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G Sitar, E Brusamolino, C Bernasconi and E Ascari
Clinica Medica II, Universita di Pavia, Italy.
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.
This article has been cited by other articles:
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| Copyright © 1989 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
