Isolation of Reed-Sternberg cells from lymph nodes of Hodgkin's disease patients.
This study describes a simple and relatively rapid method of purifying Reed-Sternberg (R-S) cells and their morphologic variants from the lymph nodes of patients affected by Hodgkin's disease. Our initial studies defined the optimal procedure for a quantitative disaggregation of Hodgkin's lymph nodes and the densities of R-S cells in several donors. These preliminary steps were helpful in the development of strategies for selectively concentrating R-S cells by density gradient centrifugation. We layered a single-cell suspension over Percoll of appropriate density, centrifuged these samples for 15 minutes, and collected a fraction enriched in R-S cells. Most of the R-S cells were distributed between densities of 1.060 and 1.072, with a peak at approximately 1.066 g/mL. R-S cells are denser than many mononuclear cells present in the lymph nodes of Hodgkin's patients and lighter than reactive cells such as eosinophils, mast cells, and neutrophils. However, the ranges of densities of these cell types overlap, making purification of R-S cells by isopyknic centrifugation impossible. Nevertheless, when this enriched fraction is further processed by velocity sedimentation in order to take advantage of the larger size of R-S cells as compared with all other cells, a substantial purification is achieved. We used three different velocity-sedimentation chambers to find the optimal conditions for obtaining the highest purity with a high final yield. The cells isolated by this method are viable, appear to be morphologically normal, and have been further characterized biologically.
Sitar
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Clinica Medica II, Università di Pavia, Italy.
Brusamolino
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Bernasconi
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Ascari
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D000954:Antigens, Surface / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D002449:Cell Aggregation; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D002500:Centrifugation, Isopycnic; D006689:Hodgkin Disease / Q000473:pathology*; D006801:Humans; D008198:Lymph Nodes / Q000473:pathology*; D010641:Phenotype; D011947:Receptors, Antigen, B-Cell / Q000032:analysis; D014407:Tumor Cells, Cultured
D000954:Antigens, Surface; D015415:Biomarkers; D011947:Receptors, Antigen, B-Cell