title: Development of a flow cytometry-based pulse-width assay for detection of aggregates in cellular therapeutics to be infused by catheter.
abstract: OBJECTIVE
  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.
authors: Aldagen, a wholly owned subsidiary of Cytomedix, Inc, Durham, North Carolina, USA. Electronic address: dhickerson13@gmail.com.
authors: Darren H M
authors: DHM
authors: Hickerson
authors: Aldagen, a wholly owned subsidiary of Cytomedix, Inc, Durham, North Carolina, USA.
authors: Hannah Storrie
authors: HS
authors: White
authors: Aldagen, a wholly owned subsidiary of Cytomedix, Inc, Durham, North Carolina, USA.
authors: Alan
authors: A
authors: Nguyen
authors: Aldagen, a wholly owned subsidiary of Cytomedix, Inc, Durham, North Carolina, USA.
authors: Elizabeth
authors: E
authors: Nieman
authors: Aldagen, a wholly owned subsidiary of Cytomedix, Inc, Durham, North Carolina, USA.
authors: Norin
authors: N
authors: Meadows
authors: CT2 Program, Duke Translational Medicine Institute, Durham, North Carolina, USA.
authors: Tracy
authors: T
authors: Gentry
authors: Triangle GxP Solutions, LLC, Morrisville, North Carolina, USA.
authors: Sandra J
authors: SJ
authors: Foster
authors: CT2 Program, Duke Translational Medicine Institute, Durham, North Carolina, USA.
authors: Andrew E
authors: AE
authors: Balber
authors: Aldagen, a wholly owned subsidiary of Cytomedix, Inc, Durham, North Carolina, USA.
authors: Michelle
authors: M
authors: Fiordalisi
authors: Unicorn Pharma Consulting, Brentwood, Tennessee, USA.
authors: James M
authors: JM
authors: Hinson
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification; D001854:Bone Marrow Cells / Q000473:pathology; D002545:Brain Ischemia / Q000175:diagnosis* / Q000276:immunology; D057785:Catheters / Q000009:adverse effects; D002449:Cell Aggregation; D064987:Cell- and Tissue-Based Therapy / Q000009:adverse effects*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D057230:Limit of Detection; D013234:Stem Cells / Q000473:pathology; D020521:Stroke / Q000473:pathology*
chemical_list: D000444:Aldehyde Dehydrogenase
keywords: bone marrow; cell aggregation; flow cytometry; pulse width; stem cells; stroke