Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometry.
A flow cytometric assay was developed for correlated measurement of DNA content and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, paraffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archival or fresh tissue/cell specimens. It is based on (a) proteolytic release of cell nuclei from deparaffinized and rehydrated 90-microm thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC-labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA staining with 4'6-diamidino-2-phenyleindole. The fluorescence was recorded with a double-beam flow cytometer equipped with a mercury arc lamp and an argon ion laser. Cytograms obtained with this assay correlated closely with those produced using nonembedded material from the same specimen. Furthermore, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional evaluation of TUNEL on (corresponding) sections (p < 0.001). Since necrotic cells can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important advantage of the assay.
Heiden
T
T
Immunopathology Laboraotry, Cancer Center Karolinska, Karolinska Institute, Stockholm, Sweden. Thomas.Heiden@onkpat.ki.se
Castaños-Vélez
E
E
Andersson
L C
LC
Biberfeld
P
P
D017209:Apoptosis; D002455:Cell Division; D002467:Cell Nucleus / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D020287:In Situ Nick-End Labeling; D016612:Paraffin Embedding; D011003:Ploidies; D015203:Reproducibility of Results; D020298:U937 Cells