Use of fluorescence threshold triggering and high-speed flow cytometry for rare event detection.

A simple rare event detection method utilizing dual-parameter flow cytometry is described, which allows quantitation of specific cellular events at the level of two cells in 10(7) total cells. Using a standard unmodified single laser flow cytometer sampling at a rate of 25,000 events/sec and a fluorescence discriminator, 10(7) total cells are processed in 7 min. The assay involves precise characterization of instrument flow rates to calculate total events processed by the cytometer rather than accumulate total events in computer memory. This method of detecting rare events is demonstrated by using a model system of breast cancer cells labeled with a metabolically activated dye and serially diluted into normal peripheral blood. Potential applications include validation of methods to detect minimum residual disease following myeloablative therapy, detection of any remaining tumor cells following purging methods, and validation of methods to detect circulating fetal cells in maternal blood.

Quantitation of human in vitro megakaryocytopoiesis by radioimmunoassay.

The isolation and characterization of human megakaryocyte growth factors has been hampered because evaluation of megakaryocyte growth in semisolid medium requires both lengthy incubation and visual quantitation. In addition, colony formation requires cell division, while most regulation of platelet production may involve individual, nonproliferating differentiating megakaryocytes. We have developed a radioimmunoassay (RIA) that makes use of an iodinated murine monoclonal antibody (MoAb) specific for platelet/megakaryocyte glycoprotein IIb/IIIa (GPIIb/IIIa) to measure megakaryocyte production in liquid marrow culture. This assay is sensitive to 3 X 10(3) platelets (roughly 30 megakaryocytes) and linear up to 1 X 10(6) platelets, and thus it provides a useful range for quantitating megakaryocyte production in in vitro marrow culture. Significant differences (threefold to fivefold) in megakaryocyte/platelet-specific GPIIb/IIIa complex are detected between stimulated and unstimulated marrow cultures by day 7, although antigen accrual in stimulated cultures continues through at least day 16. Conditions that promote megakaryocyte growth in semisolid medium (ie, aplastic plasma and PHA-LCM) have also been facilitory in liquid culture. This rapid and sensitive assay for cell-bound GPIIb/IIIa should facilitate recognition and isolation of megakaryocyte and platelet growth factors.