Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1994-05-06
2001-09-11
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007250, C435S808000, C435S968000, C435S091500, C435S173300, C435S091500, C435S800000, C435S805000
Reexamination Certificate
active
06287791
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of flow cytometry and more particularly to the analysis of hematopoietic cells from blood and bone marrow. The invention enables the calculation of blood and bone marrow differentials by multi-parameter flow cytometry.
BACKGROUND OF THE INVENTION
In U.S. Pat. No. 5,047,321, Loken and Terstappen described the multi-parameter analysis of cellular components in a body fluid. The body fluids described included blood and bone marrow. Using a combination of two nucleic acid dyes, a fluorescently labelled monoclonal antibody and two light scatter parameters, Loken and Terstappen were able to discriminate between and among various components of blood and bone marrow, count the number of cells within each component and provide a differential analysis of each.
Loken and Terstappen used LDS-751 (Exciton) as a DNA dye, Thiazole Orange (“TO”, Molecular Probes, Inc.) as an RNA dye, a fluorescently labelled anti-CD45 monoclonal antibody and forward and orthogonal light scatter on whole blood or bone marrow aspirates. Using these five parameters, they were able to detect and differentiate between erythrocytes, reticulocytes, nucleated erythrocytes, platelets, lymphocytes, monocytes, neutrophilic granulocytes, basophilic granulocytes, eosinophilic granulocytes and precursors of all nucleated cells.
Specifically, erythrocytes were characterized by light scatter and lack of fluorescence. Reticulocytes were characterized similar to erythrocytes by light scatter but could be discriminated from erythrocytes based upon their staining with Thiazole Orange. Platelets were characterized by their low light scatter and staining with LDS-751. Leukocytes were characterized by their large light scatter, LDS-751 and Thiazole Orange fluorescence and anti-CD45 fluorescence. Among the leukocytes, lymphocytes were characterized by high fluorescence intensity of anti-CD45 staining; monocytes had similar antibody fluorescence intensity with larger light scatter; neutrophilic granulocytes had dimmer antibody fluorescence intensity with large light scatter; and eosinophilic granulocytes had an antibody fluorescence intensity similar to monocytes but had a larger orthogonal light scatter and lower forward light scatter than monocytes.
While the method has utility for most analyses, a limitation in this method exists. The combination of LDS-751 with Thiazole Orange and anti-CD45 fluorescence does not permit full discrimination among the erythroid lineage (i.e., it does not permit identification of orthochromatic normoblasts, normoblasts and erythroblasts and does not permit differentiation between mature and immature reticulocytes) and does not permit the identification of proliferating myeloid cells and non-hematopoietic cells (i.e., stromal and epithelial cells).
Taking these limitations into account, there is a need for an improved method for the identification of and discrimination among the cellular components of blood and bone marrow.
SUMMARY OF THE INVENTION
The present invention comprises a method for the simultaneous, multi-parameter analysis of cells in a body fluid, such as blood and bone marrow. For each cell in a sample of cells taken from blood or bone marrow, at least two measures of light scatter are taken and at least three measures of fluorescence are taken. The three fluorescence components comprise one nucleic acid dye having a preference for RNA, a first fluorescently labelled cell surface marker which recognizes an antigen expressed in differential amounts on cells of different lineages in the hematopoietic system, and a second fluorescently labelled cell surface marker which recognizes erythrocyte precursors and proliferating cells. The fluorescence emission of the three stains must be distinguishable.
In this method, the cells are mixed separately or together with the nucleic acid dye and cell surface markers. The stained cells then are analyzed by means of flow cytometry wherein the cells are passed substantially one at a time through one or more sensing regions (wherein each of the cells is exposed separately individually to a source of light at a single wavelength and measurements of at least two light scatter parameters and measurements of at least three fluorescence emissions are separately recorded for each cell), and the data recorded for each cell is analyzed in real time or stored in a data storage and analysis means, such as a computer. U.S. Pat. No. 4,284,412 describes the configuration and use of a typical flow cytometer equipped with a single light source while U.S. Pat. No. 4,727,020 describes the configuration and use of a flow cytometer equipped with two light sources.
In a preferred embodiment of this invention, the cell surface markers comprise monoclonal antibodies. The first cell surface marker is an anti-CD45 monoclonal antibody, such as anti-HLe-1 (Becton Dickinson Immunocytometry Systems, “BDIS”). The second cell surface marker comprises one or more monoclonal antibodies that recognize proliferating cells and erythrocyte precursors. In the preferred embodiment, the antibody is an anti-CD71 monoclonal antibody such as anti-Transferrin Receptor (BDIS).
In another embodiment, a third cell surface marker can be selected from the group consisting of anti-CD61, anti-CD41 or anti-CD42, and pan platelet monoclonal antibodies. The addition of a pan platelet antibody aids in the separation of platelets and can aid in the identification of platelet precursors (i.e., megakaryocytes).
Each of these antibodies referred to herein has been given a “cluster designation” number (or “CD” number) by the International Workshop and Conference on Human Leukocyte Differentiation Antigens and many examples of each antibody have been made commercially as well as independently and submitted to the Workshop for clustering.
The antibodies can be directly conjugated to a fluorescent label or can be indirectly labelled with, for example, a goat anti-mouse antibody conjugated directly to the fluorescent label. Direct conjugation is preferred, however. Fluorescent labels which can be used in the practice of this invention include phycoerythrin (“PE”), fluorescein isothiocyanate (“FITC”), allophycocyanin (“APC”), Texas Red (“TR”, Molecular Probes, Inc.), peridinin chlorophyll complex (“PerCp”), CY5 (Biological Detection System) and conjugates thereof coupled to PE (e.g., PE/CY5, PE/APC and PE/TR). A preferred combination of labels is PE and PE/CY5. Thiazole Orange is the preferred nucleic acid dye. U.S. Pat. No. 4,520,110 describes the composition and use of PE conjugated to a monoclonal antibody, and U.S. Pat. No. 4,542,104 describes the composition and use of PE in a paired conjugate format. U.S. Pat. No. 4,876,190 describes the composition and use of PerCp.
In a preferred method, a sample of blood or bone marrow is taken and is mixed with an RNA dye, a fluorescently labelled anti-CD45 monoclonal antibody and a fluorescently labelled anti-CD71 monoclonal antibody. After mixing, the sample is run on a flow cytometer, such as a FACScan™ brand flow cytometer (BDIS) equipped with a laser light source. Cells are analyzed substantially one at a time and orthogonal light scatter, forward light scatter and three fluorescence emissions are separately recorded for each cell. The five parameters recorded for each cell are then used in one or more combinations to identify and characterize each cell.
The method of this invention can be used to both identify and discriminate between cell populations and stages of development as well as to enumerate the numbers of cells in each population and stage.
REFERENCES:
patent: 4284412 (1981-08-01), Hansen et al.
patent: 4520110 (1985-05-01), Stryer et al.
patent: 4542104 (1985-09-01), Stryer et al.
patent: 4727020 (1988-02-01), Recktenwald
patent: 4845653 (1989-07-01), Conrad et al.
patent: 4876190 (1989-10-01), Recktenwald
patent: 4957870 (1990-09-01), Lee et al.
patent: 5047321 (1991-09-01), Loken et al.
Davis et al “Clinical Flow Cytometric Reticulocyte Analysis” Pathobiology 58:99-106 1990.*
Dako Bulletin “The
Chen Chia-Huei
Terstappen Leon W M M
Becton Dickinson and Company
Highet David W.
Nguyen Bao-Thuy L.
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