Even in 7 patients in whom there was no or predominance and who had more than 2 plasma cells per high power field, the presence of such cells having the same cytoplasmic VLphenotype as that of the amyloid was evidenced by their staining with specific anti-VLmAbs (Table 1)

Even in 7 patients in whom there was no or predominance and who had more than 2 plasma cells per high power field, the presence of such cells having the same cytoplasmic VLphenotype as that of the amyloid was evidenced by their staining with specific anti-VLmAbs (Table 1). have shown that the spleen may be another source of amyloidogenic light chains. == Introduction == Systemic amyloidogenic light chainassociated (AL) amyloidosis is characterized by the deposition in the heart, kidneys, liver, nerves, and other organs or tissues Furosemide of or light chainrelated fibrils.1These molecules are the products of plasma cells deemed monoclonal based on the finding of a predominance of +or +cytoplasmic immunoglobulin (Ig), and the presence of such cells in bone marrow is one of the diagnostic hallmarks of this disorder. In comparison with multiple myeloma, patients with Furosemide AL amyloidosis Furosemide typically have a relatively low number of plasmacytes in this site, that is, less than 5% to 10%2; thus, it is not known whether this relatively sparse population secretes sufficient amounts of amyloidogenic precursor to Furosemide account for the extensive deposits that can occur throughout the body. To address this question, we have determined whether another hematopoietic organ, namely the spleen, also contains monoclonal light chainproducing plasma cells. We now report the results of immunophenotypic analyses that used monoclonal antibodies (mAbs) specific for and free light chains (FLCs),3as well as reagents reactive with the major Vand Vgene families.4Here we showed that the spleens of 4 of 8 AL and 8 of 18 AL patients had a statistically significant preponderance of plasma cells with a light chain isotype identical to that expressed by the bone marrowderived plasma cells and/or the amyloid deposits == Methods == == Patient population == The 26 patients included in this study had a diagnosis of systemic AL amyloidosis (manifested primarily by renal, cardiac, or neurologic dysfunction) based on the presence of a serum or urinary monoclonal Ig; an abnormal serum FLC / ratio, as determined by our mAb-based enzyme-linked immunosorbent assay (ELISA)5; or identification of the light chain nature of amyloid extracted from autopsy-derived tissue, as documented by amino acid sequencing and/or mass spectrometry.6,7The study was approved by the University of Tennessee Medical Center’s Institutional Review Board, and informed consent was obtained in accordance with the Declaration of Helsinki. == Immunohistochemistry == Four-micrometer-thick sections, cut from formalin-fixed, paraffin-embedded blocks of spleen, were deparaffinized and subjected to antigen retrieval by exposure in a 90C water bath for 30 minutes to a Dako Target Retrieval Solution containing citrate buffer, pH 6.0 (Dako Cytomation, Carpenteria, Rabbit Polyclonal to UTP14A CA), followed by cooling at room temperature for 20 minutes. The tissue was immunostained with a commercial antiplasma cell antibody (Dako); our murine mAbs F-C3 and F-G9, which react only with free or light chains, respectively3; and reagents specific for the major VLsubgroups (V1, 2, 3, and 4; V1, 2, 3, 6, and 8).4Immunoreactivity was visualized with the use of a streptavidin-biotin detection system (BioGenex, San Ramon, CA) and color was developed with diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA). The slides were counterstained with hematoxylin (Gill #3, Sigma-Aldrich, St Louis, MO). The number of reactive plasma cells was enumerated in 15 high-power fields using a Leitz DMRB microscope (Vashaw Scientific, Norcross, GA) fitted with a 40/0.75 dry objective and a 1.6 magnifying lens. The results were averaged and the statistical significance determined by thettest. Cytospin preparations of bone marrow obtained at the time of diagnosis also were evaluated with the same antibodies by methods detailed elsewhere.8 == Results and discussion == The predominant site of deposition and the VLnature of the amyloid in all 26 patients are given inTable 1. Immunocytochemical analyses of bone marrowderived specimens obtained at the time of diagnosis were performed in 19 cases in which clonal populations of plasma cells were found, as evidenced by their reactivity with either the antifree or reagent, as well as a particular anti-VLsubgroup mAb. In each instance, there was complete concordance between the chemical nature of the AL amyloid and the predominant plasma cell immunophenotype. Further, a striking and statistically significantly similar population was found in the spleens from 12 patients (1, 4; 1, 3; 2, 2; 6, 2; and 8, 1), as illustrated for cases 8 Furosemide (AL) and 7 (AL;Figure 1). Even in 7 patients in whom there was no or predominance and who had more than 2 plasma cells per high power field, the presence of such cells having the same cytoplasmic VLphenotype as that of the amyloid was evidenced by their staining with specific anti-VLmAbs (Table 1). Bone marrow specimens taken within 2 months of death from 6 patients also.