Environmental containment system for a flow cytometer

Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample

Reexamination Certificate

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C422S091000, C422S105000, C436S063000, C436S174000, C436S181000, C435S004000, C435S029000, C209S003100, C209S576000

Reexamination Certificate

active

06780377

ABSTRACT:

TECHNICAL FIELD
An environment containment system provides an adjustably controllable environment for cells entrained within the fluid stream generated by a flow cytometer.
BACKGROUND
A flow cytometer or cell sorter is an instrument that physically separates cells according to certain parameters. Many cell sorters use techniques to distinguish subpopulations of cells by employing a unique blend of modern technologies such as fluidics, electric fields, lasers, optics, analog and digital electronics, computers and software.
Referring to
FIG. 1
, in conventional flow cytometry (
1
), selected cells can be labeled with fluorescent molecules that bind specifically to the constituent(s) (e.g. using a fluorescently-labelled antibody to a particular surface antigen) to be measured. Certain, desired cells (a subpopulation of cells) may, for example, be fluorescently labeled while other cells in the sample of cells are not fluorescently labeled. In this example, such fluorescently labeled cells may be selectively identified from a heterogeneous cell population.
The cells and a sheath fluid are supplied to the interior volume of a droplet generator (
2
) from a cell source (
3
) through an injector tube (
4
) and a sheath fluid source (
5
) respectively. The sheath fluid confines, by hydrodynamic focusing, the sample of cells to a central core of the laminar flow that leaves the droplet generator (
2
) at an orifice (
6
) as a fluid stream (
7
). The fluid stream entraining the cells to be sorted, pass such cells, typically one by one, through a beam of excitation light from a light source (
8
) (which can be a laser or arc lamp), such that each fluorescently labeled cell emits a short flash of fluorescence. The intensity of the fluorescence may be proportional to amount of the fluorescent label on the individual cells. These flashes of fluorescence are collected by a fluorescence collection lens, which focuses the light on a sensitive fluorescence detector (
9
). The fluorescence detector (
9
) transforms the flashes of light into electrical pulses, which are measured and recorded by an analysis means (
10
), which may in part include a computer. Thus, multiple parameters including fluorescence at different wavelengths, as well as size and shape or structure, may be analyzed for each individual cell in the sample of cells.
Once the analysis of an individual cell is made, the flow cytometer (
1
) has the ability to selectively remove certain cells from the fluid stream (
7
). Droplets (
11
) are generated in the fluid stream (
7
) by the oscillation of the droplet generator (
2
), which is coupled to an oscillation generator (
12
) and an oscillation frequency controller (
13
). A droplet-separation point or break off point, which occurs shortly after the cells leave the droplet generator (
2
) through orifice (
6
) become selectively charged by a charging pulse. The charging pulse may be produced by a charging system control logic that is coupled to receive signals from the analysis means (
10
). The charging system control logic analyzes signals from the analysis means (
10
) and determines whether the cell is to become a selected cell and, if so, applies the charging pulse to the conductive fluid carrying the cells just before the droplet carrying the cell breaks off from the fluid stream (
7
). The droplet (
11
) carrying the selected cell separates as a charged droplet from the fluid stream (
7
) and falls, under the force of gravity, through a constant electric field produced by the deflecting system (
14
). If the cell is not a selected cell, then charging system control logic will not apply the charging pulse, and the droplet (
11
) containing this cell will separate from the fluid steam (
7
) as an uncharged droplet and will fall through the electric field largely unaffected by this field. Typically, one droplet (
11
) will contain only one cell so that the selective charging of a droplet will select only one cell.
The deflecting system (
14
) alters the trajectory in which the charged droplets (
11
) are traveling such that the charged droplets are physically separated from the uncharged droplets according to the value of the parameters analyzed or measured by the analysis means (
10
). After passing through the deflecting system (
14
), the droplets are collected in a cell collector (
15
) that may have several different collection receptacles or containers. Additional background information may be obtained by referring to Flow Cytometry and Sorting, Second Edition by Myron R. Melamed, Tore Lindmo, Mortimer Mendelsohn, published by Wiley-Liss New York, N.Y., 1990. Also see, for example, U.S. Pat. Nos. 5,150,313, and 3,560,754, each hereby incorporated by reference.
Although technology has led the way for a new generation of cell sorters that are simpler to operate and maintain, and significantly less expensive both to purchase and to operate, the inability to analyze, measure, or sort cells in an adjustably controllable environment significantly limits the types of applications to which flow cytometers or cell sorters may be used. And while various solutions have been suggested or proposed such as those disclosed by U.S. Pat. Nos. 5,776,781; 5,641,457; 5,083,558; and 5,200,616, each hereby incorporated by reference, significant problems remain unresolved.
A significant problem with conventional flow cytometry technology may be that aerosols, containing hazardous materials, are generated around the nozzle during and after the sorting of the cells. One aspect of this problem may be that these hazardous, dangerous, or undesirable aerosols or contaminants can be adsorbed on the surfaces of the flow cytometer resulting in premature wear. Another aspect of this problem may be that these undesirable aerosols may be inhaled or absorbed through the skin of person(s) in proximity to the flow cytometer. Moreover, these aerosols may, with respect to samples or other chemicals, represent an undesired contaminant.
Another significant problem with conventional flow cytometry technology may be that the cells, biological materials associated with the cells, or other materials being analyzed with the flow cytometer may be susceptible to contamination, modification, molecular rearrangement, or the like, by exposure to the open air; constituents of the open air; or particles, chemicals, other biological materials, such as bacteria, viruses, pollen, microscopic flora or fauna, or other pathogens that can be carried by the open air.
A controlled sterile flow cytometer environment, for example, that prevents cells from being contaminated and also maintains their viability, may allow the sorted cells to be used for further processes such as genetic modification of the sorted cells themselves, isolation or preservation of the cells in a frozen state, or culturing of the isolated cells in conventional tissue culture media, administering the isolated cells to a patient, or various permutations and combinations of these or other processes. An example of such a combination might involve removing a sample of blood from a patient, labeling certain cells (e.g. pluripotent hematopoietic stem cells) with fluorescently labeled antibodies and isolating these cells using the sterile cell sorter by observing and measuring the emitted excitation wavelengths from these fluorescently labeled antibodies, culturing these isolated certain cells and genetically modifying them (e.g. using recombinant DNA technology) to provide genetically modified cells, and culturing these cells in a tissue culture media and injecting them back into the patient to provide a cure or remedy for a disease.
Another significant problem with conventional flow cytometer technology may be that protective enclosures presently manufactured such as those disclosed by U.S. Pat. Nos. 4,063,495; 4,045,192; 3,462,920; and 3,511,162, each hereby incorporated by reference, are arranged to substantially enclose the protected area and do not permit a great deal of access for manual operations, or other features that are useful with respect to flow cytome

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