DETECTION OF INTRACELLULAR ANTIGENS BY FLOW-CYTOMETRY - COMPARISON OF2 CHEMICAL METHODS AND MICROWAVE-HEATING

Detection of intracellular antigens by flow cytometry requires effecti ve fixation and permeabilization of the cell membrane. This study comp ares three fixation/permeabilization techniques: two commercial chemic al reagents, the ORTHOPermeaFix(TM) (OPF) and the FIX&PERM Cell Permea bilization Kit(R) (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 an d rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, usin g positive- and negative-control cell lines and peripheral blood monon uclear cells. Western blotting was performed as a control of protein e xpression. For the four antigens assessed, cellular morphology, discri mination between intact cells and debris, percentage of positive cells , and mean fluorescence intensity were examined. For this last paramet er, the assessment of the MWH technique was performed using SD and a g raphical approach inspired by the concepts described by Bland and Altm an (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 199 7;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equival ent to OFF and F&P. As assessed for some of the most clinically releva nt 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. Interesting ly, this feature, which would prevent cell selection on the basis of c ombined membrane and intracellular epitopes, is associated with a decr ease of nonspecific background fluorescence.