Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0641—Erythrocytes
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/05—Inorganic components
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/14—Erythropoietin [EPO]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/70—Enzymes

Definitions

• the invention relates to a method of enriching fetal cells from maternal blood.
• the only way of diagnosing fetal disorders is to obtain cells from the amniotic fluid (amniocentesis) or the surface of the fetal sac (chorionicvillus) of the mother. These procedures are expensive and carry a risk of spontaneous abortion of between 1 ⁇ 2%-1%. Because of the risk of spontaneous abortion, these collection procedures are recommended only for women who are at a high-risk of carrying a child with a genetic defect For example, only women over age 35 are advised to have the test because the risk of Down's Syndrome is higher in that group. Some of these women refuse the test because of the risk of spontaneous abortion. Even though many of these high-risk women are tested, only a fraction of fetuses with Down's Syndrome are dete ⁇ ed.
• the high-risk women represent such a small portion of the women having children that the low- risk population still delivers most of the afflicted children. Eighty percent of those children born with Down's Syndrome are from the "low-risk," under 35-year-old group. This situation is also true of many other genetic defects or disorders.
• the present invention provides a method for enriching fetal cells that overcomes these disadvantages, and further provides other related advantages.
• the present invention is directed toward methods for enriching fetal nucleated, erythroid cells from maternal blood.
• a method comprises the steps of: incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells; removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.
• the immobilized ligand is an immobilized antibody.
• a method for enriching fetal nucleated, erythroid cells from maternal blood comprising the steps of: incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells; adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10 8 M - 1 ; removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.
• Suitable first member-second member binding pairs include biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, methotrexate-dihydrofolate reductase, 5-fluorouracil-thimydylate synthetase, and riboflavin-riboflavin binding protein.
• the first member which is chemically linked to a ligand is a biotinylated antibody and the immobilized second member is immobilized avidin.
• the method comprises the steps of: incubating a sample of maternal blood with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the first ligand to the cells, incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand; adsorbing the cells to an immobilized second member, the second member capable of binding to the first member with an affinity constant of greater than about 10 8 M - 1 , removing unbound blood products; and incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.
• the first ligand is an antibody which specifically binds to fetal nucleated, erythroid cells.
• the first member which is chemically linked to the second ligand is a biotinylated antibody.
• the immobilized second member is immobilized avidin.
• another aspect of the present invention comprises the steps of: (a) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammomum ions within the bound cells; and (b) incubating the bound cells containing accumulated ammomum ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.
• the present invention may also be performed using a combination of both erythropoietin enrichment and the method described above wherein the cells are incubated with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to .allow accumulation of ammonium ions within the bound cells, followed by the incubation of bound cells containing accumulated ammonium ions in the presence of ammoma and carbon dioxide such that selective hemolysis of maternal blood ceHs occu ⁇ .
• These two enrichment methods may be performed sequentially, and in either order.
• a method for enriching fetal nucleated, erythroid cells from maternal blood comprising the steps of: incubating a sample of maternal blood in the presence of erythropoietin such that the fetal cells are enriched; incubating the enriched cells with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to .allow specific binding of the ligand to the ceils; and removing unbound blood products.
• the enriched cells may be immobilized by incubating them with a first member chemically linked to a ligand capable of .specifically binding to the cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells, and adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10 8 M -1 .
• the enriched cells may be immobilized by incubating the enriched cells with a first ligand capable of specifically binding to the cells under conditions and for a time sufficient to allow specific binding of the first ligand to the cells; incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand; and adsorbing the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10 8 M -1 . Suitable combinations of first and second ligand are discussed in detail below.
• the methods may further comprise (subsequent to removing the unbound blood products) the steps of: incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide, such that selective hemolysis of maternal blood cells occurs.
• the cells are incubated with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells, followed by incubation of the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide, such that selective hemolysis of maternal blood cells occurs.
• ligands may be utilized, including antibodies, erythropoietin, and transferrm.
• the ligand may be immobilized on any of a variety of solid supports, such as hollow fibers, beads, magnetic beads, plates, dishes, flasks, meshes, screens, solid fibers, membranes, and dipsticks.
• a method for typing chromosomes of fetal nucleated erythroid cells comprising the steps of: incubating the fetal nucleated erythroid cells in a media containing erythropoietin under conditions and for a time sufficient to induce metaphase in the cells; fixing the DNA of the cells; staining the fixed DNA such that chromosomes may be observed; and examining the stained DNA thereby allowing the typing of the chromosomes.
• the present invention provides methods for the enrichment of fetal nucleated, erythroid cells from maternal blood.
• Maternal blood contains, among many other types of cells, both adult and fetal nucleated, erythroid cells.
• fetal nucleated, erythroid cells may be enriched from as few as 1 in 10 6 in maternal blood, to an enriched concentration of about 1 in 10 3 , and preferably, to a out 1 in 10 2 .
• nucleated erythroid cells contain a nucleus and generally include erythroblasts as well as other erythroid precursor cells.
• Maternal blood may be obtained from a pregnant female using conventional techniques well known in the art.
• peripheral blood is drawn from an easily obtainable source such as the antecubital vein (the arm vein) with conventional venipuncture techniques.
• the maternal blood Once the maternal blood has been drawn, it may be frozen using conventional techniques, or stored at 4°C for a maximum of 4 to 7 days.
• Various anticoagulants may be added to the blood as necessary, including, among others, ACD, CPDA, EDTA, and Heparin.
• the methods of the present invention comprise the steps of: (1) incubating the maternal blood with either an immobilized ligand, or a ligand which will subsequently be immobilized, such that the ligand binds to and hence immobilizes the fetal nucleated, erythroid cells; (2) removing unbound blood products; and (3) preferentially enriching the bound cells for fetal nucleated, erythroid cells.
• these basic steps may be performed in an alternative order, for example, comprising the steps of:
• the present invention may be performed utilizing a device as described in a related application entitled "Immunoselection Device and Method", attorney's docket number 200072.401, which is incorporated herein by reference.
• the maternal blood is incubated with an immobilized ligand capable of specifically binding to fet.al nucleated, erythroid cells under conditions, and for a time sufficient to allow binding of the cells to the ligand.
• an immobilized ligand capable of specifically binding to fet.al nucleated, erythroid cells under conditions, and for a time sufficient to allow binding of the cells to the ligand.
• incubation of about 15 to 30 minutes at 4°C to 37°C is preferred.
• the flow rate should be sufficiently slow to allow the cells to bind.
• the cells should be allowed at least 15 minutes in which to traverse the bed of the column.
• the ligand should be chosen such that it is capable of specifically binding fetal nucleated, erythroid cells.
• the ligand is defined to be "specifically binding” if it is capable of binding to fetal nucleated, erythroid cells, but not to more than about 10% of the maternal blood cells.
• the relative percentage of bound fetal nucleated, erythroid cells to other cells may be readily determined by analysis with a specific marker such as Alpha Fetal Protein (AFP).
• AFP Alpha Fetal Protein
• the relative percentage of fetal cells to maternal cells may be determined with either glucose oxidase or fluorescein conjugated anti- AFP antibody (see A. Kulozik and L H.
• Pawlowitzki "Fetal Cells in the Maternal Circulation: Detection by Dire ⁇ AFP-Immunofluorescence," Human Genet. 62:221, 1982). This determination is preferably performed after two or more purification steps.
• Ligands which specifically bind to fetal nucleated, erythroid cells are known in the art, including erythropoietin (Amgen, Thousand Oaks, Calif.), transferrin (Sigma Chemical Co., St Louis, Mo.) and sele ⁇ ed antibodies. Monoclonal antibodies which specifically recognize nucleated erythroid cells are particularly preferred. Monoclonal antibodies to nucleated erythroid cells such as anti-transferrin receptor antibodies may be obtained from conventional suppliers (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.).
• monoclonal anti-erythroid antibodies such as EP-1
• EP-1 may be produced using techniques well known in the art (see Yokochi et aL, "Monoclonal Antibodies Detecting Antigenic Dete ⁇ ninants With Restricted Expression On Erythroid Cells: From the Erythroid Committed Progenitor Level to the Mature Erythroblast," Blood 63:1376, 1984; see also Heddy Zola (ed.), Monoclonal Antibodies: A Manual of Tech nique s, CRC Press, Boca Raton, Fla., 1987). Briefly, cells may be generated for immunization from fetal liver clonal erythroid cultures, and enriched for progenitor cells.
• the population of cells used for antigens and for primary screening may contain immature erythroblasts, erythroblasts of an intermediate degree of maturity, and presumably, progenitor cells of BFU-E and CFU-E types. These cells may be used for intravenous immunization, followed by removal of the spleen .and fusion of the spleen cells with a myeloma line such as NSI, using standard techniques. The resulting fused cells, or hybridomas, may then be screened against the above-described cells using conventional techniques (see Yokochi et al., supra).
• the entire specifically binding antibody need not be used as the ligand. More specifically, only the binding region of the antibody is necessary to specifically bind fetal nucleated, erythroid cells. Thus, antibody fragments such as Fab or F(ab') 2 fragments may be used within the present invention.
• binding regions of the specifically binding .antibody may be incorporated into a new protein, which may be used as the ligand (see Reichmann et al., "Reshaping Human Antibodies For Therapy,” Nature 332.323-327, 1988; Verhoeyen et al., “Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science 239:1534-1536, 1989; .and Roberts et al., "Generation of an Antibody with Enhanced Affinity and Specificity for its Antigen by Protein Engineering,” Nature 3287:31-734, 1987).
• the ligand is immobilized in order to allow separation of bound cells from other blood products.
• suitable supports include, among others, hollow fibers (Amicon Corporation, Danvers, Mass.), beads (Polys ⁇ ences, Warrington, Penn.), magnetic beads (Robbin Scientific, Mountain View, Calif.), plates, dishes and flasks (Corning Glass Works, Corning, N.Y.), meshes (Becton Dickinson, Mountain View, Calif.), screens and solid fibers (see Edelman et al., U.S. Patent No. 3,843,324; see also Kuroda et al., U.S. Patent No.
• Biogel P-60TM is a porous polyacrylamide hydrogel bead.
• the beads are generally spherical, on average about 250 microns in size, and have an average pore size which excludes molecules larger than approximately 60,000 daltons.
• a ligand such as an .antibody
• a ligand may be directly coupled to the support by various methods well known in the art (see J. K. lnman, Methods ln Enzymology, Vol.34, Affinity Techniques. Enzyme Purification: Part B. W. B. Jakoby and M. Wilchek (eds.), Academic Press, New York, p. 30, 1974; see also M. Wilchek, and W. Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications," Analyt. Biochem. 171:1-3Z 1988).
• the maternal blood may be incubated with the immobilized ligand under conditions, and for a time sufficient to allow binding of the ligand to the cells.
• suitable conditions for binding to occur include incubation in a physiological buffer at about 4°C to about 37°C Particularly preferred temperatures range from about 4°C to room temperature. The time of incubation depends on the affinity and avidity of the ligand for the cell, and may be readily determined. Generally, incubation for about 15 minutes to one hour is preferred. Following incubation, unbound blood products may be removed, and fetal cells enriched using the methods described herein.
• a sample of maternal blood is incubated under suitable conditions with a ligand which is chemically linked to a first member, and then adsorbed to a second member which is immobilized on a solid support
• the first member should be capable of binding to the second member with an affinity of greater than about 10 8 M -1 .
• first member-second member binding pairs are well known in the art These include, among others, biotin-avidin, biotin-streptavidin, biocytinavidin, biocytin-streptavidin, methotrexate-dihydrofolate reductase, 5-fluorouracilthimydylate synthetase, riboflavin-riboflavin binding protein, antibody-protein A, and antibody-protein G.
• the first member is biotin and the second member is avidin.
• Either member of the above described binding pairs may function as the second member, with the complementary member functioning as the first member.
• combinations of the first member - second member binding pair may be employed.
• biotin may be linked to the ligand, as well as adsorbed to the support
• the cell, ligand, biotin-complex and biotin, support-complex may then be bound together through an incubation step with avidfo Avidin is multivarriing permitting the formation of a cell, ligand, biotin, avidin, biotin, support-complex which immobilizes the cell.
• a sample of maternal blood is incubated with a biotinylated antibody under conditions .and for a time sufficient to allow binding to occur.
• the sample is then incubated with, or passed over a support which contains immobilized avidin.
• Cells which are coupled to the biotinylated antibody are adsorbed to the immobilized avidin, thus allowing separation of cells from unbound blood products.
• unbound blood products may be removed, and fetal cells enriched using methods described below.
• a two-step method is used to immobilize the fetal nucleated, erythroid cells. Briefly, a first ligand is incubated with a sample of the maternal blood under suitable conditions as described above. Subsequently, a second ligand which has been chemically linked to a first member is added. The second ligand is capable of binding to the first ligand. The cell, first ligand, second ligand, first member-complex may then be adsorbed onto an immobilized second member, thus allowing the separation of cells from unbound blood products. Representative examples of the first member- second member binding pair have been discussed above.
• the first ligand include erythropoietin, transferrin, and selected antibodies.
• the second ligand is chosen such that it specifically recognizes and binds to the first ligand.
• the second ligand is an antibody, for example an anti-erythropoietin (Terry Fox Laboratory, Vancouver, B.C., Canada), anti-transferrin (Chemicon Intl., Inc., Temecula, Calif.), or anti-immunoglobulin antibody.
• Anti- immunogiobulin antibodies may be prepared using techniques well known in the art, or may be obtained from conventional sources, including, among others, Sigma Chemical Co., St Louis, Mo., and Becton Dickinson Immunocytometry Systems, Mountain View, Calif.
• the first ligand is an antibody which specifically recognizes fetal nucleated, erythroid cells such as an anti- transferrin receptor antibody (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.).
• the antibody is incubated with a sample of maternal blood.
• a biotinylated anti-immunoglobulin antibody such as biotinylated goat anti-mouse IgG (the second ligand which is chemically linked to a first member) is then added and incubated with the sample.
• the sample is then incubated with, or passed over a bed of material which contains the immobilized second member, in this case, immobilized avidin.
• the cell, antibody, .anti-immunoglobulin antibody, and biotin-complex will be adsorbed to the immobilized avidin, thus allowing the subsequent removal of unbound blood products.
• the immobilized cells are rinsed with a physiological buffer, thereby removing the unbound blood products.
• a physiological buffer Various methods may be used to rinse the immobilized cells, depending upon the type of support chosen. These methods include, among others: washing or flushing the support; magnetically attracting the support out of solution, followed by resuspension in a physiological buffer; and centrifugation followed by resuspension.
• physiological buffers are also well known in the art including PBS, PBS plus albumin, such as Bovine Serum Albumin (BSA), normal saline, and cell culture media.
• BSA Bovine Serum Albumin
• bound cells may be preferentially enriched for held nucleated, erythroid cells.
• at least two alternative methods may be used either separately, or together. If the two methods are performed together, either method may be performed first
• the bound cells are cultured under selected culture conditions in the presence of erythropoietin (Amgen, Thousand Oaks, Calif.) (see Emerson et al., “Developmental Regulation of Erythropoiesis by Hematopoietic Growth Fa ⁇ ors: Analysis on Populations of BFU-E From Bone Marrow, Peripheral Blood and Fetal Liver," Blood 74(1):49-55, 1989; see also Linch et al., “Studies of Circulating Hemopoietic Progenitor Cells in Human Fetal Blood,” Blood 59(5):976-979, 1982).
• Selected culture conditions generally include growth in standard cell culture media, without any other cytokines other than erythropoietin. This preferentially allows fetal, but not maternal, nucleated erythroid ceils to grow.
• Particularly preferred media includes Iscoves' Modified Dulbecco's Medium (Gibco, Grand Isl.and, N.Y.) containing a final concentration of 20% fetal bovine serum, and 2 U/ml purified urinary human EPO.
• fetal nucleated, erythroid cells are preferentially enriched based upon their uptake of ammonium ions and the selective hemolysis of maternal blood cells, or more specifically, of maternal erythroid cells (see generally Jacobs and Stewart The Role of Carbonic Anhydrase in Certain Ionic Exchanges Involving the Erythrocyte," J. Gen. Phvsiol. 25:539-552, 1942; and Maren and Wiley, "Kinetics of Carbonic Anhydrase in Whole Red Cells as Measured by Transfer of Carbon Dioxide and Ammonia," Molecular Pharmacology 6:430-440, 1970).
• the cells are incubated in the presence of ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammomum ions within the cells. This generally takes approximately 5 to 30 minutes.
• the cells are then incubated in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.
• Suitable sources of ammonia include, among others, ammonia and the ammonium salts.
• suitable sources of chloride ions include, among others, NaCl, KCl, MgCl 2 , and CaCl 2 .
• Suitable sources for carbon dioxide include, among others, carbon dioxide in solution, carbonate, and bicarbonate.
• carbonic anhydrase inhibitors are known in the art, including, among others, most monovalent anions such as cyanide and cyanate, monovalent sulfides, sulfonamides, and acetazolamide (see Lindskog et al., The Enzymes 5:587, 1971; Ward and Cull, Arch. Biochem. Biophys.150:436, 1972; and
• the carbonic anhydrase inhibitors should be selected so as to function under physiological conditions.
• Preferred carbonic anhydrase inhibitors include sulfanilamide and acetazolamide.
• Bound cells may be released by various methods either subsequent to, or prior to enrichment If bound cells are released prior to enrichment they may be subsequently enriched using the above-described methods.
• Various methods are known in the art for releasing cells.
• cells may be cultured with or without cytokines.
• Cytokines such as LL-2, can cause proliferation of cells, or changes in the surface chara ⁇ eristics of the cells, such that the cells or their progeny are released from a support
• cleavage of the ligand or of the cell-ligand bond may release the cell.
• the cells may be released by mechanical, gravitational, or electromagnetic forces.
• a particularly preferred method is mechanical agitation, for example, by agitation of the beads through pipetting, stirring, shaking, vibration, or sonication.
• fetal cells may be enriched by first incubating maternal blood with ammonia and chloride ions, and a carbonic anhydrase inhibitor. After the cells have been incubated under conditions and for a time sufficient to allow accumulation of ammonium ions, the cells are treated with ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.
• Fetal cells may also be enriched by incubation with erythropoietin as discussed above, or by a combined treatment using both erythropoietin and the method discussed above wherein the cells are treated with ammonia and carbon dioxide. Furthermore, as discussed above, the two methods may be performed in either order.
• the enriched cells remaining after the method(s) described above may then be immobilized using any of the above discussed methods, including: (1) an immobilized ligand which specifically binds to fetal nucleated, erythroid cells remaining after enrichment; (2) incubating cells remaining after enrichment with a first member linked to a ligand, the ligand being capable of specifically binding to fetal nucleated, erythroid cells, followed by adsorption of the cells to an immobilized second member, the second member being capable of binding to the first member with an affinity constant of greater than about 10 8 M -1 ; and (3) incubating cells remaining after enrichment with a first ligand, followed by incubation with a second ligand which is chemically linked to a first member which is capable of binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand, followed by adsorption of the cells to an immobilized second member, the second member being capable of binding to the first member with an
• unbound blood products may be removed using methods described above. If the cells were initially enriched using erythropoietin, subsequent to removing unbound blood products, the cells may be treated using the method wherein ammonia and carbon dioxide are used. Similarly, if these cells were initi-ally treated with the method wherein ammoma and carbon dioxide are used, subsequent to removing unbound blood products, the cells may be treated with erythropoietin.
• Fetal cells which have been enriched from maternal blood may be characterized by incubating the preferentially enriched cells with a marker capable of selectively binding to fetal cells.
• a marker selectively binds to fetal cells when its presence on the target cells is greater th.an 10-fold more than the quantity on maternal cells.
• markers are known in the art including, for example, antibodies to Alpha Fetal Protein ("AFP") (see Kulozik et al., "Fetal Cells in the Maternal Circulation: Detection by Dire ⁇ AFP- Immunofluorescence," Human Genet.62:221-224, 1982), or antibodies to antigen "i” (see Y.W.
• Antibodies to the fetal cell marker may be labeled using techniques well known in the an, and used to dete ⁇ the presence of fetal cells.
• ligand is removed from the cell surface through the method described in a related application entitled “Methods for Removing Ligands from a Particle Surface,” attorney's docket number 200072.403, which is incorporated herein by reference. Such removal may be particularly advantageous prior to chara ⁇ erization, as discussed above.
• enriched fetal cells have a variety of uses. For example, through in situ hybridization the presence of a sele ⁇ ed genetic material may be dete ⁇ ed in a fetal cell. Similarly, DNA or RNA amplification may also be used to detect a selected genetic sequence in fetal cells.
• the methods of the present invention are particularly useful for fetal cells which are enriched to such an extent that they are amenable to chromosomal typing by conventional cytogenetics techniques wherein spreads of the cells' chromosomes are examined under a microscope.
• In situ hybridization may be used as a method for detecting the presence of a sele ⁇ ed genetic material within cells (see Pinkel et al., Proc. Natl. Acad. Sci.. USA 85:9138-42, 1988; see also Hopman et al., "Detection of Numerical Chromosome Aberrations in Bladder Cancer by In Situ Hybridization, Am. J. of Path.” 135(6):1105-1117. 1989). Briefly, the genetic material associated with the preferentially enriched ceUs is first exposed using techniques well known in the art The genetic material is then incubated with a labeled probe capable of specifically hybridizing to the genetic material, under conditions and for a time sufficient to allow hybridization to occur (see, for example, K.
• genetic material is denatured .after the step of exposing.
• genetic material includes whole chromosomes, DNA, and RNA.
• sele ⁇ ed genetic sequence may be amplified using techniques well known in the art and then probed for presence of that sequence (see Kogan et al., "An Improved Method For Prenatal Diagnosis of Genetic Diseases by Analysis of Amplified DNA Sequences," The New Eng. J. of Med. 317(16):985-990, 1987; see also Witt and Erickson, "A Rapid Method for Detection of Y-Chromosomal DNA from Dried Blood Specimens by the Polymerase Chain Reaction," Human Genet. 82:271-274, 1989).
• Methods for amplification include Polymerase Chain Reaction ("PCR") (see Mullis et al., U.S. Patent No. 4,683,195; Mullis et al., U.S. Patent No.4,683,202; .and Mullis et al., U.S. Patent No.4,800,159, which are incorporated herein by reference), and RNA- based amplification techniques (see Iizardi et al., Bio/Technology 6:1197-1202, 1988; Kramer et al., Nature 339:401-402, 1989; and Lomeli et al., Clinical Chemistry 35(9):1826-1831, 1989; see also Kramer et aL, U.S. Patent No.4,786,600, which is incorporated herein by reference).
• PCR Polymerase Chain Reaction
• PCR is the most commonly used method for amplifying DNA sequences. Briefly, amplification entails adding the appropriate primer(s), enzymes and nucleotides into a reaction mixture, followed by several (20-80) cycles of denaturation and annealing in order to amplify the small amount of target DNA The DNA mixture is then separated by electrophoresis and hybridized with a labeled probe to dete ⁇ the presence of the target sequence of DNA.
• the preferentially enriched fetal cells may also be chromosomally typed (see Human Cytogenetics, D.E. Rooney and B.H. Czepulkouski (eds.), IRL Press, Oxford, U.K., 1986).
• a sample containing at least 1 fetal cell in 10 5 other cells is cultured for 2-6 days in Iscoves' Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, N.Y.) containing a final concentration of 20% fetal bovine serum (FBS) (HYCLONETM, Logan, Utah), and 2 U/ml of highly purified recombinant erythropoietin (Terry Fox Laboratory, Vancouver, B.C, Canada), in order to increase the proportion of cells in metaphase.
• IMDM Iscoves' Modified Dulbecco's Medium
• FBS fetal bovine serum
• HYCLONETM highly purified recombinant erythropoietin
• the cells are then incubated in colcemid, fixed, attached to microscope slides, trypsin-treated, and stained with Wright's stain. The slides may then be scanned microscopically for abnormal ciiromosomes.
• the beads are washed several times with distilled or deionized water, followed by several washings of PBS using a coarse glass filter conne ⁇ ed to a vacuum.
• the carboxylated gel may be stored in PBS at 4°C, and is stable for up to one year if sterilized or stored with a preservative.
• Biogel by filtering with a coarse glass filter conne ⁇ ed to a vacuum. The gel is then equilibrated in distilled or deionized water for 15-30 minutes. Equilibration in water causes an expansion of the gel to a volume of about 4 times it's previously measured amount The gel is resuspended in 10 ml of distilled or deionized water for each ml of gel (as originally measured in PBS).
• EDC-HCl l-ethyl-3-(3-dimethyla ⁇ ninopropyl) carbodiimide
• Avidin International Enzymes, Inc., Fallbrook, Calif.
• 500 ⁇ g of avidin is rapidly added for each ml of gel (as originally measured in PBS). The mixture is stirred for 15 hours.
• 2 M glycine is added to give a final concentration of 0.2 M glycine in the mixture, and stirred for an additional 1 hour.
• the gel is washed with several volumes of PBS using a coarse glass filter and vacuum, and stored in PBS with 0.1% NaN 3 at 4°C. The gel is stable for approximately one year.
• a 50 ⁇ l aliquot of mixed, anticoagulated whole blood is diluted into 3 ml of a 3% acetic acid solution. After vortexing, 7 ⁇ l samples of diluted blood are loaded into each of two chambers of a hemacytometer. After about 3 minutes to allow settling of the cells, the nuclei of cells, which are almost all lysed by the acetic acid, are counted in the four ruled fields per.
• BSA Bovine Serum Albumin
• each field representing 0.1 x 10 -3 ⁇ l sample volume.
• the average number of nuclei per field is multiplied by a dilution fa ⁇ or of 61 x 10 4 to calculate the number of nucleated cells per milliliter of whole blood. If the average is less than 10 nudei per field, the procedure is repeated except 50 ⁇ l blood is diluted into only 1 ml acetic acid, with a renting new dilution fa ⁇ or of 21 x 10 4 .
• Each tube of diluted blood is underlayered with 5 ml of Histopaque 1077 (Sigma Chemical Co.) and centrifuged at 700 g for 15 minutes at room temperature. Cells at the interface are colle ⁇ ed and washed once in PBS plus 1% BSA. The pellet is resuspended in 100 ⁇ l in PBS plus 1% BSA.
• the cells are then gently resuspended to 1 ml and 1 ⁇ g/ml of biotinylated goat anti-mouse IgG (Southern Biotech, Birmingham, Ala.) is added. The mixture is incubated for 15 minutes on ice and washed twice as described above with PBS plus 1% BSA.
• Carboxylated Biogel P-30TM (prepared as described above) is allowed to equilibrate to room temperature and placed in a K9/15 column (Ph-armada, Piscataway, N J.) to a total bed height of 1 cm. The column is washed with PBS, followed by washes with PBS plus 5% BSA. This column functions as a "precolumn.”
• the avidin column contains avidin-conjugated Biogel P-60TM, which is prepared as described above. The avidin-conjugated Biogel is allowed to equilibrate to room temperature, and placed in a K9/15 column to a total bed height of 4 cm. The column is then w.ashed with several volumes of PBS, followed by washes with PBS plus 5% BSA.
• Cells which have been prepared as described above are resuspended in PBS plus 5% BSA to a volume of 1 mL
• the cells are then gently transferred onto the top of the gel bed of the pre-column filter.
• the cells are allowed to filter through the pre-column and are washed with 1 ml PBS plus 5% BSA.
• a peristaltic pump (Cole-Parmer, Rockford, lll.) controls flow from the avidin column to a rate of about 1 ml/minute.
• 1-2 ml of PBS plus 5% BSA is added to the top of the avidin column, in order to wash out remaining cells.
• the column is washed out with 4-6 ml of PBS plus 5% BSA, followed by 4-6 ml of PBS.
• the avidin column is placed on top of a 15 ml centrifuge tube.
• the valve of the column is opened and 15 ml of RPMI 1 is added to the column with a wide bore, 9-inch transfer pipette.
• the RPMI is added to the column while the pipette is used for mechanically agitating and resuspending the cell bed, thus allowing cells to become detached from the gel matrix, and to filter into the centrifuge tube.
• the tube is then centrifuged at 400 g for 5 minutes and resuspended in cell culture media as described below.
• IMDM Iscoves' Modified Dulbecco's Medium
• FBS fetal bovine serum
• erythropoietin Terry Fox Laboratory, Vancouver, B.C., Caitada
• Cells are diluted to 5 x 10 6 nucleated cells/ml and 200 ⁇ l is plated into each well of a 96 well tissue culture plate with round bottoms (Corning Gl.ass Works, Corning, N.Y.).
• Cells which are separated from the Avidin column above are adjusted to a concentration of less than 2 X 10 7 nudeated cells/mL
• One volume of the cell mixture is chilled, and a solution at 29°C containing 18 volumes of 0.1844 M NH 4 Cl and 2 volumes of 10 ⁇ M acetazolamide is added.
• 2 volumes of 3mM NH 4 HCO 3 is rapidly added, and the whole mixture gently stirred for 3 minutes.
• Cells are washed several times with PBS by centrifugation in order to remove cellular debris.
• Enriched cells are exposed to a hypotonic solution (.075 M KCl) for 12 minutes at 37°C
• a hypotonic solution (.075 M KCl) for 12 minutes at 37°C
• the tubes are inverted once during the incubation to keep the cells suspended.
• Twenty drops of freshly prep-ared fixative (3:1 methanobacetic add) is added to the cells, vortexed, and then centrifuged for 8 minutes at approxiamtely 250 g.
• Fresh fixative is added to the cells, followed by incubation for one hour at room temperature.
• the cefls are centrifuged for 8 minutes at approximately 250 g.
• Fresh fixative is added and the process is repeated one more time.
• the cells are resuspended in a .small amount of fixative and placed at 4°C overnight
• the slides are heated in 70% fo ⁇ namide in 2x SCC (030 M NaCl, 0.030 M Na citrate) to 68°C-70°C for 2.5 minutes.
• the slides a . re then immediately placed in a rinse of 70% ethanol in water.
• the slides are placed successively in 70%, 95%, and 100% ethanol solutions for 5 minutes each.
• Each alcohol solution must be m.aintained at ⁇ -20°C.
• the slides are air dried.
• a probe is prepared from plasmid DNA according to the method of Page et al ("Single coy sequence hybridizes to polymorphic and homologous lod on human X and Y ciiromosomes" PNAS 79:5 352-5356, 1982), from American Type Culture Collection (ATCC) No 57261; except that biotin-dATP is incorporated into the probe. Twenty five microliters of the biotinylated probe (5 ⁇ g/ml) per slide is placed in a microfuge tube. The probe is heated to 70°C for 5 minutes then immediately placed on ice. Twenty microliters of the probe solution is placed onto each slide and covered with a 22 x 40 mm coverslip. The slides are placed into a box with a wet paper toweling liner and incubated at 37°C for 12-18 hours.
• Coverslips are removed from the slides and immersed into the 50% formamide solution for 30 minutes.
• the slides are then placed in 2 x SCC solution for 30 minutes with gentle rocking, then in 1 x SCC for 30 minutes with gentle rocking.
• Fluoresceinated avidin (Vector, Burlingame, Calif.) is diluted 1:1000 (1 ⁇ g/ml). The back of the slide and around cell area are wiped. Two hundred microliters of the avidin-fluorescein is added to each slide and incubated in the box for 30 minutes at room temperature. The slides are rinsed sequentially in 1) 4 x SCC for 10 minutes with rocking, 2) 4 x SCC, 0.1% Tween-20 for 10 minutes without rocking, 3) 4 x SSC for 10 minutes with rocking.
• the enriched cells are exposed to 1 ⁇ g of colcemid (Sigma, St Louis, Mo.) for one hour at 37oC.
• a hypotonic solution (0.075 M KCl) is added to the cells and incubated for 12 minutes at 37°C.
• the tubes are inverted once during the incubation to keep the cells suspended.
• Twenty drops of freshly prepared fixative (3:1 methanokacetic add) is added to the cells, vortexed, and then centrifuged for 8 minutes at approximately 250 g.
• Fresh fixative is added to the cells, followed by incubation for 1 hour at room temperature. The cells are centrifuged for 8 minutes at approximately 250 g and fresh fixative is added again.

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