A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.