SENSITIVE IMMUNOFLUORESCENCE DETECTION OF THE EXPRESSION OF P-GLYCOPROTEIN IN MALIGNANT-CELLS

Because reversal of multidrug resistance increases chemotoxicity, earl y detection of low P-glycoprotein expression is clinically relevant fo r justifying early treatment of those patients that might benefit most from reversal therapy, We elected to score P-glycoprotein in single t umor cells, because the gene is rarely amplified, mRNA levels do not n ecessarily correlate with protein levels, and many normal hematopoieti c or stroma cells within tumors and leukemic marrows also express P-gl ycoprotein. We enhanced the ''signal-to-noise'' ratio for detecting lo w P-glycoprotein levels by a novel complex made by pre-incubating mous e peroxidase-antiperoxidase, used solely to provide a stable framework for attaching multiple DTAF-labeled F(ab')(2) fragments of rabbit ant imouse IgG. We improved specificity by using both C219 and C494, which are directed against separate internal P-glycoprotein epitopes, We st andardized staining with two series of negative and positive controls, in which P-glycoprotein was quantified by immunoblot, and confirmed s ensitivity by staining a low-expression cell Line and ''mixed'' sample s containing small numbers of positive cells. We measured P-glycoprote in by flow cytometry, examining aliquots by differential interference contrast microscopy to identify malignant cells, in which we confirmed P-glycoprotein staining by fluorescence microscopy, We detected low P -glycoprotein expression in clinical samples of leukemic blasts, disti nguishing them from normal P-glycoprotein expression hematopoietic cel ls, This assay may be valuable for early diagnosis of low, but potenti ally important expression of P-glycoprotein, thereby allowing early ap plication of reversal therapy. (C) 1997 Wiley-Liss, Inc.