Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V, caspase cleavage and dUTP incorporation assays.
OBJECTIVE
To compare the performance of different assays for measuring apoptosis in cytological specimens.


METHODS
Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase-3 and -8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays.


RESULTS
Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V-positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001).


CONCLUSIONS
The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.
Division of Pathology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.
H P
HP
Dong
A
A
Holth
M G
MG
Ruud
E
E
Emilsen
B
B
Risberg
B
B
Davidson
D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D001202:Ascitic Fluid / Q000378:metabolism; D044963:Biopsy, Fine-Needle / Q000379:methods*; D020169:Caspases / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D020287:In Situ Nick-End Labeling / Q000379:methods*; D009362:Neoplasm Metastasis / Q000473:pathology; D010996:Pleural Effusion / Q000378:metabolism
D017304:Annexin A5; D020169:Caspases