Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-09-15
2001-12-11
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C436S546000, C436S547000, C436S548000, C436S172000
Reexamination Certificate
active
06329158
ABSTRACT:
BACKGROUND OF THE INVENTION
The identification of nucleated cells in multi-parameter flow cytometric analysis is important in a wide array of procedures. The importance of such identification is particularly seen in that nucleated cells preparations often contain debris or other matter that can contribute to the overall noise, thereby decreasing the system's sensitivity.
Such cells are often identified by a nucleic acid dye which stains nucleic acids within the nucleus, permitting easy detection. However, when immunofluorescent markers are used, this approach suffers from the drawback that fluorescence signals from such nucleic acid dyes generally overlap and are much brighter than those of immunofluorescent dyes, and thus, tend to interfere with the observation of the immunofluorescent signal. While nucleic acid dyes which have emission spectra which do not overlap the emission spectra of immunofluorescent dyes are known, they are not practical for use in many instances
To remedy this, researchers have tried to use lower (i.e., less than saturating) concentrations of the nucleic acid dyes, thereby reducing the magnitude of the signal (since this magnitude is a function of concentration). However, this procedure suffers from the drawback that the useful range of cell concentrations is greatly reduced as the fluorescence intensity in such circumstances is highly dependent on the number of cells in the sample. Further, when lower concentration of dye are used, the small amount of dye adsorbed onto the container surface also has a significant effect on the overall amount of dye available for staining nucleic acids, further affecting the sensitivity.
There exists a real need for a nucleic acid dye which can be used at or near saturating concentrations which will not interfere with the immunofluorescent signal.
SUMMARY OF INVENTION
This invention presents improved methodology for identification of nucleated cells in flow cytometric analysis when immunofluorescent dyes are also used. Briefly, the general method involves staining the nucleic acids with a fluorescent dye which can then be used to identify the nucleated cells by measurement of fluorescence on a flow cytometer.
The improvement presented by this invention is the use of a nucleic acid dye, or mixture of dyes, which gives low fluorescence at excitation conditions, so as not to greatly interfere with the signals of the immunofluorescent dyes, which dye or dye mixture can be used at a concentration sufficient to saturate the nucleic acid binding sites.
REFERENCES:
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patent: 4957870 (1990-09-01), Lee et al.
patent: 5047321 (1991-09-01), Loken et al.
patent: 5057413 (1991-10-01), Terstappen et al.
patent: 5436134 (1995-07-01), Haugland et al.
patent: 5437980 (1995-08-01), Haugland
Streitwieser and Heathcock “Introduction to Organic Chemistry, second edition” Macmillan Publishing Co, Inc (1981) p. 1156.*
Davis et al. “Clinical Flow Cytometric Reticulocyte Analysis”, Pathobiology 1990; 58:99-106.*
Lee et al Biosis Abstract Accession No. 87:62136, 1986.
Frey Thomas
Hoffman Robert A.
Becton Dickinson and Company
Gucker Stephen
Kunz Gary L.
Schneck David M.
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