Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
1997-05-13
2001-01-23
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S172000, C356S072000, C356S073000
Reexamination Certificate
active
06177277
ABSTRACT:
A flow cytometer is a device which is now widely used in routine diagnostics and research laboratories for analyzing and classifying cells and other particles. The cells are in liquid suspension and this suspension is pumped through a thin capillary cuvette and a laser beam is focused to the stream at a 90° angle in relation to the laser beam and detection objective lens. The laser beam can also be focused on the stream outside the capillary orifice. The sample suspension is kept in the focus of the laser beam using hydrodynamic focusing which forces the cells to flow along the centre of the axis of the capillary cuvette. Flow cytometry has recently been reviewed in many articles and e.g. by Salzman & al. in: Flow Cytometry and Sorting, Second Edition, M. R. Melamed, T. Lindmo, M. L. Mendelsohn, Eds. Wiley & Sons, Inc., New York, 1990, pp. 81-107.
The cells are normally stained with one or several fluorescent dyes or fluorescent biomolecules e.g. antibodies and flow cytometers are used for measurement of fluorescence and light scattering emitted during a course of laser excitation and these parameters are used for scoring the cells according to their characteristic features e.g. immunochemical features indicated by the fluorescent dye. The scattering signal normally indicates the size of the cell. A flow cytometer can be made for analyzing the cells or alternatively for physical separation (sorting) of cells. In the latter case the flow cytometer is combined with an electrostatic deflection device that brings the liquid droplets, which carry an electrostatic charge and include a cell being ejected from the orifice, to different cuvettes for further analysis.
The flow speed in an ordinary flow cytometer is 10-100 cm/s. Consequently, each cell is exposed by the laser beam for about 10-100 microseconds. The photon burst resulting from the fluorescence emission is detected by a photo-detector and an electric signal will be obtained from the detector having an amplitude which is directly proportional to the amount of the fluorescent dye in the cell.
The electric signals will be analyzed and registered and normally a histogram will result showing the number of cells versus fluorescence intensity.
The precision, sensitivity and reliability of flow cytometry is reduced by different factors including non-specific fluorescence, autofluorescence of the cells, fluorescence of the optics and noise generated in the photo-detectors. The sources of interference cause variation and randomly occurring signals. As a consequence, an ordinary flow cytometer is not capable of resolving small amounts of specific cells within the population of normal cells and cells that constitute less than {fraction (1/1000)} of the main population are not detectable. Examples of analysis of such “rare events” include the screening for cancer cells in the blood circulation for detection of minimum residual disease and screening of foetal erythroblasts in maternal blood circulation for early detection of genetic abnormalities. Another example is fast detection of low amounts of small cells e.g. bacteria in liquor or blood without cultivation. Erythrocytes and bacterial cells are small (1-5 &mgr;m) and they are not resolvable with ordinary flow cytometers without a strong unspecific interference. The reliable and fast detection of human and bacterial cells referred above has great potential in medicine.
Detection of “rare events” or small particles with conventional flow fluorometry is hampered by background fluorescence and scattering and it is very difficult to discriminate false signals from true signals. This invention is related to an improved device and methodology for detection of rare events or small particles in biological fluids. The term “particle” used in the text refers to any biological particle including mammalian cells, blood cells, bacterial cells, cell organelles and viruses.
BACKGROUND OF THE INVENTION
The background art of this invention is covered by many articles dealing with flow fluorometry of small particles, confocal optical microscopy, two-photon excitation, fluorescence correlation spectroscopy and single molecule detection. As background art of this invention we list the following articles relative to sensitive flow fluorometry:
Two-Photon Excitation
Denk, W., Strickler, J., and Webb, W. W. Two-photon laser microscopy. U.S. Pat. No. 5,034,613, 1991.
Lytle, F. E., Dinkel, D. M., and Fisher, W. G. Trace-Level Quantiation Via Time-Resolved Two-Photon-Excited Fluorescence. Appl. Spectrosc. 47(12):2002, 1993.
Denk, W, Strickler, J. H., and Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science. 248:73, 1990.
Sepanlak, M. J. and Yeung, E. S. Laser Two-Photon Excited Fluorescence Detection for High Pressure Liquid Chromatography. Anal.Chem. 49(11):1554-1556, 1977.
Wirth, M. J. and Fatunmbi, H. O. Very-High Detectability in Two-Photon Spectroscopy. Anal.Chem 62:973-976, 1990.
Flow Fluorometry and Cytometry
Nguyen, D. C. and Keller, R. A. Ultrasensitive laser-induced fluorescence detection in hydrodynamically focused flows. Journal of the Optical Society of America B 4(2):138, 1987.
Confocal Microscopy
Mathies, R. A. and Peck, K. Laser excited confocal microscope fluorescence scanner and method. Eur. pat. appl. 91300246.5(440 342 A3), 1991. GO1N 21/64.
Single Molecule Detection
Dovichi, N. J., Martin, J. C., Jett, J. H., Trkula, M., and Keller, R. A. Laser-Induced Fluorescence of Flowing Samples as an Approach to Single-Molecule Dectection in Liquids. Anal. Chem. 56(3):348, 1984.
Lee, Y., Maus, R. G., Smith, B. W., and Winefordner, J. D. Laser-Induced Fluorescence Detection of a Single Molecule in a Capillary. Anal.Chem. 66(23):4142, 1994.
Mathies, R. A. High Sensitivity Fluroerscent Single Particle and Single Molecule Detection Apparatus and Method. PCT/US90/02702(WO 90/14589), 1990, GO1N 21/64.
Mathies, R. A., Peck, K., and Stryer, L. Optimization of High-Sensitivity Fluorescence Detection. Anal.Chem, 62(17):1786, 1990.
Peck, K., Stryer, L., Glazer, A. N., and Mathies, R. A. Single-molecule fluorescence detection: Autocorrelation criterion and experimental realization with phycoerythrin. Proc.Natl.Acad.Sci. 86:4087, 1989.
Shera, E. B. Single molecule tracking. Eu. Pat. 88909172.4(EP 0 381, 694 B1), 1988. GO1N 21/64.
Shera, E. B., Seitzinger N. K., Davis, L. M., Keller, R. A, Soper, S. A., Detection of single fluorescent molecules, Chem Phys Letter 23:553-557, 1990.
Fluorescence Correlation Spectroscopy
Dovichi, N. J., Martin, J. C., Jett, J. H., and Keller, R. A. Attogram Detection Limit for Aqueous Dye Samples by Laser-Induced Fluorescence. Science. 219:845, 1983.
Rigler, R. and Eigen, M. Method and device for assessing the suitability of biopolymers. PCT/EP/00117 (WO94/16313), 1994. GO1N 21/64.
OBJECTS OF THE INVENTION
The object of this invention is an improved method and device for flow fluorometry. The reliability and precision of flow cytometric analysis can be improved significantly with this invention. In particular the analysis of rare events and small size particles in biological suspensions comprising a large number of particles of different kinds and sizes, can be improved and the interferences typical for the ordinary flow cytometry can be reduced. In addition, this invention allows the use of a laser of lower power and less expense than in the ordinary flow cytometry typically used today.
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Lydon James C.
Snay Jeffrey
Soini Erkki
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