JPH05507404A - Method for enrichment of fetal cells from maternal blood - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods and strained beans for enrichment of fetal cells from maternal blood The present invention relates to a method of enriching fetal cells from maternal blood.

light-skinned raw stomach member Approximately 5.3 million women become pregnant and 3.8 million give birth each year in the United States. all over the world An additional 10 million children will be born in other rich countries.

Women who are at significant risk of having a fetus with a genetic disease, such as Down syndrome Prenatal testing is used in the group.

Currently, the only way to diagnose fetal disease is to obtain maternal amniotic fluid cells (amniotic fluid puncture) or to obtain the superficial layer of the fetal sac (chorionic villi). These methods cost It is associated with a risk of sudden miscarriage of 1/2% to 1%. Sudden miscarriage Because of this, such aggregation methods are important for women who are at high risk of carrying a child with a genetic disorder. It is only permitted to do so. For example, women over the age of 35 should undergo this test. This is because the risk of Down syndrome in this population is low. This is because it is expensive. Some of these women choose not to do this due to the risk of sudden miscarriage. Refusal of testing. Even though many such high-risk women are being tested, However, only some fetuses with Down syndrome # group are detected. This high risk Women make up a small proportion of women having such children and are a low-risk group of women. The group gives birth to most of the children with disabilities. 8 children born with Down syndrome 0% were born in the "low risk" group of people under 35 years of age. This situation is caused by numerous other The same is true for genetic disorders or diseases.

Therefore, all pregnant women can be treated without risk factors and without sudden miscarriage. We address this testing dilemma by providing a safe method that may be useful for A test that can solve this problem is desired. Fetal cells are known to be circulating in the pregnant woman's bloodstream. It is known that (Kulozikand Paiylowftzki, 'Fe tal Ce1ls in the Maternal C4rulation :Detection by [1irect AFP-1mlunof! u oressence’) luman Genetics62: 221.2 24.1982), fetal cells are present in low concentrations; The methods required to isolate them are very difficult and time consuming.

For example, Hertzenberg Raji Fetal Ce1lSin the Blood of Pregnant Women: Detection and E nrichIlentbyFluorescence-ActivatedCe ll Sorting, 'Proc, Natl, cad, Sci, US A76: 1453-1455+1979 March) is fetal cells in maternal blood. A fluorescence activated cell sorter (FAC5) is used to detect. However However, the method used here is not very suitable for routine clinical testing processes; Because operating the FAC5 device requires expense and specialized skills. It is. Hertzenberg et al.'s method is also useful for routine clinical testing. defective because it requires I(LA type inspection.B ianchi RajiDjrect Hybridization to DNA Fro+m Small Numbers of Flow-5orted N ucreated Newborn Ce] Is” Q difference pka α8: 1.97 -202.1987) also uses FACS equipment to detect nucleated cells. However, the blood sample is taken from the newborn's clasp, not from the mother.

The present invention overcomes these drawbacks and provides other related advantages. Provided are methods for cell enrichment.

Light corridor 112 The present invention is directed to a method for enrichment of fetal nucleated erythroid cells from the mother. Ru. In one aspect of the invention, such a method includes; An immobilized ligand that can specifically bind to nucleated erythroid cells and Incubate under conditions and for a sufficient period of time to specifically bind the cells to the target. incubate; remove unbound blood material; and elytinate these bound cells. Preferential enrichment of these fetal cells when incubated in the presence of loboietin The process includes the steps of:

In another aspect of the invention, for enriching fetal nucleated erythroid cells from maternal blood, A method for: preparing a sample of maternal blood to specifically bind to fetal nucleated erythroid cells. The first factor is chemically bound to a ligand that can be combined with this ligand. incubating under conditions and for a sufficient period of time to cause specific binding to the cells; The cells are adsorbed to an immobilized second factor, where the second factor is approximately can bind to this first factor with an affinity constant of 10'M-' or more; and incubated the bound cells in the presence of erythroboietin. A method is provided comprising the step of preferentially leveraging fetal cells. Appropriate number Factor-second factor binding bears include biotin-avidin, biotin-streptoa Vidin, biocytin-avidin, biocytin-streptavidin, methotrex Sate-dihydrofolate reductase, 5-fluorouracil thymidylate Contains synthase and riboflavin-riboflavin binding protein 0 In one embodiment of this aspect of the invention, the The first factor is a biotinylated antibody, and this immobilized second factor is immobilized avidin.

In another aspect of the invention, the method includes: preparing a sample of maternal blood for fetal nucleated red blood cells. A first ligand that can specifically bind to cell lines and a first ligand that can specifically bind to cells of this type. Incubating under conditions and for a sufficient time to specifically bind Gant, a second ligand capable of specifically binding this sample to this first ligand; The first factor is chemically bound to the Incubate the cells under conditions and for a sufficient period of time to permit heterozygous binding; It is adsorbed onto the immobilized second factor, where the second factor is about 10"M-' can bind to this first factor with an affinity constant of Remove fluid material: and place bound cells in the presence of erythroboietin. and incubating the fetal cells to preferentially enrich the fetal cells.

In some embodiments, the first ligand specifically binds fetal nucleated erythroid cells. It is an antibody that In the preferred Bm, the second ligand is chemically bonded. This first factor is a biotinylated antibody.

In such embodiments, the immobilized second factor is immobilized avidin.

Change in the step of incubation of bound cells in the presence of erythropoietin. Another aspect of the present invention is to (a) treat the binding cells with ammonia and and chloride ions and carbonic anhydrase inhibitors, and ammonium chloride ions and carbonic anhydrase inhibitors. Under conditions and for a sufficient period of time, the murine ions accumulate in these bound cells. and (b) this bound cell containing accumulated ammonium ions. Maternal blood cells by incubation in the presence of ammonia and carbon dioxide the step of causing selective hemolysis of. Furthermore, the present invention provides erythroboi ethyne enrichment and the method described above, i.e. the cells were enriched with ammonia and chloride ions and carbonic anhydrase inhibitor and ammonium ion to this bond. Incubate under conditions and for sufficient time to accumulate in the cells, then The presence of ammonia and carbon dioxide binds cells containing accumulated ammonium ions. Both methods produce selective elution of maternal blood by incubating under It can also be carried out using a combination of. These two enrichment methods are sequentially They can be done in any order.

In another aspect of the invention, a method of enriching fetal erythroid cells from maternal blood Yes: A sample of maternal blood was incubated in the presence of erythroboietin. to enrich the fetal cells; convert the enriched cells into fetal nucleated erythroid cells. An immobilized ligand that can specifically bind and this ligand is specific to this cell. incubate under conditions and for a sufficient time to allow binding; and The method includes the step of removing unused blood material.

On the other hand, in other B cases of the present invention, this enriched cell makes them a first factor chemically bound to a ligand capable of specifically binding to; Under conditions and for a sufficient period of time that this ligand can specifically bind to these cells, incubate and adsorb the cells to the immobilized second factor (this The second factor binds to this first factor with an affinity constant of about 10'M-' or greater. can) be able to do. In a further embodiment of the invention, this enriched The cell contains a first ligand that can specifically bind to this cell and this enriched cell. conditions and sufficient conditions for this first ligand to specifically bind to this cell. fix the sample by incubating it for a certain amount of time; chemically linked to a second ligand that can specifically bind to this first ligand This second ligand specifically binds to the first factor and this first ligand. and the cells are incubated under suitable conditions and for a sufficient period of time; is adsorbed onto the immobilized second factor (here, the second factor is approximately 10'M-' can bind to this first factor with the above affinity constant).

Suitable combinations of the first and second ligands are detailed below.

Furthermore, in the above-mentioned aspect, the method further includes (removal of unbound blood substances). ): This binding cell is exposed to ammonia and chloride ions as well as carbonic anhydride. dolase inhibitors and the conditions under which ammonium ions accumulate in cells. Incubate under conditions and for sufficient time; and accumulate ammonium Incubate this cell containing on in the presence of ammonia and carbon dioxide. and causing selective hemolysis of maternal blood cells. Wear.

In another aspect of the invention, the maternal blood supply in the presence of erythropoietin As an alternative to incubating samples, the cells can be incubated with ammonia and chloride. ion as well as carbonic anhydrase inhibitor, and this binding intracellularly. Incubate under conditions and for sufficient time to accumulate ammonium ions. , then cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. maternal blood cells to selectively hemolyze maternal blood cells.

In the present invention, various antibodies including antibodies, erythroboietin and transferrin are used. Gantt is available. This ligand can be attached to any of a variety of solid phases, such as optical fibers. beads, magnetic beads, plates, dishes, flasks, meshes, screens, fixed They may be immobilized on body fibers, membranes and dipsticks.

In another aspect of the invention, a method for chromosomal classification of fetal nucleated erythroid cells. Where: Fetal nucleated erythroid cells are incubated with this in a medium containing erythroboietin. Incubate under conditions and for sufficient time to induce metaphase in cells. The DNA of this cell is fixed; the chromosomes can be observed by staining this fixed DNA. and the step of examining this stained DNA and classifying this chromosome. Provides a method comprising:

These and other aspects of the invention can be learned by reference to the detailed description below. It will become clear.

Mata Yushi's Aiming Star Escape The present invention provides methods for enrichment of maternally derived fetal nucleated erythroid cells. mother body Blood contains both adult and fetal nucleated erythroid cells, as well as numerous other cell types. Contains both sides. Through the effects of the present invention, fetal-f nuclear erythroid cells can be transferred to maternal blood. from 1 in 106 to about 1 in 103, and preferably 1 in 10 It can be enriched to a concentration of 1 in 2. In the context of the present invention, nucleated red blood cells The vacuole contains a nucleus and generally an erythroblast, as do other erythroid precursor cells. There is.

Maternal blood can be obtained from pregnant women using routine techniques well known in the art. Wear. Peripheral blood, preferably from an easily obtained source, e.g. from the antecubital vessels, is routinely used. extracted by venipuncture technique. Once the maternal blood has been collected, this is done using a routine technique. Freeze or store at 4°C for up to 4-7 days. various anticoagulants May be added to the blood if necessary, including ACD, CPDA, EDT, among others. A and heparin.

This maternal blood is then subjected to a sorting method according to the invention, in which further purification, e.g. Preferential enrichment without purification by fluorescence-activated cell sorter (FAC3) It is possible to obtain purified fetal cells. Generally, the method includes the following steps: (1) this Maternal blood with the immobilized ligand or the amount of the ligand to be immobilized subsequently. The fetal nucleated erythroid cells bind to the ligand, and the (2) remove unbound blood substances; and (3) ) preferentially enriching connective cells for fetal nucleated erythroid cells. . As mentioned above, these basic steps can be performed in an alternative order, e.g. 1) preferentially enrich maternal blood for fetal nucleated erythroid cells; (2) this enrichment The immobilized cells are treated with the immobilized ligand, or with the ligand to be subsequently immobilized. The fetal nucleated erythroid cells are then incubated with the ligand to bind to the ligand. and (3) removing unbound blood material. It can be carried out in the following manner. This invention is incorporated herein by reference. , titled “I+wnunoselection Device and Method ” (Representative's dosokenoto number 200072.401) This is done using the location.

In one aspect of the invention, the maternal blood is used to specifically bind fetal nucleated erythroid cells. An immobilized ligand that can be combined with this cell Incubate under conditions and for sufficient time to allow binding. Generally, about Incubation at 4°C to 37°C for 15 to 30 minutes is preferred. Moshiko If the incubation step is performed as the cells pass through the column, then The flow rate should be slow enough to allow cells to bind. preferred Alternatively, pass the cells through the column for at least 15 minutes.

As mentioned above, this ligand is capable of specifically binding to fetal nucleated erythroid cells. should be chosen accordingly. In the context of the present invention, this ligand is Can bind to infant nucleated red blood cells, and also binds to approximately 10% or more of maternal blood cells If there is no such thing, define it as "specific binding." Conjoined fetal nucleated red blood cells The relative percentage of lineage cell numbers to other cells is determined by specific markers, e.g. can be easily determined, for example, by alpha fetal protein (AFP) analysis. . For example, at any point in the method described below, The target percentage is the conjugate of either glucose oxidase or fluorescent material. can be measured by gated anti-AFP antibodies (A, Kulozik a nd　1. H. Pawlowitzki, “Fetal Cells int. he Materna+ C4culation: Detection by Direct AFP-Immunofluorescence,'H (see uo+an Genet, 62: 22L 1982).

This measurement is preferably carried out through two or more purification steps.

Gantts that specifically bind fetuses to nuclear erythroid cells are known in the art; This includes erythroboietin (Amgen, Thousand 0aks).

Calif, ), transferrin (Sig+wa Chemical Co , +st, Louis.

Mo,) and certain antibodies. A substance that specifically recognizes nucleated erythroid cells Clonal antibodies are particularly preferred. Monoclonal antibody against nucleated erythroid cells For example, anti-transferrin receptor antibodies are available from common commercial sources (Becton). Dickinson Immunocytometry 5ystelI’s.

Mountain View+ Calif). On the other hand, monochrome Nunal anti-erythrocyte antibodies, such as ED-1, can be produced using techniques well known in the art. (Yokochi et al., “Monoclonal Antibody s Detecting Antigenic Determinants Wi th Re5tricted Expression On Erythroi dCells: From the Erythroid Coe+m1tt ed Progenitor Level to theMature Ery See tbroblast"Blood 63: 1376, 1984. ; Furthermore, Heddy Zola ([) Monoclonal Antibo dies: A Manual of U, CRC Press, B (see Radon, Fla, 1987).

Briefly, cells were generated for immunization from fetal liver clonal red blood cell cultures. and enrich for progeny cells. This allows the antigen and the first screen to be The cell populations used for this purpose include immature red blood cells, moderately mature red blood cells, and Possibly including progenitor cells of type BFU-E and CFU-E. these cells used for intravenous immunization, then the lungs are removed and the lung cells are myelomatized. Fuse with a cell line, such as NSI, using standard techniques. obtained here The fused cells or hybridomas are then prepared using conventional techniques (Yokoch et al. , supra) to screen against the above cells.

It is not necessary to use the entire specifically binding antibody as a ligand.

More specifically, only the binding region of this antibody is specific for fetal nucleated erythroid cells. required to combine. Therefore, in the present invention antibody fragments, e.g. For example, Fab or F(ab')z fragments can be used. Furthermore, this specific A new protein that can be used as a ligand is the binding region of an antibody that binds to protein (Reichmann et al., 'Reshapi ng Human Antibodies Fortherapy”Natur e 332: 323-327. 1988; Verhoeyen et al.” ReshapingHuman Antibodies: Grafting An Antilysozyme Activity' 5science23 9: 1534-1536.1989; and Robert ts et al., “Ge r+eration of an eight body with Enhance d Affinity and 5 specificity for its An tigen by Protein Engineering” 328:731-734.1987).

In the present invention, ligands are immobilized to separate bound cells from other blood substances. to become Many suitable supports are known in the art, including, among others, optical fibers. Bar (Amicon Corporation, Danvers.

Mass), Beads (Polysciences, Warrington, Penn), magnetic beads (Robbin 5 scientific, Mou tain View, Calif), plates, dishes and flasks (Corn ing Glass Works, Corning+ N, Y,), Meso (Becton Dickinson, Mountain View, C a1if, ), screens and solid fibers (Edelman et al. , 843,324; further, Kuroda et al. U.S. Pat. No. 4,416 ．． 777), membranes (Milipore Corp, Bedfo rd, Mass, ) and Davenstick. Other than those listed , there are sources for various supports.

Particularly preferred is Btogel P-60 (trademark) (BIORAD+Ri chsondCalif). Biogel P-60 (Commercial ) are porous polyacrylamide hydrogel beads. This bead is general is spherical in shape, averages about 250 microns in size, and has a diameter of about 60,000 microns. There is an average pore size that excludes molecules larger than 0 Daltons.

A variety of methods are available for immobilizing the ligand to the support.

For example, a ligand such as an antibody can be prepared by a variety of methods well known in the art: Part B, W, B, Jakoby and M, Wilchek (W), See Academic Press+New York page 30.1974. And; furthermore, the gate, Wilchek and W, Bayer@The Avidi n-Bjotin Complex in Bioanalytical Ap plications "Revenge Suhiyo" Shidari Seishiko 171: 1-32.198 8). These methods include glutaraldehyde V, carbodiimide, carbon Rubonyldiimidazole, cyanogen bromide, tosyl chloride, biotin/avidin and Includes the use of Othin/Streptavidin. immobilize this ligand on a support Once the maternal blood is incubated with this immobilized ligand, this ligand binds to the cells. Incubate under appropriate conditions and for a sufficient amount of time. Contents of the present invention Thus, suitable conditions for binding to occur are from about 4°C to about 37°C in physiological buffer. Particularly preferred temperatures range from 4°C to room temperature, including silane incubation at 4°C. Ru. The incubation time depends on the affinity and avidity of the ligand for the cells. dependent, which can be easily determined. Ink generally lasts about 15 minutes to 1 hour After incubation, unbound blood material is removed. and enrich for fetal cells using the methods described herein.

In another aspect of the invention, a sample of maternal blood is prepared under suitable conditions for the primary cause. are chemically coupled to the molecules and incubated with the ligand, which is then immobilized on a solid phase. It is adsorbed by the second factor, which is oxidized.

This first factor can bind to this second factor with an affinity of about 10@M-' or greater. Should. A large number of suitable factor-factor-second binding pairs are well known in the art. It is. They include, inter alia, biotin-avidin, biotin-streptavidin, gin, biocytin-avidin, biocytin-streptavidin, methotrex Sade-dihydrofolate reductase, 5-fluorouracil thymidylate Synthetase, riboflavin-riboflavin-binding protein, antibody-protease protein-A, and antibody-protein G. In a preferred embodiment, this first factor is biotin and this second factor is avidin.

Any factor in the above-mentioned binding bears, with a complementary factor acting as the first factor, , can act as a second factor. Furthermore, the factor-factor-factor 2 binding pair Combinations can be used. For example, by binding biotin to Gantt, It can also be adsorbed onto a support. This cell, ligand, biotin-complex and biotin support-complex is then combined with avidin via an incubation step. can be combined. Avidin is multivalent and allows cells to be immobilized. Ligand, biotin, avidin, biotin, support-enables complex formation .

In one example of the invention, a sample of maternal blood is treated with a biotinylated antibody so that no binding occurs. Incubate under appropriate conditions and for sufficient time. Next, fix this sample. Incubate with or pass through a support containing standardized avidin. Ru. Cells bound to this biotinylated antibody adsorb to this immobilized avidin, This allows separation of cells from unbound blood material. after that, Remove unbound cells and enrich for fetal cells using the method described below. .

In another aspect of the invention, a two-step method for immobilizing fetal nucleated erythroid cells is provided. Use the tap method. Briefly, the first sample is a sample of maternal blood and Incubate under appropriate conditions as noted. then chemically binds to the first factor Add the second liquid. This second ligand binds to this first ligand. can be matched. cells, first ligand, second ligand, factor-complex Next, it is adsorbed to the immobilized second factor, which binds to the blood substance. allow for separation. Representative examples of this factor-factor-factor-second binding bear have been described above.

Representative examples of this first ligand are erythroboietin, transferrin, and certain If you choose this first ligand, which contains an antibody, you will need to specifically recognize this first ligand. Select a second ligand that recognizes and binds. In a preferred embodiment, this second link Antibodies such as anti-erythroboietin (Terry Fox Labora) tory, Vancouver.

B, C, Canada), anti-transferrin (Chemicon rntl ,,Tnc,.

Tell1ecula, Calif, ) or anti-immunoglobulin antibodies.

Anti-immunoglobulin antibodies can be prepared using techniques well known in the art), or It is usually a source, especially Sigma Chemical Co,...S T. Louis.

Mo1. and Becton [1ickinson Immunocytome Software including try Systems, MountainView, Calif Available from source.

In the preferred U configuration, this first ligand specifically recognizes fetal nucleated erythroid cells. antibodies, such as anti-transferrin receptor antibodies (Becton Dic kinson ImII unocytometry Systems, Mou tain View.

Calif, ). This antibody is incubated with a sample of maternal blood.

Biotinylated anti-immunoglobulin antibodies, e.g. biotinylated goat anti-mouse I gG (a second ligand chemically bound to the first factor) is then added. Then, incubate this sample with the immobilized sample. a bed of material containing a second factor (immobilized avidin in this case) and Incubate or pass it through. This cell, antibody, anti-immunoglobe The phosphoantibody and biotin-conjugate are adsorbed to this immobilized avidin, thereby Allows subsequent removal of unbound blood material.

As mentioned above, once the cells are fixed, unbound blood material can be removed.

In some embodiments, the fixed cells are rinsed with a physiological buffer to remove this binding. Remove unused blood substances. This rinsing of immobilized cells is performed using the selected support species. Different methods are available depending on the species. This method inter alia: washes the support or Franchize; the support is magnetically drawn out of the solution and then soaked in a physiological buffer. resuspending; and centrifuging and then resuspending. Various physiological buffers Solutions are known in the art and include PBS, albumin such as live serum albumin ( PBS containing BSA), normal saline, and cell culture medium.

Once the unbound blood material has been removed, the bound cells can be separated from the fetal nucleated red blood cells. can be enriched preferentially. As mentioned above, there are at least two options Methods can be used independently or together. If these two methods If you are doing both methods together, you can use either method first. In some embodiments, this Combined cells with erythroboietin (A+mgen, Thousand 0ak) s, Ca1if) under selected conditions (Eme rson et al., 'Developmental Regulation of Er ythropoiests by Hematopoietic Growth Factors: Analysis on Populations of BFU-E From Bone Marrow.

Peripheral Blood and Fetal Liver, “Bl” ood 74 (1): 49-55.1989; see also Lin ch et al., '5 studies of Circulating Hemopoie tic Progenitor Ce1ls in Human Fetal See Blood, “Blood 59(5): 976-979.1982. Teru). The culture conditions chosen generally include other sources other than erythroboietin. Includes growth in standard cell culture media without cytokines. This is in the mother body Instead, it preferentially proliferates fetal nucleated erythroid cells. A particularly preferred medium is 20 15cov containing % bovine serum albumin and 2U/l purified urine human FPO improved Dulbecco medium (Gibco, Grand l5land New) York).

In the second method, fetal nucleated erythroid cells are depleted of their ammonium ions. superiority based on hemolysis of maternal blood cells, or more specifically maternal erythroid cells. Pre-enrichment (generally, Jacobsand Ste@art,’T he Role of Carbonic Anhydrase in Cer tain Ionic Exchanges Involving the Er ythrocyte,' J, Gen, Ph 5io1.25:539- 552.1942; and Maren and Wtlsy, 1neti cs of Carbonic Anhydrase in Whole Red Ce1ls as Measured by Transfer of Car bon Dioxide and Ammonia, “Mo1ecular P harmacolo 6: 430-440° 1970). easy According to Accumulation of ammonium ions within cells in the presence of enzyme inhibitors Incubate under conditions and for a sufficient amount of time to allow for. This is generally It takes about 5-30 minutes. The cells are then placed in the presence of ammonia and carbon dioxide. and incubate to cause selective hemolysis of maternal blood cells.

In the context of the present invention, a number of compounds include ammonia, chloride ions and carbon dioxide. It behaves as an elementary proper origin. For example, the proper origin of ammonia is This includes ammonia and ammonium salts. A suitable source of chloride ion is Among others, NaCl, KCI.

Contains MgC+□ and CaCIz. The proper origin of carbon dioxide requires, among other things: Includes carbon dioxide, carbonate and bicarbonate in solution.

Additionally, numerous carbonic anhydrase inhibitors are known in the art. , especially for most anions such as cyanide and cyanate, Includes ruphides, sulfonamides and acetaduramides (Lindsko g et al., q 54: 58'L1971; Ward and Cu1l A rch, Biochem, Biohs, 150: 436.197 2; and Pocker and WataIlori, Biochen 12 :2475+1973). carbonic anhydrasein The inhibitor should be selected as one that functions under physiological conditions. preferable Sulfanilamide (sulfa) is a carbonic anhydrase inhibitor. niIamide) and acetazolamide is included.

Bound cells can be released by a variety of methods, either after or before enrichment. too When bound cells are released prior to enrichment, these are subsequently isolated using the method described above. It can be enriched. Various methods of releasing cells are known in the art. . In one such method, cells are also cultured with or without cytokines. can do. Cytokines, such as ■1 and -2, cause cells to proliferate, but also alters the surface properties of cells such that these cells or their progeny become free from the support. You can make things happen. In other methods, the ligand or cell-ligand Cleavage of the bond can liberate the cells. Various cleavable Gant and cleavage enzymes Known in the art and include papain and trypsin, among others. Still other methods Cells can be released by mechanical, gravitational or electromagnetic forces. specific preferences A new method is mechanical agitation, for example via pipetting, stirring, shaking, vibration or sound waves. This is the stirring of the beads.

In another aspect of the invention, the cells are first enriched and then fetal nucleated erythroid cells are enriched. A method is provided that involves adsorption and removal of unbound shoe material. To put it simply: , fetal cells are first extracted from maternal blood with ammonia and chloride ions, and carbonyl ions. Enrich by incubation with quanhydrase inhibitor.

Cells are stimulated under conditions and for a sufficient period of time to allow ammonium ion accumulation. After incubation, the cells are treated with ammonia and carbon dioxide to remove maternal blood. Causes selective hemolysis of fluid cells. Fetal cells are erythropoietic as described above. or by incubation with erythropoietin and the method described above, immediately. In other words, cells are treated with ammonia and carbon dioxide by a combination of treatments. It can also be enriched. Furthermore, as mentioned above, these two methods should be followed in which order. You can go.

The enriched cells remaining after the above methods can then be fixed using any of the above methods. (1) specifically for fetal nucleated erythroid cells remaining after enrichment; Immobilized ligand that binds heterogeneously; (2) Ligand to cells remaining after enrichment This ligand is linked to the fetal nucleated erythroid system and incubated with factor 1. ), then this cell is transferred to the immobilized second adsorption to a factor (this second factor has an affinity constant of about 106M-1 or more) and (3) cells remaining after enrichment. incubate with a first ligand and then bind to the first ligand a second ligand that is chemically bonded to the first factor; Incubate under conditions and for a sufficient amount of time to bind to the ligand, then The cells are adsorbed to an immobilized second factor (this second factor is approximately 10'M- ’ can bind to this first factor with an affinity constant greater than or equal to I'm here.

After fixation of the cells, unbound blood material is removed using the methods described above. Can be done. If the cells are first enriched with erythroboietin and then ligated, When removing uncombined blood substances, these cells are exposed to ammonia and carbon dioxide. It can be processed using a method using Similarly, if these cells It is first treated by a method using ammonia and carbon dioxide and then unbound. When removing blood substances, these cells can be treated with erythroboietin. I can do it.

Fetal cells enriched from maternal blood are used to transfer these preferentially enriched cells to fetal cells. by incubating with a marker that can selectively bind to the cells. can be characterized. A marker indicates that its target on the target cell is similar to that amount on the maternal cell. selectively binds to fetal cells when the concentration is more than 10 times higher than Various markers are known in the industry, including, for example, alpha fetal protein (rAFP J ) against antibody (1 (ulozik et al., “Fetal Cells int he　MaternalCirculation　:Detection by Direct AFP-1mmunofluorescence”Human Genet, 62: 221-224.1982) or antigens. Antibody against “i” (Y, W, Kau et al., “Concentration o f Fetal Red Blood Cells From a Mixtur e of Maternal and Fetal Blood by Ant Antibodies to ii fetal cell markers can be prepared using techniques well known in the art. It can be labeled and used for detection of the presence of fetal cells.

In preferred embodiment B of the present invention, the title rMetho, which is incorporated herein by reference, ds for! iemoving Ligands from a Part icle 5surfaceJ (agent document number 200072-403) Ligands are removed from the cell surface via methods detailed in the co-pending application. Such exclusion Decontamination may be particularly advantageous prior to characterization as described above.

As mentioned above, enriched fetal cells have a variety of uses.

For example, through in situ hybridization, The presence of genetic material can be detected in fetal cells. Similarly, DNA or l? NA amplification can also be used to detect specific gene sequences in fetal cells.

The method of the present invention is a commonly used cytogenetic method that examines the spread of chromosomes in cells under a microscope. This is particularly useful for fetal cells enriched to the extent that chromosomal classification by scientific techniques is possible. It is for use.

In situ hybridization is a method of detecting the presence of specific genetic material within cells. (Proc of Pinkel et al.)

Natl, Acad, Sci, USA 85:9138-42.198 8; Furthermore, Hop+wan at al, +”Detection of Nu s+erical Chroa+osome Aberrations in Bladder Cancer by In 5itu Hybridizati on, Am, J, of Path,' 135 (6): 1.105- 1117.1989 by Sansho]. Briefly, preferentially enriched cells The relevant genetic material is first exposed using techniques well known in the art. This legacy The genetic material is then labeled with a target that can specifically hybridize with this genetic material. identification probe and conditions and sufficient time for hybridization to occur. (e.g. K, E, Davies, Human Ge netic Diseases, IRLPress+ 0xford, U, , 1986). Finally, this hybridized labeled probe detect the presence of a block. In a preferred embodiment, this genetic material is exposed to later denatured. In the context of the present invention, genetic material includes complete chromosomes, DNA and Contains RNA.

Various methods for amplifying and detecting genetic material are well known in the art, e.g. If present, the specific gene sequence can be determined using techniques well known in the art. can be amplified and then probed for the presence of this sequence (Kogan et al. 'An Improved Method ForPrenaLal Diag nosis of Genetic Diseases by Analysis s of Amplified DNA 5 sequences, “The Ne w En, J, of Med, 317(16):985-990.1 987; and -itt and Er1ckson, 'A Rapid M method for Detection of’/-Chromosomal DNA from Dried Blood Specimens byth e Polymerase Chain Reaction, “Human” Genet, 82:271-274.1989). for amplification The method is based on the polymerase chain reaction (Mullis et al., U.S. Pat. No. 4,683.195; Mullis et al., U.S. Pat. No. 4,683, No. 202; and U.S. Patent No. 4,800.159 to Ullis et al.) , and RNA-based amplification techniques (Liza rdi et al., Bio/Technolo fi:1197-1202゜1988 ; Kramer et al. Nature 339: 401-402. 1989 ; and Lo+ne! i et al. C11nical sub'tan α35 (9) 1826-183L 1989; furthermore, Kramer et al. U.S. Patent No. 4.786 , No. 600).

PCB is the most commonly used method for amplifying DNA sequences. Ru. Briefly, amplification involves the use of a reaction mixture of appropriate primers, enzymes, and nucleotides. compound, followed by multiple denaturation and annealing steps for amplification of small amounts of target DNA. Contains several (20-80) periods. This DNA mixture is then separated by electrophoresis. , and hybridizes with a labeled probe to detect the presence of the target DIIA sequence. Ru.

Preferentially enriched fetal cells can also be chromosomally classified (Huma n Cto enetics, D, E, Rooney and B, H, C zepulkowski (W), IRL Press, 0xford, U J., 1986). Briefly, in a preferred embodiment, 105 other Calf samples containing at least one fetal cell in a final concentration of 20% Serum (FBS) (HYCLONE(TM), Logan IJtah) and 2U/1 high purity recombinant erythroboietin (Terry Fox Labor 15c including Vancouver + B, C, Canada) ove's modified Dulbecco's medium (IMDM) (Gibco, Grand Is! and, N, Y,) for 2-6 days to increase the percentage of cells in metaphase. I met. The cells were then incubated in colcemid, fixed, and microscopically sliced. The cells were plated, trypsinized, and stained with -right staining. this This slide is an example of microscopic observation of abnormal chromosomes. ■Top Carboxylation of polyacrylamide gel 17g dry BioHel p-6 0 (Trademark) (Coarse grains of 50 to 100 mesh (wet)) (BrORA I), catalog number 150-1630. Richmond.

Calif,) 0.5M NaHCO310, 5M NazCOffl, 51 Added to. The pH was adjusted to 10.5 with Na0)1 and the beads were damaged. Mixer (RZRr, Carfamo, Wiarton+) The mixture was stirred for about 20 to 30 minutes at a 1000 ml, Canada). this mixture was then placed in a 60'C water bath. After the temperature reaches 60°C, repeat this for another 2 hours. The mixture was incubated at 60° C. with occasional stirring. Next, take a hot water bath with this mixture. The temperature of the mixture was lowered to room temperature by removing it from the tank and placing it in an ice bath.

The beads were washed several times with distilled or deionized water, then several times with PBS, and then connected to a vacuum. Washed using a coarse glass filter. This carboxylated gel was prepared in PBS. can be stored at 4°C and is stable for one year under sterile conditions or with preservatives. be.

hindrance Avidin conjugation of carboxylated Biogel with a fixed amount of carboxyl First remove PBS from Biogel using a coarse glass filter connected to a vacuum. The gel was then washed with distilled or deionized water for 1 hour. Allowed to equilibrate for 5-30 minutes. Equilibration in water allows this gel to be initially quantified in Let it swell to about 4 times its volume. This gel was added per 1+il of gel (originally PBS resuspended in 10+l of distilled or deionized water.

20 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ( EDC-)ICI) (Sigma Chemical Co, Catalog number E 7750. St.

1, ouis, Mo,) was added per 11 of the measured original gels. This pH-I The temperature was quickly adjusted to 5.5 by dropping HcI. Maintain this pI (at 5.5) Be careful: pH below 5.0 or above 6.0 will cause Biogel to be activated. Significantly lower. This mixture was stirred for 5 minutes.

Avidin (International Enzymes, Inc., Fa ! 1brook, Calif,) at 10-100 mg/ml in deionized water. It was dissolved at a concentration between. Next, add 500 μg of avidin per 1++1 gel (originally (measured amount in PBS). This mixture was stirred for 1.5 hours. next Add 2M glycine such that the final concentration is 0.2M glycine in this mixture. The mixture was then stirred for an additional hour.

Wash this gel with several times the volume of PBS using a coarse glass filter and vacuum. , and stored at 4''C in PBS containing 0.1% NaNx.

This gel is stable for approximately one year.

Lord of Flames Preparation of maternal immunoadsorbent cells by two-step method 20+11 blood was collected from a pregnant woman and 1% live serum in a 50ml centrifuge tube. Albumin (BSA) (Stgwa Chemical Co, St, L ouis, Mo,) in an equal volume of PBS. Existing nuclear details per 1111 The number of cells was measured by the following method. 50μl of aliquoted mixed anticoagulated whole blood was diluted in 3 parts of 3% acetic acid solution. After stirring, add 7 μl of this diluted blood sample to It was placed in each of two tanks of a hemacytometer. Let the cells settle for about 3 minutes. After this, the cell nuclei, which were mostly lysed by acetic acid, were transferred to a modified Neuba uer ruling (VWR5cientific, 5anFrancisco , Ca1if, ) in the four ruled areas of the hemacytometer tank. It was measured using Each area is 0. I occupies a sample volume of Xl0-'μl . - Multiply the average number of nuclei per area by the dilution series of 61 x 10' and The number of nucleated cells per blood was determined. Average value is less than 10 nuclei per eN region If necessary, this process was repeated, except that 50 μm blood was mixed with only 1 ml of acetic acid. and the resulting new dilution series was 21 x 10'.

Add 5 ml of H4stopaque 10 to each tube of diluted blood. Add 71077 (Si Chemical Co,) and leave at room temperature for 15 minutes. Centrifuged at 700g. Collect cells at the interface and add PBS containing 1% BSA. I washed it once. The pellet was resuspended in 100 μm of PBS containing 1% BSA. I let it happen.

Anti-tranphnilin receptor antibody (Becton Dickinson+1+ u+unocytometry Systems, Mountain View , Ca1if, ) 20 μg/ml was added to this mixture and incubated on ice for 15 μg/ml. Incubated for minutes. Next, add 4 ml of PBS containing 1% BSA to the cells. Washed once and centrifuged at approximately 400 g for 5 minutes.

The cells were then carefully resuspended in 11 and 1 μg/ml biotinylated goat Anti-mouse IgG (Southern Biotech+ Birvingh a*, Ala,) was added. This mixture was incubated on ice for 15 minutes, It was then washed twice with PBS containing 1% BSA as described above.

Column preparation Carboxylated Biogel P-30™ (prepared as described above) was heated to room temperature. equilibrate to 9/1.5 column (Phar+macia+Pisca taieay.

N, J, ) were filled so that the total bed height was 1 cm. This column is pa s, then washed with PBS containing 5% BSA. This column is a "precolumn" Work as. Avidin column is avidin-conjugated Bioget P -60™, which was prepared as described above.

The avidin-conjugated column was equilibrated to room temperature and the K9/1 Five columns were packed with a total bed height of 4c+i. PBS this column and then several times with PBS containing 5% BSA.

Cell immunoadsorption Cells prepared as above were resuspended in PBS containing 5% BS8 to a volume of 1 ml. It was made to be the amount. Carefully remove the cells from the gel head of this pre-column filter. I moved it on top. The cells were passed through a precolumn containing 5% BSA. Washed with 1 ml of PBS. Verister pump (C(+)e-parHr, R Adjust the flow rate from the avidin column to approximately 1 ml/min with I controlled it. Once most of the cells fall onto this avidin column head, Add PBS 1-2II11 containing 5% BSA onto the avidin column. The remaining cells were washed away. This column was washed with 4-6 ml of PBS containing 5% BSA. It was then rinsed with 54-6 ml of PB.

Removal of adsorbed cells from avidin column This avidin column was placed on top of a 151 centrifuge tube. open the valve of this column , and into this column with a wide-bore 9-inch transfer pipette. 51 RPMI 1 was added. This pipette is used to mechanically agitate and recycle the cell bed. RPMI is added to this column while it is being used for suspension, thereby suspending the cells. from the gel matrix and filtered into a centrifuge tube. Next this tube The cells were centrifuged at 400 g for 5 minutes and resuspended in the following culture medium.

■Soil Preferential enrichment of nucleoerythroid cells in the fetus Enrichment of fetal cells with erythroboietin Avidin-Biogel column as described above The cells isolated from Nε (trademark), Logan, Utah) and 2U/mlO high purity recombinant Eri Troboietin (Terry Fox Laboratory + νancouv 15cove's modified Dulbecco medium containing er, B, C, + Canada) (IMDM) (Gibco, Grand l5land, N, Y,) Made it muddy. Dilute the cells to 50 x 10 nucleated cells/ml and 96-well tissue culture plate with round bottom (Corning Glass Works) , Corning+ N, Y, ) into each well.

Enrichment of fetal cells by ammonium ion differentiation separated from the above avidin column The cells were adjusted to a concentration of 2×10′ nucleated cells/1 or less. 1 capacity thin The cell mixture was chilled on ice and added with 18 volumes of 0.1844 M sH,ci and 2 volumes of A solution containing 10 MM acetazolamide at 29°C was added. 2 minutes later, 2 volumes Quickly add 3 nM (2 11.1 Stir for a minute. Cells were washed several times with PBS by centrifugation to remove cell clumps.

flames In-situ Hypuridize Shigon Enriched cells were exposed to hypotonic solution (0,075 M KCI) for 12 minutes at 37°C. Ta. During this incubation, invert the tube once to suspend the cells. I kept it in a state. Add 20 drops of fresh fixative (3:1 methanol:acetic acid) to the cells. , vortex, and centrifuge at approximately 250 g for 8 minutes. Add fresh fixative to the cells. and then incubated for 1 hour at room temperature. The cells were centrifuged at approximately 250g for 8 minutes. I was concerned. Fresh fixative was added and the process was repeated one more time. last Next, the cells were resuspended in a small amount of fixative and left overnight at 4°C.

The next day, the cells were vortexed and placed on a microscope slide (Baxter.

McGraiv Park, III). This slide is ethanol It was washed and soaked in distilled water. The slides were allowed to dry for 2 days at room temperature. .

Add this slide to 2X 5CC (0.30M NaCl, 0.030M NaCl) 68°C to 70°C for 2.5 minutes in 70% formaldehyde in citrate] It got hot for a while.

The slides were immediately rinsed with 70% ethanol in water. After rinsing, use this Place slides in 70%, 95%, and 100% ethanol solutions for 5 minutes each. There was an interval. Each alcohol solution was maintained at ≦-20°C. this slide Air dried.

Pageraji Single coy 5equence hybridizes to polymorphic and homologous Ioci o n hu+san X and Y Chromoso IIles” PNAS 79:5352-5356, 1.982), American Thai Plasmi FDNA derived from Culture Collection (ATCC) No. 57261 A probe was prepared from Ta. Add 25 μl of biotinylated probe (5 μg/a+1) per slide to the microphone. Placed in a centrifuge tube. Heat the probe at 70°C for 5 minutes and immediately place it on ice. placed. 20 μl of probe solution was placed on each slide and 22 x 40111 covered with ffi coverslip. Remove this slide using a wet paper towel. and incubated at 37°C for 12-18 hours.

A solution of 50% formaldehyde in SSC of IX was warmed to 37°C.

Remove the coverslip from the slide and add 50% formaldehyde solution. Soaked in liquid for 30 minutes. The slides were then slowly placed in a 2X SSC solution for 30 minutes. Leave it for 30 minutes while shaking it gently, then put it in the IX SSC for 30 minutes after shaking it slowly. I left it alone. Fluorescent avidin (Vector+Bur!ingame, Ca1i f,) was diluted 1:1000 (1 u g/ml). back of this slide The surface and periphery of the cell area were wiped. Add 200 μl of avidin-fluorescent material to each slide. and incubated in the box for 30 minutes at room temperature. this slide Sequentially 1) 4X SCC while shaking for 10 minutes, 2) 4X SCC, 0,1% 3) Do not shake for 10 minutes at Tween 20, 3) Do not shake for 10 minutes at 4X SCC. I rinsed it out. The back side of the slide and the surrounding head area of the cells were wiped. propiziu Anti-fade agent containing muiodine (10a+l PBS, 100mg p-phenylene diamine, 90 ml glycerol, pH 8.0, 10 μg/ml pyrovigilla 10 μl of muiodine) was added. Cover the slide with a coverslip and Stored at 4°C. This slide can be stored for several days if necessary. mark Target DNA can be observed under a microscope in the presence of fluorescence.

Another”- Classification of chromosomes Enriched cells were treated with 11/g colcemid (Stgma, st, Louis+Mo, ) for 1 hour at 37°C. The cells were treated with a hypotonic solution (MCI of 0,075 M). was added and incubated for 12 minutes at 37°C. This tube is ink The cells were kept in suspension by one inversion in tuberin. Fresh fixative (3 Add 20 drops of methanol:acetic acid) to the cells, stir, and add approximately 250 g of 8 Centrifuged for minutes. Add fresh fixative to the cells and then incubate for 1 hour at room temperature. I started. Centrifuge the cells at approximately 250g for 8 minutes and add fresh fixative again. I got it. This process was repeated one more time. Finally, fix the cells with a small amount of fixative. Resuspend and leave at 4°C overnight. The next day, the cells were stirred and microscopically mounted on mirror slides (Baxter, McGraw Park Ill.).

The slides were washed with ethanol and soaked in distilled water. this slide Air dry for approximately 3-4 days and add 0.005% trypsin (Difc).

Bactotrypsin, VWR5cientific San Fran cisco, Caltf, ) for 30 to 35 seconds. This slide is 1% Washed twice with PBS containing FBS and then with PBS alone. -right this cell t solution (Sigma Chemical Co,.

SL, Louts, Mo,) and then washed twice with deionized water. child slides were observed for metaphase cells and classified by routine techniques. .

Although the present invention has been described with respect to specific embodiments thereof for purposes of illustration, the scope of the invention has been described. Those skilled in the art will recognize that modifications and changes are possible without departing from the invention.

Accordingly, the invention is limited only by the scope of the appended claims.

A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) maternal blood; Immobilization that allows blood samples to specifically bind to fetal nucleated erythroid cells The ligand, the conditions for specifically binding this ligand to this cell, and (b) remove unbound blood substances; and (C) ink the bound cells in the presence of erythropoietin. the method comprising the step of: I will provide a.

Procedural amendment (formality) %formula% 1. Display of incident PCT/US91102789 2. Name of the invention Method for enrichment of fetal cells from maternal blood, method 3, person making corrections Relationship to the incident: Patent applicant Name: Cellpro Incoholated 4, Agent Address: 8-10-5 Toranomon-chome, Minato-ku, Tokyo 105, Date of amendment order 6. Subject of correction Translation of the description, claims, and abstract 7, contents of amendments Translations of the description, claims, and abstract (no changes to the contents) 8. List of attached documents Description, claims and abstract 1 translation each international search report international search report US9102789

Claims (72)

[Claims] 1. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. bind the immobilized ligand specifically to this cell. incubating under conditions and for a sufficient time; (b) remove unbound blood material; and (c) remove bound cells with erythrocytes. Incubate in the presence of poietin to preferentially enrich for these fetal cells. A method that involves tightening. 2. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. bind the immobilized ligand specifically to this cell. incubating under conditions and for a sufficient time; (b) remove unbound blood material; (c) remove bound cells with ammonia and and chloride ions as well as carbonic anhydrase inhibitors. conditions and sufficient time to accumulate ammonium ions in the cell. isokinibate with; and (d) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. selective hemolysis of maternal blood cells by incubation in the presence of A method comprising. 3. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. bind the immobilized ligand specifically to this cell. incubating under conditions and for a sufficient time; (b) remove unbound blood material; (c) remove bound cells with ammonia and and chloride ions as well as carbonic anhydrase inhibitors. conditions and sufficient time to accumulate ammonium ions in the cell. Incubate with; (d) Cells containing accumulated ammonium ions are treated with ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells: and (e) Incubate the bound cells in the presence of erythropoietin. to preferentially enrich fetal cells. 4. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood are treated with ammonia and chloride ions and carbonyl ions. Anhydrase inhibitor and ammonium ion accumulation in the cell incubating under conditions and for a sufficient period of time; (b) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (c) specific binding of cells remaining after hemolysis to fetal nucleated erythroid cells; an immobilized ligand capable of binding to the cell specifically. incubate under conditions and for a sufficient period of time; and (d) A method comprising removing unbound blood material. 5. After removing the unbound blood material, the cells are erythropoietic. to preferentially enrich fetal cells. 5. The method of claim 4, comprising: 6. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood were incubated in the presence of erythropoietin. (b) convert the enriched cells into fetal nucleated erythroid cells; an immobilized ligand that can specifically bind to the cell; incubate under conditions and for a sufficient time to specifically bind the compound; and hand (c) a method comprising removing unbound blood material. 7. After removing the unbound blood material, the bound cells are treated with ammonia. and chloride ion and carbonic anhydrase inhibitor, and this combination Conditions and sufficient time to accumulate ammonium ions in cells incubate with; and Cells containing accumulated ammonium ions are treated in the presence of ammonia and carbon dioxide. selectively hemolyzing maternal blood cells by incubating the maternal blood cells in a The method according to claim 6, comprising: 8. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Incubating a sample of maternal blood in the presence of erythropoietin (b) ammonia the erythropoietin-enriched cells; and chloride ions and carbonic anhydrase inhibitors; Under conditions and for a sufficient period of time to accumulate ammonium ions within cells. incubate; (c) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (d) specifically binding the enriched cells from step (c) to fetal nucleated erythroid cells; an immobilized ligand that can be used to specifically bind said ligand to this cell. incubate under conditions and for a sufficient period of time; and (d) A method comprising removing unbound blood material. 9. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood are treated with ammonia and chloride ions and carbonyl ions. Anhydrase inhibitor and ammonium ion accumulation in the cell incubating under conditions and for a sufficient period of time; (b) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (c) Enriched cells from step (b) are incubated in the presence of erythropoietin. (d) erythropoietin to preferentially enrich the fetal cells; An immobilized ligator that can specifically bind enriched cells to fetal nucleated erythroid cells. conditions and sufficient time to specifically bind the ligand to these cells. incubate with; and (e) a method comprising removing unbound blood material. 10. The ligand is more naturally present than the antibody, erythropoietin and transferrin. 10. The method according to claims 1-9, wherein the method is selected from the group consisting of: 11. After the step of removal of unbound blood material described above, the fixed cells are 10. A method according to claims 1-9, comprising liberating. 12. The method according to claim 11, wherein the cells are released by mechanical agitation. . 13. The step of removal includes rinsing by physiological hemolysis of bound cells. The method according to claims 1 to 9, comprising: 14. The physiological hemolysis includes PBS, PBS containing albumin, normal saline and 14. The method of claim 13, wherein the method is selected from the group consisting of cell culture media. 15. After a step of preferential enrichment of fetal cells, this enriched cells are specifically selected for said fetal cells. of claims, including inkinivating with markers that can be differentially bound. A method according to ranges 1 to 9. 16. The ligand may be an optical fiber, a bead, a magnetic bead, a plate, III I, flasks, meshes, screens, solid fibers, membranes and dipsticks Claims 1 to 9 are immobilized on a solid phase selected from the group consisting of the method of. 17. The carbonic anhydrase inhibitor contains sulfanilamide and Claims 2, 3, 4, 7, 8 selected from the group consisting of or the method described in 9. 18. The chloride ions are selected from NaCl, KCl, MgCl2 and CaCl2. 10. A method according to claim 2, 3, 4, 7, 8 or 9, selected from the group consisting of: 19. the ammonia is selected from the group consisting of NH4Cl and ammonium salts; The method according to claim 2, 3, 4, 7, 8 or 9. 20. the carbon dioxide is in the group consisting of carbon dioxide, carbonate and bicarbonate in solution; The method according to claim 2, 3, 4, 7, 8 or 9, selected from. 21. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. The first factor is chemically bound to a ligand that can incubating under conditions and for a sufficient period of time to allow specific binding; (b) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (c) remove unbound blood material; and (d) remove bound cells with erythropoietin. Incubation in the presence of poietin to preferentially enrich fetal cells A method comprising: 22. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) a sample of maternal blood with a ligand capable of specifically binding to said cells; The first factor is chemically bound to the cell, and the ligand is specifically bound to this cell. incubating under conditions and for a sufficient period of time; (b) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (c) removing unbound blood material; (d) removing bound cells with ammonia and and chloride ions as well as carbonic anhydrase inhibitors. conditions and sufficient time to accumulate ammonium ions in the cell. Incubate with; (e) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. selective hemolysis of maternal blood cells by incubation in the presence of A method comprising. 23. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. The first factor is chemically bound to a ligand that can incubating under conditions and for a sufficient period of time to allow specific binding; (b) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (c) removing unbound blood material; (d) removing bound cells with ammonia and and chloride ions as well as carbonic anhydrase inhibitors. conditions and sufficient time to accumulate ammonium ions in the cell. Incubate with; (e) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; and (f) incubating the bound cells in the presence of erythropoietin; , preferentially enriching the fetal cells. 24. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood are treated with ammonia and chloride ions and carbonyl ions. Anhydrase inhibitor and ammonium ion accumulation in the cell incubating under conditions and for a sufficient period of time; (b) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (c) cells remaining after hemolysis with a linker capable of specifically binding to said cells; The first factor is chemically bound to the ligand and the ligand is specifically directed to this cell. incubating under conditions and for a sufficient period of time to allow binding; (d) the cells are adsorbed to an immobilized second factor, wherein said second factor is about can bind to this first factor with an affinity constant of 108M-1 or more; and hand (e) a method comprising removing unbound blood material. 25. After removal of unbound blood material as described above, the cells are erythropoietic. to preferentially enrich these fetal cells. 25. The method of claim 24, comprising: 26. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Incubating a sample of maternal blood in the presence of erythropoietin (b) the enriched cells are specific for said cells; The first factor is chemically bound to a ligand capable of binding to Incubate under conditions and for a sufficient time to specifically bind the ligand to cells. Bate; (c) the cells are adsorbed to an immobilized second factor, wherein said second factor is about be able to bind to this first factor with an affinity constant of 16M-1 or more; and (d) A method comprising removing unbound blood material. 27. After removing the unbound blood material, the bound cells are treated with ammonia. a and chloride ions and carbonic anhydrase inhibitors, and their binding. Conditions and sufficient time to accumulate ammonium ions in the combined cells incubate under; and Cells containing accumulated ammonium ions are treated in the presence of ammonia and carbon dioxide. selectively hemolyzing maternal blood cells by incubating the maternal blood cells in a 27. The method of claim 26, comprising: 28. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Incubating a sample of maternal blood in the presence of erythropoietin (b) ammonia the erythropoietin-enriched cells; and chloride ions and carbonic anhydrase inhibitors; Under conditions and for a sufficient period of time to accumulate ammonium ions within cells. incubate; (c) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (d) the enriched cells from step (c) are capable of specifically binding to said cells; A first factor that is chemically bound to a ligand and that makes said ligand specific to this cell. incubating under conditions and for a sufficient time to bind to; (e) the cells are adsorbed to an immobilized second factor, wherein said second factor is about can bind to this first factor with an affinity constant of 108M-1 or more; and hand (f) A method comprising removing unbound blood material. 29. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood are treated with ammonia and chloride ions and carbonyl ions. Anhydrase inhibitor and ammonium ion accumulation in the cell incubating under conditions and for a sufficient period of time; (b) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (c) Enriched cells from step (b) are incubated in the presence of erythropoietin. (d) erythropoietin to preferentially enrich the fetal cells; Chemically coupling the enriched cells to a ligand capable of specifically binding to said cells the first factor, the conditions for specifically binding the ligand to the cells, and sufficient Incubate for a minute; (e) adsorbing the cells to an immobilized second factor, where the second factor is can bind to this first factor with an affinity constant of about 108 M-1 or greater; do (f) A method comprising removing unbound blood material. 30. The ligand is more naturally present than the antibody, erythropoietin and transferrin. 30. A method according to claims 21-29, wherein the method is selected from the group consisting of: 31. After the step of removal of unbound blood material described above, the fixed cells are 30. A method according to claims 21-29, comprising liberating. 32. 32. The method of claim 31, wherein the cells are released by mechanical agitation. . 33. The step of removal includes rinsing by physiological hemolysis of bound cells. 30. The method according to claims 21-29, comprising: 34. The physiological hemolysis includes PBS, PBS containing albumin, normal saline and 34. The method of claim 33, wherein the method is selected from the group consisting of cell culture media. 35. After a step of preferential enrichment of fetal cells, this enriched cells are specifically selected for said fetal cells. claimed, including incubation with a marker capable of differentially binding. The method according to ranges 21-29. 36. The ligand may be an optical fiber, a bead, a magnetic bead, a plate, III , flasks, meshes, screens, solid fibers, membranes and dipsticks Claims 21 to 29, wherein the solid phase is immobilized on a solid phase selected from the group consisting of How to put it on. 37. The carbonic anhydrase inhibitor contains sulfanilamide and Claims 22, 23, 24, 27, 28 or 29. 38. The chloride ions are selected from NaCl, KCl, MgCl2 and CaCl2. Claim 22, 23, 24, 27, 28 or 29 selected from the group consisting of the method of. 39. the ammonia is selected from the group consisting of NHCl and ammonium salts; , the method according to claim 22, 23, 24, 27, 28 or 29. 40. the carbon dioxide is in the group consisting of carbon dioxide, carbonate and bicarbonate in solution; The method according to claim 22, 23, 24, 27, 28 or 29, selected from . 41. The first factor-second factor binding pair is biotin-avidin, biotin-st leptavidin, biocytin-avidin, biocytin-streptavidin, Totrexetotodihydrofolate reductase, 5-fluorouracil-ti From midylate synthetase and riboflavin-riboflavin binding protein 30. A method according to claims 21-29, selected from the group consisting of: 42. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. A first ligand that can be produced and conditions for specifically binding the first ligand to this cell and incubate for a sufficient period of time; (b) converting the sample into a second ligand capable of specifically binding the first ligand; A first factor chemically bound to a ligand and a second factor attached to this first ligand. incubate under conditions and for a sufficient time to cause the bonds to bind; (c) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (d) remove unbound blood material; and (e) remove bound cells with erythropoietin. Incubate in the presence of poietin to preferentially enrich for these fetal cells A method comprising causing 43. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. A first ligand that can be produced and conditions for specifically binding the first ligand to this cell and incubate for a sufficient period of time; (b) converting the sample into a second ligand capable of specifically binding the first ligand; A first factor chemically bound to a ligand and a second factor attached to this first ligand. incubating under conditions and for a sufficient period of time to allow the binding of the compounds; (c) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (d) removing unbound blood material; (e) removing bound cells with ammonia and and chloride ions as well as carbonic anhydrase inhibitors. conditions and sufficient time to accumulate ammonium ions in the cell. incubate with; and (f) remove bound cells containing accumulated ammonium ions with ammonia and diacid selectively hemolyze maternal blood cells by incubating in the presence of carbon dioxide. A method comprising: 44. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood that specifically binds to fetal nucleated erythroid cells. A first ligand that can be produced and conditions for specifically binding the first ligand to this cell and incubate for a sufficient period of time; (b) converting the sample into a second ligand capable of specifically binding the first ligand; A first factor chemically bound to a ligand and a second factor attached to this first ligand. incubating under conditions and for a sufficient period of time to allow the binding of the compounds; (c) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (d) removing unbound blood material; (e) removing bound cells with ammonia and and chloride ions and carbonic anhydrase inhibitors. conditions and sufficient time to accumulate ammonium ions in the cell. Incubate with; (f) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing non-fetal cells; and (g) incubating the bound cells in the presence of erythropoietin; , preferentially enriching the fetal cells. 45. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood are treated with ammonia and chloride ions and carbonyl ions. Anhydrase inhibitor and ammonium ion accumulation in the cell incubating under conditions and for a sufficient period of time; (b) Cells containing accumulated ammonium ions are exposed to ammonia and carbon dioxide. incubating in the presence of selectively hemolyzing maternal blood cells; (c) specific binding of cells remaining after hemolysis to fetal nucleated erythroid cells; and a first ligand that can specifically bind to the cell. incubating under conditions and for a sufficient time; (d) converting the sample into a second ligand capable of specifically binding the first ligand; A first factor chemically bound to a ligand and a second factor attached to this first ligand. incubating under conditions and for a sufficient period of time to permit binding of the (e) the cells are adsorbed to an immobilized second factor, wherein said second factor is about can bind to this first factor with an affinity constant of 108M-1 or more; and hand (f) A method comprising removing unbound blood material. 46. After removal of unbound blood material as described above, the cells are erythropoietic. to preferentially enrich these fetal cells. 46. The method of claim 45, comprising: 47. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood were incubated in the presence of erythropoietin. (b) enriching the enriched cells specifically for said cells; a first ligand capable of binding to the fetal nucleated erythroid cell; (c) incubating under conditions and for a sufficient time to allow binding of the ligand; This sample is then treated with a second ligand that can specifically bind to this first ligand. The first factor is chemically bonded to the first factor, and the second ligand is attached to the first factor. incubating under conditions and for a sufficient period of time to allow binding; (d) The cells are adsorbed to an immobilized second factor, where the second factor is approximately can bind to this first factor with an affinity constant of 108M-1 or more; and hand (e) a method comprising removing unbound blood material. 48. After removing the unbound blood material, the bound cells are treated with ammonia. a and chloride ions and carbonic anhydrase inhibitors, and their binding. Conditions and sufficient time to accumulate ammonium ions in the combined cells incubate under; and Cells containing accumulated ammonium ions are treated in the presence of ammonia and carbon dioxide. selectively hemolyzing maternal blood cells by incubating the maternal blood cells in a 48. The method of claim 47, comprising: 49. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Incubating a sample of maternal blood in the presence of erythropoietin (b) the blood sample is enriched with ammonia and chloride; ion and carbonic anhydrase inhibitor, and the intracellular Incubate under conditions and for a sufficient time to accumulate ammonium ions. Toshi; (c) a first ligand capable of specifically binding the enriched cells to said cells; , conditions and sufficient time for specifically binding the first ligand to the cells; (d) incubate the sample under a first factor chemically bound to a second ligand capable of binding to this Under the conditions and for a sufficient time to bind this second ligand to the first ligand, incubate; (e) the cells are adsorbed to an immobilized second factor, wherein said second factor is about can bind to this first factor with an affinity constant of 108M-1 or more; and hand (f) A method comprising removing unbound blood material. 50. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) Samples of maternal blood are treated with ammonia and chloride ions and carbonyl ions. Anhydrase inhibitor and ammonium ion accumulation in the cell incubating under conditions and for a sufficient period of time; (b) the enriched cells are treated with a first ligand capable of specifically binding to said cells; , conditions and sufficient time for specifically binding the first ligand to the cells; (c) Enriched cells from step (b) are incubated under erythropoietic Incubate in the presence of ethyne to preferentially enrich these fetal cells. (d) converting the sample into a first ligand capable of specifically binding the first ligand; A first factor chemically bound to two ligands and a second factor attached to this first ligand. incubating under conditions and for a sufficient period of time to allow the Gunds to bind; (e) the cells are adsorbed to an immobilized second factor, wherein said second factor is about can bind to this first factor with an affinity constant of 108M-1 or more; hand (f) A method comprising removing unbound blood material. 51. The ligand is more naturally present than the antibody, erythropoietin and transferrin. 51. The method of claims 42-50, wherein the method is selected from the group consisting of: 52. After the step of removal of unbound blood material described above, the fixed cells are 51. The method of claims 42-50, comprising liberating. 53. 53. The method of claim 52, wherein the cells are released by mechanical agitation. . 54. The step of removal includes rinsing by physiological hemolysis of bound cells. 51. The method according to claims 42-50. 55. The physiological hemolysis includes PBS, PBS containing albumin, normal saline and 55. The method of claim 54, wherein the method is selected from the group consisting of cell culture media. 56. After a step of preferential enrichment of fetal cells, this enriched cells are specifically selected for said fetal cells. claimed, including incubation with a marker capable of differentially binding. A method according to ranges 1 to 9. 57. The ligand may be an optical fiber, a bead, a magnetic bead, a plate, III I, flasks, meshes, screens, solid fibers, membranes and dipsticks Claims 42 to 50 are fixed in phase selected from the group consisting of Method described. 58. The carbonic anhydrase inhibitor contains sulfanilamide and Claims 43, 44, 45, 48, 49 or 50. 59. The chloride ions are from NaCl, KCl, HgCl2 and CaCl2. Claim 43, 44, 45, 48, 49 or 50 selected from the group consisting of the method of. 60. the ammonia is selected from the group consisting of NH4Cl and ammonium salts; 51. The method according to claim 43, 44, 45, 48, 49 or 50. 61. the carbon dioxide is in the group consisting of carbon dioxide, carbonate and bicarbonate in solution; The method according to claim 43, 44, 45, 48, 49 or 50, selected from . 62. The first factor-second factor binding pair is biotin-avidin, biotin-st leptavidin, biocytin-avidin, biocytin-streptavidin, Totrexetotodihydrofolate reductase, 5-fluorouracil-ti from midylate synthetase and lipoflavin-lipoflavin binding protein 51. The method of claims 42-50, selected from the group consisting of: 63. 51. The method of claims 42-50, wherein said second ligand is an antibody. 64. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood is treated with a biotinylated anti-erythrocyte antibody and the fetal nucleated red blood cells. Incubation under conditions and for a sufficient time to allow this antibody to bind to blood cells. Toshi; (b) Pass the cells through a packed bed of porous material with avidin bound to its surface. , this biotinylated antibody is adsorbed to avidin on the surface of this porous material, and this binding fetal cells and adult cells to the porous material by; (c) rinsing the packed bed to remove unbound blood material; (d) releasing the bound cells by mechanical agitation of the packed bed; and (e) Incubate the released cells in the presence of erythropoietin. to preferentially enrich fetal cells. 65. A method for enriching fetal nucleated erythroid cells from maternal blood, comprising: (a) A sample of maternal blood is treated with a biotinylated anti-erythrocyte antibody and the fetal nucleated red blood cells. Incubation under conditions and for a sufficient time to allow this antibody to bind to blood cells. Toshi; (b) Pass the cells through a packed bed of porous material with avidin bound to its surface. , this biotinylated antibody is adsorbed to avidin on the surface of this porous material, and this binding fetal cells and adult cells to the porous material by; (c) rinsing the packed bed to remove unbound blood material; (d) releasing the bound cells by mechanical agitation of the packed bed; (e) The released cells are treated with ammonia and chloride ions and carbonic amphibians. dorase inhibitor and accumulate ammonium ions in these released cells. incubate under conditions and for a sufficient period of time to allow for accumulation; and (f) Incubate the released cells in the presence of ammonia and carbon dioxide. selectively hemolyzing maternal blood cells. 66. Enrich fetal nucleated erythroid cells from maternal blood and specific genetic material 1. A method for testing for the presence of: (a) a sample of maternal blood in a nucleated fetal An immobilized ligand that can specifically bind to erythroid cells and this Incubate under conditions and for a sufficient time to specifically bind the ligand to cells. Bate; (b) remove unbound blood material; (c) remove bound cells from erythropoiesis; incubate in the presence of Chin to preferentially enrich fetal cells; (d ) exposing genetic material for said preferentially enriched cells; (e) hybridizing genetic material related to fetal cells specifically to said genetic material; A labeled probe that can be hybridized and conditions that cause hybridization. incubate under conditions and for a sufficient period of time; and (f) detecting the presence of a hybridized labeled probe; Law. 67. After the step of exposing the genetic material, it is possible to denature this genetic material. 67. The method of claim 66, comprising: 68. Enrich fetal nucleated erythroid cells from maternal blood and specific genetic material 1. A method for testing for the presence of: (a) a sample of maternal blood in a nucleated fetal An immobilized ligand that can specifically bind to erythroid cells and this Incubate under conditions and for a sufficient time to specifically bind the ligand to cells. Bate; (b) remove unbound blood material; (c) remove bound cells from erythropoiesis; to preferentially enrich fetal cells; and (d) sorting the chromosomes for the enriched fetal cells. 69. Enrich fetal nucleated erythroid cells from maternal blood and specific genetic material 1. A method for testing for the presence of: (a) a sample of maternal blood in a nucleated fetal Chemically linked to a ligand that can specifically bind to erythroid cells the first factor, the conditions and sufficient time for specifically binding the ligand to this cell; Incubate under (b) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (c) remove unbound blood material; (d) remove bound cells from erythropoiesis; to preferentially enrich fetal cells; (e ) exposing genetic material for said preferentially enriched cells; (d) hybridizing genetic material relating to fetal cells specifically to said genetic material; A labeled probe that can be hybridized and conditions that cause hybridization. incubate under conditions and for a sufficient period of time; and (f) detecting the presence of a hybridized labeled probe; Law. 70. After the step of exposing the genetic material, it is possible to denature this genetic material. 70. The method of claim 69, comprising: 71. Enrich fetal nucleated erythroid cells from maternal blood and specific genetic material 1. A method for testing for the presence of: (a) a sample of maternal blood in a nucleated fetal Chemically linked to a ligand that can specifically bind to erythroid cells the first factor, the conditions and sufficient time for specifically binding the ligand to this cell; Incubate under (b) the cells are adsorbed to an immobilized second factor, wherein said second factor is about capable of binding to this first factor with an affinity constant of 108M or more; (c) remove unbound blood material; (d) remove bound cells from erythropoiesis; to preferentially enrich fetal cells; and (e) classifying the chromosomes associated with the enriched fetal cells. . 72. A method for classifying chromosomes of fetal nucleated erythroid cells, comprising: (a) Fetal nucleated erythroid cells are incubated in a medium containing erythropoietin. Incubate under conditions and for a sufficient period of time to induce metaphase in ; (b) fixing the DNA of the cell; (c) stain this fixed DNA so that the chromosomes can be observed; and (d) A method comprising examining the stained DNA and classifying the chromosome. .