Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-04-26
1997-05-06
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
436172, 436536, 436800, 436805, 382133, 382134, 435808, G01N 33533, G01N 33536, G01N 3349
Patent
active
056270402
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method for classifying multi-parameter data in real time (or from recorded data) into cluster groups for the purpose of defining different populations of particles in a sample. This invention is particularly useful in the field of flow cytometry wherein multi-parameter data is recorded for each cell that passes through an illumination and sensing region. It is especially useful for classifying and counting immunofluorescently labeled CD3, CD4 and CD8 lymphocytes in blood samples from AIDS patients.
BACKGROUND OF THE INVENTION
Particle analysis generally comprises the analysis of cells, nuclei, chromosomes and other particles for the purpose of identifying the particles as members of different populations and/or sorting the particles into different populations. This type of analysis includes automated analysis by means of image and flow cytometry. In either instance, the particle, such as a cell, may be labeled with one or more markers and then examined for the presence or absence of one or more such markers. In the case of a cell, such as a leukocyte, tumor cell or microorganism, the marker can be directed to a molecule on the cell surface or to a molecule in the cytoplasm. Examination of a cell's physical characteristics, as well as the presence or absence of marker(s), provides additional information which can be useful in identifying the population to which a cell belongs.
Cytometry comprises a well known methodology using multi-parameter data for identifying and distinguishing between different cell types in a sample. For example, the sample may be drawn from a variety of biological fluids, such as blood, lymph or urine, or may be derived from suspensions of cells from hard tissues such as colon, lung, breast, kidney or liver. In a flow cytometer, cells are passed, in suspension, substantially one at a time through one or more sensing regions where in each region each cell is illuminated by an energy source. The energy source generally comprises an illumination means that emits light of a single wavelength such as that provided by a laser (e.g., He/Ne or argon) or a mercury arc lamp with appropriate filters. Light at 488 nm is a generally used wavelength of emission in a flow cytometer having a single sensing region.
In series with a sensing region, multiple light collection means, such as photomultiplier tubes (or "PMT"), are used to record light that passes through each cell (generally referred to as forward light scatter), light that is reflected orthogonal to the direction of the flow of the cells through the sensing region (generally referred to as orthogonal or side light scatter) and fluorescent light emitted from the cell, if it is labeled with fluorescent marker(s), as the cell passes through the sensing region and is illuminated by the energy source. Each of forward light scatter (or FSC), orthogonal light scatter (SSC), and fluorescence emissions (FL1, FL2, etc.) comprise a separate parameter for each cell (or each "event"). Thus, for example, two, three or four parameters can be collected (and recorded) from a cell labeled with two different fluorescence markers.
Flow cytometers further comprise data acquisition, analysis and recording means, such as a computer, wherein multiple data channels record data from each PMT for the light scatter and fluorescence emitted by each cell as it passes through the sensing region. The purpose of the analysis system is to classify and count cells wherein each cell presents itself as a set of digitized parameter values. Typically, by current analysis methods, the data collected in real time (or recorded for later analysis) is plotted in 2-D space for ease of visualization. Such plots are referred to as "dot plots" and a typical example of a dot plot drawn from light scatter data recorded for leukocytes is shown in FIG. 1 of U.S. Pat. No. 4,987,086. By plotting orthogonal light scatter versus forward light scatter, one can distinguish between granulocytes, monocytes and lymphocytes in a population of le
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Bierre Pierre
Mickaels Ronald A.
Becton Dickinson and Company
Housel James C.
Parsons Nancy J.
Wark Allen W.
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