Comparison of Fully Automated and Semiautomated Systems for Protein Immunofixation Electrophoresis.

BACKGROUND: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation. METHODS: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components. RESULTS AND CONCLUSION: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care.

Immunoglobulin heavy-chain-associated amyloidosis.

Immunoglobulin- or multiple myeloma-associated amyloidosis has been distinguished by the tissue deposition of Congophilic, fibrillar protein consisting of light chains or light-chain fragments (AL amyloidosis). We now report the isolation and characterization of another form of immunoglobulin-associated amyloid obtained from a patient who had extensive systemic amyloidosis and in whom the amyloid deposits consisted not of light chains but rather of an unusual form of heavy chain. This component, isolated from splenic amyloid extracts, represented an internally deleted IgG1 heavy chain as evidenced by immunochemical, electrophoretic, and amino acid sequence analyses. A comparable immunoglobulin-related monoclonal protein, consisting only of IgG heavy chains, was present in the patient's urine. Based on serologic reactivity with a battery of anti-immunoglobulin antisera, these two immunoglobulin-related components were antigenically identical; however, when compared to normal IgG, both were deficient in Fc-associated gamma-chain determinants. The structural abnormality of the amyloid gamma-chain protein was further evidenced by SDS/PAGE and immuno-blotting analyses: An unusually low molecular mass of approximately 22 kDa was found for this material vs. the expected value of approximately 55 kDa for a normal gamma heavy chain. Despite the lack of certain Fc determinants, the amyloid and urinary heavy-chain proteins expressed the IgG1 subclass allotype marker G1m(a) located on the third constant region (CH3) domain of the internally deleted IgG1 heavy chains. That the amyloid protein contained an intact CH3 domain was established through amino acid sequence analyses of cyanogen bromide fragments and peptides generated by a lysine-specific protease. These studies also revealed that the gamma-chain amyloid protein contained the complete heavy-chain variable (VH) domain [including the diversity (DH) and joining (JH) segments] that was contiguous with the CH3 domain. The low molecular mass of the protein resulted from the total absence of the first (CH1), hinge, and second (CH2) heavy-chain constant regions. Such extensive CH deletions and the presence of a complete VH distinguish this amyloid-associated heavy chain from all other heretofore characterized gamma-heavy-chain disease proteins. This heavy-chain-related form of immunoglobulin-associated amyloidosis is tentatively designated AH amyloidosis.

An unusual case of Waldenström macroglobulinemia with half molecules of IgG in serum and urine.

Immunochemical studies are described in an unusual case of Waldenström's macroglobulinemia. Two monoclonal Igs (whole IgG1/kappa and IgG1/kappa half molecules) occurred in the serum in addition to the IgM monoclonal protein. Protein electrophoresis of the serum showed a monoclonal component in the gamma region, and the immunoelectrophoresis allowed detection of a monoclonal IgM/kappa and another abnormality represented by a double precipitin line in serum and urine, observed when antiserum anti IgG was used. The abnormal proteins were purified and further analyzed. The IgG-related proteins were whole four chains IgG monoclonal molecules, 1/2 IgG monoclonal molecules, composed of one heavy and one light chain, and residual polyclonal IgG. The half molecules were antigenically deficient with respect to normal IgG. The idiotypic analysis showed that the three monoclonal proteins shared idiotypic determinants. This patient had clinical and morphological findings of Waldenström's macroglobulinemia and, as observed in other cases, the formation of half molecules was not associated with a distinct clinical syndrome.

Generalized Castleman's disease with urinary elimination of heavy chain fragments.

A generalized form of Castleman's disease of transitional type is reported in a 66-year-old male who was found in addition to have gamma heavy chain fragments in his urine. One portion of them was constituted by Fc fragments and the other ones by fragments which reacted with anti-Fab/IgG and anti-Fc/IgG antisera but not with anti-kappa and anti-lambda antisera. The findings of the present case support an immunological basis for Castleman's disease.

Immunoselection screening methods for detection of the free heavy chains.

Immunoselection screening methods were developed for the detection of the free heavy chain determinants of biological fluids. The optimal sensitivity range of the electrosyneresis and electroimmunoassay type methods lies between 400 ng and 2 micrograms/ml and between 3 micrograms and 1 mg/ml for immunoglobulins. The sensitive, relatively rapid and cheap variants of the procedure are suitable for the detection of "heavy chain diseases" and of heavy chain fragments.

Clinical and immunochemical studies of 20 patients with amyloidosis and plasma cell dyscrasia.

Amyloidosis associated with plasma cell dyscrasia (AAPCD) is a relatively rare clinical entity (4% of our patients with PCD) and its early recognition and distinction from multiple myeloma (MM) may be of great therapeutic and prognostic significance. Laboratory parameters, such as concentrations of normal polyclonal Ig, Bence-Jones proteins and serum monoclonal components (MC) showed in our patients lower MC concentrations than in MM, lambda-L-chains and of gamma-H-chains predominating. Sequential skeletal X-ray studies and bone marrow morphology remain essential diagnostic procedures. Due to the lack of efficient therapeutic agents for AAPCD and the great progress achieved in recent years in the treatment of secondary amyloidosis, the immunochemical analysis of the isolated amyloid fibril as well as of the surrounding 'ground substance' should be pursued in AAPCD. Our data support previous observations, that in AAPCD the amyloid fibril subunit is an L-chain fragment predominantly derived from lambda-L-chains which originates from the same clone as the MC. The localization of an enzymatic cleavage point on the L-chain, the detection of a specific proteolytic enzyme and the identification of additional components in the amyloid substance, may further elucidate the etiopathogenesis of AAPCD.

Identification and incidence of a urinary fragment of IgG.

Urine from some patients, when concentrated approximately three hundred fold and immunoelectrophoresed against anti-IgG, shows an unexpected additional precipitin line in the alpha-globulin region. This reaction has been shown to be due to the presence of a low molecular weight (approximately 20 000) fragment of the heavy chain of IgG. A retrospective examination of immunoelectrophoretic plates run over a period of five years has revealed that this fragment was present in 30 out of 110 patients.

A spontaneously occurring complex of beta2-microglobulin and a fragment of gamma-chain of IgG: isolation from the urine of a patient with plasma cell leukemia and characterization.

A complex of a fragment of gamma-chain of IgG and beta2-microglobulin (beta2M) was isolated from the urine of a patient with plasma cell leukemia. The approximate m.w. of the gamma-fragment was 19,000 and this gamma-fragment was found to be associated with beta2M noncovalently. The complex could be dissociated in 7.5% (v/v) n-propanol which suggests an important role of hydrophobic bonds in the association of the gamma-fragment and beta2M. The beta2M did not bind monoclonal IgG from this patient. Several anti-beta2M antisera tested which had been prepared with free beta2M did not react with the beta2M associated with the gamma-fragment, but reacted with the free beta2M obtained by the dissociation of the complex.

Gamma heavy chain disease: clinical aspects and characterization of a deleted, noncovalently linked gamma1 heavy chain dimer (BAZ).

This report describes the clinical and immunoglobulin features of a patient with gamma heavy chain disease (HCD), who presented with a clinical picture suggestive of an underlying malignancy rather than the usual picture of lymphoma or granulomatous disease. A unique clinical feature was the nearly total replacement of the submaxillary glands by plasma cells. The patient's serum and urine contained a paraprotein, gammaHCD protein BAZ, which belongs to the gamma1 subclass and forms noncovalently linked dimers with a molecular weight of approximately 60,000 daltons. This mutant protein exhibited a deletion which encompassed most of the variable (V) region, the first constant domain (CH 1), and the hinge region. In addition, preliminary structural analyses demonstrated the replacement of alanine by glycine in position 431 of the carboxyterminal octadecapeptide. This substitution may possibly represent another allotypic marker on IgG1 proteins.

IgG half-molecules: clinical and immunologic features in a patient with plasma cell leukemia.

The clinical manifestations and immunologic features of a patient with plasma cell leukemia who produced k, IgG half-molecules are described. His serum contained both 7S myeloma protein and 4.3S half-molecules, whereas his urine contained predominantly half-molecules. The half-molecules were discovered because the serum and urine formed double precipitin lines when analyzed by commercially available IgG radial immunodiffusion plates that contained antibodies to determinants on both the Fab and Fc fragments. Immunoelectrophoresis also revealed double precipition lines with such antisera. In contrast, when antisera specific for the IgG Fc fragment were used, the serum showed only a single line formed by intact IgG, and the urine failed to react, indicating that the half-molecule was antigenically deficient in the Fc fragment. The half-molecule consisted of one covalently linked heavy and light chain, both having about normal molecular weights, suggesting that they did not have a large deletion which could have caused the half-molecule production. Comparison of the clinical manifestations of the patient with those of four other known patients who produced half-molecules suggested that half-molecule formation is not associated with a distinct clinical syndrome.