Clinical Chemistry
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Clinical Chemistry 52: 523-526, 2006; 10.1373/clinchem.2005.058883
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(Clinical Chemistry. 2006;52:523-526.)
© 2006 American Association for Clinical Chemistry, Inc.

Technical Briefs

Cell-Free Plasma DNA: A Marker for Apoptosis during Hemodialysis

Johanna Atamaniuk1,a, Katharina Ruzicka1, Karl M. Stuhlmeier2, Alireza Karimi1, Manfred Eigner3 and Mathias M. Mueller1

1 Institute of Laboratory Diagnostics, and3 First Medical Department, Dialysis Unit, Kaiser Franz Josef Hospital, Vienna, Austria;2 Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria;

aaddress correspondence to this author at: Institute of Laboratory Diagnostics, Kaiser Franz Josef Hospital, Kundratstrasse 3, A-1100, Vienna, Austria; fax 43-60191-3309, e-mail johanna.atamaniuk{at}wienkav.at


Abstract

Background: We evaluated whether cell-free plasma DNA might be an appropriate marker for cell damage during hemodialysis (HD) and whether it correlated with annexin V expression and 7-amino-actinomycin D (7AAD) nuclear staining of blood leukocytes.

Methods: Circulating DNA, annexin V, and 7AAD were measured in HD patients before HD, 20 min after start of HD, and after HD had ended. Healthy volunteers provided control measurements. Necrosis and apoptosis were monitored by gel electrophoresis.

Results: Plasma DNA concentrations were not significantly different between controls and patients before HD. Circulating DNA increased significantly (P <0.05) after 20 min of treatment with HD. Post-HD concentrations of DNA were significantly higher compared with pre-HD and controls (P <0.005). Agarose gel electrophoresis showed ladders typical of apoptosis in post-HD samples. Two subpopulations of CD45+ leukocytes were defined by flow cytometry: annexin V+/7AAD+ population for apoptosis, and annexin V+/7AAD– for early apoptosis. Compared with healthy controls, mean fluorescence (MF) of 7AAD+ apoptotic cells in the annexin V+/7AAD+ subpopulation before HD was not significantly increased. HD increased MF of 7AAD+ cells in the annexin V+/7AAD+ subpopulation. In this subpopulation, MF of annexin V+ cells was significantly higher (P <0.01). MF of annexin V+ cells in the annexin V+/7AAD+ subpopulation increased during HD.

Conclusions: During HD, cell-free plasma DNA concentrations, annexin V expression, and 7AAD uptake in leukocytes increases. The increase in plasma DNA, appearing as ladders typical of apoptosis, and the 7AAD uptake in leukocytes demonstrate that the predominant portion of circulating DNA in HD patients originates from apoptotic leukocytes.




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