Isolation of viable Hodgkin and Reed-Sternberg cells from Hodgkin disease tissues.
Hodgkin disease (HD) is characterized by a small number of malignant Hodgkin and Reed-Sternberg (H/RS) cells among a major population of nonmalignant cells. The analysis of H/RS cells has been hampered by their low frequency and fragility. Here, we describe the isolation of viable H/RS cells from HD affected tissues by high gradient magnetic cell sorting (MACS) according to expression of CD30. The cells were enriched to a purity of up to 50%. H/RS cells were distinguished from other CD30(+) cells by the expression of CD15, their size and granularity. No CD30/CD15 double-positive cells could be enriched from a lymph node affected by the lymphocyte predominant subtype of HD, activated lymph nodes or peripheral blood of healthy donors. For two cases of HD individual MACS-purified H/RS cells and H/RS cells micromanipulated from tissue sections of the same lymphoma specimens were analyzed for Ig gene rearrangements. In both cases, identical V gene rearrangements were amplified from both sources of H/RS cells, showing that H/RS cells were successfully enriched. Moreover, the finding that in both cases no additional Ig gene rearrangements other than the ones identified in the H/RS cells micromanipulated from tissue sections were amplified from the MACS-purified H/RS cells further supports the monoclonality of these cells throughout the affected lymph nodes. The isolation of viable H/RS cells ex vivo is prerequisite for a direct study of gene expression by those cells and of their interaction with cells in their vicinity.
Institute for Genetics, University of Cologne, 50931 Cologne, Germany.
J
J
Irsch
S
S
Nitsch
M L
ML
Hansmann
K
K
Rajewsky
H
H
Tesch
V
V
Diehl
A
A
Jox
R
R
Küppers
A
A
Radbruch
D000293:Adolescent; D000328:Adult; D000368:Aged; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002470:Cell Survival; D005260:Female; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D006689:Hodgkin Disease / Q000235:genetics / Q000276:immunology* / Q000473:pathology*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D016130:Immunophenotyping; D017730:Ki-1 Antigen / Q000378:metabolism; D016256:Lewis X Antigen / Q000378:metabolism; D008198:Lymph Nodes / Q000276:immunology / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016539:Reed-Sternberg Cells / Q000276:immunology* / Q000473:pathology*
D017730:Ki-1 Antigen; D016256:Lewis X Antigen