Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2001-09-20
2004-02-17
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C210S767000, C422S105000, C435S308100, C436S178000
Reexamination Certificate
active
06692968
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the field of test sample preparation and analysis. More particularly, the subject invention relates to a method and apparatus for automatically purifying a test sample and enhancing the sensitivity of the sample analysis.
BACKGROUND OF THE INVENTION
Flow cytometry is a well known technique for qualitatively and quantitatively analyzing a large number of individual cells for a specific cellular marker in a rapid manner. In a typical application, a fluorescent molecular probe that selectively binds to a predetermined cell marker, such as a fluorochrome-conjugated antibody that specifically binds an intracellular or cell surface antigen, is added to a cell sample to be analyzed so that the probe can bind or “stain” the cells within the sample that express the predetermined cell marker. The sample is then placed in flow cytometer and illuminated with a light source to enable the fluorescence associated with each cell in the sample to be quantified. The magnitude of fluorescence emitted from a particular cell correlates with the quantity of cell marker on or in that particular cell. By extrapolating this fluorescence data, the relative quantity of specific phenotypic markers expressed by cells in a sample can be rapidly and accurately determined. For an overview of flow cytometric analysis see, “Flow Cytometry and Sorting,” Myron R. Melamed, Tore Lindmo, and Mortimer L. Mendelsohn, eds., New York:Wiley-Liss, Inc., (3rd ed., 1995); Shapiro, H. M., “Practical Flow Cytometry,” New York:Wiley-Liss, Inc., (2nd ed., 1990).
Sample preparation for flow cytometric analysis is typically performed in a non-automated fashion, wherein a saturating concentration of a cell marker-specific probe is added to a cell sample by manual pipetting, and the mixture is then incubated for a period of time sufficient to allow the probe to bind the cell marker of interest. For analyses where red blood cells might cause interference (e.g., immuno-phenotyping leukocytes), the red blood cells can be removed from the sample using an agent that specifically lyses erythrocytes (for example, a hypotonic solution, ammonium chloride or carboxylic acid). Traditionally, to remove interfering unbound probe from the cell sample prior to flow cytometric analysis, the mixture is washed by adding excess buffer to the mixture, centrifuging the mixture to separate the cells from the buffer, removing the buffer containing the unbound probe, and resuspending the cells in fresh buffer. The washing procedure can be repeated multiple times to further remove any remaining unbound probe. This non-automated technique is advantageous in that it results in a relatively clean sample that contains few interferants (for example, unbound probe or cell debris) which might generate background noise or interference during the flow cytometric analysis. For many applications, however, this non-automated technique is relatively time-consuming, can result in significant cell loss due to one or more wash steps, and exposes the cells to the potentially deleterious effects (for example, activation of enzymatic processes, granule release, cell destruction, high gravity forces produced by centrifugation, etc.).
While the foregoing technique is acceptable for infrequent analyses involving a small number of samples, it is less suitable for protocols involving repeated analyses of a large number of samples. A more automated procedure is generally preferred when flow cytometric analysis is employed for clinical diagnostics, high-throughput screening, or the like. For example, in a typical clinical assay where leukocytes are immunophenotyped using flow cytometry, a sample of whole blood is placed into an apparatus that automatically processes the sample prior to analysis. One such apparatus is the COULTER® TQ-Prep™ Workstation system manufactured by Beckman Coulter, Inc. (Miami, Fla.). After adding a probe to the sample, this apparatus uses computer-controlled devices to automatically add an agent that lyses erythrocytes in the sample and a cell fixing agent (for example, paraformaldehyde). The prepared sample can then be analyzed using a flow cytometer without further processing. This automated technique is advantageous in that samples of whole blood can be prepared for analysis quickly and efficiently.
A drawback of this lysing technique can be encountered in applications requiring a high degree of sensitivity. In such applications, in the absence of a washing step, the automated technique does not remove interferants, such as unbound probe or debris from the lysed erythrocytes from the sample. The high background signal caused by the fluorescence from the unbound probe, non-specific probe binding, and/or autofluorescence from the cells and debris can obscure results generated from the analysis.
Where a fluorescently-labeled antibody is used to analyze a cell sample for a marker present in low quantities, the absence of a washing step can result in high background fluorescence caused by the unbound antibody present in the sample. Thus, if too many unbound fluorescent antibody molecules are present in the sample, the flow cytometer can not distinguish the signal emitted from the antibody-bound cells from the “noise” generated by the unbound antibody. That is, the “noise” in the sample overwhelms the “signal” emanating from the cells of interest. To avoid this, the signal to noise ratio in the sample can be improved by removing the interferants by manually washing. An example of manual washing comprises centrifuging the sample to pellet the cells, decanting the interferants contained in the supernatant, and resuspending the cells in fresh buffer. As described above for the non-automated technique, this manual washing is disadvantageous because it is time consuming, causes cell damage, and can result in significant cell loss.
A need therefore exists for an apparatus and method for quickly and efficiently removing interferants from a cell sample prior to analysis. In addition, the apparatus and method should minimize the risk of exposure to infectious blood because of operator handling of the blood cell sample. An apparatus that performs the foregoing method with only negligible cell loss, and does not expose cells to high gravitational forces or cell packing caused by centrifugation would be especially advantageous.
Similarly, there exists a need for an apparatus and method for quickly and efficiently removing interferants from a test sample prior to analysis. In addition, the apparatus and method should minimize the risk of exposure to test sample because of operator handling of the test sample. An apparatus that performs the foregoing method with only negligible loss of the composition of interest in the test sample, and does not expose test sample to high gravitational forces caused by centrifugation would be especially advantageous.
SUMMARY OF THE INVENTION
It has been discovered that filters, such as microporous hollow fiber membranes, can be utilized in sample preparation devices to quickly and efficiently remove interferants from a test sample comprising a mixture of a composition of interest and interferants. More specifically, it has been found that the use of a hollow fiber membrane having a plurality of pores with a mean diameter less than the diameter of the composition of interest can be utilized to remove interferants from a test sample containing the composition of interest and interferants to improve the signal-to-noise ratio in an assay of the composition of interest. Application of vacuum to the hollow fiber membrane permits interferants to be removed from a test sample within a lumen of the filter with little or no damage to the composition of interest. As the composition of interest does not pass through pores of the membrane, compared with conventional continuous filtration devices, clogging of the filter is less frequent, and the composition of interest is exposed to less deleterious forces. Filters within the invention can be installed in a processing apparatus such that a test
Aparicio Carlos L.
Bell Michael L.
Burshteyn Alexander
Gupta Ravinder
Insausti Maria Elena
Alter Mitchell E.
Coulter International Corp.
Howson and Howson
Ludlow Jan
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