Clinical Chemistry
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Clinical Chemistry 44: 2320-2330, 1998;
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 1998;44:2320-2330.)
© 1998 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating

Isabelle Millard1, Etienne Degrave2, Marianne Philippe1, and Jean-Luc Gala1,2,a

1 Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Clinical Biochemistry Department, Clos-Chapelle-aux-Champs, 30-UCL 30.46, 1200 Brussels, Belgium.

2 Scientific Section of the Medical Staff, Belgium Armed Forces, Queen Astrid Military Hospital, Rue Bruyn, 2, 1120 Brussels, Belgium.
a Address correspondence to this author at: Laboratoire de Technologie Moléculaire Appliquée, Service de Biochimie Clinique, Clos-Chapelle-aux-Champs, 30-UCL/30.46, B-1200 Brussels, Belgium. Fax 32-2-764-39-59; e-mail gala{at}sang.ucl.ac.be.

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFixTM (OPF) and the FIX&PERM Cell Permeabilization Kit® (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346:1085–7) as well as Petersen et al. (Clin Chem 1997;43:2039–46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.






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