Liquid purification or separation – Diverse distinct separators – Including a filter
Reexamination Certificate
1999-05-24
2001-04-03
Kim, John (Department: 1723)
Liquid purification or separation
Diverse distinct separators
Including a filter
C204S450000, C204S518000, C204S543000, C210S294000, C210S515000, C435S002000, C436S177000, C436S178000, C530S413000, C530S417000, C530S427000
Reexamination Certificate
active
06210574
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to separation of fetal erythrocytes from maternal blood samples. More particularly, the present invention provides a system and non-invasive method for enriching the population of nucleated fetal erythrocytes or nucleated fetal red blood cells (“NRBCs”) obtained from maternal blood samples by separating the NRBCs from the mother's erythrocytes, leukocytes and other blood components. More specifically, the present invention offers a system and method for enriching the population of NRBCs from a maternal blood sample which concentrates the NRBCs by electrophoresis and/or adsorption-filtration or affinity filtration.
Physicians have long sought to develop non-invasive methods for prenatal diagnosis because the available methods, amniocentesis and chorionic villus sampling (CVS) are potentially harmful to the mother and to the fetus. The rate of miscarriage for pregnant women undergoing amniocentesis is increased by 0.5-1%, and that figure is be slightly higher for CVS. Because of the inherent risks posed by amniocentesis and CVS, these procedures are offered primarily to older women, i.e., those over 35 years of age, who have a statistically greater probability of bearing children with congenital defects.
Some non-invasive methods have already been developed to diagnose specific congenital defects. For example, maternal serum alpha-fetoprotein, and levels of unconjugated estriol and human chorionic gonadotropin can be used to identify a proportion of fetuses with Downs syndrome. Similarly, ultrasonography is used to determine congenital defects involving neural tube defects and limb abnormalities.
Separation of nucleated fetal erythrocytes from maternal blood has been proposed as a viable method for facilitating prenatal diagnosis of genetic disorders. Fetal NRBCs have been separated from maternal blood by flow cytometry using a lysing reagent (European Published Patent Application No. 582736, published Feb. 16, 1994); by triple gradient discontinuous gradient gel electrophoresis (Bhat, et al, U.S. Pat. No. 5,275,933, issued Jan. 4, 1994); by separation from nucleated cells using leukocyte depletion and lysis of non-nucleated erythrocytes using ammonium chloride (Goldbard, PCT Publication WO 9417209, published Aug. 4, 1994); by use of anti-CD71 monoclonal antibody and magnetic beads and in-situ fluorescence hybridization (FISH) (Ahlert, et al, German Published Patent Application No. 4222573, published Aug. 12, 1993) or by other antibodies specific to a fetal erythrocyte antigen (Bianchi, PCT Publication WO 9107660, published May 30, 1991).
SUMMARY OF THE INVENTION
The present invention demonstrates that fetal nucleated red blood cells exhibit consistent migration patterns in an electric field according to surface charge density which are different and distinct from the migration patterns of adult enucleated red blood cells. By using a novel charge-flow separation (CFS) method, described hereinafter, the present invention is able to divide maternal blood into fractions according to the surface charge density characteristics of each cell type. As the blood cells in a maternal blood sample move through the CFS apparatus, they are focused into compartments by opposing forces, namely buffer counterflow and electric field, and then are directed into waiting collection tubes. An apparatus and method of counterflow focusing suitable for use with the system and method of the charge-flow separation of the present invention are disclosed in our U.S. Pat. Nos. 5,336,387 and 5,173,164, which are hereby incorporated by reference. A preferred embodiment of a CFS apparatus and method will be described in greater detail hereinafter.
The inventive CFS system and method has been successfully used to recover nucleated red blood cells from the peripheral circulation of pregnant women. The recovered NRBCs were identified histologically. The NRBCs exhibited consistent migration patterns whether they came from maternal blood or from umbilical cord blood collected at birth. No NRBCs were found in blood from nulliparous women.
Because the inventive system and method of charge-flow separation of NRBCs from maternal blood is based on the intrinsic physical properties of the NRBCs, there is little need for extensive preparation of the maternal blood sample. The cells may be processed at greater than or equal to 60,000 cells per second and specialized training is not required. When the inventive charge-flow separation system and method is used, the recovered cells are viable, thus raising the possibility of further enrichment by cell culture.
In conjunction with or in addition to the charge-flow separation system and method, the present invention also includes an affinity separation method for separating NRBCs from other cell populations in a maternal blood sample. The adsorption-filtration affinity method of the present invention entails layering a maternal blood sample onto a fibrous adsorption-filtration filter medium having a nominal pore size of about 8 microns and being capable of 40-80% leukocyte immobilization, with a 70-80% post-wash leukocyte retention rate, and which is extremely hydrophilic, being capable of wetting with solutions having surface tensions of up to 85-90 dynes, which has a hold up volume of 40-70 &mgr;l/cm
2
for a single layer of adsorption-filtration filter medium and which is characterized by low to medium protein binding. The preferred adsorption-filtration separation filter medium is that sold by Pall Corporation under the trademarks “LEUKOSORB” TYPES A and B or that described in U.S. Pat. Nos. 4,923,620, 4,925,572, or European Patent No. 313348, each of which is hereby incorporated by reference.
The charge-flow separation system and method and the adsorption-filtration separation system and method of the present invention may be used separately or may be used in conjunction with one another to achieve enrichment of the nucleated fetal red blood cell population in a maternal blood sample.
REFERENCES:
patent: 3829370 (1974-08-01), Bourat
patent: 3989613 (1976-11-01), Gritzner
patent: 4185964 (1980-01-01), Lancaster
patent: 4204929 (1980-05-01), Bier
patent: 4323439 (1982-04-01), O'Farrell
patent: 4362612 (1982-12-01), Bier
patent: 4588492 (1986-05-01), Bier
patent: 4673483 (1987-06-01), Mandle
patent: 4925572 (1990-05-01), Pall
patent: 4963236 (1990-10-01), Rodkey et al.
patent: 5173164 (1992-12-01), Egen et al.
patent: 5192553 (1993-03-01), Boyse et al.
patent: 5275933 (1994-01-01), Teng et al.
patent: 5336387 (1994-08-01), Egen et al.
patent: 5432054 (1995-07-01), Saunders et al.
patent: 5437987 (1995-08-01), Tens et al.
patent: 5439571 (1995-08-01), Sammons et al.
patent: 5457024 (1995-10-01), Goldbard
patent: 5676849 (1997-10-01), Sammons et al.
patent: 5906724 (1999-05-01), Sammons et al.
patent: WO90/06509 (1990-06-01), None
patent: WO94/17209 (1994-08-01), None
patent: WO94/25873 (1994-11-01), None
“Continuous Counteracting Chromatographic Electrophoresis” by Ivory, Cornelius F. and Gobie, William A.,Biotechnology Prog., vol. 6, pp. 21-32, (1990).
“Fluid Stabilization During Isoelectric Focusing in Cylindrical and Annular Columns” by Egen, N.B., Twitty, G.E., Thormann, W., and Bier, M.,Separation Science and Technology, vol. 22(5), pp. 1383-1403, (1987).
“Isolation of fetal trophoblast cells from peripheral blood of pregnant women” by Mueler, et al.Lancet, vol. 336, pp. 197-200, (1990).
“Isolating Fetal Cells From Maternal Blood, Advances in Prenatal Diagnosis Through Molecular Technology” by J.L. Simpson, M.D. and S. Elias, M.D.,JAMA, vol. 270, pp. 2357-2361, (1993).
“Practical and Theoretical Implications of Fetal/Maternal Lymphocyte Transfer” by Walknowska, et al.,Lancet, vol. 1, pp. 1119-112, (1969).
“Filterability of Erythrocytes and Whole Blood in Preterm and Full-Term Neonates and Adults” by Linderkamp, et al.Pediatric Research, vol. 20, No. 12, pp. 1269-1273 (1986).
“Trophoblast Cells in Peripheral Blood From Pregnant Women”, by Covone, et al.,Lancet, vol. 2, pp. 841-843, (1984).
Isol
Sammons David W.
Sharnez Rizwan
Twitty Garland E.
Utermohlen Joseph G.
BioSeparations, Inc.
Kim John
Rosenbaum David G.
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