobulin-gamma disease in a patient with osteosclerotic myeloma.

A 50-year-old female, heterozygous for beta-thalassaemia was found to have a lytic lesion surrounded by osteosclerotic tissue in the 1st lumbar vertebra. Aspiration of the lesion showed 100% atypical plasma cells. The bone marrow contained 17% myeloma cells. Despite normal electrophoresis and immunoelectrophoresis of serum and urine, 'rouleaux' formation was pronounced. Treatment of the serum sample with 2-mercaptoethanol and heat (56 degrees C) disclosed an uncommon pyroglobulin. Analysis of the ammonium sulphate precipitate of the serum by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis revealed a 43 kD component with higher anodic mobility than normal gamma chains. Ultrafiltration column chromatography of the serum revealed a narrow spike of approximately 4 S that contained gamma heavy chain antigenic determinants in addition to normal 7 S IgG.

Monoclonal cryoglobulins and pyroglobulins.

In a series of 1182 monoclonal Ig, the authors demonstrated 10 cases with monoclonal cryoglobulin (0.8%) and 5 cases with pyroglobulin (0.42%) in the serum; in two of these cases both thermoproteins were present simultaneously. One observation revealed mixed cryoglobulin consisting of monoclonal IgM-kappa and polyclonal IgG. In 70 Waldenström's macroglobulinemias, monoclonal cryoglobulin occurred in 10%, pyroglobulin in 5.71%. In a subgroup of 744 monoclonal IgG, monoclonal cryoglobulin was demonstrated in 3 cases (0.4%), pyroglobulin in one case (0.13%).

Structural studies on an IgM-lambda pyroglobulin.

An IgM-lambda pyroglobulin from a patient with Waldenström's syndrome was studied. Heavy and light chains were separated and their N-terminal amino acid sequence determined. The heavy chain was unblocked and belonged to the VHIII subclass, and the light chain belonged to the lambda I subclass. Factors influencing pyroprecipitability were examined through experiments designed to study some of the physical and chemical properties of an IgM-lambda pyroglobulin. Pyroprecipitability was affected by pH, ionic strength, urea, and reducing agents, suggesting an involvement of noncovalent electrostatic interactions. It was also demonstrated through recombinant experiments that it is necessary to have covalently joined homologous heavy and light chains in pentameric form for pyroprecipitation to occur. Since neither heavy nor light chains had any unique structural features, the reasons for this property remain obscure but may reflect the result of conformational factors.

IgD-plasma cell leukemia associated with pyroglobulinemia and pyroglobulinuria. New types of pyroglobulin and cytoplasmic fibrils.

New pyroglobulin variants were found in a patient with a typical plasma cell leukemia. One was found in the urine and proved to be a variant of Bence-Jones' protein with a molecular weight of approximately 68,000. The other was found in the serum and appears to be the myeloma protein of IgD. Furthermore, an ultrastructural study disclosed myeloma cells in both peripheral blood and bone marrow to have cytoplasmic fibrils which might be unique for plasma cell leukemia.