Development of a flow cytometry assay allowing minimal residual detection below the established threshold flow cytometry is the method of choice for routine assessment of minimal residual disease (mrd) in patients with haematological malignancies. Since its sensitivity in clinical samples is 10-4, most studies that stratify patients based on mrd use the threshold of 0.01% residual malignant cells. This threshold has proven to be clinically informative. However, some mrd studies seem to benefit from greater sensitivity, achieved with pcr-based techniques. In the present work, we have developed a routine mrd flow assay with a sensitivity of 10-5, by acquiring 10-fold more leucocytes than the standard 0.5×106. This technique has proved to be accurate and specific, and can be performed easily using a digital cytometer. Precision of measurements was addressed, concluding that detection of 50 malignant cells was enough in order to get coefficients of variation around 10-12%. Using this approach, we measured mrd in acute leukemia and myeloma multiple (mm) samples (n = 100 and n = 54, respectively). We found that the technique was applicable to all mm and all patients, and to 75% of aml patients. In al patients, one third of positive samples showed a mrd level below 0.01%, not detectable by standard flow cytometry. Similar results were obtained with mm samples: one third of bone marrow samples obtained from patients with negative immunofixation had mrd detectable with high-sensitivity flow assay. Taken together, these data indicate that acquiring 5 million leucocytes provides mrd flow cytometry assays with sensitivities approaching those of molecular techniques, being applicable to a majority of patients.<\/p>\n
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