Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-27
2001-04-03
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200, C435S007240, C435S007250, C435S002000, C435S325000, C435S366000, C435S372000
Reexamination Certificate
active
06210889
ABSTRACT:
BACKGROUND OF THE INVENTION
This application relates to a method and kit for isolation of fetal cells from maternal blood and to the use of same in detection of chromososomal abnormalities.
Numerical chromosomal abnormalities, particularly those in chromosomes 21, 18 and 13 and the X and Y chromosomes account for a significant portion of the genetic defects in liveborn human beings. For this reason, prenatal testing for such abnormalities is carried out in some instances, particularly where the fetus is within a high-risk group. Unfortunately, the most reliable tests involve invasive procedures, such as amniocentesis, which themselves pose some risk to the pregnancy and to the fetus. Furthermore, the cost of the procedure is substantial, which adds an additional factor to the more appropriate balancing of risk to the fetus against the likelihood of that an abnormality will be detected. Thus, it would be desirable to be able to perform reliable testing for chromosomal abnormalities using a non-invasive technique.
The existence of fetal cells in the maternal circulation has been the topic of considerable research and testing over many years. It is now understood that there are three principal types of fetal cells: lymphocytes, trophoblasts and nucleated fetal erythrocytes. These cell types are found in variable, although always very small numbers and at various times within maternal blood. Holgreve et al., “Fetal Cells in Maternal Circulation”,
J. Reproductive Med.
37: 410-418 (1992). Various proposals have been advanced for the isolation or enrichment of one or another of these cells types from a maternal blood sample, and it has been proposed to use these isolated or enriched cells for testing for chromosomal abnormalities.
Fetal cells have been enriched from maternal blood using antibody capture techniques in which an immobilized antibody that binds to fetal cells captures the fetal cells to facilitate their enrichment. Mueller et al., “Isolation of fetal trophoblasts cells from peripheral blood of pregnant women”,
The Lancet
336: 197-200 (1990); Gänshirt-Ahlert et al., “Magnetic cell sorting and the transferring receptor as potential means of prenatal diagnosis from maternal blood”
Am. J. Obstet. Gynecol.
166: 1350-1355 (1992). Fetal cells have also been labeled with antibodies and other specific binding moieties to facilitate cell sorting procedures such as flow cytometry. Herzenberg et al., “Fetal cells in the blood of pregnant women: Detection and enrichment by fluorescence-activated cell sorting”,
Proc. Natl Acad. Sci.
(
USA
) 76: 1453-1455 (1979); Bianchi et al., “Isolation of fetal DNA from nucleated erythrocytes in maternal blood”
Proc. Natl Acad. Sci.
(
USA
) 87: 3279-3283 (1990); Bruch et al., “Trophoblast-Like cells sorted from peripheral maternal blood using flow cytometry: a multiparametric study involving transmission electron microscopy and fetal DNA amplification”
Prenatal Diagnosis
11: 787-798 (1991). Price et al. “Prenatal diagnosis with fetal cells isolated from maternal blood by multiparameter flow cytometry”
Am. J. Obstet. Gynecol
165: 1731-1737 (1991). PCR techniques have also been proposed to increase the relative amount of fetal DNA to permit analysis. Bianchi et al., “Isolation of fetal DNA from nucleated erythrocytes in maternal blood”,
Proc. Natl Acad. Sci
(
USA
) 87: 3279-3283 (1990); Adkinson et al., “Improved detection of fetal cells from maternal blood with polymerase chain reaction”,
Am. J. Obstet. Gynecol.
170: 952-955 (1994); Takabayashi et al., “Development of non-invasive fetal DNA diagnosis from maternal blood”
Prenatal Diagnosis
15: 74-77 (1995).
Gradient centrifugation plays a role as a preliminary step in many of these known techniques for isolation or enrichment of fetal cells. U.S. Pat. No. 5,432,054 also describes a technique for separation of rare cells, specifically fetal nucleated red blood cells, using gradient centrifugation. In this technique, maternal blood is centrifuged in a tube having a wide top and a narrow, capillary bottom made of polyethylene, using a variable speed program which results in a stacking of red blood cells in the capillary based on density. The density fraction containing low density red blood cells (which should include any fetal cells) is recovered and then differentially hemolyzed to preferentially destroy maternal red blood cells. The hemolyzed material is then separated on a density gradient in a hypertonic medium prepared from variable amounts of a colloid (which imparts the density variation) dispersed in a meltable gel to separate red blood cells, now enriched in the fetal red blood cells from lymphocytes and ruptured maternal cells. The use of a hypertonic solution shrinks the red blood cells, making them more dense relative to lymphocytes and thus easier to separate.
The method of U.S. Pat. No. 5,432,054 in theory offers a simple approach to the preparation of enriched fetal cell preparations from maternal blood. Unfortunately, in practice trials on cell preparations made using this method have been unable to identify aneuploid chromosomal abnormalities at rates that are significantly better than random chance. Elias, S., “Multicenter NIH Clinical Trials on Fetal Cells in Maternal Blood: Description and Current Status”,
Am. J. Hum. Genet.
61 (suppl): 29 (1997)( results presented orally at the 47
th
Annual meeting of the American Society of Human Genetics, Baltimore, Md. in October, 1997). Thus, there remains a need for a method for preparing enriched fetal cells which is simple to perform, and which nonetheless provides a preparation which can be reliably used to determine genetic information about the fetus. It is an object of the present invention to provide such a method.
SUMMARY OF THE INVENTION
In accordance with the present invention, an enriched fetal cell sample is prepared from maternal blood by first separating a maternal blood sample on a three layer density gradient, and collection of the layer containing the lymphocytes and a layer immediately beneath the lymphocytes which presumably contains the nucleated fetal red blood cells and low density maternal red blood cells. The top layer in the gradient would contain the plasma and lighter components and the bottom layer would contain high density maternal red blood cells. The collected cells are then placed in a hypotonic solution and centrifuged to separate a fetal cell pellet, which contains enriched levels of fetal cells of varying types. The recovered pellet is fixed twice with Carnoy fixative and the fixed cells are then spread on a slide for FISH analysis.
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Herzenberg, L.A., et al.; “Fetal Cells in the Blood of Pregnant Women: Detection and Enrichment by Fluorescence-Activated Cell Sorting”; Proc. Natl. Acad. Sci., USA; Mar. 1979; pp. 1453-1455; vol. 76; No. 3.
Bianchi, D.W.; “Progress in the Genetic Analysis of Fetal Cells Circulating in Maternal Blood”; Current Opinion in Obstetrics and Gynecology; 1997; pp. 121-125; Rapid Science Publishers.
Hawes, C.S., et al.; “A Morphologic Study of Trophoblast Isolated From Peripheral Blood of Pregnant Women”; Am. J. Obstet. Gynecol.; May 1994; pp. 1297-1300; vol. 170; No. 5; Part 1; Mosby-Year Book, Inc.
Yeoh, S.C., et al.; “Detection of Fetal Cells in Maternal Blood”; Prenatal Diagnosis; 1991; pp. 117-123; vol. 11; John Wiley & Sons, Ltd.
Holzgreve, W., et al.; “Fetal Cells in the Maternal Circulation”; Journal of Reproductive Medicine; May 1992; pp. 410-418; vol. 37; No. 5; The Journal of Reproductive Medicine, Inc.
Adinolfi, M.; “Non- or Minimally Invasive Prenatal Diagnostic Tests on Maternal Blood Samples or Transcervical Cells”; Prenatal Diagnosis; 1995; pp. 889-896; vol. 15; John Wiley & Sons, Ltd.
Wessman, M., et al.; “Fetal Granulocytes in Maternal Venous Blood Detected by In Situ Hybridization”; Pre
Drouin Régen
Forest Jean-Claude
Masse Jacques
Myers Carla J.
Oppedahl & Larson LLP
The Universite Laval
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