Development of a flow cytometry-based pulse-width assay for detection of aggregates in cellular therapeutics to be infused by catheter.

BACKGROUND AIMS: Delivery of cell-based therapies through the carotid artery with the use of an intra-arterial catheter could introduce aggregates and cause focal ischemia in the brain. We developed a pulse-width flow cytometry method for aggregate detection and quantification. The assay was designed to be used as a cell product release assay in a clinical trial seeking to treat ischemic stroke with sorted cells brightly expressing aldehyde dehydrogenase (ALDH(br) cells) delivered through intra-arterial catheters. METHODS: The forward light scatter pulse-width axis of a flow cytometer was calibrated for particle diameter measurements through the use of traceable standard microspheres and linear regression. As a positive control, Concanavalin A-aggregated cells were counted manually and sorted onto slides to compare with pulse width-determined values. Known numbers of aggregates were spiked into purified singlet cells for quantification. A clinical standard for aggregate count and diameter was determined. The assay was used to qualify catheters with the use of ALDH(br) cells. RESULTS: The pulse-width axis was highly linear for microsphere diameter (r(2) > 0.99), which allowed for size calibration. Microscopically determined counts and diameters corresponded to pulse width-determined values. Known aggregate counts were linear with pulse width-determined aggregate counts (r(2) = 0.98). The limit of detection was determined to be 0.004%. Flow of ALDH(br) cells through catheters did not generate aggregates. The final method to be used as a release assay for the stroke clinical trial was tested successfully on samples from volunteer donors. CONCLUSIONS: The pulse-width aggregate detection assay provides a reliable, reproducible, accurate and rapid means of detection, classification and quantification of aggregates in cell therapy products.

Flow cytometry of platelets for clinical analysis.

Platelets are small, non-homogenous cells with distinctive surface features important to their essential role in hemostasis. The surface membrane is dynamic, and changes remarkably in lipid asymmetry and receptor expression on triggering of the activation process. There are also extensive and rapid intracellular changes in platelets as a result of biochemical activation through calcium fluxes, phospholipase activity, kinase activity, and phosphorylation mechanisms that lead to release of storage granule contents and generation of fast-acting prostaglandins, all in a matter of seconds after stimulation with a strong agonist. These characteristics make the platelet an interesting but difficult cell to study, and the explosion of knowledge over the last two decades has been fueled in large part by the application of flow cytometry techniques. Clinical applications of flow cytometry analysis of platelets have been pursued in individual specialized medical centers, but have not found widespread practice in clinical laboratories, mostly because of difficulties in standardization of techniques and the inherent biovariability in comparing normal to abnormal platelets. Despite these hurdles, it seems certain that flow cytometry analysis of platelets in pathological states will continue to evolve into more practical and robust procedures that will eventually become standard hematologic assays rather than specialized research tools.