CA2183657A1 - Culture and isolation of fetal cells from maternal peripheral blood - Google Patents

Abstract

Methods for culturing and isolating fetal cells, e.g., fetal erythroid cells, from a sample of peripheral blood obtained from a pregnant woman are disclosed. Cells from the sample of peripheral blood are cultured in a medium containing a cell growth factor, e.g., an erythroid growth factor, e.g., erythropoietin, to culture fetal cells. Prior to culture, non-nucleated cells can be removed from the sample of peripheral blood and/or fetal cells can be enriched. Metaphase spreads can be prepared from cultured fetal cells. A fetal cell marker or a nucleic acid sequence of interest can be detected in cultured fetal cells. The cultured fetal cells can be used to determine fetal sex and to detect disease-causing mutations and chromosomal abnormalities in a fetus.

Description

~ wo 95/26417 2 1 8 3 ~ 7 P~,l/o.,,~.lQ~906 CULTURE AND ISOLATION OF FETAL CELLS
FROM MATERNAL PERIPHERAL BLOOD
S E~ of " - l All 1~ cells are derived from pluripotent stem cells which, under a~JUlu~)lia~, conditions, ~ CIILi~lt~ along one of severa'i possible I I - Iineages.
One of tl-iese lineages, the erythroid lineage, leads to the production of red blood cells. The 10 .I~,v~ .ulll.,ll~ of mature, non-nucleated red blood cells, or elyLl..u~,yt.;" results from the c",..""ll". .,l of pluripotent stem cells to the erytilroid lineage, where they become erythroid progenitor cells, and furt_emli~ lLia~iull of erythroid progenitor cells into mature cells in response to signals received by the developing cells. An early erythroid progenitor cell is the blast forming umit-erythroid (hereafter BFU-E), which develops mto a more mature erythroid 15 progenitor cell, the colony forming unit-erytbroid (hereafter CFU-E). When stimulated by a paiticular growth factor, ~ LI.Iu,uo;~,i;ll, CFU-Es grow and d;r~.~ imto nucleated red blood cells ~ereafter nRBCs), a precurser of mature, non-nucleated red blood cells. In adults, erythroid progenitor cells are located ~JlcJulll;~al~ily in the bone marrow, where 1.~ ...AII.~,,. . ~:~ occurs in adults, and the erythroid progenitor cells do not circulate in peripheral blood. For a review of erythroid d~ U~ L see Beck, W.S. in TT - - I . 4th ~liQI~ l'Arr~l7rifip;p MA: MIT Press (Sth edition, 1991) and ~ u ~ , G., et al.
in The MolPrl~lRr pAei~ of Blood DiCPA~Pc philAAPlph;~ PA: W.B. Saumders (2nd edition, 1994), pp. 66-105.
The protein ~.yLi~upù;~iill is a growth factor necessary for erythroid dc~lu,ulll~,.lL m vivo which is produced primarily by the kidneys. E-YLIIIUIJO;., il~ was originally isolated amd purified from urine of anemic patients. Miyake, T., et al. J. Biol C~em. 252, 5558-5564 (1977). More recently, I,lya~lu~ù.~Lill has been llloL,~ uly cloned and ~ 1 Jacûbs, K, et al., Nature 313, 806-809 (1985); Lin, P., et al., Proc. Na~L
Acad Sci USA 82, 7580-7584 (1985). ElyLIIIu~ ,Lill has been used to support tne growth of erythroid progenitor cells in culh~re in vitro. For example, human adult erythroid progenitors from bone marrow and fetal erytbroid progenitor cells from umbilical cord blood or fetal liver explants have been grown in vifro by culturing cells in media containing c;lyLLluuu;~Lill.
Sutherland, H.J., et al., Blood 74, 1563-1570 (1989); Bodger, M.P., ~xp. HematoL 1~, 869-876 (1987); Peschle, A.R., et al., Bloûd 58, 565-572 (1981).
Durmg pregnancy, cells of fetal origin appear in the maternai peripheral circulation. For a review see Bianchi, D.W. and Klinger, K.W. in l'~PnPtir DisorAPrs alA'A thP
FPhlC PreVrnt;nn An~l Trprhnpnt 3rd PAiti~n Baltimore/London: The John Hopkins UniversityPress(1992). Thesecellsareapotentialsourceof;"r~"",A:;l",aboutthegender i-ind genetic makeup ûf the develûping fetus. Detectiûn, isûlatiûn and . ' on ûfilhese _ .. _ . . , _ _,, , ,,, _ _ . _ .. . . .. .. . . ..

~$3 WO95/26417 ~7 P~llu~r~906 ~, - 2 -fetal cells in a sample of maternal peripheral blood offers a non-invasive means for prenatal diagnosisandgenderi.1.,.1;~ -l;.." Thefeasgbilityofsuchanapproach,however,is hindered by a number of factors. Most. i~llUU~ L~llly, fetal cells are present in maternal blood in very limited numbers, which requires that they be enriched within a mixture of fetal and 5 maternal cells or that the fetal cells be separated in some way from maternal cells. One approach that has been used to achieve enrichment or separation of fetal cells utilizes antibodies specific for a patticular fetal cell type. For example, fetal-specific antibodies can be used in order to facilitate separation of fetal cells from maternal ~ by flowcytometry. Herzenberg, L.A., et al., Proc. NatL Acad. Sci. USA 76, 1453-1455 (1979);
Iverson, G.M., et al., Prenatal Diagnosis I, 61-73 (1981 ); Bianchi, D.W., et al., Prenatal Diagnosisll,523-528(1991). However,thisapproachrequiresveryspecializedreagents and can be costly and labor intensive. Another limitation in using fetal cells from maternal peripheral blood for diagnostic purposes is that while cells in metaphase are preferred for analysis not all fetal cells in the maternal peripheral circulation are dividing.
c of - Tnvention --The present invention provides non-invasive methods for culture and isolation of fetal cells amd for detecting nucleic acid sequences of interest in isolated fetal cells. The 20 invention is based, at :~ast in part, on the discovery that fetal cell, e.g., fetal progenitor cells, are present in the peripheral blood of a pregnant woman and can be selectively grown in vitro by culturing the cells in the presence of a cell growth factor.
In an .. ,.1 ,o.l '., ... ,1 the invention provides non-invasive methods for culture and isolation of fetal erythroid cells and for detecting nucleic acid sequences of interest in 25 isolated fetal erythroid cells. The invention is based, at least in part, on the discovery that fetal erythroid progenitor cells are present in the peripheral blood of a pregnant woman and can be selectively grown in vitro by culturing the cells in the presence of an erytbroid growth factor, e.g., ~yLluu~ui~L;Il. The methods ûfthe invention offer a non-invasive means by which to obtain fetal cells in numbers great enough to be of use .1~ ly while not 30 requiring specialized reagents such as ,.. ---~,1.. 1 antibodies or expensive equipment such as a flow cytometer. Culture of fetal erythroid progenitor cells in the presence of an erythroid growth factor, e.g., C~ UU~JO;~. ill, causes the cells to proliferate and .~ into fetal erythroid cells. Fetal er,vthroid cells can then be isolated and used in further analyses. The invention provides methods which allow for enrichment of fetal cells relative to maternal 35 cells in a peripheral blood sample, expansion of the total nuinber of fetal cells present compared to the total numbers present in the original sample, and isolation of mitotically active fetal cells. The ability to culture fetal erythroid progenitor cells from maternal peripheral blood is of great value since this approach provides a simple, non-invasive means of obtaining fetal cells for further analysis. The advantages of this approach are that it allows 21836~7 W0 9~i/26417 P~

for isolation of greater numbers of fetal cells for analysis than can easily or ~c . " ,. " ";. ,,lly be separated firom maternal blood by other methods and, since these cells are growing, also allows for isolation of fetal ce~ls in metaphase for use in diagnostic tests.
Accordingly, this invention pertains to the culture amd isolation of fetal 5 erythroid cells from a maternal peripheral blood sample as a means of obtaining fetal cells for analysis amd diagnosis. The method of the invention involves obtaining a sample of maternal peripheral blood, culturing cells in the sample in a culture medium containing am erythroid growth factor, e.g., clyLhlu~Ju;~,Lill~ which causes erythroid progenitor cells to proliferate and dirrt.cllLi.. ~, into erythroid cells, and isolating fetal erythroid cells. This method is based at 10 least upon: I ) the presence of fetal erythroid progenitor cells in the maternal peripheral circulation; 2) the scarcity of maternal erythroid progenitor cells in the maternal peripheral c;lcul~Liull, and 3) the higher sensitivity of fetal erythroid progenitors to ~,lya~u,uu;.,Li compared to the sensitivity of maternal erythroid progenitor cells to Cly LLlu~u;~Lill.
Prior to culturing, the peripheral blood sample can be treated in order to 15 remove certain cell types and/or to enrich for certain cell types so that fewer total cell numbers are utilized in the culturing step. Non-nucleated red blood cells can be removed from the sample by, for example, selective Iysis or density gradient ~Pntrifil~Atinn prior to culturing. Erythroid progenitor cells can be enriched in the sample by, for example, ru~ ;llg dual density gradient rPntrifil~Atil~nc wherein gradients of different densities are 20 utilized, and isolating a cell layer which contains erythroid progerlitor cells prior to culturing.
After culturing, erythroid cells can be isolated by picking one or more discretecolonies of cells from the culture media, for example by using a pipette to manipulate the cells. The cells of the colony can then be transferred to fresh culture medium, placed on a 111 ~1 ua~,U~C slide for further analysis, used as a source of DNA, or amalyzed further in amy 25 suitable marmer.
Arlother aspect of the invention relates to a method for identifying fetal erythroid cells after culturing cells of a maternal peripheral blood sample in an erythroid growth factor, e.g., .,ly Lluu,uu;~,Lill. This method involves detecting a fetal cell marker on erythroid cells as a means of identifying the cell as being of fetal origin. A further aspect of 30 the invention involves detection of a rlucleic acid sequence of interest in fetal nucleic acid of fetal erythroid cells after culturing. Detection of Y ~,LIullluaullldl DNA, a gene associated with a disease-causing mutation or ~h~u~u~u~ l Ahn(lrrnAlitiPe in DNA from cultured erythroid cells are all r~ithin the scope of the invention.
The invention also provides a method for isolating fetal cells in metaphase 35 from a maternal blood sample. In this method, after culture of cells in eyLh~ul~uk,.i l, cells are exposed to am agent which inhibits ,ulu~ lca~;ull of dividing cells through the cell cycle or an agent which syn~,l..u..i,~,~ gror~th of cells. Cells in metaphase can be detected i~lu~,u~icdlly and isolated. Metaphase cells can then be used for further analysis and diagnûstic te$s.

~5~
WO 9S/26~17 21~ F~l/o., The invention still further provides a method for ,u~cr~,lclllidlly isolating fetal cells from a current pregnancy in a wom~ an who has had multiple 1,l C~ S by culture of fetal ery~roid progenitor cells from a pe~ipheral blood sample obtained from the pregnant woman. This method is based upon the short life span, about 3 months, of erythroid progenitor cells such that any fetal erythroid cells that are isolated after culturing must be derived from the current fetus rather than any previous fetus.
BriPf 1~ of - Il ' _ Figure I is a schematic diagram illustrating the relative distribution of erythroid progenitor cells and nucleated red blood cells along a density gradient.
Figure 2 is a photograph of a CFU-E colony growing in methylcellulose medium containmg Cl ~/ IhlU~JUiC~ill.
Figure 3 is a photograph of an early BFU-E colony growing in methylcellulose medium containing Cly~UUpO;~,lill.
~Pt~ P.i Desc " of ~ ~ -The prPsent invention pertains to methods of culturing fetal cells from a sample of matemal peripheral blood. The methods include obtaining a sample of peripheral blood from a pregnant woman and culturing cells within the sample in a culture medium containing a cell growth factor. Fetal cells are isolated from the culture medium and a nucleic acid sequence of interest is detected in the fetal DNA.
In an ~, . ,l)o~l;, ., . ~1, the invention pertains to methods of culturing fetal erythroid cells from a sample of maternal peripheral blood. The methods involve culturing fetal erythroid progenitor cells present in the blood sample in vilro in a medium containing an erythroid cell grûvith factor, e.g., cl y ILIulJoiclill, which causes the fetal erythroid progenitor cells to proliferate and dirrt., ~ into fetal erythroid cells. Following culture, fetal erythroid cells can be isolated. In one rl~ the method of the invention comprises obtaining a sample of peripheral blood from a pregnant woman, culturing cells within the sample of peripheral blood in a culture medium containing an erythroid growth factor, e.g., cl~al~u~uh.lill, and isolating fetal erythroid cells from the culture medium.
The language "a sample of peripheral blood from a pregnant woman", also referred to herein as a maternal blood sample, is intended to include a blood sample drawn (e.g. with a needle) from a peripheral blood source (e.g. an arm vein) from a woman pregnant with a fetus. Before use in methods of the invention, the sample of peripheral blood can be treated v~ith an agent which inhibits blood ~-o~ til~n such as heparin. Preferably, the sample of peripheral blood contains d~ '/ 5-20 mls of blood. More preferably, the .

~18:~6~
W09S/26.117 P~

sample contains a,U~ll U~ a~,ly 1 O mls of blood. Al he sample may be obtained as early as 6 weeks of gestation or as late as mid-second trimester, but preferably is obtained between about 8 and about 16 weeks of gestation. Most preferably, the sample is obtained at alJ,u~ / 12 weeks of gestation. rt is known that at l 2 weeks of gestation there are about 50,000 nRBCs per milliliter circulating in fetal blood whereas by 20 weeks of gestation there are only about 1000 nRBCs per milliliter of fetal blood. See Holzgreve,W., et al. The Journal of R,f " o.lu~ Medicine 37, 410-418 (1992).
The language "culturing cells" in a 'iculture medium" is intended to refer generally to contacting cells with a culture medium apprûpriate for the survival of the cells and incubating the cells in the culture medium under conditions auulul ' ' for the survival of the cells for a period of time to allow proliferation and possibly di~~ ialiull of the cells.
The language "culture medium" is intended to include a liquid or semisolid solution or material which allows survival and growth of fetal cells, e.g., erythroid progenitor cells.
Suitable culture media generally contain a nutrient source, such as l,alboll.yl' (e.g.
sugars), general growth factors, such as those found in blood serum (e.g. fetal bovine serum), and ~ / amino acid(s), such as L-glutamine. A culture medium can also contain antibiotics, such as penicillin and ~ ullly~,;.l, a reducing agent (e.g. 2-rl.~ llA..nl), additional protein (e.g. bovine serum albumin), a buffering agent (e.g. sodium b;(.a~
and/or a pH indicator (e.g. phenol red). Conditions appropriate for survival of .,- --. "Al;A, cells in culture gener~A.lly are 37 C, 4 %-5 % CO2. The length of time of culturing can vary depending upon such factors as the number of cells desired (i.e. the extent of proliferation needed to produce the number of cells desired) and/or the degree of cellulamlirrclcll~idliul.
desired, but generally is at least about 48 hours. Preferably cells are cultured about 4 days.
More preferably, cells are cultured about 6 days.
In a preferred embodiment, the culture medium further contams a semisolid matrix material. The language "semisolid matrix material" is intended to mclude a substance which, when added to a liquid culture medium, c n convert the liquid culture medium to a gelatinous, semisolid state. A semisolid medium is one in which cells can still grow (i.e.
which is not so solid as to inhibit cell growth) but which is firm enough so that isolated cells within the medium carmot migrate from their site of growth. A semisolid matrix material can also allow growing cells to attach to the matrix material, thereby preventing their migration within the medium. Growth of a single cell in a semisolid medium results in the production of a discrete colony of cells derived, by division, from the original single cell. A cell colony cam be .l;~ l from other cell colonies by light ~ .u~ul~ic pyAnninAt~ n of the semisolid culture medium. Suitable semisolid matrix materials include methylcellulose, agargel and a plasma clot. See for example Stephenson, J.R., et al. Proc. Natl. Acad. Sci.
USA 68, 1542-1546 (1971), Iscove, N.N., et al. J. Cell PhysioL 83, 309-320 (1974); and Pike.
B.L. and Robinson, W.A. J: Cell Physiol. 76, 77 (1970).

'~1836~7 wo 95/26 A preferred culture medium for isolation of fetal erythroid cells from a maternal blood sample is Iscove's Modified Dulbecco's Medium (IMDM; I,u~ ,;ally available; Sigma, Whitaker) with added 1.1 % methylcellulose, 30 % fetal calf serum, I %
BSA, I % penecillin-~Ll~Lullly~ I % L-glutamine, 10-4 M 2~ I.IV~ Preferred culture conditions are incubation of cells at 37 C, 4 %-5 % C02 for a~,uluAilll_ cly 96 hours.
Cells are typically cultured in an appropriate container fûr the culture medium, such as a culture dish or culture flask.
The culture medium in which the cells of the matemal bloûd sample are cultured also contains a cell growth factor. The language "cell growth factor" is intended to include those factors which stimulate proliferation omlirrtlcllliaLion of fetal cells. Examples of cell growth factors include: factors which stimulate ~irrt. cllliaLivll of Iymphoid progenitor cells, e.g., interleukin-2 (IL-2), interleukin-4 (IL-4),and interleukin-7, and factors which stimulate l~ ir ~i~ progenitor cells, e.g., llclllaLùlJU;~;c cell growth factors (including erythroid growth factors), e.g., interleukin -3 (IL-3), ~;lallulouy;u-llla~,lvullàg~ colony-15 stimulating factor (GM-CSF), monocyte-la.,lu~lla~ colony-stimulating factor (M-CSF), ulocytc colony-stim~lAtinL factor (G-CSF), ~IyLlLvl~vi~.ill (EPO), and human stem cell factor (rhSCF).
The culture medium in which the cells of the maternal blood sample are cultured also can contain an erythroid growth factor. The language "ery~roid growth factor"
is intended to include .hose factors which support grow~th and proliferation of human erythroid progenitor cells. Examples include: ~;l y LLIul~o;~,~;ll (EPO), and burst promoting activity (BPA). These erythroid growth factors can be âulllilli~Ltlcd alone with each other.
The language 'lcly LLlu~ùic~ l is intended to include a preparation of the protein =Iy Llllu~uicLi~l which supports growth and proliferation of human erythroid progenitor cells. ElyLIIIv~Jv;~lill may be prepared by pllrifiAAtinn of the protein from a natural source or may be prepared by expression of the ~lyLhlul u;.,Li~l gene by lC~ DNA techmolûgy.
A crude preparation of ~lylluuuu;~,.ill isolated from human urine can be used and is CUllllll~ ,;ally available from Sigma (catalogue # E5011, 50 U/mg). Other coll~ ,l.,;ally available ~lc,ua aLiulls include Sigma # E9761 (human l~ .. l.;., --.~ 100,000 U/mg), # E9757 (from human urine; 80,000 U/mg), # E2639 (from human urine; 500 U/mg) and # E2514 (fromhumanurine;lOOU/mg). Preferably,thec.yLhlul,v.~Sillisofhumanorigin,although~lyLl~u~ùicLill from another species which is capable of supporting the ~lulif~,laLivil and dirrclcllLiaLiullofhumanerythroidprogenitorcellscanalsobeused. El~a~u~uù;~il-hasbeen 35 shown to fimction across species. For example, human and mouse cells have both been stimulated by sheep or human ~ LLu~JvicLill (see Iscove, N.N. et al. ~ Cell PhysioL 83, 309-320 (1974)). A cnn~ArAntrAti~m of cl~Lhlul,ù;~,Lill is used which is sufficient to achieve the desired result of proliferation andlomlirr~lclliiaLion of erythroid progenitor cells. A preferred n ~ range for ~,lyLIl~u~oicLill in the culture medium is about 0.25 U/ml to I U/ml.

21~3~$7 wo 95/26417 1 ~ ~/1,.. . ~906 Erythroid progenitor cells exposed to ~IyLLlu~ùi~ l in culture will be stimulated to proliferate and dirrtlc~llk.t.. ElyalluL~oi~.ill preferentially stimulates the proliferation and dirr~lcill~ia~iol~ of fetal erythroid progenitor cells in cells from a matemal blood sample for a number of reasons. First, it has been . ~ r~l that fetal erythroid 5 progenitor cells from human fetal liver are more sensitive to the ' y effects of elya--uuu;~lill than are erythroid progenitor cells from human adults (see Peschle, C., et al.
Blood58,565-572(1981)). Second,the-.. ,.. l.Al;.. ,of~,lyauu,uu;cLi.lused(e.g.,0.25U/ml to I U/ml) is lower than the optimal rr~nr~ntrAti~n for stimulation of adult erythroid AUlu~ liLula~ which is 2-4 U/ml (see Linch et al. Blood 59, 976-979 (1982)). Finally, 10 erythroid progenitor cells in human adults are found almost exclusively in the bone marrow, the site of adult l~ u,uu;..~;a, and therefore few, if any, matemal erythroid progenitor cells should be present in matemal peripheral blood.
Two types of fetal erythroid progenitor cells cam respond to the ~ lla~uly effects of ~Iy ~IIIu~uu;~ the mature, or late, BFU-E (hereafter M-BFU-E) amd the CFU-E.
15 Colonies derived from M-BFU-Es and CFU-Es can be .1;~1;,.~..;~'.. .~1 from each other and from other cell types by their IllUlUllOlo~,y, which can be detemlined ~ ,lua~u~ ,ally. BFU-E-derived colonies usually contain over 100 cells, and can contain several thousand cells, and are arranged in a diffuse "burst" or scattered pattern of cells. CFU-E-derived colonies contain about 20 to 150 cells arranged in a smaller, more compact colony. CFU-E-derived colonies 20 mature during the firs week of culture, whereas BFU-E-derived colonies may take longer to develop. Additionally, CFU-E-derived colonies may umdergo further maturation amdlirrtl~lllialion to form erythroid cells such as nucleated c~ llulJIa~la~ which display visible hrnnf Al~ hini7~1til~n which can be identified Illi~,lva~u,uil,~llly.
After culturing cells in a culture medium containing an erythroid growth 25 factor, e.g., ~IyallupOi~ erythroid cells are isolated from the culture medium. The language "isolated" is intended to refer to separation of one or more cells or colonies of cells away from other cells and/or away from the culture medium. When cells are grown in a semisolid culture medium at low enough cell density such that discrete, ~ ,":~1,,.1 ~Ir cell colorlies fomm, these cell colonies are already isolated from other cells within the semisolid 30 medium. Cells or cell colonies can be further isolated by removing the cells from the medium. For example, a discrete cell colony can be identified by examining the cell culture - umder a light ~ luacuuc and can be removed from the culture medium by picking out the cells from the culture medium. Cells can be effectively picked out of the culture medium usirlg an instrument, such as a drawn out Pasteur pipette or other suitable pipette, to 35 manipulate the cells. Once cells are separated from the culture medium, for example by picking them with a pipette, they can be transferred to a holding container (e.g. a tube), to a solid support (e.g. a Illi~,lua~,uluC slide), to fresh culture media or to any other location suitable for further growth, mAnirlllAti~n or analysis of the cells. In a preferred ~ hc~ , a single cûlûny ûf cells is picked frûm the culture medium and trarlsferred tû a IlI;elUS~,Up~ slide.

~365~
W0 95/26417 ` ~ u_,~

The language "fetal cells" lS intended to include those fetal cells present in the maternal peripheral blood circulation. The fetal cells include fetal progenitor cells, which after incubation with a cell growth factor, can dirf..l into mature cells, e.g.,granulocytes, monocytes or leukocytes. For example, fetal Iymphoid progenitor cells can be 5 cultured with IL-2, IL-7, or IL~ in order to dirf~l~ into mature T or B-ly~ /llu~y Lu~.
Other fetal progenitor cells carl be cultured with factors such as G-CSF and /or GM-CSF in order to differentiate into mature ~-a.-ulo-y L~.
The language ''erythroid cells" is intended to include those cells which are derived from erythroid progenitor cells upon culture with an erythroid growth factor, e.g., 10 clyLlllu,uuiciLill. Cultureoferythroidprogenitorcellswithanerythroidgrowthfactor,e.g., ~,ly Llu u,uù;.~ , can cause progenitor cells to dirrtll into a more mature stage of erythroid lineage development. Thus, the cells present after culture may be a mixture of cell types at different stages of the erythroid d. ~ ~IU~ .IL~I pathway. Cells at different stages of the erythroid d. ~ .,lulllll~ llLdl pathway can be .. , ~ nln~ l ly different from each other.
15 Cells or colonies of cells having a IllUI,UIlUlU~y of erythroid progenitor cells, such as BFU-E
and CFU-E colonies, or more developed erythroid cells, mcluding cells with visible hemoglobinization, such as nucleated .. y Llllul;L~al~ are detectable after culture of erythroid progenitor cells in an erythroid growth factor, e.g., ~Iy LluulJu;~ and can be isolated from the culture medium.
Before culturing cells from a sample of peripheral blood obtained from a pregnant woman, the sample can be ~ to emich for one or more types of cells within the sample. A maternal blood sample contains both nucleated cells, such as Iymphocytes, monocytes, granulocytes and, potentially, fetal cells, e.g., fetal erythroid progenitor cells, and non-nucleated mature red blood cells. Non-nucleated mature red blood cells constitute the vast majority of cells within a sample of peripheral blood and it is often desirable to remove these cells from the sample before culturing. A proportion of nucleated cells present in the sample of peripheral blood can be increased relative to a proportion of nucleated cells present in the sample of peripheral blood prior to enrichment forming a sample enriched in nucleated cells. The sample enriched in nucleated cells is then used for culture. The language "a proportion of nucleated cells" is intended to refer to the percentage of nucleated cells, relative to the total number of cells, i.e. nucleated and non-nucleated cells, in the sample. The language "increased relative to a proportion present ... prior to ~ui~lull~lL'' is intended to mean that the percentage of nucleated cells present in the sample is greater after an enrichment procedure as compared to the percentage of nucleated cells 35 present in the sample before the enrichment procedure. The percentage of nucleated cells in a sample containing both nucleated and non-nucleated cells, such as peripheral blood, can be increased relative to the starting sample by removing non-nucleated cells from the sample and/or by separating nucleated cells away from non-nucleated cells. The language "a sample enriched in nucleated cells" is intended to include a sample derived from a maternal blood ~3~
~ WO95/26417 P~ 906 _ g _ sample in which the percentage of nucleatéd ceils present in the sample has been increased relative to the percentage present in the maternal blood sample prior to an emichment procedure.
In one , ,I)c.ll;,,,. .,1 a sample enriched in nucleated cells is formed by 5 separating non-nucleated cells from nucleated cells in a sample of peripheral blood obtained from a pregnant woman. The language "~c,uala~ ;l' is intended to mean that non-nucleated cells amd nucleated cells are isolated away firom each other. Non-nucleated cells can be separated from nucleated cells by density gradient r . . ,1 . i r"~ ;. ., . The language "density gradient r . .. i r..~,.1;....~ iS intended to refer to a technique in which a mixture of cells, e.g.
10 cells of a blood sample, are cPntrifi~r d in a container, such as a centrifuge tube, through a material of a particular density to form layers containing different cell types. Under ,. . .I .; r. ~ conditions, cells travel through the density gradient material as a function of their cell size and density. Therefore, density gradient cr ntrif~l~Ptir~n cam be used to separate cells based upon differences in cell size and density. Non-nucleated red blood cells have a 15 different cell size and density than nucleated cells and thus can be separated from nucleated cells by density gradient çrntrjfi~ tion Several materials suitable for separating non-nucleatedandnucleatedcellsbydensitygradient~ .irl.~nl;r~.~are~,ulll",~"~,;allyavailable.
These include Ficoll (Pharmacia, Uppsala, Sweden), ~ tr~F -~lr (Sigma Diagnostics, St.
Louis, MO), Nycodenz (Nycomed Pharma, Oslo, Norway; available in the US from Gibco 20 BRL) and Polyl.lul~ lc,u (Nycomed Pharma, Oslo, Norway; available in the US from Gibco BRL). Appropriate ~ i r~ n~ speeds and times have been determined by the llllci:~. Generally,~ ulul c....l,irllr,n.il,.,speedsandtimesarein&erangeof 400-500 g for 20-35 minutes at room t~lll,u~,lrlLulc.
After r ...1. i r..y,,.l i.... the maternal blood sample is typically separated into at least three layers: a ~ - layer containing serum and platelets; a .. ,.. ",. ~ cell layer; and an 7 b~ l pellet which contains non-nucleated red blood cells. One or more cell layers containing nucleated cells amd devoid of most non-nucleated red blood cells (e.g.
the ".. ,.. "1. n cell layer) is removed firom the gradient to form a sample enriched in nucleated cells. The sample enriched in nucleated cells is then used for culture.
In another .. , .I~.,,li " ,. Il, non-nucleated cells cam be separated from nucleated cells by selective Iysis of the non-nucleated cells. The language "selective Iysis" is intended to refer to ~ul~,f~lclll;al destruction of one cell type, e.g. non-nucleated cells, compared to other cell types, e.g. nucleated cells. Cells in the maternal blood sample can be incubated in one of a number of hypotonic buffers knov~n to be effective, and UUII v~ ;Ullàlly used, for Iysing non-nucleated red blood cells, such as 0.17M NH4CI, 0.01M Tris, pH 7.3 (see e.g.
Tiilikainen et al. Tt , 7. It;on 17, 355 (1974) and Macera, et al. Leukemia Research 13, 729-734 (1989)). Buffers suitable for this purpose are also available Culll~ ,;ally (e.g.
"Lyse and Fix", GenTrak). After incubation in an appropriate buffer suitable for selective Iysis of non-nucleated cells, the resultant cell sarnple is a sarnple erlriched in nucleated cells.
.

WO 95/26 117 2 ~ S 3 6 ~ 7 P 11 ., ~. ~906 In a preferred embodiment, the maternal blood sample is ", ~ l before culturing cells in order to enrich for fetal cells, e.g~fetal erythroid progenitor cells, within the sample. A proportion of fetal cells, e.g., fetal erythroid progenitor cells, present in the sample of peripheral blood can be increased relative to a proportion of fetal cells, e.g., fetal erythroid 5 progenitor cells, present in the sample of peripheral blood prior to enrichment forming a sample enriched in fetal cells, e.g., fetal erythroid progenitor cells. The sample enriched in fetal cel!s is then used for culture. The language "a proportion of fetal cells, e.g., fetal erythroid progenitor cells" is intended to refer to the percentage of fetal cells, relative to the total number of cells, i.e. nucleated and non-nucleated cells, in the sample. The language 10 "increased relative to a proportion present ... prior to ~,~u;~,lull~lll" is intended to mean that the percentage of fetal cells present in the sample is greater after an enrichment procedure as compared to the percentage of fetal cells present in the sample before the enrichment procedure. The percentage of a particular type of fetal cells, e.g., fetal erythroid progenitor cells, in a sample containing other cell types, as in peripheral blood, can be increased relative 15 to the starting sample by removing other cell types, e.g. non-nucleated cells and other types of nucleated cells, from the sample and or by separating the particular type of cells, e.g., erythroid progenitor cells, away from non-nucleated cells and other types of nucleated cells.
The language "a sample enriched in fetal cells, e.g., erythroid progenitor cells" is intended to include a sample derived from a maternal blood sample in which the percentage of fetal cells, 20 e.g., fetal erythroid pr~genitor cells present in the sample has been increased relative to the percentage present in the maternal blood sample prior to an enrichment procedure.
A particular type of fetal cells, e.g., erythroid progenitor cells, can be separated from non-nucleated cells, e.g. mature red blood cells, and other types of nucleated cells, e.g. lymphocytes and " ,l."... ,. ,. l. ~. cells, by density gradient ....l,; r. ~3 ;, ' ;.." based upon 25 differences in cell size and density of selected cell type and other cell types. In one rllll.O.l;.ll~ "dual density gradient, . ,.I.ir.,~".t;.".~ is performed. The language "dual density gradient ~ .; r,.~ is intended to include techniques in which a mixture of cells, e.g.
cells of a blood sample, are centrifuged in a container, such as a centrifuge tube, through two or more materials of different densities to form layers containing different cell types or, 30 dlltlll~ "ly, a mixture of cells is centrifuged through a material of one density, one or more cell layers is removed and the removed cells are l~ ; r. l ~ ~ through a second material, preferably of a different density than the first, to form layers containing different cell types.
Dual density gradient centrifugation can be performed with the gradient materials described for single density gradient ~-rntrifil~tif~n (e.g. Ficoll, l~iCt~p~ llP Nycodenz, 35 Poly.~
A sample enriched in fetal cells, e.g., erythroid progenitor cells, can be produced, for example, by density gradient ~.. .l .; r. ~ using Polyll~ (Nycomed;
GibcoBF~Lcatalogue#100-1971). Undiluted~nti~o~gn~ trdwholebloodofamaternal blood sample can be cPntrifi~e(l through the gradient material to form the following layers 2i 3 ~i7 WO 95/26417 8 6 . ~ L A~906 (from the top to the bottom of the tube): a SllrPT:lf~nf layer containing serum and platelets; a ". ~ . ^ cell layer (the "buffy coat") comaining IY~ U~ and I~IUI10~ , a polymull,llul'u~ al cell layer containing poly ~ 1.... " .. 1. ^ leukocytes, nucleated red blood cells and erythroid progenitor cells; and a non-nucleated red blood cell pellet. The 5 poly 1 l l.., 1~1..., ... 1. ~, cell layer can be removed to form a sample enriched irl fetal erythroid progenitor cells.
Additionally, dual density gradient I~;r~ using ~TictoF~q~lP can be used to produce a sample enriched in erythroid progenitor cells. A maternal blood sample, diluted 1:1 in phosphate buffered saline, is centrifuged through â dual density gradient composed of Histopaque 1077 (density = 1.077 gm/ml) layered on top of ~Jicfnp~qllP 1119 (density = 1.119 gm/ml) to form the following layers (from the top to the bottom of the tube):
a ~ 1 layer; a ".. ,.. 1. AI cell layer (the "buffy coat"); a nucleated red blood cell layer, containing erythroid progenitor cells, at the interface between the 1.077 and 1.119 gradients; and a non-nucleated red blood cell pellet. The nucleated red blood cell layer can 15 be removed to form a sample enriched in erythroid progenitor cells. Additionally, Histopaque 1077 and 1119 can be used sequentially, as described in Example 1, to form a sample enriched in fetal cells, e.g., fetal erythroid progenitor cells.
When single or dual density gradient ~ .. ,I .; r. ~,,,.~ ,.... is performed, the cell layers may not always be discrete and j~Pnfifi ~ P Therefore, several different cell layers 20 may be removed and .,ultured separately in culture media containing a cell growth factor, e.g., an erythrûid growth factor, e.g., ~ llU~)O;Cilill, both to ensure that fetal cells, e.g.,erythroid progenitor cells have been properly selected and for ~ .... purposes (i.e., other cell layers can function as controls).
A sample enriched in fetal cells, e.g., fetal erythroid progenitor cells, can also 25 be produced using other techniques to remove non-nucleated cells and/or other cell types from a maternal blood sample or to enrich fûr erythroid progenitor cells. For example, antibodies specific for a cell surface marker present on a pa~ticular cell type can be used to either remove or enrich for that cell type within a maternal blood sample. An antibody specific for a marker present on the selected fetal cell type, e.g., fetal erythroid progenitor 30 cell, can be used to select for erythroid progenitor cells, for example by flow cytometry, panning or , .. . " ~ separation (positive selection of cells). Alternatively, an antibody specific for a marker present on a cell type other than the selected fetal cell type can be used to remove that cell type from a matemal blood sample, for example by flow cytometry, panning or; 11111111111~ separation (negative selection of cells) or by 35 ~- .. . ~1~l ,.- .I-mediated Iysis of cells which have bound the antibody.Although culture of a maternal blood sample, a sample enriched in nucleated cells or a sample enriched in fetal cells, e.g., erythroid progenitor cells, in a culture medium containing a cell growth factor, e.g., an erythroid growth factor, e.g., ~1~ U~lUIJU;.~ill, can f~ llL;~lly prûduce cûlûnies ûf fetal cells (fûr example, due tû the scarcit~v ûf maternal ~ ~ 8 3 ~ ~ 7 - ~ !
wo 9s/26~17 Pcr/usss/03so6 erythroid progenitor cells in peripheral blood and the l~igher sensitivity of fetal erythroid progenitor cells for c~yLhlui)uiclill), erythroid cells or cel~ colonies carmot be identified ulpllOlo~;cally as being fetal-derived. Therefore, to identify fetal cells as being fetal-derived, the method of the invention can further comprise detecting a fetal cell marker on or 5 in the fetal cells. The larlguage "fetal cell marker" is intended to include a cell-surface, cytoplasmic or nuclear structure or molecule present on or in a fetal cell which can be used to distinguish a fetal cell from a maternal cell.
The lamguage "detecting a fetal cell marker" is intended to include detection ofthe fetal cell marker itself or detection of another molecule which binds to the fetal cell 10 marker. A fetal cell marker can be detected on or in a fetal cell by, for example, contacting the cell, or portion thereof, with an antibody reactive against the fetal cell marker. The antibody can be labelled with a detectable substance or can be further reacted with another molecule, e.g. a second antibody reactive against the first antibody, which is labelled with detectable substance. Suitable detectable substances include enzymes, prosthetic groups, 15 fluorescent materials, 1..,~ l materials and radioactive materials. Examples of suitable enzymes include hnrrrrA~lich peroxidase, biotin, alkaline ~ or acetylch.~lin.-~r^rAc~; examples of suitable cl~y~ 'plualllcLic group complexes include streptavidin and biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein iauLllio~"~ , rhodamine, dichluluLIia~il.ykul~ fluorescein, dansyl 20 chlorideorphycoeryt~lrin;anexampleofal..,.,;,..~ materialincludesluminol;and examples of suitable radioactive material include 1 251, 1 3 11, 35S or 3H. Starldard ;1,llll."...1l;~11...1.. .~;~I~y techniques can be used to detect fetal cell marker expression using an antibody against the fetal cell marker.
Proteins which are expressed preferentially or exclusively on or in fetal cells 25 as compared to maternal cells can be used as fetal cell markers and can be detected using an antibody against the protein. Examples of suitable proteins which can be used as fetal cell markers include fetal h~m-~gl nhin and a fetal histone protein H1. Fetal ~ .. is a ~;yLoplaallfl~, protein, whereas fetal H I is a nuclear protein. Additionally, an antigenic ....; ., .1 which is ~ f~lcllLially or exclusively expressed on or in fetal erythroid cells can 30 be used as a fetal cell marker. An example of a suitable antigenic d which can be used as a fetal cell marker is the i amtigenic dr~ 11 The i antigenic,1. 1.. i.. .: is an Illlllla~ 1 membrane ~I.I,ollydl..,~, present on fetal erythroid cells. Adult erythroid cells generally do not express i but rather express the I antigenic .1. r .IllillA.Il, a branched c~ l,y~
Other examples of fetal cell markers include nucleic acid sequences which are urlique to fetal cells compared to maternal cells. Examples of unique nucleic acid sequences include Y l,hlullluaullldl DNA, in the case of fetal cells from a male fetus, and DNA
a paternal pOlylllulpllialll or polymorphic allele, such as an HLA arltigen allele.
... ... . .... ..... ..... .. . _ ... . . . _ . . _ .. .. .. . . _ _ .. ..... . .

I--wo gS/26417 218 3 ~ 7 r~l,u~J~o~so6 _ 13 --Following culture arAd isolation of fetal cells, the method of the invention canfurther comprise detecting a nucleic acid sequence of interest in fetal nucleic acid of isolated fetal cells, e.g., fetal erythroid cells. The language "a nucleic acid sequence of interest" is intended to include DNA, e.g. ~,luulllusulllal DNA or a particular gene fragment within 5 ~,h~ull~u:~u~lal DNA or amplified from ulL.ul..osu..ldl DNA, as well as RNA, e.g. mRNA or nuclear RNA. The language "fetal nucleic acid" is intended to include fetal DNA and RNA, for exa~Anple fetal ~ ul~lù~u~l~al DNA and fetal mRNA. Detection of a nucleic acid of interest in nucleic acid of fetal cells can be used to identify a fetal cell as being fetal-derived (i.e., the fetal nucleic acid can be a fetal cell marker) and also for analyses such as ~ ....;"~ of 10 fetal gender, detection of a genetic disease in the fetus and detection of a l ., . ." ,~ s~ .. ., -1 dlJllullllalily in the fetus. Fetal nucleic acid in fetal cells, cultured and isolated as described herein, can be analyæd for the occurrence of a nucleic acid of interest for diagnostic or other purposes.
Cultured fetal cells can be treated such that fetal nucleic acid present in the 15 cells is made available for detection. A nucleic acid sequence of interest can be detected in the available fetal nucleic acid by, for example, cl~y.lla~i~.dlly amplifying the nucleic acid sequence of interest and/or by hybridizing a labelled nucleic acid probe to the nucleic acid sequence of interest. The labelled probe used to detect a nucleic acid sequence of interest can be, for example, a labelled DNA probe, a labelled RNA probe or labelled .~ J ~.lides.
20 Fetal DNA can be ma Ae available by boiling the fetal cells to Iyse them, thereby releasing fetal DNA, for instance prior to hmrlifi~tir~n of fetal DNA. Fetal cells can be attached to a solid support, e.g. a IlI;~,lUal,U~)e slide, in such a way that fetal nucleic acid is made available, for example by fixing fetal erythroid cells or erythroid cell nuclei to a ,.,~ u~.. slide prior to in situ hyhriAii7~til~n Fetal nucleic acid in fetal cells, or portion thereof (e.g. nuclei), can 25 be detected directly, for example by in situ hybridization of a labelled nucleic acid probe 1'~ l ' ' '1 'l ' . I .... I h . ,~' to a nucleic acid sequence of interest or tl Ae fetal nucleic acid can be amplified prior to detection using a known,, .I .I; ri, ~ ;.... technique such as the pOI~ a~
chain reaction (PCR). Primers for PCR ,.. . ,l.l, r~ A~ are chosen which specifically amplify a DNA sequence of interest in the fetal DNA
A nucleic acid sequence which is to be detected in fetal nucleic acid of fetal erythroid cells is referred to herein as a "nucleic acid sequence of interest". In one embodiment the nucleic acid of sequence of interest is a Y ~,I Aul Aù~u~A~dl DNA sequence~
The language "a Y ul~lu~llosulllal DNA sequence" is intended to include nucleotide sequences unique to the Y CILAUII1U~UIII~ including repetitive sequences. Detection of a Y ~,I Au~l~usul~lal DNA sequence cam be used both for detection of a fetal cell marker and for fetal gender ?ntifirA~tirn In another rl~ o~ the nucleic acid sequence of interest is a sequence of a gene associated with a disease-causing mutation. The language "a sequence of a gene assûciâted with a disease-causing mutation" is intended to include nucleotide sequences ~183~
WO 95/26~17 ~ PCT/US95/03906 0 ; I Iy, all or part of a gene which may contairl a mutation which causes or is associated with a disease, or is linked to a gene which may contain a mutation which causes or is associated with a disease. That is, th~e detected gene sequence or a gene sequence linked to the detected gene sequence may contain a mutation which, if the mutation is present, 5 causes or is associated with a disease. Detection of such a sequence can be used to determine if a fetus has the disease caused by or associated with the mutation.
In yet another ~ ., . ,I.o,l;, ~ ~ ,1 the nucleic acid sequence of interest detects a CluulllOaullldl ~IbllullllaliLy. The language "detects a ~,luulllO~ àl abnormality" is intended to include nucleotide sequences which can detect changes in cluullluaulll~, number, 10 ~,luullloauuldl deletions, ~,luulllOaulllal l - ,,."~,. .". ~ and/or .,luul.lu~vlllal alterations. A
nucleic acid sequence of interest which can detect a ,IUUlllU~Ulllal ~ IIUIIIIaI;IY can be, for example, a nucleic acid sequence specific for a palticular ~,luullluaul.~e, such as a repetitive sequence from a particular uluullloaullle. Preferred cluullloaulll~,J to be examined for detection of a ~luullluaullldl ~llulllldli~y include ~ IUUIIIU~UIII~.J 13, 18, 21, X and Y.
Cluulllo~ullldl trisomies are most prevalent for these particular IUUIIIUSUIII~J~ Preparation of nucleic acid probes suitable for detecting cluullloaul,ldl 1II~ can be made by standard techniques. For example see Klinger, K., et al. Am. J. Hum. Genet. 51, 55-65 (1992).
In one rmhodimrnt a nucleic acid of interest is detected in fetal nucleic acid of fetal cells by in sit~ hybridization. In situ llyblidi~ aiiull can be used both to detect ~,luulllo,uuldl DNA and to detect eyluulaalllic mRNA. See for example Lichter, P., et al.
Hum. Genet. 80, 224-234 (1988) and U.S. Patent No. 4,888,278 to Singer et al. If in situ hybridization is to be carried out, fetal cells are separated onto a solid support, such as a llliulua~,uu~ slide, such that fetal nucleic acid is available for detection. In situ ll,yblidi~liu can be used, for example, to detect Y ~,luulllosulll~-specific sequences in fetal DNA in order to determine the gender of a fetus as described in greater detail in Example 3. In sttu llyblilli~Lioll can also be used to assess ,luulllOaulllal ~hnnrmDlitirc in a fetus, including ~,luulllOaulllal ~ , such as a trisomy, o m,lUUlllO~Ulllal 1' Al ~ or deletions,as described in greater detail in Example 5.
In another .llbo.lilll~ , fetal DNA is ~I., yllldti~,ally amplifed prior to detection of a nucleic acid sequence of interest. Fetal DNA can be amplified by PCR as described in detail in Example 4. If ~,, ,l,l; 1;, .A I ;~1l l is to be carried out, fetal erythroid cells can be Iysed by boiling and fetal DNA can then be amplified for an appropriate number of cycles of ~Ir"A I l " ,. l ;. " . and annealing (e g, a,uplu~dlll~Lt;ly 24-60). Control samples include a tube without added DNA to monitor for false positive A,. ~ With proper m~ lifirafinn of PCR conditions, more than one separate fetal gene can be amplified ~ u ~ly, Thistechnique, known as "lllul~ipl~" DmrlifiADti~An has been used with six sets of primers in the diagnosis of Duchenne's Muscular Dystrophy ((~h~ nhPrlDin J.S., et al., Prenat. Di~gnosis, 9, 349-355 (1989)). When ADmrlifirAtion is carried out, the resulting ~l~l;ii A';l 1l~ product may

2~831~7 W09!i126417 r~ u~ 906 be a mixture which contains amplified fe~al DNA of the sequence of interest (i.e., the DNA
whose presence is to be detected and/or quantitated) and other DNA sequenees. The amplified fetal DNA sequence of interest and other DNA sequences can be separated, usmg known techniques, for example by gel el~LIUI~I.U.~ . Subsequent analysis of amplified DNA can be carried out using known techniques, such as: digestion with a restriction r, ultraviolet light v;~ of ethidium bromide stained agarose gels, DNA
seq--rn~in~ or ~ bl~ iull with a labelled DNA probe? for example, specific nli~om~ rotiAf- probes (Saiki, R.K., et al, Am. .,~ Hum. Ge~ret., 43 (Suppl):A35 (1988)).
Amrlifi~ Afit~n of and hybridization to allele-specific sequences can determine whether lO polymorphic differences exist between the amplified "maternal" and "fetal" samples and can be used to identify a female fetus based upon detection of paternal poly...v.,ul~ l, in the fetal DNA. DNA sequences from the father can be identified in the autosomal UIIIUIII~SUIII~ of the fetus. C.~ ly, the method of the present invention can be used to separate and identify female fetal cells, as well as male fetal cells, from maternal blood. Thus, the method 15 can be used for all nucleic acid-based diagnostic procedures currently being achieved with other methods, such as Smnni~CPntrcic Following Amrlifi~ At~ the ,.~ " mixture can be separated on the basis of the size of the nucleic acid fragments and the resulting siæ-separated fetal nucleic acid can be contacted with an ~L~U~UIU,~ selected nucleic acid probe or probes (nucleic acid 20 sufficiently er~mrlpmf ~f~ry to the nucleic acid sequence of interest that it hybridizes to the nucleic acid sequence of interest in fetal nucleic acid under the conditions used). Generally, the nucleic acid probes are labelled (e.g., with a radioactive material, a nuulu~llul~ or other detectable material). After the size-separated fetal nucleic acid and the selected nucleic acid probes have been maintained for sufficient time under appropriate conditions for25 hybridization of .... ~ nucleic acid sequences to occur, resulting in production of fetal nucleic acid/nucleic acid probe complexes, detection of the complexes is carried out using known methods. For example, if the probe is labelled, a fetal nucleic acidllabelled nucleic acid probe complex can be detected and/or quantitated (e.g., by ~
detection of the fluorescent label). The quantity of labelled complex (and, thus, of fetal 30 nucleic acid) can be determined by ~ ." with a standard curve (i.e., a ~ulcl~relationship between the quantity of label detected and a given amount of nucleic acid present).
Fetal DNA/labelled probe complexes are ~ f ~ y detected, using a - known technique, such as ~llt~rAAio~rArhy~ Simple presence or absence of hybridization of 35 thenucleicacidprobe.""-l,l ..,- ,l, ytoanucleicacidofinterestandthefetalDNAcanbe ~PtPrminPd or the quantity of fetal DNA containing the nucleic acid sequence of interest can be APtrrminPA The result is a qualitative or U,UallLiLdLiV~ assessment of fetal DNA obtained from a maternal blood sample. For many genes which may carry a disease-causing mutation, prûbes are available whieh deteet a restrietiûn-site pOIylllu~ .ll whieh is indieative of the ~1~3f35~
wo9s/26~17 p~ l"~,c,, -_ 16 --presence of a disease-causing mutation within the gene. Detection of such a restriction site polylllu~ in fetal DNA using a nucleic acid probe specific for a gene associated with a disease of interest is indicative that the fetal DNA carries a mutation in the gene and therefore that the fetus has the disease.
The presence of fetal nucleic acid associated with diseases or conditions cam be detected and/or quantitated by the present method. In each case, an appropriate probe is used to detect the nucleic acid sequence of interest. For example, sequences from probes Stl4 (Oberle, I., et al., New Engl. J. Med., 312, 682-686 (1985)), 49a (Guerin, P., et al., NucleicAcidsRes., 16,7759(1988)~,KM-19(Gasparini,P.,etal.,Prenat. Diagnosis,9,3i9-355 (1989)), or the deletion-prone exons for the Duchenne muscular dystrophy (DMD) gene (Chamberlain,J.S.,etal.,NucleicAcidsRes., 16, 11141-11156(1988))areusedasprobes.Stl4 is a highly polymorphic sequence isolated from the long arm of the X ~,LIulllo~vlll~ that can be used to distinguish female fetal DNA from maternal DNA. It maps near the gene for Factor VIII:C and thus can also be utilized for prenatal diagnosis of II~,Illu~ ilid A. Primers cull~,uulldillg to sequences flanking the six most commonly deleted exons in the DMD gene, which have been successfully used to diagnose DMD by PCR, can also be used (Chamberlain,J.S.etal.,NucleicAcidsRes.,16,11141-11156(1988)). Otherconditions which can be diagnosed by the present method include cystic fibrosis (Newton, C.R., et al.
Lancet 2, 1481-1483 (1989)); !3-thAlS~ee~mi~ (Cai, S-P., et.al., Blood, 73:372-374 (1989));
Cai, S-P., et al.,Am. .,' Hum. Genet., 45:112-114 (1989)); Saiki, R.K., etal., New EngL ~ Med., 319, 537-541 (1988)), sickle cell anemia (Saiki, R.K., et al., New Ængl. J.
Med., 319, 537-541 (1988)), ~ ,llylk~tullul;a (DiLella, A.G., et al., Lancet, 1, 497-499 (1988)) and Gaucher disease (Theophilus, B., et al., Am. J. Hum. Genet., 45, 212- 215 (1989)). An ~)AUIU~ probe (or probes) is available for use in the present method for assessing each condition (see for example PCT Application WO 91/07660).
In another ~, ..l .o.l:,. .l the invention provides a method for isolatmg fetal erythroid cells in metaphase from a sample of peripheral blood from a pregnant woman. The method involves obtaining a sample of peripheral blood from a pregnamt woman, culturirlg cells within the sample of peripheral blood in a culture media containing an erythroid growth 30 factor, e.g., Iy~uu~ù;~,.ill, exposing cultured cells to am agent which inhibits IJ~U~;IC~ ;UII of dividing cells through the cell cycle or ~yll~,hlull;~ growth of cells and isolating fetal erythroid cells in metaphase. Agent which inhibit IJlU~;lCi~iUII of dividing cells through the cell cycle are known in the art and include colcemid, colchicine and vinblastine salt. Agents which ~YII~IUUII;L~ growth of cells are also known in the art and include bromodcu~y ~
35 nuuludeu~sy Ul idille and ethidium bromide. Following culture of cells, metaphase spreads cam be prepared by st~mdard methods and used for further analysis (see Example 2).
In amother ~mho~lim~-nt the invention provides a method for ,ulcf lclllially isolating fetal cells from a current pregnancy in a peripheral blood sample from a l~lulLi~ uu~
pregnant woman. The method involves obtaining a sample of peripheral blood from a 218~
WO 95/Z6417 P~ 906 u~ uua pregnant woman, culturing cells within the sample of peripheral blood in a culture mediurn containing an erythroid growth factor, e.g., ~ luu~uh,;i~l, amd isolating fetal erythroid cells to ~ cllLally isolate fetal cells from a current pregnancy. The language "Illl.lLi~uua pregnant woman" is intended to refer to a pregnant woman who has had one or 5 more other ~ulc~llallch,~ in addition to her current pregnancy. This method is based upon the short life span of erythroid progenitor cells (see Beck WS. Hematology, 5th edifion, MIT
Press:f.~rnhrirl~r,MA(1991)). Sincethelifespanoferythroidprogenitorcellsisaboutthree months, fetal erythroid cells cultured from a peripheral blood sample of a IllulLIJ~uuua pregnant womam will be derived from erythroid progenitor cells of a current fetus rather than 10 from a former fetus. This is an advantage of isolating fetal erythroid cells rather than another, longer lived, fetal cell type that may be present in a maternal blood sample. For example, although fetal Iylll,ullocy v ~ are also present in maternal peripheral blood, Iymphocytes may live as long as several years in peripheral blood (Schroder et al. Trnr~ nntnt~()n 17, 346 (1974); Ciaranfi et al. S~ he .~Ji~ ,. Wo~.t.~,~.,h. i/l 107, 134-138 (1977)).
15 Thus, a fetal Iymphocyte detected in a maternal blood sample of a IllulLilJaluua pregnant woman could either be from the current fetus or from a former fetus, making gender irlrntifir~tir n andlor diagnosis of the current fetus ,ulubl~,lllalic.
This invention is further illustrated by the following exarnples which should 20 not be construed as li:lliting. The contents of all references and published patents and patent orr1irotir~n~ cited throughout this application are hereby ;1.. . ,,1,,,.,.l~.l by reference.
li'XAMP1,F, 1: Culture of Erythroid P.~ ~ Cells from a Sample of Peripheral Blood from a Pregnant Woman In this exarnple, erythroid progenitor cells were cultured from samples of peripheral blood from pregnant women. The peripheral blood samples were first subjected to density gradient c . . .1. i r. ,~ ir~ns (single or dual) in order to eliminate most of the abundant non-nucleated red blood cells present in peripheral blood and to remove many of the maternally-derived nucleated cell types in the sample while also enrichung for erythroid progenitor cells prior to the culturing step. This allowed for culturing of much fewer total cell numbers than were present in the original peripheral blood sample.
Maternal peripheral blood samples were obtained by V~:ilh~)ull~ulc with infor~ned consent frorh pregnant women planning to undergo pregnancy sonogram (ultrasound) and genetic ~ c. ., . ~ c In each case, the maternal blood semple was obtained before the ~....1;. ,.,.. ,t..~ic was performed. A~u.u~i...~!Lcly 18 ml of whole peripheral blood, divided between two Vacutainer tubes containing Acid Citrate Dextrose solution A (ACD-A) as an ~ I;rr~ ,1 (Bectûn Dicksûn), was obtained from each pregnant woman.

wo 95n64l7 ~ 1 ~ 3 ~ ~ ~ PCTIUS95/03906 ~D

The maternal blood samples were received via overnight courier the day after they were obtained. After being inverted a few times to ensure mixing of the samples, 5 ml of undiluted blood was taken from each of three samples and layered atop a Polylllulul.ul~,u gradient as explained below. The remaining blood was diluted tvith an equal volume of phosphate buffered saline and 5 ml aliquofs of the dilufed blood were layered atop a Ficoll-Paque gradient, a Histopaque dual gradient, and a Nycodenz dual gradient. A schematic diagram illusfrating the typical relative f~ictrih~fi~n of erythroid progenitor cells and nucleated red blood cells along a density gradient is shown in Figure 1.
Ficoll-P~q~ Gr~li.ont A 3.5 ml aliquot of Fico~l-Paque (1.077 g/ml, Pharmacia, Uppsala, Sweden) in a 15 ml conical cenfrifuge tube was overlayered with 5 ml of diluted maternal blood. The tubes were spun for 30 minutes at 400 x g in a tabletop cenfrifuge at room t~lll,u~ ul~. The buffy coat cell layer at the interface of the plasma and the Ficoll-Paque (termed F 1) was withdrawn, rinsed, and cultured as described below.
~iet~ly~lllf D~ Tr:~irnt A 2 ml aliquot of Histopaque I . I 19 g/ml (Sigma Chemical Co.) in a 15 ml tube was carefully uY~IL~ 1 wifh 1.5 ml of Histopaque 1.077 g/ml (Sigma Chemical Co.).
A 5 ml aliquot of dilu.ed blood was then layered atop Histopaque 1.077 g/ml. The fubes were spun for 30 minufes at 400 x g in a fabletop cenfrifuge at room t~ . A Pasteur pipette was used to harvest the buffy coat at the interface of fhe plasma and the 1.077 g/ml gradient (termed Hl). To harYest the region thought to be the most likely to contain nucleated red blood cells, the entire I .119 layer from fhe 1.077/1.119 interface down to the red blood cell pellet (termed H2) was aspirated with a Pasteur pipefte. These f~vo cell fractions were placed into clean tubes, rinsed, and culfured as described below.
rO~ y~ lcv Gr~ nt A3.5mlaliquotofPolylllulyl~ul~ul~ll3glml(NycomedlGibcoBRL)inal5 ml tube was gently u ~ d wifh 5 ml of undiluted whole blood. The fubes were spun for 30 minutes at 500 x g in a fabletop cenfrifuge at room ~- IIlU~.~Lul~. As fhe red blood cells in the whole blood sample contacted the Polylllullullul~,u medium, they were ~g,lll'' ' ' by the Dexfran 500 and e~ im~nt~-d through fhe gradient under centrifugal force. This gradient medium is designed to separate two cell uul~u6~iulls by exploiting the tendency of red blood cells to lose water upon entering a II~Y~IU:IIIIUIiU ~IIV;IUIIIII~ . As expected, fhe matemal blood samples ~ d two distinct bands on the rul~ u~ull~ u gradient. The buffy coat at the plasma/Polyll,ul~l"ul~,u interface from each ofthe blood samples (termed Pl) was withdrawn with a Pasteur pipette and transferred to a clean tube. Then the poly. ". ,.1,l.. ,.,... 1. A.
cell layer (temmed P2), which theoretically confained fhe nucleated red blood cells and ., . , .. .. ... ... . .. . .. . . .. .... . . ..... . . . ... . .. .... _ .. _ .. . _ .. _ WO 95/26417 2 1 8 ~ ~ ~ 7 P~
,9 erythroid progenitors, was transferred into another clean tube. Ar~ equal volume of sterile 0.45 % NaCI in distilled H2O was added to }estore the cells to the proper osmolarity. The cells were diluted up to 12 ml in phosphate buffered saline, and cultured as described below.
S Ny. n~ n7 D~IRI GrA~ nt Nycodenz was obtained in powdered form (Gibco/BRL), and 27.6 g of Nycodenz powder was l~ lr~l up to a total volume of 100 ml in buffered medium consisting of 5 mM Tris-HCI, pH 7.5, 3 mM KCI, and 0.3 mM EDTA in distilled H2O. This 27.6 % solution, at a density of I.150 g/ml, was sterilized by dULu~,luvulg. Iso-osmotic NaCI
diluent (density 1.003 g/ml) was made by mixing 0.75 g NaCI into 100 ml of the buffered medium described above. The NaCI diluent solution was sterilized through a 0.22 llm filter.
The 27.6 % Nycodenz solution and NaCI diluent were mixed in the ratios of 3 :2 and 1:1 to yield two solutions having a density of 1.090 g/ml and 1.075 g/ml Ic*~ ly. A 2 ml aliquot of Nycûdenz 1.090 g/ml in a 15 ml tube was carefully overlayered with 1.5 ml of Nycodenz 1.075 g/ml. A 5 ml aliquot of blood diluted 1:1 in phosphate buffered saline was then layered atop Nycodenz 1.075 g/ml. The tubes were spun for 30 minutes at 1500 x g in a tabletop centrifuge at room ICIIIIJCIdlUIC. It was noted that this gradient rnmhinAtinn resulted in cell bands which were much more diffuse than on other gradients. The cell layers were 1'~ Il lln l l ~; l l. " ~1 with a large number of - - - -' ' red bloûd cells. A Pasteur pipette was used to harvest the ce!~ layer at the interface of the plasma and the 1.075 g/ml gradient (termed Nl). Most of the 1.090 layer was harvested with a Pasteur pipette (termed N2);
because there were so many cells present in this layer, not all of them were used. These cell fractions were placed into clean tubes.
r~rllAlAI;IIII of C~lt~re ~1'11illm The basic culture medium used corlsisted of I . I % methylcellulose (4000 , Sigma Chemical Co.), 30 % fetal calf serum (Sigma Chemical Co.), 0.1 mM
2~ 1u~l1.A..~.I (Sigma Chemical Co.), and I % bovine serum albumin (BSA, Fraction V;
Sigmû Chemical Co.) in Iscove's Modified Dulbecco's Medium (IMDM; Sigma Chemical30 Co.). Cultures were ,,~ with 2 units/ml of ~ auul~u;~ll (Sigma Chemical Co.) to promote erythroid cell growth and mAtllrAtinn To prepare a solution of 3.2 % methyl-cellulose in IMDM, 6.4 g of methylcellulose was weighed into a one liter dulu~,lavalJlc bottle containing 190 ml sterile IMDM. The solution was mixed on a stirring plate while the methylcellulose went slowly 35 into solution. The solution was autoclaved with the stir bar still in the bottle. After auL(J~lavillg, the methylcellulose formed a solid pink plug in the bottle. The bottle was shaken vigorously until the methylcellulose broke up mto a slurry. As it cooled to room ~ aLuuc, the methylcellulose went back into solution.

wo 95/26~17 2 1 ~ 3 ~ ~ 7 P~ 906 BSA at a final ç- nAf nfrAAtif~n of 10 % and pH of 7.6 was prepared by weighing out 10 g BSA and adding it slowly to about 60 ml distilled H20 in a small beaker on a stir plate. Meanwhile, 10 % NaHC03 was prepared by dissolving 10 g NaHC03 in distilled H20 to a final volume of 100 ml in a volumetric flask and filter sterilizing through a 0.22 ~m 5 filter. The BSA solution was titrated to pH 7.6 with l O % NaHCO3 while being c. . ~ 1y monitored with a pH meter. This solution was poured into a volumetric flask, brought up to 100 ml with distilled H20~ and filter sterilized first through a 0.45.~Lm filter and then through a 0.22 !lm filter.
Underafumehood,lO,ulof2~ u~ll,A.,..lwasaddedtolmloflMDM.
In a sterile tissue culture hoûd, 0.75 ml of this solution was added to 49.5 ml of IMDM, mixed, and filter steriliæd through a 0.22 f~m filter.
To the autoclaved bottle of 3.2 % methylcellulose/lMDM was added 5 ml of penicillan-~ ,ulullly~ ;ll antibiotic (5000 units/ml, and 5000 llg/ml; Gibco/BRL), 5 ml of L-glutamine (200 mM; Gibco/BRL), 180 ml of sterile heat-inactivated fetal calf serum (Sigma Chemical Co.), 60 ml of 10 % BSA pH 7.6, and 40 ml filtered 2-mercapto-ethanol/lMDM. The contents of the bottle were mixed thoroughly and the medium was divided into 2.3 ml aliquots in 15 ml tubes. The tubes were stored frozen at -20 C until needed.
20 PlAtin~ AAn(l Cllltl-re of r.ollc After the whole blood sample had been separated by density gradient c-,lllfiru~;aliull on a dual rTict--r qll~ gradient (1.077 g/ml over 1.119 g/ml) or on a rolylllul~ll,ulc~ gradient, the desired cell fraction was rinsed twice in phosphate buffered saline, rinsed once in IMDM, and l~ f J up to a total volume of 0.5 ml in IMDM. This 25 0.5 ml of cell suspension was added to a 2.3 ml aliquot of the methylcellulose medium in a 15 ml conical centrifuge tube along with 0.2 ml of 30 units/ml cl~lluu~uo;~ l;ll, at a final ........ lIIAI;.. , of 2 units/ml.
Tubes were vigorously vortexed to ensure complete mixing of contents, as well as to break up any cell clusters which could mimic colony formation. The final product 30 was of the ;UI~ y of thick syrup. 1.5 ml of the medium containing eells was placed in each of two covered 35 mm Falcon plastie tissue eulture dishes ~Beeton Diekson, Lmeoln Park, NJ) within a 100 mm Falcon plastic dish (Becton Dickson). A third, uncovered, 35 mm Falcon dish was placed within the 100 mm Falcon dish and partially filled with two ml of sterile distilled water to provide humidity. The eover was plæed on the 100 mm Faleon dish and the entire dish was transferred to a 37 C ineubator with a 5 % C02 atmosphere.
The more mature CFU-E IJlU~,_ll;Lul~ formed .,. .-g." ,_~ ~1. colonies of up to 150 cells within four days, and by eight days in culture virtually all of these maturing cells eontained visible hf nnnglobin Because the cl~ ILuuu;clill receptor is not expressed umtil the BFU-Es have attained a mgher level of dirf~,lcilllialion, the BFU-Es did not I ~ begin to proliferate wo 95126417 , r~ 906 in culture. By days eight to ten of culture, large scattered BFU-E colonies were visible and by day 14 these colonies were strongly hPnnngln~ hini7~1 R-"u~ Ldliv~ examples of CFU-E and BFU-E colonies are shown in Figures 2 and 3, I~ ,Li~ly (the colonies depicted in the figures were isolated from fetal livers), 5 Table I ,, ~ ` ` the number of both BFU-E and CFU-E colonies obtained from culturing each cell fraction of a blood sample from a pregnant woman, The sample was r,~
using Ficoll-Paque (F), dual Hictnp~qllr (H), Polyl.lul~.l.,ulciy (P), and dual Nycoden~ (N) density gradients, Colonies from fraction N2 could not be seen or counted due to the presence of a large number of red blood eells which obscured the culture, T~hle 1: P Pl~tive rlllnnh~ rs of pro~enitnr r~llc in fr~rf: ' whnl~ blood Density Total Number Number 15 Colonies ~Ell~ ( % ! CFU-E ( % !
Fl (1,077) 71 64 (90,2) 7 (9,8) Hl (1,077) 68 63 (92.6) 5 (7.4) H2 (1.119) 1 0 (0.0) 1 (100) Pl (1.077) 121 106 (87.6) ls (12.4) 20 P2 (1.1 13) 16 4 (25.0) 12 (75.0) Nl (1.075) 72 71 (98.6) 1 (1.4) N2(1.090) -- notanalyzable -- --Totals: 349 308 (88.3) 41 (11.7) hlXAMPI ,~, 2- Isolation of Cultured Erythroid Colonies and P. ~,... '' of ~ ~ ' Spreads In this example, erythroid cell colonies cultured as described in Example I
were isolated and used to prepare metaphase spreads of erythroid cells on a Illi~.lU:~.V~lC slide for further analysis, 35 Sli~ir Plcl"..,.l;....
The erythroid colonies were harvested directly onto Teflon-backed glass Il~i~.lU~ UlJt slides (Cel-Line Assoc, Inc,, Newfield, Nl) according to the basic method of Rajendra et al, (Human Genetics (1980) 55:363-366) and Dube et al, (Cancer Genetics and Cytogenetics (1981) 4:157-168), Because these erythroid cells are normally l~ul~Jll~ L, the 40 slides were first coated with 0,1 % poly-L-lysine to induce the cells to attach to the slides so that they would not be lost during the harvesting procedure, For poly-L-lysine treatment, 100 mg pûly-L-lysine (Sigma Chemical Co.) was dissolved m 10 ml of distilled H2O to make a WO 95~26417 ~ ~ 8 ~ r~

1 % stock solution. Aliquots of one ml each were stored froæn until used. To make a 0.1 %
working solution, 100 ,ul of 1 % stock solution was diluted with 900 !11 of distilled H20. A
drop of the working solution was placed on each~5 mm site on each slide. The slides were laid flat and ~-ul-u.~ g in a hurnid chamber (a sealed plastic box ,- ~ lllo;.L paper towels to provide humidity and a rack to keep the slides from contacting the paper towels) and incubated for two hours at 37 C. After the incubation the slides were rinsed individually in a beaker of distilled water and air dried at room t~ alu~. The coated slides were stored frozen at -20 C until ready to use.
TTqrvPetir~ofColoniP~
Cells from CFU-E colonies were best harvested from culture at two to six days because their growth peaked quickly. After this time the nuclei had either been extruded or were dense and pyknotic, rendering further analysis difficult. Cells derived from BFU-Es could be harvested at ten days in a ~lulil.,laliv~ state before extensive hPrnnglohini7qtinn had occurred, or later if larger numbers of nondividing nucleated red blood cells were desired .
Colonies were harvested onto 5 mm sites on poly-L-lysine coated slides, with four to six sites per slide. Only one colony was placed on each site except in cases where a large number of colonies arose in culture, when three or more small colonies were sometimes harvested per site. Because of differences in cell growth between samples, the total number of colonies harvested per sample .anged from three to 314.
Colonies were identified under an inverted I~ lUS~U,Ue and plucked from the medium with a Gilson Pipetman (Rainin Instrument Co., Inc., Woburn, MA) set to 4 ~
using a 10 ,ul tip. Each colony was dispersed by pipetting up and down 10 to 20 times into a 15 111 drop of IMDM containing 0.1 ,ug/ml colcemid (Gibco/BRL) on a poly-L-lysine coated slide. Slides were placed flat in a humid chamber at 37 C for 15 minutes, then 20 ,ul of 0.075 M KCI hypotonic solution was gently infused into each drop. After a further 15 minutes of incubation at 37 C in a humid box, 40 ,ul of a solution of 30 % 3:1 mPthqnnl ~lqriql acetic acid fixative/70 % 0.075M KCI was added gently to each site. The slides were incubated for five to ten minutes at room t~ ,la~ul~, then carefully tipped sideways to drain excess fluid without disturbing the cells which had adhered to the slide.
Before the slides dried, fresh 3:1 m~qth ~nr,l ~lqriql acetic acid fixative was dropped onto each slide from a pasteur pipette and the wet slides were blown on to facilitate spreading out and flattening of the cells thereon. After the slides had air dried they were soaked for five minutes in Optistain II-A (Gam Rad Corp.,Capistrano~ CA) followed by ten minutes in 3:1 mPthqnr,l glqriql acetic acid fixative. Slides were either dehydrated through an ethamol series and either used ill ~ '~/ for further analysis (e.g., as described below for nuu-~ in situ llybl;di~aliull), or were stored frozen at -20 C until they could be studied.
Erythroid cells on Ill;(,lU:~.U~ slides prepared in this manner can be used for hybridization of the cells to a nucleic acid probe (e.g., fluorescent in sifu l~bl;d;~iu~

218~7 WO95/26417 P~ 906 Wright's staining, banding or storage. Erythroid cells not alrested at the metaphase stage were prepared on ~ va~,v,ue slides in the same manner by omitting colcemid from the cell-containing solution.
s EXAMPl .~ 3: Detection of Fetal DNA in Cultured Erythroid CelTs by F` - ~ ' In Situ ~
In this example, flourescent in situ ll.ybl;di~iiull (FISH) was used to detect 10 fetal DNA within the cultured, isolated erythroid progenitor cells. Hybridi_ation of erythroid cell-derived DNA was performed ~ ~ - ly with both a Y cl..u,..vsv,,l~-specific probe and an X-~,lllvlllOaulll~ specific probe. The two probes were labeled with different fluorescent markers, allowing them to be .1;~ 1 by different colors. Since Y-specific sequences in erythroid cell-derived DNA must be of fetal origin, hybridi_ation of this probe 15 to the erythroid cell-derived DNA is used as an indicator of the fetal origin of the cells.
Fl~ rPcrPnt In Sifu Hyhri~li7:1tirn Erythroid progenitor cells were cultured as described in Example I and colonies were isolated and placed onto Illil,lva~,u,uC slides as described in Example 2. FISH
20 was performed using dn X-~,h~u~l~osullle specific probe derived from a cosmid containing a p~ vlll~;c repeat sequence of the X ~LI u~vsv~llc and a Y specific probe derived from a cosmid pDP97 containing repetitive sequences. The probes are described in further detail in Klinger et al. Am. J: Hum. Genet. 51, 55-65 (1992). The X-specific probe was labe~ed with biotin whereas the Y-specific probe was labeled with ~ Yi~Pnin The biotinylated X
25 ~,hlulllOaulll~ probe was used at a ~ l l of 2.5 ng/~ l and the digw~y~ hPIIP~I y ~,hlullluaulllc probe was used at a . ~ .~ .. 1 . ,,1 ;.. , . of 5 ng/~
The hyhri~ii7 1tirn washing, and detection protocols used were modified from Klinger et al. A~n. ~ Hum. Genet. 51, 55-65 (I 992)and were as follows. T .. 1 i.~lrl~y before llybl;di~iivll the slides were dehydrated through a 70%-80%-90%-100% ethanol series for 30 one minute each and then air dried. Probe DNA for both the X and Y ~LIvllluavlll.,~ was suspended in 50 % ~v....~..;lc/lO % dextran sulfate/6X SSC (standard sodium citrate) with 8 ~Lg/lll sonicated salmon spemm DNA (Sigma Chemical Co.) and 2 llg/lll human Cot-l DNA
(Gibco BT~L). A 2.5 ,ul drop of probe cocktail was added to each 5 mm site on the slides and - covered with a glass coverslip. The coverslips were not sealed to the slide surfaces. The 35 probe and the target nuclear DNA on glass slides were codenatured by placing the slides on a al;d~,vv~ulll~l at 80 C for five to six mirlutes. The slides were incubated in a humid box at 37 C overnight. The following day the coverslips were removed and the slides were washed once in 50 % 1.., ,..- ..i~l~/2X SSC pH 7.0 at 42 C for ten minutes, and once in O.lX SSC at 60 C fûr ten minutes. Fûllûwing a blocking step in 3 %BSA/4X SSC/O.I % Tween 20 36~7 WO 95/26~17 r~ x,~ so6 - 24 _ (polyoxyethylene-sorbitan ml-n~ ~tP; Sigma Chemical Co.) at 4Z =C for five minutes, the labeled probes hybridized to the nuclei on the slides were detected by incubation at 37 C for 15 minutes in 1 % BSA/4X SSC with llg/ml anti-l;Ov~yO~,I~, fluorescein isvlll;v~.y.. t~
(FITC) antibody (Boehringer Mannheim Corp., Tn~ rolic IN), which recognizes S diov~yO~ l and emits green nuv,-,.cl l,~e, and 2 llg/ml Cy-3 ~ C!J aViVill (Jackson ImmunoResearch Labs, Inc., West Grove, PA), which binds tightly to biotin-labeled probe and produces red nuul~,l,.,.,e. The slides were then washed once in 4X SSC/
0.1 % Tween- 20 at 42 C for ten minutes to remove excess unbound nuvlv~,LIulll~, which might otherwise result in spurious signal ba. L~lu.
Coverslips were placed on the slides atop a thin layer of 90 % glycerol containing 2.33 % DABCO antifade (1,4-vi~ab;~,y~,1O-[2.2.2]octane; Sigma Chemical Co.), 100 mM Tris pH 8.0, and 0.1 llg/ml DAPI (4,6-diamidino-2phenyl-indole; Sigma Chemical Co.). The nuulucluulllc DAPI emits blue nuvl~ ,G.l~c umder ultraviolet light when bound to AT-rich regions of the minor groove of intact DNA, and is therefore useful as a nuclear 1 .u ~ 11 (Schweizer, Ambrose, 8~ Andrle, 1978). Slides were viewed with 40X and100X oil immersion lenses on a Zeiss Axioplan el~inuulca~ e Illi~lV~ V~ (Carl Zeiss, Inc.) with a~ U~ nuvl~ ~C~IICc filters (Omega Optical, Inc., Brattleboro, VT). Filters used for DAPI nuulc~ ,c were a 365 mm excitation filter, a 410 nm dichroic filter, and à 420LP nm emission filter; for FITC nuulc~ c~ a 485/20 nm excitation filter, a 510 nm dichroic filter, and a 520-5G0 mm emi~sion filter; and for Cy-3 nuulca~ ,e, a 515-560 nm excitation filter, a 580 nm dichroic filter, and a 590LP nm emission filter. Hybridiæd nuclei were pllUi using a 1 00X oil immersion objective, with a 35 mm camera mounted directly on the llli~lU~Cv~c using Kodak Gold 400 film.
r~ ~ Bl--od SAmrlp AnAlvsi~
A total of 37 peripheral blood samples were obtairled from consenting pregnant women between 10 5~.7 weeks and 22 weeks of gestation. Cells were r .~ , I
from whole blood using either a dual Histopaque 1.077/1.119 g/ml density gradient (21 samples) or a Polymorphprep gradient (14 samples). Two samples (samples 11 and 21) were 30 frA-~ti~-n~tl~d on dual Histopaque, POIYIIIUI~ JIC~J~ Ficoll-Paque and dual Nycodenz gradients.
Fl~ ivll~cd cells were cultured with clylluv~oic~ as described in Example 1. Tn most cases, both the Hl or P I and the H2 or P2 cell fractions were cultured. Colonies of CFU-E
morphology were harvested from the H2 or P2 fractions, and occasionally were obtained from the Hl or Pl fraction as well. Colonies of BFU-E morphology which exhibited rapid 35 growth and hPTnngll~hini7~ti~\n were also harvested. Colonies were analyzed by FISH as described above. The actual karyotype of the fetuses, as determined by cytogenetic analysis, is shown in the last column (46,XY=Male, 46,XX=Female). The results are ~ I in Table 2.

218~6~7 95/26417 r~ 906 Table 2: Correlation of FISH analysis of cultures to cytogenetic ril~rrrrnin~tifln of fetal k~oty~e Total # # Male Fetal S~1~ GA~' ~nl~i~ ~l~ni~ ~I~I~ Karvotype 10 5/7 120 4 3.3 46,XY
- 2 14 5 0 0 46,XY

3 14 39 0 0 46,XY

4 14 10 0 0 46,XY
14 317 64 3 4.7 46,XY
6 14 517 96 2 2.1 46,XY
7 14 6/7 28 1 3.4 46,XY
8 15 20 0 0 46,XY
9 1 5 32 0 0 46,XY
24 0 0 46,XY
I 1 15 78 0 0 46,XY
12 15 24 0 0 46,XY
13 15 30 0 0 46,XY
14 15 45 3 6.7 46,XY
0 0 46,XY
16 16 20 0 0 46,XY
17 16 2/7 188 8 4.3 46,XY+ 46,XXtwins 18 17 3 1 33.3 46,XY
19 17 20 0 0 46,XY
17 25 0 0 46,XY
21 22 127 0 0 46,XY
22 1 1 2/7 27 0 0 46,XX
23 12 3/7 28 0 0 46,XX
24 14 314 0 0 46,XX
14 43 0 0 46,XX
26 14 3/7 8 0 0 46,XX
27 16 20 0 0 46,XX
28 16 15 0 0 46,XX
29 17 3/7 28 0 0 46,XX
17 4/7 30 0 o 46,XX
3 1 1 7 6/7 25 0 0 46,XX
32 18 4 0 0 46,XX
33 18 40 0 0 46,XX
34 18 217 7 0 0 46,XX
19 1/7 30 0 0 46,XX
36 19 2/7 26 0 0 46,XX
37 N/A 29 0 0 46,XX
i'GA= gestational age in weeks 183~7 ' wo 95126417 2 ~ 906 Of the 21 samples cultured from pregnant women whose fetuses were verifled to be male by ultrasound and/or cytogenetic analysis (including sample 17, with one male and one female twin), XY colonies were identified by FISH in seven samples, yielding a male detection rate of 33.3%. The number of XY col~niés per sample ranged from one to eight (2.1 % to 33.3% of the total number of coloniès analyzed for those cases). From the 16 pregnant women who were actually carrying female fetuses as confirmed by uyLuc~ ic analysis (not including sa~nple 17, with a male and a female twin), no XY colonies were detected, yielding a false-positive rate of 0 %. The gestational ages of the 37 ul~ .iCs analyzed varied from 10 5/7 weeks to 22 weeks. Sarnple 1, at 10 5/7 weeks, was the earliest pre~nancy with a male fetus to be analyæd, and showed male cells in four of 120 colonies.
The latest gestational age at which a male colony was cultured from a sample was 17 weeks (sample 18).
Table 3 relates the gestational age (GA) in weeks at which blood samples were obtained to the gradient fraction from which cultured colonies were harvested, the length of time (in days) that cells were cultured before harvesting, and the number and percent of the total number of colonies harvested which showed male cells by FISH.

~ 83~S7 95/264 1 7 1 ~ ~ . 906 T~hle 3: H~rvPctir~ ParamPt~ of Cllltllred Cellc from rlr~;"~ with a ~lale Frtllc GA Fraction ~ # Col-~nipc # Male ~a~

5 1 10 5/7 H2 4 120 4 (3.3) 214 H2 7 1 0 (0) H2 10 4 0 (0) 314 P2 9 9 0 (0) Pl 9 30 0 (0) 10 4 14 Hl 8 10 0 ( 0 ) 514 3/7 H2 3 64 3 (4.7) 614 5/7 H2 4 96 2 (2-1) 714 6/7 Hl 13 16 0 ( 0 ) H2 13 12 1 (8.3) 15 8 15 H2 5 20 0 (0) 915 P2 6 16 0 (0) P2 13 16 0 (0) P2 6 16 0 ( 0 ) P2 13 8 0 (0) 2011 15 Fl 8 30 0 (0) Pl 8 24 0 (0) Nl 8 24 0 (0) 12 15 Pl 7 24 0 ( 0 ) 13 15 H2 9 10 0 ( 0 ) Hl 9 20 0 (0) 14 15 P2 9 10 2 (20.0) Pl 9 35 1 ( 2.9) H2 7 5 0 (0) Hl 7 15 0 (0) 3016 16 H2 7 10 0 ( 0 ) Hl 7 10 0 (0) 17 162/7 H2 5 116 8 (6.9) H2 11 72 0 (0) 18 17 H2 4 3 1 (33.3) 3519 17 P2 8 20 0 ( 0 ) 17 H2 7 15 0 (0) Hl 7 10 0 (0) 21 æ Fl 7 28 0 ( 0 ) Pl 7 31 0 (0) P2 7 12 0 (0) Nl 7 24 0 (0) Hl 7 24 0 (0) H2 7 4 0 (0) ~1~3~7

6~17 PCT/US95/03906 F.XAMPLE 4: Use of the r~ Chain Reaction to Amplify Gene Sequences in Cultured Erythroid Progenitor Cells In this example, the polymerase chain reaction was used to amplify Y-specific 5 DNA sequences present in fetal erythroid progénitor cells cultured from maternal peripheral blood. The pol~ ., chain reaction, or PCR, which has a capacity for making I o6 copies of rare target gene sequences, is used to amplify gene sequences in cultured ery~roid progenitor cells. For fetal sex ~lf t~nnin~tinn, PCR primers specific for repeated sequences present on the Y ~L~ul~osu~l~c are used. Repeated sequences are selected because they create 10 a stronger .." ,~ ,. ", signal from a rare male fetal cell. Primers which hybridize to a regionoftheshortarmoftheY.,llu...usul,le,~,....",l.~ lbyprobeY411 (GivenbyDr.
Ulrich Muller, Children's Hospital, Boston, MA), are used. Y411 is identical to Y156 (Muller, U., et al., Nucleic Acids Res., 14, 1325-1329 (1986)), is repeated 10-60 fold, and is absolutely Y specific on Southern blots. Two Y41 I region-specific primers, primers 411-01 and 41 1-03, which are designed to amplify a 222 base pair (bp) sequence detectable with the Y cl~u~usù~c-specific probe Y411, are used. The primer sequences are described in detail in Bianchi, D.W., et al. Proc. Nall. Acad Sci. USA 87, 3279-3283 (1990). Other suitable primers are described in Wachtel, S., et al. ~um. ~eprod 6, 1466-1469 (1991).
To defiile the minimum amoumt of DNA detectable in a cell sample, a series of :~kllld~ldiLa~iUII ~ is done. DNA from male and female individuals is prepared in tenfold dilutions (I pg to I mg) and amplified using the standard reagents, including reaction buffer, in the GeneAmpkit (Perkin-Elmer Cetus catalogue #N801-0055) on a Perkin-Elmer DNA Thermal Cycler. Measures are taken to prevent aerosol ~.... l - .,;, . -;, ... of samples with 25 male DNA. All PCRs are performed under sterile conditions, wearing gloves, and using positive (l,~ pipettes. All reagents are prepared in a sterile manner amd incubated overnight prior to PCR with a restriction c~dù~u~ , having a digestion site within the target sequence. These precautions result in a significant decrease and virtual absence of false positive a.l.~,lirl~ iul., as monitored by running control reactions with all reagents but 30 no DNA. The number of Amrlifir~til~n cycles is varied between 18 and 30. Each; r~ A~ l I cycle consists of I minute at 94C, 2 minutes at 60C and 3 minutes at 72C, with a 10 minute extension in the last cycle. Amplified DNA samples are cl~ u~ullulcc~ on agarose gels, transferred to nylon filters, and hybridized to 32 P-labelled Y411 probe.
Erythroid progenitor cells are cultured and isolated as described in Example I .Prior to llmrlifi.-~tion, the cells are Iysed by boiling which makes the erythroid cell DNA
available for ~.,.l,l; l`;. ,~1;~., The erythroid cell DNA is amplified for the 222 bp sequence in probe Y411 as a .i .~ that the cells are derived from the fetus in male ~lc~ ..,h,~.
The conditions used, as described above, make it possible to detect a minimum of 100 pg of .. ... ... _ .. . , . .. . _ .. ........... ... _ _ .. .......... .. .. _ . ..... ... .. .... .... . . .... .. ..... ... . .

~ WO 95/26417 2 1 ~ ~ 6 5 7 PCT/US95/03906 fetal DNA, or the equivalent of 15 fetal cells. The ~imit of sensitivity can be improved by extending the number of cycles used in PCR. To further decrease false positive Amrlifil~fit)n and permit detection of fetal DNA at the single cell level on agarose gels, PCR is carried out using primers derived from a single copy of sequence specific for the long arm of the Y
~,luulllO~vl~ PY49a (Guerin, P., et al., Nucleic,4cids Res, 16, 7759 (1988)).
F.XA.MPI,F. 5: Detection of ~ ' in Erythroid Pl. ~ Cells Cultured from Msternal Blood In this example, in situ llyblhli~aLiull using ~luulllvsul~ specific probe sets is performed on fetal erythroid progenitor cells cultured from matemal blood in order to detect ~,luvlllOsvlllàl AhnnrmAliti~c in fetal cells. A DNA probe set specific for a particular ~,luvlllosvlll~, that provides both good signal to noise ratios and good spatial resolution of the fluorescent signals is used in in situ llybli~i~tiull. Specific probe sets have been developed for five ~luv~llv~ulll~ frequently seen as livebom . ' ivi~ ,luulllosùlll~s 13, 18, 21, X
and Y. A probe for LIUUIIIOSUlll~ I is used as a control. In cullailu.,Lil~g the probes, the general strategy was to identify a starting clone that mapped to the desired ~,luulllo:~ul~lal region by multiple genetic and physical methods, and then to use that clone to identify matching cosmid "contigs" which are then used as llybl;di~aiivll probes. The ~.luvlllu~ulllc 21 probe set is a three-coâLnid contig containing 80 kb of nullv ~,. la~l~illg DNA. The ~.lUUIIIVavlll~ 18 probe set is a three-cosmid contig containing 109 kb of IIVIIU~ Ia~ g DNA. The ~ UIIIOSVIIIC 13 probe set is a three-cosmid contig containing a,u,ul~ 'y 97 kb of llulluv~ lla~l.;l-g DNA. The X ~,IUUIIIVSVIII~ probe is a cosmid containing a p~ lUIIl~,~iC repeat sequence. The Y probe is pDP97, a repetitive clone (a 5.3 kb EcoRI Y
fragment from cosmid Y97 subcloned into EcoRI site of pUC-I 3). All the probes are described in further detail in Klinger et al. ~m. ~ Hum. Genet. 51, 55-65 (1992).
To diagnose a .,lUUlllO~vllldl abllullllali~y in a fetus, erythroid progenitor cells are cultured from matemal blood as described in Example I and ~,luunlvsulllal AhnrrmAlitit c are assessed by performing in situ hybridization using cluull~u ~v~ pecific probes such as those described above. In situ llyblidi~aliull is perfommed as described in Example 3 and in Klinger et al. ~m. J. Hum. Genet. 51, 55-65 (1992) under ~u~lc~:,iull conditions (Cremer et al., Hum Genet. 80, 235-246 (1988); Lichter et al., Hum Genet. 80, 224-234 (1988)).
Hybridization of high copy number repeat sequences is suppressed by inclusion of total 35 genomic human DNA, and the ~.luu~l~uavlllal specificity can be verified by hybridization to metaphase spreads. Probes are labelled with biotin-UTP, hybridized under :lU,~ iVII
conditions, and specific hybridization detected by conjugated streptoavidin-FITC, which shows as a single "dot" in the FITC image upon Illi~,lu~,V,uiC analysis. Altematively, if in situ llyblidiLdtiull is to be performed ~vith multiple probes, the individual probes can be .

~1~3~S~
wo 95/26~17 ~ 906 differentially labelled to al~ow for them to be ~lictin~ h~d lluu~ y. For example, one probe can be labelled with biotin-UTP and the another with ~1ivox;~rnin-uTp. The former probe can be detected with Cy-3 streptavidin while the latter can be detected with anti-,1 i v~ . ~ ,~. . ,; . .-FITC. The probes give sharp, punctate fluorescent signals in interphase cells 5 that are easily .l;~ r~l and ~ ~
Diagnosis of a ~luulllu~u~lal al"lulllldliLy is - c .. ~ l . d by comparing the hybridization of a ~ L~ulllu~ulll~-~pecific probe to DNA from fetal erythroid cells to llybl ;di~,a~ of the same probe to DNA from normal cells (a normal control). Normal cells, 10 which may be any cells which do not contain a ~lllu~l~u~ù~ldl al,llullllalily in the .,L~ulllo~ulllc(s) being examined, can be hybridized at the same time as the fetal erythroid cells to provide a normal control. For example, maternal cells can be used as a normal control Alternatively, a previously established normal control can be used for ~A common ~Lulllu~u~l~al ~l~ull~alily, a trisomy (in which three copies of a particular 15 1I1~UIIIO~UIIIC are present in a cell rather that the normal two), can be diagnosed by detection ofthreefluorescentsignalsforaparticular~,l..ul..o.u...~"e.g.commonly~ll.u~o~u~ s2l~18 or 13, in a fetal erythroid cell as compared to only two fluorescent signals for the same u~losull~ in a normal control. A suff~cient number of hybridized cells are examined to make a statistically significant ~l~ .. . ", .~ ;. .,~ of the number of fluorescent signals present per 20 cell.
EQUlVAr.F~TS
Those skilled in the art will recognize, or be able to ascertain using no more 25 than routine . ~l.. .;.,l. .~lAI;-~n, many equivalents to the specific ~ oll;~ ofthe invention descnbaherein Snoheqni~lent~eintendd~ber. ~ dbythe~ollo ingoldms

Claims (54)

1. A method for detecting a nucleic acid sequence of interest in fetal DNA of fetal cells in a sample of peripheral blood obtained from a pregnant woman, comprising:
obtaining a sample of peripheral blood from a pregnant woman;
culturing cells within the sample of peripheral blood in a culture medium containing a cell growth factor and a semisolid matrix material;
isolating fetal cells from the culture medium; and detecting a nucleic acid sequence of interest in fetal DNA of fetal cells isolated from the culture medium.

2. The method of claim 1, wherein the cell growth factor is a hemopoietic cell growth factor.

3. The method of claim 1, wherein the cell growth factor is an erythroid growth factor.

4. A method for detecting a nucleic acid sequence of interest in fetal DNA of fetal erythroid cells in a sample of peripheral blood obtained from a pregnant woman, comprising:
obtaining a sample of peripheral blood from a pregnant woman;
culturing cells within the sample of peripheral blood in a culture medium containing erythropoietin and a semisolid matrix material;
isolating fetal erythroid cells from the culture medium; and detecting a nucleic acid sequence of interest in fetal DNA of fetal erythroid cells isolated from the culture medium.

5. The method of claim 4, wherein the nucleic acid sequence of interest is a Y chromosomal DNA sequence.

6. The method of claim 4, wherein the nucleic acid sequence of interest is a sequence of a gene associated with a disease-causing mutation.

7. The method of claim 4, wherein the nucleic acid sequence of interest detects a chromosomal abnormality.

8. A method for isolating fetal cells from a sample of peripheral blood obtained from a pregnant woman, comprising:
obtaining a sample of peripheral blood from a pregnant woman;
culturing cells within the sample of peripheral blood in a culture medium containing a cell growth factor and a semisolid matrix material; and isolating fetal cells from the culture medium.

9. The method of claim 8, wherein the cell growth factor is a hemopoietic cell growth factor.

10. The method of claim 8, wherein the cell growth factor is an erythroid growth factor.

11. A method for isolating fetal erythroid cells from a sample of peripheral blood obtained from a pregnant woman, comprising:
obtaining a sample of peripheral blood from a pregnant woman;
culturing cells within the sample of peripheral blood in a culture medium containing erythropoietin and a semisolid matrix material; and isolating fetal erythroid cells from the culture medium.

12. The method of claim 11, wherein a proportion of nucleated cells present in the sample of peripheral blood obtained from the pregnant woman is increased relative to a proportion of nucleated cells present in the sample of peripheral blood prior to enrichment forming a sample enriched in nucleated cells prior to culturing the sample enriched in nucleated cells.

13. The method of claim 12, wherein the sample enriched in nucleated cells is formed by separating non-nucleated cells from nucleated cells in the peripheral blood sample forming a sample enriched in nucleated cells.

14. The method of claim 13, wherein non-nucleated cells are separated from nucleated cells by density gradient centrifugation.

15. The method of claim 14, wherein density gradient is performed with Ficoll.

16. The method of claim 14, wherein density gradient is performed with Histopaque.

17. The method of claim 14, wherein density gradient is performed with Nycodenz.

18. The method of claim 14, wherein density gradient is performed with Polymorphprep.

19. The method of claim 13, wherein non-nucleated cells are separated from nucleated cells by selective lysis of non-nucleated cells.

20. The method of claim 11, wherein a proportion of erythroid progenitor cells present in the sample of peripheral blood obtained from the pregnant woman is increased relative to a proportion of erythroid progenitor cells present in the sample of peripheral blood prior to enrichment forming a sample enriched in erythroid progenitor cells prior to culturing the sample enriched in erythroid progenitor cells.

21. The method of claim 20, wherein the sample enriched in erythroid progenitor cells is formed by separating erythroid progenitor cells from non-nucleated cells and other nucleated cells in the sample of peripheral blood forming a sample enriched in erythroid progenitor cells.

22. The method of claim 21, wherein erythroid progenitor cells are separated from non-nucleated cells and other nucleated cells in the sample of peripheral blood by density gradient centrifugation.

23. The method of claim 22, wherein density gradient centrifugation is performed with a dual density gradient.

24. The method of claim 22, wherein density gradient centrifugation is performed with Ficoll.

25. The method of claim 22, wherein density gradient centrifugation is performed with Histopaque.

26. The method of claim 22, wherein density gradient centrifugation is performed with Nycodenz.

27. The method of claim 22, wherein density gradient centrifugation is performed with Polymorphprep.

28. The method of claim 11, wherein the semisolid matrix material allows cells to attach to the semisolid matrix material.

29. The method of claim 28, wherein the semisolid matrix material is methylcellulose.

30. The method of claim 28, wherein the semisolid matrix material is agargel or a plasma clot.

31. The method of claim 11 further comprising detecting a fetal cell marker on or in the fetal erythroid cells to identify them as being fetal-derived.

32. The method of claim 31, wherein the fetal cell marker is fetal hemoglobin.

33. The method of claim 31, wherein the fetal cell marker is a fetal histone protein H1o.

34. The method of claim 31, wherein the fetal cell marker is a fetal antigenic determinant.

35. The method of claim 34, wherein the fetal antigenic determinant is an antigenic determinant i.

36. The method of claim 11, wherein fetal erythroid cells are isolated from the culture medium by picking one or more colonies of fetal erythroid cells from the culture media.

37. The method of claim 36, wherein the fetal erythroid cells are further isolated by transferring one or more colonies of erythroid cells from the culture medium to a microscope slide.

38. The method of claim 11 further comprising detecting a nucleic acid sequence of interest in fetal nucleic acid of isolated fetal erythroid cells.

39. The method of claim 38, wherein the nucleic acid sequence of interest is a Ychromosomal DNA sequence.

40. The method of claim 38, wherein the nucleic acid sequence of interest is a sequence of a gene associated with a disease-causing mutation.

41. The method of claim 38, wherein the nucleic acid sequence of interest detects a chromosomal abnormality.

42. A method for isolating fetal erythroid cells in metaphase from a sample of peripheral blood from a pregnant woman, comprising:
obtaining a sample of peripheral blood from a pregnant woman;
culturing cells within the sample of peripheral blood in a culture media containing erythropoietin and a semisolid matrix material;
exposing cultured cells to an agent which inhibits progression of dividing cellsthrough the cell cycle or synchronizes growth of cells; and isolating fetal erythroid cells in metaphase.

43. The method of claim 42, wherein the agent which inhibits progression of dividing cells through the cell cycle is colcemid.

44. The method of claim 42, wherein the agent which inhibits progression of dividing cells through the cell cycle is colchicine.

45. The method of claim 42, wherein the agent which inhibits progression of dividing cells through the cell cycle is a vinblastine salt.

46. The method of claim 42, wherein the agent which synchronizes growth of cells is bromodeoxyuridine or fluorodeoxyuridine.

47. The method of claim 42, wherein the agent which synchronizes growth of cells is ethidium bromide.

48. A method for preferentially isolating fetal cells from a current pregnancy in a peripheral blood sample from a multiparous pregnant woman, comprising:
obtaining a sample of peripheral blood from a multiparous pregnant woman;

culturing cells within the sample of peripheral blood in a culture media containing erythropoietin and a semisolid matrix material; and isolating fetal erythroid cells to preferentially isolate fetal cells from a current pregnancy.

49. A method for isolating fetal erythroid cells from a sample of peripheral blood obtained from a pregnant woman, consisting of:
obtaining a sample of peripheral blood from a pregnant woman;
separating non-nucleated cells from nucleated cells in the peripheral blood sample to form a sample enriched in nucleated cells;
culturing the sample enriched in nucleated cells in a culture medium containing erythropoietin and a semisolid matrix material; and isolating fetal erythroid cells from the culture medium.

50. The method of claim 49, wherein non-nucleated cells are separated from nucleated cells by density gradient centrifugation.

51. The method of claim 49, wherein the non-nucleated cells are separated from nucleated cells by selective lysis of non-nucleated cells.

52. The method of claim 49, wherein the semisolid matrix material is methylcellulose.

53. The method of claim 49, wherein the semisolid matrix material is agargel or a plasma clot.

54. The method of claim 49, further consists of detecting a nucleic acid sequence of interest in fetal nucleic acid of isolated fetal erythroid cells.