The Value of Laser Scatter Microscopy in Clinical Cytometry
EK Morgen, WR Geddie. University Health Network, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada
Background: Flow cytometry has traditionally lacked the ability to assess the morphology of analyzed cells. In recent years, this has been addressed by quantitative imaging cytometry (QIC), a slide-based method, and by certain flow cytometers, such as the Amnis ImageStream. The advantage of the latest generation of QIC for cell-surface-marker immunophenotyping lies in its lower cell-number requirements, ability to iteratively re-stain samples, and the high-quality images produced by laser scatter microscopy (LSM), a differential-interference-contrast-like modality. Furthermore, it captures a complete data set for the entire slide, instead of only when certain predefined criteria indicate that an "event" has occurred. Evaluation of cell morphology in cytometry is an important idea, yet the usefullness of LSM in triage and immunophenotyping remains relatively unexplored.
Design: 50 consecutive cases requiring immunophenotyping were analyzed on the CompuCyte iCys, a 2nd-generation quantitative imaging cytometer. The machine generates a series of scan fields, each 748 by 1000 pixels, by directing the microscope stage to move in the x-direction and the laser beam to scan back and forth in the y-direction. For each pixel, the iCys records laser scatter (detected with a laterally-offset photodetector that yields LSM images), light loss, and multiparametric fluorescence data. In each case, LSM was used both for initial cytology specimen triage and as an adjunct to immunophenotyping, and its utility in these roles was evaluated.
Results: LSM produced shaded relief images which clearly distinguished cells from each other, enabled assessment of cell and nuclear contours, and could provide details of chromatin pattern and cytoplasmic features. During triage, these images allowed for sufficient pattern recognition to provide a provisional diagnosis, and thus better selection of antibody panels for immunophenotyping or more appropriate ancillary studies (cell block, molecular, etc.). As an adjunct to immunophenotyping, LSM images were helpful in confirming cell lineage in gating, in excluding granulocytes from a gate (they can exhibit nonspecific fluorescence), and in excluding cell doublets or aggregates from a gate.
Conclusions: Laser scatter microscopy in quantitative imaging cytometry adds high-quality morphologic data that is useful during triage and immunophenotyping in clinical cytopathology.
Monday, March 22, 2010 1:00 PM
Poster Session II # 259, Monday Afternoon