Multiple myeloma and multiple plasmacytomas associated with free gamma heavy chain, free kappa light chain and IgGk paraproteins: an unusual triple gammopathy.

Multiple myeloma is a malignant plasma cell dyscrasia that is becoming more prevalent in an increasingly ageing population. It is a complex disease with clinical phases ranging from the premalignant monoclonal gammopathy of undetermined significance to asymptomatic (smouldering) myeloma and then symptomatic myeloma; the latter occasionally terminating in the clonal proliferation of plasma cells outside the bone marrow. We present a patient whose clonally evolved disease from monoclonal gammopathy of undetermined significance to multiple myeloma demonstrated the presence of an unusual combination of monoclonal immunoproteins. Capillary electrophoresis demonstrated the presence of three paraproteins in the gamma region (γ-region), two of which were additional to the IgGk paraprotein which migrated in the slow γ-region at initial diagnosis. Subsequent isotypic identification of the new paraproteins was not possible by immunotyping and initial immunofixation studies failed to definitively characterize the monoclonal proteins. After reduction with beta-mercaptoethanol, two paraproteins were detected by both capillary and gel electrophoresis. However, only immunofixation was able to resolve three distinct monoclonal bands, confirming the presence of free monoclonal kappa light chains in the mid-gamma region and free monoclonal heavy chains in the fast gamma region. Triple gammopathies in themselves are uncommon; this case presents a very unusual combination of paraproteins which required various electrophoretical and immunochemical techniques to identify and characterize them. The change of electrophoretic signature from the monoclonal gammopathy of undetermined significance phase to the diagnosis of multiple myeloma suggested that a number of genetically distinct subclones were present in the pretreatment clonal evolution of the disease.

Immunoelectrophoresis: a method with many faces.

Immunoelectrophoresis (IEP) was the first practical method that combined electrophoresis and -immunoprecipitation for identifying and characterizing proteins within complex mixtures. Over the years, IEP has been extended to include a variety of techniques and, as a general name, has been applied to virtually any technique that involves electrophoresis and antigen-antibody precipitin reaction for proteins. Because of the diversity in technical details of different IEP versions, the method described here deals only with classic IEP. Although it requires some manual expertise, IEP is versatile, relatively easy to customize, and economical with no need for expensive instrumentation. Further, it can discern identity, partial identity, and nonidentity of the proteins. Any low-viscosity body fluid specimen or, possibly, culture fluid and tissue extract could be tested with IEP if proper antibodies are available. With these attributes, classic IEP remains a valuable tool for clinical diagnostic testing, purity checking of biochemical and pharmaceutical products, and research.

Performance comparison of capillary and agarose gel electrophoresis for the identification and characterization of monoclonal immunoglobulins.

The objective of this study was to compare gel- and capillary-based serum protein electrophoresis methods to identify and characterize monoclonal immunoglobulins (M proteins). Five reviewers interpreted 149 consecutively ordered serum specimens following agarose gel electrophoresis (AGE), capillary electrophoresis (CE), immunofixation electrophoresis (IFE), and subtraction immunotyping (IT). As a screening test for detecting M proteins, AGE and CE displayed similar sensitivity (91% and 92%, respectively). CE was less specific (74%) than AGE (81%). An analysis of interinterpreter agreement revealed that interpretations were more consistent using gel-based methods than capillary-based methods, with 80% of the gel interpretations being in complete (5/5) agreement compared with 67% of the capillary interpretations. After implementing the capillary-based methods, the number of tests per reportable result increased (from 1.58 to 1.73). CE is an analytically suitable alternative to AGE, but laboratories implementing it will need to continue IFE testing to characterize all M proteins detected by CE.

Separation of paraproteins from human plasma by membrane chromatography.

Membrane chromatography using a commercially available blotting membrane was performed in a dead-end filtration mode to separate paraproteins from plasma of patients suffering from paraproteinemia. The affinity membrane was found to display distinct specificity to monoclonal IgG1. A dissociation constant (Kd) of 3.2 microM and a maximum binding capacity of 1.43 mg/cm2 IgG1 paraprotein were obtained from the adsorption isotherm of the affinity membrane. The membrane was found to absorb immunoglobulins species-dependently because no binding of immunoglobulins from mouse, rat and rabbit could be observed.

Subclass typing of IgG paraproteins by immunofixation electrophoresis.

We present a simple method for subclass typing of IgG paraproteins, with which we have demonstrated a large number of paraproteins that were undetected by conventional immunofixation techniques. The types and distribution of IgG subclass paraproteins were analyzed in 92 human sera in which IgG paraproteins had been demonstrated. The IgG subclass paraproteins were separated by agarose gel electrophoresis rapidly and simply and then typed with the use of sheep anti-human monospecific IgG1-IgG4 antibodies. In 24 of the sera analyzed, IgG subclass typing revealed 25 additional monoclonal bands that were not detected by conventional immunofixation electrophoresis with anti-IgG antisera. Most of these belonged to a different subclass type. The overall subclass frequencies were 68% IgG1, 13% IgG2, 16% IgG3, and 3% IgG4. The distribution of paraprotein subclasses, however, was different in monoclonal gammopathies of undetermined significance in which more IgG3 was shown, whereas in non-Hodgkin lymphomas the number of IgG2 paraproteins was greater than expected; this finding may have diagnostic and prognostic significance.

Susceptibility of monoclonal IgG paraproteins to plasmic cleavage using glycerin-stabilized human plasmin.

To shed further light on plasmin-IgG interactions a simple procedure is described that permitted 48 monoclonal IgG isolates from human serum to be profiled for their susceptibility to plasmic cleavage. In addition to anodal Fc and cathodal Fab fragments, combined immunoelectrophoresis-electrophoresis revealed transient anodal banding, as well as Fab-fragment subcleavage in many of the IgG subclass-1 isolates. The subcleavage of released Fab fragments, which bear the idiotype determinants, points to a possible ancillary role of plasmin in "idiotype processing" leading to immunoregulatory anti-idiotype networks. The cleavage of IgG by endogenous plasmin also points to a possible active role of plasmin in the "steady-state" metabolism of IgG.

Immunoglobulin clonality analysis. Resolution of ambiguities in immunofixation electrophoresis results by high-resolution, two-dimensional electrophoretic analysis of paraprotein bands eluted from agarose gels.

This article describes evaluation of the clonality of origin of immunoglobulin (Ig) chains by strategies that include qualitative analysis of paraproteins with high-resolution, two-dimensional electrophoresis (2DE) and silver staining. This approach is helpful in evaluating specimens in which standard immunoelectrophoresis or immunofixation electrophoresis (IFE) techniques do not provide definitive Ig typing results. One method the authors developed involves a "band elution" procedure, in which proteins present in high-resolution agarose gel electrophoresis bands are cut from the agarose gel, eluted with a denaturing buffer, and subjected to 2DE. The microheterogeneity patterns of the Ig light chains, heavy chains, or both are evaluated for their relationship regarding mass, charge, and, by inference, number of genes of origin. When necessary, determination of charge relationships may be aided by urea-mediated carbamylation of lysine residues, which introduces single-charge shifts to the individual protein subunits. Overall, these adjunctive techniques are particularly useful in cases with multiple bands of identical immunologic types (eg, several IgG1 bands) on IFE before and after sulfhydryl reduction (with dithiothreitol or 2-mercaptoethanol). The authors present procedural details and examples of the 2DE band elution patterns of serum and urine samples from four patients with B-cell dyscrasias, including the first reported case of POEMS syndrome with biclonal gammopathy, to the best of their knowledge.

Two IgM paraproteins bearing cryoglobulin and anti-smooth muscle activities in a patient with Waldenström's macroglobulinaemia.

A 52-year-old white male subject with typical clinical and laboratory findings of Waldenström's macroglobulinaemia is described. Two paraprotein peaks of IgM lambda class, with different physical and chemical properties and different amino acid compositions, in both heavy and light chains, were found in the patient's serum. One of the IgM components (M1) was a cryoglobulin, and the other (M2) showed strong antismooth muscle activity. As far as we know, this is the first report of double paraproteins each of which has different properties.

Paraproteins associated with low plasma sodium levels.

The mechanism of the maintenance of low plasma sodium levels seen in certain multiple myeloma cases has been attributed to the cationic nature of pathological immunoglobulins (paraproteins). This hypothesis was tested with equilibrium dialysis and polyacrylamide gel electrophoresis techniques. Citrated plasma samples and affinity chromatography purified paraproteins of three multiple myeloma patients with abnormally low plasma sodium levels were dialysed against 140 mmol/L NaCl solution at pH 7.4 for 24 hours. The electrophoresis of paraproteins was conducted under non-denaturing conditions. Low plasma sodium concentrations observed under the dialysis of the patients' plasma samples were in good agreement with earlier reports. However, the isolated paraproteins did not show any sodium exclusion during the dialysis experiment. The electrophoretic mobility of the paraproteins at pH 7.4 indicated that the isoelectric point of these molecules was below 7.4, so they cannot behave as cations at the pH of the blood. From these data it appears that the maintenance of low plasma sodium levels in certain IgG type myeloma cases cannot be explained by the previously postulated cationic nature of the paraproteins.

Incidence of three cross-reactive idiotypes on human rheumatoid factor paraproteins.

The basis for rheumatoid factor (RF) production in autoimmune or lymphoproliferative diseases cannot be understood without defining the molecular factors that dictate RF structure and specificity. Recently three different mAb (6B6.6, 17.109, and G6) have been developed that define cross-reactive idiotypes (CRI) on intact L or H chains of human monoclonal RF cryoglobulins. However, the true incidence of these CRI among RF and their relationship to each other have not been delineated. In the present experiments, a panel of 163 randomly selected IgM paraproteins was evaluated for the expression of the two kappa L chain CRI, 6B6.6 and 17.109, and the H chain CRI, G6. Among the paraproteins with kappa L chains, 14% expressed the 17.109 CRI, and 9% expressed the 6B6.6 CRI. Both ELISA and Western immunoblotting experiments showed that the two L chain CRI were mutually exclusive. Anti-IgG activity was documented in 22 of the IgM-kappa paraproteins, among which mAb 6B6.6 reacted with 7 (32%) and mAb 17.109 with 6 (27%). Both CRI were expressed exclusively by L chains within the kappaIII variable gene subgroup. Although 17.109 CRI+ paraproteins had kappaIIIb L chains, none of the 6B6.6 CRI+ paraproteins possessed L chains with this kappa sub-subgroup specific Ag. The G6 CRI was found predominantly among RF paraproteins and was frequently yet not exclusively associated with the 17.109 CRI+ L chains. Additional experiments were performed on a panel of normal adult human sera and documented the presence of 6B6.6 and 17.109 CRI on a small percentage (0.1 to 2.0%) of IgM from most individuals. These data indicate that 1) the mAb 6B6.6 and 17.109 identify two major and distinct CRI among IgM-RF paraproteins, 2) both CRI are associated exclusively with kappaIII L chains, 3) kappaIIIb and kappaIII non-b L chains are equally prevalent among IgM-RF, 4) the G6 H chain CRI is frequently associated with 17.109 CRI+ L chains, but not with 6B6.6 CRI+ L chains, and 5) although the ability to make 6B6.6 and 17.109 CRI+ kappa L chains is common in humans, these CRI are present in low concentrations in normal IgM.

IgM-kappa-lambda biclonal gammopathy elucidated by immunofixation electrophoresis.

A patient with alcoholism-related illness was found to have a rarely encountered biclonal gammopathy with IgM-kappa and IgM-lambda components. The para-protein bands were readily identified by immunofixation electrophoresis but were not by conventional immunoelectrophoresis. This patient is most appropriately classified as a biclonal gammopathy of undetermined significance, since no cause for this abnormality was found. The literature on biclonal IgM-kappa-lambda gammopathy is reviewed.

Identification and measurement of paraprotein polymers by high performance gel filtration chromatography.

An evaluation of Superose 6, a new high performance gel filtration medium, has been made for the rapid separation and quantitation of paraprotein polymers. There is a significant correlation between relative serum viscosity and the concentration of polymeric IgA as demonstrated by FPLC using the Superose 6 columns. The retention times of the columns are highly reproducible and allow good resolution of polymers and provide a simple way of separating IgG3 from albumin. Scaling up to the preparative Superose 6B could be achieved with little loss of resolution.

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.

Demonstration and partial characterization of an atypical protein in the urine of a patient with primary amyloidosis.

A paraprotein has been isolated from the urine of a patient with primary amyloidosis. Immunologically it was classified as a free lambda light chain. The molecular weight was 22500 daltons. N-terminal amino acid analysis demonstrated homology with lambda IV variable subgroup in 19 of the first 20 amino acids. Extensive homology with lambda IV chains was demonstrated also in the hypervariable region of the light chain. An antiserum produced against the paraprotein was rendered idiotype-specific by absorption with pooled human light chains. This antiserum stained tissue specimen from the rectum and liver of the patient by the indirect immunofluorescence technique. This strongly indicates that the free lambda light chains that can be isolated from the urine are also deposited in the tissues as amyloid substance.

Immunochemical analysis of the monoclonal paraprotein in scleromyxedema.

The monoclonal paraprotein from a typical case of scleromyxedema was isolated and characterized. The isolated paraprotein was of the IgG-lambda class, with a molecular weight of approximately 110,000 daltons compared with 160,000 daltons for normal IgG. Immunochemical studies indicated that the paraprotein was an incomplete IgG molecule which was missing a significant antigenic portion of the Fd fragment.