Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens.

The human amyloidoses represent a heterogeneous group of disorders characterized by the deposition of fibrillar protein in vital organs. Given the fact that at least 20 different molecules can form fibrils, the unambiguous identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these disorders. Heretofore, this information has been inferred from particular clinical features of the disease, ancillary laboratory tests, and results of immunohistochemical analyses. However, to establish unequivocally the kind of protein that is deposited as amyloid, it is necessary to determine its chemical composition through amino acid sequencing or mass spectroscopy of material extracted from fibrillar deposits. We have developed a micromethod whereby such studies can be performed readily using sections of formalin-fixed, paraffin-embedded biopsy specimens. The ability to identify precisely the nature of the tissue deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disorders.

Production and immunodiagnostic applications of antihuman light chain monoclonal antibodies.

Hybridomas producing antihuman light chain monoclonal antibodies (MoAbs) were derived from fusion of SP2/O mouse myeloma cells with splenic lymphocytes from mice repeatedly immunized with purified kappa- and lambda-type Bence Jones proteins representative of the major V kappa (V kappa I, V kappa II, V kappa III, V kappa IV) and V lambda (V lambda I, V lambda II/V, V lambda III, V lambda IV, V lambda VI) subgroups or gene families. Monoclonal antibodies were obtained that had specificity for constant-region (CL) determinants common to all kappa or lambda light chains (C kappa and C lambda, respectively) as well as for variable-region (VL) epitopes unique to each of the V kappa or V lambda subgroups. The capability of these reagents to recognize CL and VL determinants on monoclonal immunoglobulin (Ig) molecules was demonstrated in fluid-phase antigen-capturing enzyme-linked immunosorbent assay (ELISA), solid-phase ELISA, and immunoblotting. In addition, these antilight chain MoAbs were used to establish immunocytochemically the kappa or lambda type and VL-subgroup nature of light chains expressed by the cytoplasmic Ig of monoclonal plasma cell and surface Ig of B-lymphocyte populations, respectively. These antibodies facilitated the immunohistochemical detection and characterization of light-chain-associated amyloid (AL amyloid) and other types of light-chain-related tissue deposits. Furthermore, the anti-CL-specific MoAbs were used to measure serum and urinary Ig kappa and Ig lambda concentrations. Quantification of Bence Jones protein excretion, even in the presence of other urinary proteins, was possible using the highly sensitive anti-C kappa and anti-C lambda MoAbs reactive only with free light chains. The ability to identify and characterize, through the use of these antihuman light chain MoAbs, light-chain-related epitopes at the protein, cellular, and tissue level has clinical importance in the diagnosis and treatment of patients with monoclonal plasma cell and related B-cell immunoproliferative diseases.

Immunocytochemical detection of kappa and lambda light chain V region subgroups in human B-cell malignancies.

We have used a sensitive immunoperoxidase method and highly specific anti-light chain antisera to determine the light chain variable region (VL) subgroup nature of cytoplasmic (c) and cell surface (s) Ig expressed by human monoclonal plasma cells and B lymphocytes. The immunocytochemical characterization of cIg and sIg used antisera specific for the established kappa light chain V kappa subgroups (V kappa I, V kappa II, V kappa III, and V kappa IV) and the lambda light chain V lambda subgroups (V lambda I, V lambda II/V, V lambda IV, and V lambda VI). Studies were performed using cytospin preparations of bone marrow-, peripheral blood-, and lymph node-derived cells from patients with multiple myeloma, amyloidosis AL, and Waldenström's macroglobulinemia and with low-, mid-, and high-grade B-cell malignancies. The V kappa or V lambda subgroup of the cIg or sIg also could be identified after deparaffinization and enzyme treatment of formalin-fixed, paraffin-embedded specimens. For those patients who had monoclonal serum or urinary Igs, there was complete concordance between the VL subgroup of the secreted Ig and that of the cIg or sIg. The percentage distribution of V kappa or V lambda subgroups on the sIg of cells from patients with chronic lymphocytic leukemia (CLL) and other cytomorphologic types of B-cell malignancies differed from that found for kappa- or lambda-type Bence Jones proteins obtained from patients with multiple myeloma, amyloidosis AL, and Waldenström's macroglobulinemia. In contrast to the plasma cell and lymphocytoid plasma cell diseases, a relative predominance of certain VL subgroups, ie, V kappa IV, V lambda III, and V lambda IV, and the absence of the amyloid-associated V lambda VI subgroup were found in CLL and related diseases. The immunocytochemical techniques used make possible a rapid means to demonstrate B-cell monoclonality and provide further evidence for the selective expression of certain VL genes in human B-cell neoplasia.