Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-11-24
2001-03-06
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C356S036000, C435S007220, C435S007230, C435S007240, C436S063000, C436S064000, C436S523000, C436S813000
Reexamination Certificate
active
06197523
ABSTRACT:
TECHNICAL FIELD
This invention relates to a method and assembly for the detection, identification, enumeration and confirmation of circulating cancer and/or hematologic progenitor cells in an anticoagulated whole blood sample which is contained in a transparent sampling tube assembly. The detection, identification, enumeration and confirmation steps can all be performed in situ in the sampling tube assembly. More particularly, the method of this invention involves the centrifugal density-based separation of the contents of the blood sample in a manner which will ensure that any circulating cancer and/or hematologic progenitor cells in the blood sample are physically displaced by their density into a predetermined axial location in the blood sample and in the sampling tube assembly, and also into a restricted optical plane in the sampling tube assembly which is adjacent to the wall of the sampling tube, and finally into a very well-defined zone of that optical plane.
BACKGROUND ART
Cytology is the science and technology involved in the morphological characterization of mammalian cells. Cytology has clinical utility in both human and veterinary medicine. Cytology is most often used to diagnose the presence or absence of malignancy in exfoliated or harvested cells: a) that are shed into a body cavity such as the pleural space or peritoneum; b) that are shed into a body fluid that is excreted as, for example, sputum or urine; c) that are obtained by scraping or brushing a body surface, such as the uterine cervix, the uterine cavity, or bronchial mucosa; or d) that are obtained by direct needle-mediated aspiration from a tumor such as tumors of the thyroid, breast, lung, or the like. The exfoliated or harvested cells are then typically fixed, stained and visually studied, usually by bright field microscopy, and then, if needed, by immunologic stains and/or other molecular techniques.
This year approximately five hundred sixty thousand people will die from solid tumors (predominantly carcinomas) in the USA. Many of these deaths could be prevented by early diagnosis of these malignancies. Unfortunately, with the possible exception of the Prostate Specific Antigen (PSA) test for prostate cancer, there is no practical and routine methods that have been found to be effective for early detection of solid tumors through blood analysis.
Through early detection of cervical cancer, the Pap smear has decreased mortality from cervical cancer in the United States by over seventy percent. Development of an analogous test for other solid tumors could have a similar impact on overall cancer mortality.
The presence of circulating cancer cells that are spontaneously shed by cancerous tumors into the circulating blood stream which is supplying the tumors with oxygen and nutrients has been confirmed. The presence of such cells in the blood stream has been inferred for decades because of the spread of cancerous tumors by what has been described as the hematogenous route and on very rare occasions have been visualized in blood specimens. Recently sophisticated procedures which employ reverse transcriptase in conjunction with Polymerase Chain Reaction (PCR) have been able to detect the presence of tumor cells by their molecular signature in a significant number of patients with cancer, both when the cancer is localized and after it has spread.
An additional means of detecting circulating cancer cell employs a technology known as Fluorescent Activated Cell Sorting (FACS), such as that manufactured by Becton Dickinson and Company of Franklin Lakes, N.J. The FACS detection of circulating cancer cells involves detection of cancer cells by detecting fluorescent labeled antibodies which are directed against and bound to one or more epitopes that are present on or in cancer cells, and are not present on or in normal blood cells, and/or by detecting combinations of epitopes that are present on or in circulating normal blood cells and that may or may not be present on or in cancerous cells, or combinations of the aforesaid methods.
The FACS technology is thus based on cell highlighting, i.e., it is photometric and utilizes antibody-epitope specificity, and it cannot be used to morphologically analyze cells in situ in the FACS instrument. Both the reverse transcriptase/PCR, (the molecular method), and the FACS, (the immuno-phenotypic method), require that the origin of the tumor being sought be known in order to select for the specific molecular species or immuno-phenotypic signals. The aforesaid techniques have contributed to confirmation of the theory that cancer cells do circulate in the blood stream, but these techniques are not practical especially in point of care applications, by virtue of their cost and/or nature, for detecting the presence or absence of circulating tumorous cancer cells in the blood stream. Thus, there is no general or generic blood analyzing procedure for the detection and confirmation of the malignant nature of circulating cancer cells, regardless of their source, in a patient. In addition, neither the aforesaid molecular nor the immuno-phenotypic methods utilize in situ, i.e., in a closed sampling system, cytopathologically-based analyses to determine the morphometric characteristics of circulating cells which permit cancer cells to be identified and confirmed.
Since approximately eighty two percent of all cancers are epithelial in origin (seventy two percent of which are fatal), epithelial cancer cells should be detectable in circulating blood. While the presence of epithelial cells in the circulating blood stream does not, by itself, prove malignancy, it does alert the cytopathologist to the greater likelihood of malignancy since epithelial cells are not normally seen in the circulating blood stream. In certain cases, however; such as after surgery; or as a result of physical trauma; or as a result of dental flossing, or in cases of prostatitis, for example, it is possible that non-malignant epithelial cells may be found in the circulating blood stream. Visual morphological analysis of cells is currently the most reliable way to distinguish cancerous epithelial cells from benign epithelial cells which are found in the circulating blood sample. One problem which exists in connection with attempts to detect circulating cancer cells in blood via morphological analysis relates to the fact that circulating cancer cells in blood are often virtually indistinguishable from circulating hematologic progenitor cells, or blasts, by cytological analysis alone.
The paucity of cancer cells that may be present in a sample of circulating blood would require the cytopathologist to carefully examine approximately ten million nucleated blood cells in order to find one cancer cell, and that one cancer cell would be randomly located in the ten million nucleated blood cells, which in turn will themselves be homogeneously dispersed in a sea of five billion non-nucleated cellular blood constituents, i.e., the erythrocytes, plus two hundred fifty million platelets, all of which will be found in one milliliter of blood. Such a task would be very time consuming, and is thus impractical for use in analyzing a patient's blood for the presence or absence of cancer cells.
Another type of rare circulating nucleated cells which may be found in a blood sample are hematologic progenitor cells (HPC's), which include blasts, stem cells, and other progenitors of normally circulating cells are not usually present in a sample of circulating blood at levels which can be detected by the use of presently available hematotopic instruments, such as impedance counters and examination of stained peripheral blood. In patients who are receiving chemotherapy and in patients who are receiving human granulocyte colony-stimulating factor (HGCSF), and other similar cytokines, HPC's are more likely to be present, but generally at very low numbers, i.e., at about one to one thousand per ml, or less, of the sample. Thus, low concentrations of HPC's in a blood sample renders the HPC's non-detectable by routine metho
Fiedler Paul
Levine Robert A.
Rimm David L.
Wardlaw Stephen C.
Chin Christopher L.
Jones William W.
Levine Robert A.
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