Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2000-02-22
2002-09-03
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C356S036000, C435S007100, C435S007220, C435S007230, C435S007240, C436S063000, C436S064000, C436S523000, C436S810000
Reexamination Certificate
active
06444436
ABSTRACT:
TECHNICAL FIELD
This invention relates to an evacuated container and method for use in analyzing a centrifuged anticoagulated whole blood sample for the presence or absence of rare events. More particularly, this invention relates to the detection of hematological rare events such as circulating cancer cells, bacteria, hemato-parasites, or other rarely occurring visually or photometrically detectable particles in the blood sample. A preferred embodiment of the container of this invention is an evacuated rectilinear container which contains an insert which container is essentially the same length and width as a microscope slide.
BACKGROUND ART
It is known that rare events, such as cancer cells may be present in the blood stream. Co-pending U.S. patent application Ser. No. 08/976,886, filed Nov. 24, 1997 describes a method for examining a centrifuged sample of anticoagulated whole blood for the presence or absence of cancer cells or other rare events. The method described in the aforesaid patent application involves the use of a capillary tube containing a cylindrical insert or float which restricts the available space in the tube into which the white cells and platelets will settle during centrifugation. This physically elongates the buffy coat portion of the blood sample and forces any cancer cells in the blood sample toward the tube wall where they can be seen under suitable magnification. We have discovered that any cancer cells present in the blood sample will settle into the area occupied by the buffy coat, and particularly in a region above the granulocytes and below the plasma, during centrifugation. The exact location of the cancer cells in the buffy coat will be governed by the density of the cancer cells and other less well-characterized physical forces.
The capillary tubes utilized in connection with the procedure described in the aforesaid patent application are capable of holding about 111 micro liters of blood. The frequency of cancer cells in peripheral blood can be as low as about one cancer cell per milliliter, and will be relatively dependent on the stage of the cancer in the patient being tested. Stated another way, a patient in an early stage of cancer is likely to have a much lower number of circulating cancer cells in an individual's peripheral blood than a patient in advanced stages of the disease, and a sample of less than about 111 &mgr;l of blood stands about a 90% chance of missing a rare event in the blood sample when the rare event occurs at a frequency of about 1 per ml of sample. The ability to examine larger samples of peripheral blood could enable earlier detection of the disease or other rare events. A larger tube and insert could be used to enable the examination of larger blood samples, but tubes produce spherical aberrations due to the curvature of the glass tube wall. It is also noted that the use of tubular containers requires that the container be examined throughout a 360° arc for the presence or absence of the rare events. This requires rotating the tube in the microscopical examining instrument.
It would be highly desirable to be able to examine larger blood samples in the same manner as provided by the capillary tubes and inserts, without encountering visual aberrations and without the need to rotate the sample chamber, and in which a standard microscope stage could be used without significant modification. However, tubular specimen sample containers can also be used in the performance of the method aspects of this invention.
DISCLOSURE OF THE INVENTION
This invention relates to an assembly for analyzing larger samples of anticoagulated whole blood for the presence or absence of circulating cancer cells or other rare events. The assembly comprises a hollow sample container which has essentially the same shape, length and width of a microscope slide, but which is thicker than a microscope slide. The hollow interior of the container can hold from about five to about ten milliliters of blood. One end of the container is sealed and the other end is closed by an elastomeric stopper. Obviously, both ends of the container could be closed by elastomeric stoppers if so desired. A relatively flattened rectilinear insert is disposed in the container and occupies between about 80-95%, and preferably about 90% of the volume of the portion of the interior of the container in the area occupied by the insert. The specific gravity or density of the insert is such that the insert will settle into or float in the packed red cell layer and be surrounded by the buffy coat constituents during centrifugation of the blood sample in the container. The interior of the container is evacuated so that blood will flow into the container as the result of a cannula puncturing the elastomeric closure. The container can be used to draw blood directly from a patient, or can be used to obtain the blood from a larger container such as a VACUTAINER® brand container sold by Becton Dickinson and Company. When the container assembly of this invention is used to draw blood directly from a patient, the testing reagents needed to anticoagulate the blood sample and detect the rare events in the blood sample can be pre-incorporated into the sample container assembly.
Reagents needed to identify rare events can include stains such as acridine orange which can highlight cell morphology; antibodies which are specific to surface receptors on cancer cells or other rare events; and stains which, when irradiated by light of appropriate wavelengths, will emit signals at wavelengths which can be photometrically and/or visually differentially detected over the background noise of emissions from the blood sample constituents such as white cells, cytoplasm, hemoglobin, and the like, which result when the blood sample is irradiated by such light sources.
The insert can include a structure which will skew the location of the insert inside of the container so that the majority of the white cell and platelet constituents will be located on one side of the rectilinear insert thereby enabling that portion of the buffy coat to be examined for the presence or absence of cancer cells, or other rare events without the need of rotating the container about its axis. Suitable skewing structures can include rails, hemispherical bumps, fins, or the like, on the insert or on the interior of the container. The container assembly is preferably sized so as to be positionable in a conventional centrifuge, and also positionable on a conventional microscope stage.
It is therefore an object of this invention to provide an evacuated anticoagulated whole blood sampling container assembly, which can be centrifuged, which container assembly is rectilinear in configuration, and which container assembly includes a volume-occupying insert that restricts the volume available in the container assembly wherein rare events will settle during centrifugation of the blood sample in the container assembly.
It is a further object of this invention to provide a method for examining a centrifuged anticoagulated whole blood sample under suitable sample illumination conditions that will reveal the presence or absence of rare events, such as cancer cells in predetermined areas of the blood sample.
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Fiedler Paul
Levine Robert A.
Rimm David L.
Wardlaw Stephen C.
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