Reproducible flow cytometric methodology for measuring multidrug resistance in leukaemic blasts

In order to bring MDR analysis into a clinical setting, reproducible assays with clear cut off points to define MDR positivity must be used. Sensitivity can also be increased by combining the results of more than one assay. We have used a combination of flow cytometric assays to define MDR positive and negative blasts in 47 AML patients entered into MRC trials. Our primary test is a standardised and reproducible assay for anthracycline accumulation in which we use carboxylate microspheres to bind the fluorescent drug daunorubicin (dnr). Cells and beads are incubated concurrently with dnr. Cellular dnr accumulation is quantified as a cell:bead fluorescence ratio. Confirmatory assays for MDR comprise the cyclosporin modulation assay for rhodamine 123 uptake and also measurement of lung resistance protein and multidrug resistance associated protein (with LRP-56 and MRPr1 respectively). 27/47 (57%) samples had both low and accumulation and at least one positive confirmatory test (a modulated functional assay and/or protein overexpression) and were categorised as "confirmed MDR". 15/47 patients (32%) were MDR negative in all 4 assays. 5/47 (11%) patients had unconfirmed low dnr accumulation. None of the patients in this cohort had high dnr accumulation alongside overexpressed LRP or MRP or functional P-glycoprotein. We believe that this approach to MDR analysis enhances the value of the highly reproducible functional assays. The use of a primary and confirmatory tests is also likely to improve specificity.