Presented at the 1999 APIII Conference Return to 1999 Abstract Index
COMBINED IMMUNOPHENOTYPING AND DNA ANALYSIS BY LASER SCANNING CYTOMETRY
Memorial Sloan-Kettering Cancer Center
Department of Pathology
New York, New York
Drazen M. Jukic, MD,
PhD
S. Mastorides1, D. Jukic2, D. Polsky1, C.
Cordon-Cardo1
1Memorial Sloan-Kettering Cancer Center, Department of Pathology
2University of Pittsburgh Medical Center, Department of
Dermatopathology
Introduction: The Laser Scanning Cytometer (LSC) (Compucyte Corporation, Cambridge MA) is a microscope-based cytofluorometer that uses lasers to measure relative size, internal structure, and emitted fluorescence from cellular samples mounted on microscope slides and stained with fluorescent reagents. The fluorescence of individual cells is measured with a speed, precision, and sensitivity comparable to flow cytometry. A laser is used to illuminate successive areas of cells on the slide. Light resulting from the laser excitation of fluorochromed cells is detected by photo-sensors and converted into a set of digital values. The digital values are arranged as picture elements termed pixels, which are the smallest units on the display screen that can be stored, displayed, or addressed. The sets of pixels for each event are segmented so that properties such as maximum pixel, total fluorescence, and area can be determined for each cell. The result is a list of properties created for each of thousands of cells in a sample. Because LSC measurements are microscope slide-based, it is possible to record the position of each cell on the slide in list-mode fashion so that cells with given subsets of features can be viewed and correlated with specific cellular events. For example, sequential analysis of the same cells can be carried out using different immunohistochemical stains and correlated with nuclear hyperchromicity, as measured by maximum pixel fluorescence intensity, in order to identify cell types differing in DNA ploidy and cell cycle position. We demonstrate the application of this technology to the cell cycle analysis of normal human fibroblasts.
Methods: The fibroblast cells were directly cultured on slides using chambers and fixed in 80% ethanol. The tissue was incubated for 30 minutes with mouse anti-human Cyclin B1 primary antibody (.5mg/ml), then incubated for 15 minutes with FITC labeled goat anti-mouse secondary antibody. The slide was then counter-stained for 30 minutes in propidium iodide (PI) staining solution at a concentration of 5?g/ml in phosphate buffered saline (PBS) with 200?g/ml ribonuclease type A. After staining, cells were cover-slipped with a 75% glycerol/ 25% PBS mixture. LSC measurements were made with a 488nm wavelength, 20-mW argon ion laser using long red and green fluorescence detectors.
Results: Data from the LSC can be displayed as a scattergram. A single cell is represented as a dot in the scattergram and its location is the result of the measurement of two selected features. The measurement from one feature is placed on the x-axis and the measurement from another feature is placed on the y-axis. Each dot correlates one parameter with another. Propidium iodide (PI) staining is detected by the long red sensor. The Max Pixel (highest intensity of fluorescence within each cell) is the selected feature on the horizontal axis and the Integral (total amount of fluorescence within the cell) is the selected feature on the vertical axis. The nuclear PI staining that shows in the scattergram configuration is best for displaying cell cycle position and DNA ploidy analysis. Cells are segregated into G0/G1, S, G2, and M cell cycle phases. The data can also be displayed as a histogram that demonstrates one parameter on the x-axis and a count of events on the y-axis. The cells on the histogram can be segregated into G1, S, and G2/M cell cycle phases. We also demonstrate Cyclin B1 FITC staining, which is detected by the green sensor, on the x-axis and PI staining on the y-axis. The cells showing intense Cyclin B1 staining can be gated in order to generate region statistics. The cells within the gated region can also be colorized and back-gated to previous scattergrams. The normal fibroblasts which express Cyclin B1 are predominantly located in the G2 and M cell cycle phases. Objective region statistics can be generated for each gated sub-population of interest to include the total population count and a region's percentage of the total population. Events of interest can be relocated, visually confirmed, remeasured, and the images recorded.
Conclusion: Laser Scanning Cytometry is a powerful tool for simultaneous and objective measurement of multiple immunohistochemical markers. The staining intensity of the markers can be correlated with cell cycle analysis. The possibility of combining these measurements with the visual image of the corresponding cell represents an advantage since morphological structure can be preserved. Further studies examining both normal and neoplastic cell lines and tissues will be performed in the Department of Pathology at MSKCC.
